Rice cDNA Encoding PROLM is Capable of Rescuing Salt Sensitive Yeast Phenotypes G19 and Axt3K from Salt Stress

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.

Authors:

• Prasad Senadheera
• Younousse Saidi
• Frans JM Maathuis

Keywords:

• Rice seed expression
• salt stress
• prolamin
• salinitytolerance
• Oryza sativa

References:

[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.