Research ArticleIn Silico Analysis of Arabidopsis thaliana Peroxisomal6-Phosphogluconate Dehydrogenase
Aacutelvaro D Fernaacutendez-Fernaacutendez and Francisco J Corpas
Department of Biochemistry Cell and Molecular Biology of Plants Estacion Experimental del ZaidınCSIC Apartado 419 18080 Granada Spain
Correspondence should be addressed to Francisco J Corpas javiercorpaseezcsices
Received 26 December 2015 Accepted 8 February 2016
Academic Editor Leszek A Kleczkowski
Copyright copy 2016 A Fernandez-Fernandez and F J CorpasThis is an open access article distributed under theCreative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
NADPH whose regeneration is critical for reductive biosynthesis and detoxification pathways is an essential component in cellredox homeostasis Peroxisomes are subcellular organelles with a complex biochemical machinery involved in signaling and stressprocesses by molecules such as hydrogen peroxide (H
2O2) and nitric oxide (NO) NADPH is required by several peroxisomal
enzymes involved in120573-oxidation NO and glutathione (GSH) generation Plants have variousNADPH-generating dehydrogenasesone of which is 6-phosphogluconate dehydrogenase (6PGDH) Arabidopsis contains three 6PGDH genes that probably are encodedfor cytosolic chloroplasticmitochondrial and peroxisomal isozymes although their specific functions remain largely unknownThis study focuses on the in silico analysis of the biochemical characteristics and gene expression of peroxisomal 6PGDH(p6PGDH)with the aim of understanding its potential function in the peroxisomal NADPH-recycling system The data show that a group ofplant 6PGDHs contains an archetypal type 1 peroxisomal targeting signal (PTS) while in silico gene expression analysis usingaffymetrix microarray data suggests that Arabidopsis p6PGDH appears to be mainly involved in xenobiotic response growth anddevelopmental processes
1 Introduction
Peroxisomes which are present in almost all cells in eukary-otic organisms are subcellular organelles delimited by asingle membrane [1ndash3] In biochemical terms peroxisomesare characterized by a H
2O2-producing flavin oxidase and
hence their name as well as a catalase enzyme whichbreaks down this H
2O2and is exclusive to this organelle
[4 5] Plant peroxisomes are involved in important physi-ological metabolic pathways such as fatty acid 120573-oxidation(essential for germinating oilseeds mobilizing reserve lipidsand providing carbohydrates) in oxidizing the productsof photorespiration cell signaling by reactive oxygen andnitrogen species (ROS and RNS resp) and phytohormonebiosynthesis such as jasmonic acid (JA) and auxins [6ndash9]In this last case it has been shown that peroxisomal fattyacid 120573-oxidation machinery is responsible for 120573-oxidationof the carboxylic acid side chain of 12-oxophytoenoic acid(OPDA) in JA biosynthesis [9 10] On the other hand
indole-3-butyric acid (IBA) is a chain-elongated form of theactive auxin indole-3-acetic acid (IAA) and based on geneticanalysis and studies of IBA metabolism IBA conversionto IAA occurs in peroxisomes [11ndash14] In recent years theidentification of novel and unexpected peroxisomal proteinshas raised questions concerning the potential functions ofthese organelles in this intensely studied field of cell biologyFor example the presence of nitric oxide (NO) in peroxi-somes suggests that these organelles are a source of the signalmolecule NO This is supported by biochemical molecularand cellular approaches which indicate that plant peroxi-somes contain a L-arginine-dependent nitric oxide synthase(NOS) activity which is strictly dependent on NADPH andrequires calmodulin (CaM) and Ca2+ [15ndash17] Moreover thisprotein responsible to generate NO seems to be imported by aperoxisomal targeting signal type 2 (PTS-2) in a process thatdepends on the cytosolic receptor PEX7 CaM and Ca2+ [18]However little is still known about many biological aspectsof peroxisomes Unlike chloroplasts and mitochondria these
Hindawi Publishing CorporationScientificaVolume 2016 Article ID 3482760 9 pageshttpdxdoiorg10115520163482760
2 Scientifica
organelles do not contain endogenous DNA and all proteinsare encoded by nuclear genes synthesized in the cytoplasmand imported into the peroxisomes [19ndash22] While smallmolecules can access the peroxisomal matrix via passivetransport [23] peroxisomal proteins contain a peroxisomaltargeting signal (PTS) that enables them to be imported intothe organelle A group of proteins called peroxins (PEXs) areinvolved in this process with a total of 22 peroxins havingbeen identified in plants [3 24] To date two main types ofPTSs have been identified each of which has its own receptorthe protein is then transferred to a translocation complexthat mediates its transport across the peroxisomemembraneMost peroxisomalmatrix proteins are targeted by a noncleav-able tripeptide peroxisomal targeting signal 1 (PTS1) at theextreme C-terminus with the consensus sequence Ser-Lys-Leu (SKL) Other proteins are directed to peroxisomes by acleavable nanopeptide peroxisomal targeting signal 2 (PTS2)at the N terminus bearing the sequence (RK)-(LVI)-X
5-
(QH)-(LAI) [3]NADPH is an essential cofactor in many metabolic
reactions In plant peroxisomes several enzymes activitiesare strictly dependent on the presence of NADPH such asL-arginine-dependent nitric oxide synthase (NOS) whichfacilitates NO production [1 15] glutathione reductase (GR)which recycles reduced glutathione (GSH) from its oxidizedform (GSSG) [1] and 24-dienoyl-CoA reductase (DECR)which participates in the degradation of unsaturated fattyenoyl-CoA esters and has double bonds in both even- andodd-numbered positions in peroxisomes [25 26] (Figure 1)The different cell organelles are known to have multipletransporters in order to exchange metabolic intermedi-ates [27ndash29] however as to our knowledge NADPH hasno direct transport mechanism each cell compartmentmust have its own NADPH-regenerating system In higherplants during the dark phase of photosynthesis and innonphotosynthetic cells NADPH is principally regeneratedby a group of NADP-dehydrogenase enzymes includingNADP-isocitrate dehydrogenase (NADP-ICDH) the NADP-malic enzyme (NADP-ME) glucose-6-phosphate dehydro-genase (G6PDH) and 6-phosphogluconate dehydrogenase(6PGDH) the latter two belonging to the oxidative pentosephosphate pathway (PPP) [30 31] Using electronmicroscopyimmunogold labeling and biochemical techniques previousstudies have demonstrated the presence of several NADPH-generating dehydrogenases such as NADP-ICDH [32 33]and also G6PDH [34] in pea leaf peroxisomes Howeversome biochemical [34] and molecular data [26] stronglysuggest the potential localization of 6PGDH in peroxisomesThe present study therefore explores this possibility throughan in silico analysis of plant 6PGDHswith particular emphasison Arabidopsis 6PGDHs The data reveal the presence ofthe archetypal PTS1 in a group of 6PGDHs thus suggestingits peroxisomal localization while affymetrix microarraydata on the putative Arabidopsis p6PGDH suggest that it isinvolved in xenobiotic response growth and developmentalprocesses
6PG6PGDH
Rib 5-P + CO2
NADPH
24-Dienoyl-CoADECR
L-Arginine NOS ∙NO
GSSGGR
GSH
trans-3-Enoyl-CoA
Figure 1 Functions of the endogenous NADPH in plant peroxi-somes NADPH is required for several enzymatic systems includ-ing the glutathione reductase (GR) to keep the level of reducedglutathione (GSH) the L-arginine-dependent nitric oxide synthase(NOS) which generates nitric oxide (NO) and the 24-dienoyl-CoA reductase (DECR) which is necessary for the degradationof fatty acids unsaturated on odd-numbered carbons 6PGDH 6-phosphogluconate dehydrogenase 6PG 6-phosphogluconate Rib5-P ribulose 5-phosphate
2 Methods
21 Sequence Analysis Database Searches and SubcellularLocalization Predictions 6PGDH protein sequences fromcompleted genomes were retrieved from Plaza 30 (httpbioinformaticspsbugentbeplaza) [35] Blast searches werecarried out on the National Center for Biotechnology Info-rmation (NCBI) web site (httpwwwncbinlmnihgov)Alignments were performed using CLUSTAL W2 (httpwwwebiacukToolsmsaclustalw2) Localization predic-tions were made using WoLF (httpwolfpsortorg) [36]and peroxisomal targeting signal type 1 (PTS1) Predictor(httpmendelimpacatmendeljspsatpts1PTS1predictorjsp) [37ndash39] Molecular protein properties were estimatedusing the httpwwwexpasyorgproteomicsprotein charac-terisation and function Phylogenetic analyses were conduc-ted usingMEGA software version 60 (httpwwwmegasoft-warenet) [40] The molecular properties of Arabidopsis6PGDH isozymes based on their predicted amino acidsequences were used for the in silico predictions with the aidof httpwwwexpasyorgtoolsprotparamhtml
Promoter analysis and identification of cis-regulatoryelements were carried out according to the Plant Promo-ter 21 db program (httpppdbagrgifu-uacjpppdbcgi-binindexcgi)
22 Gene Expression Analyses of Peroxisomal 6PGDH underDiverse Growth Conditions and Xenobiotics Gene expressionanalyses under different conditions were carried out withthe aid of the Gene Expression Omnibus (GEO) database(httpwwwncbinlmnihgovgeo) [41] using AffymetrixMicroarray Suite 50 (MAS5)The growth conditions selectedwere as follows (i) For organ analysis Arabidopsis thaliana
Scientifica 3
plants were grown at a density of 4 plants per 127 cmsquare pot in either a growth chamber or green house setto 25∘C by day and 20∘C by night Days were set to a 16 hphotoperiod with 125 120583molmminus2 sminus1 fluorescent irradiationExpanding leaves were harvested 15 days after germinationin midphotoperiod The expanding upper 5 cm of the stemwith siliques and pedicels removed was harvested 29 daysafter germination in midphotoperiod Developed flowersand unopened buds were harvested 29 days after germina-tion in midphotoperiod (ii) For the sucrose effect on 4-day-old dark-grown seedlings Arabidopsis thaliana ecotypeColumbia was grown for 4 days in the dark at 23∘C in mul-tiwell plates containing half-strength Murashige Skoog (MS)medium without added sucrose Samples were subsequentlykept for 6 h under the same conditions with the addition of90mM sucrose (shaking thus aerobic) (iii) For herbicideeffect on 5-day-old seedlings Arabidopsis seeds were grownonhalf-strengthMSplates supplementedwith 1 sucrose andgrown at 21∘Cunder continuous light (100120583molmminus2 sminus1)withand without 5 120583M norflurazon (iv) For auxin treatmentsArabidopsis seedlings were grown for 10 d on 1x MS agar-solidifiedmedia under long-day conditions (16 8 white lightand dark cycle) Seedlings were then transferred to 2 differentliquid media containing either 01 120583M 24-D or 01 120583M 24-Dplus 1120583M brassinazole After 8 h of treatment the seedlingswere blotted with paper towels to remove excess media andsubjected to total RNA isolation
3 Results and Discussion
31 In Silico Analysis of Plant 6PGDHs Identification of Pero-xisomal Targeting Signal 1 (PTS1) At present the infor-mation available in different plant databases has increasedsignificantly and constitutes an unparalleled resource toprovide complementary data for experimental studies [42]The in silico analysis of putative peroxisomal 6PGDH aimsto describe the most important properties of this proteinin plants with particular attention paid to Arabidopsisthaliana This enzyme catalyzes the third and irreversiblereaction of the pentose phosphate pathway (PPP) whichconducts the oxidative decarboxylation of the free acid of 6-phosphogluconate to yield ribulose-5-phosphate obtainingCO2and NADPH In higher plants in comparison with the
G6PDH enzyme that regulates the PPP the 6PGDH enzymehas beenmuch less studied and is assumed to have a cytosolicand chloroplastic localization [43 44] The phylogeneticanalysis of 45 representative 6PGDH protein sequences fromdifferent organisms shows that plant 6PGDH proteins con-stitute a group clearly separated from the 6PGDH proteinsof other organisms including prokaryote and eukaryote (seeSupplemental Figure 1 in Supplementary Material availableonline at httpdxdoiorg10115520163482760) The rep-resentative 6PGDH sequences selected for this analysis aresummarized in Supplemental Table 1
With the aim of gaining a deeper understanding of thesubcellular localization of plant 6PGDHs a phylogeneticanalysis was carried out by exclusively using 51 plant 6PGDHprotein sequences Two main groups were found whichappear to be located either in chloroplasts (32 sequences) or in
peroxisomes (15 6PGDH sequences) with only four 6PGDHsequences belonging to these two main groups appearingto be located in the cytosol (Figure 2) In the peroxisomecandidate group of 6PGDH sequences a search of the maintype I and type II peroxisomal targeting signals (PTSs) wascarried out This enabled us to identify a total of 10 6PGDHprotein sequences which contain a PTS1 motif with thetripeptide SKI or SRI [22] Table 1 summarizes some charac-teristics (number of amino acids pI and molecular mass) ofthese putative peroxisomal 6PGDH proteins of higher plantswhich have a putative type 1 peroxisomal targeting signal(PTS1) The tripeptides (SKI and SRI) identified belong tothe canonical PTS1 sequence which is the tripeptide (SA)-(KR)-(LMI) at the extreme C-terminus In general theseconserved tripeptides are highly abundant in peroxisomalmatrix proteins [39] although other peroxisomalmatrix pro-teins have noncanonical C-terminal tripeptidesThe latter aremuch less conserved and generally occur in low-abundanceperoxisomal matrix proteins [45 46]
32 In Silico Analysis of Peroxisomal 6PGDH in Arabidopsisthaliana At present there aremany genetic and biochemicalinformation available on Arabidopsis thaliana Thereforethis plant has become a powerful tool to study manyaspects of higher plants [24] Analysis of the Arabidopsisdatabase shows that its genome contains three 6PGDHgenes At5g41670 At3g02360 and At1g64190 coding forproteins BAB11473 AEE73797 and AAF24560 respectivelyThe alignment of the deduced amino acid sequence ofthe three Arabidopsis 6PGDHs indicates that the proteinsequence of the three isozymes is highly conserved with 75similarity between BAB11473 and AEE73797 and 93 simi-larity between BAB11473 and AAF24560 In the sequence ofdeduced amino acids of the threeArabidopsis 6PGDHs puta-tiveNADPbinding sites with theGxGVxxGxxxG consensussequence and substrate binding sites with the LIVM-x-D-x-x-GANQS-KGTG-x-W sequence were identified(Supplemental Figure 2) These two sites are completelyconserved throughout all 6PGDH sequences from othersplant species Table 2 summarizes the principal molecularproperties of each Arabidopsis 6PGDH isozyme based onthe predicted amino acid sequences in each case By usingdifferent subcellular prediction programs it was found that6PGDH can have different locations including chloroplaststhe cytosol mitochondria and peroxisomes with subcellularlocalization showing the most significant differences amongthese 6PGDHs
The gene encoding putative peroxisomal 6PGDH(At3g02360) has a total length of 225 kb while its coordinateson chromosome 3 from A thaliana are 481898 and 484147The At3g02360 gene is transcribed in the nucleus usingtwo possible mRNAs (NM 1111035 and NM 1801712) withlengths of 1828 bp and 1829 bp respectively Both mRNAmolecules have an intron in the 51015840-UTR region and twoexons one containing part of the 51015840-UTR region and theother containing a small portion of the remaining 51015840-UTRregion CDS and 31015840-UTR (Supplemental Figure 3 andTable 3) Promoter analysis enabled us to detect a variant ofthe TATA box at positions minus565 and minus553 (Table 3) as well
4 Scientifica
gi|778707525|gi|59
0651
654|Th
gi|56616
5991
|
gi|2555799
36|Ricinus communis LKN-COOHgi|731375254|Vitis vinifera SKI-COOH
Figure 2 Evolutionary relationships of plant 6PGDHs The evolutionary history was inferred using the Neighbor-Joining method Theoptimal tree with the sum of branch length = 242428701 is shownThe tree is drawn to scale with branch lengths in the same units as those ofthe evolutionary distances used to infer the phylogenetic treeThe evolutionary distances were computed using the Poisson correctionmethodand are in the units of the number of amino acid substitutions per siteThe rate variation among sites was modeled with a gamma distribution(shape parameter = 1) The analysis involved 51 amino acid sequences All positions containing gaps and missing data were eliminated Therewere a total of 416 positions in the final dataset Evolutionary analyses were conducted in MEGA6 [30]
Scientifica 5
Table 1 Identification of 6PGDH proteins sequence of higher plants with a putative peroxisomal location for having a peroxisomal targetingsignal type 1 (PTS1) on the C-terminal The pI and MM values were calculated from their primary structure
Table 2 Genes encoding different isozymes of 6PGDH in A thaliana and molecular properties based on their predicted amino acidsequence The number of amino acids corresponds to the preprocessed protein and they were used for the in silico predictions usinghttpwebexpasyorgprotparam Transit peptide (TP) or targeting signal (TS) length is given in amino acids and molecular weight (MW)of the mature
Properties LocusAt5g41670 At3g02360 At1g64190
Protein accession number BAB11473 AEE73797 AAF24560Number of amino acids 487 486 487Subunit size (Da) 5331761 5357718 5337751pI 562 702 534Total number of negatively charged residues (Asp + Glu) 65 64 66Total number of positively charged residues (Arg + Lys) 60 64 57Stability indexlowast 2369 2686 2760120576280
(Mminus1 cmminus1) (assuming all Cys residues are reduced) 65320 63830 63830Aliphatic indexlowastlowast 8856 8710 8758Grand average of hydropathicity (GRAVY) indexlowastlowastlowast minus0278 minus0283 minus0272Transit peptide (TP)targeting signal (TS) mdash -SKI mdashSubcellular localization ChloroplastCytosol Peroxisome ChloroplastmitochondrionCytosollowastA protein with a stability index smaller than 40 is predicted as being stable with a value above 40 the protein is predicted as potentially unstablelowastlowastAliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (Ala Val Ile and Leu) A positive index indicates the increaseof thermostability of globular proteinslowastlowastlowastGRAVY (grand average of hydropathicity) index indicates the solubility of the proteins positive GRAVY (hydrophobic) negative GRAVY (hydrophilic)
as various regulatory elements identified in both transcripts(supplemental Figure 3) with the aid of the Plant Promoter21 db program
