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Research Article

volume 41 issue 5 (october 2018) : 675-680,   Doi: 10.18805/LR-386

Effects of StP5CS gene overexpression on nodulation and nitrogen fixation of vegetable soybean under salt stress conditions

X
X.W. Ren
D
D.W. Yu
S
S.P. Yang
J
J.Y. Gai
Y
Y.L. Zhu
1National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing, 210095, China.

  • Submitted06-09-2017|

  • Accepted18-05-2018|

  • First Online 20-09-2018|

  • doi 10.18805/LR-386

Cite article:- Ren X.W., Yu D.W., Yang S.P., Gai J.Y., Zhu Y.L. (2018). Effects of StP5CS gene overexpression on nodulation and nitrogen fixation of vegetable soybean under salt stress conditions. Legume Research. 41(5): 675-680. doi: 10.18805/LR-386.

ABSTRACT

Using a quartz sand culture, comparisons were made between the T5 homozygous transgenic lines (HTLs) that overexpress StP5CS (GenBank accession number: JN606861) and their wild-type (WT) host cultivar to examine the differences in the growth and development traits, the concentrations of proline in vegetable soybean [Glycine max (L.) Merrill] under salt stress conditions. Moreover, the relative expression levels of two glutamine synthetase-related genes (GmGS1â1, GmGS1â2), two nodulation-related genes (GmENOD40-1, GmENOD40-2), and one leghemoglobin gene (GmLba) were also measured. The purpose of this research was to provide a theoretical basis for elucidating the mechanisms of nodulation and nitrogen fixation in the roots of transgenic plants under salt stress conditions. Compared with WT plants, the plant height and seed weight per plant of T5 HTLs significantly increased, and the contents of proline in various tissues of T5 HTLs were also significantly elevated. Quantitative real-time PCR (qRT-PCR) analysis indicated that the expression levels of GmGS1â1, GmGS1â2, GmENOD40-1,  GmENOD40-2, and GmLba were significantly increased in T5 HTLs under salt stress conditions. These results indicate that the overexpression of StP5CS in T5 HTLs enhanced growth, nodulation and nitrogen fixation in transgenic vegetable soybean under salt stress conditions.

