The emission of isoprene from plants was first discovered in the 1950s but was relatively unknown in the plant science community until the 1990s. Isoprene is the five-carbon branched molecule that is the root member of the extensive family of isoprenoids. The amount of isoprene from plants exceeds all other hydrocarbon inputs to the atmosphere. Plant-emitted isoprene can affect ozone formation and often increases (but can decrease) growth of aerosols (particles in the atmosphere). The rate of isoprene emission is estimated using empirical or mechanistic modeling has been used to predict global emissions. Beyond its atmospheric role, isoprene can improve plant resilience to biotic and abiotic stress, likely through interactions with transcriptional networks that regulate plant growth and defense. Isoprene has all the properties of the five compounds classically described as plant hormones. These and an additional four molecules are now called small molecule plant growth regulators, and we propose that isoprene also belongs to this group. Plants previously thought to lack the capacity for isoprene emission have now been found that make isoprene in response to leaf damage. This discovery suggests that many plants once classified as non-emitters likely have the capacity to emit isoprene under specific conditions. This review summarizes past and current understanding of the biosynthesis and regulatory mechanisms, atmospheric significance, and physiological roles of isoprene emitted from plants.

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of CO2, enabling fundamental processes in organisms across all domains of life. Among all CAs, the role of mitochondrial βCA remains poorly understood. Here, we identify a mitochondrial βCA, βCA6, as a key regulator of branched-chain amino acid (BCAA) catabolism and metabolic flexibility during carbon starvation in Arabidopsis thaliana. Loss of βCA6 triggers hypersensitivity to prolonged darkness, marked by accelerated Chl degradation, early senescence, impaired BCAA degradation, and disrupted carbon-nitrogen remobilization. Transcriptomic and metabolic profiling revealed elevated expression of BCAA catabolic enzymes, as well as BCAA accumulation and reduced glutamate levels, indicating defective carbon-nitrogen remobilization. βca6 loss-of-function mutants exhibited a striking hypersensitivity to exogenous BCAAs, supporting a central role of βCA6 in BCAA homeostasis. These findings uncover a previously unrecognized function for mitochondrial CA in maintaining energy balance under dark stress. Given the evolutionary conservation of mitochondria and BCAA metabolism, our work highlights a broadly relevant mechanism by which eukaryotes integrate core metabolic pathways with environmental adaptation.

Abstract Background Leaf-level biogenic volatile organic compounds (BVOCs) emissions represent a major source of organic gases in the atmosphere, influencing both climate and air quality. These emissions are strongly driven by environmental perturbations, which affect individual plant- to ecosystem-level processes. Uncovering all the BVOCs and understanding how their emissions respond to altered environmental conditions provide critical insights into vegetation-driven changes in atmospheric chemistry. We developed a tandem instrumentation setup that integrates a proton transfer reaction time-of-flight mass spectrometer (PTR-ToF-MS) with parts-per-trillion detection limits and a photosynthetic infrared gas exchange system for the untargeted survey of all the BVOCs. This novel system enables simultaneous, real-time monitoring of BVOC emissions and photosynthetic parameters at the leaf level, offering new opportunities to disentangle the physiological and environmental drivers of VOC release. Furthermore, we established the VOC Analysis and Processing Optimization Resource (VAPOR), an open-access software tool designed for rapid data post-processing and the analysis of the variability of hundreds of BVOCs. We assessed the performance of the tandem system under varying background conditions, using standard gas mixtures and a range of environmental factors. Results Blank emissions were substantially lower for major BVOCs (e.g., isoprene) compared to those observed in plant emissions. Despite this, the observation of background-level VOCs highlights the importance of routinely acquiring and accounting for blank measurements in analyses using the coupled instrumentation. Introduction of known VOC concentrations to the system demonstrated a linear response across different compounds with varying molecular compositions, indicating minimal gas loss regardless of chemical moieties within the coupled instrumentation. We applied the optimized system to investigate the physiological mechanisms driving BVOC emissions across different genotypes of poplar and pennycress. The high mass resolution capabilities of the PTR-ToF-MS, coupled with comprehensive VAPOR-driven data analysis, enabled the identification of several important BVOCs, including methanol and methanethiol; these BVOCs displayed substantial variation across pennycress genotypes and showed concentrations ~ 100–350% higher than the blank. Moreover, isoprene emissions varied significantly among poplar genotypes grown in different potting media. Conclusions Tandem instrumentation offers a powerful tool for profiling volatile molecular markers and elucidating their genetic and environmental underpinnings. This approach enhances our ability to predict BVOC emissions in response to genotype by environmental interactions and contributes to a deeper understanding of vegetation responses to environmental changes.

