Engineered poplar for bioproduction of the triterpene squalene.

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 that have many commercial roles as specialty chemicals. Poplar is a fast growing, biomassdense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce significant quantities 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.

Molecular characteristics of isoprene synthase and its control effects on isoprene emissions from tropical trees.

The isoprene emission rate from plants is simulated by a function of light intensity and leaf temperature, and the G-93 formula is the most extensively applied algorithm for this purpose. Isoprene is biosynthesized by the enzyme isoprene synthase (IspS), and instantly emitted from the leaf. Enzyme kinetics of IspS and substrate availability are important factors involved in the short-term leaf-level control of isoprene emissions. It is thus assumed that the parameters of G-93 may correlate with the kinetics of IspSs, however, at present there is no data available on the relationship between these two parameters. In this investigation, six IspS genes from tropical trees were cloned, their properties characterized, and the relationship between the enzyme kinetics of IspSs and the parameters of G-93 examined. There was a negative correlation between the enzyme kinetics of IspS Km and parameter CT1 of G93, which is used to define the temperature dependency of isoprene emissions. However, performance constant of IspS (kcat/Km) only showed slight positive correlation with CT1.suggesting that the enzyme kinetics of IspS has limited significance in controlling the temperature response of isoprene emissions. The molecular structure of IspS was further elucidated using a molecular dynamics simulation with a focus on the active site in the 6 α-helices bundle. The simulation of the enzyme-substrate complex of IspS from B. variegata predicted a new metal binding domain in helix F (E383) and catalytic motif FXRDRLXE in the A-C loop that could involve the deprotonation of dimethylallyl diphosphate (DMADP) to form a carbocation. Notably, after the binding of a metal ion and DMADP, the active-site closure mechanism was found to involve conformational alterations in the helix H-α1 and transition from a loose to tight enclosure of the 6 α-helices bundles to tune the active pocket size. The characteristics identified for the IspSs from tropical trees could help to explain regional isoprene emissions in tropical areas.

Ozone pollution, water deficit stress and time drive poplar phyllospheric bacterial community structure.

Ground-level ozone (O3) pollution often rise in the summer and coincide with drought stress, which alters the relationships between trees and associated microbial communities in a manner that can have pronounced effects on associated biological activity and ecosystem integrity. Discerning the responses of phyllosphere microbial communities to O3 and water deficit could highlight the ability of plant-microbe interactions to either exacerbate or mitigate the effects of these stressors. Accordingly, this study was designed as the first report to specifically interrogate the impacts of elevated O3 and water deficit stress on phyllospheric bacterial community composition and diversity in hybrid poplar saplings. Significant reductions in phyllospheric bacterial alpha diversity indices were observed, with clear evidence of significant time × water deficit stress interactions. The combination of elevated O3 and water deficit stress shifted in the bacterial community composition over sampling time, resulted in significant increases in the relative abundance of the dominant Gammaproteobacteria phyla together with reductions in Betaproteobacteria. An increased prevalence of Gammaproteobacteria may represent a potential diagnostic dysbiosis-related biosignature associated with poplar disease risk. Significant positive correlations were observed between both Betaproteobacteria abundance and diversity indices and key foliar photosynthetic traits and isoprene emissions, whereas these parameters were negatively correlated with Gammaproteobacteria abundance. These findings suggest that the photosynthesis-related properties in plant leaves are closely related to the makeup of the phyllosphere bacterial community. These data provide novel insight into how plant-associated microbes can help maintain plant health and the stability of the local ecosystem in O3-polluted and dried environments.

A Novel Isoprene Synthase from the Monocot Tree Copernicia prunifera (Arecaceae) Confers Enhanced Drought Tolerance in Transgenic Arabidopsis.

