Comparative Proteomics Combined with Morphophysiological Analysis Revealed Chilling Response Patterns in Two Contrasting Maize Genotypes

Abstract

Maize yield is significantly influenced by low temperature, particularly chilling stress at the maize seedling stage. Various physiological approaches have been established to resist chilling stress; however, the detailed proteins change patterns underlying the maize chilling stress response at the seedling stage remain unknown, preventing the development of breeding-based methods to resist chilling stress in maize. Thus, we performed comprehensive physiological, comparative proteomics and specific phytohormone abscisic acid (ABA) assay on different maize inbred lines (tolerant-line KR701 and sensitive-line hei8834) at different seedling stages (the first leaf stage and third leaf stage) under chilling stress. The results revealed several signalling proteins and pathways in response to chilling stress at the maize seedling stage. Meanwhile, we found ABA pathway was important for chilling resistance of tolerant-line KR701 at the first leaf stage. Related chilling-responsive proteins were further catalogued and analysed, providing a resource for further investigation and maize breeding.

Keywords: chilling stress; differentially expressed protein; maize; proteomics; seedlings.

Figure 1

Physiological responses induced by chilling stress at different seedling stages. (A,B) Phenotypic changes in the inbred lines KR701 and hei8834 from the first leaf stage to the third leaf stage under normal condition and chilling stress condition. −chilling: uniformly growing seedlings were grown at normal condition for 24 h. +chilling: uniformly growing seedlings were grown at 4 °C for 24 h. (C) Fold change ratio of representative physiological traits (height, root length and relative leakage). Fold change ratio: +chilling/−chilling. Scale bar: 5 cm at the first leaf stage and 10 cm at the third leaf stage. The plant height and root length data are expressed as the means ± SD of 10 replicates. The “triangle”and “round” represented different repetitions. The relative leakage data are expressed as the means ± SD of three replicates. *, ** and *** denote levels of significance at p < 0.05, p < 0.01 and p < 0.001, respectively.

Figure 2

Quantitative proteomics analysis of chilling stress in maize seedlings. (A) Simple workflow for proteomics analysis. Experimental material backgrounds: tolerant- line KR701 and sensitive-line hei8834. Different stress treatments: −chilling: maize seedlings were grown at normal condition for 24 h. +chilling: maize seedlings were grown at 4 °C for 24 h. Diverse seedling stage: first leaf stage and third leaf stage. Scale bar: 10 cm. (B) Overall expression analysis of CRPs. (C) Stage-specific expression analysis of CRPs. (D) Specific classification analysis of CRPs.

Figure 3

Distribution and GO functional enrichment analysis of CRPs. (A,D) Tolerant-line KR701-specific CRPs at the first leaf stage. (B,E) Sensitive-line hei8834-specific CRPs at the first leaf stage. (C,F) Overlapping CRPs of the tolerant-line KR701 and sensitive-line hei8834 at the first leaf stage. (G,J) Tolerant-line KR701-specific CRPs at the third leaf stage. (H,K) Sensitive-line hei8834-specific CRPs at the third leaf stage. (I,L) Overlapping CRPs of the tolerant-line KR701 and sensitive-line hei8834 at the third leaf stage.

Figure 4

Network analysis of chilling stress in maize seedlings. (A) Coexpression modules identified using WGCNA. (B) Division of representative modules. At the first leaf stage, the change patterns of the tolerant-line KR701 were represented by the “MEbrown” module, and those of the sensitive-line hei8834 were represented by the “MEturquoise” module. At the third leaf stage, the change patterns of the tolerant-line KR701 were represented by the “MEmagenta” module, and those of the sensitive-line hei8834 were represented by the “MElightcyan” module. (C) Heatmap of the relative expression of 6631 genes in four representative stage-specific modules across all samples. (D–G) GO functional enrichment analysis of representative modules.

Figure 5

Metabolic profiling of maize plants subjected to chilling stress. (A) PLS analysis shows the divergence of the respective metabolomes in response to chilling stress. Different colors represent different samples. QC: quality control samples. (B) Changes in the endogenous ABA content. The y-axis is the fold change relative to the corresponding control: +chilling/−chilling. The data are expressed as the means of five replicates ±SD. * and *** denote levels of significance at p < 0.05 and p < 0.001, respectively.

Figure 6

Regulatory module of maize seedlings under chilling stress. (A) Analysis of the distribution and related functions of CRPs at the first leaf and third leaf stages. Red spots: published chilling-related proteins. Blue spots: GO predicted chilling-related proteins. Grey spots: other CRPs. (B) Ratio of stage-specific CRPs at the first leaf stage and third leaf stage. Grey square: KR701- and hei8834-overlapping CRPs. Blue square: KR701-specific CRPs. Green square: hei8834-specific CRPs. (C) Diagram showing the key signalling events during the first leaf stage to the third leaf stage. Blue box: KR701-specific response. Green box: hei8834-specific response.