Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 31793
Rice cDNA Encoding PROLM is Capable of Rescuing Salt Sensitive Yeast Phenotypes G19 and Axt3K from Salt Stress

Authors: Prasad Senadheera, Younousse Saidi, Frans JM Maathuis


Rice seed expression (cDNA) library in the Lambda Zap 11® phage constructed from the developing grain 10-20 days after flowering was transformed into yeast for functional complementation assays in three salt sensitive yeast mutants S. cerevisiae strain CY162, G19 and Axt3K. Transformed cells of G19 and Axt3K with pYES vector with cDNA inserts showed enhance tolerance than those with empty pYes vector. Sequencing of the cDNA inserts revealed that they encode for the putative proteins with the sequence homologous to rice putative protein PROLM24 (Os06g31070), a prolamin precursor. Expression of this cDNA did not affect yeast growth in absence of salt. Axt3k and G19 strains expressing the PROLM24 were able to grow upto 400 mM and 600 mM of NaCl respectively. Similarly, Axt3k mutant with PROLM24 expression showed comparatively higher growth rate in the medium with excess LiCl (50 mM). The observation that expression of PROLM24 rescued the salt sensitive phenotypes of G19 and Axt3k indicates the existence of a regulatory system that ameliorates the effect of salt stress in the transformed yeast mutants. However, the exact function of the cDNA sequence, which shows partial sequence homology to yeast UTR1 is not clear. Although UTR1 involved in ferrous uptake and iron homeostasis in yeast cells, there is no evidence to prove its role in Na+ homeostasis in yeast cells. Absence of transmembrane regions in Os06g31070 protein indicates that salt tolerance is achieved not through the direct functional complementation of the mutant genes but through an alternative mechanism.

Keywords: Rice seed expression, salt stress, prolamin, salinitytolerance, Oryza sativa

Digital Object Identifier (DOI):

