Limonene emission in transgenic tobacco downregulates foliar ascorbate peroxidase activity under drought

Limonene emission in transgenic tobacco downregulates foliar ascorbate peroxidase activity under drought | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 18 November 2025 V1 Latest version Share on Limonene emission in transgenic tobacco downregulates foliar ascorbate peroxidase activity under drought Authors : Hao Zhou 0000-0002-7010-8899 , Christoph-Martin Geilfus , Axel Mithöfer 0000-0001-5229-6913 , In-Cheol Jang 0000-0001-9408-4273 , Kirsti Ashworth , and Ian Dodd [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176344437.73520858/v1 296 views 216 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Drought stress alters the production of volatile organic compounds such as monoterpenes, but their role in regulating plant stress responses is unclear. Wild-type (WT) tobacco plants that lack monoterpene production, and transgenic plants that upregulated (−)-limonene (LG12), myrcene (MG1), or (−)-α/β-pinene (PG11) production, were grown in different chambers (to avoid non-target monoterpene effects) and allowed to dry the soil. Drought initially increased monoterpene emissions, which subsequently declined with prolonged stress. Despite maintaining higher leaf water potential and turgor than WT plants, transgenic plants had lower biomass and leaf area. Although soil drying enhanced foliar hydrogen peroxide (H 2 O 2 ) content and superoxide dismutase (SOD) activity similarly in all genotypes, (–)-limonene production downregulated ascorbate peroxidase (APX) activity, and increased malondialdehyde (MDA) and ABA concentrations under drought conditions compared to WT plants. Re-watering decreased oxidative damage within 24 hours, attenuated ABA concentrations and increased APX activity of LG12, and allowed similar seed production between genotypes. Monoterpene biosynthesis and emission modulate drought tolerance by affecting leaf water and oxidative status, but don’t alter photosynthetic processes. In species that naturally produce monoterpenes, understanding whether monoterpene biosynthesis and emission interact with native signalling pathways to regulate hormonal and antioxidant status seems warranted. Title: Limonene emission in transgenic tobacco downregulates foliar ascorbate peroxidase activity under drought Hao Zhou¹, Christoph-Martin Geilfus 2 , Axel Mithöfer 3 , In-Cheol Jang 4 , Kirsti Ashworth¹, Ian C. Dodd¹* ¹ Lancaster Environment Centre, Library Avenue, Lancaster University, Lancaster, LA1 4YQ, UK 2 Department of Soil Science and Plant Nutrition, Hochschule Geisenheim University, 65366 Geisenheim, Germany 3 Research Group Plant Defense Physiology, Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany 4 Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore *Corresponding author: [email protected] Date of submission: 16/11/2025 The number of tables: 0 The number of figures: 8 Word count: 6,915 Abstract Drought stress alters the production of volatile organic compounds such as monoterpenes, but their role in regulating plant stress responses is unclear. Wild-type (WT) tobacco plants that lack monoterpene production, and transgenic plants that upregulated (−)-limonene (LG12), myrcene (MG1), or (−)-α/β-pinene (PG11) production, were grown in different chambers (to avoid non-target monoterpene effects) and allowed to dry the soil. Drought initially increased monoterpene emissions, which subsequently declined with prolonged stress. Despite maintaining higher leaf water potential and turgor than WT plants, transgenic plants had lower biomass and leaf area. Although soil drying enhanced foliar hydrogen peroxide (H 2 O 2 ) content and superoxide dismutase (SOD) activity similarly in all genotypes, (–)-limonene production downregulated ascorbate peroxidase (APX) activity, and increased malondialdehyde (MDA) and ABA concentrations under drought conditions compared to WT plants. Re-watering decreased oxidative damage within 24 hours, attenuated ABA concentrations and increased APX activity of LG12, and allowed similar seed production between genotypes. Monoterpene biosynthesis and emission modulate drought tolerance by affecting leaf water and oxidative status, but don’t alter photosynthetic processes. In species that naturally produce monoterpenes, understanding whether monoterpene biosynthesis and emission interact with native signalling pathways to regulate hormonal and antioxidant status seems warranted. Introduction Terpenes are the largest and most structurally diverse class of plant specialised metabolites. These compounds are essential for a wide range of biological and ecological functions, including growth regulation and defence against biotic and abiotic stressors (Possell and Loreto, 2013). Among naturally occurring terpenes, monoterpenes (C 10 H 16 ) form the second-largest class and play significant roles in tropospheric chemistry and ecology (Byron et al. , 2022). They are synthesised via the plastidial methylerythritol phosphate (MEP) pathway, which converts pyruvate and glyceraldehyde-3-phosphate into the universal C₅ precursors isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), that are subsequently condensed by geranyl diphosphate synthase (GPPS) and cyclised by monoterpene synthases (TPSs) to form diverse monoterpene structures. They mediate plant responses to environmental conditions such as drought, high temperatures, and oxidative stress (Vickers et al. , 2009a; Ryan et al. , 2014; Perez-Gil et al. , 2024). Emissions of terpenes from vegetation account for approximately 0.1-2% of photosynthetically assimilated carbon, with environmental stresses causing higher emissions (5-20%) in species like poplar (Possell and Loreto, 2013). Thus, it is reasonable to hypothesise that these volatile compounds contribute beneficially to plant biology and ecosystem functioning (Sharkey et al., 2007). While monoterpene emissions respond to light and temperature and protect against these stress conditions (Niinemets et al. , 2002; Niinemets et al. , 2004; Jardine et al. , 2017; Xu et al. , 2022; Zuo et al. , 2025), their response to drought and role in drought tolerance are less understood. Drought triggers physiological and biochemical changes that strongly modulate terpene production and emissions. Typically, monoterpene emissions rise under mild to moderate drought and decline once stress intensifies (Loreto and Schnitzler, 2010). For instance, α/β-pinene concentrations in a tropical rainforest peaked threefold at 20% volumetric soil moisture before falling below 15% (Byron et al. , 2022), while in Quercus ilex a 26% reduction in soil moisture increased α-pinene and limonene emissions by 60% and 166%, respectively, but a further 5% soil moisture decrease reduced them by 20% (Mu et al. , 2018). Mediterranean species such as Cistus albidus , Q. coccifera and Pinus halepensis showed a tenfold rise in emissions as leaf water potential (Ψ leaf ) declined, yet severe water deficit (Ψ leaf < -6 MPa) caused a threefold drop (Ormeño et al. , 2007). However, these thresholds vary with soil texture and with plant available water capacity, which makes cross study comparisons difficult, and their physiological significance