Combined Metabolite and Transcriptome Profiling Reveals the Norisoprenoid Responses in Grape Berries to Abscisic Acid and Synthetic Auxin

Abstract

The abscisic acid (ABA) increase and auxin decline are both indicators of ripening initiation in grape berry, and norisoprenoid accumulation also starts at around the onset of ripening. However, the relationship between ABA, auxin, and norisoprenoids remains largely unknown, especially at the transcriptome level. To investigate the transcriptional and posttranscriptional regulation of the ABA and synthetic auxin 1-naphthaleneacetic acid (NAA) on norisoprenoid production, we performed time-series GC-MS and RNA-seq analyses on Vitis vinifera L. cv. Cabernet Sauvignon grape berries from pre-veraison to ripening. Higher levels of free norisoprenoids were found in ABA-treated mature berries in two consecutive seasons, and both free and total norisoprenoids were significantly increased by NAA in one season. The expression pattern of known norisoprenoid-associated genes in all samples and the up-regulation of specific alternative splicing isoforms of VviDXS and VviCRTISO in NAA-treated berries were predicted to contribute to the norisoprenoid accumulation in ABA and NAA-treated berries. Combined weighted gene co-expression network analysis (WGCNA) and DNA affinity purification sequencing (DAP-seq) analysis suggested that VviGATA26, and the previously identified switch genes of myb RADIALIS (VIT_207s0005g02730) and MAD-box (VIT_213s0158g00100) could be potential regulators of norisoprenoid accumulation. The positive effects of ABA on free norisoprenoids and NAA on total norisoprenoid accumulation were revealed in the commercially ripening berries. Since the endogenous ABA and auxin are sensitive to environmental factors, this finding provides new insights to develop viticultural practices for managing norisoprenoids in vineyards in response to changing climates.

Keywords: DAP-seq; abscisic acid; alternative splicing; gene expression; grape berry ripening; naphthaleneacetic acid; norisoprenoid.

Figure 1

Total soluble solids and titratable acidity in two consecutive seasons. The different shape represents the phenological stage: square (E-L 33 stage), big circle (E-L 34 stage), triangle (E-L 35 stage), rhombus (E-L 36 stage), small circle (E-L 38 stage). The lines of green, light purple, purple and light green represent Control, 800 mg/L abscisic acid (ABA) (ABA800), 1000 mg/L ABA (ABA1000), and 100 mg/L synthetic auxin 1-naphthaleneacetic acid (NAA) (NAA100), respectively. Bars represent ± standard deviation.

Figure 2

Effects of ABA and NAA on hormone accumulation and related gene expression during berry development and ripening. (A) Concentrations of endogenous ABA and indole-3-acetic acid protein (IAA) from E-L 33 to E-L 36 stage. Bars represent ± SE and different letters indicate significant differences (p = 0.05). (B) Pathways analysis of genes involved in ABA and norisoprenoid biosynthesis. Purple and red boxes indicate downregulated and upregulated genes, the colors of the boxes represent the intensity of the expression fold changes (log2). Genes with significant expression changes compared with the control groups in each developmental stage are indicated by bold margins. PSY, phytoene synthase; PDS, 15-cis-phytoene desaturase; ZISO, zeta-carotene isomerase; ZDS, zeta-carotene desaturase; CRTISO, prolycopene isomerase; LECY, lycopene epsilon-cyclase; LBCY, lycopene beta-cyclase; LUT5, beta-ring hydroxylase; BCH, beta-carotene 3-hydroxylase; LUT1, carotene epsilon-monooxygenase; ZEP, zeaxanthin epoxidase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxygenase; XDH, xanthoxin dehydrogenase; AAO, abscisic-aldehyde oxidase; CCD, carotenoid cleavage dioxygenase. (C) Pathways analysis of genes involved in auxin biosynthesis. TAR, tryptophan aminotransferase related 1; TAA1, tryptophan aminotransferase arabidopsis1; YUC, YUC flavin monooxygenase.

Figure 3

Expression of genes involved in ABA and auxin signaling pathway. Green and cyan boxes represent significantly downregulated and upregulated genes, respectively, in treated berries compared to the control.

Figure 4

Differentially expressed switch genes between treatments and control. The significance of differentially expressed genes was judged based on the False Discovery Rate ≤ 0.01 and absolute value of log2Ratio ≥ 1. Blue and cyan boxes indicate significantly downregulated and upregulated genes in treated berries compared to the control, respectively. Genes were divided into groups (a–e) according to their different responses to the treatments.

Figure 5

Concentrations of free, total and individual norisoprenoids. (A) Concentrations of free norisoprenoids in 2015. (B) Concentrations of total norisoprenoids in 2015. (C) Concentrations of free norisoprenoids in 2016. (D) Concentrations of total norisoprenoids in 2016. Statistical analysis involved the comparison of treatments at each E-L stage, and different letters represent significant differences (p = 0.05).

Figure 6

Weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) induced by ABA100 or NAA100 and the hierarchical cluster analysis of associated genes related to the accumulation of norisoprenoid. (A) Hierarchical cluster tree showing 12 merged modules of co-expressed genes. (B) Module-trait correlations and corresponding p-values. The left panel shows 12 modules and the right panel is a color scale for module trait correlation from −1 to 1.

Figure 7

Gene co-expressed and regulatory network. (A) Gene co-expressed and regulatory network related to norisoprenoid biosynthesis. The size of the nodes corresponds to the nodes ‘betweenness centrality’, which measures how often a node in the network occurs on all shortest paths between two nodes and help identify the gene that plays a ‘bridge spanning’ role in the network. Red and blue edges indicate that the interaction between two genes is identified by WGCNA and DNA affinity purification sequencing (DAP)-seq, respectively. (B) Hierarchical cluster analysis of genes in the left graph. The colors of the boxes represent the intensity of the normalized gene.

Figure 8

Qualitative RT-PCR analysis of expression of VviDXS and VviCRTISO splice variants in grape berries at specific stage under NAA100 and ABA1000 treatments. The forward and reverse primers were designed from the upstream and downstream exons of the skipped exon or retention intron, respectively.