Studies On The Mechanism Of Abscisic Acid-priming For Alkaline Stress Tolerance In Rice

Saline-alkaline?SA?stress represents a complex effect of ion toxicity from high salinity,alkalinity?high pH?,and high osmotic pressure?low water availability?;of these,alkaline stress was identified as the primary factor inhibiting rice seedling growth.Alkaline stress results in severe cellular damage to the root system and seedlings growth,and these damaging effects on rice roots have been correlated with the overaccumulation of reactive oxygen species?ROS?.The plant hormone abscisic acid?ABA?plays vital roles in enabling plants to cope with various environmental stresses.ABA has been shown to prime rice seedlings for enhanced tolerance to alkaline stress under both greenhouse and field conditions.However,the mechanism by which ABA priming protects rice seedlings from alkaline damage remains unclear.Therefore,this study was aimed to explore the physiological and molecular mechanisms of ABA priming for alkaline tolerance in rice seedlings by the clue of ROS overaccumulation damage rice roots under hydroponical.Furthermore,the effect of endogenous ABA levels on alkaline tolerance was investigated using transgenic rice lines with RNAi-mediated suppression of OsABA8ox1,a key ABA catabolic gene?OsABA8ox1-RNAi?.All rice lines were grown hydroponically and in soils with different SA levels,namely moderate?pH:7.59?,medium?pH:8.86?and severe?pH:9.29?conditions to investigate the effect and mechanisms of endogenous ABA levels on alkaline tolerance.The main results were as below:1.ABA pretreatment significantly reduced the alkalinity-induced ROS accumulation,increased the antioxidant enzyme activities and expression of ROS-scavenging genes,mitigated root damage and improved the survival rate of rice seedlings under alkaline conditions.Paraquat?2.5–25?M?was used as an intracellular generator of ROS to investigate the effect of ABA on ROS accumulation.Furthermore,ABA pretreatment reduced paraquat-induced ROS accumulation and seedling injury and mortality as shown by 23-95%decrease of O₂^·-,and17-30%decrease of H₂O₂.2.In addition,the expression of the ABA-responsive genes SalT and OsWsi18,as well as the stress tolerance-related genes OsJRL,OsPEX11,OsNAC9,OsAKT1 and OsHKT1,was superinduced by ABA pretreatment under alkaline conditions.For the two cultivars,alkaline stress-induced injury indexes of D-4 were higher than that of J-51.3.To further investigate the effect of endogenous ABA levels on alkaline tolerance,we produced transgenic rice lines with RNAi-mediated suppression of OsABA8ox1 in two different genetic backgrounds,D4 and Nipponbare?NB?.The transgenic lines with reduced OsABA8ox1 expression,and resultantly increased ABA levels,were selected and used in this study?OsABA8ox1-RNAi D4#2 and#4,and NB#4 and#7?.4.Results showed that,compared with non-transgenic wild type control plants?WT?,OsABA8ox1-RNAi lines showed enhanced tolerance to alkaline stress as shown by 5-80%increase in seedlings survival rates,11-85%increase in biomass accumulation,and reduced alkaline-induced roots growth inhibition.Furthermore,alkali-induced plasmalemma injury,ROS accumulation,and Na⁺/K⁺ratio were significantly reduced,while expression levels of ROS-scavenging genes were enhanced in OsABA8ox1-RNAi lines compared with WT plants under alkaline conditions.In addition,expression of the ABA-responsive genes,as well as the stress tolerance-related genes were superinduced in OsABA8ox1-RNAi lines under alkaline conditions.5.Results showed no appreciable difference in SA-tolerance between the OsABA8ox1-RNAi lines and WT plants under moderate and medium SA soil conditions.However,in sever SA soils,OsABA8ox1-RNAi lines showed significantly enhanced tolerance as shown by 4-60%increase in seedling survival rates,while16-19%decrease in withered seedling rates.The OsABA8ox1-RNAi lines also showed a more vigorous plant growth compared with WT plants under severe SA soil conditions;20%increase in shoot length,20-121%increase in chlorophyll content,1-3 times increase in biomass accumulation,and a significant increase in root length.At the harvest time,OsABA8ox1-RNAi lines obtained significantly higher yield components including panicle number,spikelets per panicle,branches,1000-grain weight,and percentage filled spikelets,under sever SA soil conditions.Taken together,these results suggested that ABA priming confers enhanced tolerance to alkaline stress in rice seedlings via a mechanism that potentiates the downstream antioxidant defense system,inhibit ROS overaccumulation and upregulate stress tolerance-related gene expression.Furthermore,suppression of OsABA8ox1,so as to increase endogenous ABA levels and improve rice tolerance to alkaline stress,achieving the same effect with the priming effect of exogenous ABA,provide an effective approach for improving plant growth and grain yield of rice in SA soil fields.