Seed Priming with Single-Walled Carbon Nanotubes Grafted with Pluronic P85 Preserves the Functional and Structural Characteristics of Pea Plants.

The engineering of carbon nanotubes in the last decades resulted in a variety of applications in electronics, electrochemistry, and biomedicine. A number of reports also evidenced their valuable application in agriculture as plant growth regulators and nanocarriers. In this work, we explored the effect of seed priming with single-walled carbon nanotubes grafted with Pluronic P85 polymer (denoted P85-SWCNT) on Pisum sativum (var. RAN-1) seed germination, early stages of plant development, leaf anatomy, and photosynthetic efficiency. We evaluated the observed effects in relation to hydro- (control) and P85-primed seeds. Our data clearly revealed that seed priming with P85-SWCNT is safe for the plant since it does not impair the seed germination, plant development, leaf anatomy, biomass, and photosynthetic activity, and even increases the amount of photochemically active photosystem II centers in a concentration-dependent manner. Only 300 mg/L concentration exerts an adverse effect on those parameters. The P85 polymer, however, was found to exhibit a number of negative effects on plant growth (i.e., root length, leaf anatomy, biomass accumulation and photoprotection capability), most probably related to the unfavorable interaction of P85 unimers with plant membranes. Our findings substantiate the future exploration and exploitation of P85-SWCNT as nanocarriers of specific substances promoting not only plant growth at optimal conditions but also better plant performance under a variety of environmental stresses.

Light Quality Modulates Photosynthesis and Antioxidant Properties of B. vulgaris L. Plants from Seeds Irradiated with High-Energy Heavy Ions: Implications for Cultivation in Space.

Beta vulgaris L. is a crop selected for cultivation in Space for its nutritional properties. However, exposure to ionizing radiation (IR) can alter plant photosynthetic performance and phytochemical production in the extraterrestrial environment. This study investigated if plant growth under different light quality regimes (FL-white fluorescent; RGB-red-green-blue; RB-red-blue) modifies the photosynthetic behavior and bioactive compound synthesis of plants sprouted by dry seeds irradiated with carbon or titanium high-energy ions. The study evidenced that: (i) the plant response depends on the type of heavyion; (ii) control and C-ion-irradiated plants were similar for photosynthetic pigment content and PSII photochemical efficiency, regardless of the LQ regime; (iii) under FL, net photosynthesis (AN) and water use efficiency (iWUE) declined in C- and Ti-ion plants compared to control, while the growth of irradiated plants under RGB and RB regimes offset these differences; (iv) the interaction Ti-ion× RB improved iWUE, and stimulated the production of pigments, carbohydrates, and antioxidants. The overall results highlighted that the cultivation of irradiated plants under specific LQ regimes effectively regulates photosynthesis and bioactive compound amounts in leaf edible tissues. In particular, the interaction Ti-ion × RB improved iWUE and increased pigments, carbohydrates, and antioxidant content.

Manipulation of light quality is an effective tool to regulate photosynthetic capacity and fruit antioxidant properties of Solanum lycopersicum L. cv. 'Microtom' in a controlled environment.

Light quality plays an essential role in setting plant structural and functional traits, including antioxidant compounds. This paper aimed to assess how manipulating the light spectrum during growth may regulate the photosynthetic activity and fruit bioactive compound synthesis in Solanum lycopersicum L. cv. 'Microtom' to improve plant physiological performance and fruit nutritional value. Plants were cultivated under three light quality regimes: red-green-blue LEDs (RGB), red-blue LEDs (RB) and white fluorescent lamps (FL), from sowing to fruit ripening. Leaf functional traits, photosynthetic efficiency, Rubisco and D1 protein expression, and antioxidant production in fruits were analyzed. Compared to FL, RGB and RB regimes reduced height and increased leaf number and specific leaf area, enhancing plant dwarf growth. The RGB regime improved photosynthesis and stomatal conductance despite lower biomass, favoring Rubisco synthesis and carboxylation rate than RB and FL regimes. The RB light produced plants with fewer flowers and fruits with a lower ascorbic acid amount but the highest polyphenol content, antioxidant capacity and SOD and CAT activities. Our data indicate that the high percentage of the green wavelength in the RGB regime promoted photosynthesis and reduced plant reproductive capacity compared to FL and RB. Conversely, the RB regime was the best in favoring the production of health-promoting compounds in tomato berries.

