JXB Advance Access originally published online on May 13, 2003
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Journal of Experimental Botany, Vol. 54, No. 388, pp. 1785-1787,
July 1, 2003
© 2003 Oxford University Press
Arabidopsis 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate synthase: cDNA cloning and expression analyses*
Received 10 December 2002; Accepted 24 March 2003
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1 Laboratory of Plant Nutrition, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan
2 Graduate School of Agriculture and Biosciences, Osaka Prefecture University, Sakai, 599-8531, Japan
* The nucleotide sequence reported in this paper has been submitted to DDBJ under accession number AB059683. To whom correspondence should be addressed. Fax: +81 75 753 6128. E-mail: matoh{at}kais.kyoto-u.ac.jp
Abbreviations: EST, expressed sequence tag; KDO, 3-deoxy-D-manno-oct-2-ulosonic acid; KDOS, KDO-8 phosphate synthase, RT, reverse transcription.
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The molecular characterization of two isoforms of 3-deoxy-D-manno-oct-2-ulosonate (KDO) -8-phosphate synthase (AtkdsA1 and AtkdsA2) from Arabidopsis is reported here. First, by isolating a full-length cDNA for AtkdsA1, it was confirmed that the deduced primary structures of AtkdsA1 and AtkdsA2 proteins were 93% identical. Functional expression and purification studies demonstrated the efficient catalytic activity of the AtkdsA1 enzyme to produce KDO-8-phosphate from phosphoenolpyruvate and D-arabinose-5-phosphate. RT-PCR and RNA-gel blot analysis revealed different expression profiles for both genes; the AtkdsA1 gene was predominantly expressed in the shoots, while the AtkdsA2 transcript accumulated to a higher level in the roots, implicating differential roles of these isoforms in planta.
Key words: Arabidopsis thaliana, 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate, 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate synthase, rhamnogalacturonan II.
3-Deoxy-D-manno-oct-2-ulosonic acid (KDO), one of the 2-keto-3-deoxysugars, is produced in gram-negative bacteria through the activity of KDO-8-phosphate synthase (KDOS; EC 4.1.2.16 [EC] ) catalysing the aldol condensation reaction to yield KDO-8-phosphate from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (Levin and Racker, 1959) and constitutes a link between lipid A and the O-antigen as an essential component of the lipopolysaccharides (Belunis et al., 1995; Ellwood, 1970; Rick and Osborn, 1977). The gene encoding bacterial KDOS, kdsA, has been isolated from E. coli (Woisetscheläger and Högenauer, 1987), Haemophilus influenzae (Fleischmann et al., 1995), and Chlamydia psittaci (Brabetz and Brade, 1997).
In higher plants, KDO is one of the constituent sugars of rhamnogalacturonan II (RG-II) (York et al., 1985), providing the cross-linking site for boric acid along pectic polysaccharide chains (Kobayashi et al., 1996). KDOS cDNA has recently been cloned from Pisum sativum L. (Brabetz et al., 2000) by functional complementation of a kdsA mutant strain of Salmonella enterica, and a homologue of bacterial KDOS has also been isolated from tomato (Lycopersicon esculentum, Delmas et al., unpublished results, accession number AJ294902 [GenBank] ). In this article, evidence is presented that two KDOS isoforms (AtkdsA1 and AtkdsA2) in Arabidopsis could be involved in tissue-specific KDO biosynthesis in terms of differential regulation of gene expression.
By performing TBLASTN searches (Altschul et al., 1990) with the sequence of KDOS protein from E. coli (CAA29067 [GenBank] ), two putative genes (At1g79500 and At1g16340) were found at the Arabidopsis Information Resource (http://www.arabidopsis.org/). As the sequence information of Pisum sativum L. (Brabetz et al., 2000) was not available when this work was begun, the E. coli data were used. Two expressed sequence tag (EST) clones, AA067485 [GenBank] and AI100551 [GenBank] , were also identified as being derived from the 5'-end and the 3'-end of the At1g79500 transcript, respectively, and two EST entries of AV524826 [GenBank] and AV518187 [GenBank] were predicted to correspond to the 5'-end and 3'-end of the At1g16340 transcript, respectively. These putative Arabidopsis KDOS genes were designated as AtkdsA1 (At1g79500) and AtkdsA2 (At1g16340).
First, a full-length AtkdsA1 cDNA was isolated by screening an Arabidopsis cDNA library using an oligonucleotide probe obtained by RT (reverse transcription) -PCR using total RNA from 13-d-old Arabidopsis seedlings grown on the MS media (Murashige and Skoog, 1962). The reverse transcription reaction was performed using a primer R1 (5'-GCAGGTTGTTGTAGTGAATGG-3') and Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech), and the first-strand cDNA was then amplified using a primer set of F1 (5'-GCATTCTTATGTCGCCAGAC-3') and R1. An aliquot of the reaction was subsequently subjected to a nested PCR (30 cycles of 30 s at 94 °C, 30 s at 58 °C, and 1 min at 72 °C) using a primer set of F1 and R2 (5'-ATCAGCGACAACAGGACAAT-3') and rTaq DNA polymerase (Toyobo, Osaka, Japan). The 250 bp product was thus amplified and used as the probe to screen a cDNA library from 7-d-old seedlings (Fujimori and Ohta, 1998). After two rounds of screening, a clone was obtained containing an insert of 1128 bp comprising a 20 bp 5' untranslated region (UTR), a 238 bp 3'-UTR, and an open reading frame (ORF) encoding a polypeptide of 270 amino acid residues (accession number AB059683 [GenBank] ). The determined ORF sequence was in perfect agreement with that of At1g79500 (AtKdsA1). The deduced amino acid sequence of AtKdsA1 protein was 93% identical to that of AtKdsA2 protein and shares 89% and 87% identity with the KDOS proteins from pea and tomato, respectively. Also, AtKdsA1 protein shares 43%, 44%, and 40% identity with KDOS of E. coli, H. influenzae, and C. psittaci, respectively.
