(A) Image of developmental stages used for this study. Scale bar = 2 cm. (B) Fresh weight, (C) water content, (D) anthocyanin, (E) chlorophyll, and (F) total fatty acid content in petals and sepals at different stages of development. Data are means ± SE (n = 2−5 biological replicates). * indicates significant changes (p-value<0.05) as compared to the preceding developmental stage within the given tissue.

(A) Cluster dendrogram showing samples collected at the four stages of petal and sepal development. Samples were clustered according to their gene expression profile. (B) Grouping of expressed sequence tags (ESTs) based on their developmental profile. Using a one-way ANOVA, three hypotheses were tested: H₀: µ_d-3 = µ_d1, H₀: µ_d1 = µ_d4, H₀: µ_d4 = µ_d7. A score was attributed for each test. If the gene expression was not significantly different (p-value>0.05), the score = 0. If it was significantly up-regulated (p-value<0.05), the score = 1. If it was significantly down-regulated (p-value<0.05), the score = −1. Genes were grouped based on the combination of scores for the three tests. The number of ESTs in each developmental pattern is indicated next to the graphs. Developmental expression profiles containing less than ten ESTs are not represented.

ESTs were annotated and assigned to enzymatic steps based on their homology to A. thaliana genes involved in each enzymatic step. Changes in expression of each EST are represented by three boxes corresponding to the three comparisons: d-3 vs. d1, d1 vs. d4, d4 vs. d7. Depending on the number of identified ESTs, each enzymatic step has a different number of sets of three boxes. The boxes were colored according to the change in gene expression: Red and blue boxes indicate significant up- and down-regulation (p-value<0.05), respectively, for a given comparison, while white boxes indicate no significant change in gene expression (p-value>0.05). AGPase: ADP-glucose pyrophosphorylase, FRK: fructokinase, CESA: cellulose synthase, HXK: hexokinase, INV: invertase, PGI: phosphoglucose isomerase, PGM: phosphoglucomutase, SUS: sucrose synthase, UGP: UDP-glucose pyrophosphorylase.

Developmental gene expression changes in (A) glycoylsis and the (B) pentose phosphate pathway are illustrated as in Figure 3. 6PGD: 6-phosphogluconate dehydrogenase, 6PGL: 6-phosphogluconolactonase, FBA: fructose-bisphosphate aldolase, G6PD: glucose-6-phosphate dehydrogenase, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, PFK/PFP: phosphofructokinase/6-phosphofructo-1-phosphotransferase, PGK: phosphoglycerate kinase, PGM: phosphoglycerate mutase, PK: pyruvate kinase, PPH: phosphopyruvate hydratase, RPE: ribulose-phosphate 3-epimerase, RPI: ribose-5-phosphate isomerase, TAL: transaldolase, TKL: transketolase, TPI: triose-phosphate isomerase.

Developmental gene expression changes in the (A) methylerythritol phosphate (MEP) pathway, the (B) mevalonic acid (MVA) pathway, and the (C) shikimate/phenylpropanoid pathway are illustrated as in Figure 3. AACT: acetyl-CoA acetyltransferase, ADT: arogenate dehydratase, BALDH: benzaldehyde dehydrogenase, BAMT: S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, CM: chorismate mutase, CMK: 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase, CS: chorismate synthase, DAHP: 3-deoxy-arabino-heptulonate 7-phosphate, DAHPS: 3-deoxy-7-phosphoheptulonate synthase, DHD/SHD: dehydroquinate dehydratase/shikimate dehydrogenase, DHQS: 3-dehydroquinate synthase, DMAPP: dimethylallyl pyrophosphate, DXS: 1-deoxy-D-xylulose 5-phosphate, DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase, EPSP: 3-enolpyruvyl-shikimate-5-phosphate, EPSPS: 3-phosphoshikimate 1-carboxyvinyltransferase, FPP: farnesyl pyrophosphate, FPPS: farnesyl pyrophosphate synthase, GPP: geranyl pyrophosphate, GPPS: geranyl pyrophosphate synthase, HDS: 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, HMBDP: (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate, HMG-CoA: hydroxymethylglutaryl-CoA, HMGR: hydroxymethylglutaryl-CoA reductase, HMGS: hydroxymethylglutaryl-CoA synthase, IDS: isopentenyl diphosphate synthase, IPP: isopentenyl pyrophosphate, IPPI: isopentenyl pyrophosphate isomerase, MCT: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, MECPD: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate, MECPS: 2-C- methyl-D-erythritol 2,4-cyclodiphosphate synthase, MK: mevalonate kinase, MPDC: mevalonate diphosphate decarboxylase, PAL: phenylalanine ammonia lyase, PAT: prephenate aminotransferase, PMK: phosphomevalonate kinase, SK: shikimate kinase, TPS: terpene synthase.

(A) Cluster dendrogram showing the four stages of petal and sepal development. Samples were clustered according to their individual metabolomic profiles as measured by non-targeted liquid chromatography-mass spectrometry (LC-MS). (B) Grouping of peaks detected by non-targeted LC-MS analysis based on their developmental profile. Using a one-way ANOVA, three hypotheses were tested: H₀: µ_d-3 = µ_d1, H₀: µ_d1 = µ_d4, H₀: µ_d4 = µ_d7. A score was attributed for each test. If the gene expression was not significantly different (p-value>0.05), the score = 0. If it was significantly up-regulated (p-value<0.05), the score = 1. If it was significantly down-regulated (p-value<0.05), the score = −1. Genes were grouped based on the combination of scores for the three tests. The number of peaks in each developmental pattern is indicated next to the graphs. Heatmap of (C) metabolites putatively identified by ion trap LC-MSⁿ and of (D) metabolites measured by gas chromatography-mass spectrometry in snapdragon petals. Metabolite levels were expressed relative to the average value for that metabolite in d-3 samples and log₂-transformed. Log₂-transformed values were averaged for each stage. Yellow indicates an increase in metabolite abundance and blue a decrease.

Gene-to-metabolite networks in snapdragon petals. ESTs are represented by gray circles and metabolites by white circles. Red lines indicate positive correlations (r>0.95, p-value<0.05) and blue lines negative correlations (r<0.95, p-value<0.05). Numbers in parentheses describe the numbers of nodes (metabolites or ESTs) being correlated to one or more nodes and the total number of nodes in the pathway.