Research ArticleIsolation and Expression Analysis of Novel SiliconAbsorption Gene from Roots of Mangrove (Rhizophoraapiculata) via Suppression Subtractive Hybridization
Mahbod Sahebi1 Mohamed M Hanafi12 Siti Nor Akmar Abdullah1 Mohd Y Rafii3
Parisa Azizi3 Naghmeh Nejat1 and Abu Seman Idris4
1 Laboratory of Plantations Crops Institute of Tropical Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia2 Department of Land Management Faculty of Agriculture 43400 Serdang Selangor Malaysia3 Laboratory of Food Crops Institute of Tropical Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia4 Biological Research Division GANODROP Unit Malaysia Palm Oil Board (MPOB) No 6 Persiaran Institusi Bandar Baru Bangi43000 Kajang Selangor Malaysia
Correspondence should be addressed to Mohamed M Hanafi mmhanafiagriupmedumy
Received 6 June 2013 Revised 17 October 2013 Accepted 21 October 2013 Published 1 January 2014
Academic Editor Rita Casadio
Copyright copy 2014 Mahbod Sahebi et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Silicon (Si) is the secondmost abundant element in soil after oxygen It is not an essential element for plant growth and formation butplays an important role in increasing plant tolerance towards different kinds of abiotic and biotic stressesThemolecularmechanismof Si absorption and accumulation may differ between plants such as monocotyledons and dicotyledons Silicon absorption andaccumulation in mangrove plants are affected indirectly by some proteins rich in serine and proline amino acids The expressionlevel of the genes responsible for Si absorption varies in different parts of plants In this study Si is mainly observed in the epidermalrootsrsquo cell walls of mangrove plants compared to other partsThe present work was carried out to discover further information on Sistress responsive genes in Rhizophora apiculata using the suppression subtractive hybridization technique To construct the cDNAlibrary two-month-old seedlings were exposed to 05 1 and 15mM SiO
2for 15 hrs and for 1 to 6 days resulting in a total of 360
high quality ESTs gained Further examination by RT-PCR and real-time qRT-PCR showed the expression of a candidate gene ofserine-rich protein
1 Introduction
Abiotic stresses such as drought high salinity temperaturechilling and high intensity light usually affect higher plantsby preventing plant growth Improvement in biotic andabiotic resistance in crops and trees plays a vital role inboth creation of sustainable agriculture systems throughsuppression of deleterious effects of global warming viadecreasing amounts of CO
2in the atmosphere and provision
of sufficient food sources in third-world countries Isolationof resistance-stress genes is an important key to improvestress-susceptibility in plants [1]
Mangrove plants which grow well in plant nutrient poorconditionswith high rate of salinity could be a valuable source
of antibiotic and abiotic stress genes Mangrove roots are alsoable to absorb water from anaerobic soils and in order tomaintain the absorbed water the plants need to respire easilywhich is enabled by their pneumatophores or aerial roots [2]
Mangrove forests have extremely productive ecosystemswith an average production of 2500mg C cmminus2 dayminus1 overand above a productivity factor of 4 in the shelf regionsto 40 in an open ocean [3ndash5] The high rate of organicmatter productivity and the external exchange with marineand terrestrial ecosystems via biochemical carbon cyclinghighlights the importance of the mangrove in tropical coasts[6]
Harsh environmental conditions provide for a great dealof physiological and basic adaptations in mangrove plants
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 971985 11 pageshttpdxdoiorg1011552014971985
Figure 4 Cellular components categorization of cDNA libraryresult
2
Biological process
ATP synthaseAuxin-responsive proteinCopia-type poly-protein
Equilibrative transporter
ATP synthase
Mitochondrial protein
5
2 2
89
Figure 5 Biological process categorization of the subtracted cDNAlibrary
which consequently allow them to overcome a wide rangeof abiotic stresses and survive Wetland sediments createdby rivers are significantly unsteady and anaerobic as well asfull of sulfates which lead to pressure on mangrove plants toadapt as far as possible [7]
A few efforts have been made to understand the intraspe-cific variations of mangroves and to predict the perfor-mance of mangrove ecosystems Mangrove ecosystems haveremained almost intact as a widespread gene pool because oflack of regular morphological variations between species andamong the populations although the structure of mangrove
BioMed Research International 3
72
Molecular function
ATP synthase Senescence-proteinCytochrome p450 rRNA endonucleaseProtein binding Auxin-responsive protein3-Dehydroquinatesynthase
Figure 6 Molecular function categorization of the subtractedcDNA library
Expr
essio
n ra
tio
05
1
8
16
32
64
2
4
15h 1 day 2 days 3 days 4 days 5 days 6 days
Figure 7 Relative quantity of serine-rich protein gene and actin as aninternal control Serine 15 h sample group is not different to controlgroup P value = 0169 Serine 1 day is up the regulated in samplegroup (in comparison to control group) P value = 0000 Serine2 days is up-regulated in sample group (in comparison to controlgroup) P value = 0000 Serine 3 days sample group is not differentto control group P value = 0339 Serine 4 days is up-regulated insample group (in comparison to control group) P value = 0000Serine 5 days is up-regulated in sample group (in comparison tocontrol group) P value = 0000 Serine 6 days is up-regulated insample group (in comparison to control group) P value = 0000
species population for many aquatic organisms has beenidentified [8ndash11] The major concern is to find how theirgenetic structure is organized and to determine the corre-lation between different traits which include adaptive andnonadaptive with migration of diverse genes which leads toevaluation of developmental changes in mangrove ecologicalconditions [8] Mangrove trees are capable of decreasingnutrient losses when there are changes in atmospheric
conditions by applying a variety of mechanisms includ-ing biogeochemical and physiological while exposed to awaterlogged and salty environment [12ndash14] Ion preservationimmobilization and translocation in soaked soil efficiencyof nutrient use which is the highest recorded among treesand the morphological shape of its roots probably play animportant role in establishing these mechanisms [14]
Among the plant nutrient elements in soil Si is the mostabundant after oxygen and essential for plant formationunder poor nutrient conditionsThe role of Si is not limited toplant growth as it also plays an important role in decreasingthe susceptibility of plants to different environmental stresses[15ndash19] Serine- and proline-rich proteins play a significantrole in plants with regard to Si absorption and transportation[20 21] In the present study we isolated and identified serine-rich protein genes from the roots of the mangrove plant (Rapiculata)
Currently many methods are being used to studydifferentials in the expression of genes including serialanalysis of gene expression (SAGE) differential displayreverse transcription-polymerase chain reaction (DDRT-PCR) cDNA microarray suppression subtractive hybridiza-tion (SSH) and cDNA-AFLP The false positive createdthrough the SSH technique is much lower compared to thatthrough the other methods [22 23]
2 Materials and Methods
21 Plant Materials Mangrove (Rhizophora apiculata) seedswere collected from Kuala Sepetang (04∘ 5015010158401015840N 100∘3762010158401015840) in Taiping Perak Malaysia They were grown inhydroponic culture for two months and then treated with05 1 and 15mM SiO
2for 15 hrs and for 1 to 6 days The
roots of the plants were collected immediately washed withdistilled water and frozen in liquid nitrogen to facilitate theRNA extraction process
22 Total RNA Extraction and Construction of the cDNALibrary The RNA extracted from the roots of the mangrovewas isolated using the CTAB method [24] The quality andintegrity of the extracted RNA were assessed using theNanoDrop ND-1000 spectrophotometer (NanoDrop Tech-nologies USA) Poly(A) + RNA was extracted from totalRNA using a PolyATtract mRNA Isolation Kit (PromegaUSA)
The subtracted cDNA library was constructed using thePCR-Select Subtractive Hybridization Kit (Clontech USA)following the manufacturerrsquos instructions In brief mRNAsfrom the last step for both control and treated sampleswere designated as driver and tester respectively The first-strand and double-strand cDNAs were then synthesized andthe synthesized double-strand cDNA of the tester samplewas digested with restriction enzyme Rsa I The digestedtester cDNA (blunt ends) was divided into two parts whichwere subsequently ligated with two different kinds of cDNAadaptors (long inverted terminal repeats) A and B In order tonormalize and enrich mangrove root development-related Siabsorption genes that are up- or down-regulated by Si stress
Figure 8 The nucleotide (696 bp) and deduced amino sequence (223 aa) of serine-rich protein
20015010050
minus3
minus2
minus1
0
1
2
3
Scor
e
Position
HphobKite and Doolittle
ProtScale output for user sequence
HphobKite and Doolittle
Figure 9 Analysis of hydrophilicity and hydrophobicity for theserine-rich proteinThis figure is the ProtScale output of hydrophilic-ity and hydrophobicity for the serine-rich protein
N
Amino acid composition
CH
VM
C
Q
Y
E
K
R
FA T V
I
G
P
L
S
Figure 10 Amino acid composition of serine-rich protein
CCCCCCEEEECCCCEEEEEEEEEEEEEEEEECCCCCCCCC
CCEEEEEECCCCCEEEEECCCCCCCCCCCCCCEEECCCEE
PCYLWTTVADYKQTRYYWGGRGTPLASPRRTPILLNEGKL
EEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCC
EVKPTSFLKSVPGPSYQGGGLGGSMASLLLEKMAPLELGP
CCCCCEEEECCCCEEEEEEECCCCCEEHHHHHCCCCHHHH
PDGPPLILRGKKRQIILIGVTSTSACLILLVSTTAGFFLQ
HHCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEEE
ERQNSASAMFRHTKIPIPSIASLASSPSMHSIFHVSQFRC
EECCCEEEEEEEEEEEEEEEEEC
210
Confidence of prediction
Strand Pred predicted secondary structureCoil AA target sequence
MVLSSALVYSTCSPVSVWGTVQFTGRIALPIRSFFPGSFP
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA IPRGECLAIVIVEIKFFKVCEVT
220
80706050
12011010090
160150140130
200190180170
40302010
Helix conf
Figure 11 Prediction of secondary structure for the serine-richprotein
BioMed Research International 5
Phobius posterior probabilities for serine-rich protein
TransmembraneCytoplasmic
NoncytoplasmicSignal peptide
0
50 100 200150
02
06
08
1
04
Poste
rior l
abel
prob
abili
ty
0
2
6
44
Figure 12 Prediction of subcellular location of serine-rich protein
Figure 13 3D structure of serine-rich protein The protein folds areshown in the colors of the rainbow from the N terminus (blue) tothe C terminus (red)
Figure 14 Result of the SSH cDNA library LaneM marker lane Asubtracted driver sample after second PCR lane B subtracted testersample after second PCR
(a)
(b)
Figure 15 Relative expression of serine-rich protein gene (a) andactin as an internal control (b) was amplified by semi-qRT-PCR Lmolecular ladder M untreated plants A 15 hrs silicon treated B 1-day silicon treated C 2-days silicon treated D 3-day silicon treatedE 4-day silicon treated F 5-day silicon treated and G 6-day silicontreated
two rounds of hybridizations and suppression PCR ampli-fication were processed The PCR products of secondaryPCR amplification were then purified and inserted directlyinto the pDrive UA cloning vector (Qiagen Germany) Theligated pDrive vectors were then transformed into E coliEZ cells and cultured overnight (16 hrs 37∘C) in LB agarmedium containing X-gal IPTG and ampicillin A totalof 400 independent positive white clones were picked outrandomly put in LB broth containing Amp and incubatedat 37∘C overnight to establish the mangrove root subtractivelibrary
23 EST Sequencing and Analysis About 400 positive cloneswere selected randomly and amplified using M13 primers(forward and reverse) after removal of contamination fromthe vector and primer sequence Before the assembly searchadaptors polyA tails low quality sequences short sequencesless than 100 bp in length and vector sequences wereremovedThe algorithm search of contigs and singletons wasperformed using CAP3 software This was followed by theobtained sequences being submitted to theNCBI database forhomology search
The BLASTn was used to show degree of similar-ity between the clone cDNA sequence and a knownsequence and the BLASTx (httpblastncbinlmnihgov)showed function of qualified cDNA sequences with largeORF regions Classification of cDNA sequences was basedon their E-value results in the BLAST Categories ofsequence functions are based on the Blast2GO program(httpwwwblast2goorg) [25]
Computational annotation of the mangrove EST datasetswas performed using the Blast2GO software v133 (httpwwwblast2goorg) The BLAST search was performed atNCBI [26] The degree of amino acid sequence similaritywas determined by the use of Wu-Blast from EBI [27]Hydrophilicity and hydrophobicity of serine-rich proteinwere predicted online by MemBrain TMHMM andProtScale (httpwebexpasy orgprotscale) in the toolkitof ExPASy Subcellular localization was investigated usingPSORT II Prediction and Cell-PLoc BaCelLo programThe prediction of secondary structure was carried outby (httpnpsa-pbilibcpfrcgi-binnpsa automatplpage
6 BioMed Research International
=npsa sopmahtml) and PsiPred program The prediction of3D structure was carried out by using the Pfam program
24 Amplification of Full-Length cDNA The complete CDSof serine-rich protein gene contained 696 bp and 223 aminoacids The PCR program according to KAPA HiFi Hot Startwas used as follows initial denaturation at 95∘C for 5min35 cycles of denaturation at 98∘C for 30 s annealing at575∘C for 30 s and extension at 72∘C for 1min The finalextension was 5min at 72∘C Agarose gel (15) was usedto separate PCR-amplified cDNA fragments The expectedbound about 700 bp was purified using gel purificationkit (Qiagen Germany) and 31015840-dA-overhangs (incubation72∘C for 5min) added to the blunt-ended DNA fragmentsgenerated by KAPAHiFi Hot Start DNA polymerase to ligateinto the pDrive cloning vector (Qiagen Germany) and sentfor sequencing
25 Semiquantitative RT-PCRAnalysis Reverse transcriptaseRT-PCR was performed to study the expression of theserine-rich protein gene One120583L of DNase treated (DNaseI Qiagen Germany) total RNA from each of the man-grove roots treated with Si for 15 hrs and 1 to 6 days anduntreated plants was transcribed to the first-strand cDNAusing SuperScript III (Invitrogen USA) and 500 ng oligo(dT) 18 primer in 20120583L reaction volume Reactions werethen incubated at 50∘C for 60min and heated to inactiveat 70∘C for 15min The template cDNAs for both control(untreated) and treated samples were then amplified usingserine primers as F 5-GTCATTCTGCCGAGTTCC-3 and R5-AATGCCCATTTATGTGACTTCG-3 designed accordingto the cDNA sequence homology
Actin gene as an internal control was amplified with thefollowing primers F (51015840CAC TAC TAC TGC TAA ACG GGAAA 31015840) and R (51015840ACA TCT GCT GGA AGG TGC TG 31015840)The following PCR (Tag DNA Polymerase Vivantis USA)program was used 94∘C for 2min and 35 cycles of 94∘C for30 sec 575 and 58∘C respectively for actin and serine for30 sec and 72∘C for 30 sec The PCR program was concludedwith final extension of 7min at 72∘C Actin and serine-richprotein were amplified using the same cDNA templates ThePCR products then were separated with 15 agarose gel andstained with ethidium bromide
26 Real-Time Quantitative RT-PCR Analysis Real-timeqRT-PCR was performed to evaluate the expression lev-els of candidate serine-rich protein genes in the root tis-sues in response to treatments with different concentra-tions of Si compared to these in the control plants TotalRNA was extracted from untreated and Si-treated man-grove roots Extracted RNA was then treated with DNase(DNase I Qiagen Germany) The primers for the actingene were used as in the previous section and for thesecond endogenous control ef1205721 was F 51015840 rarr 31015840 ATTGGA AAC GGA TAT GCT CCA R 51015840 rarr 31015840 TCCTTA CCT GAA CGC CTG TCA serine-rich protein geneprimer was F 51015840 rarr 31015840GCAAGTGCTATGTTCAGGCA R51015840 rarr 31015840AACAATAACGATGGCAAGGC One 120583L aliquot of
DNase treated RNA from each sample was used to prepare20120583L reaction volumes (based on the KAPA SYBER FASTOne-Step qRT-PCR) The reactions involved were a primaryincubation of 42∘C for 5min inactive RT at 95∘C for 5minfollowed by 40 cycles at 95∘C for 3 sec 60∘C for 30 sec and72∘C for 3 sec The 96-well plate was used to analyze eachsample in duplicate In this study all data was from fourindependent biological replicates A standard curve (1198772 gt095) from 10-fold serial dilutions was generated from thepurified cDNA fragments of internal and serine-rich proteingenes (102 10 1 10minus1 and 10minus2 ng120583L)
3 Results
31 Yield and Integrity of RNA The RNA appeared as a non-degraded band on 15 agarose gel containing formaldehydeThe A
260280ratios ranging from 19 to 202 indicate that
there was no protein contamination and the A260230
ratio gt1demonstrated that there is no polyphenol or polysaccharidecontamination [28]The RNA concentration was in the rangeof 05ndash12mg gminus1 The poly(A) + RNA of both treated anduntreated samples appeared as clear smears on the 1 agarosegelwith theA
260280ratio = 2 indicating high quality ofmRNA
obtained for further analysis
32 Construction of the Subtracted cDNA Library The prod-ucts of the subtracted suppression hybridization cDNAlibrary appeared on 12 agarose gel as a smear with rankingsize from 150 bp to 12 kb and 4ndash6 separate bands (Figure 14line A) which obviously differentiates them from the unsub-tracted sample driver (Figure 14 line B) The results of theSSH cDNA library indicated that the differentially expressedgenes are present in the tester or treated samples and absentor present at lower levels in the driver or untreated samplesFor further confirmation of subtraction analysis efficiencyexpression of the actin gene was examined in both the driverand control samples via 23 and 33 cycles of amplificationrespectively indicating that cDNA homologue was removedfrom both the tester and driver samples by subtraction(Figure 14)
33 ESTs Sequencing and Gene Annotation About 700 pos-itive recombinant clones were isolated from the cDNAlibrary Of those about 400 clones were randomly selectedsequenced and analyzed to isolate the gene(s) involved inSi transportation and absorption The 322 ESTs sequenceswere coalesced into 21 contigs and 13 singletons by CAP3assembly program About 195 of the ESTs resulting fromthis library did not have any significant homology to any ofthe proteins existing in the database The DNA fragmentsinvolved in the subtracted cDNA library varied in sizemore or less between 100 and 650 bp (Figure 1) Averagelength of high quality ESTs is 350 bp The most plentifulBLASTx hits correlated to species distribution related toLilium longiflorum Glycinemax Cupressus sempervirens andArabidopsis thaliana (Table 1 and Figure 2)
BioMed Research International 7