Light Spectral Composition Influences Structural and Eco-Physiological Traits of Solanum lycopersicum L. cv. 'Microtom' in Response to High-LET Ionizing Radiation.

This study evaluated if specific light quality (LQ) regimes (white fluorescent, FL; full-spectrum, FS; red-blue, RB) during plant growth modified morphological and photosynthetic traits of Solanum lycopersicum L. 'Microtom' plants irradiated at the dry seed stage with 25 Gy 48Ca ions (IR). The irradiation reduced plant size while it increased leaf dry matter content (LDMC) and relative water content (RWC) compared to the control. FS and RB light regimes determined a decrease of plant height and a rise of RWC compared to FL plants. The irradiation under FS and RB regimes favoured the development of dwarf plants and improved the leaf water status. Under the FL regime, irradiated plants showed reduced photosynthesis and stomatal conductance. The opposite behavior was observed in RB irradiated plants in which gas exchanges were significantly stimulated. RB regime enhanced Rubisco expression in irradiated plants also inducing anatomical and functional adjustments (i.e., increase of leaf thickness and incidence of intercellular spaces). Finally, 48Ca ions did not prevent fruit ripening and the achievement of the 'seed-to seed' cycle, irrespective of the LQ regime. Overall, the present study evidenced that RB light regime was the most effective in optimising growth and photosynthetic efficiency of 'Microtom' irradiated plants. These outcomes may help to develop proper cultivation protocols for the growth of dwarf tomato in Controlled Ecological Life Support Systems (CELSS).

Single-Walled Carbon Nanotubes Modify Leaf Micromorphology, Chloroplast Ultrastructure and Photosynthetic Activity of Pea Plants.

Single-walled carbon nanotubes (SWCNTs) emerge as promising novel carbon-based nanoparticles for use in biomedicine, pharmacology and precision agriculture. They were shown to penetrate cell walls and membranes and to physically interact and exchange electrons with photosynthetic complexes in vitro. Here, for the first time, we studied the concentration-dependent effect of foliar application of copolymer-grafted SWCNTs on the structural and functional characteristics of intact pea plants. The lowest used concentration of 10 mg L-1 did not cause any harmful effects on the studied leaf characteristics, while abundant epicuticular wax generation on both leaf surfaces was observed after 300 mg L-1 treatment. Swelling of both the granal and the stromal regions of thylakoid membranes was detected after application of 100 mg L-1 and was most pronounced after 300 mg L-1. Higher SWCNT doses lead to impaired photosynthesis in terms of lower proton motive force generation, slower generation of non-photochemical quenching and reduced zeaxanthin content; however, the photosystem II function was largely preserved. Our results clearly indicate that SWCNTs affect the photosynthetic apparatus in a concentration-dependent manner. Low doses (10 mg L-1) of SWCNTs appear to be a safe suitable object for future development of nanocarriers for substances that are beneficial for plant growth.

The Interplay between Light Quality and Biostimulant Application Affects the Antioxidant Capacity and Photosynthetic Traits of Soybean (Glycine max L. Merrill).

This paper evaluates the combined effect of biostimulant and light quality on bioactive compound production and seedling growth of soybean (Glycine max L. Merrill) plants. Germinated seeds pre-treated with different concentrations (0.01%, 0.05%, 0.5%) of an amino acid-based biostimulant were grown for 4 days at the dark (D), white fluorescent light (FL), full-spectrum LED (FS), and red-blue (RB) light. Potential changes in the antioxidant content of sprouts were evaluated. Part of the sprouts was left to grow at FL, FS, and RB light regimes for 24 days to assess modifications in plants' anatomical and physiological traits during the early developmental plant stage. The seed pre-treatment with all biostimulant concentrations significantly increased sprout antioxidant compounds, sugar, and protein content compared to the control (seeds treated with H2O). The positive effect on bioactive compounds was improved under FS and RB compared to D and FL light regimes. At the seedling stage, 0.05% was the only concentration of biostimulant effective in increasing the specific leaf area (SLA) and photosynthetic efficiency. Compared to FL, the growth under FS and RB light regimes significantly enhanced the beneficial effect of 0.05% on SLA and photosynthesis. This concentration led to leaf thickness increase and shoot/root ratio reduction. Our findings demonstrated that seed pre-treatment with proper biostimulant concentration in combination with specific light regimes during plant development may represent a useful means to modify the bioactive compound amount and leaf structural and photosynthetic traits.