remains uncertain because emissions are often decoupled from gas exchange (Kreuzwieser et al. , 2021). Mechanistically, emission may shift from de novo synthesis to mobilisation of storage pools, with reduced transpiration raising leaf temperature and enhancing volatility (Niinemets and Reichstein, 2003). Plants may also draw on alternative carbon sources or adjust monoterpene synthase expression to maintain production (Lüpke et al. , 2017; de Souza et al. , 2018). Yet severe and prolonged drought suppresses metabolism and substrate supply, ultimately inhibiting synthesis (Sharkey and Loreto, 1993; Loreto and Schnitzler, 2010), with turgor loss hindering compound mobilisation from storage pools to active sites of the plant (Malone et al. , 2025). Species with specialised (e.g., trichomes and oil glands) and non-specialised (e.g., foliar lipids) storage structures or high foliar concentrations can still maintain basal or delayed emissions, with water limitations sometimes producing non-linear emissions patterns (Niinemets et al. , 2004; Ormeño et al. , 2007; Byron et al. , 2022). Highly variable monoterpene emission responses reflect species (trait) differences and different studies not adopting a unified metric to determine plant and soil water deficits. Monoterpenes can provide oxidative protection, possibly by directly or indirectly acting as antioxidants. Exposing non-isoprene-emitting plants to ozone (an oxidative stressor) stimulated monoterpene synthesis (Loreto et al. , 2004), but limiting monoterpene emissions by applying non-specific chemical inhibitors such as fosmidomycin (inhibiting upstream MEP conversion) rapidly inhibited photosynthesis and increased ROS and malondialdehyde (MDA) levels, indicating lipid peroxidation (Tian et al. , 2020). Moreover, exogenous monoterpenes (< 2.5 µM) decreased foliar hydrogen peroxide (H 2 O 2 ) content, and downregulated activity of the enzymatic antioxidants superoxide dismutase, peroxidase and ascorbate peroxidase under drought stress (Zhou et al. , 2023). Endogenous isoprene and monoterpenes act as volatile signals that trigger Ca 2+ -mediated pathways, activating kinases and stress-responsive genes to enhance thermotolerance (Zuo et al. , 2025). Monoterpenes also stimulated recovery-related genes such as PEX14 and PEX16 involved in peroxisome biogenesis in heat stressed plants (Tian et al. , 2020). Thus monoterpenes protect cellular structures from oxidative damage by acting as antioxidants, directly mitigating the effects of ROS, or serving as signalling molecules assisting redox balance (Vickers et al. , 2009a; Frank et al. , 2021). Whether endogenous monoterpenes play similar protective roles during plant drought responses remains unknown. Genetically engineering the MEP pathway to consistently enhance terpene production creates useful plant materials to understand whether endogenous terpene production also has protective effects. Isoprene emitting tobacco (i.e., overexpression of isoprene synthase) showed attenuated net assimilation rate, photosynthetic efficiency and oxidative stress (ROS levels) when irrigated with half their water requirements (Ryan et al. , 2014; Pollastri et al. , 2023), heat (up to 40°C) (Pollastri et al. , 2019) and oxidative (ozone) stress (Vickers et al. , 2009b), however, with decreased biomass and leaf area (Ryan et al. , 2014). Hence, terpene overexpression may confer growth penalties with attenuated photosynthesis under heat stress (Bertamini et al. , 2021), even though lipid peroxidation was diminished under salt stress (Meng et al. , 2022). Overexpressing key enzymes such as geranyl diphosphate synthase small subunit ( GPS.SSU ) and specific monoterpene synthases increased monoterpene production in transgenic plants (Dudareva et al. , 2005). Inserting the Mentha × piperita GPS.SSU gene, along with monoTPs from Picea abies or P. sitchensis into tobacco ( Nicotiana tabacum ) increased emissions of (–)-limonene, myrcene, α-pinene, and β-pinene (Yin et al. , 2016). Tobacco plants engineered with monoterpene synthases targeted to different cellular compartments exhibited varying levels of monoterpene production, depending on enzyme localisation. In plastids, where the MEP pathway operates, limonene production was significantly higher than cytosolic localisation and not active in endoplasmic reticulum, emphasising the importance of compartmentalised biosynthesis (Ohara et al., 2003). However, no studies explicitly investigated the impacts of these monoterpene transformations on plant abiotic stress tolerance. We investigated the morphological and physiological impacts of endogenous production of specific monoterpene compounds from genetically modified tobacco plants under drought conditions. We hypothesised that genetic transformations producing specific monoterpenes, (‒)-α/β-pinene, myrcene, and (‒)-limonene, would confer distinct emission profiles on tobacco plants ( Nicotiana tabacum ), which has no specialised storage pools. After screening these genotypes (Yin et al. , 2016), we selected the most phenotypically similar genotype to the wild type for further studies. Since exogenous monoterpenes enhanced plant photosynthetic capacity, and antioxidant status by mitigating ROS accumulation and lipid peroxidation (Copolovici et al. , 2005; Zhou et al. , 2023), we further hypothesised that endogenous monoterpene would optimise photosynthesis and enhance enzymatic antioxidant responses under drought stress, depending on the prevailing stress conditions and emission levels. Methods Plant materials and experimental setup Three transgenic ( Nicotiana tabacum cv. SR1) genotypes with enhanced emissions of (−)-limonene (LG12), myrcene (MG1), and (−)-α/β-pinene (PG11) were obtained from the Temasek Life Sciences Laboratory in Singapore. Each inserted a Mentha × piperita geranyl diphosphate synthase small subunit ( MpGPS.SSU ) (AF182827) gene and a specific individual monoterpene synthase, including Picea abies (−)-limonene synthase ( PaLimS – Line LG12 ), P. abies myrcene synthase ( PaMyrS – Line MG1 ), and P. sitchensis (−)-pinene synthase ( PsPinS – Line PG11 ). Both genes were under control of the constitutively expressed Ubiquitin 10 promoter, thereby increasing endogenous monoterpene production and emission. Table S1 show the average relative expression of MpGPS.SSU and monoterpene synthase ( monoTPs ) of each line (Yin et al. , 2016). Two sets of experiments occurred (Table S2). Initially, all four genotypes (LG12, MG1, PG11 and WT) were exposed to two different water treatments (well-watered and drought) at two growth stages (before and during stem elongation). Another two independent experiments (3 & 4) comprised a factorial combination of two genotypes (WT and LG12) and two watering treatments (well-watered, WW and drought, D), resulting in four genotype × treatment combinations. In Experiments 1 and 2, WT and transgenic seeds were sown simultaneously and germinated on John Innes No. 2 compost in seedling trays (12 cells, each 3.8 × 3.8 × 5 cm; tray size: 17.8 × 14 × 5 cm) covered by transparent domes with humidity control valves. Seed trays were kept in a controlled environment (CE) room illuminated by LED growth lights (Model B150, NS1 spectra, Valoya, Helsinki, Finland) with