Study On The Molecular Mechanism Of Trehalose Regulated Rice Seed Germination

Seed germination is very sensitive to external environmental conditions.Abnormal weather will reduce the vitality of seeds and suppress protruding of radicle,resulting in low germination rate or enven not germination,which directly affects subsequent growth and seed production.Trehalose(Tre)and sucrose(Suc),as carbon sources and osmoprotectants,play an important role in plant growth and development as well as stress tolerance.Besides,a large number of studies have also shown that Tre and Suc can affect seed germination.Abscisic acid(ABA)is one of the most important hormones that regulate seed dormancy and germination,whose content is regulated by biosynthesis and catoblism.Numerous studies have shown ABA and its upstream and downstream signals were involved in regulating seed germination.However,little is known about the interacting mechanism on regulating seed germination between ABA and Trehalsoe signaling.In order to reveal the mechanism of Tre/Suc regulates seed germination,we carried out pharmacological experiments,q RT-PCR,metabolite analysis on Zhonghua 11(ZH11)in this study.we aim to reveal the possible relationship between ABA and Tre/ Suc in the regulation of seed germination.Furthermore,we have constructed Os TPP5 overexpression vector and CRISPR/Cas9 knockout vector,generated overexpression and knockout transgene lines by transforming rice callus via agrobacterium infection.The molecular mechanism of Tre regulates rice seed germination was preliminarily studied.The results of the study are as follows:(1)The effects of various hormones,Tre and Suc on the germination rate of rice seeds were analyzed.The results showed that high concentration of Tre and Suc have a significant inhibitory effect on the germination of rice seeds,which can be alleviated by exogenous gibberellin(GA).Rice seed germination is severely inhibited by ABA,and the inhibition effect of ABA combined with high concentration Tre or Suc was significantly improved,implying Tre or Suc functions upstream of ABA or GA in regulating seed germination.(2)ABA content in an ABA catabolism defective mutant(sg3 and sg11)was much higher than WT seed,resulting in lower germination rate when compared with WT.The sg3 and sg11 mutants were more sensitive to Tre/Suc teatments.Also,the content of soluble sugar in sg3 and sg11 was lower than that in WT seed,suggesting Tre/Suc might suppress the accumulation of soluble sugar as well as seed germination via ABA.(3)Then ABA content was detected and result shown that ABA level in seed treated with Tre/Suc was significantly higher than CK.Further q RT-PCR analysis result shown that expression of Os NCED3 and Os NCED5 was induced by Tre/Suc treatment.While application of fluridone(Flu),an ABA biosynthesis inhibitor,has rescued the inhibiting effect of Tre/Suc on seed germination,indicating Tre/Suc treatment inhibited seed germination by inducing ABA biosynthesis.(4)Both ABA and Tre treatments have up-regulated the expression of Os TPS and Os TPP genes,resulted in an increased metabolite flux and content of trehalose-6-phosphate(T6P)in trhalose pathway.Seed germination of tpp1,a T-DNA knockout mutant of Os TPP1 gene,was significantly delayed compared with WT.Gene expression of Os Sn RK1 a and Os Sn RK1 b in tpp1 mutant was suppressed,suggesting that elevated T6 P content has inhibited kinase activity of sucrose non-fermenting-1-related protein kinase(Sn RK1)and thus delayed seed germination.Moreover,overexpression line and knockout line of Os TPP5 gene,which is most abundant during seed germination,were generated via agrobacterium infection.Preliminary germination shown that the line OE2 which has an far more high expression of Os TPP5 gene germinated faster than WT seed,suggesting the involvement of Os TPP5 gene in seed germination regulation.In summary,this study proved that Tre and Suc induced rice endogenous ABA synthesis and accelerated T6 P accumulation which thereby inhibiting Sn RK1 activity and hindering the synthesis of soluble sugars and eventually suppressed the seed germination.