Functional and Structural Leaf Plasticity Determine Photosynthetic Performances during Drought Stress and Recovery in Two Platanus orientalis Populations from Contrasting Habitats.

In the context of climatic change, more severe and long-lasting droughts will modify the fitness of plants, with potentially worse consequences on the relict trees. We have investigated the leaf phenotypic (anatomical, physiological and biochemical) plasticity in well-watered, drought-stressed and re-watered plants of two populations of Platanus orientalis, an endangered species in the west of the Mediterranean area. The two populations originated in contrasting climate (drier and warmer, Italy (IT) population; more humid and colder, Bulgaria (BG) population). The IT control plants had thicker leaves, enabling them to maintain higher leaf water content in the dry environment, and more spongy parenchyma, which could improve water conductivity of these plants and may result in easier CO2 diffusion than in BG plants. Control BG plants were also characterized by higher photorespiration and leaf antioxidants compared to IT plants. BG plants responded to drought with greater leaf thickness shrinkage. Drought also caused substantial reduction in photosynthetic parameters of both IT and BG plants. After re-watering, photosynthesis did not fully recover in either of the two populations. However, IT leaves became thicker, while photorespiration in BG plants further increased, perhaps indicating sustained activation of defensive mechanisms. Overall, our hypothesis, that plants with a fragmented habitat (i.e., the IT population) lose phenotypic plasticity but acquire traits allowing better resistance to the climate where they became adapted, remains confirmed.

Physiological and structural adjustments of two ecotypes of Platanus orientalis L. from different habitats in response to drought and re-watering.

Platanus orientalis covers a very fragmented area in Europe and, at the edge of its natural distribution, is considered a relic endangered species near extinction. In our study, it was hypothesized that individuals from the edge of the habitat, with stronger climate constrains (drier and warmer environment, Italy, IT ecotype), developed different mechanisms of adaptation than those growing under optimal conditions at the center of the habitat (more humid and colder environment, Bulgaria, BG ecotype). Indeed, the two P. orientalis ecotypes displayed physiological, structural and functional differences already under control (unstressed) conditions. Adaptation to a dry environment stimulated constitutive isoprene emission, determined active stomatal behavior, and modified chloroplast ultrastructure, ultimately allowing more effective use of absorbed light energy for photochemistry. When exposed to short-term acute drought stress, IT plants showed active stomatal control that enhanced instantaneous water use efficiency, and stimulation of isoprene emission that sustained photochemistry and reduced oxidative damages to membranes, as compared to BG plants. None of the P. orientalis ecotypes recovered completely from drought stress after re-watering, confirming the sensitivity of this mesophyte to drought. Nevertheless, the IT ecotype showed less damage and better stability at the level of chloroplast membrane parameters when compared to the BG ecotype, which we interpret as possible adaptation to hostile environments and improved capacity to cope with future, likely more recurrent, drought stress.

Physiological significance of isoprenoids and phenylpropanoids in drought response of Arundinoideae species with contrasting habitats and metabolism.

Physiological, biochemical and morpho-anatomical traits that determine the phenotypic plasticity of plants under drought were tested in two Arundinoideae with contrasting habitats, growth traits and metabolism: the fast-growing Arundo donax, which also is a strong isoprene emitter, and the slow-growing Hakonechloa macra that does not invest on isoprene biosynthesis. In control conditions, A. donax displayed not only higher photosynthesis but also higher concentration of carotenoids and lower phenylpropanoid content than H. macra. In drought-stressed plants, photosynthesis was similarly inhibited in both species, but substantially recovered only in A. donax after rewatering. Decline of photochemical and biochemical parameters, increased concentration of CO2 inside leaves, and impairment of chloroplast ultrastructure were only observed in H. macra indicating damage of photosynthetic machinery under drought. It is suggested that volatile and non-volatile isoprenoids produced by A. donax efficiently preserve the chloroplasts from transient drought damage, while H. macra invests on phenylpropanoids that are less efficient in preserving photosynthesis but likely offer better antioxidant protection under prolonged stress.

