| Drought stress is a primary factor of reduced crop productivity due to serious disruptions in plant growth and development.Therefore,plants have evolved complex regulatory mechanisms in their defense responses to adverse conditions.All mechanisms are based on proteins that directly function in abiotic tolerance and that regulate various signaling pathways to indirectly improve abiotic tolerance.The late embryogenesis abundant(LEA)protein gene family is an important group of functional proteins to reduce cell damage and protect cells under abiotic stress conditions.we report the first comprehensive survey of the LEA gene family in “Chinese Spring” wheat(Triticum aestivum).A total of 179 TaLEA genes were identified in T.aestivum and classified into eight groups.All TaLEA genes harbored the LEA conserved motif and had few introns.TaLEA genes belonging to the same group exhibited similar gene structures and chromosomal locations.Our results revealed that most TaLEA genes contained abscisic acid(ABA)-responsive elements(ABREs)and various cis-acting elements associated with the stress response in the promoter region and were induced under ABA and abiotic stress treatments.In addition,eight genes representing each group were introduced into E.coli and yeast to investigate the protective function of TaLEA proteins under heat and salt stress.TaLEAs enhanced the tolerance of E.coli and yeast to salt and heat,indicating that these proteins have protective functions in host cells under stress conditions.In order to study the mechanism of LEA protein under drought stress,we isolated a c DNA segment of bHLH49-like protein interacting with dehydrin WZY2/Ta DHN20 promoter performed by yeast one-hybrid(Y1H)assay,TabHLH49 protein possesses a typical conserved bHLH domain and is a Myc-type bHLH transcription factor.TabHLH49 was detected in the nucleus of tobacco epidermal cells,and the amino acid sequences at the C-terminus(amino acids 323–362)is necessary for its transactivation activity.Real-time PCR analyses revealed the tissue-specific expression and drought stress-responsive expression of TabHLH49 in wheat.In addition,the verification in Y1 H and electrophoretic mobility shift assays illustrated that TabHLH49 protein can bind and interact with the promoter of the wheat WZY2 dehydrin.Furthermore,the dual-luciferase assays showed that TabHLH49 can positively regulate the expression of WZY2 dehydrin.The transient expression and BSMV-mediated gene silencing of TabHLH49 also showed that TabHLH49 positively regulates the expression of WZY2 dehydrin and improves drought stress resistance in wheat.The main results of the experiment are as following:1.A total of 179 LEA proteins were identified from wheat based on a Pfam ID search of wheat genome databases and homologous sequence alignment with LEA genes from A.thaliana and T.aestivum.The TaLEA genes were classified into eight subfamilies based on their conserved domains.The LEA?2 and DHN families were the largest,with 49 and 50 members,respectively.The LEA?1,LEA?3,LEA?4,LEA?5,and SMP families included 16,15,24,13,and 9 members,respectively.The LEA?6 family included only 3 members.The TaLEA genes encode polypeptides of 89–1062 amino acids in length,with predicted molecular weights ranging from 9.1 to 108.7?k Da.The calculated grand average of hydropathy index(GRAVY)values suggested that 151 proteins(84%)are quite hydrophilic and that 28 members(16%)are hydrophobic,with the latter group of proteins all belonging to the LEA?2 family.2.The cis-acting elements in the TaLEA gene promoters were identified by Plant CARE database.We identified the cis-elements associated with plant hormone responses,including ABRE,CGTCA motif,TGACG motif,TGA-element,ERE,GARE and TCA elements.At the same time,we also identified a large number of cis-elements related to plant stress response,such as MBS,MYC,W box,TC-rich repeats,DRE,LTRE,STRE and WRE.3.Microarray analysis of TaLEA genes indicated that the expression of 157 TaLEA genes was induced under cold stress.Among these,46 genes were upregulated by cold stress.A total of 177 TaLEA genes were induced by drought.Among these,50 TaLEAs were upregulated by drought stress.Twenty-five TaLEA genes were induced by high salinity.Under hightemperature stress,there was no obvious change in the expression of most TaLEA genes.Realtime results indicated that 22 TaLEA genes were upregulated after ABA,PEG6000,salt,heat and cold treatments.4.For the salt treatment,E.coli strains carrying TaLEA?3-3,TaLEA?4-1,TaLEA?6-2and Ta DHN43 had mean viability ratios 2-7-fold higher than those of the control strain under salt stress.For the heat treatment,E.coli strains carrying six(TaLEA?1-5,TaLEA?3-3,TaLEA?4-1,TaLEA?5-1,Ta SMP8,and Ta DHN43)of the eight TaLEA genes had mean viability ratios 2-3-fold higher than those of the control strain under heat stress.5.The verification in yeast one-hybrid(Y1H)and electrophoretic mobility shift(EMSA)assays illustrated that TabHLH49 protein can bind and interact with the promoter of the wheat WZY2 dehydrin.Furthermore,the dual-luciferase assays showed that TabHLH49 can positively regulate the expression of WZY2 dehydrin.6.TabHLH49 was detected in the nucleus of tobacco epidermal cells,and the amino acid sequences at the C-terminus(amino acids 323–362)is necessary for its transactivation activity.7.The transient expression and BSMV-mediated gene silencing of TabHLH49 also showed that TabHLH49 positively regulates the expression of WZY2 dehydrin.TabHLH49 reduced MDA content,increased relative water content and chlorophyll content under drought stress in wheat.8.Under normal growth and drought stress conditions,both the shoots and roots of wildtype wheat were longer than the transgenic lines.Wild-type(WT)wheat showed obvious wilting and curling than the transgenic lines under drought stress.Transgenic lines showed significantly lower MDA content,higher relative water content and higher chlorophyll content than WT lines under drought stress conditions... |
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