Journal of Experimental Botany, Vol. 53, No. 379, pp. 2453-2454,
December 1, 2002
© 2002 Oxford University Press
Isolation and expression analysis of the isopropylmalate synthase gene family of Arabidopsis thaliana
Received 29 July 2002; Accepted 21 August 2002
Department of Biology, Indiana University-Purdue University Fort Wayne, 2101 E. Coliseum Blvd., Fort Wayne, IN 46805-1499, USA
1 To whom correspondence should be addressed. Fax: +1 219 481 6087. E-mail: mourad{at}ipfw.edu
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Abstract |
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Isopropylmalate synthase (IPMS) is the first enzyme in the leucine biosynthetic pathway. It is the branch point in the biosynthesis of leucine and the other branched-chain amino acids. IPMS is also regulated by negative feedback inhibition by the end-product leucine. There are four highly homologous loci within the Arabidopsis thaliana genome, which contain sequences that code for IPMS. Through library screening and RT-PCR the expression patterns of three of these loci namely IMS1, IMS2, and IMS3 have been isolated and then characterized. cDNAs of IMS2 and IMS3 lacking the 5' chloroplast leader sequence were able to complement a leucine auxotroph of E. coli.
Key words: Arabidopsis thaliana, functional complementation, isopropylmalate synthase (EC 4.1.3.12), leucine biosynthesis, ribonuclease protection assay.
In Arabidopsis thaliana, the sequences and the kinetic parameters of the branched-chain amino acid regulatory enzymes, acetolactate synthase and threonine dehydratase/deaminase, responsible for the biosynthesis of valine and isoleucine are known (Mazur et al., 1987; Mourad et al., 1995, 1998, 2000). However, the sequences coding for isopropylmalate synthase (IPMS), the regulatory enzyme of the leucine branch of the branched-chain amino acid biosynthetic pathway, have not been isolated before. IPMS converts 2-ketoisovalerate into 2-isopropylmalate and forms the junction where leucine biosynthesis branches from valine biosynthesis. Since IPMS has a regulatory role at a branch site of amino acid synthesis and it functions similarly across many taxa, it has maintained certain consensus regions that are needed for its activity and the control of the leucine biosynthetic pathway. The goal of this study was 2-fold. First, to isolate members of the gene family encoding A. thaliana IPMS using consensus data accumulated from amino acid alignments in a reverse genetic approach. Second, to characterize the expression patterns of the different members of the IPMS gene family in multiple tissues and organs of A. thaliana.
A search of GenBank at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) was conducted to identify as many known IPMS amino acid sequences as possible. These sequences were aligned using MegAlign (DNASTAR Madison, Wisconsin). The conserved amino acid sequences elucidated by this amino acid alignment search were used to search all deposited A. thaliana sequences using tfastx3 software (Pearson and Lipman, 1988) at TAIR (The Arabidopsis Information Resource: www. arabidopsis.org). The first exons of the predicted coding sequence from bacterial artificial chromosome (BAC) clones F15H18 and T20O7 (Accession Nos AC013354 and AB026660, respectively) were used to prepare probes by PCR from A. thaliana genomic DNA. Probe 1, from BAC F15H18, was used to screen the A. thaliana library CD4-15 (2–3 kb) (Keiber et al., 1993) and probe 5, from BAC T20O7, was used to screen the A. thaliana library CD4-14 (1–2 kb) ( Keiber et al., 1993). Both cDNA libraries were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University. The libraries are both 
Zap II cDNA expression libraries consisting of cDNA generated from 3-d-old A. thaliana seedling hypocotyls using oligo d(T) as primer. Positive plaques isolated from the tertiary round of screening were excised using the ExAssistTM protocol (Stratagene, California) to isolate the cDNA inserts in pBluescript®. Putative positive plasmids from each library were purified then sent out for automated sequencing at Indiana University medical school. The DNA sequences were analysed, translated, and aligned with the sequences from the original alignment using the software package DNASTAR (Madison, Wisconsin).
