Leaf and root pectin methylesterase activity and ¹³C/¹²C stable isotopic ratio measurements of methanol emissions give insight into methanol production in <i>Lycopersicon esculentum</i>

New PhytologistVolume 191, Issue 4 p. 1031-1040 Full paperFree Access Leaf and root pectin methylesterase activity and 13C/12C stable isotopic ratio measurements of methanol emissions give insight into methanol production in Lycopersicon esculentum Patricia Yoshino Oikawa, Patricia Yoshino Oikawa Department of Biology, University of Virginia, Charlottesville, VA 22904, USASearch for more papers by this authorBrian M. Giebel, Brian M. Giebel Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149-1098, USASearch for more papers by this authorLeonel da Silveira Lobo O'Reilly Sternberg, Leonel da Silveira Lobo O'Reilly Sternberg Department of Biology, University of Miami, Coral Gables, FL 33124-0421, USASearch for more papers by this authorLei Li, Lei Li Department of Biology, University of Virginia, Charlottesville, VA 22904, USASearch for more papers by this authorMichael P. Timko, Michael P. Timko Department of Biology, University of Virginia, Charlottesville, VA 22904, USASearch for more papers by this authorPeter K. Swart, Peter K. Swart Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149-1098, USASearch for more papers by this authorDaniel D. Riemer, Daniel D. Riemer Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149-1098, USASearch for more papers by this authorJohn E. Mak, John E. Mak School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USASearch for more papers by this authorManuel T. Lerdau, Manuel T. Lerdau Department of Biology, University of Virginia, Charlottesville, VA 22904, USA Department of Environmental Sciences, University of Virginia, Charlottesville, VA 22904-4123, USA Xishuangbanna Tropical Botanic Garden, Melung, Xishuangbanna, Yunnan, ChinaSearch for more papers by this author Patricia Yoshino Oikawa, Patricia Yoshino Oikawa Department of Biology, University of Virginia, Charlottesville, VA 22904, USASearch for more papers by this authorBrian M. Giebel, Brian M. Giebel Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149-1098, USASearch for more papers by this authorLeonel da Silveira Lobo O'Reilly Sternberg, Leonel da Silveira Lobo O'Reilly Sternberg Department of Biology, University of Miami, Coral Gables, FL 33124-0421, USASearch for more papers by this authorLei Li, Lei Li Department of Biology, University of Virginia, Charlottesville, VA 22904, USASearch for more papers by this authorMichael P. Timko, Michael P. Timko Department of Biology, University of Virginia, Charlottesville, VA 22904, USASearch for more papers by this authorPeter K. Swart, Peter K. Swart Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149-1098, USASearch for more papers by this authorDaniel D. Riemer, Daniel D. Riemer Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149-1098, USASearch for more papers by this authorJohn E. Mak, John E. Mak School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USASearch for more papers by this authorManuel T. Lerdau, Manuel T. Lerdau Department of Biology, University of Virginia, Charlottesville, VA 22904, USA Department of Environmental Sciences, University of Virginia, Charlottesville, VA 22904-4123, USA Xishuangbanna Tropical Botanic Garden, Melung, Xishuangbanna, Yunnan, ChinaSearch for more papers by this author First published: 19 May 2011 https://doi.org/10.1111/j.1469-8137.2011.03770.xCitations: 26 Author for correspondence: Patricia Yoshino Oikawa Tel: +1 650 799 2808 Email: [email protected] AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Summary • Plant production of methanol (MeOH) is a poorly understood aspect of metabolism, and understanding MeOH production in plants is crucial for modeling MeOH emissions. Here, we have examined the source of MeOH emissions from mature and immature leaves and whether pectin methylesterase (PME) activity is a good predictor of MeOH emission. We also investigated the significance of below-ground MeOH production for mature leaf emissions. • We present measurements of MeOH emission, PME activity, and MeOH concentration in mature