JXB Advance Access originally published online on March 12, 2004
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Journal of Experimental Botany, Vol. 55, No. 399, pp. 1153-1155, May 1, 2004
© 2004 Oxford University Press
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Molecular cloning of low-temperature-inducible ribosomal proteins from soybean
Received 17 November 2003; Accepted 2 February 2004
1 Department of Plant Biotechnology, Dong-A University, Busan, 604-714, South Korea
2 School of Food and Life Science, Biohealth Products Research Centre, Inje University, Gimhae 621-749, South Korea
* To whom correspondence should be addressed. Fax: +82–51–200–6536. E-mail: jhnlee{at}donga.ac.kr
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Abstract |
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Three ribosomal protein genes induced by low-temperature treatment were isolated from soybean. GmRPS13 (742 bp) encodes a 17.1 kDa protein which has 95% identity with the 40S ribosomal protein S13 of Panax ginseng (AB043974 [GenBank] ). GmRPS6 (925 bp) encodes a 28.1 kDa protein which has 94% identity with the 40S ribosomal protein S6 of Asparagus officinalis (AJ277533 [GenBank] ). GmRPL37 (494 bp) encodes a 10.7 kDa protein which has 85% identity with the 60S ribosomal protein L37 of Arabidopsis thaliana (AF370216 [GenBank] ). The expression of these ribosomal protein genes started to increase 3 d after low-temperature treatment, whereas the cold-stress protein src1 was highly induced from the first day. Such late response of these ribosomal protein genes may be due to secondary signals during cold adaptation. The induction of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under low-temperature conditions.
Key words: Ribosomal gene, soybean, suppression subtractive hybridization.
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Low temperature is a major environmental factor that greatly influences plant growth, development, and crop yield. Most plants from temperate regions can acclimate to cold and develop freezing tolerance (Guy, 1990). Cold acclimation is a complex and global process involving many physiological and biochemical changes, such as alteration in lipid, protein, and carbohydrates composition (Bohnert et al., 1995; Somerville, 1995). To acquire freezing tolerance, changes in gene expression and de novo protein synthesis are required during cold acclimation (Guy, 1990; Thomashow, 1998). Specific signal transduction pathways are involved in cold acclimation and activation of many cold-regulated (COR) genes (Thomashow, 1998).
In Arabidopsis plants, CRT/DRE binding factors (CBF) are known to have a crucial role in freezing tolerance and to regulate the transcription of many cold-regulated (COR) genes (Jaglo-Ottosen et al., 1998; Gilmour et al., 2000). Low temperature induces rapid and transient expression of CBF1, CBF2, and CBF3 (Gilmour et al., 1998). Ectopic expression of CBF proteins in plants enhances freezing tolerance and leads to constitutive expression of COR genes in non-acclimated, transgenic Arabidopsis plants (Jaglo-Ottosen et al., 1998; Gilmour et al., 2000). ESK1 in Arabidopsis plants has been suggested to be involved in the CBF-independent pathway (Xin and Browse, 1998). Esk1 mutants are constitutively freezing-tolerant and accumulate high levels of proline. The mutation alters the transcript levels of genes involved in proline biosynthesis and degradation, but does not affect the expression of several COR genes.
De novo protein synthesis is necessary for cold response and the integrity of the translation machinery is an important factor of cold acclimation. It has been reported that several cold-sensitive mutants in yeast are defective in the assembly of ribosomal subunits (Hartwell et al., 1970; Bayliss and Ingraham, 1974). E. coli CsdA is a ribosomal-associated protein at low temperature and has helix-destabilizing activity (Jones et al., 1996). It was proposed that CspA plays an essential role in mRNA translation at low temperature in order to facilitate ribosomal function by unwinding stable secondary structures in mRNAs. The Arabidopsis los1-1 mutant is impaired in cold acclimation and defective in protein synthesis in the cold (Guo et al., 2002). This mutation specifically blocks low-temperature-induced transcription of cold-responsive genes, while it causes super-induction of CBF/DREB1 transcription factor genes. The LOS1 gene is found to encode translation elongation factor 2 and a single amino acid change in the LOS1 protein causes the incorrect functioning in the cold.
