Drought stress modulates cuticular wax composition of the grape berry

Abstract

Drought events are a major challenge for many horticultural crops, including grapes, which are often cultivated in dry and warm climates. It is not understood how the cuticle contributes to the grape berry response to water deficit (WD); furthermore, the cuticular waxes and the related biosynthetic pathways are poorly characterized in this fruit. In this study, we identified candidate wax-related genes from the grapevine genome by phylogenetic and transcriptomic analyses. Developmental and stress response expression patterns of these candidates were characterized across pre-existing RNA sequencing data sets and confirmed a high responsiveness of the pathway to environmental stresses. We then characterized the developmental and WD-induced changes in berry cuticular wax composition, and quantified differences in berry transpiration. Cuticular aliphatic wax content was modulated during development and an increase was observed under WD, with wax esters being strongly up-regulated. These compositional changes were related to up-regulated candidate genes of the aliphatic wax biosynthetic pathway, including CER10, CER2, CER3, CER1, CER4, and WSD1. The effect of WD on berry transpiration was not significant. This study indicates that changes in cuticular wax amount and composition are part of the metabolic response of the grape berry to WD, but these changes do not reduce berry transpiration.

Keywords: Vitis vinifera (grapevine); Cuticle; fruit; transpiration; triterpenoids; water deficit; wax esters.

Fig. 1.

Protein sequence phylogenetic relationships of putative grapevine (Vitis vinifera L.) homologs of Arabidopsis biosynthetic genes involved in cuticular aliphatic wax biosynthesis. PAS2 (A), KCR1 (B), CER10 (C), KCS6 (D), and CER2 (F) are involved in fatty acid elongation. CER3 and CER1 (E) are part of the alkane-forming branch, and CER4 (G) and WSD1 (H) are part of the alcohol-forming branch. BAS (I) and CYP716A (J) are part of the oleanolic acid biosynthetic pathway. Numbers represent bootstrap values (100 bootstrap replicates), with branches of values <50 being collapsed together. Bold brackets denote clades that contain the characterized Arabidopsis sequences (in bold) involved in cuticular wax synthesis, and genes that are most closely related and are the likeliest functionally related homologs. Asterisks denote genes selected for study in the water deficit experiment. Scale bars are shown in each panel.

Fig. 2.

Cuticular wax composition in berries at 27, 41, 68, 82, 96, and 111 days after anthesis (DAA) of grapevines (Vitis vinifera L.) exposed to two irrigation treatments: well-irrigated (control, CT) and deficit irrigated (water deficit, WD). Total aliphatic waxes (A), total triterpenoid waxes (B), triterpenoids:aliphatic waxes ratio (C), individual functional group classes of aliphatic waxes (D), and wax ester composition (E) are reported. Please refer to Supplementary Figs S7–S11 for detailed wax composition of other functional group classes. At 68 DAA, green and red berries were analyzed independently. Error bars represent ±SE, and significant differences between treatments were determined by two-sample t-test (*P<0.05).

Fig. 3.

Ultrastructural morphology of cuticular waxes on grape berries at 113 days after anthesis (DAA). SEM images of the cuticular wax ultrastructure on grape berries of grapevines (Vitis vinifera L.) exposed to two irrigation treatments: (A–C) well-irrigated (control, CT); (D and E) deficit irrigated (water deficit, WD). Each image represents one biological replicate. The scale bar represents 5 µm in all images.

Fig. 4.

Expression of candidate genes involved in the fatty acid elongation of the biosynthetic cuticular wax pathway in berry skins of grapevines (Vitis vinifera L.) exposed to two irrigation treatments: well-irrigated (control, CT) and deficit irrigated (water deficit, WD). Error bars represent ±SE, and significant differences between treatments were determined by two-sample t-test (*P<0.05).

Fig. 5.

Expression of candidate genes involved in the alcohol- and alkane-forming branches of the biosynthetic cuticular wax pathway in berry skins of grapevines (Vitis vinifera L.) exposed to two irrigation treatments: well-irrigated (control, CT) and deficit irrigated (water deficit, WD). Error bars represent ±SE, and significant differences between treatments were determined by two-sample t-test (*P<0.05).

Fig. 6.

Expression of candidate genes involved in the oleanolic acid biosynthesis, and transcription factors (TFs) and an E3 ubiquitin ligase involved in regulating cuticular wax development of berry skins of grapevines (Vitis vinifera L.) exposed to two irrigation treatments: well-irrigated (control, CT) and deficit irrigated (water deficit, WD). Error bars represent ±SE, and significant differences between treatments were determined by two-sample t-test (*P<0.05).

Fig. 7.

Rates of water transpiration of the whole berry at different stages of development in grapevines (Vitis vinifera L.) exposed to two irrigation treatments: well-irrigated (control, CT) and deficit irrigated (water deficit, WD). Stages of development that were measured were 27 (A), 48 (B), 75 (C), 97 (D), and 111 (E) days after anthesis (DAA). Error bars represent ±SE; significant differences in the cumulative amount of water lost for each developmental stage were determined by univariate repeated measures ANOVA (U.R.M. ANOVA). The effects of treatment, time, and their interaction are reported. In the table (F), the average transpiration rate and the significance value of the t-test at each developmental stage are reported.