and immature tissues of tomato (Lycopersicon esculentum). We also present stable carbon isotopic signatures of MeOH emission and the pectin methoxyl pool. • Our results suggest that below-ground MeOH production was not the dominant contributor to daytime MeOH emissions from mature and immature leaves. Stable carbon isotopic signatures of mature and immature leaf MeOH were similar, suggesting that they were derived from the same pathway. Foliar PME activity was related to MeOH flux, but unexplained variance suggested PME activity could not predict emissions. • The data show that MeOH production and emission are complex and cannot be predicted using PME activity alone. We hypothesize that substrate limitation of MeOH synthesis and MeOH catabolism may be important regulators of MeOH emission. Introduction Emission of methanol (MeOH) from plants is ubiquitous and plays major roles in atmospheric chemistry. MeOH is the second most abundant organic gas after methane. Annual global budgets of phytogenic MeOH emissions are estimated to be anywhere from 75 to 280 Tg yr−1 (Tg = teragrams = 1012 g), while anthropogenic emissions resulting from industrial processes range from only 4 to 8 Tg yr−1 (Singh et al., 2000; Galbally & Kirstine, 2002; Heikes et al., 2002; von Kuhlmann et al., 2003a,b; Tie et al., 2003; Jacob et al., 2005). MeOH has an atmospheric lifetime of c. 10 d (Jacob et al., 2005). This long lifetime allows MeOH to move into the upper troposphere where it can substantially lower hydroxyl radical concentrations as background concentrations of other volatile organic compounds (VOCs) in the upper troposphere are low (Singh et al., 2000, 2001; Tie et al., 2003). Studies have consistently shown that young expanding leaves emit greater amounts of MeOH than mature leaves (Macdonald & Fall, 1993; Hüve et al., 2007). Although mature leaf MeOH emissions are significantly lower than immature leaves (on average three to four times lower across species), they are still substantial and should not be ignored in modeling efforts (Macdonald & Fall, 1993; Nemecek-Marshall et al., 1995; Harley et al., 2007). Mature leaf MeOH emission can be just as significant as immature leaf emission on an annual scale. For example, after accounting for changes in leaf area index and length of time spent at each ontogenetic stage, mature and immature leaves of deciduous trees contribute approximately equal amounts to annual MeOH flux. Therefore, mature leaf MeOH emission has significant implications for atmospheric chemistry and deserves attention. Currently, the dominant biosynthetic pathway for MeOH production in mature leaves is unknown. MeOH production in immature leaves, on the other hand, is believed to be derived from the demethylation of pectin by the enzyme pectin methylesterase (PME) (Fall & Benson, 1996; Frenkel et al., 1998; Galbally & Kirstine, 2002; Keppler et al., 2004). Demethylation of pectin by PME facilitates crosslinking of pectin polymer chains and stabilizes the cell wall during expansion. As a by-product of cell growth, cumulative daily MeOH flux is known to strongly correlate with leaf expansion (Hüve et al., 2007). The PME pathway has also been directly linked to MeOH production and emission in two studies in which silencing PME genes led to significantly decreased MeOH production in tomato fruit (Frenkel et al., 1998) and significantly decreased MeOH emission response to herbivory (Korner et al., 2009). MeOH production resulting from the demethylation of DNA and protein repair pathways is believed to be small because of low activity rates; for example, PME activity rates are at least six orders of magnitude higher than protein repair enzyme activity rates (Fall & Benson, 1996). Although it is generally accepted that PME activity is the source of MeOH emissions from immature leaves, the relationship between PME activity and MeOH emissions has yet to be described. In addition, the role of PME activity in MeOH emissions from mature leaves is unknown. As mature leaves are fully expanded, foliar PME activity is expected to be low. Mature cell walls are known to have lower degrees of methyl esterification than immature cell walls and