See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/226719753 Isolation of an ILR1 auxin conjugate hydrolase homolog from Arabidopsis suecica ARTICLE in PLANT GROWTH REGULATION · JANUARY 2003 Impact Factor: 1.67 · DOI: 10.1023/A:1022528318188 CITATIONS 12 READS 22 7 AUTHORS, INCLUDING: James J Campanella Montclair State University 33 PUBLICATIONS 774 CITATIONS SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Available from: James J Campanella Retrieved on: 03 February 2016
Isolation of an ILR1 auxin conjugate hydrolase homolog from Arabidopsissuecica
James J. Campanella*, Vinela Bakllamaja, Tracie Restieri, Michael Vomacka, Jason Herron,Megan Patterson and Shahin ShahtaheriDepartment of Biology and Molecular Biology, Montclair State University, 1 Normal Avenue, Montclair, NewJersey 07043, USA; *Author for correspondence (e-mail: [email protected]; phone:973-655-4097; fax: 419-791-9834)
Received 5 February 2002; accepted in revised form 3 August 2002
We have isolated a homolog of the Arabidopsis thaliana IAA amidohydrolase ILR1 from the related speciesArabidopsis suecica. Employing PCR primers targeted to the 5� and 3� untranslated regions of ILR1, A. suecicaproduced an amplified, genomic product (sILR1) of 2027 bp. The cloned sILR1 gene was sequenced, and theDNA homology between sILR1 and ILR1 found to be 98%. The sILR1 cDNA was isolated using RT-PCR, clonedand sequenced. The sILR1 cDNA was found to have 98% homology to its homolog ILR1 both at the DNA andamino acid level. The sILR1 mRNA transcript was found to be expressed in A. suecica tissues at ages 5, 10, and15 days. Additionally, primers, designed to a � 300 bp domain conserved in bacterial and plant amidohydro-lases, were used to amplify a product from seven members of the Brassicaceae family (A. suecica, A. pumila, A.himalaica, A. griffıthiana, A. wallichii, Draba nemoroza and Capsella bursa-pastoris), Lycopersicon esculentum,and two monocot species (Zea mays and Allium cepa) using PCR; this result suggests similar genes may becharacterized in other species.
Introduction
In higher plants, the hormone indole-3-acetic acid(IAA) is stored conjugated to sugar moieties via anester linkage or to amino acids or peptides via anamide linkage (Cohen and Bandurski 1982; Bandur-ski et al. 1995; Walz et al. 2002). Over 95% of thehormone in a plant can be found in the conjugatedform, leaving only a small amount of free hormoneavailable to stimulate and control cellular growth(Hangarter and Good 1981; Campell and Town 1991;Bandurski et al. 1995; Campanella et al. 1996; Lass-well et al. 2000).
Amide conjugates account for the bulk of conju-gated IAA in dicots studied to date. IAA-Aspartate(IAA-Asp) and IAA-Glutamate (IAA-Glu) have beenidentified as natural conjugates in cucumber (Sonnerand Purvis 1985) and soybean (Cohen 1982). IAA-
Alanine (IAA-Ala) has been detected in Picea abies(Ostin et al. 1992). IAA-Ala, IAA-Asp, IAA-Leucine(IAA-Leu), and IAA-Glu have been detected in Ara-bidopsis thaliana (Barratt et al. 1998; Tam et al. 2000;Kowalczyk and Sandberg 2001), although recent data(Walz et al. 2002) suggest that these conjugates arepresent in very low abundances while IAA-peptidesprobably account for the majority of IAA-conjugates.
The overall levels of active IAA in a plant can becontrolled not only by the amount of IAA synthe-sized, but also by the quantity of IAA that is releasedfrom the conjugated state into the “free” state (Cohenand Bandurski 1982; Bandurski et al. 1995). In dicots,the active IAA hormone is released by an amidohy-drolase enzyme that cleaves the amide bond betweenthe auxin and the amino acid. Several IAA amidohy-drolases have been isolated from A. thaliana (Barteland Fink 1995; Davies et al. 1999; Lasswell et al.
2000). Each of these enzymes has a different substratespecificity. The A. thaliana IAA amidohydrolase,IAR3, is able to cleave IAA-Ala (Davies et al. 1999;Lasswell et al. 2000), while products of the otheramidohydrolase genes, known as the ILR1-like fam-ily of hydrolases (ILR1, ILL1, ILL2, ILL3 and ILL5),cleave primarily IAA-Phenylalanine (IAA-Phe) andIAA-Leucine (IAA-Leu) (Bartel and Fink 1995).
