The Combination of Abscisic Acid (ABA) and Water Stress Regulates the Epicuticular Wax Metabolism and Cuticle Properties of Detached Citrus Fruit

Abstract

The phytohormone abscisic acid (ABA) is a major regulator of fruit response to water stress, and may influence cuticle properties and wax layer composition during fruit ripening. This study investigates the effects of ABA on epicuticular wax metabolism regulation in a citrus fruit cultivar with low ABA levels, called Pinalate (Citrus sinensis L. Osbeck), and how this relationship is influenced by water stress after detachment. Harvested ABA-treated fruit were exposed to water stress by storing them at low (30-35%) relative humidity. The total epicuticular wax load rose after fruit detachment, which ABA application decreased earlier and more markedly during fruit-dehydrating storage. ABA treatment changed the abundance of the separated wax fractions and the contents of most individual components, which reveals dependence on the exposure to postharvest water stress and different trends depending on storage duration. A correlation analysis supported these responses, which mostly fitted the expression patterns of the key genes involved in wax biosynthesis and transport. A cluster analysis indicated that storage duration is an important factor for the exogenous ABA influence and the postharvest environment on epicuticular wax composition, cuticle properties and fruit physiology. Dynamic ABA-mediated reconfiguration of wax metabolism is influenced by fruit exposure to water stress conditions.

Keywords: ABA deficiency; Pinalate; fruit dehydration; gene expression; hormone application; postharvest.

Figure 1

Effect of ABA and water stress on the total epicuticular wax and individual fractions content in Pinalate fruit. The effect of ABA (1 mM) treatment was evaluated together with the influence of high (90–95%) or low (30–35%) RH conditions on the total content (µg.cm⁻²) of epicuticular wax and the individual fractions up to 3 weeks of leaving Pinalate fruit at 20 °C. Bars are means ±SD of four replicates per condition. FH: Freshly harvested fruit. For each panel, the different letters above the bars indicate significant (p < 0.05) differences among conditions according to an ANOVA analysis followed by a Tukey test (p < 0.05).

Figure 2

Effect of ABA and water stress on the proportion of epicuticular wax fractions in Pinalate fruit. The effect of ABA (1 mM) treatment on the percentage (%) of epicuticular wax fractions by 1 and 3 weeks (W) of Pinalate fruit storage at 20 °C was evaluated together with the influence of the (A) high (90–95%) or (B) low (30–35%) RH conditions. FH: Freshly harvested fruit. The values on the bars indicate the percentage of each fraction. The alkane/terpenoid and alcohol/FA ratios are indicated per condition. The same legend is used for panels (A,B).

Figure 3

Effect of ABA and water stress on epicuticular wax constituents during Pinalate fruit storage. The effects of ABA (1 mM) treatment on the content of the epicuticular wax components were evaluated together with the influence of high (90–95%) or low (30–35%) RH by 1 (A–D) and 3 (E–H) weeks at 20 °C. (A,E) Alkanes; (B,F) fatty acids; (C,G) alcohols and aldehydes; and (D,H) terpenoids. Bars are the means ±SD of four replicates per condition. Different letters above the bars indicate significant (p < 0.05) differences among conditions according to an ANOVA analysis followed by a Tukey test (p < 0.05) for each component and storage time separately.

Figure 4

Effect of ABA and water stress on cuticle properties and fruit physiological parameters in Pinalate fruit. (A) ABA content is expressed as g per g of fresh weight (FW) of the flavedo. Bars are the means ±SD of three replicates of five fruit each. (B) Cuticle thickness. Bars represent the means ±SD of about 50 measurements for all three biological replicates analyzed per condition. (C) Cuticle permeability. Bars are the means ±SD of three replicates per condition. (D) Cumulative weight loss of Pinalate fruit calculated as fruit weight loss per surface area. Bars are the means ±SD of three replicates of 10 fruit each. (E) Fruit firmness was determined according to the intact fruit compression resistance load (N) and normalized by fruit size (cm³). Bars indicate the means ±SD of three replicates of 10 fruit each. For each studied parameter, different letters indicate the statistical (p < 0.05) differences in all the conditions together according to an ANOVA analysis followed by a Tukey test (p < 0.05).

Figure 5

Clustering and correlation analyses of epicuticular wax composition, ABA content, cuticle properties and fruit physiological parameters in Pinalate fruit. (A) Hierarchical clustering analysis of the Pinalate fruit left at high (H, 90–95%) or low (L, 30–35%) RH for 1 week or 3 weeks (W), and treated (A, ABA) or not (C, control) with 1mM ABA, based on the chemical composition of their epicuticular wax layer, ABA content, cuticular properties and fruit physiology. The colors in the heatmap indicate the z-score value for each parameter and condition according to the scale in the legend. (B) Correlation matrix among the abundance of individual epicuticular wax fractions, ABA content, cuticle properties and fruit physiological parameters in the Pinalate fruit left under the above-described conditions. Numbers indicate the regression coefficient value, and only the statistically significant ones are colored according to the scale in the legend.

Figure 6

Effect of ABA and water stress on the transcriptional regulation of epicuticular wax metabolism. Relative expression levels of the genes related to the transcriptional (CsCD2) and post-transcriptional (CsCER7) regulation of the biosynthesis (CsCER6/KCS6, CsCER3, CsCER4 and CsSQS) and transport (CsABCG11/WBC11, CsABCG12/WBC12) of the wax components in the Pinalate fruit treated (+ABA) or not (−ABA) with ABA (1 mM), and left at high (90–95%) or low (30–35%) RH for up to 3 weeks at 20 °C. Gene expression values are expressed as fold change levels of all conditions as compared to the freshly harvested (FH) fruit. Values are the means of three biological replicates per condition. Different letters indicate statistical (p < 0.05) differences among all the conditions together according to an ANOVA analysis followed by a Tukey test (p < 0.05) for each gene individually.