therefore have lower potential for MeOH production via the PME pathway. Alternatively, production of MeOH in other areas of the plant may be supplying mature leaves with MeOH via the transpiration stream. Previous work has suggested that MeOH emissions from mature leaves are derived from below-ground MeOH production (Folkers et al., 2008). Experimentation with the cooling of roots, thereby decreasing metabolic activity in root tissue, indicated that some MeOH emitted from mature deciduous tree leaves is derived from MeOH production below ground (Folkers et al., 2008). The spatial heterogeneity of MeOH production in plants may therefore significantly influence MeOH emissions from mature leaves and deserves further investigation. While the instantaneous flux of MeOH from leaves is a function of leaf MeOH concentration and stomatal conductance (Niinemets & Reichstein, 2003a,b), an outstanding challenge for the field of biogenic VOC emission studies is to develop models that can predict the leaf concentration of individual VOCs (Lerdau, 1991). Such models for MeOH concentration do not yet exist. In order to help develop mechanistic MeOH emission models and address the uncertainty surrounding the role of PME activity in MeOH production, we investigated three main questions. Are MeOH emissions from mature and immature leaves derived from the same biosynthetic pathway? Is PME activity a good predictor of MeOH emissions? Do below-ground sources significantly contribute to MeOH emissions from mature leaves? We addressed the three research questions using stable carbon isotope analysis, PME activity assays, MeOH flux measurements, and MeOH extractions from mature and immature Lycopersicon esculentum. Materials and Methods Study species All Lycopersicon esculentum L. individuals were Micro Tom clones, a dwarf variety of tomato (Meissner et al., 1997). L. esculentum was chosen as a model plant because of its rapid growth and high MeOH emission behavior. MeOH emissions from mature leaves of L. esculentum, Fagus sylvatica, and Quercus robur are on average 3.6, 0.77, 0.33 nmol m−2 s−1, respectively (Folkers et al., 2008). Plants were grown in the glasshouse at the University of Virginia in Charlottesville (38°N, 78°W). Pots were placed in flats filled with 1 inch of water and illuminated during a 16 h period with natural light supplemented with high-pressure sodium lamps. Plants were fertilized every 2 wk (Scotts 20% N, 20% P, 20% K; Scotts Miracle-Gro Company, Marysville, OH, USA) and kept insect-free using a variety of insecticides. Immature leaves were sampled 3 wk past germination and mature leaves 6 wk past germination. Leaf size was measured regularly with calipers to ensure that immature leaves were rapidly expanding and mature leaves were fully expanded. Stable carbon isotope measurements Gaseous MeOH released from immature (n = 6) and mature (n = 5) leaves of L. esculentum were measured by coupling a Li-Cor LI-6400 portable gas exchange system (Li-Cor, Inc., Lincoln, NE, USA) to a heavily modified gas chromatography isotope ratio mass spectrometer (GC-IRMS; Agilent 6800 GC-Europa Scientific GEO 20-20 IRMS) capable of measuring δ13C ratios of oxygenated/biological VOCs (O/BVOCs) in air samples (Giebel et al., 2010). Measurement precisions for MeOH using this method were evaluated using a gravimetrically prepared gas-phase standard yielding a final mixing ratio of 18.6 ppbv (1.86 × 10−2 μl l−1) after dynamic dilution in zero and were ± 2.8‰ with an associated error of 2.5% compared with the raw material used to make the calibrant gas. A detailed description of the GC-IRMS system and method is available (Giebel et al., 2010); a brief review and additional details, however, are provided here. The LI-6400 enabled leaf-level gas measurements to be standardized for multiple photosynthetic variables by controlling light intensities (peak irradiance of 665 and 470 nm), temperature, and relative humidity within the cuvette. Keeping light intensities constant (photosynthetic photon flux density (PPFD) of 950 μmol photon m−2 s−1), leaf-level measurements were only taken under steady-state conditions which were, on average, as follows: leaf