34 Classification of Differentially Expressed Genes Accord-ing to gene annotation the 322 ESTs were divided intothree different groups involving biological process molecularfunction and cellular components (Figure 3) The potentialidentities of the genes are based on similarities to thosepresent in the Gen-Bank databases The genes obtained bythe SSH library classified by biological process involve 5different groups ATP synthase (889) equilibrative trans-porter (44) auxin-responsive protein (22) mitochon-drial protein (22) and copia-type polyprotein (22)those classified according to cellular components involve 3different groups The ATP synthase was highly abundantin both the cellular components and biological processcategories (Figures 4 and 5) while senescence-protein wasthe most abundant group in the molecular function category(Figure 6) Classification of the ESTs resulted in 94 ofknown 4 hypothetical and 2 unknown functions
35 Isolation of the Full-Length Serine-Rich Protein GeneOne of the ESTs sequence showing 97 similarity involvesthe ATG codon (20 query cover region) with completeCDS of the serine-rich protein gene of Arachis hypogaea(Figure 7) Full length of the gene (Figure 8) was obtainedthrough amplification of the cDNA template (First-StrandcDNA synthesis Invitrogen USA) and using gene-specificprimers as follows
serine
F 51015840 rarr 31015840 GTCATTCTGCCGAGTTCC
R 51015840 rarr 31015840AATGCCCATTTATGTGACTTCG
36 Analysis of the Differential Expression of Serine-RichProtein Gene Using Semi-qRT-PCR and Real-Time qRT-PCR The semiquantitative RT-PCR analysis showed that theexpression levels of serine-rich protein were generally higherin the Si treatment samples than in the untreated samplesThe expression level of serine-rich protein in the 3-day treatedsample was lower compared to that in the other treatedsamples with varying periods of time (Figure 15) Real-timeqRT-PCR confirmed the results of the semi quantitative RT-PCR (Figure 8) The relative transcript abundance of serine-rich protein in Si-treated plants was higher compared to thatin the untreated plants
The PCR efficiency of all reactions in this study wasbetween 87 and 98 The REST software (Qiagen HildenGermany) was employed to analyze the results of the qRT-PCRThemanufacturerrsquos instructions were followed to quan-tify the relative gene expressionDifferences among treatmentsamples were noted as being statistically significant (119875 lt005)
37 Bioinformatics Analysis The low quality regions at theend and beginning of each sequence were trimmed usinga Phred 20 cutoff value Vector screening was carried outusing the crossmatch Oligo dT tracks and other contam-inants were removed Algorithms of CAP3 [29] assemblywere used to assemble the individual ESTs into clusters of
sequences derived from the same transcript as tentative con-sensus sequences (TCs) and singletons representing uniquetranscripts Obtained sequences were submitted to NCBIdatabase
Prediction of hydrophilicity and hydrophobicity isneeded for predicting protein secondary structure anddivision of functional domain according to the theory thathydrophilicity and hydrophobicity are related to the score ofamino acid Based on the number of hydrophilic amino acidresidues we could hypothesize that the serine-rich proteinwas a hydrophilic protein (Figures 9 and 10)
38 The Prediction of Secondary Structure and FunctionDomain of Serine-Rich Protein The prediction results ofsecondary structure of serine-rich protein by PsiPred showedthat the secondary structure consisted of 11 sheets 3 helixesand 15 coils (Figure 11) Computational analysis of the cDNAclone isolated from mangrove root library indicated that its696 bp coding region codes for a protein of 223 amino acidswith a predicted molecular mass of 2421 kDa Homologysearches run with the full-length amino acid sequences
39 Subcellular Localization The prediction of subcellularlocalization with Cell-PLoc BaCelLo and WoLF PSORTshowed that serine-rich protein is likely to be localizedin chloroplast plastid and mitochondrion with a differ-ent probability of 17 5 and 1 respectively (Figure 12)Biosequence analysis of coding sequence region (CDS) ofserine-rich protein using profile hidden Markov models(HMMER) showed 60 similarity to serine-rich protein ofArachis hypogaea (TRQ0MX20 ARAHY) and 100 sim-ilarity to mitochondrial protein of Medicago truncatula(TRG7I9T8 MEDTR) (Table 2)
310 Prediction of 3D Structure In order to predict the3D structure of serine-rich protein the Phyre2 (ProteinHomology Analogy Recognition Engine) server was used[30] A Phyre2 outputmodel was generated based on the tem-plate galactose-binding domain-like Structural alignment ofserine-rich protein and galactose-binding domain-like wasperformed using theMatchmaker tool of UCSFChimera [31]The 3D structure protein of serine-rich protein gene isolatedand identified in the present study submitted to NCBI withaccession number KF211374 involves MYP bZip LCR1 andDOF transcription factor binding motifs (Figure 13)
4 Discussion
In the present study the suppression subtractive hybridiza-tion (SSH) method was used to identify differentiallyexpressed genes present in the tester sample and absent orpresent at lower levels in the driver The SSH cDNA librarywas constructed using SiO
2-treated (15 hrs and 1 to 6 days)
roots of mangrove plants whereby 321 unique genes wereobtainedThe SSHmethod provides information related onlyto global analysis of gene expression and hence semi quan-titative RT-PCR and real-time qRT-PCR were performedto evaluate the quantitative level of gene expressions over
8 BioMed Research International
Table 1 Putative identities of novel silicon-induced cDNA sequences expressed in R apiculata roots
Gen-Bankaccession Homology Organism 119864-value
Stress responseNP 1995641 Putative auxin-responsive protein Arabidopsis thaliana 500119864 minus 19
DQ8346901 Serine-rich protein mRNA Arachis hypogaea 100119864 minus 72
AAB709281 Proline-rich protein Santalum album 700119864 minus 11
TransporterAET022081 ATP synthase subunit beta Medicago truncatula 300119864 minus 44
XP 0023172511 Equilibrative nucleoside transporter Populus trichocarpa 700119864 minus 40
UnknownXP 0036143941 Hypothetical protein MTR 5g051130 Medicago truncatula 100119864 minus 38
XP 0035471291 Predicted uncharacterized proteinLOC100801 Glycine max 600119864 minus 36
XP 0022691672 Predicted uncharacterized proteinLOC100267 Vitis vinifera 100119864 minus 30
XP 0035490071 Uncharacterized protein LOC100791 Glycine max 500119864 minus 43
XP 0035412191 Uncharacterized protein LOC100796 Glycine max 500119864 minus 43
XP 0035412061 Predicted uncharacterized proteinLOC100788 Glycine max 500119864 minus 43
Cellularmetabolism
XP 0031514401 Senescence-associated protein Loa loa 800119864 minus 25
ACA048501 Senescence-associated protein Picea abies 300119864 minus 21
XP 0021396981 Senescence-associated protein Cryptosporidium hominis TU502 200119864 minus 19
AEM360701 Senescence-associated protein Mytilus eduls 200119864 minus 18
EFW131811 Senescence-associated protein Coccidioides posadasii str 700119864 minus 18
XP 0021182661 Senescence-associated protein Trichoplax adhaerens 100119864 minus 17
XP 0016105631 Senescence-associated protein Babesia bovis T2Bo 100119864 minus 16
XP 0031886401 Plant senescence-associated protein Aspergillus niger CBS 51388 500119864 minus 16
EFN584411 Senescence-associated protein Chlorella variabilis 200119864 minus 11
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 400119864 minus 10
XP 0030649921 Senescence-associated protein Micromonas pusilla CCMP1545 100119864 minus 09
EEH167211 Senescence-associated protein Paracoccidioides brasiliensis 200119864 minus 09
XP 7297621 Senescence-associated protein Plasmodium yoelii yoelii 200119864 minus 06
ABO208511 Putative senescence-associated protein Lilium longiflorum 100119864 minus 41
BAB334211 Putative senescence-associated protein Pisum sativum 900119864 minus 17
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 100119864 minus 15
BAD189051 rRNA intron-encoded endonuclease Thermoproteus sp IC-061 600119864 minus 06
XP 0025153521 Protein binding protein putative Ricinus communis 300119864 minus 34
XM 0035883071 3-Dehydroquinate synthase putative Medicago truncatula 500119864 minus 39
AES586061 Mitochondrial protein putative Medicago truncatula 300119864 minus 37
CAB710631 Copia-type polyprotein Arabidopsis thaliana 300119864 minus 13
AT4g15300 Cytochrome p450-like tbp protein Arabidopsis thaliana 200119864 minus 27
varying periods of Si treatment Most sequences recognizedfrom this subtracted library encoded different genes withhomology of genes from 13 known and 2 unknown species
One EST with known function was selected for analysisof Si-induced expression patterns in varied periods of time
using semi quantitative reverse transcriptase and real-timeqRT-PCR (Figures 15 and 7)
The SSH cDNA library of Si-treated genes was monitoredfor seven times From SSH reports it appears that somegene induction patterns are so rapid and self-motivated that
BioMed Research International 9
Table 2 Pfam domain search option for genomic and proteic annotation
Target Description Species 119864-valueQ0MX20 ARAHY Serine-rich protein Arachis hypogaea 680119864minus147
I3S491 LOTJA Uncharacterized protein Lotus japonicus 490119864 minus 05
G7I9T8 MEDTR Mitochondrial protein putative(geneMTR 1g006300) Medicago truncatula 000084
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
Figure 4 Cellular components categorization of cDNA libraryresult
2
Biological process
ATP synthaseAuxin-responsive proteinCopia-type poly-protein
Equilibrative transporter
ATP synthase
Mitochondrial protein
5
2 2
89
Figure 5 Biological process categorization of the subtracted cDNAlibrary
which consequently allow them to overcome a wide rangeof abiotic stresses and survive Wetland sediments createdby rivers are significantly unsteady and anaerobic as well asfull of sulfates which lead to pressure on mangrove plants toadapt as far as possible [7]
A few efforts have been made to understand the intraspe-cific variations of mangroves and to predict the perfor-mance of mangrove ecosystems Mangrove ecosystems haveremained almost intact as a widespread gene pool because oflack of regular morphological variations between species andamong the populations although the structure of mangrove
BioMed Research International 3
72
Molecular function
ATP synthase Senescence-proteinCytochrome p450 rRNA endonucleaseProtein binding Auxin-responsive protein3-Dehydroquinatesynthase
Figure 6 Molecular function categorization of the subtractedcDNA library
Expr
essio
n ra
tio
05
1
8
16
32
64
2
4
15h 1 day 2 days 3 days 4 days 5 days 6 days
Figure 7 Relative quantity of serine-rich protein gene and actin as aninternal control Serine 15 h sample group is not different to controlgroup P value = 0169 Serine 1 day is up the regulated in samplegroup (in comparison to control group) P value = 0000 Serine2 days is up-regulated in sample group (in comparison to controlgroup) P value = 0000 Serine 3 days sample group is not differentto control group P value = 0339 Serine 4 days is up-regulated insample group (in comparison to control group) P value = 0000Serine 5 days is up-regulated in sample group (in comparison tocontrol group) P value = 0000 Serine 6 days is up-regulated insample group (in comparison to control group) P value = 0000
species population for many aquatic organisms has beenidentified [8ndash11] The major concern is to find how theirgenetic structure is organized and to determine the corre-lation between different traits which include adaptive andnonadaptive with migration of diverse genes which leads toevaluation of developmental changes in mangrove ecologicalconditions [8] Mangrove trees are capable of decreasingnutrient losses when there are changes in atmospheric
conditions by applying a variety of mechanisms includ-ing biogeochemical and physiological while exposed to awaterlogged and salty environment [12ndash14] Ion preservationimmobilization and translocation in soaked soil efficiencyof nutrient use which is the highest recorded among treesand the morphological shape of its roots probably play animportant role in establishing these mechanisms [14]
Among the plant nutrient elements in soil Si is the mostabundant after oxygen and essential for plant formationunder poor nutrient conditionsThe role of Si is not limited toplant growth as it also plays an important role in decreasingthe susceptibility of plants to different environmental stresses[15ndash19] Serine- and proline-rich proteins play a significantrole in plants with regard to Si absorption and transportation[20 21] In the present study we isolated and identified serine-rich protein genes from the roots of the mangrove plant (Rapiculata)
Currently many methods are being used to studydifferentials in the expression of genes including serialanalysis of gene expression (SAGE) differential displayreverse transcription-polymerase chain reaction (DDRT-PCR) cDNA microarray suppression subtractive hybridiza-tion (SSH) and cDNA-AFLP The false positive createdthrough the SSH technique is much lower compared to thatthrough the other methods [22 23]
2 Materials and Methods
21 Plant Materials Mangrove (Rhizophora apiculata) seedswere collected from Kuala Sepetang (04∘ 5015010158401015840N 100∘3762010158401015840) in Taiping Perak Malaysia They were grown inhydroponic culture for two months and then treated with05 1 and 15mM SiO
2for 15 hrs and for 1 to 6 days The
roots of the plants were collected immediately washed withdistilled water and frozen in liquid nitrogen to facilitate theRNA extraction process
22 Total RNA Extraction and Construction of the cDNALibrary The RNA extracted from the roots of the mangrovewas isolated using the CTAB method [24] The quality andintegrity of the extracted RNA were assessed using theNanoDrop ND-1000 spectrophotometer (NanoDrop Tech-nologies USA) Poly(A) + RNA was extracted from totalRNA using a PolyATtract mRNA Isolation Kit (PromegaUSA)
The subtracted cDNA library was constructed using thePCR-Select Subtractive Hybridization Kit (Clontech USA)following the manufacturerrsquos instructions In brief mRNAsfrom the last step for both control and treated sampleswere designated as driver and tester respectively The first-strand and double-strand cDNAs were then synthesized andthe synthesized double-strand cDNA of the tester samplewas digested with restriction enzyme Rsa I The digestedtester cDNA (blunt ends) was divided into two parts whichwere subsequently ligated with two different kinds of cDNAadaptors (long inverted terminal repeats) A and B In order tonormalize and enrich mangrove root development-related Siabsorption genes that are up- or down-regulated by Si stress
Figure 8 The nucleotide (696 bp) and deduced amino sequence (223 aa) of serine-rich protein
20015010050
minus3
minus2
minus1
0
1
2
3
Scor
e
Position
HphobKite and Doolittle
ProtScale output for user sequence
HphobKite and Doolittle
Figure 9 Analysis of hydrophilicity and hydrophobicity for theserine-rich proteinThis figure is the ProtScale output of hydrophilic-ity and hydrophobicity for the serine-rich protein
N
Amino acid composition
CH
VM
C
Q
Y
E
K
R
FA T V
I
G
P
L
S
Figure 10 Amino acid composition of serine-rich protein
CCCCCCEEEECCCCEEEEEEEEEEEEEEEEECCCCCCCCC
CCEEEEEECCCCCEEEEECCCCCCCCCCCCCCEEECCCEE
PCYLWTTVADYKQTRYYWGGRGTPLASPRRTPILLNEGKL
EEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCC
EVKPTSFLKSVPGPSYQGGGLGGSMASLLLEKMAPLELGP
CCCCCEEEECCCCEEEEEEECCCCCEEHHHHHCCCCHHHH
PDGPPLILRGKKRQIILIGVTSTSACLILLVSTTAGFFLQ
HHCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEEE
ERQNSASAMFRHTKIPIPSIASLASSPSMHSIFHVSQFRC
EECCCEEEEEEEEEEEEEEEEEC
210
Confidence of prediction
Strand Pred predicted secondary structureCoil AA target sequence
MVLSSALVYSTCSPVSVWGTVQFTGRIALPIRSFFPGSFP
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA IPRGECLAIVIVEIKFFKVCEVT
220
80706050
12011010090
160150140130
200190180170
40302010
Helix conf
Figure 11 Prediction of secondary structure for the serine-richprotein
BioMed Research International 5
Phobius posterior probabilities for serine-rich protein
TransmembraneCytoplasmic
NoncytoplasmicSignal peptide
0
50 100 200150
02
06
08
1
04
Poste
rior l
abel
prob
abili
ty
0
2
6
44
Figure 12 Prediction of subcellular location of serine-rich protein
Figure 13 3D structure of serine-rich protein The protein folds areshown in the colors of the rainbow from the N terminus (blue) tothe C terminus (red)
Figure 14 Result of the SSH cDNA library LaneM marker lane Asubtracted driver sample after second PCR lane B subtracted testersample after second PCR
(a)
(b)
Figure 15 Relative expression of serine-rich protein gene (a) andactin as an internal control (b) was amplified by semi-qRT-PCR Lmolecular ladder M untreated plants A 15 hrs silicon treated B 1-day silicon treated C 2-days silicon treated D 3-day silicon treatedE 4-day silicon treated F 5-day silicon treated and G 6-day silicontreated
two rounds of hybridizations and suppression PCR ampli-fication were processed The PCR products of secondaryPCR amplification were then purified and inserted directlyinto the pDrive UA cloning vector (Qiagen Germany) Theligated pDrive vectors were then transformed into E coliEZ cells and cultured overnight (16 hrs 37∘C) in LB agarmedium containing X-gal IPTG and ampicillin A totalof 400 independent positive white clones were picked outrandomly put in LB broth containing Amp and incubatedat 37∘C overnight to establish the mangrove root subtractivelibrary
23 EST Sequencing and Analysis About 400 positive cloneswere selected randomly and amplified using M13 primers(forward and reverse) after removal of contamination fromthe vector and primer sequence Before the assembly searchadaptors polyA tails low quality sequences short sequencesless than 100 bp in length and vector sequences wereremovedThe algorithm search of contigs and singletons wasperformed using CAP3 software This was followed by theobtained sequences being submitted to theNCBI database forhomology search
The BLASTn was used to show degree of similar-ity between the clone cDNA sequence and a knownsequence and the BLASTx (httpblastncbinlmnihgov)showed function of qualified cDNA sequences with largeORF regions Classification of cDNA sequences was basedon their E-value results in the BLAST Categories ofsequence functions are based on the Blast2GO program(httpwwwblast2goorg) [25]
Computational annotation of the mangrove EST datasetswas performed using the Blast2GO software v133 (httpwwwblast2goorg) The BLAST search was performed atNCBI [26] The degree of amino acid sequence similaritywas determined by the use of Wu-Blast from EBI [27]Hydrophilicity and hydrophobicity of serine-rich proteinwere predicted online by MemBrain TMHMM andProtScale (httpwebexpasy orgprotscale) in the toolkitof ExPASy Subcellular localization was investigated usingPSORT II Prediction and Cell-PLoc BaCelLo programThe prediction of secondary structure was carried outby (httpnpsa-pbilibcpfrcgi-binnpsa automatplpage
6 BioMed Research International
=npsa sopmahtml) and PsiPred program The prediction of3D structure was carried out by using the Pfam program
24 Amplification of Full-Length cDNA The complete CDSof serine-rich protein gene contained 696 bp and 223 aminoacids The PCR program according to KAPA HiFi Hot Startwas used as follows initial denaturation at 95∘C for 5min35 cycles of denaturation at 98∘C for 30 s annealing at575∘C for 30 s and extension at 72∘C for 1min The finalextension was 5min at 72∘C Agarose gel (15) was usedto separate PCR-amplified cDNA fragments The expectedbound about 700 bp was purified using gel purificationkit (Qiagen Germany) and 31015840-dA-overhangs (incubation72∘C for 5min) added to the blunt-ended DNA fragmentsgenerated by KAPAHiFi Hot Start DNA polymerase to ligateinto the pDrive cloning vector (Qiagen Germany) and sentfor sequencing