Three genes encoding different ribosomal proteins were isolated from soybean (Glycine max cv. Sinpaldal2), which were induced by low-temperature stress. Low-temperature-induced cDNAs were cloned using the suppression subtractive hybridization (SSH) method. Among SSH-enriched cDNA clones, three different sequences, which showed high homology with known ribosomal protein genes, were identified. Soybean Low Temperature Inducible clones, SLTI25, SLTI98, and SLTI698 had homology with cytoplasmic ribosomal protein S13, S7, and L37, respectively. The full-length cDNAs were obtained by RACE-PCR from low-temperature-treated RNA, and designated GmRPS13 (SLTI25), GmRPS6 (SLTI98), and GmRPL37 (SLTI698).
Three ribosomal protein genes are listed in Table 1. GmRPS13 is 742 bp in length and encodes a 17.1 kDa protein which has 95% identity with the 40S ribosomal protein S13 of Panax ginseng (AB043974 [GenBank] ). The relationship between cold hardiness and the expression of ribosomal protein S7 has been studied in winter rye (Berberich et al., 2000). Short periods of cold stress sharply reduced the mRNA level while leaves of cold-hardened plants recovered the normal level of the transcripts. GmRPS6 is 925 bp in length and encodes a 28.1 kDa protein which has 94% identity with the 40S ribosomal protein S6 of Asparagus officinalis (AJ277533 [GenBank] ). Ribosomal protein S6 is located in the mRNA binding site of the 40S subunit of cytosolic ribosomes and is the major phosphoprotein of eukaryotic ribosomes (Nygärd and Nilson, 1990). The accumulation of phosphorylated RPS6 in root tips of maize was reduced in response to oxygen deprivation and heat shock, but elevated by cold stress (Williams et al., 2003). GmRPL37 is 494 bp in length and encodes a 10.7 kDa protein which has 85% identity with the 60S ribosomal protein L37 of Arabidopsis thaliana (AF370216 [GenBank] ). A yeast mutant that was defective in two rpl37 loci was unable to grow at all, indicating that RPL37 is an essential protein (Tornow and Santangelo, 1994).
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The expression of GmRPS6, GmRPS13, and GmRPL37 during low-temperature stress is shown in Fig. 1. These genes did not function during the early period of low-temperature treatment, but increased after 3 d, whereas soybean cold-stress protein src1 (Takahashi and Shimosaka, 1997) was highly induced from the first day and the strong expression level was sustained to 9 d. Such a late response of these ribosomal protein genes may be due to secondary signals during cold adaptation. The expression of src1 precedes those of the ribosomal protein genes, so the regulation pathways are likely to be different.
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The induction of ribosomal protein genes might enhance the translation process or help the proper ribosome assembly and functioning under low-temperature conditions. Recently, Brassica napus BnC24, which is induced by cold treatment and shares homology with human BBC1 (Breast Basic Conserved) protein, has been identified as ribosomal protein L13 (Sáez-Vásquez et al., 2000). Over-expression of an algal bbc1 gene, a homologue of ribosomal protein L13 increased salt and freezing tolerance in E. coli cells (Tanaka et al., 2001). Low temperature stabilizes the secondary structure of mRNA and also affects the ribosome attachment and elongation processes. Ribosomal protein S6 is located in the mRNA binding site of the 40S subunit of cytosolic ribosomes (Nygärd and Nilson, 1990), so the increased synthesis of GmRPS6 protein might enhance the translation initiation of cold-stabilized mRNA. Thus, the translation machinery may also need cold acclimation via de novo synthesis of its components such as ribosomal proteins so that it can function well under low-temperature stress.
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Acknowledgements |
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This research was partially supported by grant number R01-2000-000-00148-0 from KOSEF, and by grant number CG2122 from the Crop Functional Genomics Center of 21C New Frontier Project in Korea.
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