A single clone designated IPMS1-200-C-1 was isolated from the CD4-15 library. The nucleotide sequence of this clone was used in a BLAST search of GenBank and was found to be identical to BAC F2P9 (Table 1). Also, a single clone designated IPMS5-200-A-1 was isolated from the CD4-14 library. A BLAST search of GenBank revealed that this clone was identical to BAC MYJ24 (Table 1). Since library screening resulted in the isolation of two unexpected clones, the predicted coding regions from BAC F15H18 and BAC T20O7 were then used to design primers for use in RT-PCR to generate the remaining two cDNA clones. RT-PCR produced the desired 1521 bp fragment from primers designed to amplify the entire coding region of BAC T20O7. Sequencing verified that the fragment was indeed the full-length coding region represented by BAC T20O7, and the clone was identified as IPMS5-600 (Table 1). Attempts to generate the remaining clone by RT-PCR have been unsuccessful with RNA isolated from a variety of tissues at different developmental stages. The two clones isolated by library screening and the single clone isolated by RT-PCR have been fully sequenced and deposited in GenBank: IPMS1-200-C-1 Accession No. AF327647 (IMS1), IPMS5-200-A-1 Accession No. AF327648 (IMS2), and IPMS5-600 Accession No. AY049037 (IMS3) (Table 1).
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Using the pTrc99A (Amann et al., 1988) expression vector (Pharmacia, New Jersey), constructs were designed to express truncated versions (minus the chloroplast leader sequence at the 5' end) of the isolated cDNA sequences in the leucine-requiring E. coli strain CV512 lacking IPMS activity (Somers et al., 1973) and obtained from the Coli Genetic Stock Center (CGSC). Such truncation constructs of IMS2 and IMS3 were able to complement the E. coli leucine auxotroph and transformed cells grew in minimal medium without leucine supplementation (data not shown). These results confirmed that the truncated A. thaliana sequences are indeed expressing IPMS activity.
Ribonuclease protection assays were conducted to elucidate the expression patterns of the A. thaliana IPMS gene family. Radiolabelled antisense probes were produced using [
-32P] UTP and the MAXIscriptTM T7 Kit (Ambion, Texas). Total RNA was isolated from each tissue of A. thaliana Columbia wild type using the Plant RNA isolation aid kit of Ambion and RNeasy® Plant Mini Kit (Qiagen, California). RNA from flower and stem tissue was isolated from 1-month-old plants harbouring mature siliques. Leaf and root tissues for RNA extraction were harvested from rosette plants at the emergence of the sixth pair of leaves. The nuclease protection assay was conducted using the RPA IIITM kit (Ambion, Texas) and protected RNA fragments were electrophoresed in 6% TBE-Urea gels (Invitrogen, California) with RNA CenturyTM Marker of Ambion. IMS1 transcripts were found in very small amounts in roots, leaves, stems, and flowers. IMS2 transcripts were detected in higher amounts in roots, leaves, and stems. It was also expressed in very small amounts in flowers. IMS3 transcripts were also expressed at higher levels in roots, leaves, and stems, but IMS3 was not expressed in flowers (Fig. 1). The predicted coding sequence from BAC F15H18 was not identified in any tissue assayed.
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Full length cDNAs have been isolated for three out of four putative sequences coding for IPMS in A. thaliana. All three isolated clones and the predicted coding region of the fourth locus contain properties, both at the nucleotide and amino acid level, consistent with IPMS from other organisms. The ability of IMS2 and IMS3 sequences to revert a leucine-requiring E. coli auxotrophic strain CV512 deficient in IPMS activity to prototrophy, reinforces the identity of the sequences isolated. Expression analysis revealed that all three isolated clones are transcribed in a multitude of tissues (Fig. 1).
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Acknowledgement |
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This work was supported by a grant from Purdue Research Foundation to GM.
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