Combined Effects of Ozone and Drought on the Physiology and Membrane Lipids of Two Cowpea (Vigna unguiculata (L.) Walp) Cultivars

Abstract

The interactive effects of drought and ozone on the physiology and leaf membrane lipid content, composition and metabolism of cowpea (Vigna unguiculata (L.) Walp.) were investigated in two cultivars (EPACE-1 and IT83-D) grown under controlled conditions. The drought treatment (three-week water deprivation) did not cause leaf injury but restricted growth through stomatal closure. In contrast, the short-term ozone treatment (130 ppb 12 h daily during 14 day) had a limited impact at the whole-plant level but caused leaf injury, hydrogen peroxide accumulation and galactolipid degradation. These effects were stronger in the IT83-D cultivar, which also showed specific ozone responses such as a higher digalactosyl-diacylglycerol (DGDG):monogalactosyldiacylglycerol (MGDG) ratio and the coordinated up-regulation of DGDG synthase (VuDGD2) and ω-3 fatty acid desaturase 8 (VuFAD8) genes, suggesting that membrane remodeling occurred under ozone stress in the sensitive cultivar. When stresses were combined, ozone did not modify the stomatal response to drought and the observed effects on whole-plant physiology were essentially the same as when drought was applied alone. Conversely, the drought-induced stomatal closure appeared to alleviate ozone effects through the reduction of ozone uptake.

Keywords: cowpea; drought; membrane lipids; ozone; stress combination.

Figure A1

Detection of hydrogen peroxide using DAB staining in Vigna unguiculata leaves exposed to the following treatments: Control (C), Drought (D), Ozone (O), Ozone + Drought (OD). Leaf discs were collected and stained after 14 days of treatment. Pictures are representative of three independent biological replicates. No specific staining was observed in control leaves treated with ascorbic acid (data not shown). DAB: 3,3′-diaminobenzidine. Bar = 250 μm.

Figure 1

(a) Shoot dry weight; (b) stomatal conductance (g_s); (c) relative water content and (d) quantum yield of PSII (Φ_PSII) of Vigna unguiculata plants subjected to the following treatments: Control (C, black bars), Drought (D, dark grey bars), Ozone (O, light grey bars), Ozone + Drought (OD, white bars). Measurements were performed 7 and 14 days after the onset of the stress treatments. Means ± SEM are shown (n = 3–4). When significant differences were found, letters indicate homogenous subsets at each time point for a given cultivar (one-way ANOVA and Tukey post-hoc tests, α = 0.05).

Figure 2

(a) MGDG contents; (b) DGDG contents; (c) DGDG:MGDG ratio and (d) normalized omega-3 index in the leaves of Vigna unguiculata plants subjected to the following treatments: Control (C, black bars), Drought (D, dark grey bars), Ozone (O, light grey bars), Ozone + Drought (OD, white bars). Measurements were performed 7 and 14 days after the onset of the stress treatments. Means ± SEM are shown (n = 3). When significant differences were found, letters indicate homogenous subsets at each time point for a given cultivar (one-way ANOVA and Tukey post-hoc tests, α = 0.05). MGDG, Monogalactosyl-diacylglycerol; DGDG, digalactosyl-diacylglycerol, omega-3 index 18:3/(18:0 + 18:1 + 18:2).

Figure 3

Relative expression of genes involved in lipid metabolism in leaves of (a,b) EPACE-1 and (c,d) IT83-D plants exposed to the following treatments: Control (C, black bars), Drought (D, dark grey bars), Ozone (O, light grey bars), Ozone + Drought (OD, white bars). Measurements were performed 7 and 14 days after the onset of the stress treatments. Means ± SEM are shown (n = 3). Each value is the average of two technical replicates. Expression of target genes was normalized to the expression of the reference gene VuEF-1α. When significant differences were found, letters indicate homogenous subsets at each time point for a given cultivar (one-way ANOVA and Tukey post-hoc tests, α = 0.05). VuMGD1, type 1 monogalactosyl-diacylglycerol synthase; VuMGD2, type 2 monogalactosyl-diacylglycerol synthase; VuDGD1, type 1 digalactosyl-diacylglycerol synthase; VuDGD2, type 2 digalactosyl-diacylglycerol synthase; VuFAD7, ω-3 fatty acid desaturase 7; VuFAD8, ω-3 fatty acid desaturase 8; VuPAT1, patatin-like lipid acyl hydrolase; VuEF-1α, elongation factor 1 alpha.