average mid chamber height photosynthetic photon flux density (PPFD) at 500 μmol photons m -2 s -1 . After four weeks, seedlings were transplanted into 3 L pots (20.5 cm in height, 16 cm at the top, and 12.5 cm at the bottom) filled with 3 kg of a pre-mixed sandy substrate (70% garden sand, 30% John Innes No. 2 v/v) with limited water holding capacity (0.31 g water / g soil) to allow rapid soil drying. Next, pots were transferred to enclosed plant growth chambers as described in Stokes et al. (1993) with the door and side walls of the chamber made of 2 mm polycarbonate sheets to enhance the chamber’s strength, airtightness, and light transmission (<1.5% difference in the light quality). The supporting grid in the chamber was height-adjustable, maintaining a consistent leaf-level light intensity during plant growth at 350 ± 20 μmol photons m -2 s -1 PPFD. This light intensity was provided by a growth lamp (Powerstar HQI-BT, 600 W/D daylight, Munich, Germany) from 07:00 to 19:00 hours for a 12-hour day. Average day:night temperatures and relative humidities across the chamber were maintained at 22°C:16°C (±2°C) and 40:60% (±5%) respectively. The full light spectrum, chamber-specific, leaf-level PPFD distribution and the environmental conditions are described (Fig. S1&2). Plant pots containing individual plants of the four genotypes were placed in individual, leak-proof growth chambers. To prevent potential priming effects of volatiles across different genotypes, chamber doors were opened only for watering and placing measuring instruments. Plants were rotated inside the chambers daily and between chambers every two days to limit the effects of variable environmental conditions. Nutrients were supplied as Miracle-Gro® all-purpose soluble plant food (5 g per L of water) to the substrate every week. Two independent sets of seeds were sown to obtain plants for measurements at the pre-stem elongation (BE) and stem elongation (DE) stages. Measurements and harvesting for BE and DE occurred six and eight weeks after placing the seed for germination, respectively. All plants were well-watered daily at 08.00 and 18.00 hours, by replenishing the amount of water lost from the pots, by comparing weights at these times versus initial pot weight. Control plants were consistently well-watered (WW) daily throughout the experiment by replacing evapotranspirational losses (i.e. 100% pot capacity). To impose drought, irrigation was stopped in the fifth week to ensure plants experienced eight days of drought stress for BE and 16 days of drought for DE. Gas exchange, leaf chlorophyll content, foliar monoterpene emission and morphological and leaf water status were measured at each stage and each treatment. Additionally, four to five plants remaining after the DE measurements (exposed to 3 weeks of drought) were removed from the chambers, placed in a controlled environment, rewatered to pot capacity and then watered daily until the reproductive stage to determine seed production. Experiments 3 and 4 grew plants in 100% John Innes No. 2 substrate (allowing slower soil drying as soil water holding capacity was greater, 1.59 g water / g soil), but otherwise measurements were identical. On the day of imposing drought, baseline measurements were made and considered as Day 0 of the experiment. After 18 days of soil drying, the remaining plants were re-watered to initial pot weight and maintained well-watered. Measurements of leaf physiology, biochemistry, and foliar limonene emission occurred every three days for the drought treatments and every six days for the control, with additional measurements following re-watering to understand plant recovery (Fig. 1). Four plants of each genotype from each treatment were randomly selected using a random number generator for physiology (gas exchange, chlorophyll fluorescence) measurements with a randomised sequence to avoid potential diurnal patterns. Physiological measurements used all 4 plants, with 3 plants for biochemical and foliar monoterpene emission. Gas exchange and chlorophyll fluorescence Leaf gas exchange was measured on the newest fully developed leaf (4th or 5th from the top) using a LI-6800F portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA) equilibrated to growth chamber conditions for an hour. Leaf cuvette (6 cm 2 ) conditions were set to a PPFD of 350 μmol m -2 s -1 (90% red, 10% blue light) and 421 μmol mol -1 CO 2 , with temperature and relative humidity matching ambient chamber conditions. The airflow rate was 600 µmol s -1 , reduced to 400 µmol s -1 in drought treatments when g sw fell to 50% of the control. After securing the leaf in the cuvette and once steady-state was reached, net photosynthesis ( A net ; μmol m -2 s -1 ), stomatal conductance ( g sw ; mol m -2 s -1 ) and other pertinent physiological and environmental parameters (e.g., transpiration rate, internal CO 2 , leaf temperature) were recorded. Simultaneously, chlorophyll fluorescence was measured using the integrated Multiphase Flash fluorometer (6800-01A). An optimum flash setting based on the LI-6800 user manual was used to determine the light-adapted steady-state fluorescence ( F s ʹ ), maximum fluorescence ( F m ʹ ), and minimum fluorescence ( F o ʹ ). Dark-adapted maximum ( Fm ) and minimum ( F o ) fluorescence were measured pre-dawn. Photochemical parameters ( F v /F m , F v ’/F m ’ , ΦPSII , NPQ ) were calculated as described (Murchie and Lawson, 2013). The leaf remained in the cuvette for subsequent volatile sampling. Foliar monoterpene emission sampling and analysis Following gas exchange measurements, foliar monoterpene emissions were sampled from the cuvette’s outflow air using a modified LI-6800F port as shown in Fig. S3 (Riches et al. , 2020; Zhou et al. , 2023). Monoterpene samples were captured using sorbent tubes, which were kept in at 4℃ for further GC-MS analysis. After sampling, the LI-6800F was moved to the next randomly selected plant for the same measurements. Additional details of sampling and analysis can be found in the Supplementary Materials. The emission rate was calculated as (Niinemets et al., 2011): \(E=\ \frac{C_{\text{out}}-C_{\text{in}}}{A}F\) (5) where E is the emission rate of monoterpene (pmol m 2 s -1 ), C out and C in (pmol m ‑3 ) are the concentrations of the monoterpene at the air outlet (sampled by sorbent tubes and quantified by GCMS) and air inlet (filtered by carbon filtration capsule, assumed to be zero), respectively. F (m 3 s -1 ) is the airflow rate into the leaf cuvette, converted according to the volume of gases at STP, and A (m 2 ) is the leaf area in the cuvette. All samples were corrected based on the system blank in the same measurement. Morphology, soil, leaf water status and chlorophyll content After volatile sampling, various measurements were made on the same leaf. First, epidermal impressions were made using dental putty and nail polish casts (Matthaeus et al. , 2020). Microscope images of the adaxial and abaxial surfaces were analysed with ImageJ to determine stomatal density, stomatal size, and epidermal cell density. The stomatal index was calculated from these values. An example of the measured tobacco leaf with sample areas is presented in