Effect of long-term drought stress on leaf gas exchange and fluorescence parameters in C<Subscript>3</Subscript> and C<Subscript>4</Subscript> plants

A field study was performed on triticale, field bean, maize and amaranth, to find differences between studied species in physiological alterations resulting from progressive response as injuries and/or acclimation to long-term soil drought during various stages of plant development. The measurements of leaf water potential, electrolyte leakage, chlorophyll a fluorescence, leaf gas exchange and yield analysis were done. A special emphasis was given to the measurements of the blue, green, red and far-red fluorescence. Beside, different ratios of the four fluorescence bands (red/far-red: F ₆₉₀/F ₇₄₀, blue/red: F ₄₄₀/F ₆₉₀, blue/far-red: F ₄₄₀/F ₇₄₀ and blue/green: F ₄₄₀/F ₅₂₀) were calculated. Based on both yield analysis and measurements of physiological processes it can be suggested that field bean and maize responded with better tolerance to the water deficit in soil due to the activation of photoprotective mechanism probably connected with synthesis of the phenolic compounds, which can play a role of photoprotectors in different stages of plant development. The photosynthetic apparatus of those two species scattered the excess of excitation energy more effectively, partially through its transfer to PS I. In this way, plants avoided irreversible and/or deep injuries to PS II. The observed changes in the red fluorescence emission and in the F _v/F _m for triticale and amaranth could have occurred due to serious and irreversible photoinhibitory injuries. Probably, field bean and maize acclimatized more effectively to soil drought through the development of effective mechanisms for utilising excitation energy in the photosynthetic conversion of light accompanied by the mechanism protecting the photosynthetic apparatus against the excess of this energy.     

Superoxide dismutase activity in C<Subscript>3</Subscript> and C<Subscript>3</Subscript>/CAM intermediate species of <Emphasis Type="Italic">Clusia</Emphasis>

The C₃-CAM intermediate Clusia minor L. and the C₃ obligate Clusia multiflora H.B.K. plants were exposed for 7 d to a combination of drought stress and high irradiance of about 1200 μmol m⁻² s⁻¹ for 12 h per day. In both species under these conditions a strong decrease in stomatal conductance was observed at dawn and dusk. Changes in stomatal behaviour of C. minor were accompanied by only a low nocturnal accumulation of malate and citrate. Thus, in C. minor drought stress applied in combination with high irradiance limited CAM expression, and possibly this is the main reason why C. minor prefers semi-shaded sites in the field. The mitochondrial MnSOD, in both well watered and stressed plants of two species showed strong diurnal oscillations with maximum activity at dusk. These oscillations can be explained by the engagement of mitochondria in dissipation of an excess of reducing equivalents. In plants which are able to carry out CAM metabolism tricarboxylic acid cycle is expected to be down regulated in the dark period to prevent breakdown of the entire malate and citrate.     

Activity of C<Subscript>4</Subscript> enzymes in C<Subscript>3</Subscript>-type herbaceous plants

The activity of enzymes characteristic for C₄-type photosynthesis was determined in different organs of two herbaceous plants: Reynoutria japonica Houtt. and Helianthus tuberosus L. The activity of phosphoenolpyruvate carboxylase (PEPC) was usually higher in the roots, some of the stem tissues and petioles in comparison to the leaf blades. The highest activity of malic enzymes (NAD-ME, NADP-ME) and phosphoenolpyruvate carboxykinase (PEPCK) was in the petioles and stem tissues of both plants and the lowest in the leaf blades and the pith of Helianthus tuberosus L.     

