Characterization Of Dormancy Related MADS-box Genes In Mulberry Plants

Plant flowering is directly related to the continuation of populations.The precise flowering time of plants is the key to successful reproduction.The flowering of Arabidopsis thaliana is mainly regulated by vernalization pathway,autonomic pathway,photoperiod pathway,gibberellin pathway,and aging pathway.Vernalization is the ability to acquire or accelerate flowering by a chilling treatment.Perennials survive winter by bud dormancy and continue vegetative and reproductive growth cycle in the coming year.As both vernalization and dormancy are subjected to cold stimulation and controlled by epigenetic regulation,so vernalization and bud dormancy may share regulations.Mulberry is a perennial woody flowering plant,which is widely cultivated in Asia,Europe,Africa,and North and South America.Mulberry leaves are used for silkworm rearing.Mulberry fruits have high medicinal value and health care,and it can be used as medicine or direct edible.The fresh mulberry fruits have a short postharvest life and are prone to postharvest fungal decay.Early-maturing and late-maturing varieties are important economic traits and become important breeding objective for mulberry industry.Mulberry fruit early-and late-maturing are closely related to the release of dormancy in spring.MADS-box proteins are transcriptors in eukaryotes.MADS-box family genes participate in almost whole growth and development processes,including dormancy.In this research,firstly,we identified flowering genes in the mulberry genome.Then,mulberry MADS-box family genes were systematically analyzed using bioinformatics methods.Quantitative real-time polymerase chain reaction(qRT-PCR)was used for the expression analysis.Results showed that MaMADS33(FLC-like)was highly expressed during endodormancy and down-regulated during dormancy release.This inferred that MaMADS33 may involved in mulberry dormancy.Ectopic overexpression,subcellular localization,bimolecular fluorescence complementation system,yeast two hybrid,and double luciferase reporter system were used to further explore the gene functions.The main results are listed as follows: 1.Identification of mulberry flowering related genes and the bioinformatics analysis of mulberry MADS-box gene family.We identified 241 flowering related genes by genome-wide research in Morus notabilis genome.The 241 genes included the key genes of flowering integrator,photoperiod pathway,vernalization pathway,GA pathway,autonomic pathway,and aging pathway.Eight FT/TFL1 homologs were identified.Evolution analysis classified mulberry FT/TFL1 genes into four clades,TFL1,BFT,FT,and MFT.There were five light-labile cytoplasmic red/far-red light photoreceptor genes in A.thaliana,while only three light-labile cytoplasmic red/far-red light photoreceptor homologs were identified in mulberry.In addition,the FRI homolog was not found in the M.notabilis genome,with the exception of 13 FRL homologs,eight of which tandemly scattered on the same scaffold.In the M.notabilis genome,we identified 54 MADS-box genes,which were named as MnMADS1-54.Phylogenetic analysis showed that there were 17 type I genes and 37 type II genes.In the type I,there were 11 M? genes,two M? genes,and four M? genes.The type II genes were further classified into 13 subfamilies,AGL2,AGL6,SQUA,AG,FLC,AGl12,TM3,AGL17,STMADS11,AGL15,GLO,DEF,GGM13,and MIKC*,including three FLC-like genes(MnMADS7,MnMADS33,and MnMADS50)and four AGL24/SVP-like genes(MnMADS1,MnMADS42,MnMADS43,MnMADS4).All 54 MnMADSs were scattered on 42 scaffolds,ten of which had tandem repeat genes.We observed one AGL6-SOC1 and three SEP-AP1 tandems in mulberry.Moreover,the PI and SVP homologs and three group M? genes were tandem duplication clusters.Selective pressure analysis indicated that the tandem repeats in the M? subfamily evolved under positive selection pressure.Gene and protein structures showed that all 54 MnMADSs had a MADS-domain.Genes in the same subfamily had similar gene structures.Type I proteins coded by one exon.Most of type II proteins had the typical MIKC structure and coded by about six exons.RPKM data and qRT-PCR results indicated that the ABCDE class genes mainly expressed in the floral related organs.These results implied that the evolution of MADS-box genes were conserved in both gene structures and functions.MaMADS33 expressed highly during endodormancy,while endodormancy release resulted in the down-regulation of MaMADS33 expression.This result indicated that MaMADS33 involved in the endodormancy.2.Functional research of mulberry AGL24/SVP-like genesDAMs were homologs of A.thaliana AGL24/SVP genes.Research showed that DAMs involved in perennial dormancy.We cloned the four AGL24/SVP-like genes from mulberry resources Jinqiang 63(JQ63,Morus alba).Multi-species phylogenetic analysis indicated that MaMADS1 and MaMADS4 were close to A.thaliana SVP and AGL24,respectively.MaMADS42 and MaMADS43 were clustered with the Euphorbia esula DAM genes,all of which were gathered with StMADS11.Transient expression of mulberry AGL24/SVP-like genes fused with GFP in tobacco epidermal cells showed that MaMADS4,MaMADS42,and MaMADS43 were localized to nucleus.In addition to the nucleus,fluorescence signals of MaMADS1-GFP fused protein can be detected in the cell membrane and cytoplasm.We transformed mulberry AGL24/SVP-like genes into A.thaliana to observe flowering phenotype.Results revealed that MaMADS4 overexpression lines flower