Fig. S4. Subsequently, leaf chlorophyll content was measured at the same area used for the LI-6800F measurement using an MC-100 chlorophyll meter (Apogee Instruments, Inc., Logan, UT, USA). A leaf disc (8 mm diameter) from the same spot was obtained using a sharp cork borer, placed in a clean sample holder, and wrapped in aluminium foil to minimize water loss. The sample holder was then loaded into a C52 psychrometer chamber (Wescor Inc., Logan, UT, USA), located in a stable temperature room alongside a HR-33T dew point microvoltmeter (Wescor Inc., Logan, UT, USA). After a 3-hour incubation at 25°C, the psychrometer chamber was measured using the microvoltmeter, and leaf water potential (Ψ leaf ) calculated using a calibration table made from standard salt solutions of various concentrations. Subsequently, the leaf disc was removed using tweezers, wrapped in aluminium foil again and rapidly frozen in liquid nitrogen to disrupt cell membranes. After a brief thawing period, the leaf disc was returned to the sample holder and incubated in the psychrometer chamber for 30 minutes before determining the osmotic potential (Ψ π ). The difference between Ψ leaf and Ψ π estimates leaf turgor (P). Finally, plants were harvested. Leaf strips were sliced with a razor blade and stored in three 2 mL Eppendorf centrifuge tubes, comprising 3 samples (each 0.1 g fresh weight) for oxidative stress, antioxidant enzyme activity analysis and a spare. All tubes were immersed into liquid nitrogen and then stored in a freezer at -80°C. Plant height, shoot fresh biomass, and soil moisture (WET-2 sensor, Delta-T Devices Ltd) were recorded. Shoots were then oven-dried at 50°C to determine dry biomass. Phytohormone, H 2 O 2 , MDA and antioxidant enzyme analyses Samples collected from Experiments 3 and 4 were used for foliar hormone and biochemical analysis. Phytohormone extraction and quantification followed the descriptions in Ma et al. (2024) with calibration curve developed by using (+)-abscisic acid standard (Merck Life Science, Gillingham, UK; #90769-25MG). Foliar hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) concentrations, as indicators of oxidative stress and damage, were quantified using commercial kits. H₂O₂ was measured with the Pierce™ Quantitative Peroxide Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA; #23280) and MDA with the MDA-TBARs Assay Kit (BQC Redox Tech, Oviedo Asturias, Spain; #KB03016b). The activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were determined using the SOD Colorimetric Activity Kit (Thermo Fisher Scientific, Waltham, MA, USA; #EIASODC) and the APX Assay Kit (BQC Redox Tech, Oviedo Asturias, Spain; #KB03036), respectively. One unit of SOD is defined as the amount of enzyme causing half the maximum inhibition of the oxidation of 7.5 mM NADH in the presence of EDTA, manganese ions, and mercaptoethanol at 23°C and pH 7.4 over 15 minutes. One unit of APX is defined as the amount of enzyme that catalyses the conversion of one µmol of ascorbic acid to monodehydroascorbate per minute. According to the manufacturer, the supplied extraction buffer does not include any additional compounds (e.g. ascorbate) to stabilise the extraction. All assays were performed following manufacturer protocols with several modifications for plant tissues. Frozen leaf samples were ground to a fine powder using a Cryomill (Retsch GmbH, Haan, Germany). To prevent interference, H 2 O 2 samples were deproteinized using a TCA Deproteinising Sample Preparation Kit (Abcam Limited, Cambridge, UK; #ab204708). MDA measurements were corrected against control samples lacking thiobarbituric acid to account for interference from sugars and other compounds (Hodges et al. , 1999). Prior to analysis, samples were stored at -80°C and kept on ice during the assay procedures. Absorbance and fluorescence were measured using a FLUOstar Omega microplate reader (BMG Labtech). Concentrations and enzyme activities were calculated from standard curves using MARS data analysis software (v.5.01, BMG Labtech, Aylesbury, UK). Data analysis Data handling, graph generation, and statistical analysis were performed using R (v. 4.5.0) and R Studio (PBC, Boston, MA, USA). Regressions were defined using glm and nls functions, plots were generated using ggplot2 . Interactions and statistically significant differences ( P <0.05) among variables related to physiology, biochemistry, and leaf water status were assessed using univariate two-way or three-way ANOVA/ANCOVA. Post hoc Tukey tests compared monoterpene emission levels and variables following re-watering. Results Genetic transformation enhanced monoterpene emissions Well-watered WT plants had no detectable (−)-α/β-pinene, myrcene and (−)-limonene emissions, which became barely detectable with drought (Fig. 2). MG1 plants had no detectable pinene emissions irrespective of water treatment, but drought caused detectable limonene emissions. Increased monoterpene emissions of well-watered LG12 and PG11 plants were stable across different growth stages. PG11 had 5-fold higher (−)-α-pinene emission than (−)-β-pinene emission with little (−)-limonene emission, while LG12 had high limonene emission with barely detectable pinene emission. Drought applied before stem elongation doubled and tripled the emission rate of (−)-limonene and pinene in LG12 and PG11 respectively. With prolonged drought (16 days) during stem elongation, (−)-limonene emission significantly ( p < 0.001) decreased in LG12 compared to well-watered plants while pinene emissions dropped to well-watered levels. Overall, genotype had the strongest effect on emission rates, with drought-stimulated MT emissions decreasing at later developmental stages. Developmental stage also influenced total emissions, and drought responses varied with genotype and growth stage. Genetic transformation altered plant morphology Although all seeds germinated simultaneously, roots of three-week-old LG12 and MG1 seedlings were approximately 30% shorter, and PG11 roots over 50% shorter than WT plants. Similarly, leaf width of LG12 and MG1 was 11% less than WT plants, and significantly lower ( p < 0.001) in PG11, only half of WT plants. Before stem elongation, shoot biomass of LG12, MG1 and PG11 was 10%, 16% and 30% less than that of WT plants, respectively (Fig. 3b, Fig. S5a). Stem elongation amplified these effects of transformation, with height decreasing by 28% (averaged over all three genotypes) compared to WT (Fig. 3c). From before to during stem elongation, leaf area increased by nearly 70% in WT plants but doubled in genotypes, as these had 30% less leaf area than WT plants (Fig. S5b). Withholding water decreased shoot biomass, height and leaf area of all genotypes, but less so in transgenics (significant treatment × genotype interactions for biomass, but not height and leaf area). Although PG11 maintained similar leaf expansion and biomass accumulation, it had the lowest shoot dry weight and stem elongation. Before stem elongation, leaf chlorophyll content of well-watered MG1 and WT plants tended to be ( p >0.05) approximately 10% higher than LG12 and PG11 plants, but this variation diminished with stem elongation (Fig. 3d). Drought increased leaf chlorophyll content, particularly in LG12 and