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.

Effects of drought and recovery on soil volatile organic compound fluxes in an experimental rainforest.

Drought can affect the capacity of soils to emit and consume biogenic volatile organic compounds (VOCs). Here we show the impact of prolonged drought followed by rewetting and recovery on soil VOC fluxes in an experimental rainforest. Under wet conditions the rainforest soil acts as a net VOC sink, in particular for isoprenoids, carbonyls and alcohols. The sink capacity progressively decreases during drought, and at soil moistures below ~19%, the soil becomes a source of several VOCs. Position specific 13C-pyruvate labeling experiments reveal that soil microbes are responsible for the emissions and that the VOC production is higher during drought. Soil rewetting induces a rapid and short abiotic emission peak of carbonyl compounds, and a slow and long biotic emission peak of sulfur-containing compounds. Results show that, the extended drought periods predicted for tropical rainforest regions will strongly affect soil VOC fluxes thereby impacting atmospheric chemistry and climate.

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.

Uncovering the dominant role of root metabolism in shaping rhizosphere metabolome under drought in tropical rainforest plants.

Plant-soil-microbe interactions are crucial for driving rhizosphere processes that contribute to metabolite turnover and nutrient cycling. With the increasing frequency and severity of water scarcity due to climate warming, understanding how plant-mediated processes, such as root exudation, influence soil organic matter turnover in the rhizosphere is essential. In this study, we used 16S rRNA gene amplicon sequencing, rhizosphere metabolomics, and position-specific 13C-pyruvate labeling to examine the effects of three different plant species (Piper auritum, Hibiscus rosa sinensis, and Clitoria fairchildiana) and their associated microbial communities on soil organic carbon turnover in the rhizosphere. Our findings indicate that in these tropical plants, the rhizosphere metabolome is primarily shaped by the response of roots to drought rather than direct shifts in the rhizosphere bacterial community composition. Specifically, the reduced exudation of plant roots had a notable effect on the metabolome of the rhizosphere of P. auritum, with less reliance on neighboring microbes. Contrary to P. auritum, H. rosa sinensis and C. fairchildiana experienced changes in their exudate composition during drought, causing alterations to the bacterial communities in the rhizosphere. This, in turn, had a collective impact on the rhizosphere's metabolome. Furthermore, the exclusion of phylogenetically distant microbes from the rhizosphere led to shifts in its metabolome. Additionally, C. fairchildiana appeared to be associated with only a subset of symbiotic bacteria under drought conditions. These results indicate that plant species-specific microbial interactions systematically change with the root metabolome. As roots respond to drought, their associated microbial communities adapt, potentially reinforcing the drought tolerance strategies of plant roots. These findings have significant implications for maintaining plant health and preference during drought stress and improving plant performance under climate change.

Drought re-routes soil microbial carbon metabolism towards emission of volatile metabolites in an artificial tropical rainforest.

Drought impacts on microbial activity can alter soil carbon fate and lead to the loss of stored carbon to the atmosphere as CO2 and volatile organic compounds (VOCs). Here we examined drought impacts on carbon allocation by soil microbes in the Biosphere 2 artificial tropical rainforest by tracking 13C from position-specific 13C-pyruvate into CO2 and VOCs in parallel with multi-omics. During drought, efflux of 13C-enriched acetate, acetone and C4H6O2 (diacetyl) increased. These changes represent increased production and buildup of intermediate metabolites driven by decreased carbon cycling efficiency. Simultaneously,13C-CO2 efflux decreased, driven by a decrease in microbial activity. However, the microbial carbon allocation to energy gain relative to biosynthesis was unchanged, signifying maintained energy demand for biosynthesis of VOCs and other drought-stress-induced pathways. Overall, while carbon loss to the atmosphere via CO2 decreased during drought, carbon loss via efflux of VOCs increased, indicating microbially induced shifts in soil carbon fate.

Deep roots mitigate drought impacts on tropical trees despite limited quantitative contribution to transpiration.

Deep rooting is considered a central drought-mitigation trait with vast impact on ecosystem water cycling. Despite its importance, little is known about the overall quantitative water use via deep roots and dynamic shifts of water uptake depths with changing ambient conditions. Knowledge is especially sparse for tropical trees. Therefore, we conducted a drought, deep soil water labeling and re-wetting experiment at Biosphere 2 Tropical Rainforest. We used in situ methods to determine water stable isotope values in soil and tree water in high temporal resolution. Complemented by soil and stem water content and sap flow measurements we determined percentages and quantities of deep-water in total root water uptake dynamics of different tree species. All canopy trees had access to deep-water (max. uptake depth 3.3 m), with contributions to transpiration ranging between 21 % and 90 % during drought, when surface soil water availability was limited. Our results suggest that deep soil is an essential water source for tropical trees that delays potentially detrimental drops in plant water potentials and stem water content when surface soil water is limited and could hence mitigate the impacts of increasing drought occurrence and intensity as a consequence of climate change. Quantitatively, however, the amount of deep-water uptake was low due to the trees' reduction of sap flow during drought. Total water uptake largely followed surface soil water availability and trees switched back their uptake depth dynamically, from deep to shallow soils, following rainfall. Total transpiration fluxes were hence largely driven by precipitation input.