Deriving room temperature excitation spectra for photosystem I and photosystem II fluorescence in intact leaves from the dependence of <Emphasis Type="Italic">F</Emphasis><Subscript>V</Subscript>/<Emphasis Type="Italic">F</Emphasis><Subscript>M</Subscript> on excitation wavelength

The F ₀ and F _M level fluorescence from a wild-type barley, a Chl b-less mutant barley, and a maize leaf was determined from 430 to 685 nm at 10 nm intervals using pulse amplitude-modulated (PAM) fluorimetry. Variable wavelengths of the pulsed excitation light were achieved by passing the broadband emission of a Xe flash lamp through a birefringent tunable optical filter. For the three leaf types, spectra of F _V/F _M (=(F _M − F ₀)/F _M) have been derived: within each of the three spectra of F _V/F _M, statistically meaningful variations were detected. Also, at distinct wavelength regions, the F _V/F _M differed significantly between leaf types. From spectra of F _V/F _M, excitation spectra of PS I and PS II fluorescence were calculated using a model that considers PS I fluorescence to be constant but variable PS II fluorescence. The photosystem spectra suggest that LHC II absorption results in high values of F _V/F _M between 470 and 490 nm in the two wild-type leaves but the absence of LHC II in the Chl b-less mutant barley leaf decreases the F _V/F _M at these wavelengths. All three leaves exhibited low values of F _V/F _M around 520 nm which was tentatively ascribed to light absorption by PS I-associated carotenoids. In the 550–650 nm region, the F _V/F _M in the maize leaf was lower than in the barley wild-type leaf which is explained with higher light absorption by PS I in maize, which is a NADP-ME C₄ species, than in barley, a C₃ species. Finally, low values of F _V/F _M at 685 in maize leaf and in the Chl b-less mutant barley leaf are in agreement with preferential PS I absorption at this wavelength. The potential use of spectra of the F _V/F _M ratio to derive information on spectral absorption properties of PS I and PS II is discussed.     

Temperature determines the occurrence of CAM or C<Subscript>3</Subscript> photosynthesis in pineapple plantlets grown <Emphasis Type="Italic">in vitro</Emphasis>

Summary Ananas comosus (L.) Merr. var. Smooth Cayenne plants when grown in vitro under different temperature regimes developed as CAM or as C₃ plants. The plants used in this study were developed from the lateral buds of the nodal etiolated stem explants cultured on Murashige and Skoog medium for 3 mo. The cultures were maintained under a 16-h photoperiod for different thermoperiods. With 28°C light/15°C dark thermoperiod, as compared with constant 28°C light and dark, pineapple plants had a succulence index two times greater, and also a greater nocturnal titratable acidity and phosphoenolpyruvate carboxylase (PEPCase) activity, indicating CAM-type photosynthesis. The highest abscisic acid (ABA) level occurred during the light period, 8 h prior to maximum PEPCase activity, while the indole-3-acetic acid (IAA) peak was found during the dark period, coinciding with the time of highest PEPCase activity. These plants were also smaller with thicker leaves and fewer roots, but had greater dry weight. Their leaves showed histological characteristics of CAM plants, such as the presence of greater quantities of chlorenchyma and hypoderm. In addition, their vascular system was more conspicuous. In contrast, under constant temperature (28°C light/dark) plants showed little succulence in the leaves. There was no significant acid oscillation and diurnal variation in PEPCase activity in these plants, suggesting the occurrence of C₃ photosynthesis. Also, no diurnal variation in ABA and IAA contents was observed. The results of this study clearly indicate a role for temperature in determining the type of carbon fixation pathway in in vitro grown pineapple. Evidence that ABA and IAA participate in CAM signaling is provided.     

Long chain fatty acyl-CoA modulation of H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at mitochondrial complex I

Complex I is responsible for most of the mitochondrial H₂O₂ release, low during the oxidation of the NAD linked substrates and high during succinate oxidation, via reverse electron flow. This H₂O₂ production appear physiological since it occurs at submillimolar concentrations of succinate also in the presence of NAD substrates in heart (present work) and rat brain mitochondria (Zoccarato et al., Biochem J, 406:125–129, 2007). Long chain fatty acyl-CoAs, but not fatty acids, act as strong inhibitors of succinate dependent H₂O₂ release. The inhibitory effect of acyl-CoAs is independent of their oxidation, being relieved by carnitine and unaffected or potentiated by malonyl-CoA. The inhibition appears to depend on the unbound form since the acyl-CoA effect decreases at BSA concentrations higher than 2 mg/ml; it is not dependent on ΔpH or Δp and could depend on the inhibition of reverse electron transfer at complex I, since palmitoyl-CoA inhibits the succinate dependent NAD(P) or acetoacetate reduction.     