Cells must continuously adjust metabolic output to maintain homeostasis under changing environmental conditions, yet the mechanisms that enable rapid and reversible control of pathway activity remain largely unknown. The methylerythritol phosphate (MEP) pathway, of bacterial origin and conserved in plastid-bearing eukaryotes, including plants and apicomplexan parasites, produces isoprenoid precursors essential for growth and stress adaptation. Here, we identify methylerythritol cyclodiphosphate (MEcPP) as a dual-function metabolite that serves both as a biosynthetic intermediate and a direct modulator of enzyme activity. Genetic perturbations and high light stress revealed step-specific MEcPP accumulation independent of transcriptional regulation. Biochemical and protease-protection assays showed that MEcPP destabilizes and inhibits methylerythritol cytidylyltransferase (MCT) while modestly stabilizing hydroxymethylbutenyl diphosphate synthase (HDS). Molecular docking analyses indicate that MEcPP interacts directly with the MCT catalytic site, displacing the natural substrate and thereby attenuating enzyme activity, suggesting a competitive, feedback-like mechanism of metabolic control. These results define MEcPP as a metabolic feedback signal that translates stress-induced changes into targeted enzymatic control. This mechanism illustrates how pathway intermediates dynamically coordinate biosynthetic activity with environmental cues, representing a broadly conserved strategy for metabolite-driven control of cellular metabolism.

Isoprene is the most abundant biogenic volatile organic compound emitted by terrestrial vegetation. Here we report the impact of isoprene on root-associated microbiomes. Using isoprene-emitting (IE) transgenic tobacco and isogenic non-emitting (NE) controls, we performed co-cultivation experiments in natural soil and analysed plant phenotypes and growth alongside bacterial and fungal communities across root, rhizosphere, and soil niches. NE plants co-cultivated with IE neighbours displayed increased shoot and root biomass, suggesting interactive belowground functions of isoprene. Amplicon sequencing revealed more growth-promoting microbiota in root and rhizosphere of IE plants than NE plants. Both bacterial and fungal growth-promoting microbiota were enriched in IE and NE plants grown in the same pot. However, isoprene-fumigated plant-free soils did not replicate these shifts, indicating that plant-microbe interactions are required for the modulation of the soil microbiome. Our results suggest that isoprene acts as a belowground cue influencing microbiome assembly and indirectly enhancing growth in neighbouring plants. This work uncovers a potential ecological role for isoprene, highlighting how plant-derived isoprene can mediate plant-plant-microbiome interactions and contribute to community-level processes in the rhizosphere.

The setting of apple (Malus x domestica Borkh.) fruitlets is primarily determined by their relative sink strength, which depends on the balance between carbohydrate supply and demand and the number of competing sinks. It is generally accepted that limits in carbohydrate supply predispose small fruitlets, i.e., weak sinks, to abscission. This study examined the relationship among carbohydrate status, fruitlet growth, and abscission in ‘Gala’ and ‘Honeycrisp’ over two seasons. Fruitlets were sampled every other day after chemical thinning applications according to their flower position in the cyme (king vs. lateral) and absolute fruitlet weights and compared to untreated controls. At each sample date, fruitlets were selected from three distinct size classes: 90th, 50th, and 10thpercentile of the sample population, each with an associated fruit set prediction according to the fruitlet size distribution model (FSDM). Soluble sugars (glucose, fructose, sucrose, sorbitol) and starch were quantified at each sample date. Small fruitlets (10th percentile) consistently exhibited reduced soluble sugar concentrations and relative growth rates prior to abscission, while larger fruitlets (50thand 90th percentiles) maintained higher sugar levels; thus, having greater sink strength and a higher retention probability. Sorbitol and sucrose concentrations were often dynamic and generally reflected modeled tree-level carbon balance, based on the MaluSim model; these sugars also declined markedly after chemical thinner applications were made indicating stress. Starch accumulation further distinguished stronger from weaker sinks, with exponential accumulation in 90thpercentile fruitlets under thinning stress. Positional effects were evident, as king fruitlets generally maintained higher sugar concentrations than laterals; however, this effect was confounded by king fruit generally being larger. When position was ignored, fruit weight could explain a fruitlet’s abscission fate. When the FSDM predictions were within 90% of the actual fruit set, fruitlets destined to abscise had markedly reduced concentrations of soluble sugars and lower growth rates than those predicted to be retained, before any visible signs of fruit abscission were apparent. These data underscore the physiological basis of abscission by highlighting the central role of carbohydrate availability and sink strength that determine fruitlet survival, supported by the FSD model

