Identification of 3013 proteins in the growth zone of the maize
leaf
Maize seedlings (B73) were subjected to well-watered, mild and severe
drought conditions that inhibit leaf elongation rates by ca 30 and 60%,
respectively (Avramova et al., 2015a). Based on kinematic analyses
(Avramova et al., 2015a), we sampled meristem, elongating and mature
tissues from the 5th leaf at three days after
emergence. We prepared 4 ITRAQ labeled (Wiese et al., 2007) pools of
samples, 3 of these directly comparing the three growth conditions
(control, mild and severe drought) within each developmental zone
(meristem, elongation, and mature zone) and one contrasting the three
zones under control conditions to examine differences along the
developmental gradient (Table S1). In total, we identified 7636 peptides
which matched 3013 unique proteins.
Differences in protein levels along the developmental
gradient
We identified 1194 proteins in ITRAQ pool 4 comparing the three zones
(meristem, elongation zone, mature zone) under well-watered conditions,
which we assigned to 35 Mapman (Thimm et al., 2004) functional
categories (Figure S2) using Mercator (Lohse et al., 2014). In total,
230 proteins were differentially expressed between the zones (FDR
< 5%; Table S4) and clustered into 7 patterns (Figure 1). Two
of the clusters contained the majority of proteins (118 in cluster 1 and
66 in cluster 2), showing opposite patterns of increasing or decreasing
expression. To identify the biological processes represented by these
two clusters, we performed an enrichment analysis using Pageman (Usadel
et al., 2006; Figure S3). The biggest cluster (1) with gradually
increasing levels from the meristem to the mature zone contained 118
proteins. The categories “cell”, and “transport” were significantly
enriched in this group, while “sucrose degradation”, “protein
synthesis”, “redox”, and “RNA processing” were underrepresented.
The second largest cluster (2) of 66 proteins had the opposite pattern,
gradually decreasing protein levels from the meristem to the mature
zone. The categories “secondary metabolism”, “RNA processing”,
“protein synthesis”, and “transport” were enriched in this group and
“Calvin cycle”, “lipid metabolism”, “redox”, “RNA binding” and
“DNA synthesis” were underrepresented.
The remaining clusters contained smaller numbers of proteins (3-15;
Table S2). Clusters 3 and 4 contained 15 proteins each specifically up-
or downregulated in the meristem, respectively. Although the majority of
them was uncharacterized, proteins related to ribosomal protein
synthesis and lipid metabolism were detected as upregulated in the
meristem in cluster 3. Photosystem proteins such as chlorophyll
a-b-binding protein, and proteins related to protein degradation were
present in cluster 4. Proteins with specific up and down regulation in
the mature zone were grouped in clusters 5 (7 proteins, among which
RuBisCo’s large chain, Eukaryotic translation initiation factor and
proteasome alpha subunit) and 6 (6 proteins, among which histone H2A,
Peroxiredoxin-5, Magnesium-chelatase subunit chll and nuclear transport
factor-2), respectively. Taken together, the differential protein levels
across the growth zone reflect the developmental gradient with cell
proliferation (DNA and protein synthesis, ribosomal proteins) at the
base and photosynthesis in the mature part of the leaf connected by
active transport.
Differential protein levels in response to drought
Next, we compared control, mild and severe drought treatments in each of
the developmental zones (meristem, elongation and mature). Hierarchical
clustering resulted in two major clusters: one contained all control
samples and the other- the severe stress samples (Figure S4). Two of the
mild stress samples clustered with the control and the remaining 4 with
the severe stress samples, consistent with the intermediate character of
the treatment. Within the main clusters, the samples were largely
grouped by zone (Figure S4). In total, we identified 1791 proteins in
the meristem, 1554 in the elongation zone, and 1695 in the mature zone
of the leaf (Figure 2a). Around 31% (871) of these proteins were common
for the three zones.
In response to drought only 81 proteins were significantly affected in
the meristem, 213 in the elongation zone and one in the mature zone,
even when a relatively loose multiple testing correction was applied
(FDR < 0.1; Figure 2b; Table S2).
Enrichment analysis (Figure S5) showed that in the meristem the
categories, “protein degradation”, and “transport” were
overrepresented among upregulated genes, whereas “N-metabolism”,
“redox regulation” (thioredoxin, ascorbate and glutathione), were
overrepresented among the downregulated proteins. In the elongation
zone, the categories “photosynthesis”, “glycolysis”, “tricarboxylic
acid (TCA)/organic transformation”, “regulation of RNA
transcription”, “calcium signaling”, and “cell organization and
vesicle transport” were overrepresented in upregulated proteins, while
the categories “transport” and “protein” were underrepresented. The
categories “oxidases- copper, flavone”, “protein amino acid
activation”, and “cell vesicle transport” were overrepresented in the
downregulated proteins. In the mature zone, the only protein with
significantly downregulated expression in drought was a putative
cystatin (Table S2).