To determine if the ILR1 enzymatic activity is con-sistent across closely related species, we have identi-fied a gene from A. suecica homologous to the A.thaliana ILR1 amidohydrolase, and show that homol-ogous genes are likely to occur not only in otherclosely related Brassica species but in more phyloge-netically distant species. To do this we took advan-tage of a 300 bp conserved region found in all mem-bers of the ILR1 family.
Materials and methods
Plants and plant growth
Arabidopsis griffıthiana and Arabidopsis himalaicawere obtained from the Arabidopsis Biological Re-source Center (Ohio State University, Columbus,Ohio, USA). All other Brassica species were obtainedfrom the Sendai Arabidopsis Stock Center (MiyagiUniversity of Education, Sendai, Japan). Seeds fortomato (Lycopersicon esculentum, cv. GroundCherry) and corn (Zea mays, cv. Honey and Pearl)were purchased from Burgess Seed and Plant Com-pany and NK Lawn and Garden Company, respec-tively. Allium cepa bulbs were purchased from DutchValley Growers.
Brassica seeds were germinated and seedlings ini-tially grown under sterile conditions. Seeds were sur-face sterilized for 10 min with 500–1000 �l of ster-ilization solution (30% Clorox bleach, 0.1% TritonX-100) with agitation every 2 min during incubation.After sterilization, seeds were washed three-times in1 ml sterile, distilled water, then re-suspended in 1 mlsterile, distilled water. Seeds were then plated on toMurashige-Skoog agar medium (Sigma Co., Catalog#M-5519) under sterile conditions, cold-treated 1 wkin the dark at 4 °C, then incubated at 23 °C underconstant light (cool white, fluorescent, � 100�mol/s/m2) in a plant growth chamber (Percival Sci-entific, Model E-30B). After 2–3 wk, plants weretransplanted to soil (1:1:1, perlite:sphagnum peatmoss:vermiculite) saturated with liquid minimal me-
dia (5 mM potassium nitrate, 2.5 mM potassiumphosphate (pH 5.5), 2 mM magnesium sulfate, 2 mMcalcium nitrate, 50 �M iron-EDTA, 70 �M boricacid, 14 �M manganese chloride, 0.5 �M copper sul-fate, 1 �M zinc sulfate, 0.2 �M sodium molybdenate,10 �M sodium chloride, 0.01 �M cobalt chloride).After transplantation, pots were covered with plasticwrap and grown at 21 °C in constant light. Plantswere slowly hardened off over a one week period andfertilized with liquid minimal media every 2–3 weeksas needed.
Tomato and corn seeds were planted directly intosoil (1:1:1, perlite:sphagnum peat moss:vermiculite)saturated with liquid minimal media. Plants weregrown at 21 °C in constant light and fertilized withliquid minimal media as needed. Allium bulbs were“sprouted” partly submerged in liquid minimal mediaover a two week period at 21 °C in constant light.
For expression studies, A. suecica seeds were ger-minated in 250 ml flasks with 50 ml of liquid Mu-rashige-Skoog medium (Sigma Corporation). Theflasks were agitated at � 100 rpm at 23 °C in con-stant light (cool white, fluorescent, � 100�mol/s/m2) in a plant growth chamber (Percival Sci-entific, Model E-30B). Seedlings were collected 5,10, and 15 days after germination and stored frozenat −80 °C until RNA extraction.
DNA extraction
The DNA was extracted from 0.2–0.8 g of plant tis-sue grown 14 to 28 days of age. The tissue was fro-zen overnight at −70 °C and homogenized in an ice-cooled mortar and pestle. The cetyltrimethylammonium bromide (CTAB) method from Keller(1992) was used for DNA extraction.
RNA extraction
Total RNA was extracted from � 0.2 g of A. suecica,liquid-grown plant tissue (5, 10, or 15 day old) usingthe RNeasy RNA extraction kit (Qiagen Corporation).Before extraction, micropestles and all microfugetubes were treated with an 8% solution of RNA Se-cure (Ambion Corporation) for 10 min at 65 °C. RNAsamples were stored as aliquots until analysis.
Polymerase chain reaction
Four primers were constructed to amplify putativehomologs of ILR1 using PCR. Primers used to am-
plify the conserved � 300 bp domain of ILR1 were5�-ATTCATGAGAACCCAGAGACA-3� (ILR1F)and 5�-ACCACAAGCATGCATCTTTC-3� (ILR1R).The primers used to amplify the entire ILR1 homologwere 5�-CACCGTTGTCCTTTCTTTCA-3� (ILRF)and 5�-CAACCCGAAACCTAACCTCA-3� (ILRR).
