A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis

Abstract

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2. When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera, we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine.

Keywords: WRKY; flavonoids; grapevine; petunia; vacuolar acidification.

Figure 1

Phylogenetic tree including WRKYs belonging to different species. The analysis, based on alignments of the C-terminal WRKY domain, was performed using the neighbor-joining method by the Mega version 6 program (Tamura et al., 2013). The scale bar represents the number of substitutions per site and the numbers next to the nodes are bootstrap values from 1000 replicates. The accession numbers are reported in the Materials and methods section.

Figure 2

Analyses of primary protein structures. (A) Alignment of VvWRKY26, PhPh3, AtTTG2, and BnTTG2 predicted sequences. Identical, conserved and similar residues are shown in black, light gray and dark gray, respectively. The black lines below the alignment locate the two WRKY domains, with the conserved sequences WRKYGQK and the C2H2 zinc-finger motifs underlined by dashed lines and black dots, respectively. Triangles above the alignment indicate the position of the introns. The first three introns are conserved in all three species, while the last one close to the 3′ UTR is present only in petunia and grapevine. Red squares individuate β2, β3, and β4 in the WRKY domains. (B) Protein domain organization of Group I WRKY factors represented by colored boxes identified by MEME Suite. The consensus sequence of the motifs is reported.

Figure 3

Complementation analysis in ph3 petunia mutant with 35S:VvWRKY26. (A) Phenotype of untransformed flowers of the wild type R27 and mutant ph3 lines compared to transgenic flowers of two lines expressing VvWRKY26. (B) Total anthocyanin content (μg^*g⁻¹ fresh weight) of petal limb extracts from untransformed R27 and ph3 lines and transgenic plants determined by spectrophotometry at 540 nm. Purified malvidin 3-glucoside was used as a standard. Data represent the mean of three biological replicates ± SE. Asterisks indicate significant difference against the ph3 mutant line (^**P > 0.01). (C) The pH values of crude petal limb extracts from untransformed R27 and ph3 lines and transgenic plants. Each pH value is the mean of 10 biological replicates ± SE. Asterisks indicate significant difference against the ph3 mutant line (^*P > 0.05; ^**P > 0.01).

Figure 4

Expression analyses in petunia petals by qPCR. (A) Expression analysis of F3′H and N21 in the ph3 mutant and in VvWRKY26 expressing lines as confirmation of the microarray results. (B) Expression analysis of structural genes related to vacuolar acidification (PhPH5 and PhPH1) and to anthocyanin synthesis (PhCHS-A and PhDFR-A) in the untransformed R27 and ph3 lines and VvWRKY26 expressing plants. In all analyses the data correspond to the mean ± SE of three biological replicates (corresponding to lines 1, 2, and 5; Supplementary Figure 2) relative to an ACTIN housekeeping control and normalized against the ph3 mutant value. Abbreviations correspond to: PhF3′H, FLAVONOID-3′-MONOOXYGENASE; PhN21, NODULIN MTN21-LIKE PROTEIN; PhPH5, H⁺ P_3A-ATPASE; PhPH1, P_3B-ATPASE; PhCHS-A, CHALCONE SYNTHASE A; PhDFR-A, DIHYDROFLAVONOL 4-REDUCTASE A.

Figure 5

VvWRKY26 expression profile in grapevine. (A) VvWRKY26 expression profile in the V. vinifera cv. Corvina atlas in 45 organs/tissues during development. (B) Expression analysis of VvWRKY26 by qPCR in selected organs/stages of development of cv. Corvina Data, relative to VvUBIQUITIN1 control, are the mean of three biological replicates ± SE. For both analyses the abbreviations after organ correspond to: FS, fruit set; PFS, post fruit set; V, véraison; MR, mid-ripening; R, ripening; Bud - L, latent bud; Bud - W, winter bud; Bud - S, bud swell; Bud - B, bud burst; Bud - AB, bud after burst; Inflorescence - Y, young; Inflorescence - WD, well-developed; Flower - FB, flowering begins; Flower - F, flowering; Tendril - Y, young; Tendril - WD, well-developed; Tendril - FS, mature; Leaf - Y, young; Leaf - FS, mature; Leaf - S, senescing leaf; Stem - G, green; Stem - W, woody.

Figure 6

Localization of VvWRKY26 transcripts by in situ hybridization in berry at fruit set and post véraison stages. Sections of berry hybridized with a VvWRKY26 RNA sense probe as negative control did not show any significant signals (A–C). The antisense probe detected VvWRKY26 transcripts, resulting in a violet-gray coloration (D–I). (A–C) represent the hybridization of longitudinal section of berry at post véraison, a magnification of skin and of a vascular bundle, respectively, with a VvWRKY26 RNA sense probe (negative control). (D–F), representing the hybridization of the berry at fruit set, report the signals in the longitudinal section of the whole berry (D), in the inner integument of the seed coat (E) and in cells surrounding the vascular bundles (F). At this developmental stage no signal was detected in the skin. (G–I), representing the hybridization of the berry at post véraison, report the signals in the inner integument of a seed coat (transversal section, G), in the phloematic cells (H) and in the epidermal cell layer of the skin (I).

Figure 7

Expression analysis of CHS1, MYBPA1, PH5, and PH1 by qPCR in the control lines and VvWRKY26 expressing Sultana plantlets. The data correspond to the mean ± SE of three biological replicates relative to the VvUBIQUITIN1 control and normalized against each control value. Abbreviations correspond to: VvCHS1, CHALCONE SYNTHASE 1; VvMYBPA1, MYB regulator of PA biosynthesis; VvPH5, H⁺ P_3A-ATPASE; VvPH1, P_3B-ATPASE.