U2AF65b Functions In ABA Mediated Flowering-time Regulation Via Pre-mRNA Splling Of ABI5 And FLC In Arabidopsis

In mammalian and yeast,splicing factor U2AF65/Mud2 p functions in pre-mRNA processing.The Arabidopsis AtU2AF65 b encodes a U2AF65 homolog.However,little is known about its functions in plants.We studied the functions of AtU2AF65 b using the T-DNA inserted atu2af65 b knockout mutants harbouring early flower phenotype.Our study shows that AtU2AF65 b is involved in ABA-mediated flowering-time regulation by pre-mRNA splicing of FLC and ABI5.These results are as follows:(1)The early flower phenotype of the atu2af65 b mutant is caused by the decrease mRNA level of FLC which is a key inhibiting factor in the flowering induction process.The mRNA level of FLC was decreased significantly in the shoot apex of atu2af65 b seedlings compared with that of wild type and atu2af65 b rescued lines.The early flowering phenotype can be rescued by overexpression FLC.The atu2af65 b flc double mutants showed early flowering with a similar phenotype to atu2af65 b mutant.(2)AtU2AF65b participates in the pre-mRNA splicing of FLC in Arabidopsis.The splicing efficiency shown by the spliced and unspliced RNA levels of FLC introns in the shoot apex of atu2af65 b seedlings was significantly reduced compared with that of wild type and atu2af65 b rescued lines.(3)The mRNA level of ABI5,encoding an activator of FLC,was significantly reduced in the shoot apex of atu2af65 b seedlings.Splicing efficiency of ABI5 introns in the shoot apex of atu2af65 b seedlings was significantly decreased.The early flowering phenotype can be particily rescued by overexpression FLC.The atu2af65 b abi5 mutants exhibited a similar flowering time to atu2af65 b mutant.These data suggest that AtU2AF65 b is able to regulate the transcription activation of FLC by participating in the pre-mRNA splicing of ABI5.(4)qRT-PCR analysis and GUS histochemical staining showed that the expression level of AtU2AF65 b is regulated by both exgenous and endogenous ABA.Exogenous ABA treatment enhanced the expression level of AtU2AF65 b in Arabidopsis seedlings.The expression levels of AtU2AF65 b were significantly increased in these mutants in ABA deficient mutants aba1?aba2 and nced3,and ABA metabolism mutant cyp707a3.(5)ABA applications in soil promoted the flowering of wild-type and atu2af65 b rescued lines,but did not effact the flowring of atu2af65 b.It is indicated that At U2-AF65 b participates in the flowering time regulation process of ABA mediated.(6)No obvious alteration of the splice efficiency of FLC and ABI5 were detected inthe shoot apecies of atu2af65 b compared to the significant differences in wild type seedlings after ABA treatment.These data indicate that AtU2AF65 b functions in ABA-mediated flowering transition via pre-m RNA splicing of FLC and ABI5.