25 Semiquantitative RT-PCRAnalysis Reverse transcriptaseRT-PCR was performed to study the expression of theserine-rich protein gene One120583L of DNase treated (DNaseI Qiagen Germany) total RNA from each of the man-grove roots treated with Si for 15 hrs and 1 to 6 days anduntreated plants was transcribed to the first-strand cDNAusing SuperScript III (Invitrogen USA) and 500 ng oligo(dT) 18 primer in 20120583L reaction volume Reactions werethen incubated at 50∘C for 60min and heated to inactiveat 70∘C for 15min The template cDNAs for both control(untreated) and treated samples were then amplified usingserine primers as F 5-GTCATTCTGCCGAGTTCC-3 and R5-AATGCCCATTTATGTGACTTCG-3 designed accordingto the cDNA sequence homology
Actin gene as an internal control was amplified with thefollowing primers F (51015840CAC TAC TAC TGC TAA ACG GGAAA 31015840) and R (51015840ACA TCT GCT GGA AGG TGC TG 31015840)The following PCR (Tag DNA Polymerase Vivantis USA)program was used 94∘C for 2min and 35 cycles of 94∘C for30 sec 575 and 58∘C respectively for actin and serine for30 sec and 72∘C for 30 sec The PCR program was concludedwith final extension of 7min at 72∘C Actin and serine-richprotein were amplified using the same cDNA templates ThePCR products then were separated with 15 agarose gel andstained with ethidium bromide
26 Real-Time Quantitative RT-PCR Analysis Real-timeqRT-PCR was performed to evaluate the expression lev-els of candidate serine-rich protein genes in the root tis-sues in response to treatments with different concentra-tions of Si compared to these in the control plants TotalRNA was extracted from untreated and Si-treated man-grove roots Extracted RNA was then treated with DNase(DNase I Qiagen Germany) The primers for the actingene were used as in the previous section and for thesecond endogenous control ef1205721 was F 51015840 rarr 31015840 ATTGGA AAC GGA TAT GCT CCA R 51015840 rarr 31015840 TCCTTA CCT GAA CGC CTG TCA serine-rich protein geneprimer was F 51015840 rarr 31015840GCAAGTGCTATGTTCAGGCA R51015840 rarr 31015840AACAATAACGATGGCAAGGC One 120583L aliquot of
DNase treated RNA from each sample was used to prepare20120583L reaction volumes (based on the KAPA SYBER FASTOne-Step qRT-PCR) The reactions involved were a primaryincubation of 42∘C for 5min inactive RT at 95∘C for 5minfollowed by 40 cycles at 95∘C for 3 sec 60∘C for 30 sec and72∘C for 3 sec The 96-well plate was used to analyze eachsample in duplicate In this study all data was from fourindependent biological replicates A standard curve (1198772 gt095) from 10-fold serial dilutions was generated from thepurified cDNA fragments of internal and serine-rich proteingenes (102 10 1 10minus1 and 10minus2 ng120583L)
3 Results
31 Yield and Integrity of RNA The RNA appeared as a non-degraded band on 15 agarose gel containing formaldehydeThe A
260280ratios ranging from 19 to 202 indicate that
there was no protein contamination and the A260230
ratio gt1demonstrated that there is no polyphenol or polysaccharidecontamination [28]The RNA concentration was in the rangeof 05ndash12mg gminus1 The poly(A) + RNA of both treated anduntreated samples appeared as clear smears on the 1 agarosegelwith theA
260280ratio = 2 indicating high quality ofmRNA
obtained for further analysis
32 Construction of the Subtracted cDNA Library The prod-ucts of the subtracted suppression hybridization cDNAlibrary appeared on 12 agarose gel as a smear with rankingsize from 150 bp to 12 kb and 4ndash6 separate bands (Figure 14line A) which obviously differentiates them from the unsub-tracted sample driver (Figure 14 line B) The results of theSSH cDNA library indicated that the differentially expressedgenes are present in the tester or treated samples and absentor present at lower levels in the driver or untreated samplesFor further confirmation of subtraction analysis efficiencyexpression of the actin gene was examined in both the driverand control samples via 23 and 33 cycles of amplificationrespectively indicating that cDNA homologue was removedfrom both the tester and driver samples by subtraction(Figure 14)
33 ESTs Sequencing and Gene Annotation About 700 pos-itive recombinant clones were isolated from the cDNAlibrary Of those about 400 clones were randomly selectedsequenced and analyzed to isolate the gene(s) involved inSi transportation and absorption The 322 ESTs sequenceswere coalesced into 21 contigs and 13 singletons by CAP3assembly program About 195 of the ESTs resulting fromthis library did not have any significant homology to any ofthe proteins existing in the database The DNA fragmentsinvolved in the subtracted cDNA library varied in sizemore or less between 100 and 650 bp (Figure 1) Averagelength of high quality ESTs is 350 bp The most plentifulBLASTx hits correlated to species distribution related toLilium longiflorum Glycinemax Cupressus sempervirens andArabidopsis thaliana (Table 1 and Figure 2)
BioMed Research International 7
34 Classification of Differentially Expressed Genes Accord-ing to gene annotation the 322 ESTs were divided intothree different groups involving biological process molecularfunction and cellular components (Figure 3) The potentialidentities of the genes are based on similarities to thosepresent in the Gen-Bank databases The genes obtained bythe SSH library classified by biological process involve 5different groups ATP synthase (889) equilibrative trans-porter (44) auxin-responsive protein (22) mitochon-drial protein (22) and copia-type polyprotein (22)those classified according to cellular components involve 3different groups The ATP synthase was highly abundantin both the cellular components and biological processcategories (Figures 4 and 5) while senescence-protein wasthe most abundant group in the molecular function category(Figure 6) Classification of the ESTs resulted in 94 ofknown 4 hypothetical and 2 unknown functions
35 Isolation of the Full-Length Serine-Rich Protein GeneOne of the ESTs sequence showing 97 similarity involvesthe ATG codon (20 query cover region) with completeCDS of the serine-rich protein gene of Arachis hypogaea(Figure 7) Full length of the gene (Figure 8) was obtainedthrough amplification of the cDNA template (First-StrandcDNA synthesis Invitrogen USA) and using gene-specificprimers as follows
serine
F 51015840 rarr 31015840 GTCATTCTGCCGAGTTCC
R 51015840 rarr 31015840AATGCCCATTTATGTGACTTCG
36 Analysis of the Differential Expression of Serine-RichProtein Gene Using Semi-qRT-PCR and Real-Time qRT-PCR The semiquantitative RT-PCR analysis showed that theexpression levels of serine-rich protein were generally higherin the Si treatment samples than in the untreated samplesThe expression level of serine-rich protein in the 3-day treatedsample was lower compared to that in the other treatedsamples with varying periods of time (Figure 15) Real-timeqRT-PCR confirmed the results of the semi quantitative RT-PCR (Figure 8) The relative transcript abundance of serine-rich protein in Si-treated plants was higher compared to thatin the untreated plants
The PCR efficiency of all reactions in this study wasbetween 87 and 98 The REST software (Qiagen HildenGermany) was employed to analyze the results of the qRT-PCRThemanufacturerrsquos instructions were followed to quan-tify the relative gene expressionDifferences among treatmentsamples were noted as being statistically significant (119875 lt005)
37 Bioinformatics Analysis The low quality regions at theend and beginning of each sequence were trimmed usinga Phred 20 cutoff value Vector screening was carried outusing the crossmatch Oligo dT tracks and other contam-inants were removed Algorithms of CAP3 [29] assemblywere used to assemble the individual ESTs into clusters of
sequences derived from the same transcript as tentative con-sensus sequences (TCs) and singletons representing uniquetranscripts Obtained sequences were submitted to NCBIdatabase
Prediction of hydrophilicity and hydrophobicity isneeded for predicting protein secondary structure anddivision of functional domain according to the theory thathydrophilicity and hydrophobicity are related to the score ofamino acid Based on the number of hydrophilic amino acidresidues we could hypothesize that the serine-rich proteinwas a hydrophilic protein (Figures 9 and 10)
38 The Prediction of Secondary Structure and FunctionDomain of Serine-Rich Protein The prediction results ofsecondary structure of serine-rich protein by PsiPred showedthat the secondary structure consisted of 11 sheets 3 helixesand 15 coils (Figure 11) Computational analysis of the cDNAclone isolated from mangrove root library indicated that its696 bp coding region codes for a protein of 223 amino acidswith a predicted molecular mass of 2421 kDa Homologysearches run with the full-length amino acid sequences
39 Subcellular Localization The prediction of subcellularlocalization with Cell-PLoc BaCelLo and WoLF PSORTshowed that serine-rich protein is likely to be localizedin chloroplast plastid and mitochondrion with a differ-ent probability of 17 5 and 1 respectively (Figure 12)Biosequence analysis of coding sequence region (CDS) ofserine-rich protein using profile hidden Markov models(HMMER) showed 60 similarity to serine-rich protein ofArachis hypogaea (TRQ0MX20 ARAHY) and 100 sim-ilarity to mitochondrial protein of Medicago truncatula(TRG7I9T8 MEDTR) (Table 2)
310 Prediction of 3D Structure In order to predict the3D structure of serine-rich protein the Phyre2 (ProteinHomology Analogy Recognition Engine) server was used[30] A Phyre2 outputmodel was generated based on the tem-plate galactose-binding domain-like Structural alignment ofserine-rich protein and galactose-binding domain-like wasperformed using theMatchmaker tool of UCSFChimera [31]The 3D structure protein of serine-rich protein gene isolatedand identified in the present study submitted to NCBI withaccession number KF211374 involves MYP bZip LCR1 andDOF transcription factor binding motifs (Figure 13)
4 Discussion
In the present study the suppression subtractive hybridiza-tion (SSH) method was used to identify differentiallyexpressed genes present in the tester sample and absent orpresent at lower levels in the driver The SSH cDNA librarywas constructed using SiO
2-treated (15 hrs and 1 to 6 days)
roots of mangrove plants whereby 321 unique genes wereobtainedThe SSHmethod provides information related onlyto global analysis of gene expression and hence semi quan-titative RT-PCR and real-time qRT-PCR were performedto evaluate the quantitative level of gene expressions over
8 BioMed Research International
Table 1 Putative identities of novel silicon-induced cDNA sequences expressed in R apiculata roots
Gen-Bankaccession Homology Organism 119864-value
Stress responseNP 1995641 Putative auxin-responsive protein Arabidopsis thaliana 500119864 minus 19
DQ8346901 Serine-rich protein mRNA Arachis hypogaea 100119864 minus 72
AAB709281 Proline-rich protein Santalum album 700119864 minus 11
TransporterAET022081 ATP synthase subunit beta Medicago truncatula 300119864 minus 44
XP 0023172511 Equilibrative nucleoside transporter Populus trichocarpa 700119864 minus 40
UnknownXP 0036143941 Hypothetical protein MTR 5g051130 Medicago truncatula 100119864 minus 38
XP 0035471291 Predicted uncharacterized proteinLOC100801 Glycine max 600119864 minus 36
XP 0022691672 Predicted uncharacterized proteinLOC100267 Vitis vinifera 100119864 minus 30
XP 0035490071 Uncharacterized protein LOC100791 Glycine max 500119864 minus 43
XP 0035412191 Uncharacterized protein LOC100796 Glycine max 500119864 minus 43
XP 0035412061 Predicted uncharacterized proteinLOC100788 Glycine max 500119864 minus 43
Cellularmetabolism
XP 0031514401 Senescence-associated protein Loa loa 800119864 minus 25
ACA048501 Senescence-associated protein Picea abies 300119864 minus 21
XP 0021396981 Senescence-associated protein Cryptosporidium hominis TU502 200119864 minus 19
AEM360701 Senescence-associated protein Mytilus eduls 200119864 minus 18
EFW131811 Senescence-associated protein Coccidioides posadasii str 700119864 minus 18
XP 0021182661 Senescence-associated protein Trichoplax adhaerens 100119864 minus 17
XP 0016105631 Senescence-associated protein Babesia bovis T2Bo 100119864 minus 16
XP 0031886401 Plant senescence-associated protein Aspergillus niger CBS 51388 500119864 minus 16
EFN584411 Senescence-associated protein Chlorella variabilis 200119864 minus 11
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 400119864 minus 10
XP 0030649921 Senescence-associated protein Micromonas pusilla CCMP1545 100119864 minus 09
EEH167211 Senescence-associated protein Paracoccidioides brasiliensis 200119864 minus 09
XP 7297621 Senescence-associated protein Plasmodium yoelii yoelii 200119864 minus 06
ABO208511 Putative senescence-associated protein Lilium longiflorum 100119864 minus 41
BAB334211 Putative senescence-associated protein Pisum sativum 900119864 minus 17
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 100119864 minus 15
BAD189051 rRNA intron-encoded endonuclease Thermoproteus sp IC-061 600119864 minus 06
XP 0025153521 Protein binding protein putative Ricinus communis 300119864 minus 34
XM 0035883071 3-Dehydroquinate synthase putative Medicago truncatula 500119864 minus 39
AES586061 Mitochondrial protein putative Medicago truncatula 300119864 minus 37
CAB710631 Copia-type polyprotein Arabidopsis thaliana 300119864 minus 13
AT4g15300 Cytochrome p450-like tbp protein Arabidopsis thaliana 200119864 minus 27
varying periods of Si treatment Most sequences recognizedfrom this subtracted library encoded different genes withhomology of genes from 13 known and 2 unknown species
One EST with known function was selected for analysisof Si-induced expression patterns in varied periods of time
using semi quantitative reverse transcriptase and real-timeqRT-PCR (Figures 15 and 7)
The SSH cDNA library of Si-treated genes was monitoredfor seven times From SSH reports it appears that somegene induction patterns are so rapid and self-motivated that
BioMed Research International 9
Table 2 Pfam domain search option for genomic and proteic annotation
Target Description Species 119864-valueQ0MX20 ARAHY Serine-rich protein Arachis hypogaea 680119864minus147
I3S491 LOTJA Uncharacterized protein Lotus japonicus 490119864 minus 05
G7I9T8 MEDTR Mitochondrial protein putative(geneMTR 1g006300) Medicago truncatula 000084
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
ATP synthase Senescence-proteinCytochrome p450 rRNA endonucleaseProtein binding Auxin-responsive protein3-Dehydroquinatesynthase
Figure 6 Molecular function categorization of the subtractedcDNA library
Expr
essio
n ra
tio
05
1
8
16
32
64
2
4
15h 1 day 2 days 3 days 4 days 5 days 6 days
Figure 7 Relative quantity of serine-rich protein gene and actin as aninternal control Serine 15 h sample group is not different to controlgroup P value = 0169 Serine 1 day is up the regulated in samplegroup (in comparison to control group) P value = 0000 Serine2 days is up-regulated in sample group (in comparison to controlgroup) P value = 0000 Serine 3 days sample group is not differentto control group P value = 0339 Serine 4 days is up-regulated insample group (in comparison to control group) P value = 0000Serine 5 days is up-regulated in sample group (in comparison tocontrol group) P value = 0000 Serine 6 days is up-regulated insample group (in comparison to control group) P value = 0000
species population for many aquatic organisms has beenidentified [8ndash11] The major concern is to find how theirgenetic structure is organized and to determine the corre-lation between different traits which include adaptive andnonadaptive with migration of diverse genes which leads toevaluation of developmental changes in mangrove ecologicalconditions [8] Mangrove trees are capable of decreasingnutrient losses when there are changes in atmospheric
conditions by applying a variety of mechanisms includ-ing biogeochemical and physiological while exposed to awaterlogged and salty environment [12ndash14] Ion preservationimmobilization and translocation in soaked soil efficiencyof nutrient use which is the highest recorded among treesand the morphological shape of its roots probably play animportant role in establishing these mechanisms [14]
Among the plant nutrient elements in soil Si is the mostabundant after oxygen and essential for plant formationunder poor nutrient conditionsThe role of Si is not limited toplant growth as it also plays an important role in decreasingthe susceptibility of plants to different environmental stresses[15ndash19] Serine- and proline-rich proteins play a significantrole in plants with regard to Si absorption and transportation[20 21] In the present study we isolated and identified serine-rich protein genes from the roots of the mangrove plant (Rapiculata)
Currently many methods are being used to studydifferentials in the expression of genes including serialanalysis of gene expression (SAGE) differential displayreverse transcription-polymerase chain reaction (DDRT-PCR) cDNA microarray suppression subtractive hybridiza-tion (SSH) and cDNA-AFLP The false positive createdthrough the SSH technique is much lower compared to thatthrough the other methods [22 23]
2 Materials and Methods
21 Plant Materials Mangrove (Rhizophora apiculata) seedswere collected from Kuala Sepetang (04∘ 5015010158401015840N 100∘3762010158401015840) in Taiping Perak Malaysia They were grown inhydroponic culture for two months and then treated with05 1 and 15mM SiO
2for 15 hrs and for 1 to 6 days The
roots of the plants were collected immediately washed withdistilled water and frozen in liquid nitrogen to facilitate theRNA extraction process
22 Total RNA Extraction and Construction of the cDNALibrary The RNA extracted from the roots of the mangrovewas isolated using the CTAB method [24] The quality andintegrity of the extracted RNA were assessed using theNanoDrop ND-1000 spectrophotometer (NanoDrop Tech-nologies USA) Poly(A) + RNA was extracted from totalRNA using a PolyATtract mRNA Isolation Kit (PromegaUSA)
The subtracted cDNA library was constructed using thePCR-Select Subtractive Hybridization Kit (Clontech USA)following the manufacturerrsquos instructions In brief mRNAsfrom the last step for both control and treated sampleswere designated as driver and tester respectively The first-strand and double-strand cDNAs were then synthesized andthe synthesized double-strand cDNA of the tester samplewas digested with restriction enzyme Rsa I The digestedtester cDNA (blunt ends) was divided into two parts whichwere subsequently ligated with two different kinds of cDNAadaptors (long inverted terminal repeats) A and B In order tonormalize and enrich mangrove root development-related Siabsorption genes that are up- or down-regulated by Si stress
Figure 8 The nucleotide (696 bp) and deduced amino sequence (223 aa) of serine-rich protein
20015010050
minus3
minus2
minus1
0
1
2
3
Scor
e
Position
HphobKite and Doolittle
ProtScale output for user sequence
HphobKite and Doolittle
Figure 9 Analysis of hydrophilicity and hydrophobicity for theserine-rich proteinThis figure is the ProtScale output of hydrophilic-ity and hydrophobicity for the serine-rich protein
N
Amino acid composition
CH
VM
C
Q
Y
E
K
R
FA T V
I
G
P
L
S
Figure 10 Amino acid composition of serine-rich protein
CCCCCCEEEECCCCEEEEEEEEEEEEEEEEECCCCCCCCC
CCEEEEEECCCCCEEEEECCCCCCCCCCCCCCEEECCCEE
PCYLWTTVADYKQTRYYWGGRGTPLASPRRTPILLNEGKL
EEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCC
EVKPTSFLKSVPGPSYQGGGLGGSMASLLLEKMAPLELGP
CCCCCEEEECCCCEEEEEEECCCCCEEHHHHHCCCCHHHH
PDGPPLILRGKKRQIILIGVTSTSACLILLVSTTAGFFLQ
HHCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEEE
ERQNSASAMFRHTKIPIPSIASLASSPSMHSIFHVSQFRC
EECCCEEEEEEEEEEEEEEEEEC
210
Confidence of prediction
Strand Pred predicted secondary structureCoil AA target sequence
MVLSSALVYSTCSPVSVWGTVQFTGRIALPIRSFFPGSFP
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA IPRGECLAIVIVEIKFFKVCEVT
220
80706050
12011010090
160150140130
200190180170
40302010
Helix conf
Figure 11 Prediction of secondary structure for the serine-richprotein
BioMed Research International 5
Phobius posterior probabilities for serine-rich protein
TransmembraneCytoplasmic
NoncytoplasmicSignal peptide
0
50 100 200150
02
06
08
1
04
Poste
rior l
abel
prob
abili
ty
0
2
6
44
Figure 12 Prediction of subcellular location of serine-rich protein
Figure 13 3D structure of serine-rich protein The protein folds areshown in the colors of the rainbow from the N terminus (blue) tothe C terminus (red)
Figure 14 Result of the SSH cDNA library LaneM marker lane Asubtracted driver sample after second PCR lane B subtracted testersample after second PCR
(a)
(b)
Figure 15 Relative expression of serine-rich protein gene (a) andactin as an internal control (b) was amplified by semi-qRT-PCR Lmolecular ladder M untreated plants A 15 hrs silicon treated B 1-day silicon treated C 2-days silicon treated D 3-day silicon treatedE 4-day silicon treated F 5-day silicon treated and G 6-day silicontreated
two rounds of hybridizations and suppression PCR ampli-fication were processed The PCR products of secondaryPCR amplification were then purified and inserted directlyinto the pDrive UA cloning vector (Qiagen Germany) Theligated pDrive vectors were then transformed into E coliEZ cells and cultured overnight (16 hrs 37∘C) in LB agarmedium containing X-gal IPTG and ampicillin A totalof 400 independent positive white clones were picked outrandomly put in LB broth containing Amp and incubatedat 37∘C overnight to establish the mangrove root subtractivelibrary
23 EST Sequencing and Analysis About 400 positive cloneswere selected randomly and amplified using M13 primers(forward and reverse) after removal of contamination fromthe vector and primer sequence Before the assembly searchadaptors polyA tails low quality sequences short sequencesless than 100 bp in length and vector sequences wereremovedThe algorithm search of contigs and singletons wasperformed using CAP3 software This was followed by theobtained sequences being submitted to theNCBI database forhomology search
The BLASTn was used to show degree of similar-ity between the clone cDNA sequence and a knownsequence and the BLASTx (httpblastncbinlmnihgov)showed function of qualified cDNA sequences with largeORF regions Classification of cDNA sequences was basedon their E-value results in the BLAST Categories ofsequence functions are based on the Blast2GO program(httpwwwblast2goorg) [25]
Computational annotation of the mangrove EST datasetswas performed using the Blast2GO software v133 (httpwwwblast2goorg) The BLAST search was performed atNCBI [26] The degree of amino acid sequence similaritywas determined by the use of Wu-Blast from EBI [27]Hydrophilicity and hydrophobicity of serine-rich proteinwere predicted online by MemBrain TMHMM andProtScale (httpwebexpasy orgprotscale) in the toolkitof ExPASy Subcellular localization was investigated usingPSORT II Prediction and Cell-PLoc BaCelLo programThe prediction of secondary structure was carried outby (httpnpsa-pbilibcpfrcgi-binnpsa automatplpage
6 BioMed Research International
=npsa sopmahtml) and PsiPred program The prediction of3D structure was carried out by using the Pfam program
24 Amplification of Full-Length cDNA The complete CDSof serine-rich protein gene contained 696 bp and 223 aminoacids The PCR program according to KAPA HiFi Hot Startwas used as follows initial denaturation at 95∘C for 5min35 cycles of denaturation at 98∘C for 30 s annealing at575∘C for 30 s and extension at 72∘C for 1min The finalextension was 5min at 72∘C Agarose gel (15) was usedto separate PCR-amplified cDNA fragments The expectedbound about 700 bp was purified using gel purificationkit (Qiagen Germany) and 31015840-dA-overhangs (incubation72∘C for 5min) added to the blunt-ended DNA fragmentsgenerated by KAPAHiFi Hot Start DNA polymerase to ligateinto the pDrive cloning vector (Qiagen Germany) and sentfor sequencing
25 Semiquantitative RT-PCRAnalysis Reverse transcriptaseRT-PCR was performed to study the expression of theserine-rich protein gene One120583L of DNase treated (DNaseI Qiagen Germany) total RNA from each of the man-grove roots treated with Si for 15 hrs and 1 to 6 days anduntreated plants was transcribed to the first-strand cDNAusing SuperScript III (Invitrogen USA) and 500 ng oligo(dT) 18 primer in 20120583L reaction volume Reactions werethen incubated at 50∘C for 60min and heated to inactiveat 70∘C for 15min The template cDNAs for both control(untreated) and treated samples were then amplified usingserine primers as F 5-GTCATTCTGCCGAGTTCC-3 and R5-AATGCCCATTTATGTGACTTCG-3 designed accordingto the cDNA sequence homology
Actin gene as an internal control was amplified with thefollowing primers F (51015840CAC TAC TAC TGC TAA ACG GGAAA 31015840) and R (51015840ACA TCT GCT GGA AGG TGC TG 31015840)The following PCR (Tag DNA Polymerase Vivantis USA)program was used 94∘C for 2min and 35 cycles of 94∘C for30 sec 575 and 58∘C respectively for actin and serine for30 sec and 72∘C for 30 sec The PCR program was concludedwith final extension of 7min at 72∘C Actin and serine-richprotein were amplified using the same cDNA templates ThePCR products then were separated with 15 agarose gel andstained with ethidium bromide
26 Real-Time Quantitative RT-PCR Analysis Real-timeqRT-PCR was performed to evaluate the expression lev-els of candidate serine-rich protein genes in the root tis-sues in response to treatments with different concentra-tions of Si compared to these in the control plants TotalRNA was extracted from untreated and Si-treated man-grove roots Extracted RNA was then treated with DNase(DNase I Qiagen Germany) The primers for the actingene were used as in the previous section and for thesecond endogenous control ef1205721 was F 51015840 rarr 31015840 ATTGGA AAC GGA TAT GCT CCA R 51015840 rarr 31015840 TCCTTA CCT GAA CGC CTG TCA serine-rich protein geneprimer was F 51015840 rarr 31015840GCAAGTGCTATGTTCAGGCA R51015840 rarr 31015840AACAATAACGATGGCAAGGC One 120583L aliquot of
DNase treated RNA from each sample was used to prepare20120583L reaction volumes (based on the KAPA SYBER FASTOne-Step qRT-PCR) The reactions involved were a primaryincubation of 42∘C for 5min inactive RT at 95∘C for 5minfollowed by 40 cycles at 95∘C for 3 sec 60∘C for 30 sec and72∘C for 3 sec The 96-well plate was used to analyze eachsample in duplicate In this study all data was from fourindependent biological replicates A standard curve (1198772 gt095) from 10-fold serial dilutions was generated from thepurified cDNA fragments of internal and serine-rich proteingenes (102 10 1 10minus1 and 10minus2 ng120583L)
3 Results
31 Yield and Integrity of RNA The RNA appeared as a non-degraded band on 15 agarose gel containing formaldehydeThe A
260280ratios ranging from 19 to 202 indicate that
there was no protein contamination and the A260230
ratio gt1demonstrated that there is no polyphenol or polysaccharidecontamination [28]The RNA concentration was in the rangeof 05ndash12mg gminus1 The poly(A) + RNA of both treated anduntreated samples appeared as clear smears on the 1 agarosegelwith theA
260280ratio = 2 indicating high quality ofmRNA
obtained for further analysis
32 Construction of the Subtracted cDNA Library The prod-ucts of the subtracted suppression hybridization cDNAlibrary appeared on 12 agarose gel as a smear with rankingsize from 150 bp to 12 kb and 4ndash6 separate bands (Figure 14line A) which obviously differentiates them from the unsub-tracted sample driver (Figure 14 line B) The results of theSSH cDNA library indicated that the differentially expressedgenes are present in the tester or treated samples and absentor present at lower levels in the driver or untreated samplesFor further confirmation of subtraction analysis efficiencyexpression of the actin gene was examined in both the driverand control samples via 23 and 33 cycles of amplificationrespectively indicating that cDNA homologue was removedfrom both the tester and driver samples by subtraction(Figure 14)
33 ESTs Sequencing and Gene Annotation About 700 pos-itive recombinant clones were isolated from the cDNAlibrary Of those about 400 clones were randomly selectedsequenced and analyzed to isolate the gene(s) involved inSi transportation and absorption The 322 ESTs sequenceswere coalesced into 21 contigs and 13 singletons by CAP3assembly program About 195 of the ESTs resulting fromthis library did not have any significant homology to any ofthe proteins existing in the database The DNA fragmentsinvolved in the subtracted cDNA library varied in sizemore or less between 100 and 650 bp (Figure 1) Averagelength of high quality ESTs is 350 bp The most plentifulBLASTx hits correlated to species distribution related toLilium longiflorum Glycinemax Cupressus sempervirens andArabidopsis thaliana (Table 1 and Figure 2)
BioMed Research International 7
34 Classification of Differentially Expressed Genes Accord-ing to gene annotation the 322 ESTs were divided intothree different groups involving biological process molecularfunction and cellular components (Figure 3) The potentialidentities of the genes are based on similarities to thosepresent in the Gen-Bank databases The genes obtained bythe SSH library classified by biological process involve 5different groups ATP synthase (889) equilibrative trans-porter (44) auxin-responsive protein (22) mitochon-drial protein (22) and copia-type polyprotein (22)those classified according to cellular components involve 3different groups The ATP synthase was highly abundantin both the cellular components and biological processcategories (Figures 4 and 5) while senescence-protein wasthe most abundant group in the molecular function category(Figure 6) Classification of the ESTs resulted in 94 ofknown 4 hypothetical and 2 unknown functions
35 Isolation of the Full-Length Serine-Rich Protein GeneOne of the ESTs sequence showing 97 similarity involvesthe ATG codon (20 query cover region) with completeCDS of the serine-rich protein gene of Arachis hypogaea(Figure 7) Full length of the gene (Figure 8) was obtainedthrough amplification of the cDNA template (First-StrandcDNA synthesis Invitrogen USA) and using gene-specificprimers as follows
serine
F 51015840 rarr 31015840 GTCATTCTGCCGAGTTCC
R 51015840 rarr 31015840AATGCCCATTTATGTGACTTCG
36 Analysis of the Differential Expression of Serine-RichProtein Gene Using Semi-qRT-PCR and Real-Time qRT-PCR The semiquantitative RT-PCR analysis showed that theexpression levels of serine-rich protein were generally higherin the Si treatment samples than in the untreated samplesThe expression level of serine-rich protein in the 3-day treatedsample was lower compared to that in the other treatedsamples with varying periods of time (Figure 15) Real-timeqRT-PCR confirmed the results of the semi quantitative RT-PCR (Figure 8) The relative transcript abundance of serine-rich protein in Si-treated plants was higher compared to thatin the untreated plants
The PCR efficiency of all reactions in this study wasbetween 87 and 98 The REST software (Qiagen HildenGermany) was employed to analyze the results of the qRT-PCRThemanufacturerrsquos instructions were followed to quan-tify the relative gene expressionDifferences among treatmentsamples were noted as being statistically significant (119875 lt005)
37 Bioinformatics Analysis The low quality regions at theend and beginning of each sequence were trimmed usinga Phred 20 cutoff value Vector screening was carried outusing the crossmatch Oligo dT tracks and other contam-inants were removed Algorithms of CAP3 [29] assemblywere used to assemble the individual ESTs into clusters of
sequences derived from the same transcript as tentative con-sensus sequences (TCs) and singletons representing uniquetranscripts Obtained sequences were submitted to NCBIdatabase
Prediction of hydrophilicity and hydrophobicity isneeded for predicting protein secondary structure anddivision of functional domain according to the theory thathydrophilicity and hydrophobicity are related to the score ofamino acid Based on the number of hydrophilic amino acidresidues we could hypothesize that the serine-rich proteinwas a hydrophilic protein (Figures 9 and 10)
38 The Prediction of Secondary Structure and FunctionDomain of Serine-Rich Protein The prediction results ofsecondary structure of serine-rich protein by PsiPred showedthat the secondary structure consisted of 11 sheets 3 helixesand 15 coils (Figure 11) Computational analysis of the cDNAclone isolated from mangrove root library indicated that its696 bp coding region codes for a protein of 223 amino acidswith a predicted molecular mass of 2421 kDa Homologysearches run with the full-length amino acid sequences
39 Subcellular Localization The prediction of subcellularlocalization with Cell-PLoc BaCelLo and WoLF PSORTshowed that serine-rich protein is likely to be localizedin chloroplast plastid and mitochondrion with a differ-ent probability of 17 5 and 1 respectively (Figure 12)Biosequence analysis of coding sequence region (CDS) ofserine-rich protein using profile hidden Markov models(HMMER) showed 60 similarity to serine-rich protein ofArachis hypogaea (TRQ0MX20 ARAHY) and 100 sim-ilarity to mitochondrial protein of Medicago truncatula(TRG7I9T8 MEDTR) (Table 2)
310 Prediction of 3D Structure In order to predict the3D structure of serine-rich protein the Phyre2 (ProteinHomology Analogy Recognition Engine) server was used[30] A Phyre2 outputmodel was generated based on the tem-plate galactose-binding domain-like Structural alignment ofserine-rich protein and galactose-binding domain-like wasperformed using theMatchmaker tool of UCSFChimera [31]The 3D structure protein of serine-rich protein gene isolatedand identified in the present study submitted to NCBI withaccession number KF211374 involves MYP bZip LCR1 andDOF transcription factor binding motifs (Figure 13)
4 Discussion
In the present study the suppression subtractive hybridiza-tion (SSH) method was used to identify differentiallyexpressed genes present in the tester sample and absent orpresent at lower levels in the driver The SSH cDNA librarywas constructed using SiO
2-treated (15 hrs and 1 to 6 days)
roots of mangrove plants whereby 321 unique genes wereobtainedThe SSHmethod provides information related onlyto global analysis of gene expression and hence semi quan-titative RT-PCR and real-time qRT-PCR were performedto evaluate the quantitative level of gene expressions over
8 BioMed Research International
Table 1 Putative identities of novel silicon-induced cDNA sequences expressed in R apiculata roots
Gen-Bankaccession Homology Organism 119864-value
Stress responseNP 1995641 Putative auxin-responsive protein Arabidopsis thaliana 500119864 minus 19
DQ8346901 Serine-rich protein mRNA Arachis hypogaea 100119864 minus 72
AAB709281 Proline-rich protein Santalum album 700119864 minus 11
TransporterAET022081 ATP synthase subunit beta Medicago truncatula 300119864 minus 44
XP 0023172511 Equilibrative nucleoside transporter Populus trichocarpa 700119864 minus 40
UnknownXP 0036143941 Hypothetical protein MTR 5g051130 Medicago truncatula 100119864 minus 38
XP 0035471291 Predicted uncharacterized proteinLOC100801 Glycine max 600119864 minus 36
XP 0022691672 Predicted uncharacterized proteinLOC100267 Vitis vinifera 100119864 minus 30
XP 0035490071 Uncharacterized protein LOC100791 Glycine max 500119864 minus 43
XP 0035412191 Uncharacterized protein LOC100796 Glycine max 500119864 minus 43
XP 0035412061 Predicted uncharacterized proteinLOC100788 Glycine max 500119864 minus 43
Cellularmetabolism
XP 0031514401 Senescence-associated protein Loa loa 800119864 minus 25
ACA048501 Senescence-associated protein Picea abies 300119864 minus 21
XP 0021396981 Senescence-associated protein Cryptosporidium hominis TU502 200119864 minus 19
AEM360701 Senescence-associated protein Mytilus eduls 200119864 minus 18
EFW131811 Senescence-associated protein Coccidioides posadasii str 700119864 minus 18
XP 0021182661 Senescence-associated protein Trichoplax adhaerens 100119864 minus 17
XP 0016105631 Senescence-associated protein Babesia bovis T2Bo 100119864 minus 16
XP 0031886401 Plant senescence-associated protein Aspergillus niger CBS 51388 500119864 minus 16
EFN584411 Senescence-associated protein Chlorella variabilis 200119864 minus 11
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 400119864 minus 10
XP 0030649921 Senescence-associated protein Micromonas pusilla CCMP1545 100119864 minus 09
EEH167211 Senescence-associated protein Paracoccidioides brasiliensis 200119864 minus 09
XP 7297621 Senescence-associated protein Plasmodium yoelii yoelii 200119864 minus 06
ABO208511 Putative senescence-associated protein Lilium longiflorum 100119864 minus 41
BAB334211 Putative senescence-associated protein Pisum sativum 900119864 minus 17
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 100119864 minus 15
BAD189051 rRNA intron-encoded endonuclease Thermoproteus sp IC-061 600119864 minus 06
XP 0025153521 Protein binding protein putative Ricinus communis 300119864 minus 34
XM 0035883071 3-Dehydroquinate synthase putative Medicago truncatula 500119864 minus 39
AES586061 Mitochondrial protein putative Medicago truncatula 300119864 minus 37
CAB710631 Copia-type polyprotein Arabidopsis thaliana 300119864 minus 13
AT4g15300 Cytochrome p450-like tbp protein Arabidopsis thaliana 200119864 minus 27
varying periods of Si treatment Most sequences recognizedfrom this subtracted library encoded different genes withhomology of genes from 13 known and 2 unknown species
One EST with known function was selected for analysisof Si-induced expression patterns in varied periods of time
using semi quantitative reverse transcriptase and real-timeqRT-PCR (Figures 15 and 7)
The SSH cDNA library of Si-treated genes was monitoredfor seven times From SSH reports it appears that somegene induction patterns are so rapid and self-motivated that
BioMed Research International 9
Table 2 Pfam domain search option for genomic and proteic annotation
Target Description Species 119864-valueQ0MX20 ARAHY Serine-rich protein Arachis hypogaea 680119864minus147
I3S491 LOTJA Uncharacterized protein Lotus japonicus 490119864 minus 05
G7I9T8 MEDTR Mitochondrial protein putative(geneMTR 1g006300) Medicago truncatula 000084
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
Figure 8 The nucleotide (696 bp) and deduced amino sequence (223 aa) of serine-rich protein
20015010050
minus3
minus2
minus1
0
1
2
3
Scor
e
Position
HphobKite and Doolittle
ProtScale output for user sequence
HphobKite and Doolittle
Figure 9 Analysis of hydrophilicity and hydrophobicity for theserine-rich proteinThis figure is the ProtScale output of hydrophilic-ity and hydrophobicity for the serine-rich protein
N
Amino acid composition
CH
VM
C
Q
Y
E
K
R
FA T V
I
G
P
L
S
Figure 10 Amino acid composition of serine-rich protein
CCCCCCEEEECCCCEEEEEEEEEEEEEEEEECCCCCCCCC
CCEEEEEECCCCCEEEEECCCCCCCCCCCCCCEEECCCEE
PCYLWTTVADYKQTRYYWGGRGTPLASPRRTPILLNEGKL
EEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCC
EVKPTSFLKSVPGPSYQGGGLGGSMASLLLEKMAPLELGP
CCCCCEEEECCCCEEEEEEECCCCCEEHHHHHCCCCHHHH
PDGPPLILRGKKRQIILIGVTSTSACLILLVSTTAGFFLQ
HHCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEEE
ERQNSASAMFRHTKIPIPSIASLASSPSMHSIFHVSQFRC
EECCCEEEEEEEEEEEEEEEEEC
210
Confidence of prediction
Strand Pred predicted secondary structureCoil AA target sequence
MVLSSALVYSTCSPVSVWGTVQFTGRIALPIRSFFPGSFP
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA
ConfPredPred
AA IPRGECLAIVIVEIKFFKVCEVT
220
80706050
12011010090
160150140130
200190180170
40302010
Helix conf
Figure 11 Prediction of secondary structure for the serine-richprotein
BioMed Research International 5
Phobius posterior probabilities for serine-rich protein
TransmembraneCytoplasmic
NoncytoplasmicSignal peptide
0
50 100 200150
02
06
08
1
04
Poste
rior l
abel
prob
abili
ty
0
2
6
44
Figure 12 Prediction of subcellular location of serine-rich protein
Figure 13 3D structure of serine-rich protein The protein folds areshown in the colors of the rainbow from the N terminus (blue) tothe C terminus (red)
Figure 14 Result of the SSH cDNA library LaneM marker lane Asubtracted driver sample after second PCR lane B subtracted testersample after second PCR
(a)
(b)
Figure 15 Relative expression of serine-rich protein gene (a) andactin as an internal control (b) was amplified by semi-qRT-PCR Lmolecular ladder M untreated plants A 15 hrs silicon treated B 1-day silicon treated C 2-days silicon treated D 3-day silicon treatedE 4-day silicon treated F 5-day silicon treated and G 6-day silicontreated
two rounds of hybridizations and suppression PCR ampli-fication were processed The PCR products of secondaryPCR amplification were then purified and inserted directlyinto the pDrive UA cloning vector (Qiagen Germany) Theligated pDrive vectors were then transformed into E coliEZ cells and cultured overnight (16 hrs 37∘C) in LB agarmedium containing X-gal IPTG and ampicillin A totalof 400 independent positive white clones were picked outrandomly put in LB broth containing Amp and incubatedat 37∘C overnight to establish the mangrove root subtractivelibrary
23 EST Sequencing and Analysis About 400 positive cloneswere selected randomly and amplified using M13 primers(forward and reverse) after removal of contamination fromthe vector and primer sequence Before the assembly searchadaptors polyA tails low quality sequences short sequencesless than 100 bp in length and vector sequences wereremovedThe algorithm search of contigs and singletons wasperformed using CAP3 software This was followed by theobtained sequences being submitted to theNCBI database forhomology search
The BLASTn was used to show degree of similar-ity between the clone cDNA sequence and a knownsequence and the BLASTx (httpblastncbinlmnihgov)showed function of qualified cDNA sequences with largeORF regions Classification of cDNA sequences was basedon their E-value results in the BLAST Categories ofsequence functions are based on the Blast2GO program(httpwwwblast2goorg) [25]
Computational annotation of the mangrove EST datasetswas performed using the Blast2GO software v133 (httpwwwblast2goorg) The BLAST search was performed atNCBI [26] The degree of amino acid sequence similaritywas determined by the use of Wu-Blast from EBI [27]Hydrophilicity and hydrophobicity of serine-rich proteinwere predicted online by MemBrain TMHMM andProtScale (httpwebexpasy orgprotscale) in the toolkitof ExPASy Subcellular localization was investigated usingPSORT II Prediction and Cell-PLoc BaCelLo programThe prediction of secondary structure was carried outby (httpnpsa-pbilibcpfrcgi-binnpsa automatplpage
6 BioMed Research International
=npsa sopmahtml) and PsiPred program The prediction of3D structure was carried out by using the Pfam program
24 Amplification of Full-Length cDNA The complete CDSof serine-rich protein gene contained 696 bp and 223 aminoacids The PCR program according to KAPA HiFi Hot Startwas used as follows initial denaturation at 95∘C for 5min35 cycles of denaturation at 98∘C for 30 s annealing at575∘C for 30 s and extension at 72∘C for 1min The finalextension was 5min at 72∘C Agarose gel (15) was usedto separate PCR-amplified cDNA fragments The expectedbound about 700 bp was purified using gel purificationkit (Qiagen Germany) and 31015840-dA-overhangs (incubation72∘C for 5min) added to the blunt-ended DNA fragmentsgenerated by KAPAHiFi Hot Start DNA polymerase to ligateinto the pDrive cloning vector (Qiagen Germany) and sentfor sequencing
25 Semiquantitative RT-PCRAnalysis Reverse transcriptaseRT-PCR was performed to study the expression of theserine-rich protein gene One120583L of DNase treated (DNaseI Qiagen Germany) total RNA from each of the man-grove roots treated with Si for 15 hrs and 1 to 6 days anduntreated plants was transcribed to the first-strand cDNAusing SuperScript III (Invitrogen USA) and 500 ng oligo(dT) 18 primer in 20120583L reaction volume Reactions werethen incubated at 50∘C for 60min and heated to inactiveat 70∘C for 15min The template cDNAs for both control(untreated) and treated samples were then amplified usingserine primers as F 5-GTCATTCTGCCGAGTTCC-3 and R5-AATGCCCATTTATGTGACTTCG-3 designed accordingto the cDNA sequence homology
Actin gene as an internal control was amplified with thefollowing primers F (51015840CAC TAC TAC TGC TAA ACG GGAAA 31015840) and R (51015840ACA TCT GCT GGA AGG TGC TG 31015840)The following PCR (Tag DNA Polymerase Vivantis USA)program was used 94∘C for 2min and 35 cycles of 94∘C for30 sec 575 and 58∘C respectively for actin and serine for30 sec and 72∘C for 30 sec The PCR program was concludedwith final extension of 7min at 72∘C Actin and serine-richprotein were amplified using the same cDNA templates ThePCR products then were separated with 15 agarose gel andstained with ethidium bromide
26 Real-Time Quantitative RT-PCR Analysis Real-timeqRT-PCR was performed to evaluate the expression lev-els of candidate serine-rich protein genes in the root tis-sues in response to treatments with different concentra-tions of Si compared to these in the control plants TotalRNA was extracted from untreated and Si-treated man-grove roots Extracted RNA was then treated with DNase(DNase I Qiagen Germany) The primers for the actingene were used as in the previous section and for thesecond endogenous control ef1205721 was F 51015840 rarr 31015840 ATTGGA AAC GGA TAT GCT CCA R 51015840 rarr 31015840 TCCTTA CCT GAA CGC CTG TCA serine-rich protein geneprimer was F 51015840 rarr 31015840GCAAGTGCTATGTTCAGGCA R51015840 rarr 31015840AACAATAACGATGGCAAGGC One 120583L aliquot of
DNase treated RNA from each sample was used to prepare20120583L reaction volumes (based on the KAPA SYBER FASTOne-Step qRT-PCR) The reactions involved were a primaryincubation of 42∘C for 5min inactive RT at 95∘C for 5minfollowed by 40 cycles at 95∘C for 3 sec 60∘C for 30 sec and72∘C for 3 sec The 96-well plate was used to analyze eachsample in duplicate In this study all data was from fourindependent biological replicates A standard curve (1198772 gt095) from 10-fold serial dilutions was generated from thepurified cDNA fragments of internal and serine-rich proteingenes (102 10 1 10minus1 and 10minus2 ng120583L)
3 Results
31 Yield and Integrity of RNA The RNA appeared as a non-degraded band on 15 agarose gel containing formaldehydeThe A
260280ratios ranging from 19 to 202 indicate that
there was no protein contamination and the A260230
ratio gt1demonstrated that there is no polyphenol or polysaccharidecontamination [28]The RNA concentration was in the rangeof 05ndash12mg gminus1 The poly(A) + RNA of both treated anduntreated samples appeared as clear smears on the 1 agarosegelwith theA
260280ratio = 2 indicating high quality ofmRNA
obtained for further analysis
32 Construction of the Subtracted cDNA Library The prod-ucts of the subtracted suppression hybridization cDNAlibrary appeared on 12 agarose gel as a smear with rankingsize from 150 bp to 12 kb and 4ndash6 separate bands (Figure 14line A) which obviously differentiates them from the unsub-tracted sample driver (Figure 14 line B) The results of theSSH cDNA library indicated that the differentially expressedgenes are present in the tester or treated samples and absentor present at lower levels in the driver or untreated samplesFor further confirmation of subtraction analysis efficiencyexpression of the actin gene was examined in both the driverand control samples via 23 and 33 cycles of amplificationrespectively indicating that cDNA homologue was removedfrom both the tester and driver samples by subtraction(Figure 14)
33 ESTs Sequencing and Gene Annotation About 700 pos-itive recombinant clones were isolated from the cDNAlibrary Of those about 400 clones were randomly selectedsequenced and analyzed to isolate the gene(s) involved inSi transportation and absorption The 322 ESTs sequenceswere coalesced into 21 contigs and 13 singletons by CAP3assembly program About 195 of the ESTs resulting fromthis library did not have any significant homology to any ofthe proteins existing in the database The DNA fragmentsinvolved in the subtracted cDNA library varied in sizemore or less between 100 and 650 bp (Figure 1) Averagelength of high quality ESTs is 350 bp The most plentifulBLASTx hits correlated to species distribution related toLilium longiflorum Glycinemax Cupressus sempervirens andArabidopsis thaliana (Table 1 and Figure 2)
BioMed Research International 7
34 Classification of Differentially Expressed Genes Accord-ing to gene annotation the 322 ESTs were divided intothree different groups involving biological process molecularfunction and cellular components (Figure 3) The potentialidentities of the genes are based on similarities to thosepresent in the Gen-Bank databases The genes obtained bythe SSH library classified by biological process involve 5different groups ATP synthase (889) equilibrative trans-porter (44) auxin-responsive protein (22) mitochon-drial protein (22) and copia-type polyprotein (22)those classified according to cellular components involve 3different groups The ATP synthase was highly abundantin both the cellular components and biological processcategories (Figures 4 and 5) while senescence-protein wasthe most abundant group in the molecular function category(Figure 6) Classification of the ESTs resulted in 94 ofknown 4 hypothetical and 2 unknown functions
35 Isolation of the Full-Length Serine-Rich Protein GeneOne of the ESTs sequence showing 97 similarity involvesthe ATG codon (20 query cover region) with completeCDS of the serine-rich protein gene of Arachis hypogaea(Figure 7) Full length of the gene (Figure 8) was obtainedthrough amplification of the cDNA template (First-StrandcDNA synthesis Invitrogen USA) and using gene-specificprimers as follows
serine
F 51015840 rarr 31015840 GTCATTCTGCCGAGTTCC
R 51015840 rarr 31015840AATGCCCATTTATGTGACTTCG
36 Analysis of the Differential Expression of Serine-RichProtein Gene Using Semi-qRT-PCR and Real-Time qRT-PCR The semiquantitative RT-PCR analysis showed that theexpression levels of serine-rich protein were generally higherin the Si treatment samples than in the untreated samplesThe expression level of serine-rich protein in the 3-day treatedsample was lower compared to that in the other treatedsamples with varying periods of time (Figure 15) Real-timeqRT-PCR confirmed the results of the semi quantitative RT-PCR (Figure 8) The relative transcript abundance of serine-rich protein in Si-treated plants was higher compared to thatin the untreated plants
The PCR efficiency of all reactions in this study wasbetween 87 and 98 The REST software (Qiagen HildenGermany) was employed to analyze the results of the qRT-PCRThemanufacturerrsquos instructions were followed to quan-tify the relative gene expressionDifferences among treatmentsamples were noted as being statistically significant (119875 lt005)
37 Bioinformatics Analysis The low quality regions at theend and beginning of each sequence were trimmed usinga Phred 20 cutoff value Vector screening was carried outusing the crossmatch Oligo dT tracks and other contam-inants were removed Algorithms of CAP3 [29] assemblywere used to assemble the individual ESTs into clusters of
sequences derived from the same transcript as tentative con-sensus sequences (TCs) and singletons representing uniquetranscripts Obtained sequences were submitted to NCBIdatabase
Prediction of hydrophilicity and hydrophobicity isneeded for predicting protein secondary structure anddivision of functional domain according to the theory thathydrophilicity and hydrophobicity are related to the score ofamino acid Based on the number of hydrophilic amino acidresidues we could hypothesize that the serine-rich proteinwas a hydrophilic protein (Figures 9 and 10)
38 The Prediction of Secondary Structure and FunctionDomain of Serine-Rich Protein The prediction results ofsecondary structure of serine-rich protein by PsiPred showedthat the secondary structure consisted of 11 sheets 3 helixesand 15 coils (Figure 11) Computational analysis of the cDNAclone isolated from mangrove root library indicated that its696 bp coding region codes for a protein of 223 amino acidswith a predicted molecular mass of 2421 kDa Homologysearches run with the full-length amino acid sequences
39 Subcellular Localization The prediction of subcellularlocalization with Cell-PLoc BaCelLo and WoLF PSORTshowed that serine-rich protein is likely to be localizedin chloroplast plastid and mitochondrion with a differ-ent probability of 17 5 and 1 respectively (Figure 12)Biosequence analysis of coding sequence region (CDS) ofserine-rich protein using profile hidden Markov models(HMMER) showed 60 similarity to serine-rich protein ofArachis hypogaea (TRQ0MX20 ARAHY) and 100 sim-ilarity to mitochondrial protein of Medicago truncatula(TRG7I9T8 MEDTR) (Table 2)
310 Prediction of 3D Structure In order to predict the3D structure of serine-rich protein the Phyre2 (ProteinHomology Analogy Recognition Engine) server was used[30] A Phyre2 outputmodel was generated based on the tem-plate galactose-binding domain-like Structural alignment ofserine-rich protein and galactose-binding domain-like wasperformed using theMatchmaker tool of UCSFChimera [31]The 3D structure protein of serine-rich protein gene isolatedand identified in the present study submitted to NCBI withaccession number KF211374 involves MYP bZip LCR1 andDOF transcription factor binding motifs (Figure 13)
4 Discussion
In the present study the suppression subtractive hybridiza-tion (SSH) method was used to identify differentiallyexpressed genes present in the tester sample and absent orpresent at lower levels in the driver The SSH cDNA librarywas constructed using SiO
2-treated (15 hrs and 1 to 6 days)
roots of mangrove plants whereby 321 unique genes wereobtainedThe SSHmethod provides information related onlyto global analysis of gene expression and hence semi quan-titative RT-PCR and real-time qRT-PCR were performedto evaluate the quantitative level of gene expressions over
8 BioMed Research International
Table 1 Putative identities of novel silicon-induced cDNA sequences expressed in R apiculata roots
Gen-Bankaccession Homology Organism 119864-value
Stress responseNP 1995641 Putative auxin-responsive protein Arabidopsis thaliana 500119864 minus 19
DQ8346901 Serine-rich protein mRNA Arachis hypogaea 100119864 minus 72
AAB709281 Proline-rich protein Santalum album 700119864 minus 11
TransporterAET022081 ATP synthase subunit beta Medicago truncatula 300119864 minus 44
XP 0023172511 Equilibrative nucleoside transporter Populus trichocarpa 700119864 minus 40
UnknownXP 0036143941 Hypothetical protein MTR 5g051130 Medicago truncatula 100119864 minus 38
XP 0035471291 Predicted uncharacterized proteinLOC100801 Glycine max 600119864 minus 36
XP 0022691672 Predicted uncharacterized proteinLOC100267 Vitis vinifera 100119864 minus 30
XP 0035490071 Uncharacterized protein LOC100791 Glycine max 500119864 minus 43
XP 0035412191 Uncharacterized protein LOC100796 Glycine max 500119864 minus 43
XP 0035412061 Predicted uncharacterized proteinLOC100788 Glycine max 500119864 minus 43
Cellularmetabolism
XP 0031514401 Senescence-associated protein Loa loa 800119864 minus 25
ACA048501 Senescence-associated protein Picea abies 300119864 minus 21
XP 0021396981 Senescence-associated protein Cryptosporidium hominis TU502 200119864 minus 19
AEM360701 Senescence-associated protein Mytilus eduls 200119864 minus 18
EFW131811 Senescence-associated protein Coccidioides posadasii str 700119864 minus 18
XP 0021182661 Senescence-associated protein Trichoplax adhaerens 100119864 minus 17
XP 0016105631 Senescence-associated protein Babesia bovis T2Bo 100119864 minus 16
XP 0031886401 Plant senescence-associated protein Aspergillus niger CBS 51388 500119864 minus 16
EFN584411 Senescence-associated protein Chlorella variabilis 200119864 minus 11
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 400119864 minus 10
XP 0030649921 Senescence-associated protein Micromonas pusilla CCMP1545 100119864 minus 09
EEH167211 Senescence-associated protein Paracoccidioides brasiliensis 200119864 minus 09
XP 7297621 Senescence-associated protein Plasmodium yoelii yoelii 200119864 minus 06
ABO208511 Putative senescence-associated protein Lilium longiflorum 100119864 minus 41
BAB334211 Putative senescence-associated protein Pisum sativum 900119864 minus 17
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 100119864 minus 15
BAD189051 rRNA intron-encoded endonuclease Thermoproteus sp IC-061 600119864 minus 06
XP 0025153521 Protein binding protein putative Ricinus communis 300119864 minus 34
XM 0035883071 3-Dehydroquinate synthase putative Medicago truncatula 500119864 minus 39
AES586061 Mitochondrial protein putative Medicago truncatula 300119864 minus 37
CAB710631 Copia-type polyprotein Arabidopsis thaliana 300119864 minus 13
AT4g15300 Cytochrome p450-like tbp protein Arabidopsis thaliana 200119864 minus 27
varying periods of Si treatment Most sequences recognizedfrom this subtracted library encoded different genes withhomology of genes from 13 known and 2 unknown species
One EST with known function was selected for analysisof Si-induced expression patterns in varied periods of time
using semi quantitative reverse transcriptase and real-timeqRT-PCR (Figures 15 and 7)
The SSH cDNA library of Si-treated genes was monitoredfor seven times From SSH reports it appears that somegene induction patterns are so rapid and self-motivated that
BioMed Research International 9
Table 2 Pfam domain search option for genomic and proteic annotation
Target Description Species 119864-valueQ0MX20 ARAHY Serine-rich protein Arachis hypogaea 680119864minus147
I3S491 LOTJA Uncharacterized protein Lotus japonicus 490119864 minus 05
G7I9T8 MEDTR Mitochondrial protein putative(geneMTR 1g006300) Medicago truncatula 000084
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
Phobius posterior probabilities for serine-rich protein
TransmembraneCytoplasmic
NoncytoplasmicSignal peptide
0
50 100 200150
02
06
08
1
04
Poste
rior l
abel
prob
abili
ty
0
2
6
44
Figure 12 Prediction of subcellular location of serine-rich protein
Figure 13 3D structure of serine-rich protein The protein folds areshown in the colors of the rainbow from the N terminus (blue) tothe C terminus (red)
Figure 14 Result of the SSH cDNA library LaneM marker lane Asubtracted driver sample after second PCR lane B subtracted testersample after second PCR
(a)
(b)
Figure 15 Relative expression of serine-rich protein gene (a) andactin as an internal control (b) was amplified by semi-qRT-PCR Lmolecular ladder M untreated plants A 15 hrs silicon treated B 1-day silicon treated C 2-days silicon treated D 3-day silicon treatedE 4-day silicon treated F 5-day silicon treated and G 6-day silicontreated
