KOREAN JOURNAL OF CROP SCIENCE (한국작물학회지)

The Korean Society of Crop Science (한국작물학회)
권호 표지

Cloning and Characterization of ${\Delta}^1$-Pyrroline-5-Carboxylate Synthetase Genes and Identification of Point Mutants in Medicago truncatula

  • Published : 2007.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

To tolerate environmentally adverse conditions such as cold, drought, and salinity, plants often synthesize and accumulate proline in cells as compatible osmolytes. ${\Delta}^1$-Pyrroline-5-carboxylate synthetase(P5CS) catalyzes the rate-limiting step of proline biosynthesis from glutamate. Two complete genes, MtP5CS1 and MtP5CS2, were isolated from the model legume Medicago truncatula by cDNA cloning and bacterial artificial chromosome library screening. Nucleotide sequence analysis showed that both genes consisted of 20 exons and 19 introns. Alignment of the predicted amino acid sequences revealed high similarities with P5CS proteins from other plant species. The two MtP5CS genes were expressed in response to high salt and low temperature treatments. Semi-quantitative reverse transcription-polymerase chain reaction showed that MtP5CS1 was expressed earlier than MtP5CS2, indicating differential regulation of the two genes. To evaluate the reverse genetic effects of nucleotide changes on MtP5CS function, a Targeting Induced Local Lesions in Genomes approach was taken. Three mutants each were isolated for MtP5CS1 and MtP5CS2, of which a P5CS2 nonsense mutant carrying a codon change from arginine to stop was expected to bring translation to premature termination. These provide a valuable genetic resource with which to determine the function of the P5CS genes in environmental stress responses of legume crops.

