Function Of Rice RACK1(OsRACK1) In Salt Stress Response

RACK1 belongs to a large family of WD40 repeat proteins and is a highly conserved protein found in both animals and plants.These proteins are characterized by the presence of repeats consisting of between 40 and 60 amino acids with two internal conserved dipeptide sequences,glycine-histidine(GH) and tryptophan-aspartic acid(WD).Typically,WD40 repeat proteins contain 4 to 16 repeating regions consisting of a core of amino acids initiated by a GH dipeptide and ends with a WD dipeptide.The consensus sequence motif is {X6-94-[GH-X23-41-WD]}4-16.Because of their highly conserved structural motif,it was suggested that WD40 proteins play very diverse roles both in animals and in plants.RACK1 is now viewed as a versatile scaffold protein that can bind or act with numerous signalling molecules from diverse signal transduction pathways in a regulated fashion,including cAMP signalling,cell cycle control,Ca²⁺ release,ribosome assembly and mRNA translation regulation, cytoskeleton remodelling and proteasome degradation.RACK1 has been found to be a potential physical and functional linker between various signalling molecules.Recently,we have screened a mutant,Atrack1,from Arabidopsis thaliana that is highly tolerant to osmotic stress.Furthermore,we also found two highly homologous genes(NC008394 and NC008398) in the Oryza sativa to AtRACK1 from NCBI.The deduced amino acid sequences of these two genes-encoded proteins have 80%identity and 70%similarity to AtRACK1. However,at present,we know little about the functions of rice RACK1.Based on the results obtained from Arabidopsis,we deduced that OsRACK1 may also play important roles in the responses of rice to adverse stresses,especially to salt stress,drought stress,etc.To verify this hypothesis,we construct two kinds of transgenic rice plants,i.e.,OsRACK1 over-expression transgenic plant line and OsRACK1 inhibition transgenic plant line using RNA interference (RNAi) technique.The major results are as follows:1.We successfully constructed two kinds of transgenic rice plants by agrobacterium tumefaciens.The expression degrees of OsRACK1 gene were from 132%to 175%in over-expression transgenic plant lines and were 33%to 71%in OsRACK1 inhibition transgenic plant lines,respectively,as compared with that of non-transgenic rice plants.We chosed seeds harvested from T1 generation of one transgenic rice plant line from two kinds of two transgenic lines,respectively,in combination with those of non transgenic plants,as materials for the further analyses.2.The results from seed germination assay showed that,when exposed to salt conditions, seed germination of all genotypes was inhibited and the inhibition on seed germination was increased as the NaCl concentration increased.However,obvious differences in seed germination were observed among various genotypes,of which OsRACK1 over-expressing transgenic line was better than those of OsRACK1 inhibition transgenic plant line and non-transgenic rice seeds.Furthermore,the root length,root numbers and soluble suagr content of OsRACK1 over-expressing transgenic line were higher,but the contents of soluble protein and abseisic acid(ABA) were lower than those of OsRACK1 inhibition transgenic plant line and non-transgenic lines.These results indicated that OsRACK1 positively regulated seed germination.3.As compared to non-transgenic lines,the relative leaf water content,the contents of chlorophyll,proline and ABA,and the activities of anti-oxidases were lower in OsRACK1 over-expressing transgenic line than in OsRACK1 inhibition transgenic plant line under the same stress condition.Whereas the MDA content and the electric conductivity of OsRACK1 over-expressing transgenic line were higher than those of OsRACK1 inhibition transgenic plant line.4.Further studies on the possible physiological and molecular mechanisms of OsRACK1 gene in regulating rice salt-tolerance indicated that the K⁺/Na⁺ ratio both in roots and in leaves of OsRACK1 over-expressing transgenic line was lower than those of OsRACK1 inhibition transgenic plant line and non-transgenic lines.The abilities to absorb and transport K⁺ of OsRACK1 inhibition transgenic plant line were higher than those of OsRACK1 over-expressing transgenic line and non-transgenic lines.Salt stress significantly stimulated ABA biosynthesis and accumulation,however,this timulation was higher in OsRACK1 inhibition transgenic seedlings than in OsRACK1 over-expressing transgenic and non-transgenic seedlings.Real-time PCR analysis showed that salt stress significantly stimulated the expression of DREB1 and P5CS, as compared with that of non slat stress treatment,and no obvious differences were observed among various genotypes investigated.Exogenous ABA treatment had a stimulating effect on the expression of PSCS,but had no effect on the expression of DREB1.The expression of RACK1 was not affected by salt stress and exogenous ABA treatments.Under salt stress condition,the ABA content was negatively related to the RACK1 expression.According to the above results and other reports,it was suggested that the OsRACK1 gene exert its functions in salt-tolerance of seed germination and seedling growth with different mechanisms.OsRACK1 negatively regulated salt-tolerance of seedling growth largely through controlling ABA biosynthesis.That is,when the expression of OsRACK1 was inhibited,the ABA biosynthesis was stimulated by some unknown mechanisms,as a consequence,the tolerance to salt stress was ehnhanced.Of course,the detailed physiological and molecular mechanisms of OsRACK1 action need to be further investigation.