Integrative comparative analyses of transcript and metabolite profiles from pepper and tomato ripening and development stages uncovers species-specific patterns of network regulatory behavior

Abstract

Integrative comparative analyses of transcript and metabolite levels from climacteric and nonclimacteric fruits can be employed to unravel the similarities and differences of the underlying regulatory processes. To this end, we conducted combined gas chromatography-mass spectrometry and heterologous microarray hybridization assays in tomato (Solanum lycopersicum; climacteric) and pepper (Capsicum chilense; nonclimacteric) fruits across development and ripening. Computational methods from multivariate and network-based analyses successfully revealed the difference between the covariance structures of the integrated data sets. Moreover, our results suggest that both fruits have similar ethylene-mediated signaling components; however, their regulation is different and may reflect altered ethylene sensitivity or regulators other than ethylene in pepper. Genes involved in ethylene biosynthesis were not induced in pepper fruits. Nevertheless, genes downstream of ethylene perception such as cell wall metabolism genes, carotenoid biosynthesis genes, and the never-ripe receptor were clearly induced in pepper as in tomato fruit. While signaling sensitivity or actual signals may differ between climacteric and nonclimacteric fruit, the evidence described here suggests that activation of a common set of ripening genes influences metabolic traits. Also, a coordinate regulation of transcripts and the accumulation of key organic acids, including malate, citrate, dehydroascorbate, and threonate, in pepper fruit were observed. Therefore, the integrated analysis allows us to uncover additional information for the comprehensive understanding of biological events relevant to metabolic regulation during climacteric and nonclimacteric fruit development.

Figure 1.

Comparative analysis of the covariance structures of metabolites, transcripts, and profiles in tomato and pepper fruits. Similarity analysis is shown for the covariance structures of metabolites (A), transcripts (B), and integrated profiles (C; metabolite and transcript) over eight and 10 development and ripening stages from tomato and pepper, respectively. A similarity score was calculated for every pair of periods, each spanning four stages in development and ripening, based on the corresponding PCs (see “Materials and Methods”). The similarity score ranges from 0 to 1, with 0 indicating equality of the compared covariance structures. The results are displayed in the form of a heat map, with the color key included at the bottom.

Figure 2.

Primary metabolite levels during pepper development and ripening. Time points presented are 14, 20, 34, 51, 52, 53, 55, 57, 62, and 68 DAP. Data are normalized to the mean response calculated to the 14-DAP stage (value= 1). Values presented are means ± se of three replicates. Asterisks denote differences that were determined to be significant by Student’s t test analysis (P < 0.05) compared with the 14-DAP stage.

Figure 3.

Networks from the primary metabolite data set of tomato and pepper fruits. Networks were obtained by determining the significant correlations of the metabolite profiles from tomato (A) and pepper (B), guaranteeing a FDR of 0.05. Positive correlations are indicated with red edges, while negative correlations are displayed with blue edges. The gray edges denote the relation between the communities (clusters) of metabolites in the network. The color coding of the nodes, representing the metabolites, denotes the following compound classes: amino acids (red), organic acids (light blue), sugars and sugar alcohols (dark blue), and others (green).

Figure 4.

Functional distribution of expressed genes. Functional distribution is given for all genes showing significant (P < 0.05) expression values based on MapMan classification (Usadel et al., 2005) in pepper developmental and ripening stages (14, 20, 34, 51, 52 [breaker], 53, 55, 57, 62, and 68 DAP).

Figure 5.

Expression analysis of pepper development and ripening. A condensed PageMan display of altered pathways is shown. Gene expression data are presented as log₂ fold changes in comparison with the first harvested time point (14 DAP). The analyzed time points were 20, 34, 51, 52, 53, 55, 57, 62, and 68 DAP. The data were subjected to a Wilcoxon test in PageMan, and the results are displayed in false color. BINs colored in red are significantly up-regulated, whereas BINs colored in blue are significantly down-regulated.

Figure 6.

Quantitative PCR of cell wall-, carotenoid-, and ethylene-related genes of tomato and pepper fruits across developmental and ripening stages. Cell wall-related genes were EXP1, EXP3, XTH5, and PG. Carotenoid-related genes were PSY1, PDS, and ZDS. Ethylene-related genes were ACC oxidase (ACO1), ACC synthase (ACS4), and the Nr receptor. The values represent means ± se of four individual plants. Asterisks indicate values determined by t test to be significantly different from the first analyzed stage (P < 0.05).

Figure 7.

Networks from selected transcripts of tomato and pepper fruits. Transcripts involved in cell wall metabolism, hormone metabolism, redox regulation, major carbohydrate metabolism, protein synthesis, protein targeting, protein posttranslational modification, protein degradation, protein folding, protein assembly, and cofactor ligation were used in the analysis. Color coding for the nodes can be found in Supplemental Figure S2. Transcripts were grouped by functionality on the basis of MapMan gene ontology. Networks were obtained by determining the significant correlations of the transcript profiles from tomato (A) and pepper (B), guaranteeing a FDR of 0.05. Positive correlations are indicated with red edges, while negative correlations are displayed with blue edges. The gray edges denote the relation between the communities (clusters) of metabolites in the network. The color code for the nodes, representing the selected transcripts, corresponds to the MapMan bins and indicates the different function categories.

Figure 8.

Network-based representation of the integrated metabolomics and transcriptomic data set of tomato and pepper. An analogous procedure to that used to obtain Figures 3 and 7 was employed on the combined data set. Nodes denoting metabolites are drawn in squares, while those representing transcripts are given in circles for tomato (A) and pepper (B). The color coding of nodes represented by circles corresponds to the MapMan bins. The color coding of nodes represented by squares follows that used in Figure 3 for the network of metabolites and that of the nodes represented by circles follows that used in Figure 7 for the network of transcripts. Red edges denote positive correlations, while blue edges represent negative correlations. The gray edges denote the relation between the communities (clusters) of metabolites in the network.

Figure 9.

Experimental design. To collect pepper (cv Habanero) prior to ripening, fruits were tagged at 14 DAP and harvested at one of the following 11 time points: 14, 20, 26, 34, 51 (breaker−1; Br−1), 52 (breaker; Br), 53 (breaker+1; Br+1), 55 (breaker+2; Br+2), 57 (breaker+5; Br+5), 62 (breaker+10; Br+10), and 68 DAP (breaker+16; Br+16). For tomato (cv Ailsa Craig), the analyzed time points were 7, 17, 27, 39 (mature green [MG]), 41 (breaker−1; Br−1), 42 (breaker; Br), 43 (breaker+1; Br+1), 47 (breaker+5; Br+5), 52 (breaker+10; Br+10), and 57 DAP (breaker+15; Br+15).