two rounds of hybridizations and suppression PCR ampli-fication were processed The PCR products of secondaryPCR amplification were then purified and inserted directlyinto the pDrive UA cloning vector (Qiagen Germany) Theligated pDrive vectors were then transformed into E coliEZ cells and cultured overnight (16 hrs 37∘C) in LB agarmedium containing X-gal IPTG and ampicillin A totalof 400 independent positive white clones were picked outrandomly put in LB broth containing Amp and incubatedat 37∘C overnight to establish the mangrove root subtractivelibrary
23 EST Sequencing and Analysis About 400 positive cloneswere selected randomly and amplified using M13 primers(forward and reverse) after removal of contamination fromthe vector and primer sequence Before the assembly searchadaptors polyA tails low quality sequences short sequencesless than 100 bp in length and vector sequences wereremovedThe algorithm search of contigs and singletons wasperformed using CAP3 software This was followed by theobtained sequences being submitted to theNCBI database forhomology search
The BLASTn was used to show degree of similar-ity between the clone cDNA sequence and a knownsequence and the BLASTx (httpblastncbinlmnihgov)showed function of qualified cDNA sequences with largeORF regions Classification of cDNA sequences was basedon their E-value results in the BLAST Categories ofsequence functions are based on the Blast2GO program(httpwwwblast2goorg) [25]
Computational annotation of the mangrove EST datasetswas performed using the Blast2GO software v133 (httpwwwblast2goorg) The BLAST search was performed atNCBI [26] The degree of amino acid sequence similaritywas determined by the use of Wu-Blast from EBI [27]Hydrophilicity and hydrophobicity of serine-rich proteinwere predicted online by MemBrain TMHMM andProtScale (httpwebexpasy orgprotscale) in the toolkitof ExPASy Subcellular localization was investigated usingPSORT II Prediction and Cell-PLoc BaCelLo programThe prediction of secondary structure was carried outby (httpnpsa-pbilibcpfrcgi-binnpsa automatplpage
6 BioMed Research International
=npsa sopmahtml) and PsiPred program The prediction of3D structure was carried out by using the Pfam program
24 Amplification of Full-Length cDNA The complete CDSof serine-rich protein gene contained 696 bp and 223 aminoacids The PCR program according to KAPA HiFi Hot Startwas used as follows initial denaturation at 95∘C for 5min35 cycles of denaturation at 98∘C for 30 s annealing at575∘C for 30 s and extension at 72∘C for 1min The finalextension was 5min at 72∘C Agarose gel (15) was usedto separate PCR-amplified cDNA fragments The expectedbound about 700 bp was purified using gel purificationkit (Qiagen Germany) and 31015840-dA-overhangs (incubation72∘C for 5min) added to the blunt-ended DNA fragmentsgenerated by KAPAHiFi Hot Start DNA polymerase to ligateinto the pDrive cloning vector (Qiagen Germany) and sentfor sequencing
25 Semiquantitative RT-PCRAnalysis Reverse transcriptaseRT-PCR was performed to study the expression of theserine-rich protein gene One120583L of DNase treated (DNaseI Qiagen Germany) total RNA from each of the man-grove roots treated with Si for 15 hrs and 1 to 6 days anduntreated plants was transcribed to the first-strand cDNAusing SuperScript III (Invitrogen USA) and 500 ng oligo(dT) 18 primer in 20120583L reaction volume Reactions werethen incubated at 50∘C for 60min and heated to inactiveat 70∘C for 15min The template cDNAs for both control(untreated) and treated samples were then amplified usingserine primers as F 5-GTCATTCTGCCGAGTTCC-3 and R5-AATGCCCATTTATGTGACTTCG-3 designed accordingto the cDNA sequence homology
Actin gene as an internal control was amplified with thefollowing primers F (51015840CAC TAC TAC TGC TAA ACG GGAAA 31015840) and R (51015840ACA TCT GCT GGA AGG TGC TG 31015840)The following PCR (Tag DNA Polymerase Vivantis USA)program was used 94∘C for 2min and 35 cycles of 94∘C for30 sec 575 and 58∘C respectively for actin and serine for30 sec and 72∘C for 30 sec The PCR program was concludedwith final extension of 7min at 72∘C Actin and serine-richprotein were amplified using the same cDNA templates ThePCR products then were separated with 15 agarose gel andstained with ethidium bromide
26 Real-Time Quantitative RT-PCR Analysis Real-timeqRT-PCR was performed to evaluate the expression lev-els of candidate serine-rich protein genes in the root tis-sues in response to treatments with different concentra-tions of Si compared to these in the control plants TotalRNA was extracted from untreated and Si-treated man-grove roots Extracted RNA was then treated with DNase(DNase I Qiagen Germany) The primers for the actingene were used as in the previous section and for thesecond endogenous control ef1205721 was F 51015840 rarr 31015840 ATTGGA AAC GGA TAT GCT CCA R 51015840 rarr 31015840 TCCTTA CCT GAA CGC CTG TCA serine-rich protein geneprimer was F 51015840 rarr 31015840GCAAGTGCTATGTTCAGGCA R51015840 rarr 31015840AACAATAACGATGGCAAGGC One 120583L aliquot of
DNase treated RNA from each sample was used to prepare20120583L reaction volumes (based on the KAPA SYBER FASTOne-Step qRT-PCR) The reactions involved were a primaryincubation of 42∘C for 5min inactive RT at 95∘C for 5minfollowed by 40 cycles at 95∘C for 3 sec 60∘C for 30 sec and72∘C for 3 sec The 96-well plate was used to analyze eachsample in duplicate In this study all data was from fourindependent biological replicates A standard curve (1198772 gt095) from 10-fold serial dilutions was generated from thepurified cDNA fragments of internal and serine-rich proteingenes (102 10 1 10minus1 and 10minus2 ng120583L)
3 Results
31 Yield and Integrity of RNA The RNA appeared as a non-degraded band on 15 agarose gel containing formaldehydeThe A
260280ratios ranging from 19 to 202 indicate that
there was no protein contamination and the A260230
ratio gt1demonstrated that there is no polyphenol or polysaccharidecontamination [28]The RNA concentration was in the rangeof 05ndash12mg gminus1 The poly(A) + RNA of both treated anduntreated samples appeared as clear smears on the 1 agarosegelwith theA
260280ratio = 2 indicating high quality ofmRNA
obtained for further analysis
32 Construction of the Subtracted cDNA Library The prod-ucts of the subtracted suppression hybridization cDNAlibrary appeared on 12 agarose gel as a smear with rankingsize from 150 bp to 12 kb and 4ndash6 separate bands (Figure 14line A) which obviously differentiates them from the unsub-tracted sample driver (Figure 14 line B) The results of theSSH cDNA library indicated that the differentially expressedgenes are present in the tester or treated samples and absentor present at lower levels in the driver or untreated samplesFor further confirmation of subtraction analysis efficiencyexpression of the actin gene was examined in both the driverand control samples via 23 and 33 cycles of amplificationrespectively indicating that cDNA homologue was removedfrom both the tester and driver samples by subtraction(Figure 14)
33 ESTs Sequencing and Gene Annotation About 700 pos-itive recombinant clones were isolated from the cDNAlibrary Of those about 400 clones were randomly selectedsequenced and analyzed to isolate the gene(s) involved inSi transportation and absorption The 322 ESTs sequenceswere coalesced into 21 contigs and 13 singletons by CAP3assembly program About 195 of the ESTs resulting fromthis library did not have any significant homology to any ofthe proteins existing in the database The DNA fragmentsinvolved in the subtracted cDNA library varied in sizemore or less between 100 and 650 bp (Figure 1) Averagelength of high quality ESTs is 350 bp The most plentifulBLASTx hits correlated to species distribution related toLilium longiflorum Glycinemax Cupressus sempervirens andArabidopsis thaliana (Table 1 and Figure 2)
BioMed Research International 7
34 Classification of Differentially Expressed Genes Accord-ing to gene annotation the 322 ESTs were divided intothree different groups involving biological process molecularfunction and cellular components (Figure 3) The potentialidentities of the genes are based on similarities to thosepresent in the Gen-Bank databases The genes obtained bythe SSH library classified by biological process involve 5different groups ATP synthase (889) equilibrative trans-porter (44) auxin-responsive protein (22) mitochon-drial protein (22) and copia-type polyprotein (22)those classified according to cellular components involve 3different groups The ATP synthase was highly abundantin both the cellular components and biological processcategories (Figures 4 and 5) while senescence-protein wasthe most abundant group in the molecular function category(Figure 6) Classification of the ESTs resulted in 94 ofknown 4 hypothetical and 2 unknown functions
35 Isolation of the Full-Length Serine-Rich Protein GeneOne of the ESTs sequence showing 97 similarity involvesthe ATG codon (20 query cover region) with completeCDS of the serine-rich protein gene of Arachis hypogaea(Figure 7) Full length of the gene (Figure 8) was obtainedthrough amplification of the cDNA template (First-StrandcDNA synthesis Invitrogen USA) and using gene-specificprimers as follows
serine
F 51015840 rarr 31015840 GTCATTCTGCCGAGTTCC
R 51015840 rarr 31015840AATGCCCATTTATGTGACTTCG
36 Analysis of the Differential Expression of Serine-RichProtein Gene Using Semi-qRT-PCR and Real-Time qRT-PCR The semiquantitative RT-PCR analysis showed that theexpression levels of serine-rich protein were generally higherin the Si treatment samples than in the untreated samplesThe expression level of serine-rich protein in the 3-day treatedsample was lower compared to that in the other treatedsamples with varying periods of time (Figure 15) Real-timeqRT-PCR confirmed the results of the semi quantitative RT-PCR (Figure 8) The relative transcript abundance of serine-rich protein in Si-treated plants was higher compared to thatin the untreated plants
The PCR efficiency of all reactions in this study wasbetween 87 and 98 The REST software (Qiagen HildenGermany) was employed to analyze the results of the qRT-PCRThemanufacturerrsquos instructions were followed to quan-tify the relative gene expressionDifferences among treatmentsamples were noted as being statistically significant (119875 lt005)
37 Bioinformatics Analysis The low quality regions at theend and beginning of each sequence were trimmed usinga Phred 20 cutoff value Vector screening was carried outusing the crossmatch Oligo dT tracks and other contam-inants were removed Algorithms of CAP3 [29] assemblywere used to assemble the individual ESTs into clusters of
sequences derived from the same transcript as tentative con-sensus sequences (TCs) and singletons representing uniquetranscripts Obtained sequences were submitted to NCBIdatabase
Prediction of hydrophilicity and hydrophobicity isneeded for predicting protein secondary structure anddivision of functional domain according to the theory thathydrophilicity and hydrophobicity are related to the score ofamino acid Based on the number of hydrophilic amino acidresidues we could hypothesize that the serine-rich proteinwas a hydrophilic protein (Figures 9 and 10)
38 The Prediction of Secondary Structure and FunctionDomain of Serine-Rich Protein The prediction results ofsecondary structure of serine-rich protein by PsiPred showedthat the secondary structure consisted of 11 sheets 3 helixesand 15 coils (Figure 11) Computational analysis of the cDNAclone isolated from mangrove root library indicated that its696 bp coding region codes for a protein of 223 amino acidswith a predicted molecular mass of 2421 kDa Homologysearches run with the full-length amino acid sequences
39 Subcellular Localization The prediction of subcellularlocalization with Cell-PLoc BaCelLo and WoLF PSORTshowed that serine-rich protein is likely to be localizedin chloroplast plastid and mitochondrion with a differ-ent probability of 17 5 and 1 respectively (Figure 12)Biosequence analysis of coding sequence region (CDS) ofserine-rich protein using profile hidden Markov models(HMMER) showed 60 similarity to serine-rich protein ofArachis hypogaea (TRQ0MX20 ARAHY) and 100 sim-ilarity to mitochondrial protein of Medicago truncatula(TRG7I9T8 MEDTR) (Table 2)
310 Prediction of 3D Structure In order to predict the3D structure of serine-rich protein the Phyre2 (ProteinHomology Analogy Recognition Engine) server was used[30] A Phyre2 outputmodel was generated based on the tem-plate galactose-binding domain-like Structural alignment ofserine-rich protein and galactose-binding domain-like wasperformed using theMatchmaker tool of UCSFChimera [31]The 3D structure protein of serine-rich protein gene isolatedand identified in the present study submitted to NCBI withaccession number KF211374 involves MYP bZip LCR1 andDOF transcription factor binding motifs (Figure 13)
4 Discussion
In the present study the suppression subtractive hybridiza-tion (SSH) method was used to identify differentiallyexpressed genes present in the tester sample and absent orpresent at lower levels in the driver The SSH cDNA librarywas constructed using SiO
2-treated (15 hrs and 1 to 6 days)
roots of mangrove plants whereby 321 unique genes wereobtainedThe SSHmethod provides information related onlyto global analysis of gene expression and hence semi quan-titative RT-PCR and real-time qRT-PCR were performedto evaluate the quantitative level of gene expressions over
8 BioMed Research International
Table 1 Putative identities of novel silicon-induced cDNA sequences expressed in R apiculata roots
Gen-Bankaccession Homology Organism 119864-value
Stress responseNP 1995641 Putative auxin-responsive protein Arabidopsis thaliana 500119864 minus 19
DQ8346901 Serine-rich protein mRNA Arachis hypogaea 100119864 minus 72
AAB709281 Proline-rich protein Santalum album 700119864 minus 11
TransporterAET022081 ATP synthase subunit beta Medicago truncatula 300119864 minus 44
XP 0023172511 Equilibrative nucleoside transporter Populus trichocarpa 700119864 minus 40
UnknownXP 0036143941 Hypothetical protein MTR 5g051130 Medicago truncatula 100119864 minus 38
XP 0035471291 Predicted uncharacterized proteinLOC100801 Glycine max 600119864 minus 36
XP 0022691672 Predicted uncharacterized proteinLOC100267 Vitis vinifera 100119864 minus 30
XP 0035490071 Uncharacterized protein LOC100791 Glycine max 500119864 minus 43
XP 0035412191 Uncharacterized protein LOC100796 Glycine max 500119864 minus 43
XP 0035412061 Predicted uncharacterized proteinLOC100788 Glycine max 500119864 minus 43
Cellularmetabolism
XP 0031514401 Senescence-associated protein Loa loa 800119864 minus 25
ACA048501 Senescence-associated protein Picea abies 300119864 minus 21
XP 0021396981 Senescence-associated protein Cryptosporidium hominis TU502 200119864 minus 19
AEM360701 Senescence-associated protein Mytilus eduls 200119864 minus 18
EFW131811 Senescence-associated protein Coccidioides posadasii str 700119864 minus 18
XP 0021182661 Senescence-associated protein Trichoplax adhaerens 100119864 minus 17
XP 0016105631 Senescence-associated protein Babesia bovis T2Bo 100119864 minus 16
XP 0031886401 Plant senescence-associated protein Aspergillus niger CBS 51388 500119864 minus 16
EFN584411 Senescence-associated protein Chlorella variabilis 200119864 minus 11
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 400119864 minus 10
XP 0030649921 Senescence-associated protein Micromonas pusilla CCMP1545 100119864 minus 09
EEH167211 Senescence-associated protein Paracoccidioides brasiliensis 200119864 minus 09
XP 7297621 Senescence-associated protein Plasmodium yoelii yoelii 200119864 minus 06
ABO208511 Putative senescence-associated protein Lilium longiflorum 100119864 minus 41
BAB334211 Putative senescence-associated protein Pisum sativum 900119864 minus 17
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 100119864 minus 15
BAD189051 rRNA intron-encoded endonuclease Thermoproteus sp IC-061 600119864 minus 06
XP 0025153521 Protein binding protein putative Ricinus communis 300119864 minus 34
XM 0035883071 3-Dehydroquinate synthase putative Medicago truncatula 500119864 minus 39
AES586061 Mitochondrial protein putative Medicago truncatula 300119864 minus 37
CAB710631 Copia-type polyprotein Arabidopsis thaliana 300119864 minus 13
AT4g15300 Cytochrome p450-like tbp protein Arabidopsis thaliana 200119864 minus 27
varying periods of Si treatment Most sequences recognizedfrom this subtracted library encoded different genes withhomology of genes from 13 known and 2 unknown species
One EST with known function was selected for analysisof Si-induced expression patterns in varied periods of time
using semi quantitative reverse transcriptase and real-timeqRT-PCR (Figures 15 and 7)
The SSH cDNA library of Si-treated genes was monitoredfor seven times From SSH reports it appears that somegene induction patterns are so rapid and self-motivated that
BioMed Research International 9
Table 2 Pfam domain search option for genomic and proteic annotation
Target Description Species 119864-valueQ0MX20 ARAHY Serine-rich protein Arachis hypogaea 680119864minus147
I3S491 LOTJA Uncharacterized protein Lotus japonicus 490119864 minus 05
G7I9T8 MEDTR Mitochondrial protein putative(geneMTR 1g006300) Medicago truncatula 000084
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
=npsa sopmahtml) and PsiPred program The prediction of3D structure was carried out by using the Pfam program
24 Amplification of Full-Length cDNA The complete CDSof serine-rich protein gene contained 696 bp and 223 aminoacids The PCR program according to KAPA HiFi Hot Startwas used as follows initial denaturation at 95∘C for 5min35 cycles of denaturation at 98∘C for 30 s annealing at575∘C for 30 s and extension at 72∘C for 1min The finalextension was 5min at 72∘C Agarose gel (15) was usedto separate PCR-amplified cDNA fragments The expectedbound about 700 bp was purified using gel purificationkit (Qiagen Germany) and 31015840-dA-overhangs (incubation72∘C for 5min) added to the blunt-ended DNA fragmentsgenerated by KAPAHiFi Hot Start DNA polymerase to ligateinto the pDrive cloning vector (Qiagen Germany) and sentfor sequencing
25 Semiquantitative RT-PCRAnalysis Reverse transcriptaseRT-PCR was performed to study the expression of theserine-rich protein gene One120583L of DNase treated (DNaseI Qiagen Germany) total RNA from each of the man-grove roots treated with Si for 15 hrs and 1 to 6 days anduntreated plants was transcribed to the first-strand cDNAusing SuperScript III (Invitrogen USA) and 500 ng oligo(dT) 18 primer in 20120583L reaction volume Reactions werethen incubated at 50∘C for 60min and heated to inactiveat 70∘C for 15min The template cDNAs for both control(untreated) and treated samples were then amplified usingserine primers as F 5-GTCATTCTGCCGAGTTCC-3 and R5-AATGCCCATTTATGTGACTTCG-3 designed accordingto the cDNA sequence homology
Actin gene as an internal control was amplified with thefollowing primers F (51015840CAC TAC TAC TGC TAA ACG GGAAA 31015840) and R (51015840ACA TCT GCT GGA AGG TGC TG 31015840)The following PCR (Tag DNA Polymerase Vivantis USA)program was used 94∘C for 2min and 35 cycles of 94∘C for30 sec 575 and 58∘C respectively for actin and serine for30 sec and 72∘C for 30 sec The PCR program was concludedwith final extension of 7min at 72∘C Actin and serine-richprotein were amplified using the same cDNA templates ThePCR products then were separated with 15 agarose gel andstained with ethidium bromide