Chiral monoterpenes reveal forest emission mechanisms and drought responses.

Monoterpenes (C10H16) are emitted in large quantities by vegetation to the atmosphere (>100 TgC year-1), where they readily react with hydroxyl radicals and ozone to form new particles and, hence, clouds, affecting the Earth's radiative budget and, thereby, climate change1-3. Although most monoterpenes exist in two chiral mirror-image forms termed enantiomers, these (+) and (-) forms are rarely distinguished in measurement or modelling studies4-6. Therefore, the individual formation pathways of monoterpene enantiomers in plants and their ecological functions are poorly understood. Here we present enantiomerically separated atmospheric monoterpene and isoprene data from an enclosed tropical rainforest ecosystem in the absence of ultraviolet light and atmospheric oxidation chemistry, during a four-month controlled drought and rewetting experiment7. Surprisingly, the emitted enantiomers showed distinct diel emission peaks, which responded differently to progressive drying. Isotopic labelling established that vegetation emitted mainly de novo-synthesized (-)-α-pinene, whereas (+)-α-pinene was emitted from storage pools. As drought progressed, the source of (-)-α-pinene emissions shifted to storage pools, favouring cloud formation. Pre-drought mixing ratios of both α-pinene enantiomers correlated better with other monoterpenes than with each other, indicating different enzymatic controls. These results show that enantiomeric distribution is key to understanding the underlying processes driving monoterpene emissions from forest ecosystems and predicting atmospheric feedbacks in response to climate change.

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.

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.

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.

Hydrogen isotope response to changing salinity and rainfall in Australian mangroves.

Hydrogen isotope ratios ((2) H/(1) H, δ(2) H) of leaf waxes covary with those in precipitation and are therefore a useful paleohydrologic proxy. Mangroves are an exception to this relationship because their δ(2) H values are also influenced by salinity. The mechanisms underlying this response were investigated by measuring leaf lipid δ(2) H and leaf and xylem water δ(2) H and δ(18) O values from three mangrove species over 9.5 months in a subtropical Australian estuary. Net (2) H/(1) H fractionation between surface water and leaf lipids decreased by 0.5-1.0‰ ppt(-1) for n-alkanes and 0.4-0.8‰ ppt(-1) for isoprenoids. Xylem water was (2) H depleted relative to surface water, reflecting (2) H discrimination of 4-10‰ during water uptake at all salinities and opportunistic uptake of freshwater at high salinity. However, leaf water (2) H enrichment relative to estuary water was insensitive to salinity and identical for all species. Therefore, variations in leaf and xylem water δ(2) H values cannot explain the salinity-dependent (2) H depletion in leaf lipids, nor the 30‰ range in leaf lipid δ(2) H values among species. Biochemical changes in direct response to salt stress, such as increased compatible solute production or preferential use of stored carbohydrates, and/or the timing of lipid production and subsequent turnover rates, are more likely causes.

Holocene closure of Lib Pond, Marshall Islands.

Well-preserved sediment from closed water bodies of atolls such as Lib Pond are rare opportunities to reconstruct the past regional climate, which pieced together across a latitude and longitude range identify the range of movement patterns of wider scale climate phenomena such as the Intertropical Convergence Zone (ITCZ) and El Niño Southern Oscillation (ENSO). We conducted the first physico-chemical survey of Lib Pond, a shallow, closed-water saline lake located on remote and difficult to access Lib Island in the Marshall Islands at 8° 18' 48.99″ N, 167 22' 51.90″ E in the Pacific Ocean, in July 2009. We performed a bathymetric survey, recorded salinity, dissolved oxygen, pH, and temperature profiles, monitored the tidal variability, and conducted a vegetation survey surrounding the lake. From bathymetric data we calculated the lake volume, which we used to estimate the lake's salt budget, and ultimately the residence time of water in the lake basin. We took a series of sediment cores from the lake, cores which indicate Lib Island's changing environment and climate. Radiocarbon measurements determined sediment age, and reveal significant mixing over the last 2 ka of deposition. We conclude that prior to 3 ka, Lib Island was an atoll with a central lagoon connected to the open ocean, which was then closed off from the open ocean to form the brackish system that exists today. We predict that the sediment accumulation in Lib Pond evident today will continue. As seawater is inhibited from exchanging with fresh water, Lib Pond will become a shallower lake with increasingly fresh water.