The mobilization and transport of newly fixed carbon are driven by plant water use in an experimental rainforest under drought.

Non-structural carbohydrates (NSCs) are building blocks for biomass and fuel metabolic processes. However, it remains unclear how tropical forests mobilize, export, and transport NSCs to cope with extreme droughts. We combined drought manipulation and ecosystem 13CO2 pulse-labeling in an enclosed rainforest at Biosphere 2, assessed changes in NSCs, and traced newly assimilated carbohydrates in plant species with diverse hydraulic traits and canopy positions. We show that drought caused a depletion of leaf starch reserves and slowed export and transport of newly assimilated carbohydrates below ground. Drought effects were more pronounced in conservative canopy trees with limited supply of new photosynthates and relatively constant water status than in those with continual photosynthetic supply and deteriorated water status. We provide experimental evidence that local utilization, export, and transport of newly assimilated carbon are closely coupled with plant water use in canopy trees. We highlight that these processes are critical for understanding and predicting tree resistance and ecosystem fluxes in tropical forest under drought.

Tracing plant source water dynamics during drought by continuous transpiration measurements: An in-situ stable isotope approach.

The isotopic composition of xylem water (δX ) is of considerable interest for plant source water studies. In-situ monitored isotopic composition of transpired water (δT ) could provide a nondestructive proxy for δX -values. Using flow-through leaf chambers, we monitored 2-hourly δT -dynamics in two tropical plant species, one canopy-forming tree and one understory herbaceous species. In an enclosed rainforest (Biosphere 2), we observed δT -dynamics in response to an experimental severe drought, followed by a 2 H deep-water pulse applied belowground before starting regular rain. We also sampled branches to obtain δX -values from cryogenic vacuum extraction (CVE). Daily flux-weighted δ18 OT -values were a good proxy for δ18 OX -values under well-watered and drought conditions that matched the rainforest's water source. Transpiration-derived δ18 OX -values were mostly lower than CVE-derived values. Transpiration-derived δ2 HX -values were relatively high compared to source water and consistently higher than CVE-derived values during drought. Tracing the 2 H deep-water pulse in real-time showed distinct water uptake and transport responses: a fast and strong contribution of deep water to canopy tree transpiration contrasting with a slow and limited contribution to understory species transpiration. Thus, the in-situ transpiration method is a promising tool to capture rapid dynamics in plant water uptake and use by both woody and nonwoody species.

Elucidating Drought-Tolerance Mechanisms in Plant Roots through 1H NMR Metabolomics in Parallel with MALDI-MS, and NanoSIMS Imaging Techniques.

As direct mediators between plants and soil, roots play an important role in metabolic responses to environmental stresses such as drought, yet these responses are vastly uncharacterized on a plant-specific level, especially for co-occurring species. Here, we aim to examine the effects of drought on root metabolic profiles and carbon allocation pathways of three tropical rainforest species by combining cutting-edge metabolomic and imaging technologies in an in situ position-specific 13C-pyruvate root-labeling experiment. Further, washed (rhizosphere-depleted) and unwashed roots were examined to test the impact of microbial presence on root metabolic pathways. Drought had a species-specific impact on the metabolic profiles and spatial distribution in Piper sp. and Hibiscus rosa sinensis roots, signifying different defense mechanisms; Piper sp. enhanced root structural defense via recalcitrant compounds including lignin, while H. rosa sinensis enhanced biochemical defense via secretion of antioxidants and fatty acids. In contrast, Clitoria fairchildiana, a legume tree, was not influenced as much by drought but rather by rhizosphere presence where carbohydrate storage was enhanced, indicating a close association with symbiotic microbes. This study demonstrates how multiple techniques can be combined to identify how plants cope with drought through different drought-tolerance strategies and the consequences of such changes on below-ground organic matter composition.

Metabolic exchange between pathways for isoprenoid synthesis and implications for biosynthetic hydrogen isotope fractionation.