Photosynthetic sunfleck utilization potential of understory saplings growing under elevated CO<Subscript>2</Subscript> in FACE

Few studies have evaluated elevated CO₂ responses of trees in variable light despite its prevalence in forest understories and its potential importance for sapling survival. We studied two shade-tolerant species (Acer rubrum, Cornus florida) and two shade-intolerant species (Liquidambar styraciflua, Liriodendron tulipifera) growing in the understory of a Pinus taeda plantation under ambient and ambient+200 ppm CO₂ in a free air carbon enrichment (FACE) experiment. Photosynthetic and stomatal responses to artificial changes in light intensity were measured on saplings to determine rates of induction gain under saturating light and induction loss under shade. We expected that growth in elevated CO₂ would alter photosynthetic responses to variable light in these understory saplings. The results showed that elevated CO₂ caused the expected enhancement in steady-state photosynthesis in both high and low light, but did not affect overall stomatal conductance or rates of induction gain in the four species. Induction loss after relatively short shade periods (<6 min) was slower in trees grown in elevated CO₂ than in trees grown in ambient CO₂ despite similar decreases in stomatal conductance. As a result leaves grown in elevated CO₂ that maintained induction well in shade had higher carbon gain during subsequent light flecks than was expected from steady-state light response measurements. Thus, when frequent sunflecks maintain stomatal conductance and photosynthetic induction during the day, enhancements of long-term carbon gain by elevated CO₂ could be underestimated by steady-state photosynthetic measures. With respect to species differences, both a tolerant, A. rubrum, and an intolerant species, L. tulipifera, showed rapid induction gain, but A. rubrum also lost induction rapidly (c. 12 min) in shade. These results, as well as those from independent studies in the literature, show that induction dynamics are not closely related to species shade tolerance. Therefore, it cannot be concluded that shade-tolerant species necessarily induce faster in the variable light conditions common in understories. Although our study is the first to examine dynamic photosynthetic responses to variable light in contrasting species in elevated CO₂, studies on ecologically diverse species will be required to establish whether shade-tolerant and -intolerant species show different photosynthetic responses in elevated CO₂ during sunflecks. We conclude that elevated CO₂ affects dynamic gas exchange most strongly via photosynthetic enhancement during induction as well as in the steady state. Received: 1 April 1999 / Accepted: 16 August 1999     

Synthesis and X-ray crystal structure of [Ag(phendio)<Subscript>2</Subscript>]ClO<Subscript>4</Subscript> (phendio = 1,10-phenanthroline-5,6-dione) and its effects on fungal and mammalian cells

The Cu(II) and Ag(I) complexes, [Cu(phendio)₃](ClO₄)₂4H₂O and [Ag(phendio)₂]ClO₄ (phendio = 1,10-phenanthroline-5,6-dione), are prepared in good yield by reacting phendio with the appropriate metal perchlorate salt. The X-ray crystal structure of the Ag(I) complex shows it to have a pseudo tetrahedral structure. `Metal-free' phendio and the Cu(II) and Ag(I) phendio complexes strongly inhibit the growth of the fungal pathogen Candida albicans, and are more active than their 1,10-phenanthroline analogues. The simple Ag(I) salts, AgCH<sub>3</sub>CO<sub>2</sub>, AgNO<sub>3</sub> and AgClO<sub>4</sub>.H<sub>2</sub>O display superior anti-fungal properties compared to analogous simple Cu(II) and Mn(II) salts, suggesting that the nature of the metal ion strongly influences activity. Exposing C. albicans to `metal-free' phendio, simple Ag(I) salts and [Ag(phendio)₂]ClO₄ causes extensive, non-specific DNA cleavage. `Metal-free' phendio and [Ag(phendio)₂]ClO₄ induce gross distortions in fungal cell morphology and there is evidence for disruption of cell division. Both drugs also exhibit high anti-cancer activity when tested against cultured mammalian cells.     

