Expression Of Soybean PM1 Protein Confers High Temperature Tolerance In Yeast Mutant ?TPS2

With the effect of global warming,high temperature stress has also become an important factor affecting plant growth and would also lead to poor plant growth,crop production,and even death.Late embryogenesis abundant protein is closely related to plant stress response.It could be divided into 6 groups based on its conserved sequences and motifs.The PM1 protein belongs to the fourth group which can bind to metal ions such as copper ion?ferric ion and reduce hydroxyl radicals in plant.Meanwhile,PM1 protein showed protective effect on LDH enzyme activity under the freeze-thaw stress,which proved that PM1 protein had multifunctional protective role.However,it remains to be studied whether PM1 has the ability to withstand high temperature stress and the mechanisms of its function.In this study,yeast mutant sensitive to high-temperature,namely ?TPS2,and four yeast mutant sensitive to other stress,namely ?YAP1,?HOG1,?CNB1 and ?SOD1,were identified and selected from 15 yeast mutants.The lack of TPS2 gene leads to the loss of trehalose phosphate phosphatase(TPP)activity,which leads to the inhibition of trehalose synthesis and thus fails to induce the synthesis of large amounts of trehalose under high temperature stress causing the growth arrest of the yeast or even death.Two vectors of pYES2-CT and pYES2-PM1 were transfected into yeast mutant strain ?TPS2.The growth rate of ?TPS2/pYES2-PM1 protein in solid and liquid culture medium was significantly enhanced than that in control group,which indicated that the expression of PM1 protein increased the ability of ?TPS2 to withstand high temperature stress.At the same time,we measured the trehalose content of wild-type yeast BY4742/pYES2-CT,?TPS2/pYES2-CT and ?TPS2/pYES2-PM1 before and after heat stress.The trehalose content is unchanged regardless of the expression of PM1 before and after heat stress.The effect of PM1 protein alone enhanced the ability of ?TPS2 to resist high temperature stress rather than the synthesis of trehalose.In order to study whether the PM1 protein has the ability of protecting the membrane system and protein in vivo,the yeast soluble total protein was extracted and mixed with PM1 in the PBS system on high temperature(42? 2h).Afterwards,A340 nm was measured,and the UV absorbance A340 was calculated to evaluate the degree of aggregation.It was found that the BSA protein can not inhibit the thermal aggregation of yeast soluble total protein.The HSP protein of low concentration decreased its aggregation by 43%,while the HSP of high concentration had no protective effect.The PM1 protein of low concentration(0.5mg/ml)has no protective effect,and the PM1 protein of high concentration(2mg/ml)can protect the total protein from the yeast,the degree of aggregation decreased by 32%.Trehalose(50mg/ml)can reduced the agglomeration of soluble protein by 10%,and decreased the concentration of 22% with the unprotected low concentration PM1 protein,indicating that PM1 protein and trehalose has synergistic effect.In order to study whether the PM1 protein could protect the membrane system in vivo,we used the mitochondrial membrane potential detection dye JC-1 to stain the three recombinant yeast BY4742/pYES2-CT,?TPS2/pYES2-CT and ?TPS2/pYES2-PM1 before and after heat stress.It was found that ?TPS2/pYES2-PM1 recombinant yeast(44%)was significantly lower than that of the control ?TPS2/pYES2-CT(lesion rate 77%),indicating that the expression of the PM1 protein could protect the mitochondrial membrane potential in the yeast and that the PM1 protein could also protect the cell membrane and other membrane systems.In a word,PM1 protein can enhance the tolerance of the ?TPS2 mutant under high temperature stress.In conclusion,the PM1 protein may protect the protein from the yeast in the heat-stressed yeast,PM1 protein and trehalose has synergistic effect.Meanwhile it aslo protects the mitochondrial membrane potential of yeast under heat stress.