DNA fragments were amplified in 20 �l reactionscontaining: 10–20 ng plant DNA, 10% ThermoPolbuffer (New England Biolabs, Inc.), 5 pmol forwardand reverse primers, 200 �M dinucleotide triphos-phates (New England Biolabs, Inc.), and 0.5–1.0 unitTaq polymerase (New England Biolabs, Inc.). All am-plification was performed in a Mastercycler gradientthermocycler (Eppendorf, Inc.). The PCR productswere subjected to electrophoresis on 1–3% agarosegels and imaged using an Ultralum gel documentationsystem (Ultralum, Inc.) and Scion computer software(Scion, Inc.).
Cloning of sILR1
The sILR1 gene was isolated by PCR amplificationof genomic DNA using primers ILRF and ILRR withA. suecica DNA and an annealing temperature of 55°C. The resulting fragment was isolated from an aga-rose gel using the Concert Rapid Gel extraction kit(Gibco BRL) and blunt-end ligated into the EcoRVcloning site of the pstBLUE-1 plasmid vector(Novagen Corporation) using T4 DNA ligase(Novagen Corporation). The resulting construct,psILR1, was transformed into E. coli (DH5-�) usingheat-shock (Sambrook et al. 1989). Plasmids wereextracted from putative transformants by alkaline ly-sis (Sambrook et al. 1989), linearized by endonu-clease digestion with HindIII, and subjected to elec-trophoresis on 1% agarose gels to determine plasmidand insert sizes.
RT-PCR
Total RNA from 15 day old A. suecica plants wasused for RT-PCR to isolate an sILR1 cDNA. RT-PCRwas performed in a single tube reaction using a One-Step RT-PCR kit (Qiagen Corporation) with primersILRF and ILRR. The reverse transcriptase reactionwas incubated at 50 °C for 1 h, followed by 95 °Cfor 10 min. The PCR was performed for 40 cycles atthe following times and temperatures: 45 s at 95 °C,45 s at 58 °C, and 1 min at 72 °C, followed by a 10min extension at 72 °C at the end of the RT-PCR pro-gram.
Cloning of sILR1 cDNA
The resulting cDNA was blunt-end ligated, in-frame,into the EcoRV cloning site of the pETBlue-2 expres-sion vector (Novagen Corporation) using T4 DNA li-gase (Novagen Corporation). The resulting construct,pEcsILR1, was then transformed into E. coli (Nov-aBlue) using heat-shock (Sambrook et al. 1989) andputative transformants selected on the basis of ampi-cillin-resistance and blue-white selection (Sambrooket al. 1989). Plasmids were obtained from transfor-mants by alkaline lysis (Sambrook et al. 1989) andinsert orientation and size determined by digestionwith PvuII or EagI and electrophoresis through 1%agarose gels.
DNA Sequencing
DNA sequencing reactions were performed onpsILR1 and pEcsILR1 using dye terminator chemis-try and an Applied Biosystems 377XL sequencer (Ap-plied Biosystems, Inc.) with both T7 and SP6 primers(New England Biolabs), as well as internal primers5�-AAACATTCACTTTACCTCTTT-3� and 5�-ATG-GTGGCTATGCTTCTTGG-3� for psILR1 and 5�-CATGTCTTTCCATCGATCCC-3� for pEcsILR1.
The consensus DNA sequences for the multiplesequencing runs were determined using the BaylorCollege of Medicine (BCM) Search Launcher soft-ware (Smith et al. 1996), and the protein sequencealignments were made using BCM protein alignmentsoftware with the PIMA method (Smith and Smith1992).
The genomic and cDNA consensus sequenceswere determined on both strands, and the sequencesdeposited in GenBank (accession numbers AF385367and AF468012, respectively).
Results
Amplification of a conserved ILR1 domain
Primers were designed to a � 300 bp region betweenbases 255 and 562 of A. thaliana ILR1 that is con-served in both A. thaliana and bacterial hydrolases(Bartel and Fink 1995; Davies et al. 1999; Lasswellet al. 2000). The ILR1R and ILR1F primers were de-signed to incorporate this conserved region.