Isoprene emission aids recovery of photosynthetic performance in transgenic Nicotiana tabacum following high intensity acute UV-B exposure.

Isoprene emission by terrestrial plants is believed to play a role in mitigating the effects of abiotic stress on photosynthesis. Ultraviolet-B light (UV-B) induces damage to the photosynthetic apparatus of plants, but the role of isoprene in UV-B tolerance is poorly understood. To investigate this putative protective role, we exposed non-emitting (NE) control and transgenic isoprene emitting (IE) Nicotiana tabacum (tobacco) plants to high intensity UV-B exposure. Methanol emissions increased with UV-B intensity, indicating oxidative damage. However, isoprene emission was unaffected during exposure to UV-B radiation, but declined in the 48 h following UV-B treatment at the highest UV-B intensities of 9 and 15 Wm(-2). Photosynthesis and the performance of photosystem II (PSII) declined to similar extents in IE and NE plants following UV-B exposure, suggesting that isoprene emission did not ameliorate the immediate impact of UV-B on photosynthesis. However, after the stress, photosynthesis and PSII recovered in IE plants, which maintained isoprene formation, but not in NE plants. Recovery of IE plants was also associated with elevated antioxidant levels and cycling; suggesting that both isoprene formation and antioxidant systems contributed to reinstating the integrity and functionality of cellular membranes and photosynthesis following exposure to excessive levels of UV-B radiation.

On the use of leaf spectral indices to assess water status and photosynthetic limitations in Olea europaea L. during water-stress and recovery.

Diffusional limitations to photosynthesis, relative water content (RWC), pigment concentrations and their association with reflectance indices were studied in olive (Olea europaea) saplings subjected to water-stress and re-watering. RWC decreased sharply as drought progressed. Following rewatering, RWC gradually increased to pre-stress values. Photosynthesis (A), stomatal conductance (gs), mesophyll conductance (gm), total conductance (gt), photochemical reflectance index (PRI), water index (WI) and relative depth index (RDI) closely followed RWC. In contrast, carotenoid concentration, the carotenoid to chlorophyll ratio, water content reflectance index (WCRI) and structural independent pigment index (SIPI) showed an opposite trend to that of RWC. Photosynthesis scaled linearly with leaf conductance to CO2; however, A measured under non-photorespiratory conditions (A1%O2) was approximately two times greater than A measured at 21% [O2], indicating that photorespiration likely increased in response to drought. A1%O2 also significantly correlated with leaf conductance parameters. These relationships were apparent in saturation type curves, indicating that under non-photorespiratory conditions, CO2 conductance was not the major limitations to A. PRI was significant correlated with RWC. PRI was also very sensitive to pigment concentrations and photosynthesis, and significantly tracked all CO2 conductance parameters. WI, RDI and WCRI were all significantly correlated with RWC, and most notably to leaf transpiration. Overall, PRI correlated more closely with carotenoid concentration than SIPI; whereas WI tracked leaf transpiration more effectively than RDI and WCRI. This study clearly demonstrates that PRI and WI can be used for the fast detection of physiological traits of olive trees subjected to water-stress.

Isoprene improves photochemical efficiency and enhances heat dissipation in plants at physiological temperatures.

