The Matal-binding Properties And The Protective Effect On Biological Macromolecules Of Two Soybean LEA4 Proteins

Late embryogenesis abundant （LEA） proteins play an important role in the water-stress tolerance of plants. LEA proteins are highly expressed at the late development stage of seeds and also accumulated in vegetative tissues in response to water deficit. LEA proteins have been classified into at least seven groups according to amino acid sequence homology and specific structural features. Until now, the protective functions of group1～3 LEA proteins in plants and non-plant organisms from abiotic stresses have been proposed on the basis of those results from expression profile, localization analysis, transgenic studies and protein characterization. However, it is unclear about the protective functions and function mechanism of LEA4 proteins.The ORF of GmPM1 （accession no. M80666） encodes a polypeptide of 173 amino acid residues, and that of GmPM9 （accession no. M97285） encodes a polypeptide of 152 amino acid residues. GmPM9 shares high homology with GmPM1, except for 23 amino acid deletion. Both proteins belong to LEA4 proteins and contain high proportion of His residues, with percentages of 5.8 % and 4.6 % of the total residues, respectively. This implied that GmPM1 and GmPM9 proteins would be metal-binding proteins.GmPM1 and GmPM9 cDNAs were isolated from soybean （Glycine max L.Merr.cv Bainong 6#） immature seeds. The full-length GmPM1 and GmPM9 were cloned into pET28a vector, and then transformed into E.coli BL21 Star to get strain BL/GmPM1 and BL/GmPM9. Using the affinity chromatography technology, the proteins with 6×His tag were isolated and purified. Thrombin was added into the proteins to cleave the N-terminal 6×His peptide. The metal binding properties of GmPM1 and GmPM9 were tested using immobilized metal ion affinity chromatography （IMAC）. Fe³⁺, Ni²⁺, Cu²⁺ and Zn²⁺, bound to them, but Ca²⁺, Mg²⁺ and Mn²⁺ did not. Furthermore, the interaction between the two LEA4 proteins and metal ions was quantitated by ITC. Results indicated that the binding affinity of the GmPM1 to Fe³⁺ was stronger than that of GmPM9. Both GmPM1 and GmPM9 maintained their disordered structures after binding Fe³⁺.We tested whether the Fe-promoted Fenton reaction could be attenuated by GmPM1 and GmPM9 using the 2-deoxyribose degradation assay. The results showed that hydroxyl radicals generated by the Fe³⁺/H2O2 system were scavenged by both GmPM1 and GmPM9 in the absence or the presence of high ionic conditions （100mM NaCl）, although the scavenging activity of GmPM1 was significantly greater than that of GmPM9. The antioxidative activity of GmPM1 was approximately similar to that of BSA, one of the most efficient protein antioxidants in mammalian blood. The antioxidative activity of GmPM9, however, was most similar to that of mannitol, which was effective at high concentration. These results indicated that both GmPM1 and GmPM9 could reduce the levels of hydroxyl radical generated by the Fe³⁺/H2O2 system in site-specific assay. In non-site-specific assay, however, both GmPM1 and GmPM9 no longer inhibited the deoxyribose degradation. These results suggested that GmPM1 or GmPM9 could inhibit the deoxyribose degradation by binding Fe³⁺. Therefore, it is reasonable to speculate that binding of ions by LEA4 proteins in vivo plays an important protective role in reducing oxidative damage and ion toxicity in plant exposed to abiotic stresses.GmPM1 and GmPM9 proteins could bind Cu²⁺, Zn²⁺ and Ni²⁺ in vitro. ITC results indicated that the binding affinity of GmPM1 or GmPM9 to Zn²⁺ or Ni²⁺ was weaker （K< 105）. The binding affinity of GmPM1 to Zn²⁺ was stronger than that of GmPM9. The binding affinity of the GmPM1 to Ni²⁺ was similar to that of GmPM9. The high affinity of GmPM1 to Cu²⁺ （K > 105） was also quantitatively confirmed by ITC. Both GmPM1 and GmPM9 maintained their disordered structures after binding Cu²⁺ or Zn²⁺.Here, we further analyze the protective effects of LEA4 protein on LDH （lactate dehydrogenase） enzyme activity and Tubulin DNA in vitro freezing. LDH enzyme protection assay was performed in the presence of GmPM1 and GmPM9 at malar ratios of 10:1. After five cycles of freeze-thawing, the residual activity of LDH enzyme were 63.2% and 61.7%. GmPM1、Em （LEA1） and GmPM30 （LEA3） protein can also protect DNA unders freeze-thawing conditons.In this study, the matal-binding properties and the protective effect on biological macromolecules of two soybean LEA4 proteins have been studied. Our results provide a new insight to understand the protective functions and function mechanism of LEA4 proteins.