Effect of hypoxia on photosynthetic activity and antioxidative response in gametophores of <Emphasis Type="Italic">Mnium undulatum</Emphasis>

The effects of hypoxia caused by complete submerging of Mnium undulatum gametophores in water, on their photosynthetic activity and the activity of two antioxidative enzymes: superoxide dismutase (SOD) and catalase (CAT) were investigated. The net photosynthesis was strongly inhibited throughout the experiment, and the strong drop in the maximum quantum yield of the PSII (F_v/F_m) was also observed. Three classes of SOD: MnSOD, FeSOD, Cu/ZnSOD and three isoforms of Cu/ZnSOD were identified. A significant decrease in activity of MnSOD, FeSOD and one Cu/ZnSOD isoform was observed after 24 and 48 h of hypoxia. FeSOD activity decreased already after 1 h of submerging in water and its activity remained at the low level during whole period of the experiment. CAT activity was also strongly inhibited in response to hypoxia stress. The obtained results suggest relationships between photosynthetic activity and antioxidative system in M. undulatum gametophores under oxygen deficiency stress.     

The Regulation of TiO<Subscript>2</Subscript> Nanoparticles on the Expression of Light-Harvesting Complex II and Photosynthesis of Chloroplasts of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Recent studies demonstrated that titanium dioxide nanoparticles (TiO₂ NPs) could significantly promote photosynthesis and plant growth, but its mechanism is still unclear. In this article, we studied the mechanism of light absorption and transfer of chloroplasts of Arabidopsis thaliana caused by TiO₂ NPs treated. The results showed that TiO₂ NPs could induce significant increases of light-harvesting complex II (LHCII) b gene expression and LHCII II content on the thylakoid membrane in A. thaliana, and the increases in LHCII were higher than the non-nano TiO₂ (bulk-TiO₂) treatment. Meanwhile, spectroscopy assays indicated that TiO₂ NPs obviously increased the absorption peak intensity of the chloroplast in red and blue region, the fluorescence quantum yield near 680 nm, the excitation peak intensity near 440 and 480 nm and/or near 650 and 680 nm of the chloroplast. TiO₂ NPs treatment could reduce F ₄₈₀/F ₄₄₀ ratio and increase F ₆₅₀/F ₆₈₀ ratio and accelerate the rate of whole chain electron transport and oxygen evolution of the chloroplast. However, the photosynthesis improvement of the non-nanoTiO₂ treatment was far less effective than TiO₂ NPs treatment. Taken together, TiO₂ NPs could promote the light absorption of chloroplast, regulate the distribution of light energy from PS I to PS II by increasing LHCII and accelerate the transformation from light energy to electronic energy, water photolysis, and oxygen evolution.     

N<Subscript>2</Subscript> fixation in three perennial <Emphasis Type="Italic">Trifolium</Emphasis> species in experimental grasslands of varied plant species richness and composition

This study is the first to investigate quantitative effects of plant community composition and diversity on N₂ fixation in legumes. N₂ fixation in three perennial Trifolium species grown in field plots with varied number of neighbouring species was evaluated with the ¹⁵N natural abundance method (two field sites, several growing seasons, no N addition) and the isotope dilution method (one site, one growing season, 5 g N m⁻²). The proportion of plant N derived from N₂ fixation, pNdfa, was generally high, but the N addition decreased pNdfa, especially in species-poor communities. Also following N addition, the presence of grasses in species-rich communities increased pNdfa in T. hybridum and T. repens L., while legume abundance had the opposite effect. In T. repens, competition for light from grasses appeared to limit growth and thereby the amount of N₂ fixed at the plant level, expressed as mg N₂ fixed per sown seed. We conclude that the occurrence of diversity effects seems to be largely context dependent, with soil N availability being a major determinant, and that species composition and functional traits are more important than species richness regarding how neighbouring plant species influence N₂ fixation in legumes.     

