Protein levels correlate with enzyme activity and metabolite concentration patterns
The most prominently upregulated classes of proteins in response to drought include effects on photosynthesis, which we studied earlier (Avramova et al., 2015a). In addition, the proteome shows a prominent effect on carbon and nitrogen metabolism and redox regulation (Figure S5). To confirm the biological relevance, we measured the activity and related metabolite levels for several differentially expressed enzymes involved in photosynthesis, amino acid and carbohydrate metabolism and redox regulation.
Chlorophyll and photosynthesis-related proteins were upregulated towards the mature part of the leaf and in response to drought (Figures S3 and S5). One of these proteins was adenosine triphosphate (ATP) synthase (Table S3), which converts energy from the light reactions to ATP. Consistently, we found that enzyme activity and ATP metabolite levels increased along the leaf axis and in response to stress (Figure 3a). The protein level of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), one of the main carbon-fixing enzymes, also increased towards the mature zone (Table S3). Its small subunit was slightly downregulated by drought. We found that the corresponding enzyme activity indeed increased towards the mature part of the leaf and in response to drought (Figure 3a). An opposite pattern was found for respiratory electron transport (ferredoxin) and glycolysis (triosephosphate isomerase, TPI) protein levels and enzyme activity, which were highest in the meristem and decreased in response to drought (Table S3; Figure 3a).
In the carbon metabolism, starch synthase levels in the elongation zone increased in response to drought (Table S3). Starch synthase activity, as well as starch concentrations, were the highest in the mature part of the leaf (Figure 3b) and, in parallel with the protein levels, increased in response to drought throughout the growth zone. Sucrose synthase protein levels and activity poorly correlated. Protein levels were downregulated in the elongation zone in response to drought (Table S3), while the activity of the enzyme was increased in the mature zone in response to drought (Figure 3b).
Cellulose synthase protein levels were also upregulated with progressing development, which was confirmed by the enzyme activity and the cellulose concentrations, which additionally showed a decrease in response to drought (Table S3, Figure 3b).
Upregulation of the protein levels of enzymes involved in serine-glycine-cysteine biosynthesis from the base towards the mature leaf parts and in response to drought (Table S3, Figure 3b) were reflected in the profiles of serine, cysteine and glycine concentrations as well as the activity of cysteine synthase (Figure 3b). This could also be a result of increased RuBisCo activity in the mature zone in response to drought (Figure 3a), because it catalyzes not only carbon fixation in the maize bundle sheath cells, but also the process of photorespiration, during which glycine and serine synthesis represent an intermediate step of the synthesis of 3-phosphoglycerate (Sage et al., 2012).
Proline is a well-known stress defense molecule (AbdElgawad et al., 2015) that adjusts cellular osmotic potential, protects membranes and proteins, stabilizes photosystem II and protects plants against oxidative damage (Szabados and Savouré, 2010). DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) plays a key role in proline biosynthesis and was upregulated in response to stress in the elongation zone (Table S3). This correlates with the upregulation of proline levels predominantly in the dividing and expanding tissues (Figure 3b). Other defense mechanisms, involving redox-regulation, were also activated in response to drought. The protein levels of peroxiredoxin, glutaredoxin and thioredoxin were upregulated in the mature tissues and in response to drought stress (Table S3, Figure 3c), which closely matched their enzyme activity patterns (Figure 3c). The activation of the redox system in the mature zone (Figure 3c) could also contribute to the observed induction of the photosynthetic machinery. Peroxiredoxins, thioredoxins and glutaredoxins are not only efficient antioxidants, but also cellular signals and chaperones, protecting proteins from oxidation (Dietz, 2011; Hanschmann et al., 2013) and regulators of photosynthetic enzymes (Buchanan et al., 2002). Moreover, thioredoxins are known to activate different chloroplast enzymes, such as AGPase, a rate- limiting enzyme in starch biosynthesis (Geigenberger et al., 2005). Consistently, increases in thioredoxins correlate with starch accumulation in the mature zone of the leaves in response to drought (Figure 3b and c).
An important finding from these studies was that drought stress increased photosynthetic capacity and starch biosynthesis, resulting in elevated starch levels in the mature part of the leaf (Figure 3b). Starch biosynthesis facilitates maintenance of high photosynthetic rates by triose phosphate utilization and thereby preventing accumulation of inorganic orthophosphate (Pi), a limiting factor for ribulose-1,5-bisphosphate regeneration (Galtier et al., 1995, Paul and Foyer et al., 2001). Moreover, starch biosynthesis prevents the accumulation of soluble sugars during the photoperiod. The increase in soluble sugar levels can induce a feedback inhibition of photosynthesis by decreasing the gene expression levels of photosynthetic enzymes (e.g., PEPC, malic enzyme and RuBisCo; Jeannette et al. 2000; Sheen, 1993). Therefore, we hypothesized that increased starch synthase activity and starch levels in the mature part of drought stressed leaves are essential to facilitate the observed induction of the photosynthetic machinery and increased CO2 assimilation and growth upon re-watering (Avramova et al., 2015a).