Molecular Mechanism Of ZaARR24 Regulated Male And Female Floral Differentiation In Zanthoxylum Armatum

The flower transition from female to male seriously reduced the yield of Zanthoxylum armatum,which has been a major bottleneck in its utilization as the important economic plants.In our previous study,the cytokinin signaling pathway was significantly enriched based on RNA-seq analysis using the crucial stages of male and female floral differentiation.Meanwhile,ZaARR24,a C-type response factor involved in cytokinin signaling pathway,was shown a dominant and special expression patterns.Therefore,this study mainly focused on the regulatory function of ZaARR24 involved in the differentiation of male and female flowers,and exploried the reasons about the transition from female flowers to male flowers.Firstly,based on available transcriptome data,the complete coding sequence of the cytokinin-like regulator ZaARR24 was cloned using RACE technology.The DNA and protein sequence characteristics of ZaARR24 were analyzed.The subcellular localization of ZaARR24 protein was carried out by constructing fluorescent protein fusion vector.Subsequently,ZaARR24 was heterologous overexpressed in the wild-type Arabidopsis and analyzed for its major role in regulating male and female stamen differentiation.The potential interaction proteins with ZaARR24 was identified in Z.armatum by yeast twohybrid assay.Finally,the possible genetic regulatory pathways involved in ZaARR24 were analyzed by combining the results of transcriptome sequencing and the detection of plant sugars and endogenous hormones levels,as well as the results of exogenous treatment with6-BA.Further,the molecular mechanism and candidate exogenous regulatory factors of ZaARR24 participating in the differentiation of male and female flower in Z.armatum were preliminarily elucidated.This study provides enough evidences for revealing the underling regulations involved in floral sexy transition in Z.armatum.(1)In combination with RACE and reference genome alignment analysis,the coding sequence of the ZaARR24 gene was cloned,which encodes 125 amino acids.Compared with the published genome sequence of ZaARR24 of Z.armatum,the protein sequence was identical,although there were base differences at 207 bp and 273 bp.The bioinformatics analysis displayed that the ZaARR24 protein sequence has closer relationships with Rutaceae and Euphorbiaceae.ZaARR24 protein contains a conserved REC domain consisting of three motifs in the N-terminal amino acid region 20 to 120.In addition,protein subcellular localization indicated that ZaARR24 was located in the cytoplasm and nucleus.(2)Both vegetative and reproductive growth were significantly suppressed in ZaARR24 heterologous overexpression lines.In nutritional growth,phenotypic changes such as weaker plants,block in stem segment xylem development and atrophy of root systems were observed.In reproductive growth,phenotypic changes such as early bolting and flowering,significantly reduced amounts of flowers,the overall reduced size of floral organs,shorter fruit pod,stunted partial carpels were observed.Pollen observation and pollination treatments indicated that overexpression of ZaARR24 mainly affected the pollen and ovule development.(3)The results of the assay of 12 kinds of plant endogenous hormones showed that an overall increase in the content of cytokinin-like substances and a significant decrease in the content of auxin and jasmonate in ZaARR24 overexpression lines.Analysis of the six plant sugars showed that the contents of total sugars,reducing sugars,trehalose and sucrose were significantly increased in ZaARR24 overexpression lines,but the contents of starch and maltose were significantly decreased.The differential transcriptome analysis of inflorescence buds between wild type and ZaARR24 overexpression lines indicated that plant endogenous hormone and saccharides synthesis pathways were significantly enriched.On the other hand,cytokinin signaling,starch and maltose synthesis pathways were significantly down-regulated.The above results implied that ZaARR24 may regulate the reproductive growth process of plants by cytokinin and saccharides signaling.(4)The ZaARR24 protein without auto-activation and toxicity in yeasts was suitable for high-throughput yeast two-hybrid c DNA library screening.A total of 65 candidate interacting proteins were obtained,which were mainly involved in plant floral bud sex differentiation,sugar metabolism,phytohormone signal transduction and other related biological processes.Furthermore,ZaARR24 was dirtectly interacted with ZaAG protein comfirmed by yeast two-hybrid and subcelluar co-location analysis,which can be used for subsequent protein interaction analysis.(5)In combination with the above results,different concentrations of 6-BA were selected for treatment of flower buds of Z.armatum to analyze the expression pattern of related genes.The results of quantification of gene expression in male flower buds after treatment with different concentrations of 6-BA showed that 100 mg/L resulted in the highest expression of ZaARR24 gene and significantly reduced the expression levels of endogenous cytokinin signaling pathway.In contrast,300 mg/L significantly increased the expression levels of cytokinin synthesis and signal transduction,as well as starch and maltose biosynthesis pathways.