Quantitative Proteomic Analyses Identify ABA-Related Proteins and Signal Pathways in Maize Leaves under Drought Conditions

Abstract

Drought stress is one of major factors resulting in maize yield loss. The roles of abscisic acid (ABA) have been widely studied in crops in response to drought stress. However, more attention is needed to identify key ABA-related proteins and also gain deeper molecular insights about drought stress in maize. Based on this need, the physiology and proteomics of the ABA-deficient maize mutant vp5 and its wild-type Vp5 under drought stress were examined and analyzed. Malondialdehyde content increased and quantum efficiency of photosystem II decreased under drought stress in both genotypes. However, the magnitude of the increase or decrease was significantly higher in vp5 than in Vp5. A total of 7051 proteins with overlapping expression patterns among three replicates in the two genotypes were identified by Multiplex run iTRAQ-based quantitative proteomic and liquid chromatography-tandem mass spectrometry methods, of which the expression of only 150 proteins (130 in Vp5, 27 in vp5) showed changes of at least 1.5-fold under drought stress. Among the 150 proteins, 67 and 60 proteins were up-regulated and down-regulated by drought stress in an ABA-dependent way, respectively. ABA was found to play active roles in regulating signaling pathways related to photosynthesis, oxidative phosphorylation (mainly related to ATP synthesis), and glutathione metabolism (involved in antioxidative reaction) in the maize response to drought stress. Our results provide an extensive dataset of ABA-dependent, drought-regulated proteins in maize plants, which may help to elucidate the underlying mechanisms of ABA-enhanced tolerance to drought stress in maize.

Keywords: ABA signaling pathways; LC-MS/MS; Zea mays L.; abscisic acid (ABA); drought stress; iTRAQ; quantitative proteome.

Figure 1

iTRAQ 4-plex labeling and LC MS/MS workflow for identifying proteins in the ABA-deficient mutant vp5 and wild-type Vp5 seedling leaves under drought conditions.

Figure 2

Venn diagram showing the number of significantly expressed proteins in maize leaves under drought stress. The diagram shows the overlap between the ABA-deficient mutant vp5 and the wild-type Vp5.

Figure 3

Gene expression analysis of eight proteins in maize Vp5 and vp5 leaves under drought stress conditions. The eight proteins included chlorophyll a-b binding protein 2, chloroplast outer envelope 24 kD protein, glutathione S-transferase GST 24, glutathione S-transferase GSTU6, heat shock cognate 70 kDa protein 2, NAD(P)H-dependent oxidoreductase, photosystem II reaction center protein L, and putative RING zinc finger domain superfamily protein. Experiments were repeated at least three times.

Figure 4

Pie charts of the distribution of differentially expressed proteins based on their predicted molecular (A) biological process, (B) molecular functions, and (C) cellular components, and (D) the signaling pathways of the proteins identified in maize Vp5 leaves subjected to drought stress. In this study, 130 proteins were identified under drought stress and were classified by their known or predicted subcellular localization using Blast2Go (http://www.blast2go.com).

Figure 5

Pie charts of the distribution of differentially expressed proteins by their predicted (A) biological process, (B) molecular functions, and (C) cellular components, and (D) the signaling pathways of the proteins identified in maize Vp5 leaves under drought stress. In this study, 27 proteins were identified under drought stress, and they were classified by their known or predicted cellular localization using Blast2Go (http://www.blast2go.com).

Figure 6

Comparisons of the (A) biological process, molecular functions, cellular components, and (B) signaling pathways (from Figures 4, 5) of differentially expressed proteins identified in maize Vp5 and vp5 leaves under drought stress. The proteins were classified by their known or predicted cellular function using Blast2GO (http://www.blast2go.com).

Figure 7

Protein-protein interaction network analysis among the significantly expressed proteins in maize Vp5 and vp5 leaves under drought stress using String software.