Functional Analysis Of Cotton GbMYB5 Gene Intolerance To Drought Stress

Drought stress negatively affects plant growth and limits plant productivity. MYB transcription factors play an important regulatory role in response to environmental stress in plants including drought stress. Knowledges on drought-resistant related MYB transcription factors are particularly important to crop breeding for drought resistance.A R2R3-MYB transcription factor (GbMYB5),which was induced by PEG, ABA and GA, has been cloned from Gossypium barbadense and transformed into the genome of Nicotiana tabacum under the control of CaMV35S promoter. In this study, the roles of GbMYB5 in responses to drought stress were characterized in G. barbadense by virus-induced gene silencing (VIGS) and in transgenic tobacco plants with over-expressing GbMYB5 gene.Virus-induced gene silencing of GbMYB5 compromised cotton plantlets tolerance to drought stress.Firstly, seven days after irrigation with lOmL 18% PEG6000 solution for 2 days and watering once, the GbMYB5-silenced plants showed severe wilt. In contrast, the non-inoculated (WT) and non-silenced (empty vector CLCrVA inoculated) control plants grew well and no wilt symptom occurred at this time. Secondly, after 35 days of water-withhold treatment and 7 days post of rewatering, all of the GbMYB5-silenced plants showed severe wilt and only half of them recovered from the wilt, while approximately 90% of the non-silenced plants recovered from the drought stress.Overexpressing GbMYB5 increased drought tolerance in transgenic tobacco. Two independent overexpression lines, TP1 and TP2, each showing a high level of GbMYB5 were selected by RT-PCR for assay of drought resistance. When under PEG stress, two transgenic lines exhibited higher tolerance than the WT. After 3 weeks of culture in 1/2 MS medium containing 2% of PEG, TP1 and TP2 plantlets built bigger shoots and roots than WT plantlets. Leaf discs from TP1 and TP2 still kept green when soaked in 25% PEG solution for 7 days, in violently contrast to the yellow leaf discs from the WT. In the analysis of whole plant’s tolerance to drought stress, TP1, TP2 and WT plants were respectively water-withheld for 35 days, and all the plants wilted completely. And 7 days post re-watering once, most of transgenic plants recovered with a survival rate of 70-80%, which was significantly higher than that of 30% in WT plants.The first-to-three fully expanded leaves of TP1, TP2 and WT plants under normal growing conditions were detached and measured the rate of water loss. The two transgenic lines TP1 and TP2 showed a slower rate of water loss than the WT. In the term of relative water content (RWC), TP1, TP2 and WT plants had similar RWC (88-90%) at the beginning of water-withhold. After 7 days of drought stress, RWC in TP1 and TP2 decreased to nearly 73-74%, whereas the WT had decreased to nearly 66%. The stomatal number and stomatal size were measured in abaxial leaf surfaces collected from the TP1 and WT plants. No significant difference of stomata number was observed between the transgenic and wild type plant leaves. However, the stomatal size was markedly decreased in TP1 plants. The average stomatal dimension was 33.5 by 112.8 μm for TP1 in contrast with 38.2 by 120.6 μmfor WT. Furthermore, the ratio of opening stomata was significantly lower in TP1 than that in WT under water-withhold stress. Under normal conditions, the activities of POD and SOD were similar among TP1, TP2 and WT plants. After 7 days of drought stress, there were marked increases in the activities of POD and SOD in the overexpressing GbMYB5 plants, while the WT plants exhibited non-significant increased activities of POD and SOD. qRT-PCR analysis revealed that higher expression levels of 6 stress responsive genes including genes encode enzymes for direct ROS detoxification (NtCAT, NtSOD and NtGST), enzymes involved in biosynthesis of polyamine (NtADC1 and NtSAMDC) and stress defensive protein (NtERD10D) were significantly higher in transgenic lines than that in the WT. These results indicated that the GbMYB5 overexpressing plants tolerated the drought stress by reducing water loss under drought stress, and by higher induction of antioxidant enzyme, protective compounds (polyamines) or proteins (LEA), leading to alleviation of cellular damage when they were subjected to drought stress.Overexpressing GbMYB5 increased ABA Sensitivity in transgenic tobacco. Seeds of TP1, TP2 and WT were germinated in 1/2 MS supplemented with various concentrations (0,1,3 and 5μM) of exogenous ABA. In the absence of exogenous ABA, TP1 and TP2 germinated as well as WT. However, on the media supplemented with 3 or 5 μM of ABA, TP1 and TP2 showed hypersensitivity to ABA-elicited inhibition of seed germination, suggesting that GbMYB5 may involve in the drought reponse mediated by ABA signal transduction.