DNA was extracted from seven Brassicaceae spe-cies, chosen on the basis of phylogenetic similarity to
A. thaliana. These include A. suecica, A. pumila, A.himalaica, A. griffıthiana, A. wallichii, Capsella bur-sa-pastoris and Draba nemoroza. Additionally, to-mato and two monocots, Zea mays and Allium cepa,were examined to determine the extent of sequenceconservation. A band of the expected size, � 300 bp,was observed in each of the species examined by PCRusing primers (ILR1F and ILR1R) to the conservedregion of the A. thaliana hydrolase (Figure 1).
Isolation and characterization of sILR1 genomichomolog
To clone the entire ILR1 coding region from each ofthese members of the Brassicaceae, primers were de-signed to sequences in the 5� and 3� untranslated re-gions (UTR) of the A. thaliana ILR1. Use of theseprimers resulted in amplification of a product onlywith the A. thaliana control and A. suecica DNA (datanot shown).
Sequencing of the putative ILR1 genomic clonefrom A. suecica revealed that it was an ILR1 homologwith five exons and four introns. The homologous A.thaliana gene has a similar structure (Davies et al.1999). DNA sequence homology between the sILR1and ILR1 was 98%.
Isolation and characterization of the sILR1 cDNAhomolog
Sequence analysis of pEcsILR1 revealed that sILR1and ILR1 were 98% identical at both the cDNA andamino acid levels (Figure 2). The other A. thalianaILR1-like proteins all have much lower sequence
identity to sILR1, ranging in value from 46% identityfor ILL5 to 50% for ILL2. Both ILL3 and IAR3 re-tain a 49% identity with sILR1, and ILL1 falls be-tween with a value of 47% (data not shown).
Preliminary experiments were performed to exam-ine sILR1 expression. Total RNA was extracted fromA. suecica seedlings grown in liquid culture 5, 10, and15 days of age, and used for RT-PCR analysis. ILR1appears to be expressed in all three stages of growth(Figure 3). Differences in band size and brightnessbetween the 5-day-old tissue and the others is prob-ably due to differences in total RNA utilized in theamplification reaction and not differences in transcriptexpression.
Discussion
A family of ILR1-like hydrolases
We have isolated a homolog of the ILR1-like familyfrom A. suecica. The sILR1 gene sequence resemblesthe primary sequence of ILR1 to a high degree, andthe predicted sILR1 protein also closely resembles theILR1 gene product. However, there are six predictedamino acid (aa) differences between ILR1 and sILR1at aa sites 6, 103, 132, 143, 227 and 436. An analysisof conserved protein domains in sILR1 (Altschul etal. 1997) suggests that it is a member of the Pepti-dase M20 family with a predicted hydrolase domainbetween aa sites 60 and 316. A GenBank search ofthis domain revealed over 150 homologous sequencesin both eucaryotes and procaryotes. Four of the aminoacid differences between sILR1 and ILR1 reside inthis conserved domain.
The sILR1 gene appeared initially to be widelyconserved since the 300 bp domain amplified byprimers ILR1F/ILR1R was present among all the spe-cies examined in this study. Despite the high conser-vation of this region, even among monocots, only theA. suecica allowed PCR amplification of a completeILR1 homolog sequence. This result may indicate thatalthough the 300 bp domain is conserved, the ILR1gene itself may not be widely conserved.
There are two possible reasons for a lack of PCRamplification with ILRF/ILRR UTR primers. TheUTR sequences flanking the gene may have mutatedover evolutionary time leaving the ILR1 gene present,but not detectable by PCR with the primers em-ployed. Alternatively, the ILR1 gene sequence may
Figure 1. PCR products of DNA templates using ILR1F/ILR1Rprimers to amplify the conserved 300 bp domain of ILR1. Lane 1:molecular weight markers (Hi-Lo Marker, Minnesota Molecular),size indicated in basepairs. Lane 2: A. thaliana control. Lane 3: D.nemoroza. Lane 4: C. bursa-pastoris. Lane 5: A. himalaica. Lane6: A. griffıthiana. Lane 7: A. suecica. Lane 8: A. wallichii. Lane 9:A. pumila. Lane 10: Lycopersicon esculentum. Lane 11: Zea mays.Lane 12: Allium cepa. 3% agarose gel stained with ethidium bro-mide.
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no longer be in the other species at all due to evolu-tionary divergence.
Arabidopsis suecica is very closely related to A.thaliana, and is believed to be derived through inter-species hybridization from A. thaliana (O’Kane et al.1996). Despite the phylogenetic similarity of the twospecies, it was surprising that the 5� and 3� UTRs ofILR1 and sILR1 were conserved at 98% and 97%,respectively, since these regions tend to have a highlikelihood of sequence divergence. Within the ILR1-like family of A. thaliana, only the 5� and 3� UTRsof ILL1 and ILL2 show any identity, 91% and 83%,respectively. BLAST analysis indicates that all otherILR1-like UTRs show “no significant similarity” toeach other. Given that the ILR1 and sILR1 UTRswere so similar, this lends more importance to thedifferences in the coding region.