Keywords

References

  1. Armengaud, P., L. Thiery, N. Buhot, G. Grenier-De March, and A. Savoure. 2004. Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features. Physiol. Plant. 120 : 442-450 https://doi.org/10.1111/j.0031-9317.2004.00251.x
  2. Barker, D. G., S. Bianchi, F. Blondon, Y. Dattee, G. Duc, S. Essad, P. Flament, P. Gallusci, G. Genier, P. Guy, X. Muel, J. Tourneur, J. Denarie, and T. Huguet. 1990. Medicago truncatula, a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Plant Mol. Biol. Rep. 8 : 40-49
  3. Baulcombe, D. 2004. RNA silencing in plants. Nature 431 : 356-363 https://doi.org/10.1038/nature02874
  4. Blondon, F., D. Marie, S. Brown, and A. Kondorosi. 1994. Genome size and base composition in Medicago sativa and Medicago truncatula species. Genome 37 : 264-270 https://doi.org/10.1139/g94-037
  5. Bouche, N. and D. Bouchez. 2001. Arabidopsis gene knockout: phenotypes wanted. Curr. Opin. Plant Biol. 4 : 111-117 https://doi.org/10.1016/S1369-5266(00)00145-X
  6. Buchannan, B. B., W. Gruissem, and R. L. Jones. 2000. Biochemistry and Molecular Biology of Plants. American Society of Plant Biologists, pp. 406-409
  7. Choi, H. K., D. Kim, T. Uhm, E. Limpens, H. Lim, P. Kalo, R. V. Penmetsa, A. Seres, O. Kulikova, T. Bisseling, G. B. Kiss, and D. R. Cook. 2004. A sequence-based genetic map of Medicago truncatula and comparison of marker co-linearity with Medicago sativa. Genetics 166 : 1463-1502 https://doi.org/10.1534/genetics.166.3.1463
  8. Comai, L. and S. Henikoff. 2006. TILLING: practical single-nucleotide mutation discovery. Plant J. 45 : 684-694 https://doi.org/10.1111/j.1365-313X.2006.02670.x
  9. Csonka, L. N. and A. D. Hanson. 1991. Prokaryotic osmoregulation: genetics and physiology. Annu. Rev. Microbiol. 45 : 569-606 https://doi.org/10.1146/annurev.mi.45.100191.003033
  10. Deuschle, K., D. Funck, H. Hellmann, K. Daschner, S. Binder, W. B. Frommer. 2001. A nuclear gene encoding mitochondrial delta-pyrroline-5-carboxylate dehydrogenase and its potential role in protection from proline toxicity. Plant J. 27 : 345-356 https://doi.org/10.1046/j.1365-313X.2001.01101.x
  11. Ginzberg, I., H. Stein, Y. Kapulnik, L. Szabados, N. Strizhov, J. Schell, C. Koncz, and A. Zilberstein. 1998. Isolation and characterization of two different cDNAs of $\Delta^{1}$-pyrroline-5-carboxylate synthase in alfalfa transcriptionally induced upon salt stress. Plant Mol. Biol. 38 : 755-764 https://doi.org/10.1023/A:1006015212391
  12. Igarashi, Y., Y. Yoshiba, Y. Sanada, K. Yamaguchi-Shinozaki, K. Wada, and K. Shinozaki. 1997. Characterization of the gene for ${\Delta}^1-pyrroline-5-carboxylate$ synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L. Plant Mol. Biol. 33 : 857-865 https://doi.org/10.1023/A:1005702408601
  13. Kiyosue, T., Y. Yoshiba, K. Yamaguchi-Slnnozaki, and K. Shinozaki. 1996. A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis. Plant Cell 8 : 1323-1335 https://doi.org/10.1105/tpc.8.8.1323
  14. McCallum, C. M., L. Comai, E. A. Greene, and S. Henikoff. 2000. Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol. 123 : 439-442 https://doi.org/10.1104/pp.123.2.439
  15. Nam, Y.-W., R. V. Penmetsa, G. Endre, P. Uribe, D. Kim, and D. R. Cook. 1999. Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones for ethylene responsive genes. Theor. Appl. Genet. 98 : 638-646 https://doi.org/10.1007/s001220051115
  16. Neff, M. M., J. D. Neff, J. Chory, and A. E. Pepper. 1998. dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J. 14 : 387-392 https://doi.org/10.1046/j.1365-313X.1998.00124.x
  17. Park, S.-Y. and Y.-W. Nam. 2006. Construction of bacterial artificial chromosome library containing large BamHI genomic fragments from Medicago truncatula and identification of clones linked to hyper-nodulating genes. J. Microbiol. Biotechnol. 16 : 256-263
  18. Penmetsa, R. V. and D. R. Cook. 1997. A legume ethylene-insensitive mutant hyperinfected by its rhizobial symbiont. Science 275 : 527-530 https://doi.org/10.1126/science.275.5299.527
  19. Perry, J. A., T. L. Wang, T. J. Welham, S. Gardner, J. M. Pike, S. Yoshida, and M. Parniske. 2003. A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol. 131 : 866-871 https://doi.org/10.1104/pp.102.017384
  20. Seki, M., A. Kamei, K. Yamaguchi-Shinozaki, and K. Shinozaki. 2003. Molecular responses to drought, salinity and frost: common and different paths for plant protection. Curr. Opin. Biotechnol. 14 : 194-199 https://doi.org/10.1016/S0958-1669(03)00030-2
  21. Song, K.-H. and Y.-W. Nam. 2005. Genomic organization and differential expression of two polygalacturonase-inhibiting protein genes from Medicago truncatula. J. Plant Biol. 48 : 467-478 https://doi.org/10.1007/BF03030589
  22. Strizhov, N., E. Abraham, L. Okresz, S. Blickling, A. Zilberstein, J. Schell, C. Koncz, and L. Szabados. 1997. Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. Plant J. 12 : 557-569 https://doi.org/10.1046/j.1365-313X.1997.00557.x
  23. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22 : 4673-4680 https://doi.org/10.1093/nar/22.22.4673
  24. Till, B. J., C. Burtner, L. Comai, and S. Henikoff. 2004. Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res. 32 : 2632-2641 https://doi.org/10.1093/nar/gkh599
  25. Till, B. J., T. Zerr, L. Comai, and S. Henikoff. 2006. A protocol for TILLING and Ecotilling in plants and animals. Nat. Protocols 1 : 2465-2477 https://doi.org/10.1038/nprot.2006.329
  26. Trieu, A. T., S. H. Burleigh, I. V. Kardailsky, I. E. Maldonado-Mendoza, W. K. Versaw, L. A. Blaylock, H. Shin, T. J. Chiou, H. Katagi, G. R. Dewbre, D. Weigel, and M. J. Harrison. 2000. Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J. 22: 531-541 https://doi.org/10.1046/j.1365-313x.2000.00757.x
  27. Troll, W. and J. Lindsley. 1955. A photometric method for the determination of proline. J. Biol. Chem. 215 : 655-660
  28. VandenBosch, K. A. and G. Stacey. 2003. Summaries of legume genomics projects from around the globe. Community resources for crops and models. Plant Physiol. 131 : 840-865 https://doi.org/10.1104/pp.103.020388
  29. Yoshiba, Y., T. Kiyosue, T. Katagiri, H. Ueda, T. Mizoguchi, K. Yamaguchi-Shinozaki, K. Wada, Y. Harada, and K. Shinozaki. 1995. Correlation between the induction of a gene for $\Delta^{1}$-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J. 7 : 751-760 https://doi.org/10.1046/j.1365-313X.1995.07050751.x
  30. Zhu, H. Y., D. J. Kim, J. M. Baek, H. K. Choi, L. Ellis, H. Kuester, W. R. McCombie, H. M. Peng, and D. R. Cook. 2003. Syntenic relationships between Medicago truncatula and Arabidopsis thaliana reveal extensive divergence of genome organization. Plant Physiol. 131 : 1018-1026 https://doi.org/10.1104/pp.102.016436