Functional Study Of VaPAT1, A GRAS Gene From Vitis Amurensis, In Response To Abiotic Stresses

Grapevine (Vitis vinifera L.) is one of the most important fruit crops in our country. The publication of the grape genome sequence lays an important foundation for genome-wide identification of genes related with excellent agronomic traits, stress tolerance or disease resistance in grape. GRAS (GAI, RGA and SCR) transcriptional factor family regulates diverse processes involved in plant growth, development and stress responses. However, far less is currently known about the function of GRAS proteins in stress response in grape. In our present study, the grape GRAS transcriptional factor family members were identified and classified in genome-wide level by bioinformatics methods. Moreover, the function of VaPAT1, a GRAS gene cloned from V. amurensis, was studied by molecular biology methods, which preliminary revealed the molecular regulatory mechanism of VaPAT1 in response to abiotic stress in plants. The main results obtained are as follows:1.41 putative grape GRAS genes were identified in 12× V. vinifera cv. Pinot Noir genome and mapped onto 15 grape chromosomes except for Chr3, Chr5, Chr10 and Chr16. Meanwhile, two GRAS gene clusters were found, one is located on Chr6 and the other is located on Chrl3. Grape GRAS family were classified into 9 groups, including PAT1 (phytochrome A signal transduction), LS (lateral suppressor), SCL4/7 (scarecrow-like 4/7), HAM (hairy meristem), SHR (short-root), SCL3, SCL9, SCR (scarecrow) and DELLA.2. VaPAT1 presented close evolutionary relationship with CmsGRAS from Citrus medica and OsCIGR1 from Oryza sativa. Promoter prediction of VaPAT1 showed it contained many stress and phytohormone responsive cis-acting regulatory elements, including ARE (anaerobic inducible element), GARE (GA response element), HSE (heat stress responsive element), TCA and TC-rich repeats.3. VaPAT1 responded to ABA (abscisic acid), GA (gibberellic acid) and SA (salicylic acid) treatments. The expression of VaPAT1 decreased under GA application, similar to a typical feature of DELLA branch GRAS genes. VaPAT1 transcripts increased under cold, drought and high salinity treatments. Notably under high salinity treatment simulated by 200 mM NaCl, the VaPATl transcripts at 48 h was 22 fold higher than that before salt treatment. These results suggest that VaPATl is involved in and may play an important role in abiotic stress responsive processes.4. Yeast two-hybrid analysis revealed that VaPATl has transcriptional activation activity. The survival rates of VaPAT1 transgenic lines were significantly higher compared with WT (wild type) Arabidopsis under cold, drought and salt stress, indicating that VaPATl functions as a positive transcriptional regulator in abiotic stress responses. The VaPAT1-overexpression lines accumulated higher levels of proline and soluble sugar than WT under these stress treatments, these may be vital reasons for the enhanced stress tolerance in transgenic Arabidopsis.5. qRT-PCR analysis showed that VaPATl overexpression not only affected the expression of downstream stress-related genes, like AtCOR15A、AtRD29A and AtRD29B, but also important upstream transcription factors in stress response pathways, including AtSIZ1, AtCBF1, AtATR1/MYB34, AtMYC2. Besides, ABA synthesis related gene AtNCED3 and ABA signal transduction related gene AtCIPK20 were significantly higher in VaPATl transgenic lines than in WT, indicating that VaPATl may be involved in the ABA dependent stress response pathways.