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.