Starch is essential to maintain cell division rates under drought-stress conditions
In the growing tissues, soluble sugars are required to drive cell division and cell elongation. Under drought conditions, they are also utilized to increase the antioxidant activity that protects the dividing cells (Avramova et al., 2015a) and as an osmotic agent to maintain cell turgor. Consistently, we observed an induction of soluble sugar concentrations in the meristem. In contrast to the mature part of the leaf, the response at the leaf base of sh2 was similar to WT (Figure 5a).
Increasing soluble sugar levels in the growth zone indicate that the growth response to drought is not due to sugar limitations, but involves an additional signal. Leaf growth is typically reduced by drought in an ABA-dependent manner (Dekkers et al., 2008). Consistently, transcriptome analysis showed that ABA biosynthesis and signaling in the maize leaf growth zone were induced by drought (Avramova et al., 2015a). To investigate the effect of the mutation, we measured ABA levels throughout the growth zone of WT and mutant plants under control and drought conditions. As expected, ABA levels were strongly induced by drought and this increase was significantly higher in the meristem ofsh2 compared to the WT (Figure 7a; Figure S9a).
Drought typically induces oxidative stress in the growth zone, which partly explains the growth inhibition (Avramova et al., 2015a). Therefore, we measured redox and oxidative stress parameters in the growth zone of sh2 and WT under well-watered and drought conditions. Confirming earlier results (Avramova et al., 2015a), drought induced malondialdehyde (MDA), a marker for membrane damage, throughout the growth zone. In sh2 , this response was enhanced (Figure 7b; Figure S9b), suggesting that the mutant is less able to neutralize oxidative radicals. To test this, we measured the reactive oxygen species H2O2. As expected, H2O2 levels were induced by drought throughout the growth zone and more strongly in the mutant (Figure 7c; Figure S9c). These results show that sh2 experienced elevated oxidative stress in its growing tissues.
Both ABA and H2O2 can inhibit cell cycle progression (Humplík et al., 2017; Reichheld et al., 1999). Therefore, we expected that cell division in the mutant and thereby its leaf growth rate would be more sensitive to drought than WT. To investigate this, we performed a kinematic analysis (Sprangers et al., 2016) to quantify the response of the two genotypes to drought. The sh2 mutation reduced leaf elongation rate (LER) and final length of the 5th leaf (FLL) even under well-watered conditions. On top of that, the inhibition by mild and severe drought was stronger insh2 than in WT plants (Table 1; Table S5). The reduced growth of the mutant under well-watered conditions was due to a reduced mature cell length. Consistent with previous observations in B73 maize plants (Avramova et al., 2015a), the decrease in LER under the stress conditions was mainly due to lower cell production rates in bothsh 2 and its WT (W22). The decrease in cell production rate for the sh 2 mutant was 53% for plants grown under severe drought, while this reduction was only 31% for WT plants (Table 1). In turn, the reduced cell production was due to a strong inhibition of cell division rates. Curiously, and in contrast to B73, we observed an increase in the number of dividing cells in W22 that partly compensated for the inhibited cell division rates. This increased meristematic cell number was absent in the mutant. The increased number of cells in the meristem of the wild type was due to cells being smaller in the meristem under drought stress conditions, which compensated for a concurrent reduction of the size of the meristem as a whole. In the mutant, the size of the meristem was reduced to a greater extent than that of WT, whereas the cell size reduction of meristematic cells was similar. Taken together these results confirm the hypothesis that the mutant is more sensitive to drought than the wild type due to a stronger reduction of cell division.
Starch breakdown and growth are tightly coordinated. Starch gradually accumulates in leaves during the photoperiod and it is remobilized at night to provide energy and carbon that maintain plant growth (Thalmann and Santelia, 2017). The fact that the sh2 mutation caused a stronger growth reduction under drought conditions led us to the hypothesis that reduced starch accumulation in sh2 during the photoperiod reduces carbon and energy availability for growth during the night.
To validate this hypothesis, we measured the change in LER, starch and soluble sugar concentrations as well as starch degradation by the enzyme amylase in the leaf growth zone of sh2 and WT in response to drought at four different time points: midday (MD, 8h into the photoperiod), end of the day (ED, after 16h of light period), 2h into the night (2hN, after 2h of dark period) and at the end of the night (EN, after 8h of dark period; Figure 8; Figure S10). As expected, in the WT, starch concentrations increased until the end of the day and decreased during the night, being almost completely depleted at the end of the night (Figure 8a). Accordingly, amylase activity peaked early in the night, when starch degradation was induced (Figure 8b). Similarly, soluble sugar concentrations peaked at the end of the day and decreased during the night, due to their utilization as a carbon source for growth, but unlike starch they were not depleted by the end of the night (Figure 8c). In contrast, the sh2 mutant accumulated highly reduced starch levels during the day (Figure 8a). Consequently, soluble sugars were depleted during the night (Figure 8c). In the WT, drought induced both starch and soluble sugar accumulation during the day. Starch levels dropped at similar rates as under control conditions (Figure 8a) and soluble sugar levels were very similar to those in well-watered plants during the night (Figure 8c). In the mutant, the increase of starch levels under drought conditions was strongly reduced, whereas soluble sugar accumulation was enhanced (Figure 8a and c). In the night, sugar levels were rapidly depleted in the mutant under drought conditions (Figure 8c).
Leaf growth rates broadly mirrored the starch levels, being highest in day-time and lower during the night and in the mutant. During the night the reduction in leaf growth of sh2 was much higher (35 and 61% for WT and sh2 , respectively; Figure 8d). Although under drought stress conditions starch and soluble sugar levels were increased, growth was severely inhibited both during the day and during the night.
We reasoned that if sugar depletion caused hypersensitivity of leaf growth to drought in the mutant, supplementing the leaves with sucrose solution during the night should, at least partly, restore the mutant phenotype. Therefore, we fed sucrose through the tip of the growing leaf (Spoehr, 1942). Using this setup, we were able to reduce the decrease of the soluble sugar content in the growth zone during the dark period for both wild type and sh2 under control and drought conditions (Figure 8e; difference between 2hN and EN). The difference was larger in the drought treated leaves (45% increase due to sucrose feeding) compared to the control conditions (16% increase) and in sh2(92% increase for the controls and 143% for the drought-stressed plants) compared to the wild type, so that in sh2 under drought conditions soluble sugar levels remained essentially stable through the night (Figure 8e). In contrast to the wild type, increasing sugar content in the leaf growth zone resulted in a significant increase in leaf growth of sh2 under control conditions. During the night this fully restored leaf growth rates in sh2 to wild type levels (Figure 8f). Moreover, sugar feeding reduced the effect of drought in the wild type and almost doubled the leaf elongation rate of sh2under drought conditions (Figure 8f). Therefore, our results show that under drought conditions and in sh2 growth is limited by sugar levels in the growth zone. The mutant phenotype shows that starch accumulation during the day is crucial to sustain sugar supply to the growth zone and leaf growth both during the photoperiod and the night. Drought conditions induce starch and soluble sugar accumulation during the day and inhibit growth at the same time.

