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).