33 Gene Expression of Peroxisomal 6PGDH (p6PGDH)Figure 3 shows gene expression data for Arabidopsisp6PGDH under different growth conditions and is exposed tocertain chemicals Figure 3(a) shows that in adult plantsp6PGDH expression levels were highest in stems followedby flowers and lowest in leaves independently of the growthconditions in either the growth chamber or greenhouse Onthe other hand when Arabidopsis plants were grown underin vitro conditions and in the presence of 90mM sucrosep6PGDH gene expression was between 2-fold and 3-foldhigher (Figure 3(b)) suggesting as would be expected thatp6PGDH is involved in the carbon metabolism as thisenzyme is located in the oxidative part of the pentose phos-phate pathway [43] Additionally when plants were exposedto the herbicide norflurazon (a carotenoid biosynthesis
inhibitor) a similar 2-fold to 3-fold increase in p6PGDHgene expression was observed (Figure 3(c)) In the lattercase it is important to note that carotenoids have antioxidantproperties that help to protect chlorophyll from oxidativedamage mediated by ROS [47] as the absence of carotenoidsfacilitates chlorophyll destruction which is essential forphotosynthesisThese data are closely in line with the detoxi-fication capacity of peroxisomes which would amelioratedthe diminished antioxidant capacity of ROS to decomposechloroplasts damaged by this herbicide This is explainedby the fact that peroxisomes contain an important batteryof antioxidant enzymes including catalase superoxidedismutase and all components of the ascorbate-glutathionecycle which requires NADPH to support the regenerationof GSH by GR [1] Furthermore the relative expression ofp6PGDH was also higher in the presence of brassinazole(Figure 3(d)) a specific inhibitor of the biosynthesis ofbrassinosteroids which are a class of phytohormones thatplay an essential role in plant growth and development
Figure 3 Peroxisomal 6PGDH gene (At3g02360) expression in Arabidopsis thaliana grown under different conditions (a) Expression ofp6PGDH in leaves of 15-day-old plants stems and flowers of 29-day-old plants grown in either growth chamber or greenhouse conditions(b) Effects of a 6 h long treatment with 90mM sucrose to 4-day-old dark-grown Arabidopsis seedlings (c) Effects of 5120583M norflurazon to5-day-old continuous light Arabidopsis seedlings (d) Auxin effect on 10-day-old seedlings treated for 8 h either 01 120583M24-D or 01 120583M24-Dplus 1120583M brassinazole Data were obtained from the Gene Expression Omnibus (GEO) database and analyzed using Affymetrix MicroarraySuite 50 (MAS5) The original sample accessions (GSMxxx) are listed in the gray boxes along the bottom of the chart
Scientifica 7
Table 3 Analysis of the 51015840-UTR region At3G02360 gene coding for Arabidopsis thaliana peroxisomal 6PGDH Inr initiator TATA box anoctamer group related to TATA box Y patch an octamer group of the pyrimidine (Y) patch REG regulatory element group an octamergroup related to cis-regulatory elements CDS protein coding region UTR untranslated region
Type Sequence Genome position Position from initiation codonStrand Start End Start End
processes including promotion of stem elongation andcell division All these data suggest that p6PGDH couldbe specifically involved in growth and development sinceits gene expression is clearly induced by molecules whichblock these processes This is supported by a set of datawhich shows that sugar can promote hypocotyl elongation inArabidopsis in darkness a process which is largely dependenton brassinosteroids [48]
The reducing power (NADPH) generated by 6PGDH isknown to be important whose activity and gene expressioncan change depending on the type of stress and subcellularlocalization For example tobacco plants infected with potatovirus Y show increased cytosolic and plastidic 6PGDH activ-ity [49] Similar behavior has been observed in pepper leaveswith an increase in total 6PGDHactivity under cadmium [50]and low temperature [51] stress conditions Similar behaviorhas been described with regard to 6PGDH gene expressionThus in rice (Oryza sativa L) plants subjected to salt stress(150mM NaCl) an increase in 6PGDH transcripts in stemshas been reported [52] A subsequent study in which twogenes encoding for chloroplastic and cytosolic 6PGDH riceplants showed that both transcripts increased not only withsalinity but also with other stresses such as drought lowtemperature and treatment with abscisic acid however thetranscripts of G6PDH the first enzyme of the oxidativeportion of the pentose phosphate pathway did not undergoany change in its cytosolic and chloroplastic isoforms [53]Furthermore there is evidence to show that this enzyme isalso involved in other physiological processes for example instudies of the maturation of pepper fruits when they changefrom the green to red phenotype total 6PGDH activityincreased by more than 50 [54] In the case of tomato andspinach three genes encoding cytosolic and chloroplasticisozymes have been reported [55 56] also with respect toother plant species such as peas corn and tobacco [52 57ndash59]
4 Conclusion
In summary it is possible to conclude that there is a groupof plant 6PGDH enzymes which contain an archetypal type1 peroxisomal targeting signal Given the capacity of thisenzyme to generate NADPH in silico analysis of peroxisomal6PGDH in Arabidopsis thaliana suggests that it plays aprominent role during early seedling development in which
peroxisomes perform the key function of metabolizing lipidreserves until the seedling begins to photosynthesize [6061] for which NADPH is required Similarly at this earlyseedling stage the NADPH-dependent generation of nitricoxide in peroxisomes has also been demonstrated to beinvolved in root development [62] Finally the increase inp6PGDH in the presence of xenobiotics such as norflurazonand brassinazole is also closely in line with the induction ofthe antioxidant system present in plant peroxisomes as hasbeen demonstrated with the pea leaf peroxisomes of plantsexposed to 24-D [63]Therefore the present in silico analysissuggests the presence of a 6PGDH into peroxisomes whichwould contribute to the generation of NADPH within theseorganelles
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Research is supported by an ERDF-cofinanced grant fromthe Ministry of Science and Innovation (Recupera 2020-20134R056) and the Junta de Andalucıa (Group BIO192)
References
[1] F J Corpas J B Barroso and L A Del Rıo ldquoPeroxisomesas a source of reactive oxygen species and nitric oxide signalmolecules in plant cellsrdquo Trends in Plant Science vol 6 no 4pp 145ndash150 2001
[2] M Schrader and H D Fahimi ldquoPeroxisomes and oxidativestressrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1763 no 12 pp 1755ndash1766 2006
[3] J Hu A Baker B Bartel et al ldquoPlant peroxisomes biogenesisand functionrdquo Plant Cell vol 24 no 6 pp 2279ndash2303 2012
[4] I Pracharoenwattana and S M Smith ldquoWhen is a peroxisomenot a peroxisomerdquo Trends in Plant Science vol 13 no 10 pp522ndash525 2008
[5] F J Corpas ldquoWhat is the role of hydrogen peroxide in plantperoxisomesrdquo Plant Biology vol 17 no 6 pp 1099ndash1103 2015
[6] T G Cooper and H Beevers ldquoBeta oxidation in glyoxysomesfrom castor bean endospermrdquo The Journal of Biological Chem-istry vol 244 no 13 pp 3514ndash3520 1969
8 Scientifica
[7] S Reumann and A P M Weber ldquoPlant peroxisomes respirein the light some gaps of the photorespiratory C
2cycle have
become filledmdashothers remainrdquo Biochimica et Biophysica Actavol 1763 no 12 pp 1496ndash1510 2006
[8] A Baker and R Paudyal ldquoThe life of the peroxisome from birthto deathrdquo Current Opinion in Plant Biology vol 22 pp 39ndash472014
[9] L A del Rıo L M Sandalio F J Corpas J M Palma andJ B Barroso ldquoReactive oxygen species and reactive nitrogenspecies in peroxisomes Production scavenging and role in cellsignalingrdquo Plant Physiology vol 141 no 2 pp 330ndash335 2006
[10] A J K Koo H S Chung Y Kobayashi and G A Howe ldquoIden-tification of a peroxisomal acyl-activating enzyme involved inthe biosynthesis of jasmonic acid in ArabidopsisrdquoThe Journal ofBiological Chemistry vol 281 no 44 pp 33511ndash33520 2006
[11] B K Zolman and B Bartel ldquoAn Arabidopsis indole-3-butyricacid-response mutant defective in PEROXIN6 an apparentATPase implicated in peroxisomal functionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 6 pp 1786ndash1791 2004
[12] B K Zolman M Nyberg and B Bartel ldquoIBR3 a novelperoxisomal acyl-CoA dehydrogenase-like protein required forindole-3-butyric acid responserdquo Plant Molecular Biology vol64 no 1-2 pp 59ndash72 2007
[13] A A G Wiszniewski W Zhou S M Smith and J D BussellldquoIdentification of twoArabidopsis genes encoding a peroxisomaloxidoreductase-like protein and an acyl-CoA synthetase-likeprotein that are required for responses to pro-auxinsrdquo PlantMolecular Biology vol 69 no 5 pp 503ndash515 2009
[14] G M Spiess and B K Zolman ldquoPeroxisomes as a source ofauxin signalingmoleculesrdquo Subcellular Biochemistry vol 69 pp257ndash281 2013
[15] J B Barroso F J Corpas A Carreras et al ldquoLocalizationof nitric-oxide synthase in plant peroxisomesrdquo The Journal ofBiological Chemistry vol 274 no 51 pp 36729ndash36733 1999
[16] F J Corpas J B Barroso A Carreras et al ldquoCellular andsubcellular localization of endogenous nitric oxide in young andsenescent pea plantsrdquo Plant Physiology vol 136 no 1 pp 2722ndash2733 2004
[17] F J Corpas M Hayashi S Mano M Nishimura and J BBarroso ldquoPeroxisomes are required for in vivo nitric oxide accu-mulation in the cytosol following salinity stress of arabidopsisplantsrdquo Plant Physiology vol 151 no 4 pp 2083ndash2094 2009
[18] F J Corpas and J B Barroso ldquoPeroxisomal plant nitric oxidesynthase (NOS) protein is imported by peroxisomal targetingsignal type 2 (PTS2) in a process that depends on the cytosolicreceptor PEX7 and calmodulinrdquo FEBS Letters vol 588 no 12pp 2049ndash2054 2014
[19] L L Cross H T Ebeed and A Baker ldquoPeroxisome biogenesisprotein targeting mechanisms and PEX gene functions inplantsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular CellResearch 2015
[20] P B Lazarow andY Fujiki ldquoBiogenesis of peroxisomesrdquoAnnualReview of Cell Biology vol 1 no 1 pp 489ndash530 1985
[21] M Hayashi and M Nishimura ldquoEntering a new era of researchon plant peroxisomesrdquo Current Opinion in Plant Biology vol 6no 6 pp 577ndash582 2003
[22] S Reumann C Ma S Lemke and L Babujee ldquoAraPerox Adatabase of putative arabidopsis proteins from plant peroxi-somesrdquo Plant Physiology vol 136 no 1 pp 2587ndash2608 2004
[23] V D Antonenkov and J K Hiltunen ldquoPeroxisomal membranepermeability and solute transferrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1763 no 12 pp 1697ndash17062006
[24] M Hayashi andM Nishimura ldquoArabidopsis thalianamdashamodelorganism to study plant peroxisomesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1763 no 12 pp 1382ndash13912006
[25] C W T van Roermund E H Hettema A J Kal M vanden Berg H F Tabak and R J A Wanders ldquoPeroxisomal 120573-oxidation of polyunsaturated fatty acids in Saccharomyces cere-visiae isocitrate dehydrogenase providesNADPH for reductionof double bonds at even positionsrdquo The EMBO Journal vol 17no 3 pp 677ndash687 1998
[26] S Reumann L Babujee M Changle et al ldquoProteome analysisofArabidopsis leaf peroxisomes reveals novel targeting peptidesmetabolic pathways and defense mechanismsrdquo The Plant Cellvol 19 no 10 pp 3170ndash3193 2007
[27] M Kunze I Pracharoenwattana S M Smith and A Hartig ldquoAcentral role for the peroxisomal membrane in glyoxylate cyclefunctionrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp1441ndash1452 2006
[28] N Linka and A P M Weber ldquoIntracellular metabolite trans-porters in plantsrdquoMolecular Plant vol 3 no 1 pp 21ndash53 2010
[29] F L Theodoulou K Bernhardt N Linka and A BakerldquoPeroxisome membrane proteins multiple trafficking routesandmultiple functionsrdquo Biochemical Journal vol 451 no 3 pp345ndash352 2013
[30] F J Corpas and J B Barroso ldquoNADPH-generating dehydroge-nases their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditionsrdquoFrontiers in Environmental Science vol 2 article 55 2014
[31] M F Drincovich P Casati and C S Andreo ldquoNADP-malicenzyme from plants a ubiquitous enzyme involved in differentmetabolic pathwaysrdquo FEBS Letters vol 490 no 1-2 pp 1ndash62001
[32] F J Corpas J B Barroso L M Sandalio J M Palma J ALupianez and L A Del Rıo ldquoPeroxisomal NADP-dependentisocitrate dehydrogenase Characterization and activity regula-tion during natural senescencerdquo Plant Physiology vol 121 no 3pp 921ndash928 1999
[33] M Leterrier J B Barroso R Valderrama et al ldquoPeroxisomalNADP-isocitrate dehydrogenase is required for Arabidopsisstomatal movementrdquo Protoplasma 2015
[34] F J Corpas J B Barroso L M Sandalio et al ldquoAdehydrogenase-mediated recycling system of NADPH in plantperoxisomesrdquo Biochemical Journal vol 330 no 2 pp 777ndash7841998
[35] S Proost M van Bel L Sterck et al ldquoPLAZA a comparativegenomics resource to study gene and genome evolution inplantsrdquoThe Plant Cell vol 21 no 12 pp 3718ndash3731 2009
[36] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquoNucleic Acids Research vol 35 no 2 ppW585ndashW587 2007
[37] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoPrediction of peroxisomal targeting signal 1containing proteins from amino acid sequencerdquo Journal ofMolecular Biology vol 328 no 3 pp 581ndash592 2003
[38] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoMotif refinement of the peroxisomal targetingsignal 1 and evaluation of taxon-specific differencesrdquo Journal ofMolecular Biology vol 328 no 3 pp 567ndash579 2003
Scientifica 9
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
organelles do not contain endogenous DNA and all proteinsare encoded by nuclear genes synthesized in the cytoplasmand imported into the peroxisomes [19ndash22] While smallmolecules can access the peroxisomal matrix via passivetransport [23] peroxisomal proteins contain a peroxisomaltargeting signal (PTS) that enables them to be imported intothe organelle A group of proteins called peroxins (PEXs) areinvolved in this process with a total of 22 peroxins havingbeen identified in plants [3 24] To date two main types ofPTSs have been identified each of which has its own receptorthe protein is then transferred to a translocation complexthat mediates its transport across the peroxisomemembraneMost peroxisomalmatrix proteins are targeted by a noncleav-able tripeptide peroxisomal targeting signal 1 (PTS1) at theextreme C-terminus with the consensus sequence Ser-Lys-Leu (SKL) Other proteins are directed to peroxisomes by acleavable nanopeptide peroxisomal targeting signal 2 (PTS2)at the N terminus bearing the sequence (RK)-(LVI)-X
5-
(QH)-(LAI) [3]NADPH is an essential cofactor in many metabolic
reactions In plant peroxisomes several enzymes activitiesare strictly dependent on the presence of NADPH such asL-arginine-dependent nitric oxide synthase (NOS) whichfacilitates NO production [1 15] glutathione reductase (GR)which recycles reduced glutathione (GSH) from its oxidizedform (GSSG) [1] and 24-dienoyl-CoA reductase (DECR)which participates in the degradation of unsaturated fattyenoyl-CoA esters and has double bonds in both even- andodd-numbered positions in peroxisomes [25 26] (Figure 1)The different cell organelles are known to have multipletransporters in order to exchange metabolic intermedi-ates [27ndash29] however as to our knowledge NADPH hasno direct transport mechanism each cell compartmentmust have its own NADPH-regenerating system In higherplants during the dark phase of photosynthesis and innonphotosynthetic cells NADPH is principally regeneratedby a group of NADP-dehydrogenase enzymes includingNADP-isocitrate dehydrogenase (NADP-ICDH) the NADP-malic enzyme (NADP-ME) glucose-6-phosphate dehydro-genase (G6PDH) and 6-phosphogluconate dehydrogenase(6PGDH) the latter two belonging to the oxidative pentosephosphate pathway (PPP) [30 31] Using electronmicroscopyimmunogold labeling and biochemical techniques previousstudies have demonstrated the presence of several NADPH-generating dehydrogenases such as NADP-ICDH [32 33]and also G6PDH [34] in pea leaf peroxisomes Howeversome biochemical [34] and molecular data [26] stronglysuggest the potential localization of 6PGDH in peroxisomesThe present study therefore explores this possibility throughan in silico analysis of plant 6PGDHswith particular emphasison Arabidopsis 6PGDHs The data reveal the presence ofthe archetypal PTS1 in a group of 6PGDHs thus suggestingits peroxisomal localization while affymetrix microarraydata on the putative Arabidopsis p6PGDH suggest that it isinvolved in xenobiotic response growth and developmentalprocesses
6PG6PGDH
Rib 5-P + CO2
NADPH
24-Dienoyl-CoADECR
L-Arginine NOS ∙NO
GSSGGR
GSH
trans-3-Enoyl-CoA
Figure 1 Functions of the endogenous NADPH in plant peroxi-somes NADPH is required for several enzymatic systems includ-ing the glutathione reductase (GR) to keep the level of reducedglutathione (GSH) the L-arginine-dependent nitric oxide synthase(NOS) which generates nitric oxide (NO) and the 24-dienoyl-CoA reductase (DECR) which is necessary for the degradationof fatty acids unsaturated on odd-numbered carbons 6PGDH 6-phosphogluconate dehydrogenase 6PG 6-phosphogluconate Rib5-P ribulose 5-phosphate
2 Methods
21 Sequence Analysis Database Searches and SubcellularLocalization Predictions 6PGDH protein sequences fromcompleted genomes were retrieved from Plaza 30 (httpbioinformaticspsbugentbeplaza) [35] Blast searches werecarried out on the National Center for Biotechnology Info-rmation (NCBI) web site (httpwwwncbinlmnihgov)Alignments were performed using CLUSTAL W2 (httpwwwebiacukToolsmsaclustalw2) Localization predic-tions were made using WoLF (httpwolfpsortorg) [36]and peroxisomal targeting signal type 1 (PTS1) Predictor(httpmendelimpacatmendeljspsatpts1PTS1predictorjsp) [37ndash39] Molecular protein properties were estimatedusing the httpwwwexpasyorgproteomicsprotein charac-terisation and function Phylogenetic analyses were conduc-ted usingMEGA software version 60 (httpwwwmegasoft-warenet) [40] The molecular properties of Arabidopsis6PGDH isozymes based on their predicted amino acidsequences were used for the in silico predictions with the aidof httpwwwexpasyorgtoolsprotparamhtml
Promoter analysis and identification of cis-regulatoryelements were carried out according to the Plant Promo-ter 21 db program (httpppdbagrgifu-uacjpppdbcgi-binindexcgi)
22 Gene Expression Analyses of Peroxisomal 6PGDH underDiverse Growth Conditions and Xenobiotics Gene expressionanalyses under different conditions were carried out withthe aid of the Gene Expression Omnibus (GEO) database(httpwwwncbinlmnihgovgeo) [41] using AffymetrixMicroarray Suite 50 (MAS5)The growth conditions selectedwere as follows (i) For organ analysis Arabidopsis thaliana
Scientifica 3