temperature, 26.1 ± 0.9°C; stomatal conductance, 0.14 ± 0.05 mol H2O m−2 s−1 (mature) and 0.20 ± 0.06 mol H2O m−2 s−1 (immature); photosynthetic rates, 9.0 ± 2 μmol CO2 m−2 s−1 (mature) and 12.6 ± 3 μmol CO2 m−2 s−1 (immature); and relative humidity, 53.1 ± 4% (mature) and 56.0 ± 5% (immature) (means ± SD). Isotopic measurements were taken at temperatures similar to those previously reported for phytogenic MeOH (Keppler et al., 2004; Yamada et al., 2009). Steady-state conditions were important because MeOH emissions are tightly regulated by stomatal conductance as a result of the high solubility of MeOH (Niinemets & Reichstein, 2003a). Air was supplied to the LI-6400 at a rate of 1.0 l min−1 by a zero-air generator. The zero-air generator contained a catalytic converter which removed all hydrocarbons, including MeOH, from the air stream; however, carbon dioxide and water were unaffected. Individual leaves, with an area between 4 and 6 cm2, were placed in the cuvette of the LI-6400 and allowed to reach steady state over a period of 10–20 min before sampling. Outflow from the cuvette, with a leaf in place, was between 100 and 300 cm3 min−1 and connected directly to a custom-made preconcentration system located on the GC inlet. Approximately 1.0 l volumes were sampled directly from the cuvette outflow to the preconcentration system and controlled at a rate of 50 cm3 min−1. After sampling, the adsorbent trap was purged and subsequently back-flushed with helium carrier gas while the trap was resistively heated. Volatized MeOH was cryofocused in liquid nitrogen before being injected into the GC. Separated components in the eluant gas passed through a heated combustion column and were transferred to the open split and ion source of the IRMS (Giebel et al., 2010). For carbon, the stable isotopic composition of a sample is expressed as a ratio (R) of 13C : 12C and reported in delta (δ) notation as a per-mil (‰) difference of the sample compared with a working reference gas calibrated to the international standard Vienna PeeDee Belemnite (V-PDB). MeOH derived CO2, and that used for reference (0.1% CO2, 41.9‰), was delivered through the open split to the ion source of the IRMS. Six working reference gas injections were made during each chromatographic run and compared with the methanol peak to determine the δ13C of methanol. The stable carbon isotopic signature of pectin methoxyl groups was calculated from the isotopic signatures of untreated and demethylated apple pectin (Apple pectin, c. 70% methylated; Sigma-Aldrich). Alkaline hydrolysis (1 N NaOH) of pectin at 70°C generated demethylated pectin as in Rosenbohm et al. (2003). Carbon isotope ratios were determined in both pectin and demethylated pectin in an Isoprime IRMS (Elementar, Hanau, Germany) connected to a Eurovector elemental analyzer (Milan, Italy). The stable carbon isotopic signature of the methoxyl groups (representing 10% of all carbon in pectin) was calculated from the isotopic signature of untreated pectin (a reflection of 100% of all carbon in pectin, including both glucose and methoxyl groups) and the isotopic signature of demethylated pectin (a reflection of 90% of all carbon in pectin). Gas exchange and MeOH emission measurements Leaf-level MeOH emissions were quantified with a Li-Cor LI-6400 portable gas exchange system coupled with a proton-transfer-reaction mass spectrometer (high sensitivity PTR-MS; Ionicon Analytik, Innsbruck, Austria). PTR-MS has been described in detail elsewhere (Lindinger et al., 1998). PTR-MS requires no preconcentration or chromatography of VOCs. Instead, the air flows directly to the drift tube where VOCs undergo chemical ionization via proton-transfer reaction with H3O+. Protonated VOCs are then counted by the ion detector and can be measured down to the parts-per-trillion (ppt) level. Air exiting the LI-6400 cuvette was routed to the PTR-MS inlet via 1/4 inch Teflon tubing with a T-fitting in order to release extra flow. Flow rates through the cuvette ranged from 150 to 350 μmol s−1. Despite typically stable concentrations of MeOH in ambient air throughout the sampling periods, empty cuvette measurements were coupled with each