Isoprene-emitting plants are better protected against thermal and oxidative stresses. Isoprene may strengthen membranes avoiding their denaturation and may quench reactive oxygen and nitrogen species, achieving a similar protective effect. The physiological role of isoprene in unstressed plants, up to now, is not understood. It is shown here, by monitoring the non-photochemical quenching (NPQ) of chlorophyll fluorescence of leaves with chemically or genetically altered isoprene biosynthesis, that chloroplasts of isoprene-emitting leaves dissipate less energy as heat than chloroplasts of non-emitting leaves, when exposed to physiologically high temperatures (28-37 °C) that do not impair the photosynthetic apparatus. The effect was especially remarkable at foliar temperatures between 30 °C and 35 °C, at which isoprene emission is maximized and NPQ is quenched by about 20%. Isoprene may also allow better stability of photosynthetic membranes and a more efficient electron transfer through PSII at physiological temperatures, explaining most of the NPQ reduction and the slightly higher photochemical quenching that was also observed in isoprene-emitting leaves. The possibility that isoprene emission helps in removing thermal energy at the thylakoid level is also put forward, although such an effect was calculated to be minimal. These experiments expand current evidence that isoprene is an important trait against thermal and oxidative stresses and also explains why plants invest resources in isoprene under unstressed conditions. By improving PSII efficiency and reducing the need for heat dissipation in photosynthetic membranes, isoprene emitters are best fitted to physiologically high temperatures and will have an evolutionary advantage when adapting to a warming climate.

Ultradian variation of isoprene emission, photosynthesis, mesophyll conductance, and optimum temperature sensitivity for isoprene emission in water-stressed Eucalyptus citriodora saplings.

Water availability is a major limiting factor on plant growth and productivity. Considering that Eucalyptus spp. are among the few plant species able to produce both isoprene and monoterpenes, experiments were designed to investigate the response of isoprene emission and isoprenoid concentrations in Eucalyptus citriodora saplings exposed to decreasing fraction of transpirable soil water (FTSW). In particular, this study aimed to assess: (a) the kinetic of water stress-induced variations in photosynthesis, isoprene emission, and leaf isoprenoid concentrations during progressive soil water shortage as a function of FTSW; (b) the ultradian control of isoprene emission and photosynthesis under limited soil water availability; and (c) the optimum temperature sensitivity of isoprene emission and photosynthesis under severe water stress. The optimum temperature for isoprene emission did not change under progressive soil water deficit. However, water stress induced a reallocation of carbon through the MEP/DOXP pathway resulting in a qualitative change of the stored isoprenoids. The ultradian trend of isoprene emission was also unaffected under water stress, and a similar ultradian trend of stomatal and mesophyll conductances was also observed, highlighting a tight coordination between diffusion limitations to photosynthesis during water stress. The kinetics of photosynthetic parameters and isoprene emission in response to decreasing FTSW in E. citriodora are strikingly similar to those measured in other plant functional types. These findings may be useful to refine the algorithms employed in process-based models aiming to precisely up-scale carbon assimilation and isoprenoid emissions at regional and global scales.

Changes in photosynthesis, mesophyll conductance to CO2, and isoprenoid emissions in Populus nigra plants exposed to excess nickel.

Poplar (Populus nigra) plants were grown hydroponically with 30 and 200 µM Ni (Ni30 and Ni200). Photosynthesis limitations and isoprenoid emissions were investigated in two leaf types (mature and developing). Ni stress significantly decreased photosynthesis, and this effect depended on the leaf Ni content, which was lower in mature than in developing leaves. The main limitations to photosynthesis were attributed to mesophyll conductance and metabolism impairment. In Ni-stressed developing leaves, isoprene emission was significantly stimulated. We attribute such stimulation to the lower chloroplastic [CO2] than in control leaves. However chloroplastic [CO2] did not control isoprene emission in mature leaves. Ni stress induced the emission of cis-ß-ocimene in mature leaves, and of linalool in both leaf types. Induced biosynthesis and emission of isoprenoids reveal the onset of antioxidant processes that may also contribute to reduce Ni stress, especially in mature poplar leaves.

BVOC emissions, photosynthetic characteristics and changes in chloroplast ultrastructure of Platanus orientalis L. exposed to elevated CO2 and high temperature.