plants were grown at a density of 4 plants per 127 cmsquare pot in either a growth chamber or green house setto 25∘C by day and 20∘C by night Days were set to a 16 hphotoperiod with 125 120583molmminus2 sminus1 fluorescent irradiationExpanding leaves were harvested 15 days after germinationin midphotoperiod The expanding upper 5 cm of the stemwith siliques and pedicels removed was harvested 29 daysafter germination in midphotoperiod Developed flowersand unopened buds were harvested 29 days after germina-tion in midphotoperiod (ii) For the sucrose effect on 4-day-old dark-grown seedlings Arabidopsis thaliana ecotypeColumbia was grown for 4 days in the dark at 23∘C in mul-tiwell plates containing half-strength Murashige Skoog (MS)medium without added sucrose Samples were subsequentlykept for 6 h under the same conditions with the addition of90mM sucrose (shaking thus aerobic) (iii) For herbicideeffect on 5-day-old seedlings Arabidopsis seeds were grownonhalf-strengthMSplates supplementedwith 1 sucrose andgrown at 21∘Cunder continuous light (100120583molmminus2 sminus1)withand without 5 120583M norflurazon (iv) For auxin treatmentsArabidopsis seedlings were grown for 10 d on 1x MS agar-solidifiedmedia under long-day conditions (16 8 white lightand dark cycle) Seedlings were then transferred to 2 differentliquid media containing either 01 120583M 24-D or 01 120583M 24-Dplus 1120583M brassinazole After 8 h of treatment the seedlingswere blotted with paper towels to remove excess media andsubjected to total RNA isolation
3 Results and Discussion
31 In Silico Analysis of Plant 6PGDHs Identification of Pero-xisomal Targeting Signal 1 (PTS1) At present the infor-mation available in different plant databases has increasedsignificantly and constitutes an unparalleled resource toprovide complementary data for experimental studies [42]The in silico analysis of putative peroxisomal 6PGDH aimsto describe the most important properties of this proteinin plants with particular attention paid to Arabidopsisthaliana This enzyme catalyzes the third and irreversiblereaction of the pentose phosphate pathway (PPP) whichconducts the oxidative decarboxylation of the free acid of 6-phosphogluconate to yield ribulose-5-phosphate obtainingCO2and NADPH In higher plants in comparison with the
G6PDH enzyme that regulates the PPP the 6PGDH enzymehas beenmuch less studied and is assumed to have a cytosolicand chloroplastic localization [43 44] The phylogeneticanalysis of 45 representative 6PGDH protein sequences fromdifferent organisms shows that plant 6PGDH proteins con-stitute a group clearly separated from the 6PGDH proteinsof other organisms including prokaryote and eukaryote (seeSupplemental Figure 1 in Supplementary Material availableonline at httpdxdoiorg10115520163482760) The rep-resentative 6PGDH sequences selected for this analysis aresummarized in Supplemental Table 1
With the aim of gaining a deeper understanding of thesubcellular localization of plant 6PGDHs a phylogeneticanalysis was carried out by exclusively using 51 plant 6PGDHprotein sequences Two main groups were found whichappear to be located either in chloroplasts (32 sequences) or in
peroxisomes (15 6PGDH sequences) with only four 6PGDHsequences belonging to these two main groups appearingto be located in the cytosol (Figure 2) In the peroxisomecandidate group of 6PGDH sequences a search of the maintype I and type II peroxisomal targeting signals (PTSs) wascarried out This enabled us to identify a total of 10 6PGDHprotein sequences which contain a PTS1 motif with thetripeptide SKI or SRI [22] Table 1 summarizes some charac-teristics (number of amino acids pI and molecular mass) ofthese putative peroxisomal 6PGDH proteins of higher plantswhich have a putative type 1 peroxisomal targeting signal(PTS1) The tripeptides (SKI and SRI) identified belong tothe canonical PTS1 sequence which is the tripeptide (SA)-(KR)-(LMI) at the extreme C-terminus In general theseconserved tripeptides are highly abundant in peroxisomalmatrix proteins [39] although other peroxisomalmatrix pro-teins have noncanonical C-terminal tripeptidesThe latter aremuch less conserved and generally occur in low-abundanceperoxisomal matrix proteins [45 46]
32 In Silico Analysis of Peroxisomal 6PGDH in Arabidopsisthaliana At present there aremany genetic and biochemicalinformation available on Arabidopsis thaliana Thereforethis plant has become a powerful tool to study manyaspects of higher plants [24] Analysis of the Arabidopsisdatabase shows that its genome contains three 6PGDHgenes At5g41670 At3g02360 and At1g64190 coding forproteins BAB11473 AEE73797 and AAF24560 respectivelyThe alignment of the deduced amino acid sequence ofthe three Arabidopsis 6PGDHs indicates that the proteinsequence of the three isozymes is highly conserved with 75similarity between BAB11473 and AEE73797 and 93 simi-larity between BAB11473 and AAF24560 In the sequence ofdeduced amino acids of the threeArabidopsis 6PGDHs puta-tiveNADPbinding sites with theGxGVxxGxxxG consensussequence and substrate binding sites with the LIVM-x-D-x-x-GANQS-KGTG-x-W sequence were identified(Supplemental Figure 2) These two sites are completelyconserved throughout all 6PGDH sequences from othersplant species Table 2 summarizes the principal molecularproperties of each Arabidopsis 6PGDH isozyme based onthe predicted amino acid sequences in each case By usingdifferent subcellular prediction programs it was found that6PGDH can have different locations including chloroplaststhe cytosol mitochondria and peroxisomes with subcellularlocalization showing the most significant differences amongthese 6PGDHs
The gene encoding putative peroxisomal 6PGDH(At3g02360) has a total length of 225 kb while its coordinateson chromosome 3 from A thaliana are 481898 and 484147The At3g02360 gene is transcribed in the nucleus usingtwo possible mRNAs (NM 1111035 and NM 1801712) withlengths of 1828 bp and 1829 bp respectively Both mRNAmolecules have an intron in the 51015840-UTR region and twoexons one containing part of the 51015840-UTR region and theother containing a small portion of the remaining 51015840-UTRregion CDS and 31015840-UTR (Supplemental Figure 3 andTable 3) Promoter analysis enabled us to detect a variant ofthe TATA box at positions minus565 and minus553 (Table 3) as well
4 Scientifica
gi|778707525|gi|59
0651
654|Th
gi|56616
5991
|
gi|2555799
36|Ricinus communis LKN-COOHgi|731375254|Vitis vinifera SKI-COOH
Figure 2 Evolutionary relationships of plant 6PGDHs The evolutionary history was inferred using the Neighbor-Joining method Theoptimal tree with the sum of branch length = 242428701 is shownThe tree is drawn to scale with branch lengths in the same units as those ofthe evolutionary distances used to infer the phylogenetic treeThe evolutionary distances were computed using the Poisson correctionmethodand are in the units of the number of amino acid substitutions per siteThe rate variation among sites was modeled with a gamma distribution(shape parameter = 1) The analysis involved 51 amino acid sequences All positions containing gaps and missing data were eliminated Therewere a total of 416 positions in the final dataset Evolutionary analyses were conducted in MEGA6 [30]
Scientifica 5
Table 1 Identification of 6PGDH proteins sequence of higher plants with a putative peroxisomal location for having a peroxisomal targetingsignal type 1 (PTS1) on the C-terminal The pI and MM values were calculated from their primary structure
Table 2 Genes encoding different isozymes of 6PGDH in A thaliana and molecular properties based on their predicted amino acidsequence The number of amino acids corresponds to the preprocessed protein and they were used for the in silico predictions usinghttpwebexpasyorgprotparam Transit peptide (TP) or targeting signal (TS) length is given in amino acids and molecular weight (MW)of the mature
Properties LocusAt5g41670 At3g02360 At1g64190
Protein accession number BAB11473 AEE73797 AAF24560Number of amino acids 487 486 487Subunit size (Da) 5331761 5357718 5337751pI 562 702 534Total number of negatively charged residues (Asp + Glu) 65 64 66Total number of positively charged residues (Arg + Lys) 60 64 57Stability indexlowast 2369 2686 2760120576280
(Mminus1 cmminus1) (assuming all Cys residues are reduced) 65320 63830 63830Aliphatic indexlowastlowast 8856 8710 8758Grand average of hydropathicity (GRAVY) indexlowastlowastlowast minus0278 minus0283 minus0272Transit peptide (TP)targeting signal (TS) mdash -SKI mdashSubcellular localization ChloroplastCytosol Peroxisome ChloroplastmitochondrionCytosollowastA protein with a stability index smaller than 40 is predicted as being stable with a value above 40 the protein is predicted as potentially unstablelowastlowastAliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (Ala Val Ile and Leu) A positive index indicates the increaseof thermostability of globular proteinslowastlowastlowastGRAVY (grand average of hydropathicity) index indicates the solubility of the proteins positive GRAVY (hydrophobic) negative GRAVY (hydrophilic)
as various regulatory elements identified in both transcripts(supplemental Figure 3) with the aid of the Plant Promoter21 db program
33 Gene Expression of Peroxisomal 6PGDH (p6PGDH)Figure 3 shows gene expression data for Arabidopsisp6PGDH under different growth conditions and is exposed tocertain chemicals Figure 3(a) shows that in adult plantsp6PGDH expression levels were highest in stems followedby flowers and lowest in leaves independently of the growthconditions in either the growth chamber or greenhouse Onthe other hand when Arabidopsis plants were grown underin vitro conditions and in the presence of 90mM sucrosep6PGDH gene expression was between 2-fold and 3-foldhigher (Figure 3(b)) suggesting as would be expected thatp6PGDH is involved in the carbon metabolism as thisenzyme is located in the oxidative part of the pentose phos-phate pathway [43] Additionally when plants were exposedto the herbicide norflurazon (a carotenoid biosynthesis
inhibitor) a similar 2-fold to 3-fold increase in p6PGDHgene expression was observed (Figure 3(c)) In the lattercase it is important to note that carotenoids have antioxidantproperties that help to protect chlorophyll from oxidativedamage mediated by ROS [47] as the absence of carotenoidsfacilitates chlorophyll destruction which is essential forphotosynthesisThese data are closely in line with the detoxi-fication capacity of peroxisomes which would amelioratedthe diminished antioxidant capacity of ROS to decomposechloroplasts damaged by this herbicide This is explainedby the fact that peroxisomes contain an important batteryof antioxidant enzymes including catalase superoxidedismutase and all components of the ascorbate-glutathionecycle which requires NADPH to support the regenerationof GSH by GR [1] Furthermore the relative expression ofp6PGDH was also higher in the presence of brassinazole(Figure 3(d)) a specific inhibitor of the biosynthesis ofbrassinosteroids which are a class of phytohormones thatplay an essential role in plant growth and development
Figure 3 Peroxisomal 6PGDH gene (At3g02360) expression in Arabidopsis thaliana grown under different conditions (a) Expression ofp6PGDH in leaves of 15-day-old plants stems and flowers of 29-day-old plants grown in either growth chamber or greenhouse conditions(b) Effects of a 6 h long treatment with 90mM sucrose to 4-day-old dark-grown Arabidopsis seedlings (c) Effects of 5120583M norflurazon to5-day-old continuous light Arabidopsis seedlings (d) Auxin effect on 10-day-old seedlings treated for 8 h either 01 120583M24-D or 01 120583M24-Dplus 1120583M brassinazole Data were obtained from the Gene Expression Omnibus (GEO) database and analyzed using Affymetrix MicroarraySuite 50 (MAS5) The original sample accessions (GSMxxx) are listed in the gray boxes along the bottom of the chart
Scientifica 7
Table 3 Analysis of the 51015840-UTR region At3G02360 gene coding for Arabidopsis thaliana peroxisomal 6PGDH Inr initiator TATA box anoctamer group related to TATA box Y patch an octamer group of the pyrimidine (Y) patch REG regulatory element group an octamergroup related to cis-regulatory elements CDS protein coding region UTR untranslated region
Type Sequence Genome position Position from initiation codonStrand Start End Start End
processes including promotion of stem elongation andcell division All these data suggest that p6PGDH couldbe specifically involved in growth and development sinceits gene expression is clearly induced by molecules whichblock these processes This is supported by a set of datawhich shows that sugar can promote hypocotyl elongation inArabidopsis in darkness a process which is largely dependenton brassinosteroids [48]
The reducing power (NADPH) generated by 6PGDH isknown to be important whose activity and gene expressioncan change depending on the type of stress and subcellularlocalization For example tobacco plants infected with potatovirus Y show increased cytosolic and plastidic 6PGDH activ-ity [49] Similar behavior has been observed in pepper leaveswith an increase in total 6PGDHactivity under cadmium [50]and low temperature [51] stress conditions Similar behaviorhas been described with regard to 6PGDH gene expressionThus in rice (Oryza sativa L) plants subjected to salt stress(150mM NaCl) an increase in 6PGDH transcripts in stemshas been reported [52] A subsequent study in which twogenes encoding for chloroplastic and cytosolic 6PGDH riceplants showed that both transcripts increased not only withsalinity but also with other stresses such as drought lowtemperature and treatment with abscisic acid however thetranscripts of G6PDH the first enzyme of the oxidativeportion of the pentose phosphate pathway did not undergoany change in its cytosolic and chloroplastic isoforms [53]Furthermore there is evidence to show that this enzyme isalso involved in other physiological processes for example instudies of the maturation of pepper fruits when they changefrom the green to red phenotype total 6PGDH activityincreased by more than 50 [54] In the case of tomato andspinach three genes encoding cytosolic and chloroplasticisozymes have been reported [55 56] also with respect toother plant species such as peas corn and tobacco [52 57ndash59]
4 Conclusion
In summary it is possible to conclude that there is a groupof plant 6PGDH enzymes which contain an archetypal type1 peroxisomal targeting signal Given the capacity of thisenzyme to generate NADPH in silico analysis of peroxisomal6PGDH in Arabidopsis thaliana suggests that it plays aprominent role during early seedling development in which
peroxisomes perform the key function of metabolizing lipidreserves until the seedling begins to photosynthesize [6061] for which NADPH is required Similarly at this earlyseedling stage the NADPH-dependent generation of nitricoxide in peroxisomes has also been demonstrated to beinvolved in root development [62] Finally the increase inp6PGDH in the presence of xenobiotics such as norflurazonand brassinazole is also closely in line with the induction ofthe antioxidant system present in plant peroxisomes as hasbeen demonstrated with the pea leaf peroxisomes of plantsexposed to 24-D [63]Therefore the present in silico analysissuggests the presence of a 6PGDH into peroxisomes whichwould contribute to the generation of NADPH within theseorganelles
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Research is supported by an ERDF-cofinanced grant fromthe Ministry of Science and Innovation (Recupera 2020-20134R056) and the Junta de Andalucıa (Group BIO192)
References
[1] F J Corpas J B Barroso and L A Del Rıo ldquoPeroxisomesas a source of reactive oxygen species and nitric oxide signalmolecules in plant cellsrdquo Trends in Plant Science vol 6 no 4pp 145ndash150 2001
[2] M Schrader and H D Fahimi ldquoPeroxisomes and oxidativestressrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1763 no 12 pp 1755ndash1766 2006
[3] J Hu A Baker B Bartel et al ldquoPlant peroxisomes biogenesisand functionrdquo Plant Cell vol 24 no 6 pp 2279ndash2303 2012
[4] I Pracharoenwattana and S M Smith ldquoWhen is a peroxisomenot a peroxisomerdquo Trends in Plant Science vol 13 no 10 pp522ndash525 2008
[5] F J Corpas ldquoWhat is the role of hydrogen peroxide in plantperoxisomesrdquo Plant Biology vol 17 no 6 pp 1099ndash1103 2015
[6] T G Cooper and H Beevers ldquoBeta oxidation in glyoxysomesfrom castor bean endospermrdquo The Journal of Biological Chem-istry vol 244 no 13 pp 3514ndash3520 1969
8 Scientifica
[7] S Reumann and A P M Weber ldquoPlant peroxisomes respirein the light some gaps of the photorespiratory C
2cycle have
become filledmdashothers remainrdquo Biochimica et Biophysica Actavol 1763 no 12 pp 1496ndash1510 2006
[8] A Baker and R Paudyal ldquoThe life of the peroxisome from birthto deathrdquo Current Opinion in Plant Biology vol 22 pp 39ndash472014
[9] L A del Rıo L M Sandalio F J Corpas J M Palma andJ B Barroso ldquoReactive oxygen species and reactive nitrogenspecies in peroxisomes Production scavenging and role in cellsignalingrdquo Plant Physiology vol 141 no 2 pp 330ndash335 2006
[10] A J K Koo H S Chung Y Kobayashi and G A Howe ldquoIden-tification of a peroxisomal acyl-activating enzyme involved inthe biosynthesis of jasmonic acid in ArabidopsisrdquoThe Journal ofBiological Chemistry vol 281 no 44 pp 33511ndash33520 2006
[11] B K Zolman and B Bartel ldquoAn Arabidopsis indole-3-butyricacid-response mutant defective in PEROXIN6 an apparentATPase implicated in peroxisomal functionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 6 pp 1786ndash1791 2004
[12] B K Zolman M Nyberg and B Bartel ldquoIBR3 a novelperoxisomal acyl-CoA dehydrogenase-like protein required forindole-3-butyric acid responserdquo Plant Molecular Biology vol64 no 1-2 pp 59ndash72 2007
[13] A A G Wiszniewski W Zhou S M Smith and J D BussellldquoIdentification of twoArabidopsis genes encoding a peroxisomaloxidoreductase-like protein and an acyl-CoA synthetase-likeprotein that are required for responses to pro-auxinsrdquo PlantMolecular Biology vol 69 no 5 pp 503ndash515 2009
[14] G M Spiess and B K Zolman ldquoPeroxisomes as a source ofauxin signalingmoleculesrdquo Subcellular Biochemistry vol 69 pp257ndash281 2013
[15] J B Barroso F J Corpas A Carreras et al ldquoLocalizationof nitric-oxide synthase in plant peroxisomesrdquo The Journal ofBiological Chemistry vol 274 no 51 pp 36729ndash36733 1999
[16] F J Corpas J B Barroso A Carreras et al ldquoCellular andsubcellular localization of endogenous nitric oxide in young andsenescent pea plantsrdquo Plant Physiology vol 136 no 1 pp 2722ndash2733 2004
[17] F J Corpas M Hayashi S Mano M Nishimura and J BBarroso ldquoPeroxisomes are required for in vivo nitric oxide accu-mulation in the cytosol following salinity stress of arabidopsisplantsrdquo Plant Physiology vol 151 no 4 pp 2083ndash2094 2009
[18] F J Corpas and J B Barroso ldquoPeroxisomal plant nitric oxidesynthase (NOS) protein is imported by peroxisomal targetingsignal type 2 (PTS2) in a process that depends on the cytosolicreceptor PEX7 and calmodulinrdquo FEBS Letters vol 588 no 12pp 2049ndash2054 2014