26 Real-Time Quantitative RT-PCR Analysis Real-timeqRT-PCR was performed to evaluate the expression lev-els of candidate serine-rich protein genes in the root tis-sues in response to treatments with different concentra-tions of Si compared to these in the control plants TotalRNA was extracted from untreated and Si-treated man-grove roots Extracted RNA was then treated with DNase(DNase I Qiagen Germany) The primers for the actingene were used as in the previous section and for thesecond endogenous control ef1205721 was F 51015840 rarr 31015840 ATTGGA AAC GGA TAT GCT CCA R 51015840 rarr 31015840 TCCTTA CCT GAA CGC CTG TCA serine-rich protein geneprimer was F 51015840 rarr 31015840GCAAGTGCTATGTTCAGGCA R51015840 rarr 31015840AACAATAACGATGGCAAGGC One 120583L aliquot of
DNase treated RNA from each sample was used to prepare20120583L reaction volumes (based on the KAPA SYBER FASTOne-Step qRT-PCR) The reactions involved were a primaryincubation of 42∘C for 5min inactive RT at 95∘C for 5minfollowed by 40 cycles at 95∘C for 3 sec 60∘C for 30 sec and72∘C for 3 sec The 96-well plate was used to analyze eachsample in duplicate In this study all data was from fourindependent biological replicates A standard curve (1198772 gt095) from 10-fold serial dilutions was generated from thepurified cDNA fragments of internal and serine-rich proteingenes (102 10 1 10minus1 and 10minus2 ng120583L)
3 Results
31 Yield and Integrity of RNA The RNA appeared as a non-degraded band on 15 agarose gel containing formaldehydeThe A
260280ratios ranging from 19 to 202 indicate that
there was no protein contamination and the A260230
ratio gt1demonstrated that there is no polyphenol or polysaccharidecontamination [28]The RNA concentration was in the rangeof 05ndash12mg gminus1 The poly(A) + RNA of both treated anduntreated samples appeared as clear smears on the 1 agarosegelwith theA
260280ratio = 2 indicating high quality ofmRNA
obtained for further analysis
32 Construction of the Subtracted cDNA Library The prod-ucts of the subtracted suppression hybridization cDNAlibrary appeared on 12 agarose gel as a smear with rankingsize from 150 bp to 12 kb and 4ndash6 separate bands (Figure 14line A) which obviously differentiates them from the unsub-tracted sample driver (Figure 14 line B) The results of theSSH cDNA library indicated that the differentially expressedgenes are present in the tester or treated samples and absentor present at lower levels in the driver or untreated samplesFor further confirmation of subtraction analysis efficiencyexpression of the actin gene was examined in both the driverand control samples via 23 and 33 cycles of amplificationrespectively indicating that cDNA homologue was removedfrom both the tester and driver samples by subtraction(Figure 14)
33 ESTs Sequencing and Gene Annotation About 700 pos-itive recombinant clones were isolated from the cDNAlibrary Of those about 400 clones were randomly selectedsequenced and analyzed to isolate the gene(s) involved inSi transportation and absorption The 322 ESTs sequenceswere coalesced into 21 contigs and 13 singletons by CAP3assembly program About 195 of the ESTs resulting fromthis library did not have any significant homology to any ofthe proteins existing in the database The DNA fragmentsinvolved in the subtracted cDNA library varied in sizemore or less between 100 and 650 bp (Figure 1) Averagelength of high quality ESTs is 350 bp The most plentifulBLASTx hits correlated to species distribution related toLilium longiflorum Glycinemax Cupressus sempervirens andArabidopsis thaliana (Table 1 and Figure 2)
BioMed Research International 7
34 Classification of Differentially Expressed Genes Accord-ing to gene annotation the 322 ESTs were divided intothree different groups involving biological process molecularfunction and cellular components (Figure 3) The potentialidentities of the genes are based on similarities to thosepresent in the Gen-Bank databases The genes obtained bythe SSH library classified by biological process involve 5different groups ATP synthase (889) equilibrative trans-porter (44) auxin-responsive protein (22) mitochon-drial protein (22) and copia-type polyprotein (22)those classified according to cellular components involve 3different groups The ATP synthase was highly abundantin both the cellular components and biological processcategories (Figures 4 and 5) while senescence-protein wasthe most abundant group in the molecular function category(Figure 6) Classification of the ESTs resulted in 94 ofknown 4 hypothetical and 2 unknown functions
35 Isolation of the Full-Length Serine-Rich Protein GeneOne of the ESTs sequence showing 97 similarity involvesthe ATG codon (20 query cover region) with completeCDS of the serine-rich protein gene of Arachis hypogaea(Figure 7) Full length of the gene (Figure 8) was obtainedthrough amplification of the cDNA template (First-StrandcDNA synthesis Invitrogen USA) and using gene-specificprimers as follows
serine
F 51015840 rarr 31015840 GTCATTCTGCCGAGTTCC
R 51015840 rarr 31015840AATGCCCATTTATGTGACTTCG
36 Analysis of the Differential Expression of Serine-RichProtein Gene Using Semi-qRT-PCR and Real-Time qRT-PCR The semiquantitative RT-PCR analysis showed that theexpression levels of serine-rich protein were generally higherin the Si treatment samples than in the untreated samplesThe expression level of serine-rich protein in the 3-day treatedsample was lower compared to that in the other treatedsamples with varying periods of time (Figure 15) Real-timeqRT-PCR confirmed the results of the semi quantitative RT-PCR (Figure 8) The relative transcript abundance of serine-rich protein in Si-treated plants was higher compared to thatin the untreated plants
The PCR efficiency of all reactions in this study wasbetween 87 and 98 The REST software (Qiagen HildenGermany) was employed to analyze the results of the qRT-PCRThemanufacturerrsquos instructions were followed to quan-tify the relative gene expressionDifferences among treatmentsamples were noted as being statistically significant (119875 lt005)
37 Bioinformatics Analysis The low quality regions at theend and beginning of each sequence were trimmed usinga Phred 20 cutoff value Vector screening was carried outusing the crossmatch Oligo dT tracks and other contam-inants were removed Algorithms of CAP3 [29] assemblywere used to assemble the individual ESTs into clusters of
sequences derived from the same transcript as tentative con-sensus sequences (TCs) and singletons representing uniquetranscripts Obtained sequences were submitted to NCBIdatabase
Prediction of hydrophilicity and hydrophobicity isneeded for predicting protein secondary structure anddivision of functional domain according to the theory thathydrophilicity and hydrophobicity are related to the score ofamino acid Based on the number of hydrophilic amino acidresidues we could hypothesize that the serine-rich proteinwas a hydrophilic protein (Figures 9 and 10)
38 The Prediction of Secondary Structure and FunctionDomain of Serine-Rich Protein The prediction results ofsecondary structure of serine-rich protein by PsiPred showedthat the secondary structure consisted of 11 sheets 3 helixesand 15 coils (Figure 11) Computational analysis of the cDNAclone isolated from mangrove root library indicated that its696 bp coding region codes for a protein of 223 amino acidswith a predicted molecular mass of 2421 kDa Homologysearches run with the full-length amino acid sequences
39 Subcellular Localization The prediction of subcellularlocalization with Cell-PLoc BaCelLo and WoLF PSORTshowed that serine-rich protein is likely to be localizedin chloroplast plastid and mitochondrion with a differ-ent probability of 17 5 and 1 respectively (Figure 12)Biosequence analysis of coding sequence region (CDS) ofserine-rich protein using profile hidden Markov models(HMMER) showed 60 similarity to serine-rich protein ofArachis hypogaea (TRQ0MX20 ARAHY) and 100 sim-ilarity to mitochondrial protein of Medicago truncatula(TRG7I9T8 MEDTR) (Table 2)
310 Prediction of 3D Structure In order to predict the3D structure of serine-rich protein the Phyre2 (ProteinHomology Analogy Recognition Engine) server was used[30] A Phyre2 outputmodel was generated based on the tem-plate galactose-binding domain-like Structural alignment ofserine-rich protein and galactose-binding domain-like wasperformed using theMatchmaker tool of UCSFChimera [31]The 3D structure protein of serine-rich protein gene isolatedand identified in the present study submitted to NCBI withaccession number KF211374 involves MYP bZip LCR1 andDOF transcription factor binding motifs (Figure 13)
4 Discussion
In the present study the suppression subtractive hybridiza-tion (SSH) method was used to identify differentiallyexpressed genes present in the tester sample and absent orpresent at lower levels in the driver The SSH cDNA librarywas constructed using SiO
2-treated (15 hrs and 1 to 6 days)
roots of mangrove plants whereby 321 unique genes wereobtainedThe SSHmethod provides information related onlyto global analysis of gene expression and hence semi quan-titative RT-PCR and real-time qRT-PCR were performedto evaluate the quantitative level of gene expressions over
8 BioMed Research International
Table 1 Putative identities of novel silicon-induced cDNA sequences expressed in R apiculata roots
Gen-Bankaccession Homology Organism 119864-value
Stress responseNP 1995641 Putative auxin-responsive protein Arabidopsis thaliana 500119864 minus 19
DQ8346901 Serine-rich protein mRNA Arachis hypogaea 100119864 minus 72
AAB709281 Proline-rich protein Santalum album 700119864 minus 11
TransporterAET022081 ATP synthase subunit beta Medicago truncatula 300119864 minus 44
XP 0023172511 Equilibrative nucleoside transporter Populus trichocarpa 700119864 minus 40
UnknownXP 0036143941 Hypothetical protein MTR 5g051130 Medicago truncatula 100119864 minus 38
XP 0035471291 Predicted uncharacterized proteinLOC100801 Glycine max 600119864 minus 36
XP 0022691672 Predicted uncharacterized proteinLOC100267 Vitis vinifera 100119864 minus 30
XP 0035490071 Uncharacterized protein LOC100791 Glycine max 500119864 minus 43
XP 0035412191 Uncharacterized protein LOC100796 Glycine max 500119864 minus 43
XP 0035412061 Predicted uncharacterized proteinLOC100788 Glycine max 500119864 minus 43
Cellularmetabolism
XP 0031514401 Senescence-associated protein Loa loa 800119864 minus 25
ACA048501 Senescence-associated protein Picea abies 300119864 minus 21
XP 0021396981 Senescence-associated protein Cryptosporidium hominis TU502 200119864 minus 19
AEM360701 Senescence-associated protein Mytilus eduls 200119864 minus 18
EFW131811 Senescence-associated protein Coccidioides posadasii str 700119864 minus 18
XP 0021182661 Senescence-associated protein Trichoplax adhaerens 100119864 minus 17
XP 0016105631 Senescence-associated protein Babesia bovis T2Bo 100119864 minus 16
XP 0031886401 Plant senescence-associated protein Aspergillus niger CBS 51388 500119864 minus 16
EFN584411 Senescence-associated protein Chlorella variabilis 200119864 minus 11
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 400119864 minus 10
XP 0030649921 Senescence-associated protein Micromonas pusilla CCMP1545 100119864 minus 09
EEH167211 Senescence-associated protein Paracoccidioides brasiliensis 200119864 minus 09
XP 7297621 Senescence-associated protein Plasmodium yoelii yoelii 200119864 minus 06
ABO208511 Putative senescence-associated protein Lilium longiflorum 100119864 minus 41
BAB334211 Putative senescence-associated protein Pisum sativum 900119864 minus 17
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 100119864 minus 15
BAD189051 rRNA intron-encoded endonuclease Thermoproteus sp IC-061 600119864 minus 06
XP 0025153521 Protein binding protein putative Ricinus communis 300119864 minus 34
XM 0035883071 3-Dehydroquinate synthase putative Medicago truncatula 500119864 minus 39
AES586061 Mitochondrial protein putative Medicago truncatula 300119864 minus 37
CAB710631 Copia-type polyprotein Arabidopsis thaliana 300119864 minus 13
AT4g15300 Cytochrome p450-like tbp protein Arabidopsis thaliana 200119864 minus 27
varying periods of Si treatment Most sequences recognizedfrom this subtracted library encoded different genes withhomology of genes from 13 known and 2 unknown species
One EST with known function was selected for analysisof Si-induced expression patterns in varied periods of time
using semi quantitative reverse transcriptase and real-timeqRT-PCR (Figures 15 and 7)
The SSH cDNA library of Si-treated genes was monitoredfor seven times From SSH reports it appears that somegene induction patterns are so rapid and self-motivated that
BioMed Research International 9
Table 2 Pfam domain search option for genomic and proteic annotation
Target Description Species 119864-valueQ0MX20 ARAHY Serine-rich protein Arachis hypogaea 680119864minus147
I3S491 LOTJA Uncharacterized protein Lotus japonicus 490119864 minus 05
G7I9T8 MEDTR Mitochondrial protein putative(geneMTR 1g006300) Medicago truncatula 000084
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
34 Classification of Differentially Expressed Genes Accord-ing to gene annotation the 322 ESTs were divided intothree different groups involving biological process molecularfunction and cellular components (Figure 3) The potentialidentities of the genes are based on similarities to thosepresent in the Gen-Bank databases The genes obtained bythe SSH library classified by biological process involve 5different groups ATP synthase (889) equilibrative trans-porter (44) auxin-responsive protein (22) mitochon-drial protein (22) and copia-type polyprotein (22)those classified according to cellular components involve 3different groups The ATP synthase was highly abundantin both the cellular components and biological processcategories (Figures 4 and 5) while senescence-protein wasthe most abundant group in the molecular function category(Figure 6) Classification of the ESTs resulted in 94 ofknown 4 hypothetical and 2 unknown functions
35 Isolation of the Full-Length Serine-Rich Protein GeneOne of the ESTs sequence showing 97 similarity involvesthe ATG codon (20 query cover region) with completeCDS of the serine-rich protein gene of Arachis hypogaea(Figure 7) Full length of the gene (Figure 8) was obtainedthrough amplification of the cDNA template (First-StrandcDNA synthesis Invitrogen USA) and using gene-specificprimers as follows
serine
F 51015840 rarr 31015840 GTCATTCTGCCGAGTTCC
R 51015840 rarr 31015840AATGCCCATTTATGTGACTTCG
36 Analysis of the Differential Expression of Serine-RichProtein Gene Using Semi-qRT-PCR and Real-Time qRT-PCR The semiquantitative RT-PCR analysis showed that theexpression levels of serine-rich protein were generally higherin the Si treatment samples than in the untreated samplesThe expression level of serine-rich protein in the 3-day treatedsample was lower compared to that in the other treatedsamples with varying periods of time (Figure 15) Real-timeqRT-PCR confirmed the results of the semi quantitative RT-PCR (Figure 8) The relative transcript abundance of serine-rich protein in Si-treated plants was higher compared to thatin the untreated plants
The PCR efficiency of all reactions in this study wasbetween 87 and 98 The REST software (Qiagen HildenGermany) was employed to analyze the results of the qRT-PCRThemanufacturerrsquos instructions were followed to quan-tify the relative gene expressionDifferences among treatmentsamples were noted as being statistically significant (119875 lt005)
37 Bioinformatics Analysis The low quality regions at theend and beginning of each sequence were trimmed usinga Phred 20 cutoff value Vector screening was carried outusing the crossmatch Oligo dT tracks and other contam-inants were removed Algorithms of CAP3 [29] assemblywere used to assemble the individual ESTs into clusters of
sequences derived from the same transcript as tentative con-sensus sequences (TCs) and singletons representing uniquetranscripts Obtained sequences were submitted to NCBIdatabase
Prediction of hydrophilicity and hydrophobicity isneeded for predicting protein secondary structure anddivision of functional domain according to the theory thathydrophilicity and hydrophobicity are related to the score ofamino acid Based on the number of hydrophilic amino acidresidues we could hypothesize that the serine-rich proteinwas a hydrophilic protein (Figures 9 and 10)
38 The Prediction of Secondary Structure and FunctionDomain of Serine-Rich Protein The prediction results ofsecondary structure of serine-rich protein by PsiPred showedthat the secondary structure consisted of 11 sheets 3 helixesand 15 coils (Figure 11) Computational analysis of the cDNAclone isolated from mangrove root library indicated that its696 bp coding region codes for a protein of 223 amino acidswith a predicted molecular mass of 2421 kDa Homologysearches run with the full-length amino acid sequences
39 Subcellular Localization The prediction of subcellularlocalization with Cell-PLoc BaCelLo and WoLF PSORTshowed that serine-rich protein is likely to be localizedin chloroplast plastid and mitochondrion with a differ-ent probability of 17 5 and 1 respectively (Figure 12)Biosequence analysis of coding sequence region (CDS) ofserine-rich protein using profile hidden Markov models(HMMER) showed 60 similarity to serine-rich protein ofArachis hypogaea (TRQ0MX20 ARAHY) and 100 sim-ilarity to mitochondrial protein of Medicago truncatula(TRG7I9T8 MEDTR) (Table 2)
310 Prediction of 3D Structure In order to predict the3D structure of serine-rich protein the Phyre2 (ProteinHomology Analogy Recognition Engine) server was used[30] A Phyre2 outputmodel was generated based on the tem-plate galactose-binding domain-like Structural alignment ofserine-rich protein and galactose-binding domain-like wasperformed using theMatchmaker tool of UCSFChimera [31]The 3D structure protein of serine-rich protein gene isolatedand identified in the present study submitted to NCBI withaccession number KF211374 involves MYP bZip LCR1 andDOF transcription factor binding motifs (Figure 13)
4 Discussion
In the present study the suppression subtractive hybridiza-tion (SSH) method was used to identify differentiallyexpressed genes present in the tester sample and absent orpresent at lower levels in the driver The SSH cDNA librarywas constructed using SiO
2-treated (15 hrs and 1 to 6 days)
roots of mangrove plants whereby 321 unique genes wereobtainedThe SSHmethod provides information related onlyto global analysis of gene expression and hence semi quan-titative RT-PCR and real-time qRT-PCR were performedto evaluate the quantitative level of gene expressions over
8 BioMed Research International
Table 1 Putative identities of novel silicon-induced cDNA sequences expressed in R apiculata roots
Gen-Bankaccession Homology Organism 119864-value
Stress responseNP 1995641 Putative auxin-responsive protein Arabidopsis thaliana 500119864 minus 19
DQ8346901 Serine-rich protein mRNA Arachis hypogaea 100119864 minus 72
AAB709281 Proline-rich protein Santalum album 700119864 minus 11
TransporterAET022081 ATP synthase subunit beta Medicago truncatula 300119864 minus 44
XP 0023172511 Equilibrative nucleoside transporter Populus trichocarpa 700119864 minus 40
UnknownXP 0036143941 Hypothetical protein MTR 5g051130 Medicago truncatula 100119864 minus 38
XP 0035471291 Predicted uncharacterized proteinLOC100801 Glycine max 600119864 minus 36
XP 0022691672 Predicted uncharacterized proteinLOC100267 Vitis vinifera 100119864 minus 30
XP 0035490071 Uncharacterized protein LOC100791 Glycine max 500119864 minus 43
XP 0035412191 Uncharacterized protein LOC100796 Glycine max 500119864 minus 43
