Molecular Mechanisms Analysis Of Arabidopsis Transcription Factors VOZ1 And VOZ2 Regulating Light-induced Seed Germination

Seed germination is regulated by light.Phytochromes(Phys)act as red and far-red light photoreceptors to mediate seed germination.However,the mechanism of this process is not well understood.In this study,voz1voz2 double mutants were used to study the function and molecular mechanism of vascular plant one-zinc finger 1(VOZ1)and VOZ2 in PhyB-regulated seed germination,and obtained the following results:(1)Wild-type Col and voz1voz2 mutant seeds without cold stratification were treated with Dark,PhyB-off and PhyB-on condition.Under PhyB-off conditions,the voz1voz2 mutant had a higher germination percentage than wild type,and voz1voz2 mutant seeds germinated faster than wild type,whereas the single voz1-1 and voz2-1 mutants showed no significant differences in phenotype compared to wild type.Under dark or PhyB-on conditions,the germination percentage of voz1voz2 was close to 100%,similar to wild type.These results indicate that VOZs play a negative regulatory role in PhyB-mediated seed germination and function redundantly.At the same time,it was found that no seed germination phenotypes were observed in the VOZ1 or VOZ2 transgenic lines either in PhyB-off or PhyB-on conditions.The full function of VOZs might need to cooperate with other related proteins for its capacity.(2)Quantitative PCR(qRT-PCR)analysis showed that VOZ1 and VOZ2 expression decreased rapidly during seed imbibition,and disappeared after 48 h of imbibition.A red light pulse(PhyB-on)greatly reduced the expression of VOZ1 and VOZ2 in wild-type Col seeds,while the transcript levels of VOZ1 and VOZ2 were increased in the phyB mutant.These results indicate that PhyB mediates photo inhibition of VOZ1 and VOZ2 transcription levels during seed imbibition.(3)qRT-PCR was used to analyze the transcriptional regulatory relationship between VOZs and PIF1(Phytochrome-interacting factor 1),a key negative regulator of light-induced seed germination.The level of VOZ1 and VOZ2 expression in pif1 mutant seeds was downregulated compared with the wild type under PhyB-off condition,wheres,there was no significant difference in the expression of PIF1 in voz1voz2 mutant seeds.These findings suggest VOZ1 and VOZ2 are likely to function downstream of PIF1 in PhyB-mediated seed germination.(4)qRT-PCR was used to detect the expression of GA metabolic genes(GA3ox1,GA3ox2 and GA2ox2),DELLA genes(RGA and GAI)and ABA metabolism genes(ABA1,NCED6,NCED9 and CYP707A2)in voz1voz2 mutant.Red light irradiation and the voz1voz2 mutation caused increased expression of GA biosynthesis gene GA3ox1 during seed germination,but no significant changes in the transcription levels of DELLA genes and ABA metabolism genes.These results indicate that during seed germination,VOZs negatively regulate the expression of GA3ox1.(5)The chromatin immunoprecipitation assay(ChIP)and the electrophoresis mobility shift assay(EMSA)found VOZ2 can directly bind to two sites in the promoter region of GA3ox1.(GA3ox1-A: GCGTTAATCTTAAGG;GA3ox1-B: GTGTAGTTTTAACGC).This indicates that VOZ2 can bind to the GA3ox1 promoter in vivo and in vitro.In conclusion,VOZ1 and VOZ2 may play a negative regulatory role in the PhyB-mediated light-induced seed germination by directly regulating the expression of the GA3ox1,and suggest a new clue for further elucidating the light-and GA-mediated seed germination regulatory mechanism of plants.