[19] L L Cross H T Ebeed and A Baker ldquoPeroxisome biogenesisprotein targeting mechanisms and PEX gene functions inplantsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular CellResearch 2015
[20] P B Lazarow andY Fujiki ldquoBiogenesis of peroxisomesrdquoAnnualReview of Cell Biology vol 1 no 1 pp 489ndash530 1985
[21] M Hayashi and M Nishimura ldquoEntering a new era of researchon plant peroxisomesrdquo Current Opinion in Plant Biology vol 6no 6 pp 577ndash582 2003
[22] S Reumann C Ma S Lemke and L Babujee ldquoAraPerox Adatabase of putative arabidopsis proteins from plant peroxi-somesrdquo Plant Physiology vol 136 no 1 pp 2587ndash2608 2004
[23] V D Antonenkov and J K Hiltunen ldquoPeroxisomal membranepermeability and solute transferrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1763 no 12 pp 1697ndash17062006
[24] M Hayashi andM Nishimura ldquoArabidopsis thalianamdashamodelorganism to study plant peroxisomesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1763 no 12 pp 1382ndash13912006
[25] C W T van Roermund E H Hettema A J Kal M vanden Berg H F Tabak and R J A Wanders ldquoPeroxisomal 120573-oxidation of polyunsaturated fatty acids in Saccharomyces cere-visiae isocitrate dehydrogenase providesNADPH for reductionof double bonds at even positionsrdquo The EMBO Journal vol 17no 3 pp 677ndash687 1998
[26] S Reumann L Babujee M Changle et al ldquoProteome analysisofArabidopsis leaf peroxisomes reveals novel targeting peptidesmetabolic pathways and defense mechanismsrdquo The Plant Cellvol 19 no 10 pp 3170ndash3193 2007
[27] M Kunze I Pracharoenwattana S M Smith and A Hartig ldquoAcentral role for the peroxisomal membrane in glyoxylate cyclefunctionrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp1441ndash1452 2006
[28] N Linka and A P M Weber ldquoIntracellular metabolite trans-porters in plantsrdquoMolecular Plant vol 3 no 1 pp 21ndash53 2010
[29] F L Theodoulou K Bernhardt N Linka and A BakerldquoPeroxisome membrane proteins multiple trafficking routesandmultiple functionsrdquo Biochemical Journal vol 451 no 3 pp345ndash352 2013
[30] F J Corpas and J B Barroso ldquoNADPH-generating dehydroge-nases their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditionsrdquoFrontiers in Environmental Science vol 2 article 55 2014
[31] M F Drincovich P Casati and C S Andreo ldquoNADP-malicenzyme from plants a ubiquitous enzyme involved in differentmetabolic pathwaysrdquo FEBS Letters vol 490 no 1-2 pp 1ndash62001
[32] F J Corpas J B Barroso L M Sandalio J M Palma J ALupianez and L A Del Rıo ldquoPeroxisomal NADP-dependentisocitrate dehydrogenase Characterization and activity regula-tion during natural senescencerdquo Plant Physiology vol 121 no 3pp 921ndash928 1999
[33] M Leterrier J B Barroso R Valderrama et al ldquoPeroxisomalNADP-isocitrate dehydrogenase is required for Arabidopsisstomatal movementrdquo Protoplasma 2015
[34] F J Corpas J B Barroso L M Sandalio et al ldquoAdehydrogenase-mediated recycling system of NADPH in plantperoxisomesrdquo Biochemical Journal vol 330 no 2 pp 777ndash7841998
[35] S Proost M van Bel L Sterck et al ldquoPLAZA a comparativegenomics resource to study gene and genome evolution inplantsrdquoThe Plant Cell vol 21 no 12 pp 3718ndash3731 2009
[36] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquoNucleic Acids Research vol 35 no 2 ppW585ndashW587 2007
[37] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoPrediction of peroxisomal targeting signal 1containing proteins from amino acid sequencerdquo Journal ofMolecular Biology vol 328 no 3 pp 581ndash592 2003
[38] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoMotif refinement of the peroxisomal targetingsignal 1 and evaluation of taxon-specific differencesrdquo Journal ofMolecular Biology vol 328 no 3 pp 567ndash579 2003
Scientifica 9
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
plants were grown at a density of 4 plants per 127 cmsquare pot in either a growth chamber or green house setto 25∘C by day and 20∘C by night Days were set to a 16 hphotoperiod with 125 120583molmminus2 sminus1 fluorescent irradiationExpanding leaves were harvested 15 days after germinationin midphotoperiod The expanding upper 5 cm of the stemwith siliques and pedicels removed was harvested 29 daysafter germination in midphotoperiod Developed flowersand unopened buds were harvested 29 days after germina-tion in midphotoperiod (ii) For the sucrose effect on 4-day-old dark-grown seedlings Arabidopsis thaliana ecotypeColumbia was grown for 4 days in the dark at 23∘C in mul-tiwell plates containing half-strength Murashige Skoog (MS)medium without added sucrose Samples were subsequentlykept for 6 h under the same conditions with the addition of90mM sucrose (shaking thus aerobic) (iii) For herbicideeffect on 5-day-old seedlings Arabidopsis seeds were grownonhalf-strengthMSplates supplementedwith 1 sucrose andgrown at 21∘Cunder continuous light (100120583molmminus2 sminus1)withand without 5 120583M norflurazon (iv) For auxin treatmentsArabidopsis seedlings were grown for 10 d on 1x MS agar-solidifiedmedia under long-day conditions (16 8 white lightand dark cycle) Seedlings were then transferred to 2 differentliquid media containing either 01 120583M 24-D or 01 120583M 24-Dplus 1120583M brassinazole After 8 h of treatment the seedlingswere blotted with paper towels to remove excess media andsubjected to total RNA isolation
3 Results and Discussion
31 In Silico Analysis of Plant 6PGDHs Identification of Pero-xisomal Targeting Signal 1 (PTS1) At present the infor-mation available in different plant databases has increasedsignificantly and constitutes an unparalleled resource toprovide complementary data for experimental studies [42]The in silico analysis of putative peroxisomal 6PGDH aimsto describe the most important properties of this proteinin plants with particular attention paid to Arabidopsisthaliana This enzyme catalyzes the third and irreversiblereaction of the pentose phosphate pathway (PPP) whichconducts the oxidative decarboxylation of the free acid of 6-phosphogluconate to yield ribulose-5-phosphate obtainingCO2and NADPH In higher plants in comparison with the
G6PDH enzyme that regulates the PPP the 6PGDH enzymehas beenmuch less studied and is assumed to have a cytosolicand chloroplastic localization [43 44] The phylogeneticanalysis of 45 representative 6PGDH protein sequences fromdifferent organisms shows that plant 6PGDH proteins con-stitute a group clearly separated from the 6PGDH proteinsof other organisms including prokaryote and eukaryote (seeSupplemental Figure 1 in Supplementary Material availableonline at httpdxdoiorg10115520163482760) The rep-resentative 6PGDH sequences selected for this analysis aresummarized in Supplemental Table 1
With the aim of gaining a deeper understanding of thesubcellular localization of plant 6PGDHs a phylogeneticanalysis was carried out by exclusively using 51 plant 6PGDHprotein sequences Two main groups were found whichappear to be located either in chloroplasts (32 sequences) or in
peroxisomes (15 6PGDH sequences) with only four 6PGDHsequences belonging to these two main groups appearingto be located in the cytosol (Figure 2) In the peroxisomecandidate group of 6PGDH sequences a search of the maintype I and type II peroxisomal targeting signals (PTSs) wascarried out This enabled us to identify a total of 10 6PGDHprotein sequences which contain a PTS1 motif with thetripeptide SKI or SRI [22] Table 1 summarizes some charac-teristics (number of amino acids pI and molecular mass) ofthese putative peroxisomal 6PGDH proteins of higher plantswhich have a putative type 1 peroxisomal targeting signal(PTS1) The tripeptides (SKI and SRI) identified belong tothe canonical PTS1 sequence which is the tripeptide (SA)-(KR)-(LMI) at the extreme C-terminus In general theseconserved tripeptides are highly abundant in peroxisomalmatrix proteins [39] although other peroxisomalmatrix pro-teins have noncanonical C-terminal tripeptidesThe latter aremuch less conserved and generally occur in low-abundanceperoxisomal matrix proteins [45 46]
32 In Silico Analysis of Peroxisomal 6PGDH in Arabidopsisthaliana At present there aremany genetic and biochemicalinformation available on Arabidopsis thaliana Thereforethis plant has become a powerful tool to study manyaspects of higher plants [24] Analysis of the Arabidopsisdatabase shows that its genome contains three 6PGDHgenes At5g41670 At3g02360 and At1g64190 coding forproteins BAB11473 AEE73797 and AAF24560 respectivelyThe alignment of the deduced amino acid sequence ofthe three Arabidopsis 6PGDHs indicates that the proteinsequence of the three isozymes is highly conserved with 75similarity between BAB11473 and AEE73797 and 93 simi-larity between BAB11473 and AAF24560 In the sequence ofdeduced amino acids of the threeArabidopsis 6PGDHs puta-tiveNADPbinding sites with theGxGVxxGxxxG consensussequence and substrate binding sites with the LIVM-x-D-x-x-GANQS-KGTG-x-W sequence were identified(Supplemental Figure 2) These two sites are completelyconserved throughout all 6PGDH sequences from othersplant species Table 2 summarizes the principal molecularproperties of each Arabidopsis 6PGDH isozyme based onthe predicted amino acid sequences in each case By usingdifferent subcellular prediction programs it was found that6PGDH can have different locations including chloroplaststhe cytosol mitochondria and peroxisomes with subcellularlocalization showing the most significant differences amongthese 6PGDHs
The gene encoding putative peroxisomal 6PGDH(At3g02360) has a total length of 225 kb while its coordinateson chromosome 3 from A thaliana are 481898 and 484147The At3g02360 gene is transcribed in the nucleus usingtwo possible mRNAs (NM 1111035 and NM 1801712) withlengths of 1828 bp and 1829 bp respectively Both mRNAmolecules have an intron in the 51015840-UTR region and twoexons one containing part of the 51015840-UTR region and theother containing a small portion of the remaining 51015840-UTRregion CDS and 31015840-UTR (Supplemental Figure 3 andTable 3) Promoter analysis enabled us to detect a variant ofthe TATA box at positions minus565 and minus553 (Table 3) as well
4 Scientifica
gi|778707525|gi|59
0651
654|Th
gi|56616
5991
|
gi|2555799
36|Ricinus communis LKN-COOHgi|731375254|Vitis vinifera SKI-COOH
Figure 2 Evolutionary relationships of plant 6PGDHs The evolutionary history was inferred using the Neighbor-Joining method Theoptimal tree with the sum of branch length = 242428701 is shownThe tree is drawn to scale with branch lengths in the same units as those ofthe evolutionary distances used to infer the phylogenetic treeThe evolutionary distances were computed using the Poisson correctionmethodand are in the units of the number of amino acid substitutions per siteThe rate variation among sites was modeled with a gamma distribution(shape parameter = 1) The analysis involved 51 amino acid sequences All positions containing gaps and missing data were eliminated Therewere a total of 416 positions in the final dataset Evolutionary analyses were conducted in MEGA6 [30]
Scientifica 5
Table 1 Identification of 6PGDH proteins sequence of higher plants with a putative peroxisomal location for having a peroxisomal targetingsignal type 1 (PTS1) on the C-terminal The pI and MM values were calculated from their primary structure
Table 2 Genes encoding different isozymes of 6PGDH in A thaliana and molecular properties based on their predicted amino acidsequence The number of amino acids corresponds to the preprocessed protein and they were used for the in silico predictions usinghttpwebexpasyorgprotparam Transit peptide (TP) or targeting signal (TS) length is given in amino acids and molecular weight (MW)of the mature
Properties LocusAt5g41670 At3g02360 At1g64190
Protein accession number BAB11473 AEE73797 AAF24560Number of amino acids 487 486 487Subunit size (Da) 5331761 5357718 5337751pI 562 702 534Total number of negatively charged residues (Asp + Glu) 65 64 66Total number of positively charged residues (Arg + Lys) 60 64 57Stability indexlowast 2369 2686 2760120576280
(Mminus1 cmminus1) (assuming all Cys residues are reduced) 65320 63830 63830Aliphatic indexlowastlowast 8856 8710 8758Grand average of hydropathicity (GRAVY) indexlowastlowastlowast minus0278 minus0283 minus0272Transit peptide (TP)targeting signal (TS) mdash -SKI mdashSubcellular localization ChloroplastCytosol Peroxisome ChloroplastmitochondrionCytosollowastA protein with a stability index smaller than 40 is predicted as being stable with a value above 40 the protein is predicted as potentially unstablelowastlowastAliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (Ala Val Ile and Leu) A positive index indicates the increaseof thermostability of globular proteinslowastlowastlowastGRAVY (grand average of hydropathicity) index indicates the solubility of the proteins positive GRAVY (hydrophobic) negative GRAVY (hydrophilic)
as various regulatory elements identified in both transcripts(supplemental Figure 3) with the aid of the Plant Promoter21 db program
33 Gene Expression of Peroxisomal 6PGDH (p6PGDH)Figure 3 shows gene expression data for Arabidopsisp6PGDH under different growth conditions and is exposed tocertain chemicals Figure 3(a) shows that in adult plantsp6PGDH expression levels were highest in stems followedby flowers and lowest in leaves independently of the growthconditions in either the growth chamber or greenhouse Onthe other hand when Arabidopsis plants were grown underin vitro conditions and in the presence of 90mM sucrosep6PGDH gene expression was between 2-fold and 3-foldhigher (Figure 3(b)) suggesting as would be expected thatp6PGDH is involved in the carbon metabolism as thisenzyme is located in the oxidative part of the pentose phos-phate pathway [43] Additionally when plants were exposedto the herbicide norflurazon (a carotenoid biosynthesis
inhibitor) a similar 2-fold to 3-fold increase in p6PGDHgene expression was observed (Figure 3(c)) In the lattercase it is important to note that carotenoids have antioxidantproperties that help to protect chlorophyll from oxidativedamage mediated by ROS [47] as the absence of carotenoidsfacilitates chlorophyll destruction which is essential forphotosynthesisThese data are closely in line with the detoxi-fication capacity of peroxisomes which would amelioratedthe diminished antioxidant capacity of ROS to decomposechloroplasts damaged by this herbicide This is explainedby the fact that peroxisomes contain an important batteryof antioxidant enzymes including catalase superoxidedismutase and all components of the ascorbate-glutathionecycle which requires NADPH to support the regenerationof GSH by GR [1] Furthermore the relative expression ofp6PGDH was also higher in the presence of brassinazole(Figure 3(d)) a specific inhibitor of the biosynthesis ofbrassinosteroids which are a class of phytohormones thatplay an essential role in plant growth and development
Figure 3 Peroxisomal 6PGDH gene (At3g02360) expression in Arabidopsis thaliana grown under different conditions (a) Expression ofp6PGDH in leaves of 15-day-old plants stems and flowers of 29-day-old plants grown in either growth chamber or greenhouse conditions(b) Effects of a 6 h long treatment with 90mM sucrose to 4-day-old dark-grown Arabidopsis seedlings (c) Effects of 5120583M norflurazon to5-day-old continuous light Arabidopsis seedlings (d) Auxin effect on 10-day-old seedlings treated for 8 h either 01 120583M24-D or 01 120583M24-Dplus 1120583M brassinazole Data were obtained from the Gene Expression Omnibus (GEO) database and analyzed using Affymetrix MicroarraySuite 50 (MAS5) The original sample accessions (GSMxxx) are listed in the gray boxes along the bottom of the chart
Scientifica 7
Table 3 Analysis of the 51015840-UTR region At3G02360 gene coding for Arabidopsis thaliana peroxisomal 6PGDH Inr initiator TATA box anoctamer group related to TATA box Y patch an octamer group of the pyrimidine (Y) patch REG regulatory element group an octamergroup related to cis-regulatory elements CDS protein coding region UTR untranslated region
Type Sequence Genome position Position from initiation codonStrand Start End Start End
processes including promotion of stem elongation andcell division All these data suggest that p6PGDH couldbe specifically involved in growth and development sinceits gene expression is clearly induced by molecules whichblock these processes This is supported by a set of datawhich shows that sugar can promote hypocotyl elongation inArabidopsis in darkness a process which is largely dependenton brassinosteroids [48]
The reducing power (NADPH) generated by 6PGDH isknown to be important whose activity and gene expressioncan change depending on the type of stress and subcellularlocalization For example tobacco plants infected with potatovirus Y show increased cytosolic and plastidic 6PGDH activ-ity [49] Similar behavior has been observed in pepper leaveswith an increase in total 6PGDHactivity under cadmium [50]and low temperature [51] stress conditions Similar behaviorhas been described with regard to 6PGDH gene expressionThus in rice (Oryza sativa L) plants subjected to salt stress(150mM NaCl) an increase in 6PGDH transcripts in stemshas been reported [52] A subsequent study in which twogenes encoding for chloroplastic and cytosolic 6PGDH riceplants showed that both transcripts increased not only withsalinity but also with other stresses such as drought lowtemperature and treatment with abscisic acid however thetranscripts of G6PDH the first enzyme of the oxidativeportion of the pentose phosphate pathway did not undergoany change in its cytosolic and chloroplastic isoforms [53]Furthermore there is evidence to show that this enzyme isalso involved in other physiological processes for example instudies of the maturation of pepper fruits when they changefrom the green to red phenotype total 6PGDH activityincreased by more than 50 [54] In the case of tomato andspinach three genes encoding cytosolic and chloroplasticisozymes have been reported [55 56] also with respect toother plant species such as peas corn and tobacco [52 57ndash59]
4 Conclusion
In summary it is possible to conclude that there is a groupof plant 6PGDH enzymes which contain an archetypal type1 peroxisomal targeting signal Given the capacity of thisenzyme to generate NADPH in silico analysis of peroxisomal6PGDH in Arabidopsis thaliana suggests that it plays aprominent role during early seedling development in which