The capacity to emit isoprene, among other stresses, protects plants from drought, but the molecular mechanisms underlying this trait are only partly understood. The Arecaceae (palms) constitute a very interesting model system to test the involvement of isoprene in enhancing drought tolerance, as their high isoprene emissions may have contributed to make them hyperdominant in neotropical dry forests, characterized by recurrent and extended periods of drought stress. In this study we isolated and functionally characterized a novel isoprene synthase, the gene responsible for isoprene biosynthesis, from Copernicia prunifera, a palm from seasonally dry tropical forests. When overexpressed in the non-emitter Arabidopsis thaliana, CprISPS conferred significant levels of isoprene emission, together with enhanced tolerance to water limitation throughout plant growth and development, from germination to maturity. CprISPS overexpressors displayed higher germination, cotyledon/leaf greening, water usage efficiency, and survival than WT Arabidopsis under various types of water limitation. This increased drought tolerance was accompanied by a marked transcriptional up-regulation of both ABA-dependent and ABA-independent key drought response genes. Taken together, these results demonstrate the capacity of CprISPS to enhance drought tolerance in Arabidopsis and suggest that isoprene emission could have evolved in Arecaceae as an adaptive mechanism against drought.

CO2 -responsiveness of leaf isoprene emission: Why do species differ?

Leaf isoprene emission rate, I, decreases with increasing atmospheric CO2 concentration with major implications for global change. There is a significant interspecific variability in [CO2 ]-responsiveness of I, but the extent of this variation is unknown and its reasons are not understood. We hypothesized that the magnitude of emission reduction reflects the size and changeability of precursor pools responsible for isoprene emission (dimethylallyl diphosphate, DMADP and 2-methyl-erythritol 2,4-cyclodiphosphate, MEcDP). Changes in I and intermediate pool sizes upon increase of [CO2 ] from 400 to 1500 µmol/mol were studied in nine woody species spanning boreal to tropical ecosystems. I varied 10-fold, total substrate pool size 37-fold and the ratio of DMADP/MEcDP pool sizes 57-fold. At higher [CO2 ], I was reduced on average by 65%, but [CO2 ]-responsiveness varied an order of magnitude across species. The increase in [CO2 ] resulted in concomitant reductions in both substrate pools. The variation in [CO2 ]-responsiveness across species scaled with the reduction in pool sizes, the substrate pool size supported and the share of DMADP in total substrate pool. This study highlights a major interspecific variation in [CO2 ]-responsiveness of isoprene emission and conclusively links this variation to interspecific variability in [CO2 ] effects on substrate availability and intermediate pool size.

Growth condition controls on G-93 parameters of isoprene emission from tropical trees.

Despite its major role in global isoprene emission, information on the environmental control of isoprene emission from tropical trees has remained scarce. Thus, in this study, we examined the relationship between parameters of G-93 isoprene emission formula (CT1, CT2, and α), growth temperature and light intensity, photosynthesis (ɸ, Pmax), isoprene synthase (IspS) level, and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway metabolites using sunlit and shaded leaves of four tropical trees. The results showed that the temperature dependence of isoprene emission from shaded leaves did not differ significantly from sunlit leaves. In contrast, there was a lower saturation irradiance in shaded leaves than in sunlit leaves, the same as temperate plants. The photosynthesis rate of shaded leaves showed lower saturation irradiance, similar to the light dependence of isoprene emission. In most cases, the concentration of MEP pathway metabolites was of lower tendency in shaded leaves versus in sunlit leaves, whereas no significant difference was noted in IspS level between sunlit and shaded leaves. Correlation analysis between these parameters found that CT1 of the G-93 parameter was positively correlated with the concentration of DXP and DMADP, whereas CT2 correlated with the concentration of MEP and the average air temperature for the past 48 h. Similarly, α closely associated with the initial slope (ɸ) of photosynthesis rate, and the basal emission factor is also linked to the photon flux of past days. These results suggest that growth conditions may control the temperature dependence of isoprene emission from tropical trees via the changes in the profiles of MEP pathway metabolites, causing alteration in the parameters of the isoprene emission formula.

Plant hormone effects on isoprene emission from tropical tree in Ficus septica.

