JXB Advance Access originally published online on June 18, 2003
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Journal of Experimental Botany, Vol. 54, No. 389, pp. 1997-1999,
August 1, 2003
© 2003 Oxford University Press
Cloning and expression of a UDP-glucuronic acid decarboxylase gene in rice
Received 20 January 2003; Accepted 29 April 2003
1 Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Gakuen-cho 1-1, Sakai, Osaka 599-8531, Japan
2 Division of Pathology, Osaka Prefectural Institute of Public Health, Nakamichi 1-3-69, Higashinari-ku, Osaka, Osaka 537-0025, Japan
* To whom correspondence should be addressed. Fax: +81 72 254 9458. E-mail: skita{at}biochem.osakafu-u.ac.jp
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Abstract |
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A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-D-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-D-glucuronic acid to UDP-D-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots.
Key words: Endosperm, gene expression, Oryza sativa cv. Nipponbare (monocots), UDP-glucuronic acid decarboxylase (UXS).
UDP-xylose is an important sugar donor in the synthesis of hemicellulose and glycoproteins. Arabinoxylans in crop plants such as rice, maize, wheat, and barley are the major components of the cell wall of the starchy endosperm, as well as the aleurone layer (Izydorczyk and Bilizderis, 1995). Therefore, an investigation of UDP-xylose biosynthesis in seeds of crop plants is appropriate, since current knowledge of its production is limited. Starchy endosperm is produced in limited amounts in the seeds of Arabidopsis and, furthermore, the composition of the primary cell wall in graminaceous plants is different from that in typical dicots. In graminaceous plants, much more arabinoxylan than xyloglucan is produced in the primary wall. UDP-xylose is not only used as a substrate for xyloglucan biosynthesis (Faik et al., 2002), but also as substrate of ß-(1,4)-xylosyltransferase that catalyses the synthesis of the xylan backbone (Bailey and Hassid, 1966). In addition, the use of UDP-xylose for protein glycosylation is specific in gramineous plants. Therefore, the identification of UDP-D-glucuronic acid decarboxylase (UXS), an enzyme which is responsible for the synthesis of UDP-xylose is important.
Since the identification of a uxs gene in Filobasidiella, the fungi, was reported (Bar-Peled et al., 2001), this gene has been identified in other species as well. The uxs genes have been reported in rat of the mammalia (Moriarity et al., 2002) and in typical dicot plants, such as the pea (Kobayashi et al., 2002) and Arabidopsis (Harper and Bar-Peled, 2002). Initially an attempt was made to predict the coding for the DNA sequences of the uxs genes in Oryza sativa cv. Nipponbare by comparing rice genome sequences with sequences in the species described above using the BLAST program (Altschul et al., 1990) and by searching for open reading frame of genes using the GENSCAN program (Burge and Karlin, 1997). As a result, two candidates for uxs genes were predicted. To obtain cDNA of the uxs genes, mRNA was isolated from immature rice seeds 14 d post-anthesis, and an attempt was then made to amplify candidate genes using the RT-PCR method. Two clones containing open reading frames of 1233 and 1053 bp were successfully obtained (accession nos AB079063 [GenBank] and AB079064 [GenBank] , respectively). The clone of AB079064 [GenBank] was used for further expression analysis, since the deduced amino acid sequence of the AB079064 [GenBank] clone was more similar to the sequences identified in both pea (BAB40967 [GenBank] ) and Arabidopsis (NP_200737 [GenBank] as AtUXS3, NP_190228 [GenBank] as AtUXS5, NP_180443 [GenBank] as AtUXS6). The identity between rice (AB079064 [GenBank] ) and Arabidopsis is 85–88% and that between rice and pea is 85% (Fig. 1A). Meanwhile, the deduced amino acid sequence of the AB079063 [GenBank] clone was most similar to the sequences identified in Arabidopsis (NP_190920 [GenBank] as AtUXS1) and the identity was 71%. In the question sequence, the AB079064 [GenBank] clone was predicted to be a soluble protein by the SOSUI program (http://sosui.proteome.bio. tuat.ac.jp/sosuiframe0.html). The phylogenetic relationship of soluble UXS proteins from various taxa is shown in Fig. 1B. The UXS of rice formed a cluster with the UXSs of pea and Arabidopsis, and reed and chick-pea homologues. However, the similarity between Arabidopsis and pea is higher than that between rice and dicots. These seven proteins were subdivided between graminaceous plants (rice and reed) and dicots (pea, chick-pea and Arabidopsis).