peroxisomes perform the key function of metabolizing lipidreserves until the seedling begins to photosynthesize [6061] for which NADPH is required Similarly at this earlyseedling stage the NADPH-dependent generation of nitricoxide in peroxisomes has also been demonstrated to beinvolved in root development [62] Finally the increase inp6PGDH in the presence of xenobiotics such as norflurazonand brassinazole is also closely in line with the induction ofthe antioxidant system present in plant peroxisomes as hasbeen demonstrated with the pea leaf peroxisomes of plantsexposed to 24-D [63]Therefore the present in silico analysissuggests the presence of a 6PGDH into peroxisomes whichwould contribute to the generation of NADPH within theseorganelles
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Research is supported by an ERDF-cofinanced grant fromthe Ministry of Science and Innovation (Recupera 2020-20134R056) and the Junta de Andalucıa (Group BIO192)
References
[1] F J Corpas J B Barroso and L A Del Rıo ldquoPeroxisomesas a source of reactive oxygen species and nitric oxide signalmolecules in plant cellsrdquo Trends in Plant Science vol 6 no 4pp 145ndash150 2001
[2] M Schrader and H D Fahimi ldquoPeroxisomes and oxidativestressrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1763 no 12 pp 1755ndash1766 2006
[3] J Hu A Baker B Bartel et al ldquoPlant peroxisomes biogenesisand functionrdquo Plant Cell vol 24 no 6 pp 2279ndash2303 2012
[4] I Pracharoenwattana and S M Smith ldquoWhen is a peroxisomenot a peroxisomerdquo Trends in Plant Science vol 13 no 10 pp522ndash525 2008
[5] F J Corpas ldquoWhat is the role of hydrogen peroxide in plantperoxisomesrdquo Plant Biology vol 17 no 6 pp 1099ndash1103 2015
[6] T G Cooper and H Beevers ldquoBeta oxidation in glyoxysomesfrom castor bean endospermrdquo The Journal of Biological Chem-istry vol 244 no 13 pp 3514ndash3520 1969
8 Scientifica
[7] S Reumann and A P M Weber ldquoPlant peroxisomes respirein the light some gaps of the photorespiratory C
2cycle have
become filledmdashothers remainrdquo Biochimica et Biophysica Actavol 1763 no 12 pp 1496ndash1510 2006
[8] A Baker and R Paudyal ldquoThe life of the peroxisome from birthto deathrdquo Current Opinion in Plant Biology vol 22 pp 39ndash472014
[9] L A del Rıo L M Sandalio F J Corpas J M Palma andJ B Barroso ldquoReactive oxygen species and reactive nitrogenspecies in peroxisomes Production scavenging and role in cellsignalingrdquo Plant Physiology vol 141 no 2 pp 330ndash335 2006
[10] A J K Koo H S Chung Y Kobayashi and G A Howe ldquoIden-tification of a peroxisomal acyl-activating enzyme involved inthe biosynthesis of jasmonic acid in ArabidopsisrdquoThe Journal ofBiological Chemistry vol 281 no 44 pp 33511ndash33520 2006
[11] B K Zolman and B Bartel ldquoAn Arabidopsis indole-3-butyricacid-response mutant defective in PEROXIN6 an apparentATPase implicated in peroxisomal functionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 6 pp 1786ndash1791 2004
[12] B K Zolman M Nyberg and B Bartel ldquoIBR3 a novelperoxisomal acyl-CoA dehydrogenase-like protein required forindole-3-butyric acid responserdquo Plant Molecular Biology vol64 no 1-2 pp 59ndash72 2007
[13] A A G Wiszniewski W Zhou S M Smith and J D BussellldquoIdentification of twoArabidopsis genes encoding a peroxisomaloxidoreductase-like protein and an acyl-CoA synthetase-likeprotein that are required for responses to pro-auxinsrdquo PlantMolecular Biology vol 69 no 5 pp 503ndash515 2009
[14] G M Spiess and B K Zolman ldquoPeroxisomes as a source ofauxin signalingmoleculesrdquo Subcellular Biochemistry vol 69 pp257ndash281 2013
[15] J B Barroso F J Corpas A Carreras et al ldquoLocalizationof nitric-oxide synthase in plant peroxisomesrdquo The Journal ofBiological Chemistry vol 274 no 51 pp 36729ndash36733 1999
[16] F J Corpas J B Barroso A Carreras et al ldquoCellular andsubcellular localization of endogenous nitric oxide in young andsenescent pea plantsrdquo Plant Physiology vol 136 no 1 pp 2722ndash2733 2004
[17] F J Corpas M Hayashi S Mano M Nishimura and J BBarroso ldquoPeroxisomes are required for in vivo nitric oxide accu-mulation in the cytosol following salinity stress of arabidopsisplantsrdquo Plant Physiology vol 151 no 4 pp 2083ndash2094 2009
[18] F J Corpas and J B Barroso ldquoPeroxisomal plant nitric oxidesynthase (NOS) protein is imported by peroxisomal targetingsignal type 2 (PTS2) in a process that depends on the cytosolicreceptor PEX7 and calmodulinrdquo FEBS Letters vol 588 no 12pp 2049ndash2054 2014
[19] L L Cross H T Ebeed and A Baker ldquoPeroxisome biogenesisprotein targeting mechanisms and PEX gene functions inplantsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular CellResearch 2015
[20] P B Lazarow andY Fujiki ldquoBiogenesis of peroxisomesrdquoAnnualReview of Cell Biology vol 1 no 1 pp 489ndash530 1985
[21] M Hayashi and M Nishimura ldquoEntering a new era of researchon plant peroxisomesrdquo Current Opinion in Plant Biology vol 6no 6 pp 577ndash582 2003
[22] S Reumann C Ma S Lemke and L Babujee ldquoAraPerox Adatabase of putative arabidopsis proteins from plant peroxi-somesrdquo Plant Physiology vol 136 no 1 pp 2587ndash2608 2004
[23] V D Antonenkov and J K Hiltunen ldquoPeroxisomal membranepermeability and solute transferrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1763 no 12 pp 1697ndash17062006
[24] M Hayashi andM Nishimura ldquoArabidopsis thalianamdashamodelorganism to study plant peroxisomesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1763 no 12 pp 1382ndash13912006
[25] C W T van Roermund E H Hettema A J Kal M vanden Berg H F Tabak and R J A Wanders ldquoPeroxisomal 120573-oxidation of polyunsaturated fatty acids in Saccharomyces cere-visiae isocitrate dehydrogenase providesNADPH for reductionof double bonds at even positionsrdquo The EMBO Journal vol 17no 3 pp 677ndash687 1998
[26] S Reumann L Babujee M Changle et al ldquoProteome analysisofArabidopsis leaf peroxisomes reveals novel targeting peptidesmetabolic pathways and defense mechanismsrdquo The Plant Cellvol 19 no 10 pp 3170ndash3193 2007
[27] M Kunze I Pracharoenwattana S M Smith and A Hartig ldquoAcentral role for the peroxisomal membrane in glyoxylate cyclefunctionrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp1441ndash1452 2006
[28] N Linka and A P M Weber ldquoIntracellular metabolite trans-porters in plantsrdquoMolecular Plant vol 3 no 1 pp 21ndash53 2010
[29] F L Theodoulou K Bernhardt N Linka and A BakerldquoPeroxisome membrane proteins multiple trafficking routesandmultiple functionsrdquo Biochemical Journal vol 451 no 3 pp345ndash352 2013
[30] F J Corpas and J B Barroso ldquoNADPH-generating dehydroge-nases their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditionsrdquoFrontiers in Environmental Science vol 2 article 55 2014
[31] M F Drincovich P Casati and C S Andreo ldquoNADP-malicenzyme from plants a ubiquitous enzyme involved in differentmetabolic pathwaysrdquo FEBS Letters vol 490 no 1-2 pp 1ndash62001
[32] F J Corpas J B Barroso L M Sandalio J M Palma J ALupianez and L A Del Rıo ldquoPeroxisomal NADP-dependentisocitrate dehydrogenase Characterization and activity regula-tion during natural senescencerdquo Plant Physiology vol 121 no 3pp 921ndash928 1999
[33] M Leterrier J B Barroso R Valderrama et al ldquoPeroxisomalNADP-isocitrate dehydrogenase is required for Arabidopsisstomatal movementrdquo Protoplasma 2015
[34] F J Corpas J B Barroso L M Sandalio et al ldquoAdehydrogenase-mediated recycling system of NADPH in plantperoxisomesrdquo Biochemical Journal vol 330 no 2 pp 777ndash7841998
[35] S Proost M van Bel L Sterck et al ldquoPLAZA a comparativegenomics resource to study gene and genome evolution inplantsrdquoThe Plant Cell vol 21 no 12 pp 3718ndash3731 2009
[36] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquoNucleic Acids Research vol 35 no 2 ppW585ndashW587 2007
[37] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoPrediction of peroxisomal targeting signal 1containing proteins from amino acid sequencerdquo Journal ofMolecular Biology vol 328 no 3 pp 581ndash592 2003
[38] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoMotif refinement of the peroxisomal targetingsignal 1 and evaluation of taxon-specific differencesrdquo Journal ofMolecular Biology vol 328 no 3 pp 567ndash579 2003
Scientifica 9
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
Figure 2 Evolutionary relationships of plant 6PGDHs The evolutionary history was inferred using the Neighbor-Joining method Theoptimal tree with the sum of branch length = 242428701 is shownThe tree is drawn to scale with branch lengths in the same units as those ofthe evolutionary distances used to infer the phylogenetic treeThe evolutionary distances were computed using the Poisson correctionmethodand are in the units of the number of amino acid substitutions per siteThe rate variation among sites was modeled with a gamma distribution(shape parameter = 1) The analysis involved 51 amino acid sequences All positions containing gaps and missing data were eliminated Therewere a total of 416 positions in the final dataset Evolutionary analyses were conducted in MEGA6 [30]
Scientifica 5
Table 1 Identification of 6PGDH proteins sequence of higher plants with a putative peroxisomal location for having a peroxisomal targetingsignal type 1 (PTS1) on the C-terminal The pI and MM values were calculated from their primary structure
Table 2 Genes encoding different isozymes of 6PGDH in A thaliana and molecular properties based on their predicted amino acidsequence The number of amino acids corresponds to the preprocessed protein and they were used for the in silico predictions usinghttpwebexpasyorgprotparam Transit peptide (TP) or targeting signal (TS) length is given in amino acids and molecular weight (MW)of the mature
Properties LocusAt5g41670 At3g02360 At1g64190
Protein accession number BAB11473 AEE73797 AAF24560Number of amino acids 487 486 487Subunit size (Da) 5331761 5357718 5337751pI 562 702 534Total number of negatively charged residues (Asp + Glu) 65 64 66Total number of positively charged residues (Arg + Lys) 60 64 57Stability indexlowast 2369 2686 2760120576280
(Mminus1 cmminus1) (assuming all Cys residues are reduced) 65320 63830 63830Aliphatic indexlowastlowast 8856 8710 8758Grand average of hydropathicity (GRAVY) indexlowastlowastlowast minus0278 minus0283 minus0272Transit peptide (TP)targeting signal (TS) mdash -SKI mdashSubcellular localization ChloroplastCytosol Peroxisome ChloroplastmitochondrionCytosollowastA protein with a stability index smaller than 40 is predicted as being stable with a value above 40 the protein is predicted as potentially unstablelowastlowastAliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (Ala Val Ile and Leu) A positive index indicates the increaseof thermostability of globular proteinslowastlowastlowastGRAVY (grand average of hydropathicity) index indicates the solubility of the proteins positive GRAVY (hydrophobic) negative GRAVY (hydrophilic)
as various regulatory elements identified in both transcripts(supplemental Figure 3) with the aid of the Plant Promoter21 db program
33 Gene Expression of Peroxisomal 6PGDH (p6PGDH)Figure 3 shows gene expression data for Arabidopsisp6PGDH under different growth conditions and is exposed tocertain chemicals Figure 3(a) shows that in adult plantsp6PGDH expression levels were highest in stems followedby flowers and lowest in leaves independently of the growthconditions in either the growth chamber or greenhouse Onthe other hand when Arabidopsis plants were grown underin vitro conditions and in the presence of 90mM sucrosep6PGDH gene expression was between 2-fold and 3-foldhigher (Figure 3(b)) suggesting as would be expected thatp6PGDH is involved in the carbon metabolism as thisenzyme is located in the oxidative part of the pentose phos-phate pathway [43] Additionally when plants were exposedto the herbicide norflurazon (a carotenoid biosynthesis
inhibitor) a similar 2-fold to 3-fold increase in p6PGDHgene expression was observed (Figure 3(c)) In the lattercase it is important to note that carotenoids have antioxidantproperties that help to protect chlorophyll from oxidativedamage mediated by ROS [47] as the absence of carotenoidsfacilitates chlorophyll destruction which is essential forphotosynthesisThese data are closely in line with the detoxi-fication capacity of peroxisomes which would amelioratedthe diminished antioxidant capacity of ROS to decomposechloroplasts damaged by this herbicide This is explainedby the fact that peroxisomes contain an important batteryof antioxidant enzymes including catalase superoxidedismutase and all components of the ascorbate-glutathionecycle which requires NADPH to support the regenerationof GSH by GR [1] Furthermore the relative expression ofp6PGDH was also higher in the presence of brassinazole(Figure 3(d)) a specific inhibitor of the biosynthesis ofbrassinosteroids which are a class of phytohormones thatplay an essential role in plant growth and development
Figure 3 Peroxisomal 6PGDH gene (At3g02360) expression in Arabidopsis thaliana grown under different conditions (a) Expression ofp6PGDH in leaves of 15-day-old plants stems and flowers of 29-day-old plants grown in either growth chamber or greenhouse conditions(b) Effects of a 6 h long treatment with 90mM sucrose to 4-day-old dark-grown Arabidopsis seedlings (c) Effects of 5120583M norflurazon to5-day-old continuous light Arabidopsis seedlings (d) Auxin effect on 10-day-old seedlings treated for 8 h either 01 120583M24-D or 01 120583M24-Dplus 1120583M brassinazole Data were obtained from the Gene Expression Omnibus (GEO) database and analyzed using Affymetrix MicroarraySuite 50 (MAS5) The original sample accessions (GSMxxx) are listed in the gray boxes along the bottom of the chart
Scientifica 7
Table 3 Analysis of the 51015840-UTR region At3G02360 gene coding for Arabidopsis thaliana peroxisomal 6PGDH Inr initiator TATA box anoctamer group related to TATA box Y patch an octamer group of the pyrimidine (Y) patch REG regulatory element group an octamergroup related to cis-regulatory elements CDS protein coding region UTR untranslated region
Type Sequence Genome position Position from initiation codonStrand Start End Start End
processes including promotion of stem elongation andcell division All these data suggest that p6PGDH couldbe specifically involved in growth and development sinceits gene expression is clearly induced by molecules whichblock these processes This is supported by a set of datawhich shows that sugar can promote hypocotyl elongation inArabidopsis in darkness a process which is largely dependenton brassinosteroids [48]
The reducing power (NADPH) generated by 6PGDH isknown to be important whose activity and gene expressioncan change depending on the type of stress and subcellularlocalization For example tobacco plants infected with potatovirus Y show increased cytosolic and plastidic 6PGDH activ-ity [49] Similar behavior has been observed in pepper leaveswith an increase in total 6PGDHactivity under cadmium [50]and low temperature [51] stress conditions Similar behaviorhas been described with regard to 6PGDH gene expressionThus in rice (Oryza sativa L) plants subjected to salt stress(150mM NaCl) an increase in 6PGDH transcripts in stemshas been reported [52] A subsequent study in which twogenes encoding for chloroplastic and cytosolic 6PGDH riceplants showed that both transcripts increased not only withsalinity but also with other stresses such as drought lowtemperature and treatment with abscisic acid however thetranscripts of G6PDH the first enzyme of the oxidativeportion of the pentose phosphate pathway did not undergoany change in its cytosolic and chloroplastic isoforms [53]Furthermore there is evidence to show that this enzyme isalso involved in other physiological processes for example instudies of the maturation of pepper fruits when they changefrom the green to red phenotype total 6PGDH activityincreased by more than 50 [54] In the case of tomato andspinach three genes encoding cytosolic and chloroplasticisozymes have been reported [55 56] also with respect toother plant species such as peas corn and tobacco [52 57ndash59]
4 Conclusion
In summary it is possible to conclude that there is a groupof plant 6PGDH enzymes which contain an archetypal type1 peroxisomal targeting signal Given the capacity of thisenzyme to generate NADPH in silico analysis of peroxisomal6PGDH in Arabidopsis thaliana suggests that it plays aprominent role during early seedling development in which
peroxisomes perform the key function of metabolizing lipidreserves until the seedling begins to photosynthesize [6061] for which NADPH is required Similarly at this earlyseedling stage the NADPH-dependent generation of nitricoxide in peroxisomes has also been demonstrated to beinvolved in root development [62] Finally the increase inp6PGDH in the presence of xenobiotics such as norflurazonand brassinazole is also closely in line with the induction ofthe antioxidant system present in plant peroxisomes as hasbeen demonstrated with the pea leaf peroxisomes of plantsexposed to 24-D [63]Therefore the present in silico analysissuggests the presence of a 6PGDH into peroxisomes whichwould contribute to the generation of NADPH within theseorganelles
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Research is supported by an ERDF-cofinanced grant fromthe Ministry of Science and Innovation (Recupera 2020-20134R056) and the Junta de Andalucıa (Group BIO192)
References
[1] F J Corpas J B Barroso and L A Del Rıo ldquoPeroxisomesas a source of reactive oxygen species and nitric oxide signalmolecules in plant cellsrdquo Trends in Plant Science vol 6 no 4pp 145ndash150 2001
[2] M Schrader and H D Fahimi ldquoPeroxisomes and oxidativestressrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1763 no 12 pp 1755ndash1766 2006
[3] J Hu A Baker B Bartel et al ldquoPlant peroxisomes biogenesisand functionrdquo Plant Cell vol 24 no 6 pp 2279ndash2303 2012
[4] I Pracharoenwattana and S M Smith ldquoWhen is a peroxisomenot a peroxisomerdquo Trends in Plant Science vol 13 no 10 pp522ndash525 2008
[5] F J Corpas ldquoWhat is the role of hydrogen peroxide in plantperoxisomesrdquo Plant Biology vol 17 no 6 pp 1099ndash1103 2015
[6] T G Cooper and H Beevers ldquoBeta oxidation in glyoxysomesfrom castor bean endospermrdquo The Journal of Biological Chem-istry vol 244 no 13 pp 3514ndash3520 1969
8 Scientifica
[7] S Reumann and A P M Weber ldquoPlant peroxisomes respirein the light some gaps of the photorespiratory C
2cycle have
become filledmdashothers remainrdquo Biochimica et Biophysica Actavol 1763 no 12 pp 1496ndash1510 2006
[8] A Baker and R Paudyal ldquoThe life of the peroxisome from birthto deathrdquo Current Opinion in Plant Biology vol 22 pp 39ndash472014
[9] L A del Rıo L M Sandalio F J Corpas J M Palma andJ B Barroso ldquoReactive oxygen species and reactive nitrogenspecies in peroxisomes Production scavenging and role in cellsignalingrdquo Plant Physiology vol 141 no 2 pp 330ndash335 2006
