| Plant hormones play important roles in regulating plant growth and development,and plant hormones do not work independently,but control a certain plant physiological process in a synergistic or antagonistic way.ABA and ethylene are important plant hormones,both of which have significant roles in regulating plant seed germination and senescence.Researchers had studied the signal transduction mechanisms of ABA and ethylene,and identified several functional genes of each signal pathway,and revealed the mechanism of ABA and ethylene regulating plant growth.It was reported that ABA can affect the production of ethylene,but the role of ABA on ethylene production may be different depending on the stages of plant development.For example,exogenous ABA treatment promotes ethylene synthesis and fruit ripening in tomato,but ABA treatment reduces the ethylene production of seedling by 50%in Arabidopsis.Therefore,studying the mechanism of the effect of ABA on ethylene production will help us understand the action and regulation mechanism of plant hormones in plants.To identify the factor involved in the above process,we conducted a screen for ABA-insensitive mutants with altered ethylene release in Arabidopsis.Mutant abi4-152 was isolated in our screen,which germinated on medium containing 0.5 ?M ABA and produced less ethylene.Genetic analysis determined that abi4-152 is a dominant mutant.The mutation was mapped to?86 kb interval at the long arm on chromosome 2.DNA sequencing analysis revealed a conversion of Trp 313 of ABI4 to a premature termination codon,resulting in a removal of 16 amino acids from the C-terminal region.Two more ABI4 mutants,abi4-102 and abi4-103,were identified,both of which lost the activation domains at their C terminal regions.Production of ethylene in these two lines was higher than that in Col-0,which was different from the abi4-152.When the seedlings were grown in the dark for four days,the length of root and hypocotyls of abi4-102 and abi4-103 was shorter than that of Col-0 while the length of root and hypocotyls of abi4-152 was longer than that of Col-0.This phenotype was consistent with the ethylene production in each mutant.These results indicated that ABI4 negatively regulates the ethylene production.The results of the gene expression analysis in ethylene synthesis showed that the expression of ACS4 and ACS8 was enhanced two folds in abi4-102 and abi4-103 but was reduced significantly in the abi4-152.ABA treatment cdrastically decreased the expression of ACS4 ACS8 and AC02 in Col-0,however,the transcript level of ACS4 and ACS8 in abi4-102 and abi4-103 was higher than that of Col-0,and the transcript levels of AC2 were not affected.These results were in agreement with the phenotype of ethylene production of the mutants.When treated with 5 ?M ABA,the wild type Col-0 exhibited about 50%deduction of ethylene,but the ethylene production was partly resistant to the inhibition of ABA in abi4-102 and abi4-103.ChIP(chromatin immunoprecipitation)assay showed that ABI4 binds to the promoters of these genes directly and that ABA enhances the interaction.ABI4 is regulated at post-transcription level.We analysed the levels of the ABI4-Myc fusion protein in the OEM(ABI4-152,the truncated ABI4)and OE(the full length ABI4)transgenic plants by Western Blotting assay,and the results showed that the truncated protein in the OEM was very stable.Also,the ABI4-GFP in the transgenic plants was not detected,but the truncated ABI4 form with GFP fusion was readily detectable.Therefore,the 16 amino acids at the C terminal region are important in the stability of the ABI4.These results demonstrate that,with a more stably expressed ABI4-152 protein,the mutant abi4-152 disrupts the ethylene production by reinforcing the function of the wild-type protein.Taken together,our data indicated that ABA negatively regulates ethylene production through ABI4-mediated transcription repression of ethylene biosynthesis genes ACS4 and ACS8 in Arabidopsia. |
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