WT plants, with the latter significantly higher than MG1 and PG11 by over 20%. Stem elongation and prolonged drought amplified this increase such that chlorophyll content of PG11 more than doubled and was approximately 40% higher than in LG12 and MG1 plants. Three-way ANOVA confirmed significant effects of treatment, genotype, stage and their interactions on leaf chlorophyll content, highlighting both genotype- and stage-dependence of the drought response. Variation in soil and leaf water status did not cause these genotypic differences (Fig. S6). Although withholding water reduced seed yield by an average of 11% in all genotypes (no significant treatment × genotype interaction – Fig. 3e), genetic transformation didn’t affect seed production. Genetic transformation had minimal effects on stomatal distribution and development under WW conditions (Fig. S7). Stomatal density increased with stem elongation by ~10% without significant differences between genotypes. Abaxial stomatal density was significantly higher than adaxial with significant genotype × stage ( p = 0.047) and stage × leaf side interactions ( p = 0.006, Table. S3). While MG1 and PG11 plants had higher adaxial stomatal index (but not size), drought diminished these effects. Drought altered foliar emission pattern of (−)-limonene Genetic transformation altered leaf water status and gas exchange In Experiment 3 and 4, LG12 leaves emitted (−)-limonene, whereas WT leaves did not. No detectable (−)-limonene was found in leaf extracts under either watering regime, suggesting specific storage pools were absent. Drought had a highly significant (P < 0.001) effect on (−)-limonene emission patterns in LG12, showing an approximately quartic relationship with soil moisture (Fig. 4a), but a more scattered relationship with leaf water potential (Ψ leaf ; Fig. 4b). As soil dried, (−)-limonene emission rates (E Lim ) initially increased 3- to 4-fold, reaching a maximum nearly 6-fold higher than baseline before soil moisture fell below 20% (i.e. Ψ leaf ~ -1.0 MPa). This peak occurred while Ψ leaf declined from −0.5 MPa to around −1.0 MPa. With continuing drought, E Lim dropped sharply back to initial levels, indicating that plants could not sustain high (−)-limonene emissions once soil moisture fell below 20%. Beyond this threshold, plants maintained only basal E Lim as Ψ leaf continued to decline from approximately −1.0 to just below −2.0 MPa. Following re-watering (RW), E Lim sustained at basal levels comparable to those observed at the end of drought, approximately 70% lower than in well-watered plants on the same day (Fig. 4a). Normalised E Lim based on emission from well-watered plants on the same day (Fig. S8) showed a similar response pattern. Genetic transformation altered leaf water status and gas exchange Experiments 3 and 4 sowed LG12 seeds three days later than WT to minimise genotypic differences in morphology at the onset of soil drying (see Fig. S9). Both leaf water potential (Ψ leaf ) and turgor (P) significantly ( p <0.001) declined as soil moisture decreased in both genotypes (Fig. 5a&b). Soil moisture explained most of the variation in Ψ leaf and P, highlighting the strong dependence of both parameters on substrate water availability. Ψ leaf declined exponentially with soil moisture from -0.5 MPa to nearly -2.0 MPa as soil moisture reached a minimum of 10% (Fig. 5a), with a higher Ψ leaf (by an average of 0.2 MPa) of LG12 than WT plants when soil moisture ranged between ~15 and 35%. Leaf Ψ π of both genotypes responded similarly to drought (Fig. 5b). Leaf turgor was approximately 0.58 MPa in well-watered plants of both genotypes, and declined linearly with soil moisture in both genotypes but at twice the rate in WT plants (Fig. 5c). Thus, Ψ leaf of LG12 plants was less sensitive to soil drying irrespective of morphology. Net photosynthesis rate ( A net ) and stomatal conductance ( g s ) consistently decreased as Ψ leaf declined in both genotypes (Fig. 5d&e) in both experiments. Well-watered plants had similar photosynthesis rates (approximately 11 µmol m -2 s -1 ) but variable g sw (0.16 – 0.35 mol m -2 s -1 ). A net of LG12 tended to decline more rapidly than WT at intermediate leaf water status (Ψ leaf of -1.0 to -1.5 MPa), while A net of WT plants decreased almost linearly (at a rate of 7.5 µmol m -2 s -1 MPa -1 ). Stomatal response to Ψ leaf significantly differed between genotypes (genotype × Ψ leaf: p = 0.049 - Fig. 5e), as WT plants maintained 10-40% higher g sw when Ψ leaf was lower than -1.0 MPa. However, intrinsic water-use-efficiency ( A net / g sw ) increased similarly in both genotypes by approximately 4-fold (Fig. 5f). Genetic transformation had minimal impact on photosynthetic efficiency under drought compared to WT plants (Fig. S10). (−)-limonene transformation affects oxidative and hormone status Drought enhanced H 2 O 2 accumulation, the activity of enzymatic antioxidants and lipid peroxidation in both LG12 and WT plants. Withholding water increased H 2 O 2 content 2.5-fold, which stabilised at Ψ leaf < -1.0 MPa (Fig. 6a). Coincident with H 2 O 2 accumulation, SOD activity increased nearly 7.5-fold, similarly in both genotypes (Fig. 6b). Conversely, foliar APX activity approximately doubled in LG12 plants but increased more than 3-fold in WT plants (Fig. 6c). Foliar MDA concentration in WT plants increased 3.5-fold before stabilising at Ψ leaf < -1.5 MPa, with values approximately 30% higher in LG12 (Fig. 6d). Thus, (−)-limonene transformation did not modify SOD-catalysed transformation of superoxide radicals to H 2 O 2 , but lower APX activity of LG12 plants was associated with greater lipid damage under water stress. Foliar H₂O₂ levels and APX activity rose in parallel with SOD activity in both genotypes, yet the resulting APX : SOD ratio was same H 2 O 2 and APX level, LG12 leaves accumulated significantly more MDA with the rate of MDA accumulation roughly double that of WT (Fig. 6f, g). Thus, (−)-limonene expression weakens the APX-related enzymatic antioxidant system, intensifying lipid peroxidation. Foliar ABA concentration did not differ between genotypes under well-watered conditions, but prolonged soil drying increased it by 10-fold and 30-fold in WT and LG12 leaves respectively (Fig. 6h), with a significant Ψ leaf × genotype interaction ( p <0.001). Thus, (−)-limonene transformation boosted ABA accumulation, especially when Ψ leaf was < -1.5 MPa. Both genotypes showed similar physiological responses following re-watering but did not recover to the well-watered level, with A net lower by ~35% (Fig. 7a) and g sw by over 60% (Fig. 7b) compared with well-watered controls at the same stage. One day after re-watering, both genotypes had significantly higher ABA levels than well-watered plants, with twice as much ABA in LG12 than WT leaves causing a significant genotype × treatment interaction (Fig. 7c). Both genotypes had a similarly lower Ψ leaf than well-watered plants at this time (Fig. 7d). Thus, re-watering did not fully restore leaf gas exchange, foliar ABA and Ψ leaf levels to well-watered values. Differences in foliar oxidative stress and antioxidant activity occurred between genotypes. Re-watering returned H 2 O 2 content of WT plants to well-watered