Molecular Mechanism Of Arabidopsis Blue Light Receptor CRY1 Regulates GA Signal During Photomorphogenesis

Plants on earth are affected by many environmental factors in the whole life cycle from seed germination to flowering.Light not only provides the necessary energy source for photosynthesis,but also provides regulatory signals for growth and development.Plants have evolved a variety of light receptors to sense the quality,quantity and direction of light,and then regulate and balance their growth and development.Cryptochrome CRY1 mediates blue light to promote light morphogenesis,such as inhibition of hypocotyl elongation of seedlings.Gibberellin(GA),a plant endogenous hormone,is involved in inhibiting photomorphogenesis and promoting hypocotyl elongation.At present,the antagonistic mechanism of blue light and GA is not completely clear.Therefore,in-depth exploration of how cryptochrome CRY1 regulates GA signal during photomorphogenesis is of great significance to clarify the molecular mechanism of how blue light signal antagonizes GA signal to regulate plant photomorphogenesis.In this paper,a series of biochemical and genetic phenotypic analysis experiments have been carried out to solve this scientific problem,and the following research results have been obtained:(1)The regulatory effect of CRY1 on GA signal transduction and the stability of signal inhibitor DELLA protein during photomorphogenesis have been clarified.By analyzing the response of hypocotyl elongation of related genotypes to GA under blue light,the expression of GA response genes PRE1 and EXP8,and the stability of DELLA protein,which is the negative regulator of GA signal,it was found that the response of hypocotyl elongation of cry1 mutant seedlings to exogenous GA was enhanced,while the sensitivity of hypocotyl elongation of CRY1 overexpression seedlings to exogenous GA was weakened,the results indicate that CRY1 negatively regulates GA signal during photomorphogenesis;Under exogenous GA treatment,the expression of GA response genes PRE1 and EXP8 in cry1 mutant seedlings were significantly higher than those in wild-type seedlings,which further proved that CRY1 negatively regulates GA signal.Under Exogenous GA treatment,the degradation rate of RGA-Flag or GAI-Flag fusion protein in the cry1 mutant background was accelerated,but its degradation rate in the background of GFP-CRY1 overexpression was slower,indicating that CRY1 mediates blue light to inhibited GA-induced DELLA protein degradation.(2)The interaction characteristics and genetic relationships between CRY1 and DELLA protein have been analyzed.The interaction between CRY1 and GA signal negative regulator DELLA protein RGA and GAI were confirmed by bimolecular fluorescence complementarity(Bi FC),semi in-vitro GST pull-down and Co-IP.Analyzing the structural domains of CRY1 and RGA/GAI interaction,it was proved that the CNT1 and CCT1 domains of CRY1 both interact with RGA/GAI,and the DELLA and GRAS domains of RGA/GAI proteins interact with CRY1.Analyzing the genetic relationships between CRY1 and RGA/GAI,it was found that the hypocotyls of cry1/RGA-Flag,cry1/GAI-Flag and cry1/gai mutant seedlings under blue light were shorter than those of cry1 mutant seedlings,and the transcription level of cell elongation related genes(PRE1,PRE5,EXP3,EXP5,and IAA19)in cry1/gai seedlings under blue light was lower than those in cry1 mutant seedlings.These results indicate that CRY1 is located upstream of RGA/GAI,to promote light morphogenesis and inhibit the expression of genes related to cell elongation.(3)The interaction characteristics and genetic relationships between CRY1 and GA receptor GID1 have been analyzed.Using Bi FC experiment,it was found that CRY1 interacted with GA receptors GID1 a,GID1b,and GID1 c.Furthermore,semi in-vitro GST pull-down and Co-IP experiments confirmed that CRY1 interacted with GID1 in a blue light dependent manner,and the CNT1 and CCT1 domains of CRY1 interacted with GID1.By analyzing the genetic relationships between CRY1 and GID1,it was found that gid1b/c double mutation partially inhibited the long hypocotyl phenotype of cry1 mutant seedlings under blue light,and GID1b-Flag overexpression partially inhibited the short hypocotyl phenotype of Myc-CRY1 overexpression seedlings;Compared with cry1 mutant seedlings,the transcription levels of cell elongation related genes(PRE1,PRE5,EXP3,EXP5,and IAA19)in cry1/gid1b/c mutant seedlings were decreased.These results suggest that CRY1 and GID1 antagonistically regulate light morphogenesis and the expression of downstream cell elongation related genes.(4)The molecular mechanism of CRY1 and GID1 antagonistic regulation of photomorphogenesis has been clarified.Through GST pull-down and Co-IP experiments in-vitro and in-vivo,it was proved that CRY1 interacted directly with GID1 in a blue light dependent manner,and then inhibited the interaction between GID1 and DELLA protein.It shows that CRY1 can inhibit the formation of GA-GID1-DELLA complex by binding with GID1,relieve the degradation of DELLA by GA,and then promote photomorphogenesis.In conclusion,this paper reveals a new mechanism that blue light receptor CRY1 can directly bind to GA receptor GID1 and DELLA protein,a key inhibitor of GA signal,in a blue light dependent manner,so as to inhibit GA signal and promote light morphogenesis.