Research On AsHSP26.8-mediated Response To Abiotic Stress In Creeping Bentgrass

In recent years,extreme environments such as high temperature,drought and salinity have led to the decline of crop yield and quality.It is an urgent problem for researchers to explore the mechanism of plant response to abiotic stress and enhance the ability of crops to cope with stress.Small heat shock proteins(sHSPs)are ubiquitous stress expressed proteins in plants,which play an important role in plant growth and development and abiotic stress response.AsHSP26.8 is a small heat shock protein gene located at chloroplast in creeping bentgrass.In this study,AsHSP26.8 was successfully introducing into creeping bentgrass by Agrobacterium-mediated transformation.The transgenic creeping bentgrass was taken as the research object to explore the regulation mechanism of AsHSP26.8-mediated plant response to high temperature,salinity and drought stress.The main results are as follows:1.AsHSP26.8 has previously been categorized as a chloroplast-localized sHSP.To confirm this,an AsHSP26.8-GFP fusion construct was introduced into rice protoplasts.Similar to its homologous gene in Arabidopsis,AtHSP21,green fluorescence was observed localized in chloroplasts and appeared to be associated with thylakoid membranes.2.We evaluated the performance of transgenic(TG)plants overexpressing AsHSP26.8 under heat stress.After 10 days of heat treatment,AsHSP26.8 TG plants exhibited severe leaf damage and became largely wilted and yellow,while wild-type(WT)controls showed only minor heat-elicited symptom.In addition,very few TG plants survived from the heat stress,while all the WT controls completely recovered and grew normally after 28 days of recovery.TG plants have significantly lower chlorophyll content than WT controls under normal conditions.After 2 or 4 days of heat stress,the total chlorophyll content in TG plants remained significantly lower than that in WT controls.In addition,the RWC in TG plants was also significantly lower than that in WT controls,indicating a lack of water retention capacity in TG plants under heat stress.After 4 days of heat treatment,TG plants have significantly higher electrolyte leakage(EL)value than WT controls,indicating a more severe cell membrane injury in TG plants than in WT controls under heat stress.Although no significant difference in O2-content was observed between WT and TG plants under heat stress,the H2O2 content in TG plants was significantly higher than that in WT controls under heat stress,suggesting that TG plants have higher level of heat-elicited ROS accumulation than WT controls under heat stress.Quantitative real-time RT-PCR analysis showed that AsHSP17 is expressed at a higher level,whereas AsHSFA2 expressed at a lower level in AsHSP26.8 TG plants than in WT controls.These results suggest that AsHSP26.8 might affect the expression of other heat-responsive genes including HSFs and HSPs and thus contribute to the increased creeping bentgrass plant sensitivity to heat stress.3.We examined the performance of the AsHSP26.8-overexpressing TG plants and WT controls under salinity stress(200 mM NaCl).TG plants exhibited severe damage after 28 days of salt treatment;the leaf tissue was largely wilted with the loss of green colour,while WT controls remained green with much less damage.After 38 days of recovery,most WT plants recovered and became normal in growth,while only few TG plants survived the salt stress but also grew much slower than WT controls.The recovered WT controls have significantly longer shoot and higher biomass(fresh and dry weight of both shoot and root)than the survived TG plants.These results indicate that overexpression of AsHSP26.8 both increases plant susceptibility to and inhibits the plant growth recovery from salt stress.TG plants had significantly lower RWC than WT controls during salt stress treatment and at the early stages of plant recovery.The chlorophyll content in TG plants was lower than WT controls under normal condition.This difference became significantly more pronounced upon salt treatment.In addition,TG plants had significantly higher EL content and H2O2 accumulation than WT controls under salt treatment.TG plants accumulated significantly more Na+than WT controls at 2 and 4 days of salt treatment.Although there was a tendency of more K+ uptake in TG plants than in WT controls upon salt stress,especially at 4 days of salt treatment,lower K+:Na+ratio in TG plants than in WT controls was observed at both 2 and 4 days of salt treatment.HKT1 was significantly down-regulated,while SOS1 was slightly up-regulated in HSP26.8 TG plants compared to WT controls.These results suggest that HSP26.8 might regulate plant salt susceptibility by affecting Na+accumulation and Na+/K+ homeostasis in plant tissue.4.We examined the performance of the AsHSP26.8-overexpressing TG plants and WT controls under drought stress.Serious drought-elicited damage was observed in WT control plants after 14 days of drought treatment and most of the WT plants died from the water deficiency,while less severe symptoms caused by water withholding occurred in TG plants and most TG plants recovered from the drought-elicited damage.Plant water retention and cell membrane integrity analyses showed that AsHSP26.8 TG plants had significantly higher RWC and lower EL values than WT controls during the period of drought treatment and recovery.TG plants had significantly lower chlorophyll content than WT controls under normal condition,but after 14 days of drought treatment,the chlorophyll content in WT plants declined dramatically and became significantly lower than that in TG plants.These results indicate the role AsHSP26.8 plays in triggering plant drought response by coordinating enhanced capacity of water retention,maintenance of cell membrane integrity and chlorophyll biosynthesis under water-deficiency condition.AsABI5,AsDREB1B and AsDREB1C were up-regulated,while AsPP2C11 was down-regulated(WT/TG＝1/0.00026)in HSP26.8 overexpression transgenic creeping bentgrass compared to wild-type controls.These results indicate that AsHSP26.8 promotes the drought tolerance by modifying the expression of genes involved in both ABA-dependent and independent signalling pathways to coordinate the overall response in plants.