Light and growth temperature alter carbon isotope discrimination and estimated bundle sheath leakiness in C<Subscript>4</Subscript> grasses and dicots

We combined measurements of short-term (during gas exchange) and long-term (from plant dry matter) carbon isotope discrimination to estimate CO₂ leakiness from bundle sheath cells in six C₄ species (three grasses and three dicots) as a function of leaf insertion level, growth temperature and short-term irradiance. The two methods for determining leakiness yielded similar results (P > 0.05) for all species except Setaria macrostachya, which may be explained by the leaf of this species not being accommodating to gas exchange. Leaf insertion level had no effect on leakiness. At the highest growth temperature (36°C) leakiness was lower than at the two lower growth temperatures (16°C and 26°C), between which no differences in leakiness were apparent. Higher irradiance decreased leakiness in three species, while it had no significant effect on the others (there was an opposite trend in two species). The inverse response to increasing irradiance was most marked in the two NAD-ME dicots (both Amaranthus species), which both showed almost 50% leakiness at low light (300 μmol quanta m⁻² s⁻¹) compared to about 30% at high light (1,600 μmol quanta m⁻² s⁻¹). NADP-ME subtype grasses had lower leakiness than NAD-ME dicots. Although there were exceptions, particularly in the effect of irradiance on leakiness in Sorghum and Boerhavia, we conclude that conditions favourable to C₄ photosynthesis (high temperature and high light) lead to a reduction in leakiness.     

A H<Subscript>2</Subscript>O<Subscript>2</Subscript>-producing glyoxal oxidase is required for filamentous growth and pathogenicity in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (<C4) and produces H₂O₂. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by P. J. PuntThe first two authors contributed equally to this workWe dedicate this work to the memory of Jeff Schell, a charismatic and outstanding person who loved science and respected people     

Foliar quality influences tree-herbivore-parasitoid interactions: effects of elevated CO<Subscript>2</Subscript>, O<Subscript>3</Subscript>, and plant genotype

This study examined the effects of carbon dioxide (CO₂)-, ozone (O₃)-, and genotype-mediated changes in quaking aspen (Populus tremuloides) chemistry on performance of the forest tent caterpillar (Malacosoma disstria) and its dipteran parasitoid (Compsilura concinnata) at the Aspen Free-Air CO₂ Enrichment (FACE) site. Parasitized and non-parasitized forest tent caterpillars were reared on two aspen genotypes under elevated levels of CO₂ and O₃, alone and in combination. Foliage was collected for determination of the chemical composition of leaves fed upon by forest tent caterpillars during the period of endoparasitoid larval development. Elevated CO₂ decreased nitrogen levels but had no effect on concentrations of carbon-based compounds. In contrast, elevated O₃ decreased nitrogen and phenolic glycoside levels, but increased concentrations of starch and condensed tannins. Foliar chemistry also differed between aspen genotypes. CO₂, O₃, genotype, and their interactions altered forest tent caterpillar performance, and differentially so between sexes. In general, enriched CO₂ had little effect on forest tent caterpillar performance under ambient O₃, but reduced performance (for insects on one aspen genotype) under elevated O₃. Conversely, elevated O₃ improved forest tent caterpillar performance under ambient, but not elevated, CO₂. Parasitoid larval survivorship decreased under elevated O₃, depending upon levels of CO₂ and aspen genotype. Additionally, larval performance and masses of mature female parasitoids differed between aspen genotypes. These results suggest that host-parasitoid interactions in forest systems may be altered by atmospheric conditions anticipated for the future, and that the degree of change may be influenced by plant genotype.     

Characterization of Myelin Sheath F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP Synthase and its Regulation by IF<Subscript>1</Subscript>

F_oF₁-ATP synthase is the nanomotor responsible for most of ATP synthesis in the cell. In physiological conditions, it carries out ATP synthesis thanks to a proton gradient generated by the respiratory chain in the inner mitochondrial membrane. We previously reported that isolated myelin vesicles (IMV) contain functional F_oF₁-ATP synthase and respiratory chain complexes and are able to conduct an aerobic metabolism, to support the axonal energy demand. In this study, by biochemical assay, Western Blot (WB) analysis and immunofluorescence microscopy, we characterized the IMV F_oF₁-ATP synthase. ATP synthase activity decreased in the presence of the specific inhibitors (olygomicin, DCCD, FCCP, valynomicin/nigericin) and respiratory chain inhibitors (antimycin A, KCN), suggesting a coupling of oxygen consumption and ATP synthesis. ATPase activity was inhibited in low pH conditions. WB and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F₁ (IF₁), a small protein that binds the F₁ moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath. Data are discussed in terms of the role of IF₁ in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events. Overall, data are consistent with an energetic role of myelin sheath, and may shed light on the relationship among demyelination and axonal degeneration.     

