Physiological And Biochemical Mechanisms Of Salt Tolerance In Rice Mutants Sst And Hst1

Salt stress is considered a major threat to worldwide food production because it negatively affects growth of important crops such as rice.Developing rice genotypes with enhanced salt tolerance is an important approach to overcome salt stress challenge.Two genes,SST and HST1,have recently been found to control salt tolerance in rice,of which the loss-of-function mutants,sst and hstl,are both highly resistant to salt stress.Although these two genes have been cloned,the mechanisms of salt tolerance at physiological and biochemical level conferred by them have not yet been fully investigated and well understood.For this reason,physiological and biochemical analyses were performed using sst and hstl mutants in this study.The sst mutant was previously obtained from an indica rice cultivar R401 in our lab.To obtain hstl mutant,I knocked out the HST1 gene in R401 using CRISPR/Cas9 approach.With the sst and hstl mutants from R401,I compared them with the wild-type(WT)R401 under normal and 150-mM NaCl stress conditions by analysing a number of related traits,including vegetative growth(growth rate and biomass),photosynthesis characteristics,ion contents,organic solutes,lipids peroxidation,cell electrolyte leakage,leaf water maintenance and transcription level of genes involved in antioxidant defence.For vegetative growth,root length,shoot length and leaf length,number of green leaves,and fresh and dry weights of roots and shoots were evaluated.For leaf water maintenance,relative water content(RWC)was assessed.Photosynthesis characteristics of chlorophylls a and b and total chlorophyll were evaluated using spectrophotometry methods.To estimate membrane status,electrolyte leakage(EL)was assessed.For lipid peroxidation,malondialdehyde(MDA)was assessed.For organic solutes,Proline contents were analysed.For ion contents,potassium(K+)and sodium(Na+)contents and their ratios were evaluated.Using qRT-PCR system,expression of genes encoding enzymes involved in antioxidant biosynthesis,including superoxide dismutases A and B(OsSODA and OsSODB),ascorbate peroxidases 1 and 2(OsAPX1 and OsAPX2),and Catalases B and C(OsCATB and OsCatC)were analyzed.The main results are listed as follows:(1)In the HST1 gene editing experiment,a total of 60 transgenic(T0)seedlings were obtained,of which 58(96.7%)were positive in hygromycin testing.Sequencing showed that 47(81%)of the 58 positive transgenic plants had nucleotide insertion/deletion mutations in the target gene.Twelve of the mutant plants obtained were homozygous and 35 were heterozygous.(2)All plants showed reduction in growth rate and biomass production under salt stress condition.The reduction in WT plants was much greater than that in hst1 and sst plants.sst showed a slightly higher growth rate and biomass than hstl under normal condition,but not under salt stress condition.Growth rate and biomass of rice mutant plants were largely similar to those of WT plants(p>0.05)under normal condition.(3)RWC under normal condition was similar in all genotypes but significantly higher in sst and hstl than in WT(p<0.05)under salt stress.Chlorophyll a contents in sst and hstl were similar to those in WT under normal condition,but all contents(a,b and total)were higher than those in WT under salt stress(p<0.05).Chlorophyll b was found lower than chlorophyll a.(4)Electrolyte leakage(EL)and the contents of MDA and proline were increased under salt stress but significantly lower in sst and hstl than in WT(p<0.05).The differences among all genotypes were minor under normal condition(p>0.05).(5)Under salt stress,the mutants had higher K+/Na+ ratios than WT,with hstl at the maximum index of 0.706,followed by sst at 0.354 and WT at 0.154.OsAPXl,OsAPX2 and OsSODA antioxidant genes were highly expressed in sst and hstl as compared to WT(p<0.05)under salt stress condition.OsSODB and OsCATB expression levels increased in sst but not in hst1 and WT after salt stress.OsCatC expression level greatly increased in hst1 but not in sst and WT(p<0.05)after salt stress.In mature(35-day-old)plants treated with different salt amounts for 36 hours,OsSODB and OsAPXl expression in sst and hstl was high under moderate stress(150 mM NaCl)and was at maximum in hstl.Under excess salts(200 mM NaCl),expression was high in sst as compared to hstl and WT.Taken together,vegetative growth,photosynthesis,cell leakage,water maintenance,ion contents,lipid peroxidation level,organic solutes,antioxidant enzyme activities were all generally found at a better level in sst and hst1 mutants.Antioxidant activities in sst were improved more than in hstl,suggesting that the salt tolerance of sst might probably come from the pathway of antioxidant activities.Ion contents(K+/Na+)were improved more in hstl than in sst,suggesting that hstl might get salt tolerance through ion regulation pathway.These results will provide a foundation for in-depth research of salt tolerance mechanisms of SST and HST1 genes in rice,and the knowledge obtained will support on-going salt tolerance studies and breeding program in the future.