| Abscisic acid(ABA)is the main phytohormone regulating seed dormancy and germination.The seed ABA content and the sensitivity of embryo to ABA together regulate seed dormancy and germination process.In the early stage,our team discovered that Arabidopsis IQM4 was involved in ABA-regulated seed germination,but its mechanism was not clear(Xiao Wenhui,2016).In this paper,five aspects will be studied:(1)analyzing the dormancy level of the wild type Col and IQM4 mutants of Arabidopsis and determining the endogenous ABA and GA3 content of fresh seeds to find whether IQM4 participates in the regulation of seed dormancy;(2)RNA-Seq analysis of Col and iqm4-1 seed is used to study how IQM4 participate in the regulation of seed dormancy and germination on the transcriptional level;(3)analyzing IQM4 expression profiles using RT-qPCR to explore the functional site of IQM4 on transcriptional level;(4)analyzing gene expressions of seed maturation transcription factor,ABA biosynthesis and catabolism enzyme and ABA signal transduction by RT-qPCR,to find how IQM4 regulates seed maturation,dormancy and germination;(5)p35S:IQM4-HA transgenic plants were constructed to provide experimental materials for the study of IQM4 interacting proteins.Main research results are as follows:(1)Compared with Col,the ABA content of the mutant iqm4 seeds decreased significantly,but there was no significant difference in GA3,meanwhile there was no significant difference between ABA and GA3 in seeds of the over-expression lines.The germination rate of fresh iqm4 seeds treated without stratification was significantly higher than that of Col,but the over-expression lines had no differences from Col,while all seeds were germinated after low temperature stratification.The results showed that IQM4 regulates seed dormancy by affecting ABA accumulation.(2)The differentially expressed genes of the RNA-Seq involve ABA anabolism and signal transduction,GA anabolism and signal transduction,cell wall structure composition,heat shock protein and heat shock transcription factors.(3)IQM4 expresses in different organs of Arabidopsis thaliana,among which the expression in fresh and dry seeds was the highest.After seed storage and low temperature absorption and germination,the expression of IQM4 decreased in varying degrees.(4)IQM4 mutation inhibited the expression of NCED6,NCED9,LEC1,LEC2,ABI3 and ABI5 in the mid-stage development of the seed,and promoted the expression of CYP707A3,but the over-expression of IQM4 inhibited the expression of LEC1 and LEC2.At the late stage of seed development,IQM4 deletion inhibited the expression of NCED6 and NCED9,and over-expression of IQM4 enhanced the expression of NCED3,NCED9,CYP707A2,CYP707A3,ABI4 and ABI5.The results showed that IQM4 mutation changed the transcription level of ABA accumulation and signal transduction in the developing seed,and IQM4 promote the accumulation of ABA during seed development.(5)In the iqm4-1 seedlings treated by ABA,the expression of ABA negative regulator WRKY40 was significantly increased,but the expression of negative factors of the ABA signaling ABI2,HAB1,HAB2 as well as positive factors of the ABA signaling ABI5 and SnRK2.2 was significantly down-regulated,which showed that IQM4 regulates the ABA signal transduction process during seed germination.(6)Two p35S:IQM4-HA homozygous lines were successfully constructed to provide experimental materials for further study of IQM4 interacting proteins.To sum up,IQM4 regulates seed dormancy by promoting the accumulation of endogenous ABA in Arabidopsis seed,and regulates the sensitivity of seed germination to ABA by inhibiting the negative regulatory factor WRKY40 and promoting the expression of positive regulatory factor ABI5.IQM4 plays an important role in seed dormancy and germination,and is a positive regulator of the ABA signaling pathway,whose specific molecular mechanism needs further study. |
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