Plant hormones and the circadian rhythm have been implicated in coordinated control of isoprene emission in plants. To gain insights into the signalling networks, foliar application of plant hormones was conducted in a native emitter, Ficus septica. Spraying of 50 µM jasmonic acid (JA) gradually decreased isoprene emission by 88% compared with initial levels within 5 days, and emission increased after relief from JA application. We further explored the molecular regulatory mechanism of isoprene emission by analysing photosynthetic rate, gene expression of 2-C-methyl-D-erythrytol 4-phosphate (MEP) pathway, hormone signalling and circadian rhythm processes, and metabolite pool sizes of MEP pathway. Results show that isoprene emission strongly correlated with isoprene synthase (IspS) gene expression and IspS protein levels over the period of JA treatment, indicating transcriptional and possible translational modulation of IspS by JA. Application of JA coordinately modulated genes in the auxin, cytokinin (CK), and circadian rhythm signal transduction pathways. Among the transcriptional factors analysed, MYC2 (JA) and LHY (circadian clock) negatively correlated with isoprene emission. Putative cis-elements predicted on IspS promoter (G-box for MYC2 and circadian for LHY) supports our proposal that isoprene emission is regulated by coordinated transcriptional modulation of IspS gene by phytohormone and circadian rhythm signalling.

The capacity to emit isoprene differentiates the photosynthetic temperature responses of tropical plant species.

Experimental research shows that isoprene emission by plants can improve photosynthetic performance at high temperatures. But whether species that emit isoprene have higher thermal limits than non-emitting species remains largely untested. Tropical plants are adapted to narrow temperature ranges and global warming could result in significant ecosystem restructuring due to small variations in species' thermal tolerances. We compared photosynthetic temperature responses of 26 co-occurring tropical tree and liana species to test whether isoprene-emitting species are more tolerant to high temperatures. We classified species as isoprene emitters versus non-emitters based on published datasets. Maximum temperatures for net photosynthesis were ~1.8°C higher for isoprene-emitting species than for non-emitters, and thermal response curves were 24% wider; differences in optimum temperatures (Topt ) or photosynthetic rates at Topt were not significant. Modelling the carbon cost of isoprene emission, we show that even strong emission rates cause little reduction in the net carbon assimilation advantage over non-emitters at supraoptimal temperatures. Isoprene emissions may alleviate biochemical limitations, which together with stomatal conductance, co-limit photosynthesis above Topt . Our findings provide evidence that isoprene emission may be an adaptation to warmer thermal niches, and that emitting species may fare better under global warming than co-occurring non-emitting species.

Differential regulation of volatile emission from Eucalyptus globulus leaves upon single and combined ozone and wounding treatments through recovery and relationships with ozone uptake.

Both ozone and wounding constitute two key abiotic stress factors, but their interactive effects on plant constitutive and stress-elicited volatile (VOC) emissions are poorly understood. Furthermore, the information on time-dependent modifications in VOC release during recovery from a combined stress is very limited. We studied the modifications in photosynthetic characteristics and constitutive and stress-induced volatile emissions in response to single and combined applications of acute ozone (4, 5, and 6 ppm) and wounding treatments through recovery (0.5-75 h) in a constitutive isoprene and mono- and sesquiterpene emitter Eucalyptus globulus. Overall, the photosynthetic characteristics were surprisingly resistant to all ozone and wounding treatments. Constitutive isoprene emissions were strongly upregulated by ozone and combined ozone and wounding treatments and remained high through recovery phase, but wounding applied alone reduced isoprene emission. All stress treatments enhanced emissions of lipoxygenase pathway volatiles (LOX), mono- and sesquiterpenes, saturated aldehydes (C7-C10), benzenoids, and geranylgeranyl diphosphate (GGDP) pathway volatiles. Once elicited, GGDP volatile, saturated aldehyde and benzenoid emissions remained high through the recovery period. In contrast, LOX emissions, and total mono- and sesquiterpene emissions decreased through recovery period. However, secondary rises in total sesquiterpene emissions at 75 h and in total monoterpenes at 25-50 h were observed. Overall, acute ozone and wounding treatments synergistically altered gas exchange characteristics and stress volatile emissions. Through the treatments and recovery period, stomatal ozone uptake rate and volatile emission rates were poorly correlated, reflecting possible ozone-scavenging effect of volatiles and thus, reduction of effective ozone dose and elicitation of induced defense by the acute ozone concentrations applied. These results underscore the important role of interactive stresses on both constitutive and induced volatile emission responses.

Disproportionate photosynthetic decline and inverse relationship between constitutive and induced volatile emissions upon feeding of Quercus robur leaves by large larvae of gypsy moth (Lymantria dispar).

