Integrative Proteome and Phosphoproteome Profiling of Early Cold Response in Maize Seedlings

Abstract

Cold limits the growth and yield of maize in temperate regions, but the molecular mechanism of cold adaptation remains largely unexplored in maize. To identify early molecular events during cold shock, maize seedlings were treated under 4 °C for 30 min and 2 h, and analyzed at both the proteome and phosphoproteome levels. Over 8500 proteins and 19,300 phosphopeptides were quantified. About 660 and 620 proteins were cold responsive at protein abundance or site-specific phosphorylation levels, but only 65 proteins were shared between them. Functional enrichment analysis of cold-responsive proteins and phosphoproteins revealed that early cold response in maize is associated with photosynthesis light reaction, spliceosome, endocytosis, and defense response, consistent with similar studies in Arabidopsis. Thirty-two photosynthesis proteins were down-regulated at protein levels, and 48 spliceosome proteins were altered at site-specific phosphorylation levels. Thirty-one kinases and 33 transcriptional factors were cold responsive at protein, phosphopeptide, or site-specific phosphorylation levels. Our results showed that maize seedlings respond to cold shock rapidly, at both the proteome and phosphoproteome levels. This study provides a comprehensive landscape at the cold-responsive proteome and phosphoproteome in maize seedlings that can be a significant resource to understand how C₄ plants respond to a sudden temperature drop.

Keywords: TMT-labeling; cold stress; maize; phosphoproteome; photosynthesis; proteome; seedlings; spliceosome.

Figure 1

Workflow for global proteome and phosphoproteome profiling of maize seedlings under rapid temperature drop. Five-leaf-stage B73 maize seedlings were cold treated at 4 °C for 30 min and 2 h. Protein samples were labeled with individual TMT reagents, combined, and fractionated into 15 fractions for global proteome analysis or 7 fractions for phosphoproteome analysis.

Figure 2

PCA analysis of the global proteome (A) and phosphoproteome (B) samples. The control samples (CK), the 30 min, and the 2 h samples are well separated on the PCA plots in both the global proteome and the phosphoproteome analyses.

Figure 3

Volcano plot visualization of cold-responsive proteins (A,B) and phosphopeptides (C,D) under 30 min (A,C) or 2 h (B,D) of cold treatment. Cold-responsive proteins or phosphopeptides are depicted in red. X-axis is the Log2 of fold change (treatment /control) and Y-axis is the negative Log10 of the p value for independent t-test adjusted by the Benjamini–Hochberg procedure. Vertical dashed lines denote a fold change cutoff in either direction. The horizontal dashed line represents a cutoff of an adjusted p value of 0.01.

Figure 4

Venn diagram showing numbers of cold-responsive proteins (A,B) and phosphopeptides (C,D) of maize seedlings under different cold-shock durations (30 min and 2 h). Numbers of up-regulated (A,C) and down-regulated (B,D) ones were listed separately. Color in each region is coded according to the count of observations contained therein.

Figure 5

Motif analysis of cold up-regulated phosphopeptides. (A) Significantly enriched phosphorylation motifs of maize B73 under 30 min of cold stress. (B) Significantly enriched phosphorylation motifs of maize B73 under 2 h of cold stress.

Figure 6

Functional enrichment of cold-responsive proteins and phosphoproteins using GO terms of biological process (A), molecular function (B), and cellular component (C), or using KEGG pathway terms (D).

Figure 7

Venn diagrams showing the overlap between proteins observed in the proteome and in the phosphoproteome analyses (A), and between cold-responsive proteins at protein abundance levels and at site-specific phosphorylation levels (B) in maize seedlings during cold shock.

Figure 8

Schematic diagram of photosynthetic complexes on the thylakoid membrane. Cold-responsive proteins at protein abundance levels are shown in colors, while those unquantified or not-responsive are shown in gray. Proteins responsive to cold at site-specific phosphorylation levels are shown in red fonts.

Figure 9

Expression profiles of cold-responsive spliceosome proteins at protein abundance (Total) or site-specific phosphorylation levels (Phos). Protein and phosphopeptide intensities were log2 transformed and scaled for display. Dark blue to dark red color gradient denotes lower to higher expression. One representative phosphopeptide of each phosphoprotein is shown. The accession numbers and the respective phosphosites are listed on the right of the panel. “NA” indicates the phosphosites were not confidently determined (ptmRS site probability < 75%).