Transcriptome Analyses Reveal The Involvement Of Both C And N Termini Of Cryptochrome 1 In Its Regulation Of Phytohormone-responsive Gene Expression In Arabidopsis

Light not only provides plants with energy to maintain life,but also supplies plants with signal to regulate their developmental processes in the whole life cycle.Cryptochromes(CRY)are blue-light photoreceptors that mediate various light responses in plants and animals.The C-termini of CRY1 and CRY2(CCT1 and CCT2)are known to mediate CRY1 signaling,since transgenic plants expressing CCT1 or CCT2 fused to ?-glucuronidase(GUS)(GUS-CCT1 or GUS-CCT2,abbreviated as CCT1 or CCT2)show a constitutively photomorphogenic phenotype.It has long been demonstrated that Arabidopsis CCT1 and CCT2 mediate light signaling through direct interaction with COP1.Most recently,CRY1 N terminus(CNT1)has been found to be involved in CRY1 signaling independent of CCT1,and implicated in the inhibition of gibberellin acids(GA)/brassinosteroids(BR)/auxin-responsive gene expression.Although it has been found that CNT1 and CCT1 are both involved in mediating CRY1 signaling,it is worth investigating how they are involved in regulating phytohormone-responsive gene expression at the transcriptomic level.Here,we performed RNA-Seq assay using CCT1,and compared the results with those obtained previously from cry1cry2 mutant and the transgenic plants expressing CNT1 fused to nuclear localization signal sequence(NLS)-tagged YFP(CNT1-NLS-YFP,abbreviated as CNT1),which display enhanced responsiveness to blue light.We found that 2,903(67.85%)of the CRY-regulated genes are regulated by CCT1 and that 1,095 of these CCT1-regulated genes are also regulated by CNT1.After annotating the gene functions,we found that CCT1 is involved in mediating CRY1 regulation of phytohormone-responsive genes,like CNT1,and that about half of the up-regulated genes by GA/BR/auxin are down-regulated by CCT1 and CNT1,consistent with the antagonistic role for CRY1 and these phytohormones in regulating hypocotyl elongation.qRT-PCR analyzes the genes that are relative to hypocotyl elongation and up-regulated by GA/BR/auxin,and found these genes were indeed down-regulated by blue light,CRY,CCT1 and CNT1.We further found that the identified genes specifically regulated by CCT1 indeed expressed at a lower level in CCT1 than in CNT1 seedlings exposed to blue light,such as ACS5.Similarly,the identified CNT1-specific genes expressed at a lower level in CNT1 than in CCT1 seedlings exposed to blue light,such as SAUR15.Physiological studies showed that both CCT1 and CNT1 are likely involved in mediating CRY1 reduction of seedlings sensitivity to GA under blue light.Furthermore,protein expression studies demonstrate that the inhibition of GA promotion of HY5 degradation by CRY1 is likely mediated by CCT1,but not by CNT1.To further examine the functional relevance of CCT1 and CNT1 with COP1,we also performed RNA-seq analysis of cop1 mutant and compared the genes,particularly those responsive to GA/BR/auxin,which are related to CCT1,CNT1 and COP1.We found that CCT1 and CNT1 co-regulate the expression of a large number of genes responsive to GA/BR/auxin with COP1 in an opposite direction.We also found that blue light inhibit seedlings sensitivity to BR in a CRY1 dependent manner,and qRT-PCR analyses reveal that the expression of BR response genes are also inhibited by CRY1.These results provide genome-wide transcriptome information concerning the signaling mechanism of CRY1,unraveling possible involvement of its C and N termini in its regulation of response of GA,BR and likely other phytohormones,and provide clue to further explore the molecular mechanisms of CRY1 signaling,which may be involve in regulation of phytohormone-responsive gene expression.