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The ORF region of the rice uxs gene was inserted into a pET expression vector 20b (Novagen), and the resulting plasmid was then introduced into bacterial host strain BL21 (DE3) pLysS (Novagen). After a 3 h exposure to isopropyl thio-ß-D-galactoside (IPTG) at a final concentration of 1.0 mM at 37 °C, the bacterial cells were harvested, and then solubilized by sonication. An approximately 40 kDa band in crude extract that had been induced by the addition of 1 mM IPTG was detected (Fig. 2A). This band was not detected in control specimens in the absence of IPTG induction. The supernatant from the crude extract after centrifugation in 9000 g for 15 min was obtained. When the supernatant was applied to SDS-PAGE, the dense, 40 kDa band was observed, indicating that the protein is soluble, and is not formed as an inclusion body. In addition, it was confirmed that a portion of the amino acid sequences of the 40 kDa protein was identical to the deduced sequences. When the enzymatic reaction of the soluble crude extract (10 mg ml–1) was investigated at 23 °C for 15 min using 1 mM UDP-glucuronic acid with 40 mM TRIS-HCl (pH 7.5), UDP-xylose was found in the reaction mixture by HPLC analysis using a DEAE-2SW column (Tosoh). After a 15 min reaction, the ratio between the synthesized UDP-xylose and UDP-glucuronic acid approached 1:1 (Fig. 2B). This reaction was similar to the case of 1 mM nicotinamide adenine dinucleotide. UDP-xylose was identified by comparison of its HPLC peak with that of any authentic standard. After enzyme reaction with 1 mM UDP-glucuronic acid, a solution of phenol and chloroform (1:1, v/v) was added to the sample followed by mixing. The sample was subsequently neutralized with HCl and centrifuged at 22 000 g for 15 min at 4 °C, and the supernatant was collected. Large amounts of protein were removed by passing the sample through a Toyopearl CM-Sepharose (Tosoh) column with 0.1 M TRIS-HCl buffer (pH 6.0), and the void fractions were collected. The sample was fractionated using a Toyopearl Super-Q 650M (Tosoh) column with a gradient of 0.05–0.8 mM sodium dihydrogen phosphate solution. After HPLC analysis of the fractions, only the fractions containing a peak having the same retention time as standard UDP-xylose were collected, desalted using Bio-gel P-2 column (Bio-Rad) and lyophilized. The chemical shifts of the sample dissolved in D2O by 500 MHz 1H-NMR spectroscopy (JNM-A500, JEOL) were identical to those of standard UDP-xylose.
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The expression of the uxs gene by a non-quantitative RT-PCR method using primers specific to the uxs was also examined. The findings show that the uxs gene is expressed not only in 14 d after flowering, but in harvested rice seeds as well. Therefore, mRNA of the uxs was maintained in the rice grains even after cell wall formation had ceased. The uxs gene would be necessary at the beginning of the germination period, since it is present in young leaves and roots 5 d after sowing.
A study of the putative membrane-bound uxs gene (AB079063 [GenBank] ) is currently underway. Differences in the localization and function of soluble vis-à-vis putative membrane-bound UXSs will be needed to develop a better understanding of the role of these enzymes in plant growth and development.
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Acknowledgements |
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We thank Professor Y Tsumuraya (Saitama University) for generously providing a sample of UDP-xylose. This work was supported, in part, by Research Fellowships from the Japanese Society for the Promotion of Science for Young Scientists to KS (No. 07529).
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