Function And Signaling Mechanism Of CRYPTOCHROME In The Liverwort Marchantia Polymorpha

Cryptochromes have been found in various plants ranging from aquatic algae to land plants,but related reports on its signal transduction mechanism are mainly concentrated in the higher plant Arabidopsis.Marcantia polymorpha,as the earliest branch of land plants,has been widely used as a tool in molecular genetics for its micro-sized and unduplicated genome of haploid with low functional redundancy.The cryptochrome in liverwort has not been studied,and the effect of blue light on the growth and development of liverwort is rarely reported.This study mainly investigated the phylogenetic relationship and subcellular location of the CRY protein in liverwort.We successfully obtained the mutant of Mpcry using genome editing mediated by CRISPR/Cas9 and analyze the transcriptome data of MpCRY-knockout mutants in M.polymorpha with wild type(WT).This article mainly studies the function and signal transduction mechanism of MpCRY under blue light.The main results are as follows:1.The primary protein structure of MpCRY and its phylogenetic relationship to other cryptochromesAccording to the analysis of the geneious software,the MpCRY protein sequence has a conservative PHR domain at the N-terminal and a conserved DAS domain at the C-terminal.The evolutionary analysis of CRYs of 8 different species from aquatic plants to land plants shows that all land plants except for M.polymorpha contain multiple cryptochromes,indicating that M.polymorpha CRY has not undergone sub-functionalization and new functionalization of genes repeat.2.Mpcry-knockout mutants blue light specifically forms thallus asymmetric growthMpCRY-knockout mutants were constructed using the CRISPR/Cas9 system,and the phenotypes were analyzed under the irradiation of white light,blue light,and red light.The wild-type and MpCRY-knockout mutants of M.polymorpha showed slender thallus,with asymmetric branches,and grew vertically relative to the medium when irradiated by blue light,indicating that the growth and development of M.polymorpha were affected by blue light.Moreover,the proportion of thallus asymmetric growth under the blue light of the MpCRY-knockout mutants was higher than that of the wild type.This indicates that blue light could mediate the symmetry of thallus in liverwort via MpCRY.3.The Phenotype of hypocotyls in complementation of Arabidopsis cry1 with MpCRY and the effect of Mpcry-knockout mutants in growth-phase transition.We Constructed the AtCRY1pro:MpCRY-Myc overexpression vector and transformed it into the Arabidopsis wild-type and cry1 mutant.The complementary plants of the liverwort CRY gene in the Arabidopsis mutant cry1 background were obtained.The results showed that AtCRY1pro:MpCRY/cry1 does not restore the long hypocotyl phenotype of cry1 mutants.The formation time of first visible gametangiophore in Mpcry-knockout mutants was slightly later than that of the wild type by 4-5 days.The results showed that MpCRY practically did not have a light-dependent function of promoting growth-phase transition.4.CRY mediates photosynthesis to regulate asymmetric growth of thallus under blue light.Transcriptome analysis of wild-type and MpCRY mutants of M.polymorpha under blue and white light shows that there are a total of 1086 differentially changed genes under blue and white light.According to the comparison between genes differentially expressed in blue light and that in white light,900 down-regulated genes and 968 up-regulated genes were identified in WT;meanwhile 990 down-regulated genes and 1,116 up-regulated genes were identified in Mpcry.Through GO enrichment analysis and finally differentially expressed genes are primarily enriched in the photosynthesis pathways of light reaction and carbon reaction,as well as the chlorophyll synthesis pathway.A further KEGG analysis of differentially expressed genes indicates a significant downregulation of Mpcry under blue light when compared with most of the genes under white light in the photosynthesis and chlorophyll biosynthesis pathways but presents no change in WT.Therefore,we predicted that Mpcry plays an important role in photosynthesis.Sugar is a product of photosynthesis,then we acquired that sucrose added into the culture medium could reduce the proportion of individuals with asymmetric thallus.We then predicted the enrichment of upstream transcription factors according to the differential expression of those 1086 genes and found that some of the transcription factors are homologous with those regulated by CRY in Arabidopsis,which further indicates the conservativeness of CRY on certain functions.5.The interaction between MpCRY and MpSPA regulating the thallus asymmetric growth under blue light conditionIt is found that MpSPA of M.polymorpha also has an N-terminal kinase-like domain,a coiled-coil domain,and a C-terminal WD40 domain.Mpspa Crisp/Cas9 knockout mutants,like Mpcry knockout mutants,have more than 40% thallus asymmetric growth under blue light,while the proportion of Mpcryspa double mutants in thallus asymmetric growth is about 40%.This shows that MpSPA can also promote the symmetrical growth under blue light,and in the same pathway with MpCRY.Meanwhile,yeast two-hybrid indicated MpSPA can interact with MpCRY through the N-terminal domain in MpSPA.The blue light-independent interaction was also proved by Bi FC in Arabidopsis protoplast and co-immunoprecipitation in HEK293 T cell.Co-expressed MpSPA and MpCRY in M.polymorpha.It was found that MpSPA and MpCRY were co-localized in the nucleus and photobody was formed under blue light.Finally,the result of q-PCR assay proves that MpSPA and MpCRY can positively regulate the expression of genes involved in photosynthesis.The results of all the above indicted MpSPA and MpCRY can interact with each other which forms photobody under blue light and regulate the expression of photosynthesis genes.6.MpSPA and MpCOP can form a complex and this complex can interact with MpHY5Performing BLAST analysis in the M.polymorpha database using the amino acid sequence of AtCOP1 shows that M.polymorpha has a single COP in the M.polymorpha genome database.MpCOP interacts with the N-terminal domain of MpSPA through its N-terminal domain by the yeast two-hybrid experiment.The Co-IP experiment of HEK293 T cells and the Bi FC experiment of Arabidopsis protoplasts proved the interaction of MpCOP and MpSPA.The yeast two-hybrid indicated MpSPA can interact strongly with the N-terminal and C-terminal of MpHY5.The HEK293 T cell experiment proved that MpCOP and MpHY5 have a weak interaction,and MpSPA can enhance the binding of MpCOP and MpHY5.7.The molecular mechanism of MpCRY regulating the function of MpSPA/MpCOP protein complexThe yeast three-hybrid experiment indicated the interaction between MpCRY and MpCOP depends on MpSPA.MpCRY can inhibit the interaction of MpSPA and MpHY5 through the Co-IP experiment in HEK293 T cells.The MpHY5 protein abundance in Mpspa and Mpcry mutants was lower than that in the wild type under blue light.It is proved that MpCRY and MpSPA can inhibit the degradation of MpHY5.By observing the liverwort sporestransferred to MpSPA-Citrine and MpCRY-Tdtomato,it is found that blue light can promote the accumulation of MpSPA and MpCRY.Therefore,this study found out that under blue light,MpCRY affects the photosynthesis of M.polymorpha by regulating the expression of genes involved in photosynthesis,resulting in an increase in the proportion of the thallus asymmetric growth.And further studied the molecular mechanism of MpCRY,which is MpCRY can interact with MpSPA to form photobody under blue light and inhibit its own degradation.The increased content of MpCRY can inhibit the interaction between MpSPA and MpHY5,thereby inhibiting the combination of MpCOP and MpHY5,leading to the accumulation of MpHY5.It provides new evidence for studying the conservation of cryptochromes in evolutionary functions.