Stability and activity of rubisco in chestnut plantlets transferred to <Emphasis Type="Italic">ex vitro</Emphasis> conditions under elevated CO<Subscript>2</Subscript>

Summary Heterotrophic plantlets obtained by in vitro propagation are biochemically different compared to autotrophic plantlets. When heterotrophic plantlets are transferred to ex vitro conditions, higher irradiance levels are generally applied. Irradiance levels higher than those used in vitro lead to oxidative stress symptoms, that can be counteracted by CO₂ concentrations above normal. We analyzed the stability and activity of Rubisco and leaf-soluble sugars and starch contents in chestnut plantlets transferred from in vitro to ex vitro conditions under four treatments obtained by associating two irradiances of 150 (low light, LL) and 300 (high light, HL) μmolm⁻²s⁻¹, respectively three and six times in vitro irradiance, with two CO₂ levels of 350 (low CO₂, LCO₂) and 700 (high CO₂, HCO₂) μll⁻¹. In in vitro plantlets it was possible to immunodetect apparent products of degradation of Rubisco large subunit (LSU). In ex vitro plantlets, these degradation products were no longer dtected except under LL associated with LCO₂. The decrease in soluble sugars and starch in plantlets under HL HCO₂ gave an indication of a faster acquisition of autotrophic characteristics. However, under the same treatment, a down-regulation of Rubisco activity was observed. From the results taken as a whole, two aspects seem to be confirmed: HL HCO₂ is more efficient in inducing an autotrophic behavior in chestnut ex vitro plantlets; actively growing systems as ex vitro plantlets reflect the down-regulation of Rubisco by HCO₂ without accumulation of carbohydrates.     

Physiological and biochemical aspects and molecular mechanisms of plant adaptation to the elevated concentration of atmospheric CO<Subscript>2</Subscript>

The review of publications concerning the impact of increasing CO₂ concentration in the Earths atmosphere (C_a) on higher terrestrial plants. The physiological changes in plants induced by increasing C_a, including growth and biochemical composition, the characteristics of photosynthesis and respiration, as well as the molecular mechanisms of the regulation of the activity of most important biosynthetic enzymes at early and late stages of the exposure to elevated C_a are under consideration. Various concepts of metabolic regulation during acclimation to increasing CO₂ concentration are critically reviewed. The pathways of possible involvement of carbonic anhydrase-mediated systems of CO₂ transport and concentration during C₃ photosynthesis of higher plants, the metabolic and signal mechanisms of photosynthesis inhibition by carbohydrates and the role of ethylene at elevated C_a are presented. The effect of elevated C_a on plant development and source-sink relations, as well as its interaction with other environmental factors, such as mineral, primarily nitrogen nutrition, light, temperature, and water regime, are discussed in with the context of potential forecasting of the consequences of increase in C_a and temperature for the activities of various higher plant forms in the rapidly changing climate.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 129–145.Original Russian Text Copyright © 2005 by Romanova.     

Gain of function of stomatal movements in rooting <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. plants: regulation by H<Subscript>2</Subscript>O<Subscript>2</Subscript> is independent of ABA before the protruding of roots