Gypsy moth (Lymantria dispar L., Lymantriinae) is a major pest of pedunculate oak (Quercus robur) forests in Europe, but how its infections scale with foliage physiological characteristics, in particular with photosynthesis rates and emissions of volatile organic compounds has not been studied. Differently from the majority of insect herbivores, large larvae of L. dispar rapidly consume leaf area, and can also bite through tough tissues, including secondary and primary leaf veins. Given the rapid and devastating feeding responses, we hypothesized that infection of Q. robur leaves by L. dispar leads to disproportionate scaling of leaf photosynthesis and constitutive isoprene emissions with damaged leaf area, and to less prominent enhancements of induced volatile release. Leaves with 0% (control) to 50% of leaf area removed by larvae were studied. Across this range of infection severity, all physiological characteristics were quantitatively correlated with the degree of damage, but all these traits changed disproportionately with the degree of damage. The net assimilation rate was reduced by almost 10-fold and constitutive isoprene emissions by more than 7-fold, whereas the emissions of green leaf volatiles, monoterpenes, methyl salicylate and the homoterpene (3E)-4,8-dimethy-1,3,7-nonatriene scaled negatively and almost linearly with net assimilation rate through damage treatments. This study demonstrates that feeding by large insect herbivores disproportionately alters photosynthetic rate and constitutive isoprene emissions. Furthermore, the leaves have a surprisingly large capacity for enhancement of induced emissions even when foliage photosynthetic function is severely impaired.

Interaction of drought and ozone exposure on isoprene emission from extensively cultivated poplar.

The combined effects of ozone (O3 ) and drought on isoprene emission were studied for the first time. Young hybrid poplars (clone 546, Populus deltoides cv. 55/56 x P. deltoides cv. Imperial) were exposed to O3 (charcoal-filtered air, CF, and non-filtered air +40 ppb, E-O3 ) and soil water stress (well-watered, WW, and mild drought, MD, one-third irrigation) for 96 days. Consistent with light-saturated photosynthesis (Asat ), intercellular CO2 concentration (Ci ) and chlorophyll content, isoprene emission depended on drought, O3 , leaf position and sampling time. Drought stimulated emission (+38.4%), and O3 decreased it (-40.4%). Ozone increased the carbon cost per unit of isoprene emission. Ozone and drought effects were stronger in middle leaves (13th-15th from the apex) than in upper leaves (6th-8th). Only Asat showed a significant interaction between O3 and drought. When the responses were up-scaled to the entire-plant level, however, drought effects on total leaf area translated into around twice higher emission from WW plants in clean air than in E-O3 . Our results suggest that direct effects on plant emission rates and changes in total leaf area may affect isoprene emission from intensively cultivated hybrid poplar under combined MD and O3 exposure, with important feedbacks for air quality.

Isoprenoids and phenylpropanoids are part of the antioxidant defense orchestrated daily by drought-stressed Platanus × acerifolia plants during Mediterranean summers.

The hypothesis was tested that isoprenoids and phenylpropanoids play a prominent role in countering photooxidative stress, following the depletion of antioxidant enzyme activity in plants exposed to severe drought stress under high solar irradiance and high temperatures. Platanus × acerifolia, a high isoprene-emitting species, was drought-stressed during summer (WS) and compared with unstressed controls (WW). Water relations and photosynthetic parameters were measured under mild, moderate, and severe drought stress conditions. Volatile and nonvolatile isoprenoids, antioxidant enzymes, and phenylpropanoids were measured with the same time course, but in four different periods of the day. Drought severely inhibited photosynthesis, whereas it did not markedly affect the photochemical machinery. Isoprene emission and zeaxanthin concentration were higher in WS than in WW leaves, particularly at mild and moderate stresses, and during the hottest hours of the day. The activities of catalase and ascorbate peroxidase steeply declined during the day, while the activity of guaiacol peroxidase and the concentration of quercetin increased during the day, peaking in the hottest hours in both WW and WS plants. Our experiment reveals a sequence of antioxidants that were used daily by plants to orchestrate defense against oxidative stress induced by drought and associated high light and high temperature. Secondary metabolites seem valuable complements of antioxidant enzymes to counter oxidative stress during the hottest daily hours.