levels, causing slightly (~13%) lower levels of rewatered LG12 plants (Fig. 7e). SOD activity remained elevated in re-watered WT plants, ~25% higher than other treatments (Fig. 7f). By contrast, APX activity increased substantially upon re-watering, nearly doubling in WT and rising a further concentration, which was approximately 40% lower in LG12 than WT (Fig. 7h). Thus, (−)-limonene emission in LG12 plants enhanced APX activity after re-watering, contributing to less lipid peroxidation than WT. Principal component analysis (Fig. S11) accounted for 84.9% of the variance (PC1 = 79.3%, PC2 = 5.6%) and separated the genotypes mainly along PC1. Variation in leaf turgor, ABA content, APX activity and MDA content were the most discriminating traits between LG12 and WT plants. Discussion Genetically enhancing volatile organic compound synthesis and emissions can improve plant stress resilience, but such transformations often incur growth and metabolic trade-offs (Vickers et al. , 2009b; Ryan et al. , 2014; Yin et al. , 2016). Conferring tobacco foliar (−)-limonene and (−)-α/β-pinene emissions in both LG12 and PG11 genotypes suppressed growth and biomass accumulation. (−)-limonene emission more clearly correlated with soil drying than leaf water status, suggesting signals from drying soil modulate emission behaviour. Although LG12 better maintained leaf water status with soil drying, it showed a steeper decline in A net and lower g sw at intermediate stress, and APX activity increased less than in WT. Re-watering enhanced LG12 APX activity and decreased MDA accumulation relative to WT. Although enhanced monoterpene emission imposed transient growth and oxidative penalties under drought, reproductive success was maintained after re-watering. Foliar monoterpene emission decreased biomass irrespective of gas exchange Wild-type tobacco emitted no monoterpenes under well-watered conditions, consistent with the lack of native production in N. tabacum (cv. SR1) (Lücker et al. , 2004; Yin et al. , 2016). Thus, transgenic emissions likely reflected de novo synthesis with immediate release. Across developmental stages, LG12 and PG11 emitted (−)-limonene and (−)-α/β-pinene (Fig. 2), respectively, indicating substrate-driven plasticity under stress (Jardine et al. , 2017). In PG11, the (−)-α:(−)-β-pinene ratio (1:4) matched earlier reports (Yin et al. , 2016), consistent with MEP-flux enhancement via GPS.SSU activity (Lücker et al. , 2004; Vickers et al. , 2011). By contrast, myrcene emissions from MG1 ( MpGPS.SSU + P. abies myrcene synthase) were below our detection limit, likely reflecting the effects of transgene silencing in this later-generation (T3) or inherent enzymatic limitations. Soil moisture strongly affected (−)-limonene emission rate (E Lim ), rising transiently at intermediate stress then declining sharply under prolonged drought, as in Quercus spp. (Ormeño et al. , 2007; Mu et al. , 2018). Emissions increased >4-fold during drying (Fig. 4) coinciding with the soil moisture range over which Ψleaf declined from about −0.5 to −1.0 MPa (Fig. 5a) and then returned to baseline as LG12 neared wilting. Soil water status better explained this pattern than Ψ leaf , and E Lim sensitivity to soil moisture (≈ 20%; Fig. 4a) and leaf water status (≈ −1.0 MPa; Fig. 4b) thresholds. This soil moisture-mediated emission response aligns with evidence that de novo emissions in species lacking storage pools are largely independent of stomata and persist even when photosynthesis halves (Staudt et al. , 2002; Niinemets et al. , 2004; Tattini et al. , 2015). Consistently, (−)-limonene emission was not tightly linked to physiological limitation (i.e., A net and gsw ; Fig. 5). Intensifying drought returned terpene emissions to baseline levels, and they did not act as alternative energy sinks or protect photosynthesis, as PSII efficiency and NPQ did not change (Fig. S10). In contrast, some monoterpenes (e.g. α-pinene, camphor) sustain photosynthetic efficiency under heat stress with up to 40℃ leaf temperature (Copolovici et al. , 2005; Bertamini et al. , 2021; Xu et al. , 2022). Excess electron transport and reducing power (ATP/NADPH) under drought can fuel terpenoid synthesis when net assimilation rate (NAR) is limited under drought conditions with species-specific thresholds (Dani et al. , 2014). Despite soil drying decreasing Ci and assimilation, electron transport and starch/glucose breakdown (de Souza et al. , 2018) can sustain the resources required, estimated to comprise al., 2002; Perez-Gil et al. , 2024). LG12 plants may downregulate NAR to increase ETR-NAR ratio and boost constitutive (−)-limonene production, although A net decline seemed driven by more sensitive g sw under intermediate Ψ leaf (Fig. 5d&e). Whether tobacco’s constitutive MT expression mirrors feedback regulation of native emitters, and how biochemical (energy partitioning) and physiological (stomatal) processes interact to constrain Anet during drought, remain unclear. Ultimately, turgor loss, downregulated MEP activity, and restricted lipid–air partitioning limit (−)-limonene under severe drought. After re-watering, LG12 E Lim remained photosynthesis (Fig. 7a). After severe deficit, metabolic imbalance or oxidative damage can restrict de novo emissions to 20–60% of unstressed levels (Lüpke et al. , 2017). Constitutive transgene expression likely sustains a basal (−)-limonene level, partly decoupling biosynthesis from photosynthetic limits even after recovery. Growth penalties indicate that monoterpene overproduction imposes developmental and metabolic constraints. During stem elongation, well-watered transgenics had lower biomass (Fig. 3b) and leaf area (Fig. S5b) than WT plants. Overproduction in non-native producers can reduce growth by 30–60% by depleting IPP (Lange and Ahkami, 2013), a crucial precursor for hormone (e.g., abscisic acid) and pigment (e.g. carotenoids, chlorophyll) synthesis (Dudareva et al. , 2005; Vickers et al. , 2011). Because <0.1% of assimilated carbon was lost as monoterpenes under WW conditions in our experiment, growth reductions are unlikely to reflect direct carbon costs. Instead, competition for plastidial IPP could disturb pigment accumulation or hormone balance (Lyu et al. , 2022), constraining development and environmental responses (Boncan et al. , 2020). Although MpGPS.SSU transformants had slightly longer flowering stems and higher leaf gibberellin content (Yin et al. , 2016), WT plants remained taller than transgenics (Fig. 3c) and likely intercepted more photon energy, boosting biomass. Repeated leaf-level photon-flux measurements would test this hypothesis (Zhou et al. , 2022). Despite phenotypic differences, reproductive output was similar across genotypes (Fig. 3e). Under water- and carbon-limitation, downregulating growth conserves carbohydrates to support survival and reproduction (Huang et al. , 2019). With soil drying, Ψ leaf declined exponentially (Fig. 5a), independently of stomatal distribution (Fig. S7) or response (Fig. 5e). However, transgenic plants maintained higher Ψ leaf (Fig. 5a, Fig. S6b), with similar Ψ π in LG12 and WT (Fig. 5b) suggesting leaf water status wasn’t maintained by additional osmolyte accumulation. Higher turgor (Fig. 