Hydrogen isotope ratios of plant lipids are used for paleoclimate reconstruction, but are influenced by both source water and biosynthetic processes. Measuring 2 H : 1 H ratios of multiple compounds produced by different pathways could allow these effects to be separated, but hydrogen isotope fractionations during isoprenoid biosynthesis remain poorly constrained. To investigate how hydrogen isotope fractionation during isoprenoid biosynthesis is influenced by molecular exchange between the cytosolic and plastidial production pathways, we paired position-specific 13 C-pyruvate labeling with hydrogen isotope measurements of lipids in Pachira aquatica saplings. We find that acetogenic compounds primarily incorporated carbon from 13 C2-pyruvate, whereas isoprenoids incorporated 13 C1- and 13 C2-pyruvate equally. This indicates that cytosolic pyruvate is primarily introduced into plastidial isoprenoids via glyceraldehyde 3-phosphate and that plastidial isoprenoid intermediates are incorporated into cytosolic isoprenoids. Probably as a result of the large differences in hydrogen isotope fractionation between plastidial and cytosolic isoprenoid pathways, sterols from P. aquatica are at least 50‰ less 2 H-enriched relative to phytol than sterols in other plants. These results provide the first experimental evidence that incorporation of plastidial intermediates reduces 2 H : 1 H ratios of sterols. This suggests that relative offsets between the 2 H : 1 H ratios of sterols and phytol can trace exchange between the two isoprenoid synthesis pathways.

Heatwave frequency and seedling death alter stress-specific emissions of volatile organic compounds in Aleppo pine.

Biogenic volatile organic compounds (BVOC) play important roles in plant stress responses and can serve as stress indicators. While the impacts of gradual environmental changes on BVOCs have been studied extensively, insights in emission responses to repeated stress and recovery are widely absent. Therefore, we studied the dynamics of shoot gas exchange and BVOC emissions in Pinus halepensis seedlings during an induced moderate drought, two four-day-long heatwaves, and the combination of drought and heatwaves. We found clear stress-specific responses of BVOC emissions. Reductions in acetone emissions with declining soil water content and transpiration stood out as a clear drought indicator. All other measured BVOC emissions responded exponentially to rising temperatures during heat stress (maximum of 43 °C), but monoterpenes and methyl salicylate showed a reduced temperature sensitivity during the second heatwave. We found that these decreases in monoterpene emissions between heatwaves were not reflected by similar declines in their internal storage pools. Because stress intensity was extremely severe, most of the seedlings in the heat-drought treatment died at the end of the second heatwave (dark respiration ceased). Interestingly, BVOC emissions (methanol, monoterpenes, methyl salicylate, and acetaldehyde) differed between dying and surviving seedlings, already well before indications of a reduced vitality became visible in gas exchange dynamics. In summary, we could clearly show that the dynamics of BVOC emissions are sensitive to stress type, stress frequency, and stress severity. Moreover, we found indications that stress-induced seedling mortality was preceded by altered methanol, monoterpene, and acetaldehyde emission dynamics.

Leaf-level metabolic changes in response to drought affect daytime CO2 emission and isoprenoid synthesis pathways.

In the near future, climate change will cause enhanced frequency and/or severity of droughts in terrestrial ecosystems, including tropical forests. Drought responses by tropical trees may affect their carbon use, including production of volatile organic compounds (VOCs), with implications for carbon cycling and atmospheric chemistry that are challenging to predict. It remains unclear how metabolic adjustments by mature tropical trees in response to drought will affect their carbon fluxes associated with daytime CO2 production and VOC emission. To address this gap, we used position-specific 13C-pyruvate labeling to investigate leaf CO2 and VOC fluxes from four tropical species before and during a controlled drought in the enclosed rainforest of Biosphere 2 (B2). Overall, plants that were more drought-sensitive had greater reductions in daytime CO2 production. Although daytime CO2 production was always dominated by non-mitochondrial processes, the relative contribution of CO2 from the tricarboxylic acid cycle tended to increase under drought. A notable exception was the legume tree Clitoria fairchildiana R.A. Howard, which had less anabolic CO2 production than the other species even under pre-drought conditions, perhaps due to more efficient refixation of CO2 and anaplerotic use for amino acid synthesis. The C. fairchildiana was also the only species to allocate detectable amounts of 13C label to VOCs and was a major source of VOCs in B2. In C. fairchildiana leaves, our data indicate that intermediates from the mevalonic acid (MVA) pathway are used to produce the volatile monoterpene trans-ß-ocimene, but not isoprene. This apparent crosstalk between the MVA and methylerythritol phosphate pathways for monoterpene synthesis declined with drought. Finally, although trans-ß-ocimene emissions increased under drought, it was increasingly sourced from stored intermediates and not de novo synthesis. Unique metabolic responses of legumes may play a disproportionate role in the overall changes in daytime CO2 and VOC fluxes in tropical forests experiencing drought.

Qualitative and quantitative characterization of volatile organic compound emissions from cut grass.

