The Regulation Of ABA Response By An F-box Gene AHF In Arabidopsis Thaliana

The regulation of ABA response by an F-box gene AHF in Arabidopsis thaliana The phytohormone abscisic acid(ABA)regulates many aspects of plant growth and development and the responses to various environmental stresses.After binding to ABA,the PYR1/PYL/RCAR receptor interacts with the clade A type 2C protein phosphatases,which are the core negative regulators in ABA signaling,and relieves their inhibition on the Sn RK2 protein kinases.The activated Sn RK2 kinases recognize a variety of substrates,including the transcription factors and the anion channels,then regulate the expression of ABA-responsive genes and the physiological responses.The ubiquitin proteasome pathway widely regulates the biological processes of eukaryotic cells,including the regulation of ABA responses.The E3 ubiquitin ligases are responsible for recognizing specific substrate proteins in the ubiquitination process.SCF is a kind of Multimeric RING-Finger E3 Ligases,which has the most members in this superfamily.The SCF complex includes four subunits,of which the F-box subunit is responsible for recognition and interacts with the various substrates through its variable C-terminal domain.In this study,we identified an F-box gene which is related to ABA.Overexpression of this gene causes hypersensitive phenotypes of ABA response,and more tolerant to drought in the trnasgenic Arabidopsis,so it was named as AHF(ABA hypersensitive F-box).The results are as follows:The N-terminus of the protein encoded by the AHF gene contains a conserved F-box domain,and the C-terminus contains 5 kelch repeats.There are no definite reports on the functions of the homologues that are closely related to AHF.The coding sequence of AHF fused with GFP was inserted into the vector p EGAD and the transgenic Arabidopsis overexpressing AHF gene was generated.Exogenous ABA treatment significantly inhibited the degradation of AHF-GFP protein in the transgenic plants.Overexpression of AHF gene resulted in the hypersensitive resposes to ABA in the seed germination and root growth of seedling in the transgenic Arabidopsis.The transgenic plants at rosette stage showed more tolerant to drought.These results indicate that AHF gene positively regulates ABA responses.The expression of ABA-responsive genes RD29 A,RD22,RAB18 and ABA synthesis enzyme genes NCED3,NCED6 were up regulated by ABA application in both wild type and AHF-overexpresed transgenic plants,while the up-regulated expression was more obvious in the overexpression lines,such as the expression levels of RD22,NCED3 and NCED3,which were 2 or 3 times as much as in the wild-type.Through screening the yeast c DNA library encoding Arabidopsis genes From 11 different tissues and the yeast two-hybrid assay,four AHF-interacting proteins were found,including the adaptor protein ASK1 in the SCF complex,and three other proteins respectively belonging to the ERECTA,MAPK kinase families and the6-phosphogluconate dehydrogenase family.The homozygote of a T-DNA insertion mutant of AHF gene was obtained through three-primer identification method.Through RT-PCR and ABA-related phenotypic identification,the T-DNA insertion mutant of AHF gene was found to be a gain-of-function mutant.