To investigate the interactive effects of increasing [CO(2)] and heat wave occurrence on isoprene (IE) and methanol (ME) emissions, Platanus orientalis was grown for one month in ambient (380 micromol mol(-1)) or elevated (800 micromol mol(-1)) [CO(2)] and exposed to high temperature (HT) (38 degrees C/4 h). In pre-existing leaves, IE emissions were always higher but ME emissions lower as compared to newly-emerged leaves. They were both stimulated by HT. Elevated [CO(2)] significantly reduced IE in both leaf types, whereas it increased ME in newly-emerged leaves only. In newly-emerged leaves, elevated [CO(2)] decreased photosynthesis and altered the chloroplast ultrastructure and membrane integrity. These harmful effects were amplified by HT. HT did not cause any unfavorable effects in pre-existing leaves, which were characterized by inherently higher IE rates. We conclude that: (1) these results further prove the isoprene's putative thermo-protective role of membranes; (2) HT may likely outweigh the inhibitory effects of elevated [CO(2)] on IE in the future.

The impact of blue light on leaf mesophyll conductance.

Blue light has many direct and indirect effects on photosynthesis. The impact of blue light on mesophyll conductance (g(m)), one of the main diffusive limitation to photosynthesis, was investigated in leaves of Nicotiana tabacum and Platanus orientalis, characterized by high and low g(m), respectively. Leaves were exposed to blue light fractions between 0% and 80% of incident light intensity (300 micromol photons m(-2) s(-1)), the other fraction being supplied as red light. Leaves exposed to blue light showed reduced photosynthesis and unaltered stomatal conductance. The g(m), measured using the chlorophyll fluorescence-based method, was strongly reduced in both plant species. Such a reduction of g(m) may not be real, as several assumptions used for the calculation of g(m) by fluorescence may not hold under blue light. To assess possible artefacts, the electron transport rate measured by fluorescence (J(f)) and by gas-exchange (J(c)) were compared in leaves exposed to different fractions of blue light under non-photorespiratory conditions. The two values were only equal, a prerequisite for correct g(m) measurements, when the illumination was totally provided as red light. Under increasing blue light levels an increasing discrepancy was observed, which suggests that J(f) was not correctly calculated, and that such an error could also upset g(m) measurements. Blue light was not found to change the absorbance of light by leaves, whereas it slightly decreased the distribution of light to PSII. To equate J(f) and J(c) under blue light, a further factor must be added to the J(f) equation, which possibly accounted for the reduced efficiency of energy transfer between the pigments predominantly absorbing blue light (the carotenoids) and the chlorophylls. This correction reduced by about 50% the effect of blue light on g(m). However, the residual reduction of g(m) under blue light was real and significant, although it did not appear to limit the chloroplast CO(2) concentration and, consequently, photosynthesis. Reduction of g(m) might be caused by chloroplast movement to avoid photodamage, in turn affecting the chloroplast surface exposed to intercellular spaces. However, g(m) reduction occurred immediately after exposure to blue light and was complete after less than 3 min, whereas chloroplast relocation was expected to occur more slowly. In addition, fast g(m) reduction was also observed after inhibiting chloroplast movement by cytochalasin. It is therefore concluded that g(m) reduction under blue light is unlikely to be caused by chloroplast movement only, and must be elicited by other, as yet unknown, factors.

Trapping of the quenched conformation associated with non-photochemical quenching of chlorophyll fluorescence at low temperature.

The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures between 5 and 25 degrees C and during rapid jumps of the leaf temperature. At 5 degrees C, NPQ relaxed very slowly in the dark and was sustained for up to 30 min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to 25 degrees C, the quenched state relaxed within 1 min, characteristic for qE, the energy-dependent component of NPQ. Measurements of the membrane permeability (delta A515) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending the model of Horton and co-workers (FEBS Lett 579:4201-4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE.

Singlet oxygen quenching by phenylamides and their parent compounds.

This paper demonstrates for the first time that plant metabolites of the phenylamide type, conjugates of putrescine with hydroxycinnamic acids (p-coumaric, caffeic and ferulic), possess 1O2 quenching properties. Data were obtained confirming that their acidic parent compounds were also able to quench 1O2, as did polyamines (putrescine, spermidine and spermine), and that this ability depends on the number of amino groups. Potentiation of the 1O2 quenching ability of the conjugates relative to both parent components was established. The importance of polyamines and phenylamides in the plant non-enzymatic antioxidant defence at sites of intensive 1O2 generation, such as the photosynthetic centers, was suggested.