REFERENCES

  1. Alyemeni, M.N., Hayat, Q., Hayat, S., Faizan, M., and Faraz, A., (2016). Exogenous proline application enhances the efficiency of nitrogen fixation and assimilation in chickpea plants exposed to cadmium. Legume Research, 39: 221-227.
  2. Bates, L.S., Waldren, R.P., and Teare, I.D., (1973). Rapid determination of free proline for water-stress studies. Plant Soil, 39: 205-207.
  3. Broughton, W.J. and Dilworth, M.J., (1971). Control of leghaemoglobin synthesis in snake beans. Biochemical Journal, 125: 1075-    1080.
  4. diCenzo, G.C., Zamani, M., Cowie, A., and Finan, T.M., (2015). Proline auxotrophy in Sinorhizobium meliloti results in a plant-    specific symbiotic phenotype. Microbiology, 161: 2341-2351.
  5. Ferguson, B.J. and Mathesius, U., (2014). Phytohormone regulation of legume-rhizobia interactions. Journal of Chemical Ecology, 40: 770-790.
  6. Guan, D., Stacey, N., Liu, C., Wen, J., Mysore, K.S., Torres-Jerez, I., Vernie, T., Tadege, M., et al., (2013). Rhizobial infection is associated with the development of peripheral vasculature in nodules of Medicago truncatula. Plant Physiology, 162: 107-115.
  7. Kumar, D., Kumar, P., Alka, (2008). Effect of saline water irrigation on ions uptake and forage yield of berseem (Trifolium alexandrinum L.) during seedling stage. Legume Research, 31: 297-299
  8. Kim, G.B. and Nam, Y.W., (2013). A novel Ä1-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation. Journal of Plant Physiology, 170: 291-302.
  9. King, N.D., Hojnacki, D., and O’Brian, M.R., (2000). The Bradyrhizobium japonicum proline biosynthesis gene proC is essential for symbiosis. Applied and Environmental Microbiology, 66: 5469-5471.
  10. Kouchi, H. and Hata, S., (1993). Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Molecular and General Genetics, 238: 106-119.
  11. Le, D.T., Aldrich, D.L., Valliyodan, B., Watanabe, Y., Van Ha, C., Nishiyama, R., et al., (2012). Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions. PLoS ONE, 7: e46487.
  12. Libault, M., Thibivilliers, S., Bilgin, D.D., Radwan, O., Benitez, M., Clough, S.J., and Stacey, G., (2008). Identification of four soybean reference genes for gene expression normalization. Plant Genome, 1: 44-54.
  13. Masalkar, P., Wallace, I.S., Hwang, J.H., and Roberts, D.M., (2010). Interaction of cytosolic glutamine synthetase of soybean root nodules with the C-terminal domain of the symbiosome membrane nodulin 26 aquaglyceroporin. Journal of Biological Chemistry, 285: 23880-23888.
  14. Morais, M.C., Panuccio, M.R., Muscolo, A., and Freitas, H., (2012). Salt tolerance traits increase the invasive success of Acacia longifolia in Portuguese coastal dunes. Plant Physiology and Biochemistry, 55: 60-65.
  15. Nandhini, D.U., Somasundaram, E., Amanullah, M.M., (2017). Effect of rhizobial nod factors (lipochitooligosaccharide) on seedling growth of blackgram under salt stress. Legume Research, 41: 159-162.
  16. Ott, T., van Dongen, J.T., Gunther, C., Krusell, L., Desbrosses, G., Vigeolas, H., Bock, V., et al., (2005). Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development. Current Biology, 15: 531-535.
  17. Pham, V.T., Rediers, H., Ghequire, M.G., Nguyen, H.H., De Mot, R., Vanderleyden, J., and Spaepen, S., (2017). The plant growth-    promoting effect of the nitrogen-fixing endophyte Pseudomonas stutzeri A15. Archives of Microbiology, 199: 513-517.
  18. Phang, T.H., Shao G.H., and Lam, H.M., (2008). Salt tolerance in soybean. Journal of Integrative Plant Biology, 50: 1196-1212.
  19. Ramana, G.V., Padhy, S.P., Chaitanya, K.V., (2012). Differential responses of four soybean (Glycine max L.) cultivars to salinity stress. Legume Research, 35: 185-193.
  20. Rivero, R.M., Mestre, T.C., Mittler, R., Rubio, F., Garcia-Sanchez, F., and Martinez, V., (2014). The combined effect of salinity and heat reveals a specific physiological, biochemical and molecular response in tomato plants. Plant Cell and Environment., 37: 1059-1073.
  21. Schmittgen, T.D. and Livak, K.J., (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3: 1101-    1108.
  22. She, Q., Sandal, N.N., Stougaard, J., and Marcker, K.A., (1993). Comparative sequence analysis of cis elements present in Glycine max L. leghemoglobin lba and lbc3 genes. Plant Molecular Biology, 22: 931-935.
  23. Surekha, C., Nirmala Kumari, K., Aruna, L., Suneetha, G., Arundhati, A., and Kavi Kishor, P.B., (2014). Expression of the Vigna aconitifolia P5CSF129A gene in transgenic pigeonpea enhances proline accumulation and salt tolerance. Plant Cell Tissue and Organ Culture, 116: 27-36.
  24. Szabados, L. and Savoure, A., (2010). Proline: a multifunctional amino acid. Trends in Plant Science, 15: 89-97.
  25. Verdoy, D., De la Pena, T.C., Redondo, F.J., Lucas, M.M., and Pueyo, J.J., (2006). Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress. Plant Cell and Environment, 29: 1913-1923.
  26. Zhang, G.C., Zhu, W.L., Gai, J.Y., Zhu, Y.L., and Yang, L.F., (2015). Enhanced salt tolerance of transgenic vegetable soybeans resulting from overexpression of a novel D-pyrroline-5-carboxylate synthetase gene from Solanum torvum Swartz. Horticulture Environment and Biotechnology, 56: 94-104.
  27. Zhang, G.W., Hu, Q.Z., Xu, S.C., Gong, Y.M., (2013). Polyamines play a positive role in salt tolerant mechanisms by activiting antioxidant enzymes in roots of vegetable soybean. Legume Research, 36: 234-240.
  28. Zhu, W.L., Yang, L.F., Yang, S.P., Gai, J.Y., and Zhu, Y.L., (2016). Overexpression of rice phosphate transporter gene OsPT2 enhances nitrogen fixation and ammonium assimilation in transgenic soybean under phosphorus deficiency. Journal of Plant Biology, 59: 172-181. 
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    Research Article

    volume 41 issue 5 (october 2018) : 675-680,   Doi: 10.18805/LR-386

    Effects of StP5CS gene overexpression on nodulation and nitrogen fixation of vegetable soybean under salt stress conditions

    X
    X.W. Ren
    D
    D.W. Yu
    S
    S.P. Yang
    J
    J.Y. Gai
    Y
    Y.L. Zhu
    1National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing, 210095, China.

