Ecophysiological responses of Betula pendula, Pinus uncinata and Rhododendron ferrugineum in the Catalan Pyrenees to low summer rainfall.

Climate change is producing modifications in the intensity and frequency of rainfall in some regions of the planet. According to predictions, annual rainfall distribution in Western Europe will result in an increase in episodes of drought, thereby negatively affecting nutrient availability. Since high mountain systems will be particularly vulnerable, the physiological and nutritional responses to changes in summer rainfall were monitored over the course of two consecutive summers on three species, which are representative of subalpine forests: birch (Betula pendula Roth.), rhododendron (Rhododendron ferrugineum L.) and mountain pine (Pinus uncinata Mill.). Birch was especially susceptible to scarce precipitation showing alterations in leaf morphology and a decline in net photosynthesis (A) due to stomatal closure, which led to photoinhibition and to early leaf senescence as shown by the photosynthetic nitrogen-use efficiency (PNUE), carbon/nitrogen (C/N) ratio, foliar N and 13C isotope discrimination (Δ13C) results. The Δ13C of the soluble fraction is a good estimator of intrinsic water-use efficiency in this species. Rhododendron and mountain pine had sclerophyllous leaves, as indicated by leaf mass per area, Δ13C, PNUE and C/N results. Rhododendron was particularly affected by short periods of scarce rainfall, which negatively affected gas exchange and photochemistry, and reduced the remobilization of leaf N and P. Mountain pine was the most tolerant species since alterations of gas exchange, photochemistry and Δ13C were not observed. Its highest investment of N in RuBisCo and highest potassium, iron and magnesium leaf concentration contributed to the highest A rates observed.

Effect of environmental stress factors on ecophysiological traits and susceptibility to pathogens of five Populus clones throughout the growing season.

The variability of ecophysiological traits associated with productivity (e.g., water relations, leaf structure, photosynthesis and nitrogen (N) content) and susceptibility to fungal and insect infection were investigated in five poplar clones (Populus deltoides Batr.-Lux clone; Populus nigra L.-58-861 clone and Populus × canadensis Mönch.-Luisa Avanzo, I-214 and Adige clones) during their growing season. The objective of the study was to determine their physiological responses under summer constraints (characteristic of the Mediterranean climate) and to propose clone candidates for environmental restoration activities such as phytoremediation. Relative water content, the radiometric water index and (13)C isotope discrimination (Δ(13)C) results reflected improved water relations in Adige and Lux during summer drought. Leaf structural parameters such as leaf area, leaf mass per area, density (D) and thickness (T) indicated poorer structural adaptations to summer drought in clone 58-861. Nitrogen content and Δ(13)C results pointed to a stomatal component as the main limitant of photosynthesis in all clones. Adige and Lux showed enhanced photoprotection as indicated by the size and the de-epoxidation index of the xanthophyll-cycle pool, and also improved antioxidant defence displayed by higher ascorbate, reduced glutathione, total phenolics and α-tocopherol levels. Photoprotective and antioxidative responses allowed all clones to maintain a high maximum quantum yield of PSII (Fv/Fm) with the exception of Luisa Avanzo and 58-861 which experienced slight photoinhibition in late spring. The study of susceptibility to rust (Melampsora sp.) and lace bug (Monosteira unicostata Muls. and Rey) infections showed Adige and Lux to be the most tolerant. Overall, these two clones presented high adaptability to summer conditions and improved resistance to abiotic and biotic stress, thereby making them highly commendable clones for use in environmental remediation programmes.

Stomatal patchiness in the Mediterranean holm oak (Quercus ilex L.) under water stress in the nursery and in the forest.

The evergreen holm oak Quercus ilex L. is the most representative tree in Mediterranean forests. Accurate estimation of the limiting factors of photosynthesis for Q. ilex and the prediction of ecosystem water-use efficiency by mechanistic models can be achieved only by establishing whether this species shows heterogenic stomatal aperture, and, if so, the circumstances in which this occurs. Here, we collected gas-exchange and chlorophyll fluorescence data in Q. ilex leaves from a nursery to measure the effects of stomatal oscillations on PSII quantum yield (Φ(PSII)) under water stress. Stomatal conductance (g(s)) was used as an integrative indicator of the degree of water stress. Images of chlorophyll fluorescence showed heterogeneous Φ(PSII) when g(s) was <50 mmol H(2)O m(-2) s(-1), representative of severe drought and corresponding to a container capacity <45%. Stomatal patchiness was related to a coefficient of variation (CV) of Φ(PSII) values >2.5%. A parallel study in the forest confirmed heterogeneous Φ(PSII) values in leaves in response to declining water availability. Three kinds of Q. ilex individuals were distinguished: those resprouting after a clear-cut (resprouts, R); intact individuals growing in the same clear-cut area as resprouts (controls, C); and intact individuals in a nearby, undisturbed area (forest controls, CF). Patchiness increased in C and CF in response to increasing drought from early May to late July, whereas in R, Φ(PSII) values were maintained as a result of their improved water relations since the pre-existing roots were associated with a smaller aerial biomass. Patchiness was related to a % CV of Φ(PSII) values >4 and associated in the summer with mean g(s) values of 30 mmol H(2)O m(-2) s(-1). Under milder drought in spring, Φ(PSII) patchiness was less strictly related to g(s) variations, pointing to biochemical limitants of photosynthesis. The occurrence of heterogenic photosynthesis caused by patchy stomatal closure in Q. ilex during severe drought should be taken into account in ecosystem modelling in which harsher water stress conditions associated with climate change are predicted.