[10] A J K Koo H S Chung Y Kobayashi and G A Howe ldquoIden-tification of a peroxisomal acyl-activating enzyme involved inthe biosynthesis of jasmonic acid in ArabidopsisrdquoThe Journal ofBiological Chemistry vol 281 no 44 pp 33511ndash33520 2006
[11] B K Zolman and B Bartel ldquoAn Arabidopsis indole-3-butyricacid-response mutant defective in PEROXIN6 an apparentATPase implicated in peroxisomal functionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 6 pp 1786ndash1791 2004
[12] B K Zolman M Nyberg and B Bartel ldquoIBR3 a novelperoxisomal acyl-CoA dehydrogenase-like protein required forindole-3-butyric acid responserdquo Plant Molecular Biology vol64 no 1-2 pp 59ndash72 2007
[13] A A G Wiszniewski W Zhou S M Smith and J D BussellldquoIdentification of twoArabidopsis genes encoding a peroxisomaloxidoreductase-like protein and an acyl-CoA synthetase-likeprotein that are required for responses to pro-auxinsrdquo PlantMolecular Biology vol 69 no 5 pp 503ndash515 2009
[14] G M Spiess and B K Zolman ldquoPeroxisomes as a source ofauxin signalingmoleculesrdquo Subcellular Biochemistry vol 69 pp257ndash281 2013
[15] J B Barroso F J Corpas A Carreras et al ldquoLocalizationof nitric-oxide synthase in plant peroxisomesrdquo The Journal ofBiological Chemistry vol 274 no 51 pp 36729ndash36733 1999
[16] F J Corpas J B Barroso A Carreras et al ldquoCellular andsubcellular localization of endogenous nitric oxide in young andsenescent pea plantsrdquo Plant Physiology vol 136 no 1 pp 2722ndash2733 2004
[17] F J Corpas M Hayashi S Mano M Nishimura and J BBarroso ldquoPeroxisomes are required for in vivo nitric oxide accu-mulation in the cytosol following salinity stress of arabidopsisplantsrdquo Plant Physiology vol 151 no 4 pp 2083ndash2094 2009
[18] F J Corpas and J B Barroso ldquoPeroxisomal plant nitric oxidesynthase (NOS) protein is imported by peroxisomal targetingsignal type 2 (PTS2) in a process that depends on the cytosolicreceptor PEX7 and calmodulinrdquo FEBS Letters vol 588 no 12pp 2049ndash2054 2014
[19] L L Cross H T Ebeed and A Baker ldquoPeroxisome biogenesisprotein targeting mechanisms and PEX gene functions inplantsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular CellResearch 2015
[20] P B Lazarow andY Fujiki ldquoBiogenesis of peroxisomesrdquoAnnualReview of Cell Biology vol 1 no 1 pp 489ndash530 1985
[21] M Hayashi and M Nishimura ldquoEntering a new era of researchon plant peroxisomesrdquo Current Opinion in Plant Biology vol 6no 6 pp 577ndash582 2003
[22] S Reumann C Ma S Lemke and L Babujee ldquoAraPerox Adatabase of putative arabidopsis proteins from plant peroxi-somesrdquo Plant Physiology vol 136 no 1 pp 2587ndash2608 2004
[23] V D Antonenkov and J K Hiltunen ldquoPeroxisomal membranepermeability and solute transferrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1763 no 12 pp 1697ndash17062006
[24] M Hayashi andM Nishimura ldquoArabidopsis thalianamdashamodelorganism to study plant peroxisomesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1763 no 12 pp 1382ndash13912006
[25] C W T van Roermund E H Hettema A J Kal M vanden Berg H F Tabak and R J A Wanders ldquoPeroxisomal 120573-oxidation of polyunsaturated fatty acids in Saccharomyces cere-visiae isocitrate dehydrogenase providesNADPH for reductionof double bonds at even positionsrdquo The EMBO Journal vol 17no 3 pp 677ndash687 1998
[26] S Reumann L Babujee M Changle et al ldquoProteome analysisofArabidopsis leaf peroxisomes reveals novel targeting peptidesmetabolic pathways and defense mechanismsrdquo The Plant Cellvol 19 no 10 pp 3170ndash3193 2007
[27] M Kunze I Pracharoenwattana S M Smith and A Hartig ldquoAcentral role for the peroxisomal membrane in glyoxylate cyclefunctionrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp1441ndash1452 2006
[28] N Linka and A P M Weber ldquoIntracellular metabolite trans-porters in plantsrdquoMolecular Plant vol 3 no 1 pp 21ndash53 2010
[29] F L Theodoulou K Bernhardt N Linka and A BakerldquoPeroxisome membrane proteins multiple trafficking routesandmultiple functionsrdquo Biochemical Journal vol 451 no 3 pp345ndash352 2013
[30] F J Corpas and J B Barroso ldquoNADPH-generating dehydroge-nases their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditionsrdquoFrontiers in Environmental Science vol 2 article 55 2014
[31] M F Drincovich P Casati and C S Andreo ldquoNADP-malicenzyme from plants a ubiquitous enzyme involved in differentmetabolic pathwaysrdquo FEBS Letters vol 490 no 1-2 pp 1ndash62001
[32] F J Corpas J B Barroso L M Sandalio J M Palma J ALupianez and L A Del Rıo ldquoPeroxisomal NADP-dependentisocitrate dehydrogenase Characterization and activity regula-tion during natural senescencerdquo Plant Physiology vol 121 no 3pp 921ndash928 1999
[33] M Leterrier J B Barroso R Valderrama et al ldquoPeroxisomalNADP-isocitrate dehydrogenase is required for Arabidopsisstomatal movementrdquo Protoplasma 2015
[34] F J Corpas J B Barroso L M Sandalio et al ldquoAdehydrogenase-mediated recycling system of NADPH in plantperoxisomesrdquo Biochemical Journal vol 330 no 2 pp 777ndash7841998
[35] S Proost M van Bel L Sterck et al ldquoPLAZA a comparativegenomics resource to study gene and genome evolution inplantsrdquoThe Plant Cell vol 21 no 12 pp 3718ndash3731 2009
[36] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquoNucleic Acids Research vol 35 no 2 ppW585ndashW587 2007
[37] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoPrediction of peroxisomal targeting signal 1containing proteins from amino acid sequencerdquo Journal ofMolecular Biology vol 328 no 3 pp 581ndash592 2003
[38] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoMotif refinement of the peroxisomal targetingsignal 1 and evaluation of taxon-specific differencesrdquo Journal ofMolecular Biology vol 328 no 3 pp 567ndash579 2003
Scientifica 9
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
Table 1 Identification of 6PGDH proteins sequence of higher plants with a putative peroxisomal location for having a peroxisomal targetingsignal type 1 (PTS1) on the C-terminal The pI and MM values were calculated from their primary structure
Table 2 Genes encoding different isozymes of 6PGDH in A thaliana and molecular properties based on their predicted amino acidsequence The number of amino acids corresponds to the preprocessed protein and they were used for the in silico predictions usinghttpwebexpasyorgprotparam Transit peptide (TP) or targeting signal (TS) length is given in amino acids and molecular weight (MW)of the mature
Properties LocusAt5g41670 At3g02360 At1g64190
Protein accession number BAB11473 AEE73797 AAF24560Number of amino acids 487 486 487Subunit size (Da) 5331761 5357718 5337751pI 562 702 534Total number of negatively charged residues (Asp + Glu) 65 64 66Total number of positively charged residues (Arg + Lys) 60 64 57Stability indexlowast 2369 2686 2760120576280
(Mminus1 cmminus1) (assuming all Cys residues are reduced) 65320 63830 63830Aliphatic indexlowastlowast 8856 8710 8758Grand average of hydropathicity (GRAVY) indexlowastlowastlowast minus0278 minus0283 minus0272Transit peptide (TP)targeting signal (TS) mdash -SKI mdashSubcellular localization ChloroplastCytosol Peroxisome ChloroplastmitochondrionCytosollowastA protein with a stability index smaller than 40 is predicted as being stable with a value above 40 the protein is predicted as potentially unstablelowastlowastAliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (Ala Val Ile and Leu) A positive index indicates the increaseof thermostability of globular proteinslowastlowastlowastGRAVY (grand average of hydropathicity) index indicates the solubility of the proteins positive GRAVY (hydrophobic) negative GRAVY (hydrophilic)
as various regulatory elements identified in both transcripts(supplemental Figure 3) with the aid of the Plant Promoter21 db program
33 Gene Expression of Peroxisomal 6PGDH (p6PGDH)Figure 3 shows gene expression data for Arabidopsisp6PGDH under different growth conditions and is exposed tocertain chemicals Figure 3(a) shows that in adult plantsp6PGDH expression levels were highest in stems followedby flowers and lowest in leaves independently of the growthconditions in either the growth chamber or greenhouse Onthe other hand when Arabidopsis plants were grown underin vitro conditions and in the presence of 90mM sucrosep6PGDH gene expression was between 2-fold and 3-foldhigher (Figure 3(b)) suggesting as would be expected thatp6PGDH is involved in the carbon metabolism as thisenzyme is located in the oxidative part of the pentose phos-phate pathway [43] Additionally when plants were exposedto the herbicide norflurazon (a carotenoid biosynthesis
inhibitor) a similar 2-fold to 3-fold increase in p6PGDHgene expression was observed (Figure 3(c)) In the lattercase it is important to note that carotenoids have antioxidantproperties that help to protect chlorophyll from oxidativedamage mediated by ROS [47] as the absence of carotenoidsfacilitates chlorophyll destruction which is essential forphotosynthesisThese data are closely in line with the detoxi-fication capacity of peroxisomes which would amelioratedthe diminished antioxidant capacity of ROS to decomposechloroplasts damaged by this herbicide This is explainedby the fact that peroxisomes contain an important batteryof antioxidant enzymes including catalase superoxidedismutase and all components of the ascorbate-glutathionecycle which requires NADPH to support the regenerationof GSH by GR [1] Furthermore the relative expression ofp6PGDH was also higher in the presence of brassinazole(Figure 3(d)) a specific inhibitor of the biosynthesis ofbrassinosteroids which are a class of phytohormones thatplay an essential role in plant growth and development
Figure 3 Peroxisomal 6PGDH gene (At3g02360) expression in Arabidopsis thaliana grown under different conditions (a) Expression ofp6PGDH in leaves of 15-day-old plants stems and flowers of 29-day-old plants grown in either growth chamber or greenhouse conditions(b) Effects of a 6 h long treatment with 90mM sucrose to 4-day-old dark-grown Arabidopsis seedlings (c) Effects of 5120583M norflurazon to5-day-old continuous light Arabidopsis seedlings (d) Auxin effect on 10-day-old seedlings treated for 8 h either 01 120583M24-D or 01 120583M24-Dplus 1120583M brassinazole Data were obtained from the Gene Expression Omnibus (GEO) database and analyzed using Affymetrix MicroarraySuite 50 (MAS5) The original sample accessions (GSMxxx) are listed in the gray boxes along the bottom of the chart
Scientifica 7
Table 3 Analysis of the 51015840-UTR region At3G02360 gene coding for Arabidopsis thaliana peroxisomal 6PGDH Inr initiator TATA box anoctamer group related to TATA box Y patch an octamer group of the pyrimidine (Y) patch REG regulatory element group an octamergroup related to cis-regulatory elements CDS protein coding region UTR untranslated region
Type Sequence Genome position Position from initiation codonStrand Start End Start End
processes including promotion of stem elongation andcell division All these data suggest that p6PGDH couldbe specifically involved in growth and development sinceits gene expression is clearly induced by molecules whichblock these processes This is supported by a set of datawhich shows that sugar can promote hypocotyl elongation inArabidopsis in darkness a process which is largely dependenton brassinosteroids [48]
The reducing power (NADPH) generated by 6PGDH isknown to be important whose activity and gene expressioncan change depending on the type of stress and subcellularlocalization For example tobacco plants infected with potatovirus Y show increased cytosolic and plastidic 6PGDH activ-ity [49] Similar behavior has been observed in pepper leaveswith an increase in total 6PGDHactivity under cadmium [50]and low temperature [51] stress conditions Similar behaviorhas been described with regard to 6PGDH gene expressionThus in rice (Oryza sativa L) plants subjected to salt stress(150mM NaCl) an increase in 6PGDH transcripts in stemshas been reported [52] A subsequent study in which twogenes encoding for chloroplastic and cytosolic 6PGDH riceplants showed that both transcripts increased not only withsalinity but also with other stresses such as drought lowtemperature and treatment with abscisic acid however thetranscripts of G6PDH the first enzyme of the oxidativeportion of the pentose phosphate pathway did not undergoany change in its cytosolic and chloroplastic isoforms [53]Furthermore there is evidence to show that this enzyme isalso involved in other physiological processes for example instudies of the maturation of pepper fruits when they changefrom the green to red phenotype total 6PGDH activityincreased by more than 50 [54] In the case of tomato andspinach three genes encoding cytosolic and chloroplasticisozymes have been reported [55 56] also with respect toother plant species such as peas corn and tobacco [52 57ndash59]
4 Conclusion
In summary it is possible to conclude that there is a groupof plant 6PGDH enzymes which contain an archetypal type1 peroxisomal targeting signal Given the capacity of thisenzyme to generate NADPH in silico analysis of peroxisomal6PGDH in Arabidopsis thaliana suggests that it plays aprominent role during early seedling development in which
peroxisomes perform the key function of metabolizing lipidreserves until the seedling begins to photosynthesize [6061] for which NADPH is required Similarly at this earlyseedling stage the NADPH-dependent generation of nitricoxide in peroxisomes has also been demonstrated to beinvolved in root development [62] Finally the increase inp6PGDH in the presence of xenobiotics such as norflurazonand brassinazole is also closely in line with the induction ofthe antioxidant system present in plant peroxisomes as hasbeen demonstrated with the pea leaf peroxisomes of plantsexposed to 24-D [63]Therefore the present in silico analysissuggests the presence of a 6PGDH into peroxisomes whichwould contribute to the generation of NADPH within theseorganelles
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Research is supported by an ERDF-cofinanced grant fromthe Ministry of Science and Innovation (Recupera 2020-20134R056) and the Junta de Andalucıa (Group BIO192)
References
[1] F J Corpas J B Barroso and L A Del Rıo ldquoPeroxisomesas a source of reactive oxygen species and nitric oxide signalmolecules in plant cellsrdquo Trends in Plant Science vol 6 no 4pp 145ndash150 2001
[2] M Schrader and H D Fahimi ldquoPeroxisomes and oxidativestressrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1763 no 12 pp 1755ndash1766 2006
[3] J Hu A Baker B Bartel et al ldquoPlant peroxisomes biogenesisand functionrdquo Plant Cell vol 24 no 6 pp 2279ndash2303 2012
[4] I Pracharoenwattana and S M Smith ldquoWhen is a peroxisomenot a peroxisomerdquo Trends in Plant Science vol 13 no 10 pp522ndash525 2008
[5] F J Corpas ldquoWhat is the role of hydrogen peroxide in plantperoxisomesrdquo Plant Biology vol 17 no 6 pp 1099ndash1103 2015
[6] T G Cooper and H Beevers ldquoBeta oxidation in glyoxysomesfrom castor bean endospermrdquo The Journal of Biological Chem-istry vol 244 no 13 pp 3514ndash3520 1969
8 Scientifica
[7] S Reumann and A P M Weber ldquoPlant peroxisomes respirein the light some gaps of the photorespiratory C
2cycle have
become filledmdashothers remainrdquo Biochimica et Biophysica Actavol 1763 no 12 pp 1496ndash1510 2006
[8] A Baker and R Paudyal ldquoThe life of the peroxisome from birthto deathrdquo Current Opinion in Plant Biology vol 22 pp 39ndash472014
[9] L A del Rıo L M Sandalio F J Corpas J M Palma andJ B Barroso ldquoReactive oxygen species and reactive nitrogenspecies in peroxisomes Production scavenging and role in cellsignalingrdquo Plant Physiology vol 141 no 2 pp 330ndash335 2006
[10] A J K Koo H S Chung Y Kobayashi and G A Howe ldquoIden-tification of a peroxisomal acyl-activating enzyme involved inthe biosynthesis of jasmonic acid in ArabidopsisrdquoThe Journal ofBiological Chemistry vol 281 no 44 pp 33511ndash33520 2006
[11] B K Zolman and B Bartel ldquoAn Arabidopsis indole-3-butyricacid-response mutant defective in PEROXIN6 an apparentATPase implicated in peroxisomal functionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 6 pp 1786ndash1791 2004
[12] B K Zolman M Nyberg and B Bartel ldquoIBR3 a novelperoxisomal acyl-CoA dehydrogenase-like protein required forindole-3-butyric acid responserdquo Plant Molecular Biology vol64 no 1-2 pp 59ndash72 2007
[13] A A G Wiszniewski W Zhou S M Smith and J D BussellldquoIdentification of twoArabidopsis genes encoding a peroxisomaloxidoreductase-like protein and an acyl-CoA synthetase-likeprotein that are required for responses to pro-auxinsrdquo PlantMolecular Biology vol 69 no 5 pp 503ndash515 2009
[14] G M Spiess and B K Zolman ldquoPeroxisomes as a source ofauxin signalingmoleculesrdquo Subcellular Biochemistry vol 69 pp257ndash281 2013
[15] J B Barroso F J Corpas A Carreras et al ldquoLocalizationof nitric-oxide synthase in plant peroxisomesrdquo The Journal ofBiological Chemistry vol 274 no 51 pp 36729ndash36733 1999
[16] F J Corpas J B Barroso A Carreras et al ldquoCellular andsubcellular localization of endogenous nitric oxide in young andsenescent pea plantsrdquo Plant Physiology vol 136 no 1 pp 2722ndash2733 2004
[17] F J Corpas M Hayashi S Mano M Nishimura and J BBarroso ldquoPeroxisomes are required for in vivo nitric oxide accu-mulation in the cytosol following salinity stress of arabidopsisplantsrdquo Plant Physiology vol 151 no 4 pp 2083ndash2094 2009
[18] F J Corpas and J B Barroso ldquoPeroxisomal plant nitric oxidesynthase (NOS) protein is imported by peroxisomal targetingsignal type 2 (PTS2) in a process that depends on the cytosolicreceptor PEX7 and calmodulinrdquo FEBS Letters vol 588 no 12pp 2049ndash2054 2014
[19] L L Cross H T Ebeed and A Baker ldquoPeroxisome biogenesisprotein targeting mechanisms and PEX gene functions inplantsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular CellResearch 2015
[20] P B Lazarow andY Fujiki ldquoBiogenesis of peroxisomesrdquoAnnualReview of Cell Biology vol 1 no 1 pp 489ndash530 1985
[21] M Hayashi and M Nishimura ldquoEntering a new era of researchon plant peroxisomesrdquo Current Opinion in Plant Biology vol 6no 6 pp 577ndash582 2003
[22] S Reumann C Ma S Lemke and L Babujee ldquoAraPerox Adatabase of putative arabidopsis proteins from plant peroxi-somesrdquo Plant Physiology vol 136 no 1 pp 2587ndash2608 2004
[23] V D Antonenkov and J K Hiltunen ldquoPeroxisomal membranepermeability and solute transferrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1763 no 12 pp 1697ndash17062006
[24] M Hayashi andM Nishimura ldquoArabidopsis thalianamdashamodelorganism to study plant peroxisomesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1763 no 12 pp 1382ndash13912006
