MOESM1 of A 13C isotope labeling method for the measurement of lignin metabolic flux in Arabidopsis stems

Additional file 1. Figure S1. A simplified pathway illustrating the enzymes and metabolites involved in lignin biosynthesis. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate CoA ligase; HCT, hydroxycinnamoyl CoA:shikimatehydroxycinnamoyl transferase; C3′H, p-coumaroyl shikimate 3′-hydroxylase; CSE, caffeoyl shikimate esterase; CCoAOMT, caffeoyl CoA O-methyltransferase; F5H, ferulate5-hydroxylase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl CoA reductase; CAD, cinnamyl alcohol dehydrogenase. SALDH, sinapaldehydedehydrogenase. Figure S2. Excised stems incubated in tubes with MS medium in growth chamber. (A) An Arabidopsis stem was excised and placed into a 1.5 mL tube containing liquid MS medium. (B) Arabidopsis stems incubated in MS medium were placed in a rack to perform feeding experiment (picture taken from side). (C) Stems were sitting away from each other to mimic their growth in the soil (picture taken from top). Figure S3. Medium absorbed by the excised stems during the feeding process. The loss of medium from each tube with an excised stem was measured after feeding for 0, 40, 90, 180, and 240 min. Data represented mean ± SD (n = 45). Figure S4. Hierarchical clustering of labeling percentage profiles of soluble phenylpropanoids from the base of Arabidopsis stems supplied with 1 mM [13C6]-Phe over the feeding time course. The averaged labeling percentage data of each metabolite over the time course from Figure 5 were clustered based on squared Euclidian distance. Figure S5. Metabolic profiles of soluble phenylpropanoids from the base of Arabidopsis stems supplied with 1 mM [13C6]-Phe over the feeding time course. Sum of endogenous and 13C6 labeled compounds was quantified with LC/MS-MS and normalized to fresh weight of stem tissue. The plot of each metabolite measured was placed above its name on the pathway. Dashed lines mean multiple steps. Data represent mean ± SD (n = 3)

Gene Ontology term enrichment analysis for up-regulated genes contained in the petal developmental clusters {0,1,0}, {1,0,0}, and {1,1,0}.

a<p>GO terms with FDR corrected p-value less than 0.05 are shown.</p

Gene to metabolite correlation network for amino acid pathways, the TCA cycle, and the phenylporpanoid and terpenoid network.

<p>Gene-to-metabolite networks in snapdragon petals. ESTs are represented by gray circles and metabolites by white circles. Red lines indicate positive correlations (<i>r</i>>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.</p

Remodeling of the petal and sepal metabolomes over flower development.

<p>(<b>A</b>) 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>B</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 (<b>C</b>) metabolites putatively identified by ion trap LC-MSⁿ and of (<b>D</b>) 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.</p

Developmental rearrangements in gene expression of snapdragon petals and sepals.

<p>(<b>A</b>) Cluster dendrogram showing samples collected at the four stages of petal and sepal development. Samples were clustered according to their gene expression profile. (<b>B</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.</p

Gene ontology terms enriched in genes that show significant positive correlation with the metabolite classes “Phe, Tyr, phenylpropanoids”, “Terpenoids”, “TCA cycle intermediates”, and “AA”.

a<p>GO terms with FDR corrected p-value less than 0.05 are shown.</p

Gene expression in glycolysis and the pentose phosphate pathway over petal development.

<p>Developmental gene expression changes in (<b>A</b>) glycoylsis and the (<b>B</b>) pentose phosphate pathway are illustrated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040381#pone-0040381-g003" target="_blank">Figure 3</a>. 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.</p

Gene expression in sucrose metabolism over petal development.

<p>ESTs were annotated and assigned to enzymatic steps based on their homology to <i>A. thaliana</i> genes involved in each enzymatic step. Changes in expression of each EST are represented by three boxes corresponding to the three comparisons: d-3 <i>vs</i>. d1, d1 <i>vs</i>. d4, d4 <i>vs</i>. 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.</p

Gene Ontology term enrichment analysis for down-regulated genes contained in the petal developmental clusters {0,−1,0}, {−1,0,0}, and {−1,−1,0}.

a<p>GO terms with FDR corrected p-value less than 0.05 are shown.</p

Changes in phenotype and biomass composition during snapdragon flower development.

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