Study On The Function Of Transcription Factor MaJOINTLESS And Upstream Regulatory Factors MaABI5 And MaABF1 During Fruit Abscission In Mulberry(Morus Alba L.)

Mulberry(Morus alba L.)is one of the ten most economically valuable trees in the world.Its leaves can not only be used to feed the silkworm,but the mulberry fruit also has important economic and medicinal values.However,mulberry is easy to fall off in the process of ripening,making it soft and black,affecting its industrial value.The preharvest shedding of mulberries has a significant impact on the industry and value.The Abscission Zone(AZ)is the tissue area where abscission of plant organs occurs.Abscission zone development is regulated by a large number of transcription factors,among which Sl JOINTLESS is an important transcription factor in the model plant tomato.JOINTLESS is a transcription factor with MADS-box structure,which can promote the expression of abscission-related genes.MaJOINTLESS is a homologous gene of Sl JOINTLESS.The MADS-box family gene of mulberry has been identified,but the specific function of MaJOINTLESS has not been studied.The basic leucine zipper transcription factor(bZIP)is a transcription factor widely found in eukaryotes,and its structural domain mainly consists of a basic region(N-X7-R/K)and a leucine zipper region with diverse functions.It has been reported that bZIP is involved in abscisic acid signaling pathway and abiotic stresses.For example,the ABF1 in Arabidopsis has a regulatory role for seed dormancy and germination;The ABI5 in potato can respond to ABA or osmotic stress;The Hv ABI5 has the ability to regulate drought response and seed germination;In addition,bZIP can also respond to ABA signaling by regulating the plant biological clock.Notably,ABA signaling pathway is one of the important regulatory pathways for fruit abscission.In this study,we analyzed the MaJOINTLESS promoter and identified the upstream regulators MaABF1 and MaABI5.Then the initial analysis of the two regulatory factors was followed by purification to obtain MaABF1 and MaABI5 proteins,and the binding of the regulatory factors to the MaJOINTLESS promoter was verified by electrophoretic mobility shift assay(EMSA).Subsequently,through dual luciferase reporter experiment,transient overexpression in tobacco leaves,and virus-induced gene silencing,we further studied the role of regulatory factors on MaJOINTLESS and the regulatory expression of fruit abscission related genes;Finally,the differentially expressed genes were analyzed by identifying bZIP family genes and transcriptome sequencing to explore the expression and synergistic relationship of endogenous genes in mulberry during abscission.The main findings were as follows:1.Cloning,tissue expression and subcellular localization of upstream regulators of MaJOINTLESSFirst,we cloned the MaJOINTLESS promoter and performed promoter activity analysis.Histochemical GUS assays revealed that MaJOINTLESS promoter activity was strongest after abscisic acid treatment.We predicted the regulators upstream of the promoter and obtained MaABI5 and MaABF1,which were then cloned from the white mulberry variety " Hongguo 2 hao ".Bioinformatics analysis revealed that both MaABF1 and MaABI5 contain the BRLZ structural domain,which is a conserved domain unique to the bZIP family;therefore,both MaABF1 and MaABI5 are bZIP family transcription factors;Tissue expression analysis revealed that MaABF1 was mainly expressed in the leaf and AZ,while MaABI5 was mainly expressed in the winter bud and AZ,and the period expression showed opposite expression trends,and it was tentatively speculated that these two genes might have opposite roles in mulberry fruit abscission;Subcellular localization indicated that MaABF1 and MaABI5 proteins were localized in the nucleus.2.Regulation of MaJOINTLESS by regulatory factors MaABF1 and MaABI5To verify how MaABF1 and MaABI5 regulate MaJOINTLESS,Prokaryotic expression and purification of the core structural domain proteins BRLZF1 and BRLZI5 from MaABF1 and MaABI5,and then performed EMSA experiments,which showed that the structural domain proteins BRLZF1 and BRLZI5 can bind to the MaJOINTLESS promoter.suggesting that MaABF1 and MaABI5 proteins can also bind to the MaJOINTLESS promoter;Dual luciferase assay showed that MaABF1 could transcriptionally activate MaJOINTLESS,while MaABI5 could transcriptionally repress MaJOINTLESS;Next,we silenced the homologous genes NbABF1 and NbABI5 in tobacco using VIGS technology to detect changes in NbJOINTLESS as well as abscissionrelated genes.It was found that when NbABF1 was down-regulated in expression,NbABI5,NbJOINTLESS and other abscission-related genes were