Functional Analysis Of Arabidopsis Thaliana F-box Gene TFF

F-box proteins are a core component of SCF(SKP1-Cullin-F-box)-complex in Arabidopsis thaliana.Genetic analysis revealed that F-box proteins play a role in multiple signaling pathways,including hormone signaling,light signal response,circadian clock,flowering time and disease resistance.Arabidopsis thaliana TFF(TIR1 family F-box)is a recently discovered F-box gene whose biological function has not been formally identified.It is not clear that TFF functions in what signaling pathway in plants.To explore action mechanisms of TFF in auxin signaling pathway,loss-of-function mutants tff,were successfully isolated and characterized by bioinformatics,genetics and cell biology approaches.The genetic function of TFF gene and the polar localization of auxin efflux transporter PIN were analyzed.The detailed results were as follows:(1)Bioinformatics analysis showed that the protein encoded by TFF contained a conserved F-box and an unknown functional domain at the C-terminal.Analysis of the upstream promoter of the start codon by plant CARE software revealed that the promoter region contains a large number of auxin elements.Amino acid sequence homology analysis showed that there exists 81% homology between TFF protein and auxin receptor TIR1/AFB family.Likely TFF functions as F-box in auxin signaling pathway to regulate plant growth and development.(2)Genetic analysis showed that loss of TFF function affected the vegetative growth and reproductive development of plants.During vegetative growth period,tff mutants displayed short roots,unopened cotyledons and dwarf phenotypes.During the reproductive development period,homozygous mutants exhibited shorter siliques,high seed abortion rate and thereby low seed setting rate relative to the wild type.These results suggested that TFF plays a critical role during plant growth and development.(3)Microscopy analysis showed that,the loss of TFF function affected the embryo development,and thereby a higher proportion of aborted seeds and heteromorphic embryos appeared in homozygous mutants.The statistical results of embryo stages indicated that embryo abortion occurred in the transition stage to the heart-shaped stage,suggesting that TFF is necessary for embryo development.(4)Cell biological analysis showed that the expression levels of auxin-responsive molecular marker DR5-GFP were reduced in tff roots,and that polarity of auxin efflux transporters PIN1,PIN3 and PIN7 and auxin influx transporter AUX1 was not altered in tff mutants,in addition to reduced fluorescence intensities in stele cells,,indicating that the loss of TFF function does not affect the direction of polar auxin transport,but likely may affect auxin biosynthesis.Taken together,these results suggested that Arabidopsis TFF may act as F-box functions that regulate plant growth and development via modulating auxin signaling.Loss of TFF function leads to severe defects in plant growth and development,especially effects on embryo development.Likely,TFF gene may be involved in the regulation of embryonic development.These results would be important to further dissecting molecular action mechanisms of TFF and provide new insights for further investigation on the molecular mechanism by which F-box proteins regulating embryonic development in plants.