Volatile isoprenoids mainly include isoprene and monoterpenes, which improve the thermotolerance of the emitting plant by lowering reactive oxygen species (ROS) levels, preserving chloroplast membrane ultrastructure, maintaining photosynthesis and primary metabolism, inducing heat shock proteins, and preserving growth and development. Recent data showed that isoprenoids can act as signaling molecules to improve plant thermotolerance by altering related gene expression through Ca2+-mediated signaling pathways. To promote further understanding of isoprenoid-mediated thermotolerance mechanisms, we review current understanding of isoprenoid-induced plant thermotolerance, along with new findings describing the corresponding underlying mechanisms and putative signaling pathways. This information is beneficial for the potential utilization of isoprenoids for enhancing crop tolerance to global warming either by enhancing the emission of isoprenoids or by using isoprenoid-inspired anti-high temperature agents.

In leaves, major CO₂ fluxes alternate between fixation by the Calvin-Benson-Bassham (CBB) cycle during light and release by the tricarboxylic acid (TCA) cycle in darkness. The speed at which leaf metabolism transitions between these pathways likely influences plant tolerance to fluctuating light conditions. To investigate these rapid metabolic shifts, we exposed leaves to ¹³CO₂ for 20 min to establish a quasi-steady state before abruptly turning off the light while maintaining ¹³CO₂ feeding. Within 10 s of dark transition, 3-phosphoglycerate levels rose significantly while most other CBB cycle intermediates decreased by more than 90

Understanding plant responses to the environment is based on research performed across several scales and subdisciplines. However, the interpretation and repeatability of experimental results depend on careful reporting of experimental procedures and environmental conditions. These conditions include light intensity and quality, temperature, relative humidity and vapor pressure deficit, soil water potential or volumetric water content, and pot size, which interact on plant physiological responses across biological and experimental scales regardless of whether they are the focus of the experiment. To ascertain how effectively and consistently these conditions are reported, we reviewed more than 200 plant science research articles on vascular plants published from 2020 through 2024. Environmental condition data were often not reported, including cases where the specific environmental variable was the focus of the study. This situation hampers both replicability and interpretability of results and hinders progress in understanding plant physiological responses across subdisciplines. The Environmental and Ecological Plant Physiology section of the American Society of Plant Biologists recommends several best practices to measure and report environmental conditions in plant physiology experiments, such as measuring and reporting actual environmental conditions, especially of control variables, to enable replication and comparative interpretation among experiments. These guidelines can aid authors in experimental design and manuscript preparation and assist reviewers in evaluating submitted manuscripts. Following such guidelines will enhance the dynamic progress of sound plant science within our community by improving replicability and enabling cross-disciplinary interpretation of results.

Extreme heat events are increasingly common, and if these align with pollen development, they can alter pollen nutrient composition. However, no studies have examined how the timing of heat relative to bud development affects the role of pollen in plant pollination and bee health. To explore this, we exposed highbush blueberry plants to extreme heat (37.5 °C) or normal (25 °C) conditions for 4 h across several floral bud stages. Pollen was analyzed for protein, carbohydrate, and amino acid content. We found that blueberry floral buds vary in their sensitivity to heat, with bud swell being the most heat-sensitive developmental stage with significant reductions in pollen protein, total and several individual amino acids. When pollen from blueberry plants exposed to the same conditions was fed to Osmia lignaria larvae, we found that individuals fed heat-stressed pollen were 7 times more likely to die compared to those fed non-stressed pollen. Blueberry flowers exposed to the same conditions were used for a hand pollination study, where we observed a 39% reduction in fruit set following heat stress at bud swell. This study reveals how extreme heat can disrupt both plant pollination and bee survival through changes in pollen nutritional composition.

Isoprene is the most abundant nonmethane biogenic hydrocarbon emitted by some plants, mostly trees. It plays critical roles in atmospheric chemistry by contributing to ozone and aerosol formation. Isoprene also benefits plants, particularly under stress, through its signaling roles. Legume crops like soybean were thought to have evolutionarily lost isoprene synthase (ISPS) and are typically considered nonemitters. Here, we report that damage to soybean leaves by wounding or burning triggered a burst of isoprene emission from the undamaged part of the leaves. In silico analysis identified intact ISPS genes in the soybean genome, with features similar to known ISPSs. Protein made from these gene sequences catalyzed isoprene production in the presence of dimethylallyl diphosphate. Isoprene emission in soybeans was linked to reduced photosynthesis rates and stomatal conductance. Metabolomic analysis showed that leaf damage caused a surge in glyceraldehyde 3-phosphate and pyruvate levels, leading to an increase of most of the methylerythritol 4-phosphate pathway metabolites.