Effect of elevated CO2 on carbon partitioning in young Quercus ilex L. during resprouting.

Stored carbon (C) represents a very important C pool with residence times of years to decades in tree organic matter. With the objective of understanding C assimilation, partitioning and remobilization in 2-year-old Quercus ilex L., those trees were exposed for 7 months to different [CO(2)] (elevated: 700 µmol mol(-1) ; and ambient: 350 µmol mol(-1) CO(2)). The (13)C-isotopic composition of the ambient CO(2) (ca.-12.8‰) was modified (to ca.-19.2‰) under the elevated CO(2) conditions in order to analyze C allocation and partitioning before aerial biomass excision, and during the following regrowth (resprouting). Although after 7 months of growth under elevated [CO(2)], Q. ilex plants increased dry matter production, the absence of significant differences in photosynthetic activity suggests that such an increase was lower than expected. Nitrogen availability was not involved in photosynthetic acclimation. The removal of aboveground organs did not enable the balance between C availability and C requirements to be achieved. The isotopic characterization revealed that before the cutting, C partitioning to the stem (main C sink) prevented leaf C accumulation. During regrowth the roots were the organ with more of the labelled C. Furthermore, developing leaves had more C sink strength than shoots during this period. After the cutting, the amount of C delivered from the root to the development of aboveground organs exceeded the requirements of leaves, with the consequent carbohydrate accumulation. These findings demonstrate that, despite having a new C sink, the responsiveness of those resprouts under elevated [CO(2)] conditions will be strongly conditioned by the plant's capacity to use the extra C present in leaves through its allocation to other organs (roots) and processes (respiration).

Oxidative stress induced in tobacco leaves by chloroplast over-expression of maize plastidial transglutaminase.

As part of a project aiming to characterize the role of maize plastidial transglutaminase (chlTGZ) in the plant chloroplast, this paper presents results on stress induced by continuous chlTGZ over-expression in transplastomic tobacco leaves. Thylakoid remodelling induced by chlTGZ over-expression in young leaves of tobacco chloroplasts has already been reported (Ioannidis et al. in Biochem Biophys Acta 1787:1215-1222, 2009). In the present work, we determined the induced alterations in the photosynthetic apparatus, in the chloroplast ultrastructure, and, particularly, the activation of oxidative and antioxidative metabolism pathways, regarding ageing and functionality of the tobacco transformed plants. The results revealed that photochemistry impairment and oxidative stress increased with transplastomic leaf age. The decrease in pigment levels in the transformed leaves was accompanied by an increase in H(2)O(2) and lipid peroxidation. The rise in H(2)O(2) correlated with a decrease in catalase activity, whereas there was an increase in peroxidase activity. In addition, chlTGZ over-expression lead to a drop in reduced glutathione, while Fe-superoxide dismutase activity was higher in transformed than in wild-type leaves. Together with the induced oxidative stress, the over-expressed chlTGZ protein accumulated progressively in chloroplast inclusion bodies. These traits were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections. Consequently, the electron transport between photosystems decrease in the old leaves. In spite of these alterations, transplastomic plants can be maintained and reproduced in vitro. These results are discussed in line with chlTGZ involvement in chloroplast functionality.

Alternative methods for sampling and preservation of photosynthetic pigments and tocopherols in plant material from remote locations.

