The Roles Of Arabidopsis ACS5 During NaCl Inhibiting Seed Germination

Seed germination is one of the most important stages influenced by external and internal factors during plant growth and development.High salt stress,as an abiotic stress,could inhibit seed germination significantly,resulting in attenuated crop yields.The application of exogenous ethylene could promote seed germination and alleviate the inhibition of seed germination by NaCl treatment.It has been reported that1-amino cyclopropanecarboxylic acid?ACC?is the precursor of ethylene synthesis.ACC and NaCl influence the stability of HY5 protein and the regulation of ABI5 by controlling the accumulation of COP1in nucleus and orchestrate the germination of seed.ACC content is also affected by various factors during seed germination and the mechanism as well as the effect on seed germination remains to be clarified.ACS?ACC synthase?is the rate-limiting enzyme of ACC synthesis pathway.AtACS family has 12members,which are expressed with various spatiotemporal specificities.In each phase during the growth and development of plants,the expression of ACS5,among all the ACS family genes,reach the highest level in the seed germination stage.It remains to be revealed what is the relationship between NaCl stress,seed germination and ACS5.It was found that under salt treatment,the seed germination time of acs5-1mutant was earlier than that of WT.Therefore,this project was aimed at exploring the mechanism of the ACS5-mediated inhibition of seed germination by NaCl.The complementary lines(acs5/acs5-1^#1and acs5/acs5-1^#2),ACS5 overexpressed lines?ACS5-OE?and ProACS5::GUS transgenic lines were constructed.Under NaCl treatment,the result of histochemical localization of ACS5 indicated that ACS5 mRNA expression presented high-level at the germination stage,and ACS5 was significantly accumulated in seed germination stage induced by NaCl.It was suggested that ACS5 participated in the inhibition of seed germination mediated by NaCl.The following observation showed that in control conditions,there was no significant difference of germination time between WT,acs5-1 and acs5-2 seeds.However,under NaCl treatment,compared with WT,the germination times of acs5-1,acs5-2 and ACS5-OE were advanced and the germination rates were enhanced.The WT,acs5/acs5-1^#11 and acs5/acs5-1^#22 materials showed similar phenotype,suggesting that acs5 mutation alleviated the inhibitory effect of NaCl on seed germination.It has been shown that the accumulation of Methionine?Met?and S-adenosylmethionine?SAM?has a positive regulatory effect on seed germination.The content of Met and SAM in acs5-1 was significantly higher than that in WT.It is speculated that this may be one of the reasons that why the acs5 mutant germinate earlier than WT under salt stress.At the same time,it was found that in control conditions,ACC contents of acs5 and WT has no significant difference.When treat the seeds with NaCl by two days,the ACC content in acs5-1 is higher than that in WT.In order to explore the reasons for the increase of ACC content,the activity changes of other ACS family members in acs5-1 mutants during seed germination under NaCl treatment were detected.The result elucidated that the transcription levels of ACS2 and ACS7,which could form inactive dimmers on the second and third day of germination acs5-1 with ACS5,significantly increased compared with WT.ACS2 and ACS7 vectors carrying GFP tags were constructed and transferred into WT and acs5-1.Western Blot results showed that ACS2 and ACS7 proteins in acs5-1were significantly accumulated compared with that in WT under NaCl treatment.Further observations of the acs2-1 acs5-1 double mutant phenotype:NaCl treatment delayed the germination time of acs2-1 acs5-1double mutant,and decreased the germination rate compared with that in WT.It was indicated that when the content of ACS2 protein decreased in acs5-1 mutants,the germination was delayed under salt treatment.In summary,under NaCl treatment,the germination of acs5 was earlier than that of WT,which could be attributed to two reasons.Firstly,the accumulation of Met and SAM in acs5 mutants was higher than that of WT;secondly,ACS5 affected the change of ACC content in acs5 mutants by affecting the accumulation of ACS2 protein,leading to alleviated inhibitory effect of NaCl on seed germination.