5c) of LG12 may reflect altered hydraulic or mesophyll conductance, or root structure and functioning (Parkash and Singh, 2020). ABA accumulation induces stomatal closure and decreases leaf hydraulic conductance (Pantin et al. , 2013) while sustaining root growth when soil water availability declines (Huntenburg et al. , 2022). Both LG12 and WT increased foliar ABA as soil dried but enhanced stomatal closure in LG12 at Ψ leaf between -1.0 – -1.5 MPa did not parallel additional ABA accumulation. When Ψ leaf fell below −1.5 MPa, LG12 had twice as much ABA as the WT (Fig. 6g) when stomata were nearly closed. Isoprene-emitting tobacco did not exhibit enhanced foliar ABA accumulation (Ryan et al. , 2014), likely because Ψ leaf never dropped below −1.5 MPa. The additional ABA in LG12 leaves no longer affects stomata but likely reflects altered plastid metabolism. When monoterpene production declines, precursor accumulation (IPP, DMAPP, GPP) may stimulate MEP-pathway flux (Wang et al. , 2019), elevating ABA under severe drought. Measuring carotenoid precursors in LG12 may explain its substantial ABA accumulation and adaptive significance. In transgenic tomato, terpene-synthase overexpression enhances ABA-mediated salt adaptation by promoting osmolytes (proline, soluble proteins and soluble sugars) and modulating ABA-responsive transcription (Meng et al. , 2022). ABA differences between LG12 and WT were greatest when E Lim fell to basal levels, suggesting stress signalling was prioritised over terpene biosynthesis (Marešová et al. , 2022). However, similar osmotic potential of both genotypes (Fig. 5b) suggests context-specific osmolyte pathways in salt versus drought (Zhu, 2002). Thus, elevated ABA of LG12 most likely reflects an enhanced flux through MEP pathway toward carotenoid derived ABA biosynthesis without detectable osmotic adjustment. The MEP pathway also mediates stress-induced retrograde signalling. Methylerythritol cyclodiphosphate (MEcPP), the precursor to IPP and DMAPP, induces nuclear-encoded plastid proteins, including factors that down-regulate light-harvesting chlorophyll a/b–binding gene expression (Xiao et al. , 2012). MEcPP is also involved in oxidative stress sensing, signalling and responses (Perez-Gil et al. , 2024). This shifts the balance between photosynthesis-associated nuclear (PhANGs) and plastid genes to conserve resources during water deficit (Lv et al. , 2019) and may explain lower chlorophyll and biomass in transgenic tobacco. Enhanced MEP flux may reinforce MEcPP signalling, reallocating resources from growth to defence. Oxidative status during drought and recovery Drought increased H 2 O 2 accumulation and lipid peroxidation in both LG12 and WT plants. Exogenous (i.e., exceeding 2.5 mM) (Ibrahim et al. , 2006; Zhou et al. , 2023) and high endogenous (i.e., estimated at 1 mM) (Widhalm et al. , 2015) monoterpene exposures influence ROS dynamics and membrane permeability in a concentration-dependent manner, whereas at lower concentrations some monoterpenes can mitigate H 2 O 2 accumulation. APX activity increased as Ψ leaf declined, potentially due to turgor loss. Although LG12 plants had similar H 2 O 2 content (Fig. 6a) and SOD activity (Fig. 6b), their diminished APX activity (Fig. 6c) likely caused higher foliar MDA content under drought (Fig. 6d). Downregulating APX isomers at various subcellular structures in transformed and mutant plants also exacerbates ROS-mediated signalling and redox balance (Foyer et al. , 2017). Since photosynthesis is the primary source of ROS under drought and light stress, specifically downregulating chloroplastic APX isoforms such as thylakoid-bound APX (tAPX), would be particularly impactful (Foyer, 2018). Lower APX activity increases foliar lipid peroxidation and MDA content, with APX4 knockdown exacerbating lipid damage and programmed cell death under high light-induced oxidative stress (Kuo et al. , 2020). Failure to detoxify H 2 O 2 at its primary site of production allows it to diffuse and initiate lipid peroxidation cascades in the thylakoid and envelope membranes, causing MDA accumulation. This chloroplastic water-water cycle relies on both APX and 2-cysteine peroxiredoxins (PRXs), and is essential in preventing photo-oxidative stress (Foyer, 2018). Thus, diminished APX activity of LG12 likely disrupts the delicate redox balance across multiple subcellular compartments, altering the spatiotemporal dynamics of H 2 O 2 as a damaging agent and a critical signalling molecule. Consistent with this, re-watering increased APX activity in LG12 and lowered MDA relative to WT withing 24 hours (Fig. 7), indicating partial restoration of antioxidant capacity during recovery. LG12’s continuous limonene biosynthesis and emission may disrupt development and redox balance by modifying enzymatic (APX) activity and unknown signalling pathways. Despite elevated MDA content, a clear marker of oxidative damage, LG12 plants maintained PSII photosynthetic efficiency suggesting additional mechanisms attenuate oxidative damage. Plants with lower APX activity compensate to maintain oxidative homeostasis. For instance, APX4-deficient rice had higher foliar H 2 O 2 levels but increased photochemical efficiency and photosynthetic proteins, while simultaneously activating the ascorbate/glutathione cycle, nuclei and mitochondria signalling pathways to mitigate oxidative damage (Sousa et al. , 2018). The water-water cycle is not a critical sink for excess electrons when photosynthesis is limited (Driever and Baker, 2011). Presumably, the continuous metabolic investment in limonene biosynthesis in LG12 enhances alternative photoprotective mechanisms (such as other antioxidant systems or cyclic electron flow) that protect the core photosynthetic machinery even as lipid membranes sustain some damage under drought conditions. The suppression of APX activity could be a direct consequence of limonene accumulation acting as a negative feedback signal, or it could be an unintended pleiotropic effect of the transgene insertion itself. Distinguishing between these possibilities is essential. Correlating monoterpene synthase expression levels from different lines and emission flux (Yin et al. , 2016) with isoform-specific APX activity measurements across different transgenic lines would help establish a dose-response relationship. Ultimately, these investigations will reveal whether there is an inherent metabolic cost to engineering high-level monoterpene production, a critical insight for the future development of stress-resilient plants. Drought-induced loss of lipid integrity and reduced APX capacity in LG12 likely alters limonene partitioning between lipid and aqueous phases and decreases chloroplast and cellular retention (Niinemets and Reichstein, 2002), meaning that altered membrane stabilisation or membrane solubility could both occur, yet, the contribution of endogenous (–)-limonene is unknown. Future work should examine lipid composition, subcellular membrane oxidative damage in MEP-pathway–modified genotypes with reduced APX activity, and whether specific monoterpenes interact with ROS to cause phytotoxicity. After rewatering, LG12 plants had lower MDA content and upregulated APX, but unchanged SOD activity