During photosynthesis, CO₂ uptake is counterbalanced by concurrent CO₂-releasing processes, complicating the interpretation of gas exchange measurements. While photorespiration accounts for a significant portion of this CO₂ release, emerging evidence indicates that there are additional metabolic pathways that release CO2 during photosynthesis. This metabolism-termed day respiration (often Rd) or respiration in the light (RL)-is now recognized as an independent and significant source of CO2 emission during photosynthesis. Here we revisit classical models of photosynthesis and incorporate new insights from isotopic labeling and metabolic flux analysis (MFA) to investigate the biochemical basis of RL. We identified the cytosolic glucose-6-phosphate (G6P) shunt through the oxidative pentose phosphate pathway (OPPP) as the predominant contributor to RL. This shunt explains some long-standing anomalies in Calvin-Benson-Bassham (CBB) cycle labeling. Under non-stressed conditions, RL remains stable across varying CO₂ concentrations and light intensities. Under heat stress, RL shifts toward a plastidial source. Together, these findings resolve longstanding questions about carbon flux during photosynthesis and improve our understanding of RL by explaining its metabolic origin, physiological significance in carbon balance during photosynthesis, and regulation under varying environmental conditions.

Isoprene, emitted by some plants, deters insect herbivory. However, the associated biochemical and physiological responses that confer herbivory resistance remain unknown. We used engineered isoprene-emitting (IE) and non-emitting (NE) control tobacco plants to interpret isoprene-mediated defense against herbivory in plants. Hornworm larvae raised on IE plants exhibited stunted growth compared to those raised on NE plants. Worms preferred to feed on NE rather than IE leaves, indicating deterrent effects of isoprene on insect feeding. Worm feeding induced a greater increase in jasmonic acid (JA), a crucial hormone for insect resistance, in IE leaves compared to that in NE leaves. Assimilation rates were stably maintained in IE plants, suggesting a protective role of isoprene in preserving photosynthetic efficiency during insect herbivory. Wound-induced increase in isoprene emission correlated with the elevation of key metabolites of the isoprene biosynthesis pathway. Our results highlight JA-priming functions of isoprene and provide insights into isoprene-mediated defense against insect herbivory.

In the future, plants may encounter increased light and elevated CO 2 levels. How consequent alterations in photosynthetic rates will impact fluxes in photosynthetic carbon metabolism remains uncertain. Respiration in light ( R L ) is pivotal in plant carbon balance and a key parameter in photosynthesis models. Understanding the dynamics of photosynthetic metabolism and R L under varying environmental conditions is essential for optimizing plant growth and agricultural productivity. However, measuring R L under high light and high CO 2 (HLHC) conditions poses challenges using traditional gas exchange methods. In this study, we employed isotopically nonstationary metabolic flux analysis (INST-MFA) to estimate R L and investigate photosynthetic carbon flux, unveiling nuanced adjustments in Camelina sativa under HLHC. Despite numerous flux alterations in HLHC, R L remained stable. HLHC affects several factors influencing R L , such as starch and sucrose partitioning, v o / v c ratio, triose phosphate partitioning, and hexose kinase activity. Analysis of A/C i curve operational points reveals that HLHC's major changes primarily stem from CO 2 suppressing photorespiration. Integration of these fluxes into a simplified model predicts changes in CBC labeling under HLHC. This study extends our prior discovery that incomplete CBC labeling is due to unlabeled carbon reimported during R L , offering insights into manipulating labeling through adjustments in photosynthetic rates.

Building sustainable platforms to produce biofuels and specialty chemicals has become an increasingly important strategy to supplement and replace fossil fuels and petrochemical-derived products. Terpenoids are the most diverse class of natural products which have many commercial roles as specialty chemicals. Poplar is a fast growing, biomass dense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce large amounts of other terpenes. Here we aimed to engineer poplar with optimized pathways to produce squalene, a triterpene commonly used in cosmetic oils, a potential biofuel candidate, and the precursor to the further diversified classes of triterpenoids and sterols. The squalene production pathways were either re-targeted from the cytosol to plastids or co-produced with lipid droplets in the cytosol. Squalene and lipid droplet co-production appeared to be toxic, which we hypothesize to be due to disruption of adventitious root formation, suggesting a need for tissue specific production. Plastidial squalene production enabled up to 0.63 mg/g fresh weight in leaf tissue, which also resulted in reductions in isoprene emission and photosynthesis. These results were also studied through a technoeconomic analysis, providing further insight into developing poplar as a production host.