leaf measurement in order to control for fluctuations in background MeOH. All measurements were taken between 10:00 and 16:00 h. PTR-MS measurements were recorded for 20 cycles for a total sampling time of c. 3 min. All measurements were taken under steady-state conditions at a PPFD of 750 μmol m−2 s−1, a leaf temperature of 31 ± 1.9°C, stomatal conductances of 0.09 ± 0.04 mol H2O m−2 s−1 (mature) and 0.15 ± 0.06 mol H2O m−2 s−1 (immature), photosynthetic rates of 7.0 ± 2 μmol CO2 m−2 s−1 (mature) and 10.5 ± 2 μmol CO2 m−2 s−1 (immature), and relative humidities of 55.6 ± 3% (mature) and 58.2 ± 4% (immature) (means ± SD). The leaf surface area enclosed in the cuvette was measured using a Li-Cor Leaf Area Meter. The portion of leaf enclosed in the cuvette was weighed directly after being removed from the plant. MeOH emission rates are expressed on a per unit FW basis (nmol g−1 FW s−1). Four point calibrations were made regularly throughout the sampling period with dilutions of a gravimetrically prepared MeOH gas standard provided by the Riemer laboratory (University of Miami) containing 3 ppmv (3 μl l−1) ± 2% MeOH in nitrogen gas. The accuracy of MeOH measurements was estimated to be c. 20% (based on the accuracy of calibration measurements) and reproducibility of c. 10%. MeOH emission measurements were made on 10 immature and 10 mature L. esculentum leaves. PME enzyme activity rates Directly following MeOH emission measurement, sampled leaves were excised and frozen in liquid nitrogen. A portion of the sampled plant's root mass was rinsed and also frozen in liquid nitrogen. Frozen samples were assayed for PME enzyme activity via a titration technique previously developed for L. esculentum (Anthon & Barrett, 2006). Plant tissue was ground in a mortar and pestle to a fine powder, weighed, and mixed in equal weight with a solution composed of 50% 2 M NaCl and 50% 10 mM phosphate buffer (pH 7.5). Samples were then centrifuged at 8000 g for 5 min. A 25 μl quantity of plant supernatant was added to 2.5 ml of pectin solution containing 0.5% pectin, 0.2 M NaCl, 0.1 mM phosphate buffer (pH 7.5). The sample solution pH was adjusted to 7.5 using small amounts of 0.1 M NaOH (in 1–5 μl). Once the solution dropped back down to pH 7, 1–5 μl 0.1 M NaOH was added until the solution pH reached 7.3. The time for the solution to drop back down to pH 7 was recorded. The demethylation of pectin by PME acidifies the solution. PME activity is therefore expressed in nmol g−1 FW s−1 based on the change in pH for a given amount of fresh tissue over time. Measuring change in pH over time is a proxy for PME activity and not a direct measurement of enzyme activity, but this change in pH has been shown to be a highly repeatable proxy for enzyme activity (Anthon & Barrett, 2006). A total of 10 immature and 10 mature L. esculentum were assayed for PME enzyme activity. MeOH extractions MeOH extraction was conducted on stem and leaf L. esculentum tissue. Whole plants were frozen in a liquid nitrogen bath before removal of the midstem and an adjacent mature leaf. Tissues were weighed and ground in 5 × equal weight EDTA with a mortar and pestle (Leegood, 1993; Nemecek-Marshall et al., 1995). Samples were then centrifuged at 3000 g for 4 min before removing the top layer and neutralizing with NaOH. Samples were then injected into a gas chromatograph coupled with a flame ionization detector (GC-FID). A three-point calibration was made with dilutions of pure MeOH in deionized water. An additional calibration curve was made with aliquots of pure MeOH added to plant extract, which produced a standard equation similar to the DI water calibration curve. MeOH concentration was measured with an uncertainty of 4%. Tissues from 12 immature and nine mature plants were measured for MeOH concentration. Statistical analysis Differences between mature and immature mean MeOH δ13C values were examined with a t-test (Proc TTEST, SAS 9.1; SAS Institute Inc., Cary, NC, USA). Linear regression was used to assess how well the independent variables leaf enzyme activity, root enzyme activity, and leaf type (mature and immature) predict MeOH emissions (Proc GLM, SAS 9.1; SAS Institute