[25] C W T van Roermund E H Hettema A J Kal M vanden Berg H F Tabak and R J A Wanders ldquoPeroxisomal 120573-oxidation of polyunsaturated fatty acids in Saccharomyces cere-visiae isocitrate dehydrogenase providesNADPH for reductionof double bonds at even positionsrdquo The EMBO Journal vol 17no 3 pp 677ndash687 1998
[26] S Reumann L Babujee M Changle et al ldquoProteome analysisofArabidopsis leaf peroxisomes reveals novel targeting peptidesmetabolic pathways and defense mechanismsrdquo The Plant Cellvol 19 no 10 pp 3170ndash3193 2007
[27] M Kunze I Pracharoenwattana S M Smith and A Hartig ldquoAcentral role for the peroxisomal membrane in glyoxylate cyclefunctionrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp1441ndash1452 2006
[28] N Linka and A P M Weber ldquoIntracellular metabolite trans-porters in plantsrdquoMolecular Plant vol 3 no 1 pp 21ndash53 2010
[29] F L Theodoulou K Bernhardt N Linka and A BakerldquoPeroxisome membrane proteins multiple trafficking routesandmultiple functionsrdquo Biochemical Journal vol 451 no 3 pp345ndash352 2013
[30] F J Corpas and J B Barroso ldquoNADPH-generating dehydroge-nases their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditionsrdquoFrontiers in Environmental Science vol 2 article 55 2014
[31] M F Drincovich P Casati and C S Andreo ldquoNADP-malicenzyme from plants a ubiquitous enzyme involved in differentmetabolic pathwaysrdquo FEBS Letters vol 490 no 1-2 pp 1ndash62001
[32] F J Corpas J B Barroso L M Sandalio J M Palma J ALupianez and L A Del Rıo ldquoPeroxisomal NADP-dependentisocitrate dehydrogenase Characterization and activity regula-tion during natural senescencerdquo Plant Physiology vol 121 no 3pp 921ndash928 1999
[33] M Leterrier J B Barroso R Valderrama et al ldquoPeroxisomalNADP-isocitrate dehydrogenase is required for Arabidopsisstomatal movementrdquo Protoplasma 2015
[34] F J Corpas J B Barroso L M Sandalio et al ldquoAdehydrogenase-mediated recycling system of NADPH in plantperoxisomesrdquo Biochemical Journal vol 330 no 2 pp 777ndash7841998
[35] S Proost M van Bel L Sterck et al ldquoPLAZA a comparativegenomics resource to study gene and genome evolution inplantsrdquoThe Plant Cell vol 21 no 12 pp 3718ndash3731 2009
[36] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquoNucleic Acids Research vol 35 no 2 ppW585ndashW587 2007
[37] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoPrediction of peroxisomal targeting signal 1containing proteins from amino acid sequencerdquo Journal ofMolecular Biology vol 328 no 3 pp 581ndash592 2003
[38] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoMotif refinement of the peroxisomal targetingsignal 1 and evaluation of taxon-specific differencesrdquo Journal ofMolecular Biology vol 328 no 3 pp 567ndash579 2003
Scientifica 9
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
Figure 3 Peroxisomal 6PGDH gene (At3g02360) expression in Arabidopsis thaliana grown under different conditions (a) Expression ofp6PGDH in leaves of 15-day-old plants stems and flowers of 29-day-old plants grown in either growth chamber or greenhouse conditions(b) Effects of a 6 h long treatment with 90mM sucrose to 4-day-old dark-grown Arabidopsis seedlings (c) Effects of 5120583M norflurazon to5-day-old continuous light Arabidopsis seedlings (d) Auxin effect on 10-day-old seedlings treated for 8 h either 01 120583M24-D or 01 120583M24-Dplus 1120583M brassinazole Data were obtained from the Gene Expression Omnibus (GEO) database and analyzed using Affymetrix MicroarraySuite 50 (MAS5) The original sample accessions (GSMxxx) are listed in the gray boxes along the bottom of the chart
Scientifica 7
Table 3 Analysis of the 51015840-UTR region At3G02360 gene coding for Arabidopsis thaliana peroxisomal 6PGDH Inr initiator TATA box anoctamer group related to TATA box Y patch an octamer group of the pyrimidine (Y) patch REG regulatory element group an octamergroup related to cis-regulatory elements CDS protein coding region UTR untranslated region
Type Sequence Genome position Position from initiation codonStrand Start End Start End
processes including promotion of stem elongation andcell division All these data suggest that p6PGDH couldbe specifically involved in growth and development sinceits gene expression is clearly induced by molecules whichblock these processes This is supported by a set of datawhich shows that sugar can promote hypocotyl elongation inArabidopsis in darkness a process which is largely dependenton brassinosteroids [48]
The reducing power (NADPH) generated by 6PGDH isknown to be important whose activity and gene expressioncan change depending on the type of stress and subcellularlocalization For example tobacco plants infected with potatovirus Y show increased cytosolic and plastidic 6PGDH activ-ity [49] Similar behavior has been observed in pepper leaveswith an increase in total 6PGDHactivity under cadmium [50]and low temperature [51] stress conditions Similar behaviorhas been described with regard to 6PGDH gene expressionThus in rice (Oryza sativa L) plants subjected to salt stress(150mM NaCl) an increase in 6PGDH transcripts in stemshas been reported [52] A subsequent study in which twogenes encoding for chloroplastic and cytosolic 6PGDH riceplants showed that both transcripts increased not only withsalinity but also with other stresses such as drought lowtemperature and treatment with abscisic acid however thetranscripts of G6PDH the first enzyme of the oxidativeportion of the pentose phosphate pathway did not undergoany change in its cytosolic and chloroplastic isoforms [53]Furthermore there is evidence to show that this enzyme isalso involved in other physiological processes for example instudies of the maturation of pepper fruits when they changefrom the green to red phenotype total 6PGDH activityincreased by more than 50 [54] In the case of tomato andspinach three genes encoding cytosolic and chloroplasticisozymes have been reported [55 56] also with respect toother plant species such as peas corn and tobacco [52 57ndash59]
4 Conclusion
In summary it is possible to conclude that there is a groupof plant 6PGDH enzymes which contain an archetypal type1 peroxisomal targeting signal Given the capacity of thisenzyme to generate NADPH in silico analysis of peroxisomal6PGDH in Arabidopsis thaliana suggests that it plays aprominent role during early seedling development in which
peroxisomes perform the key function of metabolizing lipidreserves until the seedling begins to photosynthesize [6061] for which NADPH is required Similarly at this earlyseedling stage the NADPH-dependent generation of nitricoxide in peroxisomes has also been demonstrated to beinvolved in root development [62] Finally the increase inp6PGDH in the presence of xenobiotics such as norflurazonand brassinazole is also closely in line with the induction ofthe antioxidant system present in plant peroxisomes as hasbeen demonstrated with the pea leaf peroxisomes of plantsexposed to 24-D [63]Therefore the present in silico analysissuggests the presence of a 6PGDH into peroxisomes whichwould contribute to the generation of NADPH within theseorganelles
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Research is supported by an ERDF-cofinanced grant fromthe Ministry of Science and Innovation (Recupera 2020-20134R056) and the Junta de Andalucıa (Group BIO192)
References
[1] F J Corpas J B Barroso and L A Del Rıo ldquoPeroxisomesas a source of reactive oxygen species and nitric oxide signalmolecules in plant cellsrdquo Trends in Plant Science vol 6 no 4pp 145ndash150 2001
[2] M Schrader and H D Fahimi ldquoPeroxisomes and oxidativestressrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1763 no 12 pp 1755ndash1766 2006
[3] J Hu A Baker B Bartel et al ldquoPlant peroxisomes biogenesisand functionrdquo Plant Cell vol 24 no 6 pp 2279ndash2303 2012
[4] I Pracharoenwattana and S M Smith ldquoWhen is a peroxisomenot a peroxisomerdquo Trends in Plant Science vol 13 no 10 pp522ndash525 2008
[5] F J Corpas ldquoWhat is the role of hydrogen peroxide in plantperoxisomesrdquo Plant Biology vol 17 no 6 pp 1099ndash1103 2015
[6] T G Cooper and H Beevers ldquoBeta oxidation in glyoxysomesfrom castor bean endospermrdquo The Journal of Biological Chem-istry vol 244 no 13 pp 3514ndash3520 1969
8 Scientifica
[7] S Reumann and A P M Weber ldquoPlant peroxisomes respirein the light some gaps of the photorespiratory C
2cycle have
become filledmdashothers remainrdquo Biochimica et Biophysica Actavol 1763 no 12 pp 1496ndash1510 2006
[8] A Baker and R Paudyal ldquoThe life of the peroxisome from birthto deathrdquo Current Opinion in Plant Biology vol 22 pp 39ndash472014
[9] L A del Rıo L M Sandalio F J Corpas J M Palma andJ B Barroso ldquoReactive oxygen species and reactive nitrogenspecies in peroxisomes Production scavenging and role in cellsignalingrdquo Plant Physiology vol 141 no 2 pp 330ndash335 2006
[10] A J K Koo H S Chung Y Kobayashi and G A Howe ldquoIden-tification of a peroxisomal acyl-activating enzyme involved inthe biosynthesis of jasmonic acid in ArabidopsisrdquoThe Journal ofBiological Chemistry vol 281 no 44 pp 33511ndash33520 2006
[11] B K Zolman and B Bartel ldquoAn Arabidopsis indole-3-butyricacid-response mutant defective in PEROXIN6 an apparentATPase implicated in peroxisomal functionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 6 pp 1786ndash1791 2004
[12] B K Zolman M Nyberg and B Bartel ldquoIBR3 a novelperoxisomal acyl-CoA dehydrogenase-like protein required forindole-3-butyric acid responserdquo Plant Molecular Biology vol64 no 1-2 pp 59ndash72 2007
[13] A A G Wiszniewski W Zhou S M Smith and J D BussellldquoIdentification of twoArabidopsis genes encoding a peroxisomaloxidoreductase-like protein and an acyl-CoA synthetase-likeprotein that are required for responses to pro-auxinsrdquo PlantMolecular Biology vol 69 no 5 pp 503ndash515 2009
[14] G M Spiess and B K Zolman ldquoPeroxisomes as a source ofauxin signalingmoleculesrdquo Subcellular Biochemistry vol 69 pp257ndash281 2013
[15] J B Barroso F J Corpas A Carreras et al ldquoLocalizationof nitric-oxide synthase in plant peroxisomesrdquo The Journal ofBiological Chemistry vol 274 no 51 pp 36729ndash36733 1999
[16] F J Corpas J B Barroso A Carreras et al ldquoCellular andsubcellular localization of endogenous nitric oxide in young andsenescent pea plantsrdquo Plant Physiology vol 136 no 1 pp 2722ndash2733 2004
[17] F J Corpas M Hayashi S Mano M Nishimura and J BBarroso ldquoPeroxisomes are required for in vivo nitric oxide accu-mulation in the cytosol following salinity stress of arabidopsisplantsrdquo Plant Physiology vol 151 no 4 pp 2083ndash2094 2009
[18] F J Corpas and J B Barroso ldquoPeroxisomal plant nitric oxidesynthase (NOS) protein is imported by peroxisomal targetingsignal type 2 (PTS2) in a process that depends on the cytosolicreceptor PEX7 and calmodulinrdquo FEBS Letters vol 588 no 12pp 2049ndash2054 2014
[19] L L Cross H T Ebeed and A Baker ldquoPeroxisome biogenesisprotein targeting mechanisms and PEX gene functions inplantsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular CellResearch 2015
[20] P B Lazarow andY Fujiki ldquoBiogenesis of peroxisomesrdquoAnnualReview of Cell Biology vol 1 no 1 pp 489ndash530 1985
[21] M Hayashi and M Nishimura ldquoEntering a new era of researchon plant peroxisomesrdquo Current Opinion in Plant Biology vol 6no 6 pp 577ndash582 2003
[22] S Reumann C Ma S Lemke and L Babujee ldquoAraPerox Adatabase of putative arabidopsis proteins from plant peroxi-somesrdquo Plant Physiology vol 136 no 1 pp 2587ndash2608 2004
[23] V D Antonenkov and J K Hiltunen ldquoPeroxisomal membranepermeability and solute transferrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1763 no 12 pp 1697ndash17062006
[24] M Hayashi andM Nishimura ldquoArabidopsis thalianamdashamodelorganism to study plant peroxisomesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1763 no 12 pp 1382ndash13912006
[25] C W T van Roermund E H Hettema A J Kal M vanden Berg H F Tabak and R J A Wanders ldquoPeroxisomal 120573-oxidation of polyunsaturated fatty acids in Saccharomyces cere-visiae isocitrate dehydrogenase providesNADPH for reductionof double bonds at even positionsrdquo The EMBO Journal vol 17no 3 pp 677ndash687 1998
[26] S Reumann L Babujee M Changle et al ldquoProteome analysisofArabidopsis leaf peroxisomes reveals novel targeting peptidesmetabolic pathways and defense mechanismsrdquo The Plant Cellvol 19 no 10 pp 3170ndash3193 2007
[27] M Kunze I Pracharoenwattana S M Smith and A Hartig ldquoAcentral role for the peroxisomal membrane in glyoxylate cyclefunctionrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp1441ndash1452 2006
[28] N Linka and A P M Weber ldquoIntracellular metabolite trans-porters in plantsrdquoMolecular Plant vol 3 no 1 pp 21ndash53 2010
[29] F L Theodoulou K Bernhardt N Linka and A BakerldquoPeroxisome membrane proteins multiple trafficking routesandmultiple functionsrdquo Biochemical Journal vol 451 no 3 pp345ndash352 2013
[30] F J Corpas and J B Barroso ldquoNADPH-generating dehydroge-nases their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditionsrdquoFrontiers in Environmental Science vol 2 article 55 2014
[31] M F Drincovich P Casati and C S Andreo ldquoNADP-malicenzyme from plants a ubiquitous enzyme involved in differentmetabolic pathwaysrdquo FEBS Letters vol 490 no 1-2 pp 1ndash62001
[32] F J Corpas J B Barroso L M Sandalio J M Palma J ALupianez and L A Del Rıo ldquoPeroxisomal NADP-dependentisocitrate dehydrogenase Characterization and activity regula-tion during natural senescencerdquo Plant Physiology vol 121 no 3pp 921ndash928 1999
[33] M Leterrier J B Barroso R Valderrama et al ldquoPeroxisomalNADP-isocitrate dehydrogenase is required for Arabidopsisstomatal movementrdquo Protoplasma 2015
[34] F J Corpas J B Barroso L M Sandalio et al ldquoAdehydrogenase-mediated recycling system of NADPH in plantperoxisomesrdquo Biochemical Journal vol 330 no 2 pp 777ndash7841998
[35] S Proost M van Bel L Sterck et al ldquoPLAZA a comparativegenomics resource to study gene and genome evolution inplantsrdquoThe Plant Cell vol 21 no 12 pp 3718ndash3731 2009
[36] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquoNucleic Acids Research vol 35 no 2 ppW585ndashW587 2007
[37] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoPrediction of peroxisomal targeting signal 1containing proteins from amino acid sequencerdquo Journal ofMolecular Biology vol 328 no 3 pp 581ndash592 2003
[38] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoMotif refinement of the peroxisomal targetingsignal 1 and evaluation of taxon-specific differencesrdquo Journal ofMolecular Biology vol 328 no 3 pp 567ndash579 2003
Scientifica 9
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
Table 3 Analysis of the 51015840-UTR region At3G02360 gene coding for Arabidopsis thaliana peroxisomal 6PGDH Inr initiator TATA box anoctamer group related to TATA box Y patch an octamer group of the pyrimidine (Y) patch REG regulatory element group an octamergroup related to cis-regulatory elements CDS protein coding region UTR untranslated region
Type Sequence Genome position Position from initiation codonStrand Start End Start End
processes including promotion of stem elongation andcell division All these data suggest that p6PGDH couldbe specifically involved in growth and development sinceits gene expression is clearly induced by molecules whichblock these processes This is supported by a set of datawhich shows that sugar can promote hypocotyl elongation inArabidopsis in darkness a process which is largely dependenton brassinosteroids [48]
The reducing power (NADPH) generated by 6PGDH isknown to be important whose activity and gene expressioncan change depending on the type of stress and subcellularlocalization For example tobacco plants infected with potatovirus Y show increased cytosolic and plastidic 6PGDH activ-ity [49] Similar behavior has been observed in pepper leaveswith an increase in total 6PGDHactivity under cadmium [50]and low temperature [51] stress conditions Similar behaviorhas been described with regard to 6PGDH gene expressionThus in rice (Oryza sativa L) plants subjected to salt stress(150mM NaCl) an increase in 6PGDH transcripts in stemshas been reported [52] A subsequent study in which twogenes encoding for chloroplastic and cytosolic 6PGDH riceplants showed that both transcripts increased not only withsalinity but also with other stresses such as drought lowtemperature and treatment with abscisic acid however thetranscripts of G6PDH the first enzyme of the oxidativeportion of the pentose phosphate pathway did not undergoany change in its cytosolic and chloroplastic isoforms [53]Furthermore there is evidence to show that this enzyme isalso involved in other physiological processes for example instudies of the maturation of pepper fruits when they changefrom the green to red phenotype total 6PGDH activityincreased by more than 50 [54] In the case of tomato andspinach three genes encoding cytosolic and chloroplasticisozymes have been reported [55 56] also with respect toother plant species such as peas corn and tobacco [52 57ndash59]
4 Conclusion
In summary it is possible to conclude that there is a groupof plant 6PGDH enzymes which contain an archetypal type1 peroxisomal targeting signal Given the capacity of thisenzyme to generate NADPH in silico analysis of peroxisomal6PGDH in Arabidopsis thaliana suggests that it plays aprominent role during early seedling development in which
peroxisomes perform the key function of metabolizing lipidreserves until the seedling begins to photosynthesize [6061] for which NADPH is required Similarly at this earlyseedling stage the NADPH-dependent generation of nitricoxide in peroxisomes has also been demonstrated to beinvolved in root development [62] Finally the increase inp6PGDH in the presence of xenobiotics such as norflurazonand brassinazole is also closely in line with the induction ofthe antioxidant system present in plant peroxisomes as hasbeen demonstrated with the pea leaf peroxisomes of plantsexposed to 24-D [63]Therefore the present in silico analysissuggests the presence of a 6PGDH into peroxisomes whichwould contribute to the generation of NADPH within theseorganelles
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Research is supported by an ERDF-cofinanced grant fromthe Ministry of Science and Innovation (Recupera 2020-20134R056) and the Junta de Andalucıa (Group BIO192)
References
[1] F J Corpas J B Barroso and L A Del Rıo ldquoPeroxisomesas a source of reactive oxygen species and nitric oxide signalmolecules in plant cellsrdquo Trends in Plant Science vol 6 no 4pp 145ndash150 2001
[2] M Schrader and H D Fahimi ldquoPeroxisomes and oxidativestressrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1763 no 12 pp 1755ndash1766 2006
[3] J Hu A Baker B Bartel et al ldquoPlant peroxisomes biogenesisand functionrdquo Plant Cell vol 24 no 6 pp 2279ndash2303 2012