Isoprene prevents the negative consequences of high temperature stress in Platanus orientalis leaves.

The phenomenon of enhanced plant thermotolerance by isoprene was studied in leaves of the same age of 1- or 2-year-old Platanus orientalis plants. Our goals were to determine whether the isoprene emission depends on the age of the plant, and whether different emission rates can influence heat resistance in plants of different age. Two-year-old plants emit greater amounts of isoprene and possess better capacity to cope with heat stress than 1-year-old plants. After a high temperature treatment (38°C for 4 h), photosynthetic activity, hydrogen peroxide content, lipid peroxidation and antiradical activity were preserved in isoprene emitting leaves of 1- and 2-year-old plants. However, heat inhibited photosynthesis and PSII efficiency, caused accumulation of H2O2, and increased all indices of membrane damage and antioxidant capacity in leaves of plants of both ages in which isoprene was inhibited by fosmidomycin. In isoprene-inhibited leaves fumigated with exogenous isoprene during the heat treatment, the negative effects on photosynthetic capacity were reduced. These results further support the notion that isoprene plays an important role in protecting photosynthesis against damage at high temperature. It is suggested that isoprene is an important compound of the non-enzymatic defence of plants against thermal stress, possibly contributing to scavenging of reactive oxygen species (ROS) and membrane stabilising capacity, especially in developed plants.

Localized ozone fumigation system for studying ozone effects on photosynthesis, respiration, electron transport rate and isoprene emission in field-grown Mediterranean oak species.

We used a localized ozone (O3) fumigation (LOF) system to study acute and short-term O(3) effects on physiological leaf traits. The LOF system enabled investigation of primary and secondary metabolic responses of similarly and differently aged leaves on the same plant to three different O3 concentrations ([O3]), unconfounded by other influences on O3 sensitivity, such as genetic, meteorological and soil factors. To simulate the diurnal cycle of O3 formation, current-year and 1-year-old Quercus ilex (L.) and Quercus pubescens (L.) leaves were fumigated with O3 at different positions (and hence, different leaf ages) on the same branch over three consecutive days. The LOF system supplied a high [O3] (300+/-50 ppb) on leaves appressed to the vents, and an intermediate, super-ambient [O3] (varying between 120 and 280 ppb) on leaves less than 30 cm from the vent. Leaves more than 60 cm from the O3 vent were exposed to an [O3] comparable with the ambient concentration, with a 100 ppb peak during the hottest hours of the day. Only leaves exposed to the high [O3] were affected by the 3-day treatment, confirming that Mediterranean oak are tolerant to ambient and super-ambient [O3], but may be damaged by acute exposure to high [O3]. Stomatal and mesophyll conductance and photosynthesis were all reduced immediately after fumigation with high [O3], but recovered to control values within 72 h. Both the intercellular and chloroplast CO2 concentrations ([CO2]) remained constant throughout the experiment. Thus, although treatment with a high [O3] may have induced stomatal closure and consequent down-regulation of photosynthesis, we found no evidence that photosynthesis was limited by low [CO2] at the site of fixation. One-year-old leaves of Q. ilex were much less sensitive to O3 than current-year leaves, suggesting that the low stomatal conductance observed in aging leaves limited O3 uptake. No similar effect of leaf age was found in Q. pubescens. Dark respiration decreased during the treatment period, but a similar decrease was observed in leaves exposed to low [O3], and therefore may not be an effect of O3 treatment. Light respiration, on the other hand, was mostly constant in ozone-treated leaves and increased only in leaves in which photosynthesis was temporarily inhibited by high [O3], preventing them from acting as strong sinks that recycle respiratory CO2 in the leaves. There was no evidence of photochemical damage in Q. ilex leaves, whereas Q. pubescens leaves exposed to a high [O3] showed limited photochemical damage, but recovered rapidly. Biochemical markers were affected by the high [O3], indicating accumulation of reactive oxygen species (ROS) and increased denaturation of lipid membranes, followed by activation of isoprene biosynthesis in Q. pubescens leaves. We speculate that the high isoprene emissions helped quench ROS and normalize membrane stability in leaves recovering from O3 stress.