Inc.). Differences between mature and immature PME activities in root and leaf tissue were examined with t-tests. Differences between mature and immature MeOH emission rates were also examined with a t-test. Data used in regression analyses and t-tests were log-transformed to meet normality and homogeneity of variance assumptions. Nonparametric regression was used to determine whether or not MeOH concentration in stem tissue was a good predictor of MeOH concentration in leaf tissue (Proc GAM, SAS 9.1; SAS Institute Inc.). A Wilcoxon two-sample exact test was used to compare MeOH concentrations measured in leaf tissue between leaf types (Proc NPAR1WAY, SAS 9.1; SAS Institute Inc.). Three outlier points were detected according to Cook's D influence statistic and were removed from the analysis. Results Stable carbon isotope analysis of MeOH emissions was used to test the hypothesis that the stable carbon isotopic signatures of MeOH from mature and immature leaves are similar. The measured δ13C of MeOH emissions from mature and immature L. esculentum leaves were not significantly different (t = −1.08, df = 8, P = 0.31); the values for mature and immature leaves were, on average, −19.0 and −21.5‰, respectively (Fig. 1). We interpret this similarity to support the hypothesis that the dominant biosynthetic pathway for MeOH production in plants, PME activity, is conserved as leaves develop. Stable carbon isotope analysis of pectin and the pectin methoxyl pool was used to test the hypothesis that the isotopic signature of the pectin methoxyl pool is similar to the signature of MeOH emissions. We measured the δ13C values of purified apple pectin and apple pectin methoxyl groups. The δ13C of purified apple pectin (−26.2‰) was enriched in 13C relative to the pectin methoxyl groups (−38‰; Fig. 1). The depletion of the 13C pectin methoxyl pool is biosynthetically reasonable because the methyl donor to pectin is S-adenosyl-methionine (SAM), which has a δ13C of −39.2‰ (as measured in caffeine by Weilacher et al., 1996). The apple pectin methoxyl pool was isotopically distinct from apple pectin, previously measured tomato pectin (Park & Epstein, 1961), and MeOH emissions from tomato (Fig. 1). We interpret the difference in isotopic signature between the pectin methoxyl pool and MeOH emissions as evidence that an enrichment process (e.g. MeOH catabolism) may occur during the production and emission of MeOH in plants. Figure 1Open in figure viewerPowerPoint Average δ13C values for methanol (MeOH) emissions from mature (n = 5) and immature (n = 6) Lycopersicon esculentum, tomato pectin measured by Park & Epstein (1961), apple pectin, and pectin methoxyl groups. Values are means ± SE. No significant differences were found between the two leaf types (t = −1.08, df = 8, P = 0.31). Enzyme activity is known to be a good predictor for mechanistic VOC emission models (Fall & Wildermuth, 1998; Logan et al., 2000). Flux measurements were taken in conjunction with enzyme activity rate measurements in leaves to test if PME activity in leaves and roots were good predictors of MeOH emission. Foliar PME activity was significantly related to MeOH emission across both leaf types (F = 6.24, P = 0.022; Fig. 2a), but only explained a small amount of the variance in MeOH emission (R2 = 0.26). Additionally, no significant relationship between PME activity and MeOH emission was detected within leaf type (F = 1.66, P = 0.22; Fig. 2a). We interpret these results as evidence that, although foliar PME activity was related to MeOH emission, other factors must also be considered when predicting MeOH emission. Root PME activity did not correlate with MeOH emission across leaf types (F = 0.52, P = 0.48) or within leaf type (F = 0.25, P = 0.63), indicating that below-ground PME activity was not related to foliar MeOH emission (Fig. 2b). These data were graphed on log scale plots as they were log-transformed for statistical analysis (Fig. 2a,b). Figure 2Open in figure viewerPowerPoint The relationship between leaf pectin methylesterase (PME) activity and leaf methanol (MeOH) flux (regression; F = 6.24, P = 0.022 across leaf type; F = 1.66, P = 0.22 within leaf type) (a) and