Phytogenic biosynthesis and emission of methyl acetate.

Acetylation of plant metabolites fundamentally changes their volatility, solubility and activity as semiochemicals. Here we present a new technique termed dynamic (13) C-pulse chasing to track the fate of C1-3 carbon atoms of pyruvate into the biosynthesis and emission of methyl acetate (MA) and CO2 . (13) C-labelling of MA and CO2 branch emissions respond within minutes to changes in (13) C-positionally labelled pyruvate solutions fed through the transpiration stream. Strong (13) C-labelling of MA emissions occurred only under pyruvate-2-(13) C and pyruvate-2,3-(13) C feeding, but not pyruvate-1-(13) C feeding. In contrast, strong (13) CO2 emissions were only observed under pyruvate-1-(13) C feeding. These results demonstrate that MA (and other volatile and non-volatile metabolites) derive from the C2,3 atoms of pyruvate while the C1 atom undergoes decarboxylation. The latter is a non-mitochondrial source of CO2 in the light generally not considered in studies of CO2 sources and sinks. Within a tropical rainforest mesocosm, we also observed atmospheric concentrations of MA up to 0.6 ppbv that tracked light and temperature conditions. Moreover, signals partially attributed to MA were observed in ambient air within and above a tropical rainforest in the Amazon. Our study highlights the potential importance of acetyl coenzyme A (CoA) biosynthesis as a source of acetate esters and CO2 to the atmosphere.

Pronounced differences in diurnal variation of carbon isotope composition of leaf respired CO2 among functional groups.

The first broad species survey of diurnal variation in carbon (C) isotope signatures of leaf dark-respired CO(2) (delta(13)C(res)) is presented here and functional differences and diurnal dynamics are linked to fractionation in different respiratory pathways, based on (13)C-labelling experiments. delta(13)C(res) was analysed with a rapid in-tube incubation technique in 16 species. A large diurnal increase in delta(13)C(res) (4-8 per thousand) occurred in evergreen, slow-growing and aromatic species and correlated significantly with cumulative photosynthesis, whereas no variation occurred in herbaceous, fast-growing plants or temperate trees. The diurnal increase in delta(13)C(res) declined almost proportionally to reductions in cumulative light and was reduced in growing compared with mature leaves. Pyruvate positional labelling provided direct evidence that functional groups differ in C allocation between respiratory pathways owing to different metabolic demands for growth, maintenance and secondary metabolism. Diurnal increase in C flux through pyruvate dehydrogenase (for investment in, for example, isoprene or aromatic compounds) combined with consistently low Krebs cycle activity resulted in pronounced increase in delta(13)C(res) in evergreen and aromatic species. By contrast, fast growing herbs with high respiratory demand exhibited no diurnal changes since C was fully respired. Hence, diurnal delta(13)C(res) pattern may provide information for C allocation in plants.

Short-term dynamics of isotopic composition of leaf-respired CO2 upon darkening: measurements and implications.

Recent advances in understanding the metabolic origin and the temporal dynamics in delta(13)C of dark-respired CO(2) (delta(13)C(res)) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in delta(13)C(res) have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in delta(13)C of respiratory substrates; second, post-photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post-illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in delta(13)C(res) (ranging from 0 to 10 per thousand diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in delta(13)C(res) was negligible in all investigated species. Further, rapid post-illumination changes in delta(13)C(res) (30 min), which increased from 2.6 per thousand to 5 per thousand over the course of the day, were examined by positional (13)C-labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in delta(13)C(res). However, a Rayleigh fractionation of (12)C-discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post-illumination peak in delta(13)C(res). These results are highly relevant since delta(13)C(res) is used in large-scale carbon cycle studies.

Metabolic Fate of the Carboxyl Groups of Malate and Pyruvate and their Influence on δ(13)C of Leaf-Respired CO2 during Light Enhanced Dark Respiration.

