Comparative Proteomics and Physiological Analyses Reveal Important Maize Filling-Kernel Drought-Responsive Genes and Metabolic Pathways

Abstract

Despite recent scientific headway in deciphering maize (Zea mays L.) drought stress responses, the overall picture of key proteins and genes, pathways, and protein-protein interactions regulating maize filling-kernel drought tolerance is still fragmented. Yet, maize filling-kernel drought stress remains devastating and its study is critical for tolerance breeding. Here, through a comprehensive comparative proteomics analysis of filling-kernel proteomes of two contrasting (drought-tolerant YE8112 and drought-sensitive MO17) inbred lines, we report diverse but key molecular actors mediating drought tolerance in maize. Using isobaric tags for relative quantification approach, a total of 5175 differentially abundant proteins (DAPs) were identified from four experimental comparisons. By way of Venn diagram analysis, four critical sets of drought-responsive proteins were mined out and further analyzed by bioinformatics techniques. The YE8112-exclusive DAPs chiefly participated in pathways related to "protein processing in the endoplasmic reticulum" and "tryptophan metabolism", whereas MO17-exclusive DAPs were involved in "starch and sucrose metabolism" and "oxidative phosphorylation" pathways. Most notably, we report that YE8112 kernels were comparatively drought tolerant to MO17 kernels attributable to their redox post translational modifications and epigenetic regulation mechanisms, elevated expression of heat shock proteins, enriched energy metabolism and secondary metabolites biosynthesis, and up-regulated expression of seed storage proteins. Further, comparative physiological analysis and quantitative real time polymerase chain reaction results substantiated the proteomics findings. Our study presents an elaborate understanding of drought-responsive proteins and metabolic pathways mediating maize filling-kernel drought tolerance, and provides important candidate genes for subsequent functional validation.

Keywords: Zea mays L.; drought stress; filling kernel; heat shock proteins; iTRAQ; proteomes.

Figure 1

Phenotypic characterization of the two maize inbred lines (sensitive MO17, S; and tolerant YE8112, T) ears’ responses to drought stress. Observations and measurements were made at 26 days post pollination (DPP) under both water-sufficient (control, C) and water-deficit (drought, D) conditions. (A,B) Ear phenotypes; (C) ear length; (D) ear bare tip length; (E) kernel rows per ear; (F) kernel number per row. Data are presented as mean ± standard errors (n = 3). Different letters on error bars mean significant difference at p < 0.05. (C–F) Each replication is an average for the measurement of 10 ears. Scale bars = 4 cm for both Figure 1A,B.

Figure 2

Physiological changes in the kernels of two contrasting maize inbred lines (sensitive MO17 and tolerant YE8112) in response to drought stress. Physiological changes were measured at different time points (13, 14, 17, 20, 23, and 26 days post pollination; DPP) under both water-sufficient (control) and water-deficit conditions. (A) Relative water content; (B) proline content; (C) peroxidase (POD) activity; (D) malonaldehyde (MDA) content. Data are presented as mean ± standard errors (n = 3). Different letters above line graphs show significant difference (p ≤ 0.05) among treatments at a given stress time point.

Figure 3

Analysis of differentially abundant proteins (DAPs) identified in four experimental comparisons. (A) Total number of DAPs identified in each experimental comparison group, by expression type. Up-regulated means DAPs with increased differential abundance. Down-regulated means DAPs with decreased differential abundance. (B) Venn diagram analysis of DAPs. Overlapping regions of the Venn diagrams indicate DAPs shared between or among corresponding groups. DAPs uniquely expressed in TC_TD (I), SD_TD (II), and TC_TD and SD_TD (III) are indicated with arrows. Area IV shows 105 overlapping DAPs within line.

Figure 4

Clustering analysis of differentially abundant proteins (DAPs). (A) Heat map of DAPs overlapping in in SD_TD experimental comparison. Each row represents a significantly abundantly expressed protein. SD1-3 refers to the biological replicate number for MO17, whilst TD1-3 refers to the replicate number for YE8112. The DAPs were clustered based on the differentially expressed levels. The scale bar indicates the logarithmic value (log 2) expression of the DAPs, up-regulated (red) and down-regulated (green); (B) volcano plot showing the (log 2; −log 10 false discovery rate, FDR) expression of the DAPs in SD_TD comparison. Purple bubbles represent differentially expressed proteins and black bubbles represent proteins with non-differential expression.

Figure 5

Gene ontology (GO) functional classification of differentially abundant proteins (DAPs). (A) Most significantly enriched GO terms (top 20) in tolerant line YE8112 under drought conditions; (B) most significantly enriched GO terms in sensitive line MO17 under drought conditions. The number above each bar graph shows the enrichment factor of each GO term (rich factor ≤ 1).

Figure 6

KEGG pathway enrichment analysis of the DAPs. (A) Most significantly enriched pathway in TD_TC; (B) most significantly enriched pathways in SD_SC, based on the hypergeometric test, p < 0.05. The color gradient represents the size of the p value; the color is from orange to red, and the nearer to red represents a smaller p value, and a higher significance level of enrichment of the corresponding KEGG pathway. The label above the bar graph shows the enrichment factor (rich factor ≤ 1).

Figure 7

Protein–protein interaction analysis of the maize kernel drought-responsive differentially abundant proteins (DAPs). (A) DAPs differentially expressed in YE8112 after drought treatment (TC_TD). (B) MO17-specific DAPs. String database (version 10.5; http://www.string-db.org/) was used to construct the network. The nodes represent proteins, and the thickness of connectors between nodes represents the strength of the supporting data.

Figure 8

Quantitative real-time PCR (qRT-PCR) analysis results of the maize kernel drought-responsive genes encoding differentially abundant proteins (DAPs) from different experimental comparisons. (A) DAPs unique to TC_TD; (B) DAPs specific to SD_TD; (C) DAPs shared between SD_TD and TC_TD; (D) common or overlapping DAPs between TC_TD and SC_SD. All negative expression level values mean that the genes were down-regulated. GAPDH (accession No. X07156) was used as the house keeping gene. Error bars represent the SE (n = 3).