The physiology and proteomics of drought tolerance in maize: early stomatal closure as a cause of lower tolerance to short-term dehydration?

Abstract

Understanding the response of a crop to drought is the first step in the breeding of tolerant genotypes. In our study, two maize (Zea mays L.) genotypes with contrasting sensitivity to dehydration were subjected to moderate drought conditions. The subsequent analysis of their physiological parameters revealed a decreased stomatal conductance accompanied by a slighter decrease in the relative water content in the sensitive genotype. In contrast, the tolerant genotype maintained open stomata and active photosynthesis, even under dehydration conditions. Drought-induced changes in the leaf proteome were analyzed by two independent approaches, 2D gel electrophoresis and iTRAQ analysis, which provided compatible but only partially overlapping results. Drought caused the up-regulation of protective and stress-related proteins (mainly chaperones and dehydrins) in both genotypes. The differences in the levels of various detoxification proteins corresponded well with the observed changes in the activities of antioxidant enzymes. The number and levels of up-regulated protective proteins were generally lower in the sensitive genotype, implying a reduced level of proteosynthesis, which was also indicated by specific changes in the components of the translation machinery. Based on these results, we propose that the hypersensitive early stomatal closure in the sensitive genotype leads to the inhibition of photosynthesis and, subsequently, to a less efficient synthesis of the protective/detoxification proteins that are associated with drought tolerance.

Figure 1. The gas exchange and water use characteristics of the leaves of drought-stressed maize genotypes.

The net photosynthetic rate (P_N) (A), intercellular CO₂ concentration (c_i) (B), net transpiration rate (E) (C), stomatal conductance (g_S) (D), water use efficiency (WUE) (E) and relative water content (RWC) (F) in the leaves of two maize genotypes (2023 and CE704) that were subjected to 6 days of drought (solid bars) or normally watered (hatched bars). The means ± SD (n  = 18) are shown. The letters a-c denote the statistical significance (as determined by the Tukey-Kramer test) of the differences between genotypes/water treatments (only those marked with different letters differ significantly at p≤0.05).

Figure 2. The morphology and biomass characteristics of drought-stressed maize genotypes.

The dry mass of the shoot (DMS) (A), dry mass of the roots (DMR) (B), specific weight of the 4^th leaf (SLW) (C) and plant height (D) of two maize genotypes (2023 and CE704) subjected to 6 days of drought (solid bars) or normally watered (hatched bars). The means ± SD (n  = 20) are shown. The letters a-c denote the statistical significance (as determined by the Tukey-Kramer test) of the differences between genotypes/water treatments (only those marked with different letters differ significantly at p≤0.05).

Figure 3. The activities of antioxidant enzymes in the leaves of drought-stressed maize genotypes.

The activities of ascorbate peroxidase (APX) (A), glutathione reductase (GR) (B), superoxide dismutase (SOD) (C) and catalase (CAT) (D) in the leaves of two maize genotypes (2023 and CE704) subjected to 6 days of drought (solid bars) or normally watered (hatched bars). The means ± SD (n  = 8) are shown. The letters a-b denote the statistical significance (as determined by the Tukey-Kramer test) of the differences between genotypes/water treatments (only those marked with different letters differ significantly at p≤0.05).

Figure 4. The functional classification of differentially expressed drought-related proteins from maize leaves.

The number of proteins identified by the iTRAQ method in two maize genotypes (2023 and CE704) with up-regulated (A) or down-regulated (B) levels is shown; only those proteins whose levels changed due to drought in at least one genotype by at least twofold were included. ET: proteins of the photosynthetic electron-transport chain and chlorophyll synthesis; SM: proteins participating in photosynthetic carbon fixation and saccharide metabolism; MT: membrane proteins participating in transport; LM: proteins participating in lipid metabolism; AM: proteins participating in amino acid metabolism; DX: detoxification proteins; ST: stress proteins; DH: dehydrins; CP: chaperones; SG: proteins involved in cell signaling; PT: proteases and their inhibitors; GE: proteins participating in gene expression and its regulation; MS: miscellaneous proteins.

Figure 5. The functional classification of differentially expressed proteins from maize leaves with genotype-dependent contrasting responses to drought.

The number of proteins identified by the iTRAQ method that were up-regulated in one genotype and down-regulated in the other genotype or vice versa is shown in panel (A); the number of proteins belonging to this category with different levels in control plants of both genotypes is shown in panel (B). Only proteins whose levels changed differentially in the two genotypes by at least twofold were included. 2023> CE704: up-regulation of protein levels in the 2023 genotype and down-regulation in the CE704 genotype (A) or higher level in control plants of the CE704 genotype compared with the 2023 genotype (B); CE704>2023: up-regulation of protein levels in the CE704 genotype and down-regulation in the 2023 genotype (A) or higher level in control plants of the 2023 genotype compared with the CE704 genotype (B); ET: proteins of the photosynthetic electron-transport chain and chlorophyll synthesis; SM: proteins participating in photosynthetic carbon fixation and saccharide metabolism; MT: membrane proteins participating in transport; LM: proteins participating in lipid metabolism; AM: proteins participating in amino acid metabolism; DX: detoxification proteins; ST: stress proteins; DH: dehydrins; CP: chaperones; SG: proteins involved in cell signaling; PT: proteases and their inhibitors; GE: proteins participating in gene expression and its regulation; MS: miscellaneous proteins.

Figure 6. The 2D gels showing the leaf proteomes of drought-stressed and control plants of two maize genotypes.

S: drought-stressed; C: control; 2023: sensitive genotype; CE704: tolerant genotype. Only selected regions of the gels are shown; the frames mark the differences in the representation of two isoforms of the heat-shock protein HSP26 (spots nos. 4 and 5) in the drought-stressed plants of both genotypes. The protein spots that are differentially represented between genotypes and water treatments are marked by arrows and the respective numbers (1–11; N … unidentified protein) refer to the notation used in Table 3.

Figure 7. Phenotypic representation of drought-stressed and control plants of two maize genotypes.

2023: sensitive genotype; CE704: tolerant genotype.