    • Submitted06-09-2017|

    • Accepted18-05-2018|

    • First Online 20-09-2018|

    • doi 10.18805/LR-386

    Cite article:- Ren X.W., Yu D.W., Yang S.P., Gai J.Y., Zhu Y.L. (2018). Effects of StP5CS gene overexpression on nodulation and nitrogen fixation of vegetable soybean under salt stress conditions. Legume Research. 41(5): 675-680. doi: 10.18805/LR-386.

    ABSTRACT

    Using a quartz sand culture, comparisons were made between the T5 homozygous transgenic lines (HTLs) that overexpress StP5CS (GenBank accession number: JN606861) and their wild-type (WT) host cultivar to examine the differences in the growth and development traits, the concentrations of proline in vegetable soybean [Glycine max (L.) Merrill] under salt stress conditions. Moreover, the relative expression levels of two glutamine synthetase-related genes (GmGS1â1, GmGS1â2), two nodulation-related genes (GmENOD40-1, GmENOD40-2), and one leghemoglobin gene (GmLba) were also measured. The purpose of this research was to provide a theoretical basis for elucidating the mechanisms of nodulation and nitrogen fixation in the roots of transgenic plants under salt stress conditions. Compared with WT plants, the plant height and seed weight per plant of T5 HTLs significantly increased, and the contents of proline in various tissues of T5 HTLs were also significantly elevated. Quantitative real-time PCR (qRT-PCR) analysis indicated that the expression levels of GmGS1â1, GmGS1â2, GmENOD40-1,  GmENOD40-2, and GmLba were significantly increased in T5 HTLs under salt stress conditions. These results indicate that the overexpression of StP5CS in T5 HTLs enhanced growth, nodulation and nitrogen fixation in transgenic vegetable soybean under salt stress conditions.