Current methods for the study of pigments involve freezing in liquid nitrogen and storage at -80 degrees C or lyophilization until HPLC analysis. These requirements greatly restrict ecophysiological research in remote areas where such resources are hardly available. We aimed to overcome such limitations by developing several techniques not requiring freezing or lyophilization. Two species with contrasting foliar characteristics (Olea europaea and Taraxacum officinale) were chosen. Seven preservation methods were designed, optimized and tested in a field trial. These protocols were compared with a control immediately frozen after collection. Pigments and tocopherols were analysed by HPLC. Main artefacts were chlorophyll epimerization or phaeophytinization, carotenoid isomerization, altered de-epoxidation index and tocopherol degradation. Among all methods, sample desiccation in silica gel provides robust samples (pigment composition was unaffected by storage time or temperature) and almost unaltered pigment profiles, except for a shift in epoxidation state. Although liquid nitrogen freezing and subsequent lyophilization or freezer storage were preferred, when these facilities are either not available or not suitable for long-distance transport, desiccation with silica gel, passive extraction in acetone and/or storage of fresh samples in water vapour saturated atmospheres enable a complete pigment characterization. Silica gel is advisable for long-term sample conservation.

Remodeling of tobacco thylakoids by over-expression of maize plastidial transglutaminase.

Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing epsilon-(gamma-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIalpha) at the expense of centers located mainly in stroma thylakoids (PSIIbeta). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.

Response of transglutaminase activity and bound putrescine to changes in light intensity under natural or controlled conditions in Quercus ilex leaves.

In order to further study a previously observed relationship between polyamine (PA) content and changes in irradiation, we examined the level of free and bound PAs, the activity of transglutaminase (TGase, EC 2.3.2.13) and chlorophyll fluorescence in holm oak (Quercus ilex L.) leaves in response to different levels of light intensity and amount. A diurnal trend of free and bound putrescine (F-Put and B-Put, respectively) and TGase activity was observed in plants under natural conditions in the forest, with the highest value corresponding to the maximum light intensity and amount of light received by the leaves. In another set of experiments, potted Q. ilex plants in experimental fields were subjected to a range of periods of natural photosynthetic photon flux density (PPFD) by covering or not covering the whole trees. Under a natural photoperiod (uncovered leaves), B-Put content and TGase activity paralleled the diurnal PPFD pattern, reaching a maximum at the highest PPFD; prior to this maximum, free PAs showed a significant rise. Plants that were in darkness until midday and suddenly exposed to high light intensity showed enhanced TGase activity, resulting in the maximum accumulation of B-Put. The involvement of the accumulation of B-Put reflected in the changes of the B-Put/bound spermidine ratio during the photoprotective responses to high light stress in forest plants is discussed in relation to the chlorophyll fluorescence parameters observed.

Functional role of red (retro)-carotenoids as passive light filters in the leaves of Buxus sempervirens L.: increased protection of photosynthetic tissues?

Red (retro)-carotenoids accumulate in chloroplasts of Buxus sempervirens leaves during the process of winter leaf acclimation. As a result of their irregular presence, different leaf colour phenotypes can be found simultaneously in the same location. Five different colour phenotypes (green, brown, red, orange, and yellow), with a distinct pattern of pigment distribution and concentration, have been characterized. Leaf reddening due to the presence of anthocyanins or carotenoids, is a process frequently observed in plant species under photoinhibitory situations. Two main hypotheses have been proposed to explain the function of such colour change: antioxidative protection exerted by red-coloured molecules, and green light filtering. The potential photoprotective role of red (retro-) carotenoids as light filters was tested in Buxus sempervirens leaves. In shade leaves of this species the upper (adaxial) mesophyll of the lamina was replaced by the equivalent upper part of a different colour phenotype. These hybrid leaves were exposed to a photoinhibitory treatment in order to compare the photoprotective effect exerted by adaxial parts of phenotypes with a different proportion of red (retro)-carotenoids in the lower mesophyll of a shade leaf. The results indicated that the presence of red (retro)-carotenoids in the upper mesophyll did not increase photoprotection of the lower mesophyll when compared with chlorophyll, and the best protection was achieved by an upper green layer. This was due to the fact that the extent of photoinhibition was proportional to the amount of red light transmitted by the upper mesophyll and/or to the chlorophyll pool located above. These results do not exclude a protective function of carotenoids in the upper leaf layer, but imply that, at least under the conditions of this experiment, the accumulation of red pigments in the outer leaf layers does not increase photoprotection in the lower mesophyll.

Leaf morphology, photochemistry and water status changes in resprouting Quercus ilex during drought.