Reactive oxygen species (ROS), namely superoxide radical (O₂ ⁻) and hydrogen peroxide (H₂O₂) are generated when plant tissues endure a variety of environmental stresses, including light stress. The extremely short life times of ROS makes the study of their production in planta very difficult. The use of ROS-specific tracer dyes, 3-3′ diaminobenzidine and nitroblue tetrazolium, together with high-resolution imaging provides the opportunity to identify sites of photooxidative stress response by ROS accumulation. This technique was applied to grapevine during the first 7 days after transfer from in vitro to ex vitro under an irradiance 4-fold higher than in vitro. ROS accumulation was detected in the first days of analysis, which gradually decreased to levels comparable to greenhouse leaves. O₂ ⁻ was uniformly distributed while H₂O₂ accumulated preferentially in veins, wounds and stomatal guard and surrounding cells. To evaluate the role of H₂O₂ in stomatal functioning and its crosstalk with abscisic acid (ABA) we focused on the percentage of coloured structures, stomatal aperture and ABA concentration. We propose that the high H₂O₂ level triggered by increased light is responsible for the activation of a signalling pathway over stomatal cells, in a process apparently irrespective of ABA regulation prior to root protrusion. This could explain the gain of function of a low yet consistent percentage of stomatal cells, essential for plant survival during the ontogenic period in analysis.     

Correlation between photorespiration,CO<Subscript>2</Subscript>-assimilation and spatiotemporal dynamics of photosynthesis in leaves of the C<Subscript>3</Subscript>-photosynthesis/crassulacean acid metabolism-intermediate species <Emphasis Type="Italic">Clusia minor</Emphasis> L. (Clusiaceae)

The tree Clusia minor L. (Clusiaceae) operates with different modes of photosynthesis in response to different combinations of environmental parameters. Here plants were subjected to experimental conditions eliciting performance of C₃-photosynthesis and crassulacean acid metabolism (CAM), respectively. A combination of instruments was used to determine CO₂ and water vapour gas exchange, relative quantum use efficiency of photosynthesis (Φ_PSII) and for the first time in such studies also photorespiration simultaneously with the other parameters. In the C₃-mode photorespiration was constant during the light period, where oxygenase activity of ribulose-bis-phosphate carboxylase/oxygenase (RubisCO) was ranging between 32.1 and 35.7% of total RubisCO activity. In the CAM-mode photorespiration depended on the CAM phases. In phase II in the morning was 15.6%. In phase IV in the afternoon initially it was 37.9% and then declined to 17.6% of total RubisCO activity towards the evening. Anatomically leaves of C. minor are differentiated in palisade and spongy parenchyma with an internal air space of 9.3% of the total volume and therefore could be structurally homobaric. However, heterogeneity of Φ_PSII under both non-photorespiratory and photorespiratory conditions in the C₃- and CAM-mode indicated that lateral diffusion of CO₂ and O₂ were subject to limitations showing that leaves are functionally heterobaric.     

Involvement of phospholipase A<Subscript>2</Subscript> in the response of <Emphasis Type="Italic">Solanum</Emphasis> species to an elicitor from <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Changes in activity of phospholipase A₂ (PLA₂), a key enzyme in lipid metabolism and signal network in defence mechanisms, were investigated in Solanum species and Phytophthora infestans interaction. We have compared PLA₂ activity in response to an elicitor, a culture filtrate (CF) derived from P. infestans, in non-host resistant Solanum nigrum var. gigantea, field resistant S. tuberosum cv Bzura and susceptible S. tuberosum clone H-8105. To elucidate the contribution of specific forms of PLA₂ to plant defence mechanism reasonably selective PLA₂ inhibitors, haloenol lactone suicide substrate (HELSS) and p-bromophenacyl bromide (BPB), which discriminate between Ca⁺²-independent PLA₂ (iPLA₂) and Ca⁺²-dependent secretory PLA₂ (sPLA₂), were used. The in vivo and in vitro effects of the inhibitors on PLA₂ activity and on generation of reactive oxygen species (ROS) induced by CF in the studied plants were assayed. We found that PLA₂ activity increased in response to CF treatment, displaying various kinetics and intensity depending on the resistance status of a given genotype. Differences among the genotypes in the effects of each inhibitor on CF-induced PLA₂ activity and on ROS production may reflect the diversity of PLA₂ isoforms in plants. Contrary to BPB, the inhibitory effect of HELSS was observable mainly on CF-induced PLA₂ activity, which suggests that iPLA₂ participates in signal transduction in defence reactions. Various effects of the two inhibitors on PLA₂ activity and ROS production suggest different contribution of sPLA₂ and iPLA₂ to modulation of defence reactions in the interaction between Solanum genotypes and P. infestans.