A recombinant AtKdsA1 protein was expressed in E. coli as a fusion protein with the maltose-binding protein (MBP). The full-length of the AtkdsA1 cDNA was cloned into a plasmid pMAL-c2 (New England Biolabs), and an E. coli strain, BL21, was used as the host for the recombinant protein expression. KDOS activity was determined at 30 °C in 0.15 ml reactions containing 50 mM potassium phosphate buffer (pH 7.5), 100 mM KCl, 0.5 mM dithiothreitol, 3 mM PEP, 3 mM D-arabinose-5-phosphate, and the enzyme protein. The reaction was terminated by adding cold 10% (w/v) trichloroacetic acid and the KDO formed was determined by the periodate-thiobarbituric method (Ray, 1980). KDOS activity of the lysate of the transformed cells increased from 7.6x10–3 units mg–1 protein to 20.1x10–3 units mg–1 protein after induction with isopropyl-ß-D-thiogalactopyranoside (60 µM) for 6 h at 28 °C. To confirm that the recombinant protein has KDOS activity, the fusion protein was purified to homogenity by amylose resin column chromatography according to the manufacturer’s instruction, using a column buffer of 20 mM potassium phosphate, pH 7.5, 200 mM NaCl, 1 mM EDTA, and 5 mM 2-mercaptoethanol. The fusion protein was eluted with 20 mM potassium phosphate, pH 7.5, 5 mM 2-mercaptoethanol and 10 mM maltose. The purified MBP-AtKdsA1 fusion protein exhibited the efficient KDOS activity (827 mU mg–1), which was as high as that from the purified carrot enzyme (495 mU mg–1; Matsuura, 2002). In this experiment, the MBP domain could not be completely removed by the incubation with the Factor Xa protease (New England Biolabs). However, MBP alone, which was split from the fusion protein and purified by Mono-Q (Amersham Biosciences) column chromatography, did not show any KDOS activity, and the KDOS activity observed with the MBP-AtKdsA1 fusion protein was ascribed to the AtKdsA1 domain, confirming that AtKdsA1 is the functional homologue of bacterial KDOS. From the sequence identity of 93% at the amino acid level, it is thought that AtKdsA2 represents the KDOS isozyme in Arabidopsis.
With the aim of clarifying the differential physiological roles, if any, of AtKdsA1 and AtKdsA2, the tissue specificity of the transcript accumulation of the AtkdsA1 and AtkdsA2 genes (Fig. 1) was examined. Probe labelling, hybridization (50 °C) and detection were carried out using a DIG-High Prime DNA Labeling and Detection Kit (Roche Molecular Biochemicals, Mannheim, Germany). Hybridization probes were prepared by PCR. Thus, the 870 bp covering the entire ORF of AtkdsA1 cDNA was amplified using the primers of F1 and R1. This AtkdsA1 ORF probe should hybridize to both AtkdsA1 and AtkdsA2 transcripts, because their ORF sequence share 86% identity at the nucleotide level. On the other hand, a 550 bp fragment from the AtkdsA1 3'-UTR region was also amplified by RT-PCR using a primer set of 5'-TCAGTGATC GCGAGGTTG-3' and 5'-GCATCCAACGCAAATCAAGCAC-3'. In this 3'-UTR region, the sequence similarity was only 45% between AtkdsA1 and AtkdsA2. With the full-length ORF fragment of AtkdsA1 cDNA as the probe, a single hybridization signal of 1.7 kb was detected, and the intensity was notably stronger in the root than in the shoot (Fig. 1, left panel). On the other hand, the hybridization signal visualized with the AtkdsA1 3'-UTR probe was more intense in the shoot than in the roots (Fig. 1, right panel). These results indicated that the signal detected in the roots (Fig. 1, left panel) was primarily attributable to the AtkdsA2 gene transcript, and semi-quantitative RT-PCR analyses have confirmed these observations. As shown in Fig. 2, the PCR fragment derived from AtkdsA1 mRNA was detected only in the shoot, and virtually no PCR amplification was observed in the roots. On the other hand, the PCR amplification reflecting the AtkdsA2 gene expression was detected in both shoots and roots, and the accumulation of the PCR product was slightly higher in the root than in the shoot. These results were consistent with the findings from the northern hybridization analysis (Fig. 1) that the AtkdsA1 gene was mainly transcribed in the shoots while AtkdsA2 was mainly transcribed in the roots, implicating a specific role of AtkdsA1 in the shoots.
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