XP 0035412061 Predicted uncharacterized proteinLOC100788 Glycine max 500119864 minus 43
Cellularmetabolism
XP 0031514401 Senescence-associated protein Loa loa 800119864 minus 25
ACA048501 Senescence-associated protein Picea abies 300119864 minus 21
XP 0021396981 Senescence-associated protein Cryptosporidium hominis TU502 200119864 minus 19
AEM360701 Senescence-associated protein Mytilus eduls 200119864 minus 18
EFW131811 Senescence-associated protein Coccidioides posadasii str 700119864 minus 18
XP 0021182661 Senescence-associated protein Trichoplax adhaerens 100119864 minus 17
XP 0016105631 Senescence-associated protein Babesia bovis T2Bo 100119864 minus 16
XP 0031886401 Plant senescence-associated protein Aspergillus niger CBS 51388 500119864 minus 16
EFN584411 Senescence-associated protein Chlorella variabilis 200119864 minus 11
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 400119864 minus 10
XP 0030649921 Senescence-associated protein Micromonas pusilla CCMP1545 100119864 minus 09
EEH167211 Senescence-associated protein Paracoccidioides brasiliensis 200119864 minus 09
XP 7297621 Senescence-associated protein Plasmodium yoelii yoelii 200119864 minus 06
ABO208511 Putative senescence-associated protein Lilium longiflorum 100119864 minus 41
BAB334211 Putative senescence-associated protein Pisum sativum 900119864 minus 17
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 100119864 minus 15
BAD189051 rRNA intron-encoded endonuclease Thermoproteus sp IC-061 600119864 minus 06
XP 0025153521 Protein binding protein putative Ricinus communis 300119864 minus 34
XM 0035883071 3-Dehydroquinate synthase putative Medicago truncatula 500119864 minus 39
AES586061 Mitochondrial protein putative Medicago truncatula 300119864 minus 37
CAB710631 Copia-type polyprotein Arabidopsis thaliana 300119864 minus 13
AT4g15300 Cytochrome p450-like tbp protein Arabidopsis thaliana 200119864 minus 27
varying periods of Si treatment Most sequences recognizedfrom this subtracted library encoded different genes withhomology of genes from 13 known and 2 unknown species
One EST with known function was selected for analysisof Si-induced expression patterns in varied periods of time
using semi quantitative reverse transcriptase and real-timeqRT-PCR (Figures 15 and 7)
The SSH cDNA library of Si-treated genes was monitoredfor seven times From SSH reports it appears that somegene induction patterns are so rapid and self-motivated that
BioMed Research International 9
Table 2 Pfam domain search option for genomic and proteic annotation
Target Description Species 119864-valueQ0MX20 ARAHY Serine-rich protein Arachis hypogaea 680119864minus147
I3S491 LOTJA Uncharacterized protein Lotus japonicus 490119864 minus 05
G7I9T8 MEDTR Mitochondrial protein putative(geneMTR 1g006300) Medicago truncatula 000084
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
Table 1 Putative identities of novel silicon-induced cDNA sequences expressed in R apiculata roots
Gen-Bankaccession Homology Organism 119864-value
Stress responseNP 1995641 Putative auxin-responsive protein Arabidopsis thaliana 500119864 minus 19
DQ8346901 Serine-rich protein mRNA Arachis hypogaea 100119864 minus 72
AAB709281 Proline-rich protein Santalum album 700119864 minus 11
TransporterAET022081 ATP synthase subunit beta Medicago truncatula 300119864 minus 44
XP 0023172511 Equilibrative nucleoside transporter Populus trichocarpa 700119864 minus 40
UnknownXP 0036143941 Hypothetical protein MTR 5g051130 Medicago truncatula 100119864 minus 38
XP 0035471291 Predicted uncharacterized proteinLOC100801 Glycine max 600119864 minus 36
XP 0022691672 Predicted uncharacterized proteinLOC100267 Vitis vinifera 100119864 minus 30
XP 0035490071 Uncharacterized protein LOC100791 Glycine max 500119864 minus 43
XP 0035412191 Uncharacterized protein LOC100796 Glycine max 500119864 minus 43
XP 0035412061 Predicted uncharacterized proteinLOC100788 Glycine max 500119864 minus 43
Cellularmetabolism
XP 0031514401 Senescence-associated protein Loa loa 800119864 minus 25
ACA048501 Senescence-associated protein Picea abies 300119864 minus 21
XP 0021396981 Senescence-associated protein Cryptosporidium hominis TU502 200119864 minus 19
AEM360701 Senescence-associated protein Mytilus eduls 200119864 minus 18
EFW131811 Senescence-associated protein Coccidioides posadasii str 700119864 minus 18
XP 0021182661 Senescence-associated protein Trichoplax adhaerens 100119864 minus 17
XP 0016105631 Senescence-associated protein Babesia bovis T2Bo 100119864 minus 16
XP 0031886401 Plant senescence-associated protein Aspergillus niger CBS 51388 500119864 minus 16
EFN584411 Senescence-associated protein Chlorella variabilis 200119864 minus 11
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 400119864 minus 10
XP 0030649921 Senescence-associated protein Micromonas pusilla CCMP1545 100119864 minus 09
EEH167211 Senescence-associated protein Paracoccidioides brasiliensis 200119864 minus 09
XP 7297621 Senescence-associated protein Plasmodium yoelii yoelii 200119864 minus 06
ABO208511 Putative senescence-associated protein Lilium longiflorum 100119864 minus 41
BAB334211 Putative senescence-associated protein Pisum sativum 900119864 minus 17
ACJ096341 Putative senescence-associated protein Cupressus sempervirens 100119864 minus 15
BAD189051 rRNA intron-encoded endonuclease Thermoproteus sp IC-061 600119864 minus 06
XP 0025153521 Protein binding protein putative Ricinus communis 300119864 minus 34
XM 0035883071 3-Dehydroquinate synthase putative Medicago truncatula 500119864 minus 39
AES586061 Mitochondrial protein putative Medicago truncatula 300119864 minus 37
CAB710631 Copia-type polyprotein Arabidopsis thaliana 300119864 minus 13
AT4g15300 Cytochrome p450-like tbp protein Arabidopsis thaliana 200119864 minus 27
varying periods of Si treatment Most sequences recognizedfrom this subtracted library encoded different genes withhomology of genes from 13 known and 2 unknown species
One EST with known function was selected for analysisof Si-induced expression patterns in varied periods of time
using semi quantitative reverse transcriptase and real-timeqRT-PCR (Figures 15 and 7)
The SSH cDNA library of Si-treated genes was monitoredfor seven times From SSH reports it appears that somegene induction patterns are so rapid and self-motivated that
BioMed Research International 9
Table 2 Pfam domain search option for genomic and proteic annotation
Target Description Species 119864-valueQ0MX20 ARAHY Serine-rich protein Arachis hypogaea 680119864minus147
I3S491 LOTJA Uncharacterized protein Lotus japonicus 490119864 minus 05
G7I9T8 MEDTR Mitochondrial protein putative(geneMTR 1g006300) Medicago truncatula 000084
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
they may not be detected by Si stress applied The resultsof the SSH library constructed in this study contain themajority of induced genes such as serine-rich protein whichwas upregulated and recovered from the roots of two-monthold mangrove seeds after Si treatment Previous studiesidentified Lsi1 Lsi2 and Lsi6 as the genes responsible forSi transportation and accumulation isolated from roots andleaves of rice and cornThe capability of an influx transporteron one side and an efflux transporter on the other side of the
cell to permit effective transcellular transport of the nutrientswas revealed after identification of Lsi1 and Lsi2
The protein encoded by these genes is localized like Lsi1on the plasma membrane of cells in both the exodermisand the endodermis However in contrast to Lsi1 which islocalized on the distal side Lsi2 is localized on the proximalside of the same cells [32] Meanwhile Yamaji et al [33]reported on the role of Lsi6 in plant nutrient redirection atthe node I part A corollary to Yamajirsquos [33] result is that the
10 BioMed Research International
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
role of Lsi6 could be as a transporter involved in intravascularSi transportation In the present study it is reported thatthe serine-rich protein could have a similar role in mangroveroots
The presence of a trichloroethyl-ester-protecting groupand an alcoholic-hydroxyl-group-protecting group in serineis easy to strip down [20] when subjected to modificationwith higher Si content Furthermore serine derivatives canselectively take part in a plurality of chemical reactions andgenerate derivatives that can perform multiple derivatizationreactions [20] Apart from serine-rich proteins some otherglycoproteins and polysaccharides may have roles in Si trans-portation and accumulation For instance Kauss et al [21]argued that the results of Si deposition did not follow an equalindependency from accumulation of phenolics in every plantMoreover Perumalla and Heath [34] mentioned that 221015840-dipyridl an iron-chelating inhibitor of proline hydroxylationdecreased the number of Si deposition sites Hence there is arelationship between availability of proline and deposition ofSi in plants
Mangroves in association with Si may involve manyglycoproteins and polysaccharides enriched by many aminoacids including threonine proline serine glycine glutamicacid and aspartic acids which are OH-terminated The SSHlibrary was successful in identifying 4 ESTs associated withthe serine-rich protein mRNA the sequence submitted tothe Gen-Bank (Access number DQ8346901) and 2 ESTsassociated with proline-rich proteins the sequence submittedto the Gen-Bank (Access number AAB709281) It has beenreported that these polysaccharides play an important role asstable intermediates in Si accumulation transportation andnucleation [20 21]
Many ESTs associated with transcription regulatory andsignal transduction were isolated from the Si-induced man-grove SSH library including ATP synthase subunit betamitochondrial protein and auxin-responsive protein
Several ESTs isolated were homologous to senescence-associated proteins Senescence-associated protein functionsas a defense mechanism in response to diseases causedby fungi bacteria and viruses [35] Several ESTs isolatedfrom the SSH library were homologous to cytochromep450-like tbp protein involved in molecular functions suchas hydroxylation and molecular trajectories for hydroxyla-tion [36] The other less abundant ESTs identified by theSSH library involved one EST which was homologous tothe equilibrative nucleoside transporter (Gen-Bank Accessnumber XP 0023172511) four ESTs associated with rRNAintron-encoded endonuclease (Gen-Bank Access numberBAD189051) one EST homologous to protein binding pro-tein putative (Gen-Bank Access number XP 0025153521)one EST associated with 3-dehydroquinate synthase puta-tive (Gen-Bank Access number XM 0035883071) oneEST homologous to mitochondrial protein putative (Gen-Bank Access number AES586061) and one EST homolo-gous to copia-type polyprotein (Gen-Bank Access numberCAB710631)
The analysis and EST data presented here are a first globaloverview of Si absorption genes inmangroveThe annotationof these ESTs has identified many genes associated with or
having a potential role in Si absorption These genes providea starting point for understanding the nature of molecularmechanisms of plantrsquos Si absorption Energy conversionprocesses of the protein of interested gene are involved inincreasing the amount of some amino acids such as serineand glutamic acid as we observed in the other study whichhas been done over a functional study of the transgenic Ara-bidopsis thaliana using the HPLCmethod On the other handit has been reported that the biosilica formation in differentorganisms is under control by increase in the amount of someamino acids such as serine and proline Hence we concludedthat transformation of serine-rich protein can be effective in Siabsorption and accumulation
In conclusion most of Si-induced SSH mangrove rootswere observed to have a putative or known function whereassome of the ESTs isolated from the SSH have never beenrecorded from other plant species Unrecorded genes mayhave different functions that play different roles in plantsand understanding of their functions would be useful inidentifying the mechanisms involved in plant breeding anddevelopment In the next phase of experiments the full-lengthcopy of the serine-rich protein will be cloned following trans-genic Arabidopsis thaliana and its function as Si transportergene analyzed
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The senior author (Mahbod Sahebi) would like to expresssincere gratitude to Universiti Putra Malaysia (UPM) for thefinancial support provided through the RUGS research Grantno 9327800 and research facilities
References
[1] S Tanaka K Ikeda M Ono and H Miyasaka ldquoIsolationof several anti-stress genes from a mangrove plant Avicenniamarinardquo World Journal of Microbiology and Biotechnology vol18 no 8 pp 801ndash804 2002
[2] Y Kitaya K Yabuki M Kiyota A Tani T Hirano and I AigaldquoGas exchange and oxygen concentration in pneumatophoresand prop roots of four mangrove speciesrdquo Trees vol 16 no 2-3pp 155ndash158 2002
[3] E Ong ldquoMangroves and aquaculture in Malaysiardquo Ambio vol11 no 5 pp 252ndash257 1982
[4] W H Berger V Smetacek and G Wefer ldquoOcean productiv-ity and paleoproductivity-an overviewrdquo in Productivity of theOceans Present and Past Report of the Dahlem Workshop onProductivity of the Ocean Life Sciences Research Reports pp1ndash34 John Wiley amp Sons Chichester UK 1989
[5] J S Bunt ldquoHow can fragile marine ecosystems best be con-servedrdquo in Use and Misuse of the Seafloor K J Hsu andJ Thiede Eds Dahlem Workshop Reports EnvironmentalScience Research Report 11 pp 229ndash242 John Wiley amp SonsChichester UK 1991
BioMed Research International 11
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
[6] T C Jennerjahn and V Ittekkot ldquoRelevance of mangroves forthe production and deposition of organic matter along tropicalcontinentalmarginsrdquoNaturwissenschaften vol 89 no 1 pp 23ndash30 2002
[7] R S Dodd and Z Afzal Rafii ldquoEvolutionary genetics ofmangroves continental drift to recent climate changerdquo Treesvol 16 no 2-3 pp 80ndash86 2002
[8] R S Dodd Z A Rafii and A Bousquet-Melou ldquoEvolutionarydivergence in the pan-Atlantic mangroveAvicennia germinansrdquoNew Phytologist vol 145 no 1 pp 115ndash125 2001
[9] T L Maguire P Saenger P Baverstock and R HenryldquoMicrosatellite analysis of genetic structure in the mangrovespecies Avicennia marina (Forsk) Vierh (Avicenniaceae)rdquoMolecular Ecology vol 9 no 11 pp 1853ndash1862 2001
[10] N C Duke J A H Benzie J A Goodall and E R BallmentldquoGenetic structure and evolution of species in the mangrovegenus Avicennia (avicenniaceae) in the Indo-West PacificrdquoEvolution vol 52 no 6 pp 1612ndash1626 1998
[11] M Lakshmi S Rajalakshmi M Parani C S Anuratha and AParida ldquoMolecular phylogeny of mangroves I Use of molecularmarkers in assessing the intraspecific genetic variability inthe mangrove species Acanthus ilicifolius Linn (Acanthaceae)rdquoTheoretical and Applied Genetics vol 94 no 8 pp 1121ndash11271997
[12] M C Ball ldquoEcophysiology of mangrovesrdquo Trees vol 2 no 3pp 129ndash142 1988
[13] M C Ball S S Mulkey R L Chazdon and A P SmithldquoComparative ecophysiology of mangrove forest and tropicallowlandmoist rainforestrdquo inTropical Forest Plant EcophysiologyS S Mulkey R L Chazdonand and A P Smith Eds pp 461ndash496 Champan and Hall New York NY USA 1996
[14] D M Alongi B F Clough P Dixon and F Tirendi ldquoNutrientpartitioning and storage in arid-zone forests of the mangrovesRhizophora stylosa and Avicennia marinardquo Trees vol 17 no 1pp 51ndash60 2003
[15] E A Dannon and K Wydra ldquoInteraction between siliconamendment bacterial wilt development and phenotype ofRalstonia solanacearum in tomato genotypesrdquo Physiological andMolecular Plant Pathology vol 64 no 5 pp 233ndash243 2004
[16] R V C Diogo and K Wydra ldquoSilicon-induced basal resistancein tomato against Ralstonia solanacearum is related to modifi-cation of pectic cell wall polysaccharide structurerdquo Physiologicaland Molecular Plant Pathology vol 70 no 4ndash6 pp 120ndash1292007
[17] F A R Peters L E Datnoff G H Korndorfer K W Seeboldand M C Rush ldquoEffect of silicon and host resistance on sheathblight development in ricerdquoPlant Disease vol 85 no 8 pp 827ndash832 2001
[18] E A Waraich R Ahmad S Saifullah M Y Ashraf and EEhsanullah ldquoRole of mineral nutrition in alleviation of droughtstress in plantsrdquo Australian Journal of Crop Science vol 5 no 6pp 764ndash777 2011
[19] W Sun J Zhang Q Fan G Xue Z Li and Y LiangldquoSilicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrierrdquoEuropean Journal of Plant Pathology vol 128 no 1 pp 39ndash492010
[20] N Sahai and J A Tossell ldquoFormation energies and NMRchemical calculated for putative serine-silicate complexes insilica biomineralizationrdquoGeochimica et CosmochimicaActa vol65 no 13 pp 2043ndash2053 2001
[21] H Kauss K Seehaus R Franke S Gilbert R A Dietrich andN Kroger ldquoSilica deposition by a strongly cationic proline-richprotein from systemically resistant cucumber plantsrdquoThe PlantJournal vol 33 no 1 pp 87ndash95 2003
[22] W W Zhang G L Jian T F Jiang S Z Wang F J Qi and SC Xu ldquoCotton gene expression profiles in resistant Gossypiumhirsutum cv Zhongzhimian KV1 responding to Verticilliumdahliae strain V991 infectionrdquo Molecular Biology Reports vol39 no 10 pp 9765ndash9774 2012
[23] M Sahebi X T Zhang C Tezara et al ldquoComparison of supres-sion subtractive hybridization with other methods used toidentify differentially expressed genes in plantsrdquo in EngineeringResearch Method N M Adam and S Sorooshian Eds pp 15ndash24 Faculty of Engineering Universiti Putra Malaysia SerdangSelangor Malaysia 2012
[24] M SahebiMMHanafi SNAAbdullahNNejatM Y Rafiiand P Azizi ldquoExtraction of total RNA frommangrove plants toidentify different genes involved in its adaptability to the varietyof stressesrdquo Pakistan Journal of Agricultural Science vol 50 no4 pp 1ndash9 2013
[25] A Conesa S Gotz J M Garcıa-Gomez J Terol M Talonand M Robles ldquoBlast2GO a universal tool for annotationvisualization and analysis in functional genomics researchrdquoBioinformatics vol 21 no 18 pp 3674ndash3676 2005
[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997
[27] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[28] D J Schultz R Craig D L Cox-Foster R O Mumma and JI Medford ldquoRNA isolation from recalcitrant plant tissuerdquo PlantMolecular Biology Reporter vol 12 no 4 pp 310ndash316 1994
[29] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999
[30] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo NatureProtocols vol 4 no 3 pp 363ndash371 2009
[31] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004
[32] J F Ma N Yamaji N Mitani et al ldquoAn efflux transporter ofsilicon in ricerdquo Nature vol 448 no 7150 pp 209ndash212 2007
[33] N Yamaji N Mitatni and J F Ma ldquoA transporter regulatingsilicon distribution in rice shootsrdquoThe Plant Cell vol 20 no 5pp 1381ndash1389 2008
[34] C J Perumalla and M C Heath ldquoThe effect of inhibitors ofvarious cellular processes on the wall modifications inducedin bean leaves by the cowpea rust fungusrdquo Physiological andMolecular Plant Pathology vol 38 no 4 pp 293ndash300 1991
[35] C Espinoza C Medina S Somerville and P Arce-JohnsonldquoSenescence-associated genes induced during compatible viralinteractions with grapevine and Arabidopsisrdquo Journal of Exper-imental Botany vol 58 no 12 pp 3197ndash3212 2007
[36] P RO deMontellanoCytochrome P450 StructureMechanismand Biochemistry Springer Berlin Germany 2004
