Comparative transcriptome analysis of diploid Zhuosexiang and its autotetraploid grape Ziqiuxiang under salt stress and preliminary identification of salt-tolerant gene VvTPS21

【Objective】This study aimed to clarify the differences in salt tolerance between the diploid grape cultivar Zhuosexiang and its homozygous tetraploid counterpart Ziqiuxiang through phenotypic observation. Transcriptome sequencing technology was employed to obtain relevant data, which were then subjected to in-depth mining and analysis using bioinformatics tools. This process identified key genes that enhance grape salt tolerance under polyploidy effects. Furthermore, qRT- PCR technology was utilized to determine the dynamic expression patterns of these key genes.【Methods】We used the diploid grape cultivar Zhuosexiang and the homozygous tetraploid grape cultivar Ziqiuxiang as experimental materials. Salt stress was induced by irrigating with 200 mmol· L- 1 NaCl solution, while irrigation with pure water was set as the control. Combined with observations of grape plant phenotypic changes, transcriptome analysis was performed on leaves 7 d post- salt treatment. Differential expression analysis was conducted using DESeq26 software, with a significance threshold of P- value＜0.05 and fold change ratios ≥2 (|log2FC| ≥ 1) set as the criteria for identifying differentially expressed genes. Leaf samples were collected at 0, 3, 6, 12 and 24 h after salt treatment for qRT-PCR verification of the salt-tolerant gene VvTPS21.【Results】Under salt stress conditions, grape seedlings showed salt damage symptoms by the 7th day of treatment, with yellowing gradually progressing upward from the base of the plants. However, the Ziqiuxiang variety showed less extensive leaf yellowing and fewer instances of it compared with the Zhuosexiang variety. By day 14 of treatment, the lower leaves of Zhuosexiang plants had largely withered and lost physiological activity. Although some leaves of Ziqiuxiang plants also exhibited wilting, the whole plant maintained a certain level of physiological activity. Furthermore, analysis of the salt damage index showed that Ziqiuxiang grapes had stronger salt tolerance than Zhuosexiang grapes. RNA-Seq analysis showed that the gene expression data of the Zhuosexiang and Ziqiuxiang grape groups had distinct distributions, indicating that the two varieties would have significant differences in gene expression levels. Under salt stress treatment, the number of differentially expressed genes in Zhuosexiang grapes is lower than that in Ziqiuxiang grapes. Compared with the control (CK), among the differentially expressed genes identified in the Zhuosexiang grape after salt treatment, 2508 genes were upregulated and 1223 genes were downregulated. In contrast, among the differentially expressed genes identified in the Ziqiuxiang grape after salt treatment, 2427 genes were upregulated and 2875 genes were downregulated. When comparing the differentially expressed genes between the salttreated Zhuosexiang and Ziqiuxiang grape varieties, we found 658 downregulated genes and 232 upregulated genes. To explore the biological functions of differentially expressed genes in Zhuosexiang and Ziqiuxiang grapes, we performed GO enrichment analysis. In the BP module, differentially expressed genes are mainly concentrated in functions related to defense response and abscisic acid response, while in the CC module, they are mainly concentrated in the nucleus. In the MF module, the differentially expressed genes are mainly concentrated in the terms related to binding with specific proteins and ATP. KEGG metabolic pathway enrichment analysis indicated that under salt stress, more differentially expressed genes in Zhuosexiang grapes were involved in the biosynthetic pathways of secondary metabolites. In contrast, the differentially expressed genes in Ziqiuxiang grapes were enriched not only in the biosynthetic pathways of secondary metabolites, but also more substantially in plant hormone signal transduction pathways. To further investigate the gene expression characteristics of Zhuosexiang and Ziqiuxiang grapes after salt treatment, we identified 361 highly expressed genes in Zhuosexiang grapes under salt stress. The expression levels of these genes showed no significant change in Ziqiuxiang grapes. Among these 361 genes, 22 transcription factors were identified, belonging to WRKY, MYB, AP2/ERF, LBD and other gene families. We further identified 892 genes that are highly responsive to salt stress in Ziqiuxiang grapes, while the expression levels of these genes showed no significant change in Zhuosexiang grapes under salt stress. Among the 892 genes, 45 transcription factors were identified, belonging to gene families including MYB, AP2/ERF, bHLH, and bZIP. Based on transcriptome data analysis, screening of the TPS gene family revealed that Zhuosexiang exhibited four significantly differentially expressed TPS genes under salt stress, including two upregulated genes and two downregulated genes. While Ziqiuxiang exhibited eight differentially expressed TPS genes under salt stress, with three genes up-regulated and five down-regulated. Further analysis of the differentially expressed TPS genes revealed that three genes were commonly differentially expressed in both grape varieties. Subsequently, a qRT-PCR experiment identified the salt-tolerant gene VvTPS21. The expression of this gene was significantly up- regulated in Ziqiuxiang grapes under salt stress, but significantly down- regulated in Zhuosexiang grapes. Subsequently, we analyzed the promoter of VvTPS21 and identified ABRE, a cis-acting element involved in the abscisic acid response. Additionally, through ELISA testing, we detected that the abscisic acid content in Ziqiuxiang grape is higher than that in Zhuosexiang.【Conclusion】The regulatory mechanisms of gene expression in response to salt stress may differ among grape varieties with different ploidy levels. The tetraploid grape cultivar Ziqiuxiang exhibits stronger salt tolerance than the diploid cultivar Zhuosexiang. Under salt stress, a larger number of differentially expressed genes were de-tected in Ziqiuxiang, and these genes were more significantly enriched in functions related to defense response and abscisic acid signaling, while more metabolic pathways related to plant hormone signaling were enriched as well. The identified salt-tolerant gene VvTPS21 may play an important role in the response of polyploid grapes to salt stress. We speculate that it functions through the abscisic acid signaling pathway, but the specific salt-tolerant mechanism requires further study.