The Physiology and Proteomics of Drought Tolerance in Maize: Early Stomatal Closure as a Cause of Lower Tolerance to Short-Term Dehydration?

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

The disadvantages of being a hybrid during drought: A combined analysis of plant morphology, physiology and leaf proteome in maize

<div><p>A comparative analysis of various parameters that characterize plant morphology, growth, water status, photosynthesis, cell damage, and antioxidative and osmoprotective systems together with an iTRAQ analysis of the leaf proteome was performed in two inbred lines of maize (<i>Zea mays</i> L.) differing in drought susceptibility and their reciprocal F1 hybrids. The aim of this study was to dissect the parent-hybrid relationships to better understand the mechanisms of the heterotic effect and its potential association with the stress response. The results clearly showed that the four examined genotypes have completely different strategies for coping with limited water availability and that the inherent properties of the F1 hybrids, <i>i</i>.<i>e</i>. positive heterosis in morphological parameters (or, more generally, a larger plant body) becomes a distinct disadvantage when the water supply is limited. However, although a greater loss of photosynthetic efficiency was an inherent disadvantage, the precise causes and consequences of the original predisposition towards faster growth and biomass accumulation differed even between reciprocal hybrids. Both maternal and paternal parents could be imitated by their progeny in some aspects of the drought response (<i>e</i>.<i>g</i>., the absence of general protein down-regulation, changes in the levels of some carbon fixation or other photosynthetic proteins). Nevertheless, other features (<i>e</i>.<i>g</i>., dehydrin or light-harvesting protein contents, reduced chloroplast proteosynthesis) were quite unique to a particular hybrid. Our study also confirmed that the strategy for leaving stomata open even when the water supply is limited (coupled to a smaller body size and some other physiological properties), observed in one of our inbred lines, is associated with drought-resistance not only during mild drought (as we showed previously) but also during more severe drought conditions.</p></div

Five proteins with the strongest contrast in the response to drought between 2023 and CE704 maize genotypes.

<p>The number in the column “CE704”, resp. “2023”, represents the n-fold increase or decrease in the protein content after 6 days of drought, derived from the ratio S_CE704/C_CE704 (resp. S₂₀₂₃/C₂₀₂₃) in case of the increased protein content and from the formula: –1/(S_CE704/C_CE704) (resp. –1/[S₂₀₂₃/C₂₀₂₃]) in case of the decreased protein content. The number in the column “Contrast” represents the difference between genotypes according to the ratio (S_CE704/C_CE704)/(S₂₀₂₃/C₂₀₂₃) in case of the higher protein up-regulation in the CE704 genotype/higher protein down-regulation in the 2023 genotype (upper part of the table). For the opposite situation (lower part of the table), the formula: –1/([S_CE704/C_CE704]/[S₂₀₂₃/C₂₀₂₃]) was used. AT  =  <i>Arabidopsis thaliana</i> (L.) Heynh.; ETC  =  electron transport chain; ZM  =  <i>Zea mays</i> L.</p

The chlorophyll <i>a</i> fluorescence kinetics (O-J-I-P) measured in dark-adapted leaves of drought-stressed maize genotypes.

<p>Direct transients (<b><i>A</i></b>), the relative variable fluorescence and the difference kinetics W_OI = (F_t-F₀)/(F_I-F₀) and ΔW_OI = (W_{OI Stress}-W_{OI Control}) (<b><i>B</i></b>), W_OJ = (F_t-F₀)/(F_J-F₀) and ΔW_OJ = (W_{OJ Stress}-W_{OJ Control}) (<b><i>C</i></b>), W_OK = (F_t-F₀)/(F_K-F₀) and ΔW_OK = (W_{OK Stress}-W_{OK Control}) (<b><i>D</i></b>), W_IP = (F_t-F_I)/(F_P-F_I) (<b><i>E</i></b>) and the part of W_OI between 30 and 300 ms (<b><i>F</i></b>) in leaves of maize inbred lines 2023 and CE704 and their F1 hybrids 2023×CE704 and CE704×2023 subjected to 10 days of drought (Stress) or normally watered (Control). The relative variable fluorescence is plotted on left vertical axes using open symbols, the difference kinetics is plotted on right vertical axes using solid symbols. F_t represents the fluorescence intensity measured at any time during the recording period, F_I the fluorescence intensity at the I-step, F_J the fluorescence intensity at the J-step, F_K the fluorescence intensity at the K-step, F_P the maximum fluorescence intensity, and F₀ the initial fluorescence intensity. Mean values (n = 20) are shown for each genotype/water treatment combination.</p

Five proteins that showed the highest negative uniparental heterosis in maize F1 hybrids 2023×CE704 or CE704×2023 under conditions of normal water supply.

<p>Five proteins that showed the highest negative uniparental heterosis in maize F1 hybrids 2023×CE704 or CE704×2023 under conditions of normal water supply.</p

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

<p>The number of fully developed leaves (<b><i>A</i></b>), the plant height (<b><i>B</i></b>), the total area of the photosynthetically active leaves (<b><i>C</i></b>), the leaf area ratio (LAR) (<b><i>D</i></b>), the shoot fresh mass (FM) (<b><i>E</i></b>), the shoot dry mass (DM) (<b><i>F</i></b>), the root fresh mass (<b><i>G</i></b>) and the root dry mass (<b><i>H</i></b>) of maize inbred lines 2023 (23) and CE704 (04) and their F1 hybrids 2023×CE704 (23×04) and CE704×2023 (04×23) subjected to 10 days of drought (solid bars) or normally watered (hatched bars). Means ± SD (n = 20) are shown. The letters <i>A-C</i> denote the statistical significance of the differences between genotypes under control conditions, the letters <i>a-c</i> denote the statistical significance of the differences between genotypes under drought conditions (only those marked with different letters differ significantly at p ≤ 0.05). Asterisks indicate significant differences between control and drought-stressed plants of the respective genotype (p ≤ 0.05).</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038017#pone-0038017-t003" target="_blank">Table 3</a>.</p

The differences in leaf proteins observed either between the genotypes or between control (C) and drought-stressed (S) plants of 2023 and CE704 maize genotypes, as evaluated by the 2D-electrophoresis method.

<p>AT  =  <i>Arabidopsis thaliana</i> (L.) Heynh.; ETC  =  electron transport chain; OEC  =  oxygen evolving complex of photosystem II; OS  =  <i>Oryza sativa</i> L.; LE  =  <i>Lycopersicon esculentum</i> Mill.; TA  =  <i>Triticum aestivum</i> L.; ZM  =  <i>Zea mays</i> L. The following symbols indicate the quantity of individual spots: –  =  absence, +/−  =  very weak intensity, +  =  medium intensity, ++  =  high intensity.</p

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

<p>The number of proteins identified by the iTRAQ method in two maize genotypes (2023 and CE704) with up-regulated (<b><i>A</i></b>) or down-regulated (<b><i>B</i></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.</p