down-regulated in expression.While NbABI5 gene was down-regulated,NbABF1,NbJOINTLESS and other abscission-related genes were up-regulated in expression;To verify whether MaABF1 and MaABI5 regulators bind to each other,we performed bimolecular fluorescence complementation experiments,which showed that MaABF1 and MaABI5 regulators interacted at the nucleus,implying that MaABF1 and MaABI5 jointly regulate MaJOINTLESS;These results suggest that ABF1 positively regulates abscission-related genes,while ABI5 negatively regulates abscission-related genes.3.Identification and analysis of bZIP family genes in mulberryTo analyze the overall situation of mulberry bZIP family genes,we identified bZIP family genes of Morus alba by bioinformatics methods such as homology search,structural domain prediction,and phylogenetic tree,and a total of 50 MabZIP family genes were identified;The bZIP proteins of mulberry can be divided into 12 subfamilies with reference to the bZIP branching of Arabidopsis,among which the number of A subfamily proteins is the largest;cis-acting element analysis revealed that MabZIP family genes contain abscisic acid-inducible elements(ABRE),which account for 88% of the MabZIPs family and are mainly involved in the abscisic acid response,indicating that mulberry bZIP family genes can be involved in abscisic acid signaling;Tissue expression analysis showed that most of the bZIP family genes of mulberry were expressed in roots,stems,leaves and fruits,and a few genes were expressed only in specific tissues.Through the overall analysis of bZIP family genes of mulberry,we found that MaABF1 and MaABI5 correspond to MabZIP29 and MabZIP6,respectively,of the bZIP family genes,both of which are members of subfamily A and have similar structures and motifs to At ABF1 and At ABI5 of Arabidopsis.These results imply that MaABF1 and MaABI5 may function as members of subfamily A of the bZIP family of Arabidopsis,acting in the ABA signaling pathway.4.Transcriptome analysis of normal and deciduous fruit stalks at the young and mature stagesTo systematically explore the molecular mechanism of fruit abscission in mulberry,transcriptome sequencing of mulberry abscission zone was performed to study genomewide gene expression profiles of mulberry AZ at the young and mature stages,and to compare the differentially expressed gene clusters of drop and non-drop fruits.The results revealed that 451 differentially expressed genes were identified after fruit drop at the young fruit stage,293 genes were up-regulated and 158 genes were down-regulated.At maturity,144 differentially expressed genes were identified,of which 142 genes were upregulated and 2 genes were down-regulated;The GO functional enrichment analysis of differentially expressed genes showed that the most enriched genes were those with catalytic activity and binding function.It indicates that there may be many important enzymes involved in the process of fruit drop in mulberry,resulting in a series of catalytic reactions;KEGG pathway enrichment analysis showed that most of the differentially expressed genes were concentrated in MAPK signaling pathway,flavonoid biosynthesis,citric acid cycle,phytohormone signaling,amino acid biosynthesis,glycolysis and other pathways;Map Man genome annotation analysis revealed that a series of key enzyme genes such as glucosyltransferase,antioxidant enzymes,oxygenases,and dehydrogenases are involved in physiological fruit abscission of mulberry.Finally,we examined the expression of MaABI5,MaABF1 and MaJOINTLESS,and the results showed that these three genes were down-regulated in the young fruit drop-prone stage and significantly up-regulated in the ripe fruit drop-prone stage,indicating that these three genes play an significant role in the fruit ripening and abscission process.In addition,we identified several regulatory factors involved in mulberry abscission,such as germin-like proteins GLPs,pathogen infection-related NHL(NDR1/HIN1-like)family genes,alkaline phosphatase-encoding gene ALP1,glucosidase gene,copine family gene BON1,and acidic endogenous chitinase-like genes.In summary,we cloned and characterized the transcription factors MaJOINTLESS,MaABI5 and MaABF1 in mulberry,and found that MaABI5 and MaABF1 have regulatory effects on MaJOINTLESS and fruit drop-related genes.Analysis of endogenous gene expression changes in mulberry fruit abscission by analyzing the overall MabZIPs family of genes and transcriptome sequencing showed that transcription factors MaABI5,MaABF1 and MaJOINTLESS are involved in fruit drop in mulberry.These results provide a reference for our future research on the regulatory mechanism of fruit abscission in mulberry.