Enzymatic activity in sILR1
Four of the predicted amino acid differences [aa 103(Cys>Gly), aa 132 (Asp>His), aa 143 (Tyr>His) andaa 227 (His>Tyr)] between ILR1 and sILR1 occur inthe putative peptidase domain (Figure 2). The aminoacids at sites 103 and 143 are conserved betweenIAR3 and sILR1, as well as ILL1, ILL2, ILL3 and
Figure 2. Predicted amino acid alignment of IAA-conjugate hydrolases ILR1, sILR1 and IAR3. Protein sequences were aligned with theBCM Search Launcher by using the PIMA method (Smith and Smith 1992). Amino acids in bold are those that either differ between ILR1and sILR1 or remain conserved between sILR1 and IAR3. The underlining indicates where the conserved 300 bp region encodes.
Figure 3. Steady state expression of sILR1 transcript in variousages of A. suecica whole seedlings. Coupled one-step RT-PCR am-plification of sILR1 cDNA from A. suecica total RNA. Lanes 1 and5 are molecular weight standards (Hi-Lo Marker, Minnesota Mo-lecular). Lane 2: 5 day old tissue. Lane 3: 10 day old tissue. Lane4: 15 day old tissue. This is a 0.7% agarose gel stained with ethid-ium bromide.
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ILL5 (data not shown). This conservation may indi-cate that sILR1 is functionally more similar to theseother hydrolases than to ILR1. Alternatively, sILR1may also differ functionally from the other ILR1 fam-ily members since the unconserved, predictedchanges at aa 132 (Asp>His) and aa 227 (His>Tyr) insILR1 may alter enzymatic activity or substrate spec-ificity. All these changes make it difficult to anticipatehow the enzymatic activity of sILR1 may differ.
NNPREDICT (Kneller et al. 1990) can predict thedifferences in protein secondary structure betweengiven sequences. Using NNPREDICT, we found thatfour major putative alterations occur between sILR1and ILR1 structures (Figure 4). Three of the predictedchanges occur in the presumptive peptidase region ofsILR1. Three residues (107–109) are changed fromturn elements to beta strand elements, while two oth-ers (130–131) become alpha helices. A third structuralalteration (227) lengthens an already present betastrand. The fourth change (436 and 439) occurs at thecarboxyl end of the protein, elongating an alpha helixnear the predicted endoplasmic reticulum localizationsignal (Davies et al. 1999) and potentially affectingcellular localization of sILR1.
Most likely the sILR1 gene was mutated in recentevolutionary history, since A. thaliana hybridized toArabidopsis arenosa (O’Kane et al. 1996), but thistells us nothing about whether the sILR1’s presentactivity is utilized and required by A. suecica. All theamino acid alterations in sILR1 arose from singlebasepair mutations, so changes in the protein are dueto minor sequence alterations between the species.Further analysis of ILR1 homologs in other speciesmay help to determine how much selection pressurethere is to maintain the function of this hydrolase.
We are presently examining expression of thesILR1 protein itself and its enzymatic activity. If theA. suecica sILR1 gene actually arose from the ILR1gene of A. thaliana in an interspecies hybridization,it may give us a unique opportunity to observe mo-lecular evolution’s direct effect on enzymatic activityand function. Our further analysis of sILR1 gene ex-pression and protein product will provide a morecomplete picture of IAA-conjugate function and me-tabolism.
Figure 4. Secondary structure comparison between sILR1 and ILR1 using NNPREDICT (Kneller et al. 1990). Periods (.) indicate structuralhomology between the sequences. “H’s” indicate alpha helices. “E’s” indicate beta strand elements. Dashes (−) indicate turn elements. Boldletters indicate structural differences.
The authors wish to gratefully acknowledge the helpand suggestions of John Smalley and Quinn Vega. Wethank Lisa Campanella for her great help in editing.We also wish to thank the Arabidopsis Biological Re-source Center at Ohio State University for their quickresponse to our seed requests. Additionally, we wouldlike to thank Nobuharu Goto for his kind donation ofseed and Allyson Hubers of the Novagen Corporationfor her technical expertise. This work was supportedprimarily by a Sokol grant for undergraduate researchfrom Montclair State University.
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