Molecular Mechanism For The Enhanced Salt Tolerance Of Arabidopsis Thaliana, Saccharomyces Cerevisiae And Escherichia Coli By Soybean Em (LEA1) Protein

Salt, drought and cold stress are the important adverse environmental factors to restrict plant growth, and plant has developed various molecular mechanisms to cope with different stresses during evolution. Some proteins in plant are induced to attenuate the damage caused by stress, e.g. late embryogenesis abundant (LEA) proteins produced in response to dehydration. There are at least six groups of LEA proteins by virtue of structural features and conserved motifs. Group 1 LEA (LEA1) proteins are highly conserved and found not only in plants but also in animals and bacteria. LEA1 proteins are extremely hydrophilic and have been proposed to act as water binding proteins, water replacement or hydration buffers to regulate water status to protect cells under dehydration stress. It has been shown that expression of exogenous LEA1 proteins conferred drought and salt tolerance for plants. However, the protective mechanisms of LEA1 proteins remains poorly understood.In this study, soybean Em (LEA1) gene form Glycine max L. Merr.cv Bainong 6# have been cloned, which contains three DNA fragments coding different conserved 20 amino acid motifs (N-motif, M-motif and C-motif) in the N-terminal, middle and C-terminal domains. At first, the full-length Em gene and three domains (Em-N, En-2M and Em-C) DNA fragments covering the above motifs were transformed into Escherichia coli. The survival rates of three recombinant bacteria in 500 mM NaCl or 500 mM KCl culture medium were tested. Our results show that not only the full-length Em proteins but also its three domains are able to confer the salt tolerance of bacteria and their ability are different: Em>Em-C≈Em-2M>Em-N.Then, we transformed the full-length Em gene and Em-2M domain DNA fragment into Saccharomyces cerevisiae. The growth of recombinant yeast in 1500 mM NaCl or 700 mM MgCl2 culture medium were monitored. The results demonstrate that the introduction of the full-length Em gene and Em-2M domain DNA fragment are capable of improving the salt tolerance of recombinant yeast and their ability are alike.Furthermore, the full-length Em gene and Em-2M domain DNA fragment are also transformed into higher model plant Arabidopsis thaliana. The seed germination rate of the transgenic plant in 150 mM NaCl culture medium and the root growth in 125 mM NaCl culture medium are recorded. Our results illustrate that not only the full-length Em gene but also Em-2M domain DNA fragment can enhance the salt tolerance of transgenic Arabidopsis thaliana. Therefore, Soybean Em proteins and its conserved domains are able to confer the salt tolerance of Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana under salt stress.In in vitro experiments, we analyzed the protective function of soybean Em proteins and its domain peptides on proteins under freezing in vitro. The results show that soybean Em proteins and it three domain peptides are able to prevent LDH from inactivation, aggregation and deleterious conformational changes by freeze-thaw, but their protective ability is different: Em>Em-C>Em-2M>Em-N.CD spectroscopy reveals that Em-N had a high potential to formα-helix while Em, Em-2M and Em-C contained bothα-helix and polyproline II (PII) structure. These data imply that both structures may involve in the protective effect of Em protein against freezing stress. To test the functions ofα-helix and PII structure of LEA1 proteins, five Em-2M mutants and one Em-N mutant were made through site mutation to contain different content of PII structure andα-helix. CD spectroscopy further reveals that at least two PII structures exist in soybean Em protein, one PII helical structure located between the fifty-third and sixty first amino acid in the M-motif and another in C-terminal domain. Then, the enzyme protective effect of these mutant peptides on LDH is also tested.When PII structure was absent, the protective effect was decreased compared to the original domain peptide despite an increase inα-helical content. On the other hand, when PII structure was formed after mutation, the protective effect was increased despite a reduced tendency to formα-helix. These data clearly demonstrate the involvement of PII structure in the enzyme protective function of the soybean Em domain peptides. In addition, when PII content remained unchanged, the protective effect was correlated in line with the tendency to formα-helix.In summary, not only the expression of soybean Em proteins but also that of its conserved domains are able to confer the salt tolerance of Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana, indicating that LEA1 proteins which are highly conserved during evolution may protect prokaryotic and eukaryotic cells by the similar mechanism. The protective faction of LEA1 proteins on cells is to protect the activity and stabilize the structure of proteins (enzymes) in cells by the interaction of PII structure andα-helix formed under stress.