The Characteristics Of Soybean C1 Protein C-terminal Short Peptide Forming Oligomers And Their Protective Functions In Cu²⁺ Stress

Abiotic stresses such as frozen, drought and heavy metal ions are very common in the process of plant growth; they often lead to plant damage, crop failure, and even death. Many plants can express and accumulate of LEA protein?late embryogenesis abundant protein? to deal with these stresses. Researches have shown the close relationship between LEA proteins and the tolerance to abiotic stress. LEA proteins can be divided into 6 groups according to the feature of their sequences. The PM1 protein of soybean belongs to the fourth group and mainly exists in the radicle. PM1 protein can bind to Cu²⁺ and improve the tolerance to Cu²⁺. However, the protective functions and mechanisms of PM1 protein need further study.The histidines of PM1 protein mainly exist in the C-terminal, and the ?-Helix only exists in N-terminal. According to these characteristics, we designed and constructed recombinant vectors for E.coli and yeast. Gel filtration chromatography was used to detecte the apparent molecular weight of PM1 protein and its peptide fragment. We found that only PM1 protein and the peptides in the C-terminal can form dimer. The heat-stable protein of soybean radicle and the recombinant yeasts??cup2/pYES2-PM1, ?cup2/pYES2-PM1-N and ?cup2/pYES2-PM1-C? were detected by Western blot, and oligomers were found in PM1 protein and PM1-C peptide. These suggested that PM1 protein and PM1-C peptide can form oligomers in and outside cells.When treated with crosslinking agent, oligomers or polymer could be found in PM1 and peptides such as PM1-C, PM1-C1 and PM1-C2, but only monomer could be found in PM1-N. These results confirmed the important role of C-terminal in the formation of PM1 oligomers.Histidine was also proved to be the key amino acid by the fact that the ability of PM1 protein and PM1-C peptide to form oligomers greatly reduced when treated by DEPC. PM1 protein and PM1-C peptide could form more oligomers and polymers when binded to Cu²⁺, considering the fact that histidine in PM1 protein was the main binding site of Cu²⁺, we conjectured that PM1 could reduce the concentration and toxic of Cu²⁺ by binding to Cu²⁺ and forming oligomers and polymers.The recombinant yeasts ?cup2/pYES2-PM1 and ?cup2/pYES2-PM1-C grew much faster than the control ?cup2/pYES2-CT in solid medium and fluid medium. This indicated that PM1 protein and PM1-C peptide could resist the stress causing by Cu²⁺ alone.Our research showed that PM1 protein and PM1-C peptide could form oligomers in vitro and in vivo, furthermore, histidine was the key amino acid in the formation of these oligomers. We suggested that there was a balance in the monomer, oligomers and polymers of PM1 protein, more oligomers and polymers would appear when the Cu²⁺ in cell excessived and this process would reduce the content of free Cu²⁺. When the Cu²⁺ in cell reduced, PM1 protein would act in the opposite way to keep the balance of Cu²⁺ in the cell.