Multi-protective Functions Of GmASR Protein-a Soybean Intrinsically Disordered Protein

The abiotic stresses, such as salt, drought and heavy metal ions can seriously restrict plantgrowth and development. Some proteins would be induced to reduce the damage when theplants under stresses. The plant LEA （Late embryogenesis abundant） protein is one group ofthe most important proteins related to the stresses. The LEA proteins are divided into7groupsby the virtue of conserved motifs. The ASR （Abscisic acid stress and ripening-induce） proteinsare considered to be the7member of LEA protein （LEA7）. Transgenic plants expressingheterologous ASR genes showed higher drought and salt tolerance than wild types. However，theprotective functions and mechanisms of ASR protein need further study.The soybean GmASR gene, which was cloned from the Glycine max L. Merr.cv Bainong6#, encodes a polypeptide of236amino acid residues and contains high proportion of Hisresidues, with percentages of9.2%of the total residues. The yeast expression plasmids ofpYES2/GmASR was constructed and was transformed into the copper-sensitive yeast mutantCUP2, zinc-sensitive yeast mutant ZRC1or salt-and osmotic-sensitive mutant HOG tocreate recombinants of CUP2/GmASR、 ZRC1/GmASR and HOG/GmASR. Theexpressions of GmASR gene in that recombinants were verified by RT-PCR, and the growth ofrecombinants were monitored under different stresses. The results indicated that the expressionof GmASR protein could confer Cu²⁺tolerance in yeast CUP2and Zn²⁺tolerance in yeastZRC1. We also proved that the GmASR protein could elevate the NaCl tolerance but did notenhance the osmotic stress of yeast HOG.The GmASR gene was inserted into the pCAMBIA1300vector and transformed intotobacco BY-2cell. The growth of transgenic BY2cells in noraml or100μM Cu²⁺culture weremonitored. The results demonstrate that the expression of the GmASR protein could confer theCu²⁺tolerance in tobacco cell BY-2.The full-length GmASR gene was cloned into pET28a vector, and then transformed intoE.coli BL21Star to gain strain BL/GmASR which expressed the un-tagged, full-length GmASRprotein. The GmASR protein was induced by IPTG and purified by using affinitychromatography technology. Bioinformatics and biochemical analysis showed that GmASRprotein is disordered in aqueous solutions.The metal-binding characteristics of GmASR wereinvestigated by using immobilized metal ion affinity chromatography （IMAC）. The resultsindicated that the GmASR protein could bind Fe³⁺, Ni²⁺, Cu²⁺and Zn²⁺but did not bind Ca²⁺,Mg²⁺and Mn²⁺. The direct binding properties of GmASR to Fe³⁺/Zn²⁺were further confirmed byintrinsic fluorescence assays. Furthermore, the interactions between the GmASR protein and theFe³⁺/Zn²⁺were quantitated by ITC. Results indicated that the GmASR protein have three Fe³⁺binding sites, but only two Zn²⁺binding sites. Natively disordered in aqueous solution, GmASR remained disordered in the presence of Fe³⁺, but was found to aggregate in the presence of Zn²⁺.The aggregated GmASR protein could be partially resolubilized after Zn²⁺was chelated byEDTA.To determine whether the GmASR protein can inhibit hydroxyl radical generation, theeffect of GmASR on hydroxyl radicals generated by Fe³⁺was analyzed by measuring the degreeof deoxyribose degradation. These results indicate that GmASR protein can reduce the levels ofhydroxyl radical generated by the Fe³⁺/H2O2system via a site-specific mechanism. Theanti-oxidative activity of the GmASR protein was found to be much higher than that of BSA. Wealso proved that the GmASR protein could protect DNA against oxidative damage. Because thehydroxyl radicals was generated by EDTA-Fe³⁺/H2O2system when EDTA was added, theGmASR protein had no protective effect （non site-specific way）. These results indicate that theGmASR protein blocks the generation of hydroxy1radicals by chelating Fe³⁺ions.In in vitro experiments, we analyzed the protective function of GmASR protein on LDHenzyme udner freeze-thaw, heat and dehydration stresses. The results indicated that the GmARprotein is capable of preventing the inactivation and aggragation of LDH enzyme underfreeze-thaw stress. The GmASR protein also could proctect the LDH enzyme activity from thedehydration stress, but had no proctecting roles in heat stress.In conclusion, the expression of GmASR protein could enhance the heavy metal toleranceof tobacco BY-2and yeast cells. The IDP characteristics, metal-binding properties andprotective functions of GmASR protein have been studied. Our results could help to clarify theprotective functions and the mechanism of GmASR protein, which also provide the evidencefor the hypothesis that the IDP protein could have multifunction.