The enhanced CO2 release of illuminated leaves transferred into darkness, termed "light enhanced dark respiration (LEDR)", is often associated with an increase in the carbon isotope ratio of the respired CO2 (δ(13)CLEDR). The latter has been hypothesized to result from different respiratory substrates and decarboxylation reactions in various metabolic pathways, which are poorly understood so far. To provide a better insight into the underlying metabolic processes of δ(13)CLEDR, we fed position-specific (13)C-labeled malate and pyruvate via the xylem stream to leaves of species with high and low δ(13)CLEDR values (Halimium halimifolium and Oxalis triangularis, respectively). During respective label application, we determined label-derived leaf (13)CO2 respiration using laser spectroscopy and the (13)C allocation to metabolic fractions during light-dark transitions. Our results clearly show that both carboxyl groups (C-1 and C-4 position) of malate similarly influence respiration and metabolic fractions in both species, indicating possible isotope randomization of the carboxyl groups of malate by the fumarase reaction. While C-2 position of pyruvate was only weakly respired, the species-specific difference in natural δ(13)CLEDR patterns were best reflected by the (13)CO2 respiration patterns of the C-1 position of pyruvate. Furthermore, (13)C label from malate and pyruvate were mainly allocated to amino and organic acid fractions in both species and only little to sugar and lipid fractions. In summary, our results suggest that respiration of both carboxyl groups of malate (via fumarase) by tricarboxylic acid cycle reactions or by NAD-malic enzyme influences δ(13)CLEDR. The latter supplies the pyruvate dehydrogenase reaction, which in turn determines natural δ(13)CLEDR pattern by releasing the C-1 position of pyruvate.

Dynamic carbon allocation into source and sink tissues determine within-plant differences in carbon isotope ratios.

Organs of C3 plants differ in their C isotopic signature (δ13C). In general, leaves are 13C-depleted relative to other organs. To investigate the development of spatial δ13C patterns, we induced different C allocation strategies by reducing light and nutrient availability for 12 months in the Mediterranean shrub Halimium halimifolium L. We measured morphological and physiological traits and the spatial δ13C variation among seven tissue classes during the experiment. A reduction of light (Low-L treatment) increased aboveground C allocation, plant height and specific leaf area. Reduced nutrient availability (Low-N treatment) enhanced C allocation into fine roots and reduced the spatial δ13C variation. In contrast, control and Low-L plants with high C allocation in new leaves showed a high δ13C variation within the plant (up to 2.5‰). The spatial δ13C variation was significantly correlated with the proportion of second-generation leaves from whole-plant biomass (R2=0.46). According to our results, isotope fractionation in dark respiration can influence the C isotope composition of plant tissues but cannot explain the entire spatial pattern seen. Our study indicates a foliar depletion in 13C during leaf development combined with export of relatively 13C-enriched C by mature source leaves as an important reason for the observed spatial δ13C pattern.

High intraspecific ability to adjust both carbon uptake and allocation under light and nutrient reduction in Halimium halimifolium L.

The allocation of recently assimilated carbon (C) by plants depends on developmental stage and on environmental factors, but the underlying mechanisms are still a matter of debate. In the present study, we investigated the regulation of C uptake and allocation and their adjustments during plant growth. We induced different allocation strategies in the Mediterranean shrub Halimium halimifolium L. by a reduction of light (Low L treatment) and nutrient availability (Low N treatment) and analyzed allocation parameters as well as morphological and physiological traits for 15 months. Further, we conducted a (13)CO2 pulse-labeling and followed the way of recently assimilated carbon to eight different tissue classes and respiration for 13 days. The plant responses were remarkably distinct in our study, with mainly morphological/physiological adaptions in case of light reduction and adjustment of C allocation in case of nutrient reduction. The transport of recently assimilated C to the root system was enhanced in amount (c. 200%) and velocity under nutrient limited conditions compared to control plants. Despite the 57% light reduction the total biomass production was not affected in the Low L treatment. The plants probably compensated light reduction by an improvement of their ability to fix C. Thus, our results support the concept that photosynthesis is, at least in a medium term perspective, influenced by the C demand of the plant and not exclusively by environmental factors. Finally, our results indicate that growing heterotrophic tissues strongly reduce the C reflux from storage and structural C pools and therefore enhance the fraction of recent assimilates allocated to respiration. We propose that this interruption of the C reflux from storage and structural C pools could be a regulation mechanism for C translocation in plants.