earlier than wild-type.Overexpression of MaMADS1 resulted in late flowering and abnormal floral organs.Yeast two hybridization experiments were carried out to test whether the abnormal floral organs were related to the A,B,and E class proteins.Furthermore,mulberry and A.thaliana flowering related proteins were also tested.The results indicated that MaMADS1 could interact with B and E class proteins of A.thaliana,especially the E class proteins.MaMADS1 could interact with the mulberry A,B,and E class proteins.The interactions between MaMADS1 and flowering related proteins FLC,SOC1,and AGL24/SVP of mulberry and A.thaliana were also observed.In addition,MaMADS1 could form homologous dipolymer.These results inferred the functional conservation of MaMADS1 in floral organ development and flowering.The qRT-PCR results showed that MaMADS1 was relatively highly expressed in one-year bark,mature leaves,terminal buds,mature petiols,and bracts,especially in mature petiols,and bracts.The expression profiles of MaMADS42 and MaMADS43 were similar,which expressed highly in one-year bark and mature leaves.Comparing with MaMADS1,MaMADS4 also highly expressed in one-year bark,mature leaves,mature petiols,and bracts,while the expression of MaMADS4 was relatively lower in terminal buds.In the case of the annual floral buds,the expression profiles of four mulberry AGL24/SVP-like genes tended to be consistent,which peaked in July and then decreased.This results suggested that the mulberry AGL24/SVP-like genes may involved in the paradormancy.3.Functional analysis of mulberry FLC-like genesWe cloned the three FLC-like gene in JQ63,and named as MaMADS33,MaMADS50,and MaMADS7,respectively.Protein domain analysis showed that MaMADS33 and MaMADS50 had complete MIKC structures,while the C-terminal of MaMADS7 had lower conservation.Evolution reconstruction of 86 FLC homologs from multi-species indicated that MaMADS33,MaMADS50,and MaMADS7 were clustered in a clade and closed to the FLC homologs of Mimulus.The subcellular localization analysis of mulberry FLC-like-GFP fused proteins indicated that MaMADS50 and MaMADS7 were localized to the nucleus.MaMADS33 was mainly localized to the nucleus,while MaMADS33 was also detected in the cytoplasm and cell membrane of tobacco epidermal cells.Overexpression of mulberry FLC-like genes in A.thaliana delayed flowering with down-regulated expression of FT and SOC1.These results indicated that mulberry FLC-like genes inhibited flowering,which was similar to the function of A.thaliana FLC gene in floral regulation.In our research,we found four CArG-box candidates in the promoter of MaFT.We detected the regulation relationship of mulberry FLC-like and MaFT by double luciferase reporter system.Results showed that both MaMADS33,50,and 7 can directly or indirectly bound to the promoter of MaFT gene to down-regulate the downstream report genes in vivo.Gene expression profiles in annual floral buds of JQ63 showed that the expression of MaMADS33 was fluctuated from April to September,and was lowest in October.MaMADS33 was peaked in November and then down-regulated to the lowest in February and March.The expression of MaMADS50 was highest in September,and relatively low after November.The expression of MaMADS7 was high in May and it was fluctuated in most of time.The expression level of MaFT was relatively high during June and October,and then sharply declined in November.In February and March,the expression of MaFT had a small increase.The expression profiles of annul floral buds inferred that MaMADS33 and MaFT were dormancy related genes.The expression of MaMADS33 was peaked at the tenth day after chilling treatment.The expression pattern of MaFT in the chilling treatment was opposite to the expression of MaMADS33.In the ABA treated floral buds,MaMADS33 and MaFT were all up-regulated,then the expression of MaMADS33 raised stably,while the expression of MaFT was down-regulated with the increasing of treatment duration.All these results suggested that MaMADS33 may involve in dormancy by down-regulating the expression of MaFT.We detected the expression profile of MaMADS33 in different mulberry resources with different chilling requirements.The results indicated that the expression of MaMADS33 positively correlated with the chilling requirements.To further explore the function of MaMADS33,mulberry yeast two hybrid library was constructed to screen MaMADS33 interaction proteins.Twenty positive clones were screened,including the mulberry winter accumulating protein 18(MaWAP18).The gene expression level and the protein level of MaWAP18 were high in the dormancy.We cloned the full length sequence of MaWAP18 to verify the yeast two hybrid result.In addition,double molecular fluorescence complementary experiment was conducted to further confirm the interaction between MaMADS33 and MaWAP18.Results showed that the strongest fluorescent signal was detected in the guard cells.Integrated the functional analysis of dormancy related MADS-box genes,mulberry DAM-like genes may participate in the paradormancy.FLC homolog MaMADS33 can induced by cold.MaMADS33 expression was down-regulated when endodormancy release.MaMADS33 and MaFT present an opposite expression pattern during endodormancy.In addition,MaMADS33 can interact with winter accumulated protein MaWAP18.This study provides new data for the involvement of FLC homolog in endodormancy.The results support the speculation that vernalization and endodormancy share the regulation.