Mechanical wounding of plants triggers the release of a blend of reactive biogenic volatile organic compounds (BVOCs). During and after mowing and harvesting of managed grasslands, significant BVOC emissions have the potential to alter the physical and chemical properties of the atmosphere and lead to ozone and aerosol formation with consequences for regional air quality. We show that the amount and composition of BVOCs emitted per unit dry weight of plant material is comparable between laboratory enclosure measurements of artificially severed grassland plant species and in situ ecosystem-scale flux measurements above a temperate mountain grassland during and after periodic mowing and harvesting. The investigated grassland ecosystem emitted annually up to 130 mg carbon m(-2) in response to cutting and drying, the largest part being consistently represented by methanol and a blend of green leaf volatiles (GLV). In addition, we report the plant species-specific emission of furfural, terpenoid-like compounds (e.g., camphor), and sesquiterpenes from cut plant material, which may be used as tracers for the presence of given plant species in the ecosystem.

Ecosystem fluxes during drought and recovery in an experimental forest.

Severe droughts endanger ecosystem functioning worldwide. We investigated how drought affects carbon and water fluxes as well as soil-plant-atmosphere interactions by tracing 13CO2 and deep water 2H2O label pulses and volatile organic compounds (VOCs) in an enclosed experimental rainforest. Ecosystem dynamics were driven by different plant functional group responses to drought. Drought-sensitive canopy trees dominated total fluxes but also exhibited the strongest response to topsoil drying. Although all canopy-forming trees had access to deep water, these reserves were spared until late in the drought. Belowground carbon transport was slowed, yet allocation of fresh carbon to VOCs remained high. Atmospheric VOC composition reflected increasing stress responses and dynamic soil-plant-atmosphere interactions, potentially affecting atmospheric chemistry and climate feedbacks. These interactions and distinct functional group strategies thus modulate drought impacts and ecosystem susceptibility to climate change.

Source partitioning of atmospheric methane using stable carbon isotope measurements in the Reuss Valley, Switzerland.

Measurements of methane ( CH4 ) mole fractions and δ13 C-CH4 that resolve the diel cycle in the agriculturally dominated Reuss Valley, Switzerland, were used to quantify the contributions of different CH4 sources to the atmospheric CH4 source mix. Both a nocturnal (NBL) and a diurnal convective boundary layer (CBL) approach were employed. A diel course of CH4 mole fractions was found with a daytime minimum (background around 1900 ppb) and a nocturnal maximum (up to 3500 ppb). The δ13 C value in CH4 only showed small variations during the day (9-21 hours CET, -45.0±0.2 ‰ mean±SE ) when the atmosphere was well mixed, but decreased by -4.8±0.1 ‰ during the night. Biogenic emissions dominated in both approaches (ranging from 60 to 94%), but non-biogenic sources were rather important (42.2% and 46.0% with CBL, 5.8% and 40% with NBL approach in 2011 and 2012, respectively, of total emissions). The CH4 sink, dominated by tropospheric OH oxidation and only to a minor extend by soil surface uptake, was quantified at roughly 4% of local emissions.

Biotic, abiotic and management controls on methanol exchange above a temperate mountain grassland.

Methanol (CH3OH) fluxes were quantified above a managed temperate mountain grassland in the Stubai Valley (Tyrol, Austria) during the growing seasons 2008 and 2009. Half-hourly methanol fluxes were calculated by means of the virtual disjunct eddy covariance (vDEC) method using 3-dimensional wind data from a sonic anemometer and methanol volume mixing ratios measured with a proton-transfer-reaction mass spectrometer (PTR-MS). During (undisturbed) mature and growing phases methanol fluxes exhibited a clear diurnal cycle with close-to-zero fluxes during nighttime and emissions, up to 10 nmol m-2 s-1, which followed the diurnal course of radiation and air temperature. Management events were found to represent the largest perturbations of methanol exchange at the studied grassland ecosystem: Peak emissions of 144.5 nmol m-2 s-1 were found during/after cutting of the meadow reflecting the wounding of the plant material and subsequent depletion of the leaf internal aqueous methanol pools. After the application of organic fertilizer, elevated methanol emissions of up to 26.7 nmol m-2 s-1 were observed, likely reflecting enhanced microbial activity associated with the applied manure. Simple and multiple linear regression analyses revealed air temperature and radiation as the dominant abiotic controls, jointly explaining 47 % and 70 % of the variability in half-hourly and daily methanol fluxes. In contrast to published leaf-level laboratory studies, the surface conductance and the daily change in the amount of green plant area, used as ecosystem-scale proxies for stomatal conductance and growth, respectively, were found to exert only minor biotic controls on methanol exchange.