LEA Proteomic Analysis And Functional Study In Wheat(Triticum Aestivum) Under Drought Stress

Drought is a major factor affecting wheat growth and yield.The level of drought tolerance of plants has been reported to positively correlate with the expression of drought-induced genes.Some important functions in drought tolerance have been suggested for drought-induced genes.Late embryogenesis abundant?LEA?proteins are a group of extremely hydrophilic polypeptides,which accumulate to high levels in seeds during the late stage of embryogenesis and in vegetative tissues under dehydration stress conditions such as drought,high salt and low temperature.LEA proteins are thought to protect proteins,nucleic acids and membrane lipids under water-deficit conditions.Studies on the expression pattern,structure and function of LEA proteins can provide theoretical basis for further revealing the molecular mechanism of drought resistance of wheat and genetic breeding.In this study,the expression patterns of LEA protein were analyzed in two wheat varieties with different drought-tolerant under drought stress by label-free quantitative proteomics,and LEA proteins related to drought-resistance of wheat were identified.The physicochemical properties and functions of LEA proteins identifed were analyzed.According to the dates of proteomics analysis,a new TaLea14-A gene of LEA5C family and two genes encoding LEA-like proteins,i.e.wsh1 and wsh2,were cloned in wheat.The structure and functions in abiotic stresses of TaLea14-A and WSH1 protein were analyzed.The main conclusions of the study are as follows:1.Proteomics analysis on wheat leaves two genotypes of two genotypes with different drought tolerance was performed.A total of 38 LEA proteins and homologs of LEA proteins were identified,which are classified into 8 different subfamilies,i.e.Dehydrin,LEA₄,LEA₁,LEA₂,ABA_WDS,LEA₄-like,a novel class of LEA proteins and unclassified.The results showed that the classifications and abundance of LEA and non-LEA proteins expressed were related to the growth conditions and genotypes of wheat.Of the 38 LEA proteins identified,32 were constitutively expressed in Shaanhe 6 or both two genotypes of wheat,indicating that they were involved in the normal growth and development of wheat seedlings.The abundance of 20 LEA proteins changed significantly after drought stress,indicating that they were related to drought-tolerance of wheat.2.Transcription levels of 13 representative LEA genes were analyzed by RT-qPCR.After drought stress,the transcription levels of all LEA genes identified in Shaanhe 6 wheat were coincident with the expression levels of their encoding proteins.In Zhengyin 1 wheat,the situation is different.Three LEA genes,O65216,M7ZBB0 and M7YN16,showed different trends in transcription and coding protein expression.3.LEA related genes TaLea14-A and wsh1 were cloned from wheat leaves under drought stress.Analysis showed that TaLea14-A protein belongs to the LEA5C subfamily,with a WHY domain and is weakly hydrophilic.WSH1 protein has a high homologous similarity with LEA proteins without any known domain of LEA protein families,which is highly hydrophilic and has a coiled coil domain.WSH1 protein is a new LEA-like protein from wheat with similar properties with LEA proteins.Both TaLea14-A and WSH1 protein were located in nucleus,cytoplasm and cell membrane in the protoplast of rice cell.4.As RT-qPCR showed,the expression of TaLea14-A in wheat was induced by ABA,drought,high salt and low temperature stress.While,the expression of wsh1 was induced by ABA,drought and high salt stress,but inhibited by low temperature stress.5.As FTIR spectroscopy indicated,TaLea14-A in D₂O formed more?-sheet elements,as well as?-helix and random coil elements.After fully dehydration,the aggregation of TaLea14-A protein was not induced,but the content of?-sheet and?-turn elements significantly increased.The?-helix elements were prominently in hehydrated WSH1.While,the structure of dry WSH1 changed and formed more?-helix elements,as well as?-sheet and?-turn elements.It indicated that the structures of TaLea14-A and WSH1 changed after dehydration.6.In vitro,TaLea14-A effectively inhibited the inactivation of LDH under freeze-thaw,high temperature and dehydration stress.Opposite to TaLea14-A,WSH1 protein reduced activity of LDH and increased damage to LDH under freeze-thaw,high temperature and dehydration stress.As FTIR spectroscopy showed,a handful of intermolecular?-sheet aggregates were formed in dry LDH,suggesting that aggregation was not the main factor caused inactivation of LDH under dehydration condition.TaLea14-A might protected LDH activity by additional mechanism,in addition to by inhibiting aggregates.7.The overexpression of TaLea14-A in escherichia coli enhanced the tolerance of escherichia coli to low temperature,high temperature,high salt,oxidative and PEG mimicked drought stress,and promoted the growth of escherichia coli under normal condition.Studies on transgenic arabidopsis thaliana showed that transgenic plants showed higher drought-tolerance than wild type under drought stress.Such as proline accumulation,antioxidant enzyme activity increased.In vivo prokaryotic overexpression proved that WSH1 showed inhibition on the growth of escherichia coli under drought,high salt and oxidation stress and has stronger inhibition compared to under normal condition.Moreover,WSH1 reduced the viability of escherichia coli under low and high temperature stress.These indicate that WSH1 protein is a protein with antibacterial activity.