Functional and morphological (structural) characteristics of Quercus ilex L. leaves under drought stress were studied in the forest and in a nursery. We compared undisturbed individuals (controls) with resprouts emerging after clear-cut or excision. When soil water availability was high, gas-exchange was similar in resprouts and controls, despite higher midday leaf water potential, midday leaf hydration and relative water content (RWC). In moderate drought, stomatal closure was found to limit photosynthesis in controls, and in severe drought non-stomatal limitations of photosynthesis were also greater than in resprouts. Leaf structure and chemical composition changed under drought stress. Leaves tended to be smaller in controls with increasing drought, and resprouts had larger leaves and lower leaf mass area (LMA). The relationship between nitrogen (N) content and LMA implied lower N investment in photosynthetic components in controls, which could be responsible for their increased non-stomatal limitation of photosynthesis. Changes were more apparent in leaf density (D) and thickness (T), components of LMA. Decreases in D were related to reductions in cell wall components: hemicellulose, cellulose and lignin. In resprouts, reduced D and leaf T accounted for the higher mesophyll conductance (gmes) to CO2 measured.

Stomatal limitation to CO2 assimilation and down-regulation of photosynthesis in Quercus ilex resprouts in response to slowly imposed drought.

Holm oak (Quercus ilex L.) is native to hot, dry Mediterranean forests where limited water availability often reduces photosynthesis in many species, and forest fires are frequent. Holm oaks resprout after a disturbance, with improved photosynthetic activity and water relations compared with unburned plants. To better understand the role of water availability in this improvement, watering was withheld from container-grown plants, either intact (controls) or resprouts after excision of the shoot, to gradually obtain a wide range of soil water availabilities. At high water availability, gas exchange rates did not differ between controls and resprouts. At moderate soil dryness, net photosynthesis of control plants decreased as a result of increased stomatal limitation, whereas gas exchange rates of resprouts, which had higher midday and predawn leaf water potentials, were unchanged. Under severe drought, resprouts showed a less marked decline in gas exchange than controls and maintained photosystem II integrity, as indicated by chlorophyll fluorescence measurements. Photosynthesis was down-regulated in both plant types in response to reduced CO2 availability caused by high stomatal limitation. Lower non-stomatal limitations in resprouts than in control plants, as evidenced by higher carboxylation velocity and the capacity for ribulose-1,5-bisphosphate regeneration, conferred greater drought resistance under external constraints similar to summer conditions at midday.

Variations in Quercus ilex chloroplast pigment content during summer stress: involvement in photoprotection according to principal component analysis.

We examined chloroplast pigment variation in holm oak (Quercus ilex L.) leaves for two periods under two climatic conditions, at midday during summer. We compared variation between control (unburned) plants and plants burned the preceding summer, since post-fire resprouts show higher photosynthetic rates and lower thermal energy dissipation. Principal component (PC) analysis was performed on nine pigment-content variables for the two periods separately. Two PC factors (PC1 and PC2) explained 83 and 84% of the variance of the data for each period. In both periods, PC1 was marked by positive loading of pigments associated with light absorption or structural function namely neoxanthin, lutein, ß-carotene, chlorophyll a, and chlorophyll b. These pigments were only affected by leaf age. In contrast, PC2 was marked by high loadings of xanthophyll-cycle pigments (associated with photoprotection), and lutein-5,6-epoxide. Leaf content of these pigments was affected by climatic conditions. In the situations considered in PC analysis (leaf types, periods), the lutein-5,6-epoxide content presented a variation pattern similar to that of violaxanthin, and was significantly correlated with thermal dissipation of excess energy (represented by non-photochemical quenching or NPQ). These results suggest a relationship of lutein and lutein-5,6-epoxide with photoprotection.

Carbon isotope discrimination in Quercus ilex resprouts after fire and tree-fell.

Ecophysiological differences related to photosynthesis were compared in holm oak Quercus ilex leaves from undisturbed holm-oak vegetation, resprouts after fire and resprouts after tree-fell. No significant differences in any parameter measured were observed between the two kinds of resprout throughout the first growing season following disturbance. Resprouting leaves showed lower carbon isotope discrimination (Δ) and intercellular CO2 concentration (p i), and higher photosynthesis, leaf conductance and transpiration rates than leaves from undisturbed stands. Nitrogen, soluble protein content and ribulose bisphosphate carboxylase (RuBPCase) activity were 88%, 96% and 45% higher respectively, in both kinds of resprout. The results indicate that photosynthetic capacity, rather than stomatal conductance, is the limiting factor in photosynthesis in resprouts, Chlorophyll content and chlorophyll a/b ratio did not differ between resprouts and undisturbed leaves, indicating that the observed differences were not a result of differences in light environment during leaf development. Leaf mass per area (LMA), was 80% higher in the resprouts, and was negatively related (r=-0.86) to Δ and positively related (r=0.87) to N content. Enhanced carbon assimilation after disturbances resulted in higher water use efficiency, as indicated by lower Δ values in the resprouts. We conclude that the cause of defoliation was not relevant in the physiology of the resprouts, suggesting the importance of underground organs.