Mapping, Cloning And Functional Analysis Of A Salt-sensitive Mutant Gene Rss2 In Rice (Oryza Sativa L.)

Salinity is the major environmental factor limiting crop growth and productivity.Rice is a salt-sensitive crop,particularly at the seedling and reproductive stages.To clarify the genetic control of salt tolerance in rice,lots of QTL controlling the salt-related traits in rice have been identified by the researchers,but most of their total phenotypic variations are poor.An alternative strategy for identifying genes involved in salt tolerance is to isolate and characterize mutants with altered salt tolerance.The genetic screening of Nipponbare population mutagenized with ethyl methanesulfonate?EMS?treated with NaCl was performed and a saltsensitive mutant named as rice salt sensitive 2?rss2?was isolated.To obtain a better understanding of rss2mutant,salt-related physiological mechanism,QTL mapping,cloning and functional analysis experiments was progressed.Withen salt stress,rss2 mutant displayed salt sensitivity at the seed germination,which exhibited quicker and severer wilting than wild-type plants.The wild-type plants exhibited better recovery than rss2 mutant following the salt recovery treatment.Under normal hydroponic conditions,no significant difference was observed in the contents of K⁺ and Na⁺ in both the roots and shoots of rss2 mutant and wild-type plants.In contrast,under salt stress,rss2 mutant accumulated more Na⁺ in the shoots compared with wild-type plants,Na⁺ was focused on the blade in particular.However,we observed no significant differences in K⁺ contents in both the roots and shoots and Na⁺ contents in the roots between rss2 mutant and wild-type plants.The rss2 mutant?20 d,40 d and Heading stages?showed salt sensitivity compared with wild-type plants under soil salt stress.Wild-type plants and rss2 mutant were grown in medium supplemented with either NaCI,Na₂SO₄,NaNO₃,Na⁺,Cl-,KCl,K₂SO₄ or KNO₃.The results suggested that rss2 mutant overaccumulated K⁺,Na⁺ or Cl-compared with wild-type plants,respectively.When treated with sorbitol,mannitol or PEG6000,no significant difference was observed in the wilting of rss2 mutant and wild-type plants.These results indicated that reduced salt tolerance of rss2 mutant might be due to a enhancement of Na⁺ toxicity in the shoots.Genetic analysis indicated that the increase in the shoot Na⁺ content of rss2 mutant was controlled by a single recessive gene.Further genome-wide analysis of the linkage map constructed from the F2 population of rss2/ZYQ8 showed that two quantitative trait loci?QTL?on chromosomes 1 and 6 were responsible for the Na⁺ concentration in shoots,which explained 14.5%and 53.3%,respectively,of the phenotypic variance,respectively.The locus on chromosome 1,but not that on chromosome 6,was also detected in the F2population of Nipponbare/ZYQ8,suggesting that the QTL on chromosome 6 was responsible for the salt sensitivity in rss2 mutant.By analyzing 2453 individuals of an enlarged mapping population of rss2/ZYQ8,the RSS2 locus was subsequently fine-mapped to a 26.6-kb region between IM21980 and IM22006,there are three candidate genes in this region.A comparison of the nucleotide sequences of the three genes in rss2 mutant and wild-type plants revealed that one nucleotide of Os06g0554800?OsPDR12?,known to encode a PDR transporter,substitution lead to amino acid substitution:glycine?G1376?to aspartic acid?D?.This amino acid substitution lies in the transmembrane segment,according to the available RSS2 protein structure prediction.This region is relatively highly conserved among the known rice members of the PDR gene family.The three corresponding OsPDR12 genes were designated respectively as RSS2.1,RSS2.2 and RSS2.3,whose ORF were respectively 3501 bp,3504 bp and 4503 bp.RSS2animo acid mutation site was located at the stacking area of the three transcripts.Under salt stress,ospdrl2 mutant displayed salt sensitivity at the seedling growth.A complementation test showed that only transfer of a wild-type DNA fragment containing the RSS2.3 promoter region or the cauliflower mosaic virus?CaMV?35S promoter and the entire ORF?4503bp?into rss2 mutant conferred physiological characteristics similar to those of wild-type plants,including the contents of Na⁺ in the shoots,sensitivity to salt stresses.These results demonstrated that RSS2.3 was indeed responsible for the higher contents of Na⁺ in the shoots found in rss2 mutant under salt stress.The RSS2.3-GFP fusion protein under the control of CaMV 35S promoter was expressed in onion epidermal cells and transgenic rice plants,the results confirmed that the fusion protein specifically localized to the plasma membrane.Expression of RSS2.3-GFP fusion protein in X.laevis oocytes showed also RSS2.3-GFP were equally expressed in the plasma membrane.RSS2.3 promoter-GUS was expressed only in the phloem tissues.Relative to total RNA,The level of RSS2.3 mRNA was much higher in the leaf blade than that of in the root and leaf sheath.It did not change signifcantly in response to NaCl stress.Under salt stress,no significant difference was observed in the contents of Na⁺ in the phloem sap of rss2 mutant and wild-type plants.Under normal conditions,Na⁺ contents in xylem sap were not different between rss2 mutant and wild-type plants;However,The xylem sap of rss2 mutant contained more Na⁺ than that of wild-type plants under salt stress,consistent with the results of shoot Na⁺ analysis.Taken together,these results support the idea that RSS2.3 was involved in regulating Na⁺ content in the shoots.Ion permeability of RSS2.3 was tested using yeast?Na⁺ sensitivity mutant G19,K⁺ uptake defective strain CY162 and gefl mutation?complementary experiments,the results do not suggest that RSS2.3 was permeble to Na⁺,K⁺ and Cl-.Besides,voltage clamp experiments was applied for studying RSS2.3.RSS2.3 in X.laevis oocytes was expressed as positive control and recorded no Na⁺,K⁺ and Cl-currents mediated by RSS2.3 in oocytes.We isolated a novel OsPDR12 gene by map-based cloning.RSS2.3 was specifically localized to the plasma membrane and was expressed only in the phloem tissues.Physiological analysis suggested that RSS2,3 was involved in regulating Na⁺ content in the shoots under salt stress.The obtaining of the novel OsPDR12 gene will be useful for exploring the salt-tolerantic molecular mechanism of rss2 mutant.