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).