Characterization And Functional Analysis Of Trihelix Transcription Factors Responsive To Abiotic Stresses In Cotton

1. ObjectivePlant growth and productivity are under constant threat from environmental changes in theform of biotic and abiotic stress factors. The most common abiotic stresses are high salinity, lowtemperature and drought, which limit the crop productivity, reducing average yields for mostmajor crop plants by more than50%. In molecular breeding, a key transcription factor cansimultaneously regulate the expression of many functional genes to improve tolerance of crops tovarious stresses. Trihelix transcription factors are a small family only identified in plants. Theycan interact with cis-elements present in the promoter regions of several stress-related genes andregulate their expression to enhance plant stress tolerance. Therefore, trihelix transcription factorsplay a critical role in stress signaling transduction pathways. Cotton is an important crop plant forboth textile and oil, but its growth, yield and fiber quality were affected by various abiotic stresses,such as drought, salinity and low-temperature, doing great harm to cotton production.Understanding the molecular mechanisms of stress tolerance in cotton will lay the foundation forimproving the stress tolerance of cotton by gene manipulation. Based on the EST sequences, wecloned several GT genes responsive to abiotic stresses from cotton and their functions were furtheranalyzed.2. Methods and resultsBy doing BLAST(the Basic Local Alignment Search Tool) search the cotton ESTs database,28GT unigenes, named GhGT1to GhGT26, GhGT29and GhGT30, were obtained. Theexpression profiles of these unigenes under the treatments of drought, salinity, cold and exogenoushormone ABA in Xin-lu-mian26were investigated by both RT-PCR and qPCR method. SevenGhGT unigenes including GhGT7,11,18,23,26,29and30were chosen for further analysis. Thefull-length cDNA of these GhGTs genes were cloned using the method of Rapid amplification ofcDNA Ends (RACE). Based on the deduced protein domains and conserved amino acid residues,these GhGT proteins were classified into different subfamilies: GhGT7and GhGT30belong toGT-2; GhGT11and GhGT23belong to SIP1; GhGT26belongs to GT-1; GhGT29belongs to SH4subfamily and GhGT18belongs to MYB transcription factor family. Although7GhGT genes were ubiquitously expressed in root, stem, leaf, flower, ovule (0DPA) and fiber (12DPA), theirexpression patterns were different. The highest expression level was observed in leaves forGhGT7, in ovules for GhGT18, in fibers for GhGT23and in flowers and fibers for GhGT26,GhGT29and GhGT30. The sub-cellular localization assay in the Arabidopsis protoplasts andonion epidermal cell system revealed that GhGT7,11,18,23,26,29and30proteins werelocalized in the nucleus, indicating that they are nuclear proteins. Using the Arabidopsisprotoplasts assay system, we found that GhGT29protein had trans-activation activity, GhGT18showed trans-repression activity, while no trans-activation activity was detected for GhGT7andGhGT23. Electrophoretic Mobility Shift Assay（EMSA）showed that GhGT7,11,23and26proteins could bind to both GT and MYB elements. Specifically, GhGT7protein could bind toGT-1box, GT2-Box, GT-3b, MBS1, MRE1, MRE3and MRE4; GhGT11protein could bind toSite1, GT-1box, GT2-Box, GT3-Box, GT-3b and MRE3; GhGT23protein could bind to Box,Site1, GT-1box, GT2-Box, GT3-Box, GT-3b, MBS1and MRE4, and GhGT26protein could bindto Site1, MRE3, MRE4, respectively. Employing agrobacterium-mediated transformationtechnique, GhGT7,11,18,23,26and29genes were successfully introduced into Arabidopsisthanliana to generate transgenic plants, respectively. The expression level of transgene wasanalyzed for each homozygous transgenic line. Performance of transgenic plants overexpressingGhGT11,18,23and26genes under abiotic stresses were studied, respectively. Under normalgrowth condition, no visible phenotype difference between wild-type plants and transgenic plantsoverexpressing each GhGT gene was observed. Overexpression of GhGT23and GhGT26significantly enhanced the tolerance of transgenic Arabidopsis plants to high-salinity and droughtstresses compared with wild-type plants, respectively. However transgenic plants overexpressingGhGT11and GhGT18showed sensitivity to high salinity and drought stresses, respectively. Underdrought and high salinity treatments, the performance of root growth of wild-type plants wasbetter than that of transgenic plants overexpressing GhGT18and GhGT11, while the performanceof root growth of transgenic plants overexpressing GhGT23and GhGT26was better than that ofwild-type plants. Compared to wild-type plants, ABA treatment promoted the root growth oftransgenic plants overexpressing GhGT23, but inhibited the root growth of transgenic plantsoverexpressing GhGT11. Transgenic plants overexpressing GhGT18and GhGT26were notsensitive to ABA treatment. Overexpression of GhGT23improved the tolerance of transgenicplants to abiotic stresses probably through up-regulating the expression of downstream genesincluding DREB1B, COR6.6, COR47, RD22, SAP18, COR15A, DREB2A, DREB2B, STZ, AP2andDREB2C.3. ConclusionIn short, seven GhGTs genes responsive to abiotic stresses were identified and cloned, andbiochemical function of their proteins were characterized. Overexpression of each of the four GhGTs genes including GhGT11, GhGT18, GhGT23and GhGT26affected the tolerance oftransgenic plants to abiotic stresses. The possible mechanism of GhGT23-mediated improvementof transgenic plants to abiotic stresses was further discussed.