Study on the molecular mechanism of promotion of inflorescence formation of CCC treatment for the second fruiting in grape

Abstract: 【Objective】The study aimed to explore the main biological processes and important genes in-volved in inflorescence formation for the second fruiting of‘Summer Black’grape after chlormequat chloride (CCC) treatment through screening the differential expression genes (DEGs) in the key node buds for the second fruiting at different development stages by transcriptome sequencing.【Methods】 Leaves of the‘Summer Black’grape were sprayed with 0.5 g·L -1 CCC on the 10th and 1st day before full blooming. The control plants were sprayed with purified water. In the control and CCC treatment, when the 14th leaf of the main shoot unfolded, the top of the shoot was removed and all the auxiliary shoots were wiped out. The latent buds on the 5th and 6th nodes were used as experimental materials, and these buds were collected on the 3rd day before full blooming, 4th day after full blooming (DAF) and 11th DAF of the first season fruit of‘Summer Black’grape, respectively. The samplings were car- ried out on 7th, 14th and 21th day after the first CCC treatment. The total RNA of latent buds was ex- tracted, and the genomic DNA was removed. The differences in transcript abundance values among the  samples were calculated using the ratio of Fragments Per Kilobase of transcript per million mapped reads (FPKM), and the significance of the differences was computed using the false discovery rate (FDR). Genes with a log2 ratio ≥ 2 and FDR significance score < 0.01 were regarded as DEGs. Gene Ontology (GO) function enrichment and KEGG pathway enrichment analysis of DEGs were performed using Plant Met Gen MAP. The K-means clustering were performed using Gene-E to reveal the expres- sion patterns of the DEGs at the three time points.【Results】The number of DEGs on the 3rd DAF, 4th DAF and 11th DAF post CCC treatment was 395, 557 and 97, respectively, compared with the control, among them 52, 21 and 39 genes were up regulated and 343, 536 and 58 genes were down regulated, re-spectively. GO analysis results showed that in the process of inflorescence primordium formation in the latent buds, the DEGs were mainly enriched in cell components, cell processes, signal biological pro- cesses, metabolic processes and stimulation responses. Further analysis of KEGG metabolic pathway showed that the DEGs were significantly enriched in phenylpropanoid synthesis, plant hormone signal transduction; CCC treatment could decrease the expression levels of the DEGs in the pathways of phen- ylpropanoid synthesis and flavonoid synthesis on the 3rd DAF and 4th DAF, while on the 11th DAF, the number of up- regulated genes post CCC treatment was more than those on the 3rd DAF and 4th DAF, indicating the two pathways might be involved in the regulation of the inflorescence formation for the second fruiting. The differentiation time of uncommitted meristems might be prolonged by down regu- lating the expression of related genes in the phenylpropanoid synthesis and flavonoid synthesis pathway between the 3rd DAF and 4th DAF, and the uncommitted meristems could be prevented from differenti-ating into tendril primordium on the 11th DAF. Besides phenylpropanoid synthesis and flavonoid syn- thesis pathways, the expression levels of genes involved in abscisic acid signal transduction, auxin sig- nal transduction, cytokinin signal transduction, gibberellin signal transduction, ethylene signal transduc- tion, circadian rhythm, positive regulation of flower development, photoperiodism, negative regulation of flower development and flower development, as well as some transcription factors were up- or down- regulated at one or more time points post CCC treatment, indicating these genes were also in- volved in the inflorescence formation for the second fruiting of‘Summer Black’grape.【Conclusion】 Through transcriptome sequencing and qRT-PCR, the expression levels of PODN1 and POD47 in phen- ylpropanoid synthesis pathway, STS6 in flavonoid synthesis pathway, IAA26 in auxin signal transduc- tion pathway, GARP6 and GA3OX1 in gibberellin signal transduction pathway were significantly changed in the key period of latent buds differentiation (formation of uncommitted lateral meristems and differentiation of uncommitted lateral meristems) for the second fruiting of Summer Black grape af-ter CCC treatment. As a chemical measure, CCC treatment might promote inflorescence formation by changing the hormone contents and ratios in the tree, as well as the synthesis and metabolism of second-ary metabolites. It would be a complex gene regulatory networks responding to CCC treatment to pro- mote the inflorescence formation for the second fruiting of‘Summer Black’grape.