A mechanistic model for the role of starch biosynthesis in the
regulation of leaf growth under drought stress
Our proteome analysis suggested a role for starch synthesis in the
response to drought. We tested this hypothesis by studying sh2 , a
knockout allele of AGPase, a rate-limiting enzyme for starch
biosynthesis. It was previously shown that Sh2 is expressed in
leaves (Hannah et al., 2012), but its mutation has only a minor effect
on starch content of leaves (Boehlein et al., 2018). However, these
observations were made in mature leaves and in well-watered conditions.
We showed that Sh2 expression is significantly higher in
expanding tissues than in the mature part of the leaf. In addition,
drought stress induces Sh2 expression predominantly in the
meristem (Figure S11), suggesting that the gene plays a role in the
drought response of leaf growth. Consistent with the hypothesis, the
mutation strongly reduced starch accumulation in the meristem, but to a
higher extent in the mature zone. Moreover, the mutation increased
sensitivity of leaf growth to drought and suppressed the growth
compensation normally observed during recovery.
We integrated our results in a mechanistic model describing the effect
of drought in mature and meristematic cells (Figure 9, black arrows). In
the mature cells, drought stress inhibits photosynthesis, however
chlorophyll content and photosynthetic potential increase, which
facilitates a superior CO2 acquisition upon re-watering
(Avramova et. al., 2015a). While net CO2 assimilation
(Anet) is reduced due to stomatal closure, presumably
driven by increased ABA levels, an induction of the two photosystems,
chlorophyll levels, and CO2 fixation capacity by
upregulation of RuBisCo and PEPC is observed in the mature cells under
drought stress conditions. Increased protein levels of ATP synthase
facilitate increased conversion of light energy into ATP. Additionally,
increased thioredoxin activity stimulates the Calvin cycle (Buchanan et
al., 2002), which leads to higher biosynthesis of glucose and fructose.
At the transcript level, we observed an upregulation of sucrose
synthesis (Avramova et al., 2015a), which is consistent with the
increased activities of the enzymes sucrose phosphate synthase (SPS),
AGPase (additionally activated by the induced thioredoxin activity;
Geigenberger et al., 2005) and starch synthase (SSY), which convert the
hexoses to sucrose and starch, respectively. When CO2uptake decreases due to stomatal closure, starch in the mature part of
the leaf is degraded to sucrose and transported to the meristem.
Consistently, sucrose transporter genes were upregulated under drought
(Figure S12), which contributes to the accumulation of sugars in the
growing tissues. In the meristematic cells, sucrose is converted into
hexoses by cytoplasmic invertase (INV), whose activity was induced by
drought (Figure 9). Hexoses function as osmoprotectants and signals to
induce plant stress responses and reduce ROS damage (Thalmann and
Santelia, 2017). They contribute to maintenance of turgor pressure,
required for cell expansion, and to synthesize cell-wall components,
including cellulose (Thalmann and Santelia, 2017). However, the elevated
levels of hexoses in the growth zone were possibly localized in the
vacuole for osmoregulation and therefore unavailable for growth
(Clifford et al., 1998). This could explain the downregulation of
cellulose synthase activity and lower cellulose levels, particularly in
the meristem in response to drought. Increased ABA levels most likely
also mediate the growth inhibition under stress conditions (Kempa et
al., 2008). ABA induces the cyclin-dependent kinase (CDK) inhibitors
kip-related proteins (KRPs) and inhibits the expression of the mitotic
B-type cyclins (CYCB; Humplík et al., 2017). Consistently, our
transcriptome analysis showed induced expression of krp2 and
downregulation of cdk and cycb in the meristem, explaining
the cell cycle inhibition under drought conditions (Avramova et al.,
2015a). ABA induces ROS production by plasma membrane-associated NADPH
oxdixases (Kwak et al., 2003) and mitochondria (He et al., 2012).
Drought induced ROS accumulation could be an additional factor
contributing to cell cycle inhibition (Reichheld et al., 1999, Waszczak
et al., 2018).
By inhibiting AGPase activity, sh2 limits starch biosynthesis
from hexoses resulting from photosynthesis and largely prevents starch
accumulation in the mature part of the leaves normally occurring under
drought conditions (Figure 9, red arrows), which leads to increased
accumulation of soluble sugars. The ratio soluble sugars/starch
increases significantly in the mature parts of the leaf in response to
drought (Figure S13), inducing a feedback inhibition of photosynthetic
genes (Figure 9). Such a feedback was shown for glucose and sucrose,
which negatively regulates photosynthetic gene expression (Carvalho et
al., 2010; Paul and Foyer, 2001; Sheen, 1993). Soluble sugars, including
glucose and sucrose exert specific repression of the promoter activity
of several maize photosynthetic genes e.g., RuBisCo, chlorophyll-binding
protein and PEP carboxylase (Sheen, 1990). The reduced ability ofsh2 to accumulate starch results in accumulation of
triose-6-phosphate (Figure 9), which inhibits RuBisCo and ATP synthase
activities (Paul and Foyer, 2001; Yang et al., 2016) and therefore
reduces photosynthetic capacity. Due to lower CO2fixation capacity by PEPC and RuBisCo, Anet decreases in
the mutant in absence of a difference in ABA levels and stomatal
conductance. This prevents the induction of the photosynthetic machinery
(also chlorophyll contents in the two photosystems) during leaf
development in stress conditions and the recovery of CO2assimilation upon re-watering, despite the stomatal re-opening (Figure
5).
Similarly, in Arabidopsis thaliana a positive correlation between
the rate of photosynthesis and starch biosynthesis was observed in thetl25 mutant, with a mutation in the gene encoding the small
subunit of AGPase, (Sun et al., 1999). Unlike the wild type, thetl25 mutant was unable to increase photosynthesis in response to
in high light, high CO2 and low O2,
demonstrating the requirement for starch synthesis in adjusting
photosynthetic capacity to environmental conditions. Moreover,
overexpression of an altered maize AGPase large subunit (Sh2r6hs) in
wheat (Triticum aestivum L. ) increased CO2assimilation rate in source tissue and consequently carbon metabolites
in sink tissue and seed, which led to an increased grain yield
(Smidansky et al., 2007).
In the meristematic cells, the sh2 mutation increased sensitivity
to drought by increasing ABA levels and ROS accumulation, but no
increase in hexose-induced osmoprotection (Figure 9). Cheng et al.,
(2002) and Dekkers et al., (2008) indicated that increased ABA
biosynthesis reduces seedling growth. In addition, reduced starch
biosynthesis possibly directs the soluble sugars produced by
photosynthesis to mitochondria, which increases mitochondrial metabolism
including electron transport chain (ETC) activity with potentially
harmful consequences such as ROS production (Moller, 2001; Pastore et
al., 2007). These observations might explain the increased
H2O2 levels in the sh2 mutant.
Therefore, despite the overall higher accumulation of sugars in thesh2 mutant in the mature zone, the mutation leads to stronger
inhibition of cell division in the meristem and consequently higher
growth impairment in response to drought.