root PME activity and leaf MeOH flux (regression; F = 0.52, P = 0.48 across leaf type; F = 0.25, P = 0.63 within leaf type) (b) in mature (n = 10) and immature (n = 10) Lycopersicon esculentum. (a, b) Closed circles, mature; open circles, immature. Lines are fitted to all data. Data are shown on a log–log scale. Mature leaf PME activity was higher than expected based on our knowledge of PME activity in fully expanded leaves (average ± SE PME activity rates were 6.4 ± 1.7 and 11.9 ± 2.1 nmol g−1 FW s−1 for mature and immature leaves, respectively; Fig. 3). Based on mean PME activity rates measured in mature and immature leaves, average mature leaf MeOH flux was lower than expected. Mature leaf PME activity was c. 50% of immature leaf PME activity, while MeOH flux from mature leaves was c. 33% of MeOH flux from immature leaves (average ± SE MeOH flux rates were 0.03 ± 0.01 and 0.09 ± 0.02 nmol g−1 FW s−1 for mature and immature leaves, respectively; Fig. 3). We interpret relatively high PME activity and low MeOH emission from mature leaves as possibly indicative of a MeOH sink. Mature and immature leaves did not have significantly different concentrations of MeOH (P = 0.28, Wilcoxon exact; average ± SE MeOH concentrations were 0.74 ± 0.17 and 0.93 ± 0.20 mg g−1 FW for mature and immature leaves, respectively), indicating that although immature leaf MeOH emission was high, immature leaf MeOH concentration was not. We interpret relatively high MeOH emissions without high MeOH concentrations in immature leaves as also congruent with a MeOH sink. Figure 3Open in figure viewerPowerPoint Leaf pectin methylesterase (PME) activity, root PME activity and leaf methanol (MeOH) flux for mature (black bars; n = 10) and immature (gray bars; n = 10) Lycopersicon esculentum. Values are means ± SE. Immature leaves tended to have higher PME activity (t-test; t = 2.03, df = 18, P = 0.057) and higher MeOH flux (t-test; t = 3.35, df = 18, P = 0.0036) than mature leaves. **, significant difference at P < 0.01. No significant difference was detected between PME activity in immature and mature root tissues (t-test; df = 18, t = 0.32, P = 0.76). In order to assess whether the transpiration stream was the dominant contributor of MeOH to leaves, we tested whether or not MeOH concentrations in stems could predict concentrations in leaves. In contrast to our hypothesis, concentrations of MeOH in stems were not good predictors for concentrations of MeOH in leaves (χ2 = 3.28, P = 0.35, across leaf types; χ2 = 6.51, P = 0.10, mature only; χ2 = 2.20, P = 0.53, immature only; Fig. 4). We interpret this result as evidence that MeOH transported in the transpiration stream was likely not the dominant source of MeOH to leaves. Figure 4Open in figure viewerPowerPoint The relationship between methanol (MeOH) concentrations in stem and leaf tissue from mature (closed circles; n = 9) and immature (open circles; n = 12) Lycopersicon esculentum (nonparametric regression; across leaf type, χ2 = 3.28, P = 0.35; mature only, χ2 = 6.51, P = 0.10; immature only, χ2 = 2.20, P = 0.53). Discussion Although it is believed that MeOH emission from immature leaves is derived from the PME pathway, the relationship between PME activity and immature leaf MeOH emission has not previously been described. Furthermore, the role of PME activity in MeOH production in mature leaf tissue has remained unstudied. Because of the fully expanded nature of mature leaves and a previous study indicating that some mature leaf MeOH is derived from below-ground MeOH production (Folkers et al., 2008), we predicted that mature leaf MeOH would be mainly derived from PME activity in root tissue. We also hypothesized that if the dominant source of MeOH in mature leaves is below-ground production, the concentrations of MeOH in stems would predict concentrations in leaves. In contrast to our hypothesis, root PME activity was not related to MeOH flux, and MeOH extractions from mature stem and leaf tissue showed that MeOH in the transpiration stream could not predict MeOH in leaf tissue. These results provide strong evidence that below-ground MeOH production through the PME pathway was unlikely to be the do