Transcriptional regulatory network prediction of eugenol- loaded chitosan nanoparticles inhibiting SO2-induced postharvest abscission in Kyoho grape

Abstract:【Objective】Sulfur dioxide (SO2) is widely used for postharvest preservation of table grapes, it can effectively control grape infection with Botrytis cinerea during storage, but it will also lead to berry abscission and affect the economic value of table grapes. To find a method to inhibit SO2-induced postharvest grape berry abscission, and to explore its molecular mechanism, in this study, the effect of eugenol- loaded chitosan nanoparticles as edible materials on SO2- induced berry abscission was explored, and the key candidate genes that played a role in inhibiting grape berry abscission were screened, and the transcriptional regulatory molecular mechanism of inhibiting grape abscission was explored.【Methods】The Kyoho grapes were treated with SO2 and SO2 + eugenol-loaded chitosan nanopar-ticles (SN). The grape samples stored for 2 d, 4 d and 6 d were collected as experimental materials. The postharvest grape phenotype was photographed and the berry abscission rate was recorded every 2 d. The berry abscission of the control and treatment was observed and calculated. RNA sequencing (RNAseq) was performed on different grape samples collected, and the differential expression between control and treatment was analyzed by DESeq2 software to obtain differentially expressed genes (DEGs). GO enrichment and TCseq analysis were used to analyze the DEGs, and the TFs and binding sites of target genes were predicted through PlantTFDB database to explore the possible transcriptional regulation relationship in the process of inhibiting SO2-induced berry abscission during storage. Finally, the DEGs expression was verified by quantitative real-time PCR (qRT-PCR).【Results】By observing the phenotype and recording berry abscission rate, we found that the berry abscission rate with eugenol-loaded chitosan nanoparticles treatment was significantly lower than that with SO2 control at 6 d during postharvest storage, indicating that eugenol- loaded chitosan nanoparticles significantly inhibited SO2- induced berry abscission. According to the standards of |log2FC| ≥ 0.585 and P-adjust ＜ 0.05, 978, 141 and 946 DEGs were detected at 2, 4 and 6 d, respectively, including 57, 4 and 50 TFs. Among them, 35 up- regulated genes and 15 down- regulated genes were expressed at 2, 4 and 6 d during storage. GO functional enrichment analysis showed that DEGs down-regulated on the 2th day and up-regulated on the 6th day were enriched into more GO term. It mainly involved organic substance catabolic process, carbohydrate metabolic process, defense response, phenylpropanoid metabolic process, cellulose metabolic process, pectin metabolic process, lignin catabolic process, abscisic acid-activated signaling pathway, cell wall organization and other related term, among which cellulose metabolism process, pectin metabolism process and lignin catabolic process were related to cell wall metabolism. According to the expression patterns of DEGs, 1766 DEGs were divided into 6 Clusters by TCseq analysis. The average distribution of log2FC of DEGs in Cluster 1-6 in different periods was generally consistent with the expression change patterns of log2FC. Log2FC heat maps of all transcription factors (TFs) in Cluster 1-6 showed that eugenol-loaded chitosan nanoparticles-induced differential changes occurred in the expression of many types of TFs, mainly including ERF, NAC, bHLH, bZIP, WRKY, DOF, HB- other, HDZIP, MYB and MYB_related, etc. The binding sites between TFs and target genes in Cluster 1-6 were predicted by PlantTFDB database, and 22 key candidate TFs and 203 target genes were further screened according to the conditions of correlation coefficient (r) ＞ 0.8 and P-adjust < 0.01. The transcription factor regulatory network map further revealed the regulatory relationship between 22 TFs and 203 target genes. Log2FC heat maps of 22 TFs showed that eugenol-loaded chitosan nanoparticles induced the continuous up- regulation of MYB61, DOF5.6, DIV and ERF4 genes; the expressions of bZIP53, WRKY24, WRKY65, ARF6, GAMYB, bHLH66 and HFB2B were continuously down- regulated; ERF92 and NAC87 were first up-regulated and then down-regulated; KAN2, MYB15, RAP2-7, WRKY11, ANL2 and WRKY53 decreased first and then increased. Eight DEGs were randomly selected from the transcriptional regulation network diagram for qRT-PCR to verify the reliability of transcriptome data, and the qRT-PCR results were consistent with the transcriptome data. After eugenol-loaded chitosan nanoparticles were treated, the expression of bZIP53, WRK24, WRK14, GAPDH and HT1 was up-regulated, the expression trend of ARF and ERF5 was up-regulated first and then down-regulated, and CSE was downregulated first and then up- regulated. And HT1 and GAPDH were regulated by transcription factors bZIP53 and WRKY24, respectively. After treatment with eugenol-loaded chitosan nanoparticles, the expression of HT1 and bZIP53 showed the same trend, and the change trend of GAPDH and WRKY24 was consistent, which speculated that bZIP53 and WRK24 may participate in the inhibition of SO2-induced berry abscission by regulating target genes related to energy metabolism and sugar metabolism.【Conclusion】In this study, we screened out the key transcription factors and corresponding target genes of eugenol-loaded chitosan nanoparticles in inhibiting SO2-induced grape berry abscission, predicted the possible transcriptional regulatory network in the process of inhibiting SO2- induced berry abscission, and laid a theoretical foundation for revealing the molecular mechanism of edible coating inhibiting grape berry abscission.