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1732


[1] Pearson, G.A. & Bernstein, L. (1959). Salinity effects at several growth stages of rice. Agronomy Journal, 51,654-657.
[2] Akbar, M., Yabuno, T. & Nakao, S. (1972). Breeding for saline resistant varieties of rice; Variability for salt tolerance among some rice varieties. Japan Journal of Breeding, 22, 277-284.
[3] Abdullah, Z., Khan, M. A. & Flowers, T.J. (2001). Causes of Sterility in Seed Set of Rice under Salinity Stress. Journal of Agronomy and Crop Science, 187(1), 25-32.
[4] Sultana, N., Ikeda, T. & Itoh, R. (1999). Effect of NaCl salinity on photosynthesis and dry matter accumulation in developing rice grains Environmental and Experimental Botany. Volume 42, Issue 3, Pages 211-220.
[5] Serrano, R. & Gaxiola, R. (1994). Microbial models and salt stress tolerance in plants. Critical Review in Plant Science, 13, 121-138.
[6] Serrano, R. (1991). Transport across yeast vacuolar and plasma membranes, p.523-585. In J. R. Pringle, J. R. Broach, and E. W. Jones (ed.), The molecular and cellular biology of the yeast Saccharomyces. Cell cycle and cell biology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[7] Serrano, R., and. Villalba J. M. (1995). Expression and localization of plant membrane proteins in Saccharomyces. Methods in Cell Biology,50, 481-496.
[8] Ramos, J. (1999). Contrastingsal t tolerance mechanisms in Saccharomyces cerevisiae and Debaryomyces hansenii. Recent Research Development Microbiology, 3, 377-390.
[9] Greenway, H., & Munns R. (1980). Mechanisms of salt tolerance in non-halophytes. Annual Review of Plant Physiology, 31,149-190.
[10] Marschner, H. (1995). Mineral nutrition of higher plants. Springer, Berlin, Germany.
[11] Serrano, R. 1996. Salt tolerance in plants and microorganisms: toxicity targets and defense responses. Int. Rev. Cytol. 165:1-51.
[12] Gaber, R. F., Styles, C. A. & Fink, G. R. (1988) TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Molecular and Cellular Biology, 8, 2848- 2859.
[13] Ko, C. H. & Gaber R. F.(1991). TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae. Molecular and Cellular Biology, 11, 4266-4273.
[14] Mendoza, I., Rubio, F., Rodríguez-Navarro, A. & Pardo, J. M. (1994). The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. Journal of Biological Chemistry, 269:8792- 8796.
[15] Rudolph, H.K, Antebi, A., Fink, G.R., Buckley, C.M., Dorman, T.E., LeVitre, J., Davidow, L.S., Mao, J.I. & Moir, D.T. (1989). The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family. Cell, 58(1), 133-145.
[16] Martinez, R., Latreille, M.T. & Mirande, M. (1991). A PMR2 tandem repeat with a modified C-terminus is located downstream from the KRS1 gene encoding lysyl-tRNA synthetase in Saccharomyces cerevisiae. Molecular and General Genetics, 227(1), 149-154.
[17] Rodríguez-Navarro, A., Quintero, F.J. & Garciadeblás, B. (1994). Na(+)-ATPases and Na+/H+ antiporters in fungi. Biochimica et Biophysica Acta, 1187(2), 203-205.
[18] Haro, R., Bañuelos, M. A., Quintero, F. J., Rubio, F. & Rodríguez- Navarro, A. (1993) Genetic basis of Sodium exclusion and Sodium tolerance in yeast. A model for plants. Plant Physiology, 89, 868-874.
[19] Garciadeblas, B., Rubio, F., Quintero, F.J., Banuelos, M.A. & Haro, R. (1993). Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae. Molecular & General Genetics, 236, 363-368.
[20] Andre, B. (1995). An overview of membrane transport proteins in Saccharomyces cerevisiae. Yeast, 11, 1575-1611.
[21] Prior, C., Potier, S., Souciet J et al. Characterization of the NHA1 gene encoding a Na+/H+-antiporter of the yeast Saccharomyces cerevisiae. FEBS Letter, 1996; 387:89-93.
[22] Nass, R. & Rao, R. (1998). Novel localization of a Na+/H+ exchanger in a late endosomal compartment of yeast. Journal of Biological Chemistry, 273: 21054-21060.
[23] Mulet, J.M., Leube, M.P., Kron, S.J., Rios, G., Fink, G.R. & Serrano, R. (1999). A Novel Mechanism of Ion Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the Trk1-Trk2 Potassium Transporter, Molecular and cellular biology, 19( 5), 3328- 3337.
[24] Pérez-Valle J., Jenkins H., Merchan S., Montiel V., Ramos J., Sharma S., Serrano R.& Yenush L. (2007). Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters. Molecular and Cellular Biology, 27(16)5725-36.
[25] Mulet, J.M., Alejandro, S., Romero, C., Serrano R., Munson, A.M., Haydon, D.H., Love, S.L., Fell, G.L., Palanivel, V.R. & Rosenwald, A.G.(2004).Yeast ARL1 encodes a regulator of K+ influx. Journal of Cell Sciences, 117(11):2309-20.
[26] Casado, C., Yenush, L., Melero, C., Ruiz Mdel, C., Serrano, R., Pérez- Valle, J., Ari├▒o, J. & Ramos J. (2010). Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase, FEBS Letters, 584(11), 2415-2420.
[27] Forment, J., Mulet, J.M., Vicente, O. & Serrano, R. (2002). The yeast SR protein kinase Sky1p modulates salt tolerance, membrane potential and the Trk1,2 potassium transporter. Biochimica et Biophysica Acta,1565(1):36-40.
[28] Rios, G., Ferrando, A. & Serrano, R. (1997). Mechanisms of salt tolerance conferred by overexpression of the HAL1 gene in Saccharomyces cerevisiae. Yeast, 13(6), 515-28.
[29] Munsona, A.M., Love, S.L., Shu, J., Palanivel, V.R. & Rosenwald, A.G., (2004). ARL1 participates with ATC1/LIC4 to regulate responses of yeast cells to ions, Biochemical and Biophysical Research Communications, 315( 3), 617-623.
[30] Ferrando, A., Kron, S. J., Rios, G., Fink, G. R. & Serrano, R. (1995). Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3. Molecular and Cellular Biology, 15, 5470-5481.