How light, temperature, and measurement and growth [CO2] interactively control isoprene emission in hybrid aspen.

Plant isoprene emissions have been modelled assuming independent controls by light, temperature and atmospheric [CO2]. However, the isoprene emission rate is ultimately controlled by the pool size of its immediate substrate, dimethylallyl diphosphate (DMADP), and isoprene synthase activity, implying that the environmental controls might interact. In addition, acclimation to growth [CO2] can shift the share of the control by DMADP pool size and isoprene synthase activity, and thereby alter the environmental sensitivity. Environmental controls of isoprene emission were studied in hybrid aspen (Populus tremula × Populus tremuloides) saplings acclimated either to ambient [CO2] of 380 µmol mol(-1) or elevated [CO2] of 780 µmol mol(-1). The data demonstrated strong interactive effects of environmental drivers and growth [CO2] on isoprene emissions. Light enhancement of isoprene emission was the greatest at intermediate temperatures and was greater in elevated-[CO2]-grown plants, indicating greater enhancement of the DMADP supply. The optimum temperature for isoprene emission was higher at lower light, suggesting activation of alternative DMADP sinks at higher light. In addition, [CO2] inhibition of isoprene emission was lost at a higher temperature with particularly strong effects in elevated-[CO2]-grown plants. Nevertheless, DMADP pool size was still predicted to more strongly control isoprene emission at higher temperatures in elevated-[CO2]-grown plants. We argue that interactive environmental controls and acclimation to growth [CO2] should be incorporated in future isoprene emission models at the level of DMADP pool size.

A model of plant isoprene emission based on available reducing power captures responses to atmospheric CO2.

We present a unifying model for isoprene emission by photosynthesizing leaves based on the hypothesis that isoprene biosynthesis depends on a balance between the supply of photosynthetic reducing power and the demands of carbon fixation. We compared the predictions from our model, as well as from two other widely used models, with measurements of isoprene emission from leaves of Populus nigra and hybrid aspen (Populus tremula × P. tremuloides) in response to changes in leaf internal CO2 concentration (C(i)) and photosynthetic photon flux density (PPFD) under diverse ambient CO2 concentrations (C(a)). Our model reproduces the observed changes in isoprene emissions with C(i) and PPFD, and also reproduces the tendency for the fraction of fixed carbon allocated to isoprene to increase with increasing PPFD. It also provides a simple mechanism for the previously unexplained decrease in the quantum efficiency of isoprene emission with increasing C(a). Experimental and modelled results support our hypothesis. Our model can reproduce the key features of the observations and has the potential to improve process-based modelling of isoprene emissions by land vegetation at the ecosystem and global scales.

Relationship between Cumulative Temperature and Light Intensity and G93 Parameters of Isoprene Emission for the Tropical Tree Ficus septica.

The most widely used isoprene emission algorithm, G93 formula, estimates instantaneous leaf-level isoprene emission using the basal emission factor and light and temperature dependency parameters. The G93 parameters have been suggested to show variation depending on past weather conditions, but no study has closely examined the relationship between past meteorological data and the algorithm parameters. Here, to examine the influence of the past weather on these parameters, we monitored weather conditions, G93 parameters, isoprene synthase transcripts and protein levels, and MEP pathway metabolites in the tropical tree Ficus septica for 12 days and analyzed their relationship with cumulative temperature and light intensity. Plants were illuminated with varying (ascending and descending) light regimes, and our previously developed Ping-Pong optimization method was used to parameterize G93. The cumulative temperature of the past 5 and 7 days positively correlated with CT2 and α, respectively, while the cumulative light intensity of the past 10 days showed the highest negative correlation with α. Concentrations of MEP pathway metabolites and IspS gene expression increased with increasing cumulative temperature. At best, the cumulative temperature of the past 2 days positively correlated with the MEP pathway metabolites and IspS gene expression, while these factors showed a biphasic positive and negative correlation with cumulative light intensity. Optimized G93 captured well the temperature and light dependency of isoprene emission at the beginning of the experiment; however, its performance significantly decreased for the latter stages of the experimental duration, especially for the descending phase. This was successfully improved through separate optimization of the ascending and descending phases, emphasizing the importance of the optimization of formula parameters and model improvement. These results have important implications for the improvement of isoprene emission algorithms, particularly under the predicted increase in future global temperatures.

