Enzyme activity profiles during fruit development in tomato cultivars and Solanum pennellii

Abstract

Enzymes interact to generate metabolic networks. The activities of more than 22 enzymes from central metabolism were profiled during the development of fruit of the modern tomato cultivar Solanum lycopersicum 'M82' and its wild relative Solanum pennellii (LA0716). In S. pennellii, the mature fruit remains green and contains lower sugar and higher organic acid levels. These genotypes are the parents of a widely used near introgression line population. Enzymes were also profiled in a second cultivar, S. lycopersicum 'Moneymaker', for which data sets for the developmental changes of metabolites and transcripts are available. Whereas most enzyme activities declined during fruit development in the modern S. lycopersicum cultivars, they remained high or even increased in S. pennellii, especially enzymes required for organic acid synthesis. The enzyme profiles were sufficiently characteristic to allow stages of development and cultivars and the wild species to be distinguished by principal component analysis and clustering. Many enzymes showed coordinated changes during fruit development of a given genotype. Comparison of the correlation matrices revealed a large overlap between the two modern cultivars and considerable overlap with S. pennellii, indicating that despite the very different development responses, some basic modules are retained. Comparison of enzyme activity, metabolite profiles, and transcript profiles in S. lycopersicum 'Moneymaker' revealed remarkably little connectivity between the developmental changes of transcripts and enzymes and even less between enzymes and metabolites. We discuss the concept that the metabolite profile is an emergent property that is generated by complex network interactions.

Figure 1.

A and B, Growth characteristics of fruits harvested at different time points during development of the red-fruited S. lycopersicum ‘M82’ (A) and the green-fruited S. pennellii (B). The DAA and DAF are depicted. The phases and stages of fruit development of S. lycopersicum ‘M82’ are assigned according to Gillaspy et al. (1993). C and D, Scatterplots and regression plots of the fruit fresh weight (g) and the fruit volume (cm³) of the red-fruited S. lycopersicum ‘M82’ (C) and the green-fruited S. pennellii (D). The black solid lines represent the regression line of a linear fit according to y = mx with the slope m. The red lines indicate the 95% confidence band, a measure of certainty. The colors and symbols used in each plot indicate the monitored or assigned DAA (for details, see below): 28 = dark blue triangle (up), 35 = light blue triangle (down), 42 = light green diamond, 49 = yellow hexagon, 56 = orange square, 63 = light red circle, and 70 = dark red circle. E and F, Boxplot graphs illustrating the changes in fruit volume during the development of the red-fruited S. lycopersicum ‘M82’ (E) and the green-fruited S. pennellii (F). Flowers of S. lycopersicum ‘M82’ and S. pennellii from two independent experiments growing under greenhouse conditions were tagged, and fruits were harvested at the indicated time points after anthesis (DAA). Fruit height and diameter were measured to calculate the fruit volume (cm³). The number of samples used is indicated at the bottom of each graph. The median and the mean are indicated by solid and dashed lines in each box, respectively. To aid interpretation, the median and the mean values of each DAA are connected by a black solid line and a gray dashed line, respectively. Outlier values are depicted by solid black circles.

Figure 2.

Overview of the enzyme activities mapped onto metabolic pathways in the red-fruited S. lycopersicum ‘M82’ (red bars), S. lycopersicum ‘MM’ (yellow bars), and the green-fruited S. pennellii (blue bars). Enzyme activities (expressed as nmol g⁻¹ fresh weight min⁻¹; compare with Supplemental Tables S3–S5) for the majority of the 28 determined enzymes are depicted as bar diagrams including se bars according to the DAA group, namely 35, 42, 49, 56, and 63 DAA (left to right). The very early (28 DAA) and the very late (70 DAA) stages are not visualized and statistically assessed due to few measurements being available at these time points for S. lycopersicum ‘M82’. The growth and harvest of S. lycopersicum ‘M82’ and S. pennellii were performed at the same season and time, whereas S. lycopersicum ‘MM’ was grown separately. The data, including statistical assessments, are available in Supplemental Tables S3 to S5.

Figure 3.

Principal component analysis of the shrunk data set comprising 22 enzyme activities in all three genotypes. A, Separation between the tomato cultivars and the wild relative for principal components 1 and 2. B, Contribution of enzymes for group separation. The genotypes depicted in A are color coded as follows: red circles = S. lycopersicum ‘M82’, yellow squares = S. lycopersicum ‘MM’, and blue diamonds = S. pennellii, with color shades according to early (bright color with crossed shape), mid (medium color with dotted shape), and late (dark color) groups of fruit development as determined by clustering and described in Table I. The enzymes depicted in B are color coded according to their functional/pathway assignment as follows: blue = amino acid metabolism, green = Suc and starch metabolism, light red = glycolysis, dark red = glycolysis and Suc and starch metabolism, and yellow = tricarboxylic acid cycle.

Figure 4.

Venn diagrams of positive (P_adj < 0.05; A) and negative (P_adj < 0.05; B) significant correlations found in a genotype-specific data set and their overlaps for 22 enzymes reliably measured in all three genotypes. The numbers are extracted from the lower triangle (i.e. enzyme correlations between A versus B and B versus A were counted just once) without the diagonal (enzyme against itself) of the symmetric correlation matrix (Fig. 5). The number of enzymes is 22, and the number of all possible correlations is 231. The Venn diagrams show the total number of positive or negative significant correlations for each genotype-specific data set. The total number of significant correlations is shown outside the Venn sectors, the number of unique correlations of each genotype is shown in italics in the genotype-specific sector, and the number of shared significant correlations observed in two or all three data sets is depicted in italics in each of the overlaps.

Figure 5.

Heat map of unique and overlapping significant pairwise enzyme activity correlations. The analysis is restricted to 22 enzymes for which complete data sets were available for all three genotypes, using a significance threshold of P_adj < 0.05. Enzymes that show a significant pairwise correlation in only one genotype are shaded gray, in both of the two cultivars are shaded red, between one cultivar and S. pennellii are shaded orange, and between all three genotypes are shaded yellow. The shading and the letter code for the significant pairwise correlation are depicted in the key within the heat map graph. The matrix diagonal (i.e. correlations of enzymes between themselves) and nonsignificant correlations are shaded dark gray. For counting the overlap among genotypes, only the half-matrix without the diagonal was considered (compare with Fig. 4), whereas the full symmetric correlation matrix is depicted here.

Figure 6.

Heat map of the correlation matrix between enzymes and metabolites. The plot summarizes the Pearson correlation coefficients between enzyme activities (Supplemental Table S4) and metabolites (Carrari et al., 2006) in S. lycopersicum ‘MM’, measured in the samples from the same material. The parameters are grouped with enzyme activities in the left/top section and metabolites in the right/bottom section. Sectors corresponding to enzyme-enzyme, metabolite-metabolite, and enzyme-metabolite pairs are indicated. The color code for negative correlation is as follows: red if P < −0.01 and orange if −0.01 < P < −0.05; the color code for positive correlation is as follows: light blue if 0.01 < P < 0.05 and dark blue if P < 0.01. The full data set is given in Supplemental Table S7.