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.