The Mechanism Of Transcription Factor AcHZP45 In Regulating Chlorophyll Metabolism In Kiwifruit

Chlorophyll is one of the major pigments that presenting color for plants,the degradation of which during fruit development promotes the exposure of more ‘ripe’red or yellow/orange color,attracting wild dispersers of seeds as well as the human consumer.Commercially cultivated kiwifruit also show the wide variety of flesh color,which can be generally classified into green-and yellow-fleshed cultivars,including most red-cored cultivars.However,the underlying mechanisms for the differential formation of flesh color between green-and yellow-fleshed kiwifruit are still unclear.In present study,the fruits of green-fleshed ‘Xuxiang’(XX)and yellow-fleshed ‘Jinshi No.1’(JS)were selected as the experimental materials,and the research methods such as RNA-sequencing(RNA-seq),weighted gene co-expression network analysis(WGCNA),tobacco transient overexpression,kiwifruit homologous genenic transformation,dual-luciferase assay,electrophoretic mobility shift assay(EMSA)and DNA affinity purification sequencing(DAP-seq),were employed to identify the key genes related to the kiwifruit chlorophyll metabolism and elucidate the transcriptional regulatory mechanisms of differential chlorophyll metabolism during green-and yellow-fleshed kiwifruit development.The main results are as follows:1.The metabolism of chlorophyll during fruit development were significantly different between green-fleshed ‘XX’ and yellow-fleshed ‘JS’.The pigments quantification and comparative analysis for developing fruits of these two cultivars were conducted and found that chlorophyll degradation rate in yellow-fleshed ‘JS’ was obviously higher than that in green-fleshed ‘XX’,indicating that variation in chlorophyll might mainly contribute to differences in flesh color.The conjoint analysis between RNA-seq data and physiological traits(chlorophyll,carotenoid and chlorophyll/carotenoid ratio)were applied based on the WGCNA and identified four candidate genes(Ac CRD1 and Ac POR1 driving chlorophyll biosynthesis,and Ac SGR1 and Ac SGR2 responsible for chlorophyll degradation)which were highly related to the differential chlorophyll metabolism between these two kiwifruit cultivars.Comparative sequence analysis indicated that protein sequences of the four hub genes in the two cultivars were very similar,thus expression difference of these four genes might contribute to the difference in chlorophyll metabolism in the fruit of the two cultivars.2.Functional analysis of four hub genes in chlorophyll metabolism.The measurement of intermediate metabolites in the developing fruits from ‘XX’ and ‘JS’showed that prochlorophyllide(Pchlide)and chlorophyllide(Chlide),the catalytic products of Ac CRD1 and Ac POR1,were gradually increased during ‘XX’ fruit ripening,but significantly decreased in ‘JS’,suggesting that obviously declined expression of Ac CRD1 and Ac POR1 in yellow-fleshed ‘JS’ might lead to the blockage of chlorophyll biosynthesis.Moreover,transient expression of Ac SGR1 and Ac SGR2 in tobacco as well as overexpression of them in kiwifruit both resulted in the significant reduction of chlorophyll content,suggesting the essential role of Ac SGR1 and Ac SGR2 in promoting chlorophyll degradation.Thus,the differences in chlorophyll metabolism between the two cultivars might attribute to both chlorophyll synthesis and degradation pathways.3.Transcription factor AcHZP45 promoted kiwifruit de-greening through bidirectionally regulating the expression of chlorophyll biosynthetic genes(CBGs)Ac CRD1 and Ac POR1 as well as the chlorophyll catabolic genes(CCGs)Ac SGR1 and Ac SGR2.Based on the WGCNA analysis,12 differentially expressed transcription factor(DETFs)were identified which showed high correlation with Ac CRD1,Ac POR1,Ac SGR1 and Ac SGR2.Among of them,AcHZP45 belonging to the homeobox-leucine zipper(HD-Zip II)was verified to obviously repress the activities of promoters of Ac CRD1 and Ac POR1 and activate the activities of promoters of Ac SGR1 and Ac SGR2 through dual-luciferase assay.The direct bound of AcHZP45 on the promoters of these four targets was further validated through EMSA.Gene expression profile showed the different expression patterns of AcHZP45 in ‘XX’ and ‘JS’,with more stable expression in the green-fleshed ‘XX’ and significantly increased expression during yellow-fleshed‘JS’ fruit ripening.Transient expression of AcHZP45 in tobacco could induce chlorophyll degradation through regulating the expression of endogenous related genes in tobacco.In addition,differential expression of AcHZP45 between the inner and outer pericarp of ‘JS’ might contribute to the differential chlorophyll degradation in the two tissues by differentially inducing the expression of Ac SGR1 and Ac SGR2.4.Identification of other target genes downstream of AcHZP45.A total of 8,862 peaks located in the promoter region were identified through DAP-seq analysis and KEGG analysis for the peaks-related genes revealed that 38 genes were significantly enriched in chlorophyll metabolism and photosynthesis-related pathways.Based on the previous results from RNA-seq and WGCNA analysis,the expression of these 38 genes during ‘XX’ and ‘JS’ fruit development and their correlation with chlorophyll were analyzed.One CBG(Ac POR2)as well as three photosystem-associated genes(PAGs)(Ac Lhca1.2,Ac Lhcb5 and Ac PSAO)were further screened and might be involved in regulating differential chlorophyll metabolism in green-and yellow-fleshed kiwifruit.Dual luciferase assay showed that AcHZP45 also could significantly repress the promoters of these four genes(Ac POR2,Ac Lhca1.2,Ac Lhcb5 and Ac PSAO).Taken together,this study proposed a transcriptional regulation model HZP45-CBGs/CCGs/PAGs,in which the key transcription factor AcHZP45 functions as a regulatory hub.The differential expression of these genes between two cultivars(greenfleshed ‘XX’ and yellow-fleshed ‘JS)and/or different tissues(inner and outer pericarp of ‘JS’)differentially regulate the contrary dynamics of chlorophyll biosynthesis and degradation,as well as the photosynthesis-related processes,coordinating the difference in chlorophyll content in kiwifruit flesh.These results provide new insights into the regulatory mechanisms of kiwifruit chlorophyll metabolism and enrich the biological functions of HD-Zip family members in fruit chlorophyll metabolism.