Triose phosphate utilization (TPU) limitation is one of the three biochemical limitations of photosynthetic CO2 assimilation rate in C3 plants. Under TPU limitation, abrupt and large transitions in light intensity cause damped oscillations in photosynthesis. When plants are salt-stressed, photosynthesis is often down-regulated particularly under dynamic light intensity, but how salt stress affects TPU-related dynamic photosynthesis is still unknown. To elucidate this, tomato (Solanum lycopersicum) was grown with and without sodium chloride (NaCl, 100 mM) stress for 13 d. Under high CO2 partial pressure, rapid increases in light intensity caused profound photosynthetic oscillations. Salt stress reduced photosynthetic oscillations in leaves initially under both low- and high-light conditions and reduced the duration of oscillations by about 2 min. Besides, salt stress increased the threshold for CO2 partial pressure at which oscillations occurred. Salt stress increased TPU capacity without affecting Rubisco carboxylation and electron transport capacity, indicating the up-regulation of end-product synthesis capacity in photosynthesis. Thus salt stress may reduce photosynthetic oscillations by decreasing leaf internal CO2 partial pressure and/or increasing TPU capacity. Our results provide new insights into how salt stress modulates dynamic photosynthesis as controlled by CO2 availability and end-product synthesis.

Isotopically nonstationary metabolic flux analysis (INST-MFA) is a powerful technique for studying plant central metabolism, which involves introducing a 13CO2 tracer to plant leaves and sampling the labeled metabolic intermediates during the transient period before reaching an isotopic steady state. The metabolic intermediates involved in the C3 cycle have exceptionally fast turnover rates, with some intermediates turning over many times a second. As a result, it is necessary to rapidly introduce the label and then rapidly quench the plant tissue to determine concentrations in the light or capture the labeling kinetics of these intermediates at early labeling time points. Here, we describe a rapid quenching (0.1-0.5 s) system for 13CO2 labeling experiments in plant leaves to minimize metabolic changes during labeling and quenching experiments. This system is integrated into a commercially available gas exchange analyzer to measure initial rates of gas exchange, precisely control ambient conditions, and monitor the conversion from 12CO2 to 13CO2.

Photorespiration is an essential process related to photosynthesis that is initiated following the oxygenation reaction catalyzed by rubisco, the initial enzyme of the Calvin-Benson-Bassham cycle. This reaction produces an inhibitory intermediate that is recycled back into the Calvin-Benson-Bassham cycle by photorespiration which requires the use of energy and the release of a portion of the carbon as CO2. The energy use and CO2 release of canonical photorespiration form a foundation for biochemical models used to describe and predict leaf carbon exchange and energy use (ATP and NAPDH). The ATP and NADPH demand of canonical photorespiration is thought to be different than that of the Calvin-Benson-Bassham cycle, requiring increased flexibility in the ratio of ATP and NADPH from the light reactions. Photorespiration requires many reactions across the chloroplasts, mitochondria and peroxisomes and involves many intermediates. Growing evidence indicates that these intermediates do not all stay in photorespiration as typically assumed and instead feed into other aspects of metabolism and leave as glycine, serine, and methylene-THF. Here we discuss how alternative flux through and from canonical photorespiration alters the ATP and NADPH requirements of metabolism following rubisco oxygenation using additional derivations of biochemical models of leaf photosynthesis and energetics. Using these new derivations, we determine that the ATP and NADPH demands of photorespiration are highly sensitive to alternative flux in ways that fundamentally changes how photorespiration contributes to the ratio of total ATP and NADPH demand. Specifically, alternative flows of carbon through photorespiration could reduce ATP and NADPH demand ratio to values below what is produced from linear electron transport.

Isoprene, a volatile hydrocarbon, is typically emitted from the leaves of many plant species. Given its well-known function in plant growth and defense aboveground, we examined its effects on root physiology. We used isoprene-emitting (IE) lines and a non-emitting (NE) line of Arabidopsis and investigated their performance by analyzing root phenotype, hormone levels, transcriptome, and metabolite profiles under both normal and salt stress conditions. We show that IE lines emitted tiny amounts of isoprene from roots and showed an increased root/shoot ratio compared with NE line. Isoprene emission exerted a noteworthy influence on hormone profiles related to plant growth and stress response, promoting root development and salt-stress resistance. Methyl erythritol 4-phosphate pathway metabolites, precursors of isoprene and hormones, were higher in the roots of IE lines than in the NE line. Transcriptome data indicated that the presence of isoprene increased the expression of key genes involved in hormone metabolism/signaling. Our findings reveal that constitutive root isoprene emission sustains root growth under saline conditions by regulating and/or priming hormone biosynthesis and signaling mechanisms and expression of key genes relevant to salt stress defense.