Cloning And Functional Analysis Of Circadian Core Clock Genes OsCCA1 And OsPRR1 In Rice

Circadian clock is an internal～24 hour(h)time keeping system that play a critical role in regulating almost all metabolic process of plant growth and development.Plant circadian clock comprises one central feedback loop interconnected with morning and evening loops,the central loop comprises three transcription factor genes CIRCADIAN CLOCK ASSOCIATED1(CCA1),LATE ELONGATED HYPOCOTYL(LHY)and their reciprocal regulator TIMING OF CAB EXPRESSION1(TOC1),also known as PSEUDORESPONSE REGULATOR1(PRR1).The central oscillator CCA1 directly binds to the CBS(CCA1-binding site)/EE(evening element)of the clock and downstream genes to regulate their expression and output traits.As one of the most important food crops in the world,rice(Oryza sativa)also have been considered as model for monocots.So,research the function of the rice circadian core clock gene OsCCA1 and OsPRR1 will help to clarify the clock regulation mechanism of monocot development and provide important theoretical basis for crop breeding.We obtained the full-length gene OsCCAl and OsPRR1 by the homology cloning method.OsCCA1 encodes 719 amino acids with conserved MYB domain at the N-terminus and is most closely related to maize ZmCCA1.OsPRRl is predicted to contain the N-terminus REC signal receiver domain and the C-terminus CCT nuclear localization signal domains and is grouped into the monocot lineage closely related to ZmTOC1,indicating that the circadian clock genes are functional conseaved in plants.Quantitative PCR analysis showed that OsCCA1 and OsPRR1 were expressed in many different rice tissues,such as leaf,shoot,tiller bud,and endosperm and exhibited diurnal expression patterns in these tissues.Similar to Arabidopsis,the expression of OsCCA1 peaked at dawn,gradually declined until dusk,and increased at night,which was inversely correlated with expression patterns of OsPRR1.Notably,both OsCCAl and OsPPR1 were expressed at much higher levels in the leaf and tiller bud than in the root and endosperm,indicating robust circadian regulation in the tiller buds as in the leaves.Using reverse genetic approaches,we generated OsCCAl and OsPRR1 transgenic lines respectively.Compared to the transgenic control(TC)plants,overexpression of OsCCA1 in rice was associated with a reduction of tiller numbers and inhibition of bud outgrowth.On the contrary,the OsCCA1 antisense suppression lines and loss-of-function mutant oscca1 produced a large number of tillers,accelerated bud outgrowth and increased bud length.Likewise,in OsPRR1-OE lines,OsCCA1 was downregulated,and tiller number and bud length were increased,whereas the OsPRR1-AS lines showed opposite changes in tiller numbers and bud length.These results demonstrate that OsCCAl and OsPRR1 affect rice tillering by regulating tiller bud outgrowth.To elucidate the regulatory mechanism of OsCCA1 for rice tiller development,we performed electrophoretic mobility shift assays(EMSA)and demonstrated that OsCCAl could directly bind to the promoter of DWARF 10(D10)and TEOSINTE BRANCHED 1(OsTB1)which associated with strigolactones(SLs)biosynthesis and signal transduction respectively.Using a yeast one-hybrid experiment,we confirmed functional consequence of OsCCA1-binding to the promoters of OsTB1 and D10 in vivo.Moreover,the expression of D10 and OsTB1 exhibited diurnal patterns and their diurnal expression were altered in OsCCA1 and OsPRR1 transgenic plants.These results indicate that D10 and OsTB1 are the direct targets of OsCCA1.To further confirm the the regulatory mechanism,we detected the endogenous SL(epi-5DS)levels in the root exudate of OsCCA1-AS lines and oscca1 mutant using LC/MS-MS technique.Results show that the amount of epi-5DS was dramatically reduced in OsCCA1-AS lines and was undetectable in oscca1 mutant and application of a strigolactone analog(GR24)could not rescue the high-tillering phenotype of OsCCA1-AS lines and oscca1 mutant,indicating that,except SL biosynthesis,OsCCA1 also plays an important role in the SL signaling pathway.In addition,we also demonstrate that photosynthetic sugars promote tiller bud outgrowth through modulating OsCCA1 expression.