    REFERENCES

    1. Alyemeni, M.N., Hayat, Q., Hayat, S., Faizan, M., and Faraz, A., (2016). Exogenous proline application enhances the efficiency of nitrogen fixation and assimilation in chickpea plants exposed to cadmium. Legume Research, 39: 221-227.
    2. Bates, L.S., Waldren, R.P., and Teare, I.D., (1973). Rapid determination of free proline for water-stress studies. Plant Soil, 39: 205-207.
    3. Broughton, W.J. and Dilworth, M.J., (1971). Control of leghaemoglobin synthesis in snake beans. Biochemical Journal, 125: 1075-    1080.
    4. diCenzo, G.C., Zamani, M., Cowie, A., and Finan, T.M., (2015). Proline auxotrophy in Sinorhizobium meliloti results in a plant-    specific symbiotic phenotype. Microbiology, 161: 2341-2351.
    5. Ferguson, B.J. and Mathesius, U., (2014). Phytohormone regulation of legume-rhizobia interactions. Journal of Chemical Ecology, 40: 770-790.
    6. Guan, D., Stacey, N., Liu, C., Wen, J., Mysore, K.S., Torres-Jerez, I., Vernie, T., Tadege, M., et al., (2013). Rhizobial infection is associated with the development of peripheral vasculature in nodules of Medicago truncatula. Plant Physiology, 162: 107-115.
    7. Kumar, D., Kumar, P., Alka, (2008). Effect of saline water irrigation on ions uptake and forage yield of berseem (Trifolium alexandrinum L.) during seedling stage. Legume Research, 31: 297-299
    8. Kim, G.B. and Nam, Y.W., (2013). A novel Ä1-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation. Journal of Plant Physiology, 170: 291-302.
    9. King, N.D., Hojnacki, D., and O’Brian, M.R., (2000). The Bradyrhizobium japonicum proline biosynthesis gene proC is essential for symbiosis. Applied and Environmental Microbiology, 66: 5469-5471.
    10. Kouchi, H. and Hata, S., (1993). Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Molecular and General Genetics, 238: 106-119.
    11. Le, D.T., Aldrich, D.L., Valliyodan, B., Watanabe, Y., Van Ha, C., Nishiyama, R., et al., (2012). Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions. PLoS ONE, 7: e46487.
    12. Libault, M., Thibivilliers, S., Bilgin, D.D., Radwan, O., Benitez, M., Clough, S.J., and Stacey, G., (2008). Identification of four soybean reference genes for gene expression normalization. Plant Genome, 1: 44-54.
    13. Masalkar, P., Wallace, I.S., Hwang, J.H., and Roberts, D.M., (2010). Interaction of cytosolic glutamine synthetase of soybean root nodules with the C-terminal domain of the symbiosome membrane nodulin 26 aquaglyceroporin. Journal of Biological Chemistry, 285: 23880-23888.
    14. Morais, M.C., Panuccio, M.R., Muscolo, A., and Freitas, H., (2012). Salt tolerance traits increase the invasive success of Acacia longifolia in Portuguese coastal dunes. Plant Physiology and Biochemistry, 55: 60-65.
    15. Nandhini, D.U., Somasundaram, E., Amanullah, M.M., (2017). Effect of rhizobial nod factors (lipochitooligosaccharide) on seedling growth of blackgram under salt stress. Legume Research, 41: 159-162.
    16. Ott, T., van Dongen, J.T., Gunther, C., Krusell, L., Desbrosses, G., Vigeolas, H., Bock, V., et al., (2005). Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development. Current Biology, 15: 531-535.
    17. Pham, V.T., Rediers, H., Ghequire, M.G., Nguyen, H.H., De Mot, R., Vanderleyden, J., and Spaepen, S., (2017). The plant growth-    promoting effect of the nitrogen-fixing endophyte Pseudomonas stutzeri A15. Archives of Microbiology, 199: 513-517.
    18. Phang, T.H., Shao G.H., and Lam, H.M., (2008). Salt tolerance in soybean. Journal of Integrative Plant Biology, 50: 1196-1212.
    19. Ramana, G.V., Padhy, S.P., Chaitanya, K.V., (2012). Differential responses of four soybean (Glycine max L.) cultivars to salinity stress. Legume Research, 35: 185-193.
    20. Rivero, R.M., Mestre, T.C., Mittler, R., Rubio, F., Garcia-Sanchez, F., and Martinez, V., (2014). The combined effect of salinity and heat reveals a specific physiological, biochemical and molecular response in tomato plants. Plant Cell and Environment., 37: 1059-1073.
    21. Schmittgen, T.D. and Livak, K.J., (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3: 1101-    1108.
    22. She, Q., Sandal, N.N., Stougaard, J., and Marcker, K.A., (1993). Comparative sequence analysis of cis elements present in Glycine max L. leghemoglobin lba and lbc3 genes. Plant Molecular Biology, 22: 931-935.
    23. Surekha, C., Nirmala Kumari, K., Aruna, L., Suneetha, G., Arundhati, A., and Kavi Kishor, P.B., (2014). Expression of the Vigna aconitifolia P5CSF129A gene in transgenic pigeonpea enhances proline accumulation and salt tolerance. Plant Cell Tissue and Organ Culture, 116: 27-36.
    24. Szabados, L. and Savoure, A., (2010). Proline: a multifunctional amino acid. Trends in Plant Science, 15: 89-97.
    25. Verdoy, D., De la Pena, T.C., Redondo, F.J., Lucas, M.M., and Pueyo, J.J., (2006). Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress. Plant Cell and Environment, 29: 1913-1923.
    26. Zhang, G.C., Zhu, W.L., Gai, J.Y., Zhu, Y.L., and Yang, L.F., (2015). Enhanced salt tolerance of transgenic vegetable soybeans resulting from overexpression of a novel D-pyrroline-5-carboxylate synthetase gene from Solanum torvum Swartz. Horticulture Environment and Biotechnology, 56: 94-104.
    27. Zhang, G.W., Hu, Q.Z., Xu, S.C., Gong, Y.M., (2013). Polyamines play a positive role in salt tolerant mechanisms by activiting antioxidant enzymes in roots of vegetable soybean. Legume Research, 36: 234-240.
    28. Zhu, W.L., Yang, L.F., Yang, S.P., Gai, J.Y., and Zhu, Y.L., (2016). Overexpression of rice phosphate transporter gene OsPT2 enhances nitrogen fixation and ammonium assimilation in transgenic soybean under phosphorus deficiency. Journal of Plant Biology, 59: 172-181. 
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