Figure 3. The effect of drought stress on metabolite and biochemical activities in the growth zone of the maize leaf.
Enzyme activities and metabolite concentration patterns related to photosynthesis (a), amino acid and sugar metabolism (b) and redox regulation (c) were compared in response to mild and severe drought stress in each developmental zone of the growing maize leaf (meristem, elongation zone, and mature zone). Metabolite and biochemistry measurements were performed in each centimeter (from 1 to 10) of the growth zone of the 5th leaf of plants, grown in well-watered conditions (C1–C10) and plants, subjected to mild (M1–M10) and severe (S1–S10) drought. Data are means of at least 3 biological replications (each consisting of three pooled plants). Data are hierarchically clustered and mean-centered, which effectively removed the differences in concentration and activity values between the different metabolites and enzymes to highlight the magnitude of differences across the growth zones and in response to the treatments. The lengths of the different zones along the leaf axis are based on Avramova et al (2015a) and indicated in the bottom row. Syn, synthase; TPI, Triosephosphate isomerase
Figure 4. The effect of drought on starch metabolism and photosynthetic enzyme activities and pigment levels in the shrunken2 (sh2) maize mutant and its wild type (W22).
Well-watered control plants (C) were compared to severely drought (D) stressed plants (more detailed analysis including mild drought stress is presented in Figure S6). Measurements were done in each 1 cm of the leaf growth zone. (a) Activity of the enzyme ADP-glucose pyrophosphorylase (AGPase); (b) Starch synthase activity; (c) Starch concentration; (d) Phosphoenolpyruvate carboxylase (PEPC) activity; (e) Ribulose-1,5-bisphospate carboxylase/oxygenase (RuBisCo) activity; (f) Chlorophyll a concentration; (g) Chlorophyll b concentration. Data are means ± SE (n=3). FW, Fresh weight; 3-PGA, 3-phosphoglycerate. A three-factor ANOVA was used as a statistical test, and P -values are present in Table S4. The lengths of the different zones along the leaf axis are based on the kinematic analysis in Table 1 and indicated in the bottom row.
Figure 5. The effect of drough on the sugar metabolism of theshrunken2 (sh2) maize mutant and its wild type (W22).Well-watered control plants (C) were compared to severely drought (D) stressed plants. More detailed analysis including mild drought stress is presented in Figure S7. Measurements were done in each 1 cm of the leaf growth zone. (a) Soluble sugar concentration; (b) Sucrose concentration; (c) Glucose concentration; (d) Fructose concentration; (e) Invertase activity; (f) Sucrose phosphate synthase activity; Data are means ± SE (n=3). FW, Fresh weight. A three-factor ANOVA was used as a statistical test, and P -values are present in Table S4. The lengths of the different zones along the leaf axis are based on the kinematic analysis in Table 1 and indicated in the bottom row.
Figure 6. The effect of drought on photosynthesis (Anet) and stomatal conductance (gs) in the shrunken2 (sh2) maize mutant and its wild type (W22).The rates of net photosynthesis (a) and stomatal conductance (b) of young seedlings, grown in severe drought (D) conditions before, one day after and one week after re-watering (RW). Unstressed plants of the same age as the plants that were subjected to stress and allowed to recover were included as a control (C) for ontogenetic differences. More detailed analysis including mild drought stress is presented in Figure S8. A three-factor ANOVA and Tukey test were used for statistical analysis, and significant differences (P < 0.05) are marked with different letters. Data are averages ± SE (n = 5).
Figure 7. The effect of drought on stress parameters inshrunken2 (sh2) maize mutant and its wild type (W22).Well-watered control plants (C) were compared to severely drought (D) stressed plants. More detailed analysis including mild drought stress is presented in Figure S9. Measurements were done in each 1 cm of the leaf growth zone. (a) Abscisic acid (ABA) concentration; (b) Malondialdehyde (MDA) concentration; (c) Hydrogen peroxide (H2O2) concentration. Data are means ± SE (n=3). FW, Fresh weight. A three-factor ANOVA was used as a statistical test, and P -values are present in Table S4. The lengths of the different zones along the leaf axis are based on the kinematic analysis in Table 1 and indicated in the bottom row.
Figure 8. The effect of drought on starch metabolism in theshrunken2 (sh2) maize mutant and its wild type (W22).Well-watered control (C) and severely drought (D) stressed plants were compared in terms of (a) Starch concentration, (b) Amylase activity and (c) Soluble sugar concentration at four different timepoints: Midday (MD), End of the Day (ED), 2 hours into the night (2hN) and End of the Night (EN); Data are means ± SE (n=3). A three-factor ANOVA was used as a statistical test, and P -values are present in Table S4. (d) Leaf elongation rate (LER) was measured during the day and during the night. Data are means ± SE (n=6). Part of the plants in the experiment were fed with 0.3 M sucrose during the night and soluble sugar concentration (e) and LER (f) were measured at two time points: 2hN (two hours after feeding) and EN. Data are means ± SE (n=5). A three-factor ANOVA and Tukey’s test were used as a statistical analyses and significant differences (P < 0.05) are marked with different letters above the bars. More detailed analysis including mild drought stress is presented in Figure S10. FW, Fresh weight; HS, hydrolyzed starch.
Figure 9. Model of the metabolic regulation of the drought response and the effect of the sh2 mutation, encoding the starch biosynthesis limiting enzyme ADP-glucose pyrophosphorylase (AGPase), on the drought response in meristematic and mature cells of maize leaves.Up or down regulated gene expression, protein levels, enzyme activities, or metabolite concentrations are indicated by a black (drought stress effect) or a red (effect of sh2 mutation on drought response) arrow. Blue arrows indicate activation or inhibition and purple arrows indicate transport. Interrupted arrows indicate indirect regulation, while uninterrupted arrows indicate direct regulation. Interrupted red lines indicate hypothesis, based on literature. Red question mark indicates that the assumption is made based on the literature due to lack of own data. FBI, feedback inhibition; Anet, net photosynthesis (net CO2 assimilation); PEPC, phosphoenolpyruvate carboxylase; ABA, abscisic acid; Fd, ferredoxin; FDR, ferredoxin reductase; Thx, thioredoxin; PS, photosystem; ATP SYN, ATP synthase; Tri-6-P, triose-6-phosphate; SPS, sucrose phosphate synthase; SSY, starch synthase; SUC, sucrose; INVs, invertases; ROS, reactive oxygen species; CDK, cyclin-dependent kinase.