A unifying conceptual model for the environmental responses of isoprene emissions from plants.

BACKGROUND AND AIMS: Isoprene is the most important volatile organic compound emitted by land plants in terms of abundance and environmental effects. Controls on isoprene emission rates include light, temperature, water supply and CO2 concentration. A need to quantify these controls has long been recognized. There are already models that give realistic results, but they are complex, highly empirical and require separate responses to different drivers. This study sets out to find a simpler, unifying principle. METHODS: A simple model is presented based on the idea of balancing demands for reducing power (derived from photosynthetic electron transport) in primary metabolism versus the secondary pathway that leads to the synthesis of isoprene. This model's ability to account for key features in a variety of experimental data sets is assessed. KEY RESULTS: The model simultaneously predicts the fundamental responses observed in short-term experiments, namely: (1) the decoupling between carbon assimilation and isoprene emission; (2) a continued increase in isoprene emission with photosynthetically active radiation (PAR) at high PAR, after carbon assimilation has saturated; (3) a maximum of isoprene emission at low internal CO2 concentration (ci) and an asymptotic decline thereafter with increasing ci; (4) maintenance of high isoprene emissions when carbon assimilation is restricted by drought; and (5) a temperature optimum higher than that of photosynthesis, but lower than that of isoprene synthase activity. CONCLUSIONS: A simple model was used to test the hypothesis that reducing power available to the synthesis pathway for isoprene varies according to the extent to which the needs of carbon assimilation are satisfied. Despite its simplicity the model explains much in terms of the observed response of isoprene to external drivers as well as the observed decoupling between carbon assimilation and isoprene emission. The concept has the potential to improve global-scale modelling of vegetation isoprene emission.

Ozone impairs the response of isoprene emission to foliar nitrogen and phosphorus in poplar.

Tropospheric ozone (O3) impairs physiological processes of plants while nitrogen (N) deposition may cause imbalances in soil N and other nutrients such as phosphorus (P) suggesting an increase of P demand for plants. However, the combined effect of O3, soil N and P on isoprene emission from leaves has never been tested. We therefore examined isoprene emission in leaves of Oxford poplar clone exposed to O3 (ambient, AA [35.0 nmol mol-1 as daily mean]; 1.5 × AA; 2.0 × AA), soil N (0 and 80 kg N ha-1) and soil P (0, 40 and 80 kg P ha-1) in July and September in a Free-Air Controlled Exposure (FACE) facility. We also investigated the response of isoprene emission to foliar N, P and abscisic acid (ABA) contents in September because the 2-C-methylerythritol-5-phosphate (MEP) pathway of isoprenoid biosynthesis produces ABA. We found that O3 increased isoprene emission in July, which was associated to increased dark respiration, suggesting an activation of metabolism against O3 stress as an initial response. However, O3 decreased isoprene emission in September which was associated to reduced net photosynthesis. In September, isoprene emission was positively correlated with leaf N content and negatively correlated with leaf P content in AA. However, no response of isoprene emission to foliar N and P was found in elevated O3, suggesting that the isoprene responses to foliar N and P depended on the O3 exposure levels. Isoprene emission rate in 1.5 × AA and 2.0 × AA increased with increasing leaf ABA content, indicating accelerated senescence of injured leaves to favor new leaf growth when high O3 and nutritional availability in the soil were combined. Even though foliar N and P usually act as a proxy for isoprene emission rate, the impact of recent abiotic factors such as O3 should be always considered for modeling isoprene emission under climate change.

Response of isoprene emission from poplar saplings to ozone pollution and nitrogen deposition depends on leaf position along the vertical canopy profile.

