The basic leucine zipper transcription factor ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 is an important transcriptional regulator of abscisic acid-dependent grape berry ripening processes

Abstract

In grape (Vitis vinifera), abscisic acid (ABA) accumulates during fruit ripening and is thought to play a pivotal role in this process, but the molecular basis of this control is poorly understood. This work characterizes ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 (VvABF2), a grape basic leucine zipper transcription factor belonging to a phylogenetic subgroup previously shown to be involved in ABA and abiotic stress signaling in other plant species. VvABF2 transcripts mainly accumulated in the berry, from the onset of ripening to the harvesting stage, and were up-regulated by ABA. Microarray analysis of transgenic grape cells overexpressing VvABF2 showed that this transcription factor up-regulates and/or modifies existing networks related to ABA responses. In addition, grape cells overexpressing VvABF2 exhibited enhanced responses to ABA treatment compared with control cells. Among the VvABF2-mediated responses highlighted in this study, the synthesis of phenolic compounds and cell wall softening were the most strongly affected. VvABF2 overexpression strongly increased the accumulation of stilbenes that play a role in plant defense and human health (resveratrol and piceid). In addition, the firmness of fruits from tomato (Solanum lycopersicum) plants overexpressing VvABF2 was strongly reduced. These data indicate that VvABF2 is an important transcriptional regulator of ABA-dependent grape berry ripening.

Figure 1.

Sequence analysis of VvABF2. Full-length sequence comparison of VvABF2 and its closest orthologs from Arabidopsis, AtABF2 (AF093445), AtABF3 (AF093546), AtABF4 (AF093547), and AtABF1 (AF093544), using the Clustal Omega program (Sievers et al., 2011). Conserved residues are shaded in black, conserved residues in four out of five of the sequences are indicated in dark gray shading, and conserved residues in three out of five of the sequences are shown by a light gray shading. The basic regions and the Leu repeats are indicated by white rectangles and arrows, respectively. The Gln-rich region, commonly found in transcriptional activation domains, is underlined (dashed line) for VvABF2. The recognition sites for calmodulin-dependent protein kinase II (XRXXSX) and casein kinase II [X(S/T)XX(D/E)X] are indicated, respectively, by gray and black lines on the top of the alignment. Putative phosphorylated amino acids in the VvABF2 sequence are marked by stars.

Figure 2.

Phylogenetic analysis of VvABF2. The phylogenetic tree represents VvABF2 (black circle) and its orthologs (boldface) from the A subgroup of bZIP transcription factors in Arabidopsis (AT) and grape (VIT). The closest ortholog of VvABF2 from tomato (SlAREB1; Bastías et al., 2011) and representative Arabidopsis bZIP transcription factors from other bZIP subgroups (subgroup G, AtGBF2; H, AtHY5; I, AtbZIP29; E, AtbZIP34; D, AtTGA3; S, AtATB2; B, bZIP28; and C, AtBZO2H2) are also reported according to Jakoby et al. (2002) and Corrêa et al. (2008). The gene identifiers as well as synonyms (published names) are given. Multiple sequence alignments were generated using Clustal Omega (Sievers et al., 2011) on full-length proteins as implemented by MEGA software, version 5.0 (Tamura et al., 2011). The phylogenetic tree was constructed by neighbor joining with complete deletions as implemented by MEGA. Reliability values at each branch represent bootstrap samples (2,000 replicates).

Figure 3.

Quantitative real-time RT-PCR analysis of VvABF2 expression patterns in grapevine cv Cabernet Sauvignon plants and ABA-treated cells. A, VvABF2 expression in grapevine organs: roots (R), stems (S), leaves (L), inflorescences (I), and ripening berries at 11 WAF (RB). Error bars were calculated as sd for three independent experiments. Gene expression was normalized with VvEF1γ (V. vinifera ELONGATION FACTOR1; Q9SPF8). B, VvABF2 expression at different stages of berry development, from 2 WAF to mature berries at 15 WAF. The arrow indicates the véraison stage. Error bars were calculated as the sd for four replicates from two independent experiments (summer 2006 and 2009). Gene expression was normalized with VvEF1γ. C, VvABF2 expression in different tissues from ripening berries at 11 WAF. Error bars were calculated as the sd for three independent experiments. Gene expression was normalized with VvEF1γ. D, VvABF2 transcript accumulation in cv Cabernet Sauvignon suspension cells treated with 20 μm ABA (gray bars) or with the same amount of ethanol (control; black bars). Error bars were calculated as sd for three independent experiments. Gene expression was normalized with VvEF1γ.

Figure 4.

Subcellular localization and transactivation ability of VvABF2. A, Nuclear localization of the GFP-VvABF2 fusion protein in tobacco leaf protoplasts. These confocal microscopy images indicate, from left to right, protoplast sections analyzed for GFP fluorescence, the same sections analyzed for chloroplast autofluorescence, and the transmission light image from the same protoplast sections. B, Promoter activation by VvABF2 of selected ABA-regulated genes (Wang et al., 2011), VvLEA (VIT_08s0007g04240), VvNAC (VIT_19s0014g03290), and VvBenzoR (VIT_07s0005g00140), in tobacco protoplasts. pr35S::GUS was used as a positive control. White bars indicate GUS activity without additional construct or treatment, gray bars indicate GUS activity after transformation with the 35S::VvABF2 construct, dotted bars indicate GUS activity after 20 μm ABA treatment, and black bars indicate GUS activity after transformation with the 35S::VvABF2 construct coupled with 20 μm ABA treatment. Data from three independent experiments were pooled and analyzed. Error bars indicate sd. Statistical significance was assessed by one-way ANOVA followed by Tukey’s honestly significant difference post-hoc test (P ≤ 0.05).

Figure 5.

Relative expression level of VvABF2 in transgenic grape 41B cells. VvABF2 transcript level was assessed by quantitative real-time PCR in control (pFB8 empty vector) and VvABF2-overexpressing (35S::VvABF2) 41B cell lines, treated or not with 20 μm ABA for 1 h. Gene expression was normalized with VvEF1γ. Data are means of three independent experiments, and error bars are sd.

Figure 6.

Overlap, expression profile clustering, and functional categorization of the 1,722 differentially expressed genes in the three experimental conditions. A, Three-way Venn diagram showing the overlap of differentially expressed genes in the three experimental conditions: condition 1 (control + ABA versus control), condition 2 (35S::VvABF2 versus control), and condition 3 (35S::VvABF2 + ABA versus control). B, Ten clusters have been created using MapMan (Thimm et al., 2004) on transcript ratios for the 1,722 differentially expressed genes under condition 1, condition 2, and condition 3. C, Classification of the 1,722 differentially expressed genes within selected MapMan ontology classes. The x axis indicates the number of genes within the different functional categories (y axis) for each condition: condition 1, condition 2, and condition 3. MISC, Miscellaneous.

Figure 7.

Time course of stilbene production (nmol g⁻¹ dry weight [DW]) in the culture medium of control and 35S::VvABF2 transgenic cells supplied with 20 μm ABA. Total trans-piceid (gray bars) and trans-resveratrol (black bars) were measured in the extracellular medium. Values represent means ± sd of triplicate assays of one representative experiment out of three.