The sh2 starch-deficient mutant is hypersensitive to drought
To validate this hypothesis, we studied the response of theshrunken2 mutant (sh2 ) to drought and subsequent re-watering. The Sh2 gene encodes the large subunit of ADP-glucose pyrophosphorylase (AGPase), the rate-limiting enzyme, providing the substrate ADP glucose for starch biosynthesis. Consistently, the endosperm of sh2 is deficient in starch (Dickinson and Preiss, 1969; Tsai and Nelson, 1966). In addition to the seed, Sh2 is expressed in leaves (Hannah et al., 2012), makingsh2 suitable for studying the role of starch synthesis in the response of leaf development to drought. We expected the reduced AGPase activity in sh2 to reduce starch accumulation under normal and drought stress conditions, inhibiting photosynthesis, growth, and recovery from drought. To test this, we first measured AGPase, starch synthase activities and starch levels in the leaf growth zone (Figure 4, Figure S6). Although in well-watered conditions sh2 had significantly lower AGPase (Figure 4a) and starch synthase (Figure 4b) activities compared to WT across the leaf growth zone, starch levels where not affected (Figure 4c). Severe drought stress significantly increased the activity of these enzymes and led to starch accumulation in the mature zone of the wild-type. In the mutant, however, the induction of starch synthase activity was significantly reduced and absent for AGPase activity, virtually abolishing the drought-induced starch accumulation, with the exception of the region at 3 - 6 cm from the base (Figure 4a, b and c). Thus, starch synthesis in the mutant is compromised throughout the growth zone, preventing the induction of starch levels in response to drought.
According to our hypothesis, the reduced ability of the mutantsh2 to synthesize starch would lead to accumulation of soluble sugars in the mature zone of the maize leaf, causing a feedback inhibition of the photosynthetic machinery. To test this idea, we analyzed sugar levels of sh2 and its WT in the growth zone of plants grown in well-watered and drought conditions (Figure 5; Figure S7) during the photoperiod. Drought-stress induced soluble sugar accumulation along the leaf growth zone in WT. As expected, reduced starch synthesis induced soluble sugar accumulation in sh2 , particularly in the mature zone (Figure 5a-d). The activities of invertase, catalyzing the sucrose breakdown to glucose and fructose, and sucrose phosphate synthase, catalyzing the opposite reaction, showed contrasting patterns. Invertase activity was the highest in the elongation zone, while the activity of sucrose phosphate synthase gradually increased towards the mature zone (Figure 5e and f), suggesting that sucrose synthesis occurred predominantly in the photosynthetic tissues, while sucrose degradation was localized in the growing tissues. The activities of both enzymes were increased by about 50% throughout the growth zone in drought conditions. Sucrose phosphate synthase activity was elevated in the mature part of the sh2leaves, consistent with enhanced export of the sugars that, cannot be stored as starch in the mutant. Nevertheless, soluble sugars accumulated in the mature part of sh2 leaves to higher levels compared to WT.
To test if this sugar accumulation led to feedback inhibition of photosynthesis, we investigated the effect of the compromised starch synthesis on photosynthetic capacity by measuring chlorophyll content and the activities of the main CO2-fixing enzymes (PEPC and RuBisCo) of sh2 and WT. PEPC activity was very similar in WT and sh2 in well-watered conditions, but in drought the enzyme activity was less increased in the mutant (Figure 4d). RuBisCo activity was lower in sh2 compared to WT in control conditions and significantly induced by drought only in WT (Figure 4e). Chlorophyll levels were similar in sh2 and WT in control conditions, but were also induced more strongly in WT by drought (Figure 4e and f). These results confirm our hypothesis that the reduced ability of the mutant to accumulate starch under drought stress impairs the induction of CO2 fixing enzymes and chlorophyll accumulation by drought (Avramova et al., 2015a).
We then tested if this reduced induction of photosynthetic capacity under drought would affect rates of photosynthesis and compromise the recovery of the mutant plants upon re-watering. Gas-exchange measurements showed that in well-watered condition net photosynthesis (Anet) was significantly lower in the mutant (Figure 6a), whereas stomatal conductance (gs) was unaffected by the mutation. As expected, under mild and severe drought, Anet and gs were severely reduced only in WT (Figures 6 and S8). However, the reduction in Anetwas significantly stronger in sh2 compared to WT (Figure 6a), while no significant difference between the genotypes was observed in the response of gs (Figures 6b and S8b). Starch concentration in the leaves of these WT plants increased in response to drought, while in the mutant, no significant changes were detected (Figure 6c). One day after re-watering, WT plants increased the rate of photosynthesis and gs to the level of the control plants, while starch content increased to levels that were even significantly higher than in the control plants. Photosynthesis of thesh2 mutant, however, only returned to control levels a week after re-watering, despite gs being fully restored on the first day after re-watering. Consistently, no change in its leaf starch levels was detected. In accordance with earlier findings (Avramova et al., 2015a), a week after re-watering, photosynthesis in WT plants that had been exposed to drought conditions was significantly higher than controls that remained well-watered throughout the experiment. This compensation was not observed for sh2 , where photosynthesis of drought exposed plants barely recovered to the level of the control plants. These results show that photosynthesis during drought and upon recovery was compromised in sh 2 due to its reduced ability to upregulate starch synthesis, confirming our hypothesis that starch synthesis plays an important role in the induction of the photosynthetic machinery under drought and increased CO2 assimilation and growth upon re-watering.