We investigated isoprene (ISO) emission and gas exchange in leaves from different positions along the vertical canopy profile of poplar saplings (Populus euramericana cv. '74/76'). For a growing season, plants were subjected to four N treatments, control (NC, no N addition), low N (LN, 50 kg N ha-1year-1), middle N (MN, 100 kg N ha-1year-1), high N (HN, 200 kg N ha-1year-1) and three O3 treatments (CF, charcoal-filtered ambient air; NF, non-filtered ambient air; NF + O3, NF + 40 ppb O3). Our results showed the effects of O3 and/or N on standardized ISO rate (ISOrate) and photosynthetic parameters differed along with the leaf position, with larger negative effects of O3 and positive effects of N on ISOrate and photosynthetic parameters in the older leaves. Expanded young leaves were insensitive to both treatments even at very high O3 concentration (67 ppb as 10-h average) and HN treatment. Significant O3 × N interactions were only found in middle and lower leaves, where ISOrate declined by O3 just when N was limited (NC and LN). With increasing light-saturated photosynthesis and chlorophyll content, ISOrate was reduced in the upper leaves but on the contrary increased in middle and lower leaves. The responses of ISOrate to AOT40 (accumulated exposure to hourly O3 concentrations > 40 ppb) and PODY (accumulative stomatal uptake of O3 > Y nmol O3 m-2 PLA s-1) were not significant in upper leaves, but ISOrate significantly decreased with increasing AOT40 or PODY under limited N supply in middle leaves but at all N levels in lower leaves. Overall, ISOrate changed along the vertical canopy profile in response to combined O3 and N exposure, a behavior that should be incorporated into multi-layer canopy models. Our results are relevant for modelling regional isoprene emissions under current and future O3 pollution and N deposition scenarios.

Ozone exposure, nitrogen addition and moderate drought dynamically interact to affect isoprene emission in poplar.

Ozone (O3) pollution can induce changes in plant growth and metabolism, and in turn, affects isoprene emission (ISO), but the extent of these effects may be modified by co-occurring soil water and nitrogen (N) availability. To date, however, much less is known about the combined effects of two of these factors on isoprene emission from plants. We investigated for the first time the combined effects of O3 exposure (CF, charcoal-filtered air; EO3, non-filtered air plus 40 ppb of O3), N addition (N0, no additional N; N50, 50 kg ha-1 year-1 of N) and moderate drought (WW, well-watered; WR, 40% of WW irrigation) on photosynthetic carbon assimilation and ISO emission in hybrid poplar at both leaf- and plant-level over time. Consistent with leaf-level photosynthesis (Pnleaf) and ISO (ISOleaf) responses, plant-level ISO (ISOplant) responses to O3, N addition and moderate drought were more marked after long exposure (September) than short exposure duration (July). EO3 significantly decreased ISOleaf and Pnleaf, while WR and N50 significantly increased them. Although O3 and water interacted significantly to affect Pnleaf over the exposure duration, neither N50 nor WR mitigated the negative effects of EO3 on ISOleaf. When ISO was scaled up to the plant level, the WR-induced increase in ISOleaf under EO3 was offset by a reduction in total leaf area. By contrast, effects of EO3 on ISOplant were not changed by N addition. Our results highlight that the dynamic effects on ISO emission change over the exposure duration depending on involved co-occurring factors and evaluation scales.

Impact of high or low levels of phosphorus and high sodium in soils on productivity and stress tolerance of Arundo donax plants.

The potential of Arundo donax to grow in degraded soils, characterized by excess of salinity (Na+), and phosphorus deficiency (-P) or excess (+P) also coupled with salinity (+NaP), was investigated by combining in vivo plant phenotyping, quantification of metabolites and ultrastructural imaging of leaves with a transcriptome-wide screening. Photosynthesis and growth were impaired by + Na, -P and + NaP. While + Na caused stomatal closure, enhanced biosynthesis of carotenoids, sucrose and isoprene and impaired anatomy of cell walls, +P negatively affected starch production and isoprene emission, and damaged chloroplasts. Finally, +NaP largely inhibited photosynthesis due to stomatal limitations, increased sugar content, induced/repressed a number of genes 10 time higher with respect to + P and + Na, and caused appearance of numerous and large plastoglobules and starch granules in chloroplasts. Our results show that A. donax is sensitive to unbalances of soil ion content, despite activation of defensive mechanisms that enhance plant resilience, growth and biomass production of A. donax under these conditions.