A Preliminary Exploration On The Mechanism Of OsNCED Gene Modulating Salt Tolerance In Weedy Rice

As a companion weed of cultivated rice,weedy rice(Oryza sativa F.spontanea)often competes with cultivated rice for various natural resources in paddy field.Weedy rice can be a good germplasm resource for rice crop improvement,on the one hand,has a complex genetic base;on the other hand,through long-term natural and artificial selection,it has multiple resistance compared with cultivated rice.As a stress hormone in plants.Abscisic acid(ABA)known as a stress hormone in plants and 9-cis-epoxycarotenoid dioxygenase(NCED)is a key rate-limiting enzyme in the ABA biosynthesis pathway in higher plants.However,how does NCED gene modulate rice salt tolerance and used it to develop salt tolerance rice are still challenge.Here,we use the Osnced3 and Osnced5 transgenic lines(knockout lines of OsNCEDs from salt-tolerant weedy rice WR04-6),to analyze the differenent sensitivity under exogenous ABA between WR04-6 and transgenic slines,combining the differences in morphological,physiological and expression of related anti-stress genes between WR04-6 and transgenic strains under salt stress.Based on these,we initially explore the mechanism of NCED gene modulation of salt tolerance in weedy rice and shed a light for molecular improvement on salt tolerance rice.The main findings are as follows:1.Evaluation of salt tolerance of Osnced3 transgenic linesIndoor hydroponic experiments reveal that,compared with non-transgenic wild type control plants(WT),nced3-90 mutant shows enhance ABA sensitivity as show by 0.89%-23.99%increase in reduction of plant height under different concentrations of exogenous ABA treatment.Under different concentrations of salt stress,nced3-90 mutant shows enhance tolerance to salt stress as shown by 8.33%-28.10%increase in seedlings survival rates,0.32%-19.42%increase in biomass accumulation and reduce salt-induced shoot growth inhibition.In addition,0.19%-14.51%increase in osmoregulatory substance content and 0.18%-16.5%increase in antioxidant enzyme activity.Furthermore,salt-induced membrane peroxidation,ROS accumulation,and Na+/K+ratio are significantly reduced,while expression levels of related anti-stress genes are enhanced in nced3-90 mutant compared with WT plants.However,under different concentrations of exogenous ABA treatment,nced3-17 mutant shows reduce ABA sensitivity as show by 0.01%-7.76%decrease in reduction of plant height.Under different concentrations of salt stress,nced3-17 mutant shows reduce tolerance to salt stress as shown by 0.33%-28.15%decrease in seedlings survival rates,2.62%-36.14%decrease in biomass accumulation and enhance salt-induced shoot growth inhibition.In addition,0.85%-16.76%decrease in osmoregulatory substance content and 0.07%-18.87%decrease in antioxidant enzyme activity.Furthermore,salt-induced membrane peroxidation,ROS accumulation,and Na+/K+ratio are significantly enhanced,while expression levels of related anti-stress genes are decreased in nced3-17 mutant compared with WT plants.2.Evaluation of salt tolerance of Osnced5 transgenic linesResults show that,compared with non-transgenic wild type control plants(WT),Osnced5 transgenic lines show enhance ABA sensitivity as show by 2.23%-20.37%increase in reduction of plant height under different concentrations of exogenous ABA treatment.Under different concentrations of salt stress,Osnced5 transgenic lines show enhance tolerance to salt stress as shown by 2%-64.8%increase in seedlings survival rates,2.16%-34.80%increase in biomass accumulation and reduce salt-induced shoot growth inhibition.In addition,0.51%-16.35%increase in osmoregulatory substance content and 0.16%-18.78%increase in antioxidant enzyme activity.Furthermore,salt-induced membrane peroxidation,ROS accumulation,and Na+/K+ratio are significantly reduced,while expression levels of related anti-stress genes are enhanced in Osnced5 transgenic lines compared with WT plants.3.Gene redundancy of OsNCED family may modulates rice salt toleranceIn this study,the gene expression levels of other members of the OsNCED gene family in seedling leaves of WR04-6,Osnced3 and Osnced5 transgenic lines are detected under salt stress.The results show that the expression level of OsNCED4 gene are changed,compared with WT,the OsNCED4 gene expression in seedling leaves of nced3-90 mutant and Osnced5 transgenic lines increase by 37.64%-92.08%and 0.98%-190.51%,respectively.OsNCED4 gene expression of nced3-17 mutant decrease 1.07%-84.29%.Therefore this study hypothesize that gene redundancy in the OsNCED family may modulate rice salt tolerance and that salt stress-induced OsNCED4 gene expression is responsible for the altered salt tolerance of weedy rice.