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-β-ocimene, but not isoprene. This apparent crosstalk between the MVA and methylerythritol phosphate pathways for monoterpene synthesis declined with drought. Finally, although trans-β-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

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 H-2 : H-1 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 C-13-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 parts per thousand less H-2-enriched relative to phytol than sterols in other plants. These results provide the first experimental evidence that incorporation of plastidial intermediates reduces H-2/H-1 ratios of sterols. This suggests that relative offsets between the H-2 : H-1 ratios of sterols and phytol can trace exchange between the two isoprenoid synthesis pathways

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 H-2 : H-1 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 C-13-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 parts per thousand less H-2-enriched relative to phytol than sterols in other plants. These results provide the first experimental evidence that incorporation of plastidial intermediates reduces H-2/H-1 ratios of sterols. This suggests that relative offsets between the H-2 : H-1 ratios of sterols and phytol can trace exchange between the two isoprenoid synthesis pathways

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

Abstract Nonstructural 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 belowground. Drought effects were more pronounced in conservative canopy trees with limited supply of new photosynthates and relatively constant water-status than 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

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 13 C-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

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