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.