[4] I Pracharoenwattana and S M Smith ldquoWhen is a peroxisomenot a peroxisomerdquo Trends in Plant Science vol 13 no 10 pp522ndash525 2008
[5] F J Corpas ldquoWhat is the role of hydrogen peroxide in plantperoxisomesrdquo Plant Biology vol 17 no 6 pp 1099ndash1103 2015
[6] T G Cooper and H Beevers ldquoBeta oxidation in glyoxysomesfrom castor bean endospermrdquo The Journal of Biological Chem-istry vol 244 no 13 pp 3514ndash3520 1969
8 Scientifica
[7] S Reumann and A P M Weber ldquoPlant peroxisomes respirein the light some gaps of the photorespiratory C
2cycle have
become filledmdashothers remainrdquo Biochimica et Biophysica Actavol 1763 no 12 pp 1496ndash1510 2006
[8] A Baker and R Paudyal ldquoThe life of the peroxisome from birthto deathrdquo Current Opinion in Plant Biology vol 22 pp 39ndash472014
[9] L A del Rıo L M Sandalio F J Corpas J M Palma andJ B Barroso ldquoReactive oxygen species and reactive nitrogenspecies in peroxisomes Production scavenging and role in cellsignalingrdquo Plant Physiology vol 141 no 2 pp 330ndash335 2006
[10] A J K Koo H S Chung Y Kobayashi and G A Howe ldquoIden-tification of a peroxisomal acyl-activating enzyme involved inthe biosynthesis of jasmonic acid in ArabidopsisrdquoThe Journal ofBiological Chemistry vol 281 no 44 pp 33511ndash33520 2006
[11] B K Zolman and B Bartel ldquoAn Arabidopsis indole-3-butyricacid-response mutant defective in PEROXIN6 an apparentATPase implicated in peroxisomal functionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 6 pp 1786ndash1791 2004
[12] B K Zolman M Nyberg and B Bartel ldquoIBR3 a novelperoxisomal acyl-CoA dehydrogenase-like protein required forindole-3-butyric acid responserdquo Plant Molecular Biology vol64 no 1-2 pp 59ndash72 2007
[13] A A G Wiszniewski W Zhou S M Smith and J D BussellldquoIdentification of twoArabidopsis genes encoding a peroxisomaloxidoreductase-like protein and an acyl-CoA synthetase-likeprotein that are required for responses to pro-auxinsrdquo PlantMolecular Biology vol 69 no 5 pp 503ndash515 2009
[14] G M Spiess and B K Zolman ldquoPeroxisomes as a source ofauxin signalingmoleculesrdquo Subcellular Biochemistry vol 69 pp257ndash281 2013
[15] J B Barroso F J Corpas A Carreras et al ldquoLocalizationof nitric-oxide synthase in plant peroxisomesrdquo The Journal ofBiological Chemistry vol 274 no 51 pp 36729ndash36733 1999
[16] F J Corpas J B Barroso A Carreras et al ldquoCellular andsubcellular localization of endogenous nitric oxide in young andsenescent pea plantsrdquo Plant Physiology vol 136 no 1 pp 2722ndash2733 2004
[17] F J Corpas M Hayashi S Mano M Nishimura and J BBarroso ldquoPeroxisomes are required for in vivo nitric oxide accu-mulation in the cytosol following salinity stress of arabidopsisplantsrdquo Plant Physiology vol 151 no 4 pp 2083ndash2094 2009
[18] F J Corpas and J B Barroso ldquoPeroxisomal plant nitric oxidesynthase (NOS) protein is imported by peroxisomal targetingsignal type 2 (PTS2) in a process that depends on the cytosolicreceptor PEX7 and calmodulinrdquo FEBS Letters vol 588 no 12pp 2049ndash2054 2014
[19] L L Cross H T Ebeed and A Baker ldquoPeroxisome biogenesisprotein targeting mechanisms and PEX gene functions inplantsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular CellResearch 2015
[20] P B Lazarow andY Fujiki ldquoBiogenesis of peroxisomesrdquoAnnualReview of Cell Biology vol 1 no 1 pp 489ndash530 1985
[21] M Hayashi and M Nishimura ldquoEntering a new era of researchon plant peroxisomesrdquo Current Opinion in Plant Biology vol 6no 6 pp 577ndash582 2003
[22] S Reumann C Ma S Lemke and L Babujee ldquoAraPerox Adatabase of putative arabidopsis proteins from plant peroxi-somesrdquo Plant Physiology vol 136 no 1 pp 2587ndash2608 2004
[23] V D Antonenkov and J K Hiltunen ldquoPeroxisomal membranepermeability and solute transferrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1763 no 12 pp 1697ndash17062006
[24] M Hayashi andM Nishimura ldquoArabidopsis thalianamdashamodelorganism to study plant peroxisomesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1763 no 12 pp 1382ndash13912006
[25] C W T van Roermund E H Hettema A J Kal M vanden Berg H F Tabak and R J A Wanders ldquoPeroxisomal 120573-oxidation of polyunsaturated fatty acids in Saccharomyces cere-visiae isocitrate dehydrogenase providesNADPH for reductionof double bonds at even positionsrdquo The EMBO Journal vol 17no 3 pp 677ndash687 1998
[26] S Reumann L Babujee M Changle et al ldquoProteome analysisofArabidopsis leaf peroxisomes reveals novel targeting peptidesmetabolic pathways and defense mechanismsrdquo The Plant Cellvol 19 no 10 pp 3170ndash3193 2007
[27] M Kunze I Pracharoenwattana S M Smith and A Hartig ldquoAcentral role for the peroxisomal membrane in glyoxylate cyclefunctionrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp1441ndash1452 2006
[28] N Linka and A P M Weber ldquoIntracellular metabolite trans-porters in plantsrdquoMolecular Plant vol 3 no 1 pp 21ndash53 2010
[29] F L Theodoulou K Bernhardt N Linka and A BakerldquoPeroxisome membrane proteins multiple trafficking routesandmultiple functionsrdquo Biochemical Journal vol 451 no 3 pp345ndash352 2013
[30] F J Corpas and J B Barroso ldquoNADPH-generating dehydroge-nases their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditionsrdquoFrontiers in Environmental Science vol 2 article 55 2014
[31] M F Drincovich P Casati and C S Andreo ldquoNADP-malicenzyme from plants a ubiquitous enzyme involved in differentmetabolic pathwaysrdquo FEBS Letters vol 490 no 1-2 pp 1ndash62001
[32] F J Corpas J B Barroso L M Sandalio J M Palma J ALupianez and L A Del Rıo ldquoPeroxisomal NADP-dependentisocitrate dehydrogenase Characterization and activity regula-tion during natural senescencerdquo Plant Physiology vol 121 no 3pp 921ndash928 1999
[33] M Leterrier J B Barroso R Valderrama et al ldquoPeroxisomalNADP-isocitrate dehydrogenase is required for Arabidopsisstomatal movementrdquo Protoplasma 2015
[34] F J Corpas J B Barroso L M Sandalio et al ldquoAdehydrogenase-mediated recycling system of NADPH in plantperoxisomesrdquo Biochemical Journal vol 330 no 2 pp 777ndash7841998
[35] S Proost M van Bel L Sterck et al ldquoPLAZA a comparativegenomics resource to study gene and genome evolution inplantsrdquoThe Plant Cell vol 21 no 12 pp 3718ndash3731 2009
[36] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquoNucleic Acids Research vol 35 no 2 ppW585ndashW587 2007
[37] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoPrediction of peroxisomal targeting signal 1containing proteins from amino acid sequencerdquo Journal ofMolecular Biology vol 328 no 3 pp 581ndash592 2003
[38] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoMotif refinement of the peroxisomal targetingsignal 1 and evaluation of taxon-specific differencesrdquo Journal ofMolecular Biology vol 328 no 3 pp 567ndash579 2003
Scientifica 9
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
[7] S Reumann and A P M Weber ldquoPlant peroxisomes respirein the light some gaps of the photorespiratory C
2cycle have
become filledmdashothers remainrdquo Biochimica et Biophysica Actavol 1763 no 12 pp 1496ndash1510 2006
[8] A Baker and R Paudyal ldquoThe life of the peroxisome from birthto deathrdquo Current Opinion in Plant Biology vol 22 pp 39ndash472014
[9] L A del Rıo L M Sandalio F J Corpas J M Palma andJ B Barroso ldquoReactive oxygen species and reactive nitrogenspecies in peroxisomes Production scavenging and role in cellsignalingrdquo Plant Physiology vol 141 no 2 pp 330ndash335 2006
[10] A J K Koo H S Chung Y Kobayashi and G A Howe ldquoIden-tification of a peroxisomal acyl-activating enzyme involved inthe biosynthesis of jasmonic acid in ArabidopsisrdquoThe Journal ofBiological Chemistry vol 281 no 44 pp 33511ndash33520 2006
[11] B K Zolman and B Bartel ldquoAn Arabidopsis indole-3-butyricacid-response mutant defective in PEROXIN6 an apparentATPase implicated in peroxisomal functionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 6 pp 1786ndash1791 2004
[12] B K Zolman M Nyberg and B Bartel ldquoIBR3 a novelperoxisomal acyl-CoA dehydrogenase-like protein required forindole-3-butyric acid responserdquo Plant Molecular Biology vol64 no 1-2 pp 59ndash72 2007
[13] A A G Wiszniewski W Zhou S M Smith and J D BussellldquoIdentification of twoArabidopsis genes encoding a peroxisomaloxidoreductase-like protein and an acyl-CoA synthetase-likeprotein that are required for responses to pro-auxinsrdquo PlantMolecular Biology vol 69 no 5 pp 503ndash515 2009
[14] G M Spiess and B K Zolman ldquoPeroxisomes as a source ofauxin signalingmoleculesrdquo Subcellular Biochemistry vol 69 pp257ndash281 2013
[15] J B Barroso F J Corpas A Carreras et al ldquoLocalizationof nitric-oxide synthase in plant peroxisomesrdquo The Journal ofBiological Chemistry vol 274 no 51 pp 36729ndash36733 1999
[16] F J Corpas J B Barroso A Carreras et al ldquoCellular andsubcellular localization of endogenous nitric oxide in young andsenescent pea plantsrdquo Plant Physiology vol 136 no 1 pp 2722ndash2733 2004
[17] F J Corpas M Hayashi S Mano M Nishimura and J BBarroso ldquoPeroxisomes are required for in vivo nitric oxide accu-mulation in the cytosol following salinity stress of arabidopsisplantsrdquo Plant Physiology vol 151 no 4 pp 2083ndash2094 2009
[18] F J Corpas and J B Barroso ldquoPeroxisomal plant nitric oxidesynthase (NOS) protein is imported by peroxisomal targetingsignal type 2 (PTS2) in a process that depends on the cytosolicreceptor PEX7 and calmodulinrdquo FEBS Letters vol 588 no 12pp 2049ndash2054 2014
[19] L L Cross H T Ebeed and A Baker ldquoPeroxisome biogenesisprotein targeting mechanisms and PEX gene functions inplantsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular CellResearch 2015
[20] P B Lazarow andY Fujiki ldquoBiogenesis of peroxisomesrdquoAnnualReview of Cell Biology vol 1 no 1 pp 489ndash530 1985
[21] M Hayashi and M Nishimura ldquoEntering a new era of researchon plant peroxisomesrdquo Current Opinion in Plant Biology vol 6no 6 pp 577ndash582 2003
[22] S Reumann C Ma S Lemke and L Babujee ldquoAraPerox Adatabase of putative arabidopsis proteins from plant peroxi-somesrdquo Plant Physiology vol 136 no 1 pp 2587ndash2608 2004
[23] V D Antonenkov and J K Hiltunen ldquoPeroxisomal membranepermeability and solute transferrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Cell Research vol 1763 no 12 pp 1697ndash17062006
[24] M Hayashi andM Nishimura ldquoArabidopsis thalianamdashamodelorganism to study plant peroxisomesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1763 no 12 pp 1382ndash13912006
[25] C W T van Roermund E H Hettema A J Kal M vanden Berg H F Tabak and R J A Wanders ldquoPeroxisomal 120573-oxidation of polyunsaturated fatty acids in Saccharomyces cere-visiae isocitrate dehydrogenase providesNADPH for reductionof double bonds at even positionsrdquo The EMBO Journal vol 17no 3 pp 677ndash687 1998
[26] S Reumann L Babujee M Changle et al ldquoProteome analysisofArabidopsis leaf peroxisomes reveals novel targeting peptidesmetabolic pathways and defense mechanismsrdquo The Plant Cellvol 19 no 10 pp 3170ndash3193 2007
[27] M Kunze I Pracharoenwattana S M Smith and A Hartig ldquoAcentral role for the peroxisomal membrane in glyoxylate cyclefunctionrdquo Biochimica et Biophysica Acta vol 1763 no 12 pp1441ndash1452 2006
[28] N Linka and A P M Weber ldquoIntracellular metabolite trans-porters in plantsrdquoMolecular Plant vol 3 no 1 pp 21ndash53 2010
[29] F L Theodoulou K Bernhardt N Linka and A BakerldquoPeroxisome membrane proteins multiple trafficking routesandmultiple functionsrdquo Biochemical Journal vol 451 no 3 pp345ndash352 2013
[30] F J Corpas and J B Barroso ldquoNADPH-generating dehydroge-nases their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditionsrdquoFrontiers in Environmental Science vol 2 article 55 2014
[31] M F Drincovich P Casati and C S Andreo ldquoNADP-malicenzyme from plants a ubiquitous enzyme involved in differentmetabolic pathwaysrdquo FEBS Letters vol 490 no 1-2 pp 1ndash62001
[32] F J Corpas J B Barroso L M Sandalio J M Palma J ALupianez and L A Del Rıo ldquoPeroxisomal NADP-dependentisocitrate dehydrogenase Characterization and activity regula-tion during natural senescencerdquo Plant Physiology vol 121 no 3pp 921ndash928 1999
[33] M Leterrier J B Barroso R Valderrama et al ldquoPeroxisomalNADP-isocitrate dehydrogenase is required for Arabidopsisstomatal movementrdquo Protoplasma 2015
[34] F J Corpas J B Barroso L M Sandalio et al ldquoAdehydrogenase-mediated recycling system of NADPH in plantperoxisomesrdquo Biochemical Journal vol 330 no 2 pp 777ndash7841998
[35] S Proost M van Bel L Sterck et al ldquoPLAZA a comparativegenomics resource to study gene and genome evolution inplantsrdquoThe Plant Cell vol 21 no 12 pp 3718ndash3731 2009
[36] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquoNucleic Acids Research vol 35 no 2 ppW585ndashW587 2007
[37] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoPrediction of peroxisomal targeting signal 1containing proteins from amino acid sequencerdquo Journal ofMolecular Biology vol 328 no 3 pp 581ndash592 2003
[38] G Neuberger S Maurer-Stroh B Eisenhaber A Hartig andF Eisenhaber ldquoMotif refinement of the peroxisomal targetingsignal 1 and evaluation of taxon-specific differencesrdquo Journal ofMolecular Biology vol 328 no 3 pp 567ndash579 2003
Scientifica 9
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
[39] S Reumann D Buchwald and T Lingner ldquoPredPlantPTS1 aweb server for the prediction of plant peroxisomal proteinsrdquoFrontiers in Plant Science vol 3 article 194 2012
[40] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013
[41] T Barrett S EWilhite P Ledoux et al ldquoNCBIGEO archive forfunctional genomics data setsmdashupdaterdquoNucleic Acids Researchvol 41 no 1 pp D991ndashD995 2013
[42] X G Zhu J P Lynch D S LeBauer A J Millar M Stittand S P Long ldquoPlants in silico why why now and whatmdashanintegrative platform for plant systems biology researchrdquo PlantCell and Environment 2015
[43] N J Kruger andA von Schaewen ldquoThe oxidative pentose phos-phate pathway structure and organisationrdquo Current Opinion inPlant Biology vol 6 no 3 pp 236ndash246 2003
[44] G Spielbauer L Li L Romisch-Margl et al ldquoChloroplast-localized 6-phosphogluconate dehydrogenase is critical formaize endosperm starch accumulationrdquo Journal of Experimen-tal Botany vol 64 no 8 pp 2231ndash2242 2013
[45] G Chowdhary A R A Kataya T Lingner and S ReumannldquoNon-canonical peroxisome targeting signals identificationof novel PTS1 tripeptides and characterization of enhancerelements by computational permutation analysisrdquo BMC PlantBiology vol 12 article 142 2012
[46] C Williams E Bener Aksam K Gunkel M Veenhuis and IJ van der Klei ldquoThe relevance of the non-canonical PTS1 ofperoxisomal catalaserdquo Biochimica et Biophysica Acta vol 1823no 7 pp 1133ndash1141 2012
[47] H Cao J Wang X Dong et al ldquoCarotenoid accumula-tion affects redox status starch metabolism and flavonoidanthocyanin accumulation in citrusrdquo BMC Plant Biology vol15 article 27 2015
[48] Y Zhang Z Liu J Wang Y Chen Y Bi and J He ldquoBrassinos-teroid is required for sugar promotion of hypocotyl elongationin Arabidopsis in darknessrdquo Planta vol 242 no 4 pp 881ndash8932015
[49] L Sindelar M Sindelarova and L Burketova ldquoChanges inactivity of glucose-6-phosphate and 6-phosphogluconate dehy-drogenase isozymes upon potato virus Y infection in tobaccoleaf tissues and protoplastsrdquo Plant Physiology and Biochemistryvol 37 no 3 pp 195ndash201 1999
[50] A M Leon J M Palma F J Corpas et al ldquoAntioxidativeenzymes in cultivars of pepper plants with different sensitivityto cadmiumrdquo Plant Physiology and Biochemistry vol 40 no 10pp 813ndash820 2002
[51] M Airaki M Leterrier R M Mateos et al ldquoMetabolism ofreactive oxygen species and reactive nitrogen species in pepper(Capsicum annuum L) plants under low temperature stressrdquoPlant Cell and Environment vol 35 no 2 pp 281ndash295 2012
[52] J Huang H Zhang J Wang and J Yang ldquoMolecular cloningand characterization of rice 6-phosphogluconate dehydroge-nase gene that is up-regulated by salt stressrdquoMolecular BiologyReports vol 30 no 4 pp 223ndash227 2003
[53] F-Y Hou J Huang S-L Yu and H-S Zhang ldquoThe 6-phosphogluconate dehydrogenase genes are responsive to abi-otic stresses in ricerdquo Journal of Integrative Plant Biology vol 49no 5 pp 655ndash663 2007
[54] R M Mateos D Bonilla-Valverde L A Del Rıo J M Palmaand F J Corpas ldquoNADP-dehydrogenases from pepper fruits
effect of maturationrdquo Physiologia Plantarum vol 135 no 2 pp130ndash139 2009
[55] S D Tanksley and G D Kuehn ldquoGenetics subcellular local-ization and molecular characterization of 6-phosphogluconatedehydrogenase isozymes in tomatordquo Biochemical Genetics vol23 no 5-6 pp 441ndash454 1985
[56] K Krepinsky M Plaumann W Martin and C Schnarren-berger ldquoPurification and cloning of chloroplast 6-phospho-gluconate dehydrogenase from spinach cyanobacterial genesfor chloroplast and cytosolic isoenzymes encoded in eukaryoticchromosomesrdquo European Journal of Biochemistry vol 268 no9 pp 2678ndash2686 2001
[57] C Schnarrenberger A Oeser and N E Tolbert ldquoTwo isoen-zymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leavesrdquoArchives ofBiochemistry and Biophysics vol 154 no 1 pp 438ndash448 1973
[58] C Schnarrenberger A Flechner and W Martin ldquoEnzymaticevidence for a complete oxidative pentose phosphate pathwayin chloroplasts and an incomplete pathway in the cytosol ofspinach leavesrdquo Plant Physiology vol 108 no 2 pp 609ndash6141995
[59] P M Debnam and M J Emes ldquoSubcellular distribution ofenzymes of the oxidative pentose phosphate pathway in rootand leaf tissuesrdquo Journal of Experimental Botany vol 50 no340 pp 1653ndash1661 1999
[60] J Schwender J B Ohlrogge and Y Shachar-Hill ldquoA flux modelof glycolysis and the oxidative pentosephosphate pathwayin developing 119861119903119886119904119904119894119888119886119899119886119901119906119904 embryosrdquo The Journal of BiologicalChemistry vol 278 no 32 pp 29442ndash29453 2003
[61] D Hutchings S Rawsthorne and M J Emes ldquoFatty acidsynthesis and the oxidative pentose phosphate pathway indeveloping embryos of oilseed rape (Brassica napus L)rdquo Journalof Experimental Botany vol 56 no 412 pp 577ndash585 2005
[62] M Schlicht J Ludwig-Muller C Burbach D Volkmann and FBaluska ldquoIndole-3-butyric acid induces lateral root formationvia peroxisome-derived indole-3-acetic acid and nitric oxiderdquoThe New Phytologist vol 200 no 2 pp 473ndash482 2013
[63] I McCarthy-Suarez M Gomez L A Del Rıo and J M PalmaldquoRole of peroxisomes in the oxidative injury induced by 24-dichlorophenoxyacetic acid in leaves of pea plantsrdquo BiologiaPlantarum vol 55 no 3 pp 485ndash492 2011