Discussion

Drought impacts energy generating processes in the maize leaf growth zone.

Leaf growth is one of the most drought sensitive developmental processes and a broad range of studies across a wide variety of species have addressed the effects of drought on growth rates, cell division and expansion, physiological and molecular levels (Avramova et al., 2015a, 2016; Walter et al., 2009). Because the results were obtained in different experimental systems, we still lack an integrated, systems level understanding of these responses. This would require integration of observations at multiple organizational levels in the same experimental system (Ghatak et al., 2017). The linear organization of the developmental processes, the size of the growth zone, availability of a fully sequenced genome and a wide collection of genetic tools make the maize leaf an ideal model system for such integrative studies (Avramova et al., 2015b). Therefore, to extend earlier cellular, transcriptome, biochemical and metabolite studies (Avramova et al, 2015a, 2017), we performed a proteome analysis of the effect of standardized drought treatments in the maize leaf growth zone.
In accordance with transcriptome data (Li et al., 2010; Avramova et al., 2015a), “protein synthesis”, “RNA processing” and “transport”, were enriched in the meristem. In contrast, the proteome of the mature part was dominated by energy generating processes, including photosynthesis and carbohydrate metabolism such as glycolysis and TCA cycle. Consistent with the protein levels, the enzyme activities and metabolite analyses showed the induction of processes related to the light reaction and the carbon fixation, as well as starch and sugar biosynthesis and redox regulation in the mature part of the growth zone. We confirmed the upregulation of the photosynthetic machinery by increased chlorophyll content (Avramova et al., 2015a), increased activities of RuBisCo and ATP synthase and ATP metabolite concentrations in the mature cells (current study). The consistency of the photosynthetic response between protein (Table S3), mRNA levels (Avramova et al., 2015a), enzyme activities and metabolites (Figure 3a), supports transcriptional regulation of this process (Ponnala et al., 2014).