relative to WT plants (Fig. 7), suggesting SOD-APX mis-regulation and altered redox balance in transgenic plants during recovery. Together, our data indicate that during drought, limonene synthase overexpression and endogenous (–)-limonene emission in LG12 reduced leaf chlorophyl content but don’t affect photosynthesis. LG12 plant has APX-independent feedback that maintains PSII functionality with higher ABA concentration but does not prevent lipid peroxidation (Fig. 8). Upon re-watering, the redox network upregulates APX activity to detoxify H 2 O 2 , lower MDA and re-establish oxidative homeostasis. Thus, endogenous monoterpenes modulate oxidative stress by regulating ABA/ROS signalling during drought and enzymatically enhancing the water-water cycle after re-watering, rather than acting as universal antioxidants. Conclusions Altered MEP-regulated hormonal responses including ABA- and APX-related signalling, rather than direct carbon loss via monoterpene emission, likely restricts biomass in MEP-transformed tobacco. Despite higher drought-induced foliar lipid peroxidation (i.e. MDA concentration) in the (–)-limonene producer, similar carbon assimilation and photosynthetic efficiency indicate compensatory mechanisms that regulate redox homeostasis, attenuate adverse effects to the photosystem and maintain leaf water status. Thus interactions between monoterpene metabolism, growth and oxidative defence maintain leaf performance under stress and secure reproductive output after re-watering. 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Box plots present the (−)-α-pinene, (−)-β-pinene, (−)-limonene and total monoterpene emission rate of three transgenic genotypes and WT plants, with mean value under each box. Post hoc Tukey tests results indicate significant differences between treatment and genotypes by letters. Three-way ANOVA results ( p Values presented) in the table on the right. Fig. 3. Appearance of 3-week-old seedlings (a), shoot dry weight (b), height (c), leaf chlorophyll content (d) of well-watered (WW, blue) and droughted (D, orange) plants before (BE) and during (DE) stem elongation, and seed dry weight of plants exposed to these drought treatments then re-watered until maturity (e), from Experiments 1 and 2. Table and bar plots (±SE) with post hoc Tukey test indicating significant differences between treatments and genotypes by different letters. Three-way ANOVA results ( p values presented) in (b-d), and two-way ANOVA results in (e). Fig. 4. Relationships between (−)-Limonene emission rate ( E Lim ) and soil moisture (a) and leaf water potential (b). Each symbol is an individual plant from Experiment 2 with regression curves fitted in (a & b) with generalised additive model. Histograms with error bars (±SE) located on the top right corner of (a) present the data for LG12 plants after re-watering (LGRW) and well-watered plants (LGWW) on the same day. T-test shows the significant difference between LGRW and LGWW ( P < 0.01, indicated by **). Fig. 5. Leaf water potential (a) osmotic potential (b) and turgor pressure (c) responses to soil moisture (%), net photosynthesis rate ( A net , d) and stomatal conductance ( gsw , e) responses to leaf water potential (Ψ leaf ) and the relationship between A net and gsw (f) for LG12 (green) and wild-type (WT, yellow) plants from Experiments 3 (square and solid line) and 4 (circle and dashed line). ANCOVA results reveal the significant impact of soil moisture or Ψ leaf , experiment (E), genotype (G) and their interactions on the corresponding variables. A single regression line was fitted when no significant effects of genotype, replicate experiments or interactions were reported (b, d, f), otherwise each genotype (a, e) and experiment (c) received a unique regression line, with R 2 reported. Exponential models were used for a, b, d, e, and f, and a linear model for c. Fig. 6. H 2 O 2 (a), SOD (b), APX (c), and MDA (d) response to leaf water potential (Ψ leaf ), and the relationships between APX & SOD (e), MDA & H 2 O 2 (f), MDA & APX (g), ABA & Ψ leaf of LG12 (green) and wild-type (WT, yellow) plants under water deficit, from Experiment 4. ANCOVA results reveal the significant impact of Ψ leaf , SOD, H 2 O 2 or APX, and genotype (G) and their interactions on the corresponding variables. A single regression line was fitted when no significant effects of genotype or interactions were reported (a, b), otherwise each genotype (c – h) received a unique regression line, with R 2 reported. Exponential models were used for a, c, and d, and a linear model for b, e, f, g, and h. Fig. 7. Net photosynthesis ( A net , a), stomatal conductance ( g sw , b), ABA concentration (c), leaf water potential (d), H 2 O 2 content (e), SOD activity (f), APX activity (g) and MDA content (h) of tobacco plants after re-watering from Experiment 4. Boxplots (±SE) present the data for both LG12 and wildtype plants after re-watering (LGRW, WTRW) and well-watered plants (LGWW, WTWW) on the same day, post hoc Tukey test results indicate the significant differences between treatments by letters. Two-way ANOVA results ( p -values) reveal the significant impact of water treatment (T), genotype (G) and their interactions Fig. . Conceptual summary of drought–recovery responses in wild-type (WT) and (−)-limonene–emitting tobacco (LG12) at varying soil moisture %. Only LG12 emitted (−)-limonene, and had decreased leaf chlorophyll content. During initial drough (40-20% soil moisture)t, LG12 displayed a transient emission peak (~20% soil moisture), slightly higher leaf water potential, low APX activity and rapid MDA accumulation. As drought continued (<20% soil moisture), LG12 maintained turgor with elevated ABA but emission rates dropped while MDA remained high with low APX. After re-watering, LG12 increased APX activity without increasing SOD, reduced MDA levels, and maintained higher ABA levels than WT. Information & Authors Information Version history V1 Version 1 18 November 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords antioxidant enzymes apx drought mep pathway monoterpene oxidative stress tobacco volatile emissions water relations Authors Affiliations Hao Zhou 0000-0002-7010-8899 Lancaster University Lancaster Environment Centre View all articles by this author Christoph-Martin Geilfus Hochschule Geisenheim View all articles by this author Axel Mithöfer 0000-0001-5229-6913 Max-Planck-Institut fur chemische Okologie View all articles by this author In-Cheol Jang 0000-0001-9408-4273 Temasek Life Sciences Laboratory Ltd View all articles by this author Kirsti Ashworth Lancaster University Lancaster Environment Centre View all articles by this author Ian Dodd [email protected] Lancaster University Lancaster Environment Centre View all articles by this author Metrics & Citations Metrics Article Usage 296 views 216 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Hao Zhou, Christoph-Martin Geilfus, Axel Mithöfer, et al. Limonene emission in transgenic tobacco downregulates foliar ascorbate peroxidase activity under drought. Authorea . 18 November 2025. DOI: https://doi.org/10.22541/au.176344437.73520858/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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