[31] de Nadal, E., Clotet, J., Posas, F., Serrano, R., G├│mez, N. & Ari├▒o, J. (1998). The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppzlp Ser/Thr protein phosphatase. Proceedings of Natational Academy of Science, USA, 95,7357-7362.
[32] Schachtman, D.P. & Schroeder. J.I.(1994). Structure and transport mechanism of a high-affinity potassium uptake transporter from higher plants. Nature, 370, 655-658.
[33] Igarashi, Y., Yoshiba, Y., Sanada, Y., Yamaguchi-Shinozaki, K., Wada, K. & Shinozaki, K.(1997) Characterization of the gene for N1-pyrroline- 5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L. Plant molecular Biology,33(5), 857-865.
[34] Obata, T., Kitamoto, H.K., Nakamura, A., Fukuda, A., Tanaka, Y., (2007) Rice Shaker Potassium Channel OsKAT1 Confers Tolerance to Salinity Stress on Yeast and Rice Cells. Plant Physiology,144, 1978- 1985.
[35] Fukuda, A., Nakamura, A., Tagiri, A., Tanaka, H., Miyao, A., Hirochika, H.& Tanaka, Y. (2004) Function, intracellular Localization and the Importance in Salt Tolerance of a Vacuolar Na+/H+ Antiporter from Rice. Plant Cell Physiology, 45(2), 146-159.
[36] Gietz, D., St Jean, A., Woods, R.A. & Schiestl, R.H. (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Research, 20, 1425.
[37] Chen, D., Yang, B., Kuo, T. (1992). One-step transformation of yeast in stationary phase. Current Genetic 21: 83-84.
[38] Quintero, F. J., Garciadeblas, B. & Rodr─▒'guez-Navarro, A. (1996). The SAL1 gene of Arabidopsis, encoding an enzyme with 3 (2), 5 bisphosphate nucleotidase and inositol 1-phosphatase activities, increases salt tolerance in yeast. Plant Cell, 8, 529-537.
[39] Gobert, A., Park, G., Amtmann, A., Sanders, D. & Maathuis, F.J.M. (2006). Arabidopsis thaliana cyclic nucleotide gated channel 3 forms a nonselective ion transporter involved in germination and cation transport. Journal of Experimental Botany, 57: 791-800.
[40] Wieland, J., Nitsche, A.M., Strayle, J., Steiner, H. & Rudolph, H.K. (1995). The PMR2 gene cluster encodes functionally distinct isoforms of a putative Na1 pump in the yeast plasma membrane. EMBO J 14, 3870- 3882. O. Young, "Synthetic structure of industrial plastics (Book style with paper title and editor)," in Plastics, 2nd ed. vol. 3, J. Peters, Ed. New York: McGraw-Hill, 1964, pp. 15-64.
[41] Banuelos, M.A., Sychrova, H., Bleykasten-Grosshans, C., Souciet, J.L. & Potier, S. (1998). The Nha1 antiporter of Saccharomyces cerevisiae mediates sodium and potassium efflux. Microbiology, 144, 2749-2758.
[42] Darley, C.P., Wuytswinkel, O.C.M., Woude, K., Mager, W.H. & De Boer, A.H. (2000). Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na1/H1 exchangers. Biochemical Journal, 351, 241-249.
[43] Borst-Pauwels, G. W. F. H. (1981). Ion transport in yeast. Biochimica et Biophysica Acta, 650, 88-127.
[44] de Nadal, E., Calero, F., Ramos, J. & Ari├▒o, J. (1999) Biochemical and Genetic Analyses of the Role of Yeast Casein Kinase 2 in Salt Tolerance, Journal of Bacteriology,181(20), 6456-6462.
[45] Kawai, S., Suzuki, S., Mori, S. & Murata K. (2001). Molecular cloning and identification of UTR1 of a yeast Saccharomyces cerevisiae as a gene encoding an NAD kinase. FEMS Microbiology Letters, 200(2), 181-184.
[46] Batard Y, Hehn A, Nedelkina S, Schalk M, Pallett K, Schaller H, Werck-Reichhart D (2000) Increasing expression of P450 and P450- reductase proteins from monocots in heterologous systems. Arch Biochem Biophys 379:161-169.
[47] Tusnády, G.E. & Simon, I. (2001). The HMMTOP transmembrane topology prediction server. Bioinformatics, 17, 849-850.
[48] Sundaram R. M., Sakthivel K., Hariprasad A. S., Ramesha M. S., Viraktamath B. C., Neeraja C. N., Balachandran S. M., Shobha Rani N., Revathi P. Sandhya P., et al. (2010) Molecular Breeding Development and validation of a PCR-based functional marker system for the major wide-compatible gene locus S5 in rice 26, 719-727.
[49] Marchler-Bauer A et al. (2011), "CDD: a Conserved Domain Database for the functional annotation of proteins.", Nucleic Acids Res.39(D)225- 9.
[50] Hruz, T., Laule, O., Szabo, G., Wessendorp, F., Bleuler, S., Oertle, L., Widmayer, P., Gruissem, W. & Zimmermann, P (2008).Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes. Advances in Bioinformatics, 2008, 420747.
[51] Pandit, A., Rai, V., Bal, S., Kumar, V., Chauhan, M., Gautam, R.K., Singh, R., Sharma, P.C. & Singh, K., (2010). Combining QTL mapping and transcriptome profiling of bulked RILs for identification of functional polymorphism for salt tolerance genes in rice (Oryza sativa L.), Molecular Genetics and Genomics, 284( 2), 121-136.
[52] Walia, H., Wilson, C., Condamine, P., Liu, X., Ismail, A.M., Zeng, L., Wanamaker, S.I., Mandal, J., Xu, J., Cui, X. & Close T.J., (2005) Comparative transcriptional profiling of two contrasting rice genotypes under salinity stress during the vegetative growth stage. Plant Physiology 139:822-835.
[53] Cotsaftis, O., Plett, D., Johnson, A.A., Walia, H., Wilson, C., Ismail, A.M., Close, T.J., Tester, M. & Baumann U. (2011) Root-specific transcript profiling of contrasting rice genotypes in response to salinity stress. Molecular Plant, 4(1),25-41.
[54] Senadheera, P., Singh, R. K. & Maathuis, F.J. M., (2009). Differentially expressed membrane transporters in rice roots may contribute to cultivar dependent salt tolerance, Journal of Experimental Botany, 60(9): 2553- 2563.
[55] Charoenlappanit S, Roytrakul S , Teerakathiti T, Juntawong N (2010) Proteome analysis of salt tolerant and salt sensitive rice suspension cells in response to NaCl stress. 36th Congress on Science and Technology of Thailand.