DataSheet_1_Biochemical analysis of the TPS-a subfamily in Medicago truncatula.pdf

Terpenes are important mediators of plant chemical response to environmental cues. Here, we describe the genome-wide identification and biochemical characterization of TPS-a members in Medicago truncatula, a model legume crop. Genome mining identified thirty-nine full-length terpene synthases with a significant number predicted to produce monoterpenes and sesquiterpenes. Biochemical characterization of the TPS-a subfamily associated with sesquiterpene biosynthesis revealed such compounds, that exhibit substantial biological activity in other plants. Gene expression analysis using qPCR and the Medicago gene atlas illustrated distinct tissue and time-based variation in expression in leaves and roots. Together our work establishes the gene-to-metabolite relationships for sesquiterpene synthases in M. truncatula. Understanding the biosynthetic capacity is a foundational step to defining the ecological roles of this important family of compounds.</p

Quartiles of MMP protein expression.

<p>Minimum to 25% represented quartile 1 (Q1), 26% to Median represented quartile 2 (Q2), Median to 75% represented quartile 3 (Q3) and 75% to Maximum represented quartile 4 (Q4).</p

Correlation between plasma MMP/TIMP levels and % change in FEV1.

<p>Plasma protein levels for MMP-1, -9 and TIMP-1 were correlated with the % change from baseline in FEV1 at 6, 9 and 18 months of follow up. The coefficient of determination (R²) was calculated for each value at each time point.</p

Correlation between BALF MMP/TIMP levels and % change in FEV1.

<p>BALF protein levels for MMP-1, -9 and TIMP-1 were correlated with the % change from baseline in FEV1 at 6, 9 and 18 months of follow up. The coefficient of determination (R²) was calculated for each value at each time point.</p

Association between quartile of plasma protein expression and clinical parameters.

<p>The mean values of age, pack years, FEV1% predicted, FEV1/FVC % predicted, TLC % predicted, CT score and St. George questionnaire total score were calculated for each quartile of MMP-1, -9 and TIMP-1 plasma protein expression. Bold indicates p<0.05 compared to quartile 1. Parentheses () indicate standard deviation.</p

Correlation between MMPs and DL_CO or FEV1/FVC % predicted.

<p>Linear regression analyses correlated baseline BALF MMP-1, -9, -12 and TIMP-1 lavage levels with change in DL_CO or the FEV1/FVC % predicted ratio at 9-month follow up. R² = coefficient of correlation.</p

MMP and TIMP-1 levels in the plasma of normal smokers and emphysema subjects.

<p>Luminex multiplex assays for MMP-1 (top panel), MMP-9 (center panel) and TIMP-1 (bottom panel) were done on age-matched plasma samples from non-smoking controls, normal smokers and subjects with moderate to severe emphysema. MMP-12 was measured but levels were below the limit of detection.</p

Coefficients of correlation (R²) between elastase and collagenase activity and COPD disease parameters.

<p>The coefficient of correlation (R²) was calculated for elastase and collagenase activity and age, pack years, FEV1, FEV1/FVC, TLC, CT score, D_LCO and St. George total score.</p><p>PFT parameters are reported in % predicted post bronchodilator.</p

Emphysema characteristics.

<p>All data are presented as mean ± standard deviation. Data for forced expiratory volume in one second (FEV₁), forced vital capacity (FVC) total lung capacity, residual volume and diffusion capacity for carbon monoxide (DLCO) are all presented as percent predicted. FEV1/FVC ratio is presented as %. Bronchodilator responsiveness is presented as % change in FEV1.</p

BALF MMP and TIMP-1 protein levels and protease activity in controls, smokers and emphysema subjects.

<p>BALF was performed on 72 normal control subjects, 16 smokers, and 101 subjects with moderate to severe emphysema who had refrained from smoking for at least six months. (A) MMP-1, (B) MMP-9, (C) MMP-12 and (D) TIMP-1 protein levels were recorded. p<0.0001 by Kruskal-Wallis one-way analysis of variance by ranks followed by a Dunn’s multiple comparison test. <i>p</i> values shown, comparing both treatments connected by a line. (E) Collagenase and (F) elastase activity were measured in the BALF of 26 normal controls, 9 smokers without emphysema and 60 emphysema subjects. Differences between cohorts was assessed by ANOVA; p<0.05. (G) MMP-1/TIMP-1 BALF ratios and (H) MMP-9/TIMP-1 BALF ratios were determined in the BALF of 82 normal controls, 18 smokers without emphysema and 98 emphysema subjects. Differences between cohorts was assessed by ANOVA; p<0.05.</p