Ethylene response factor Sl-ERF.B.3 is responsive to abiotic stresses and mediates salt and cold stress response regulation in tomato.

Sl-ERF.B.3 (Solanum lycopersicum ethylene response factor B.3) gene encodes for a tomato transcription factor of the ERF (ethylene responsive factor) family. Our results of real-time RT-PCR showed that Sl-ERF.B.3 is an abiotic stress responsive gene, which is induced by cold, heat, and flooding, but downregulated by salinity and drought. To get more insight into the role of Sl-ERF.B.3 in plant response to separate salinity and cold, a comparative study between wild type and two Sl-ERF.B.3 antisense transgenic tomato lines was achieved. Compared with wild type, Sl-ERF.B.3 antisense transgenic plants exhibited a salt stress dependent growth inhibition. This inhibition was significantly enhanced in shoots but reduced in roots, leading to an increased root to shoot ratio. Furthermore, the cold stress essay clearly revealed that introducing antisense Sl-ERF.B.3 in transgenic tomato plants reduces their cell injury and enhances their tolerance against 14 d of cold stress. All these results suggest that Sl-ERF.B.3 gene is involved in plant response to abiotic stresses and may play a role in the layout of stress symptoms under cold stress and in growth regulation under salinity.

Light-mediated K(leaf) induction and contribution of both the PIP1s and PIP2s aquaporins in five tree species: walnut (Juglans regia) case study.

Understanding the response of leaf hydraulic conductance (K(leaf)) to light is a challenge in elucidating plant-water relationships. Recent data have shown that the effect of light on K(leaf) is not systematically related to aquaporin regulation, leading to conflicting conclusions. Here we investigated the relationship between light, K(leaf), and aquaporin transcript levels in five tree species (Juglans regia L., Fagus sylvatica L., Quercus robur L., Salix alba L. and Populus tremula L.) grown in the same environmental conditions, but differing in their K(leaf) responses to light. Moreover, the K(leaf) was measured by two independent methods (high-pressure flow metre (HPFM) and evaporative flux method (EFM)) in the most (J. regia) and least (S. alba) responsive species and the transcript levels of aquaporins were analyzed in perfused and unperfused leaves. Here, we found that the light-induced K(leaf) value was closely related to stronger expression of both the PIP1 and PIP2 aquaporin genes in walnut (J. regia), but to stimulation of PIP1 aquaporins alone in F. sylvatica and Q. robur. In walnut, all newly identified aquaporins were found to be upregulated in the light and downregulated in the dark, further supporting the relationship between the light-mediated induction of K(leaf) and aquaporin expression in walnut. We also demonstrated that the K(leaf) response to light was quality-dependent, K(leaf) being 60% lower in the absence of blue light. This decrease in K(leaf) was correlated with strong downregulation of three PIP2 aquaporins and of all the PIP1 aquaporins tested. These data support a relationship between light-mediated K(leaf) regulation and the abundance of aquaporin transcripts in the walnut tree.

Effect of moderate and high light on photosystem II function in Arabidopsis thaliana depleted in digalactosyl-diacylglycerol.

The response of the heat-sensitive dgd1-2 and dgd1-3 Arabidopsis mutants depleted in the galactolipid DGDG to photoinhibition of chloroplasts photosystem II was studied to verify if there is a relationship between heat stress vulnerability due to depletion in DGDG and the susceptibility to photoinhibitory damage. Non-photochemical quenching (NPQ) is known to dissipate excessive absorbed light energy as heat to protect plants against photodamage. The main component of NPQ is dependent of the transthylakoid pH gradient and is modulated by zeaxanthin (Zx) synthesis. These processes together with chlorophyll fluorescence induction were used to characterize the response of the genotypes. The mutants were more sensitive to photoinhibition to a small extent but this was more severe for dgd1-3 especially at high light intensity. It was deduced that DGDG was not a main factor to influence photoinhibition but other lipid components could affect PSII sensitivity towards photoinhibition in relation to the physical properties of the thylakoid membrane. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Characterization and subcellular localization of geranylgeranyl diphosphate synthase from Catharanthus roseus.

The enzyme geranylgeranyl diphosphate synthase (GGPS: EC 2.5.1.1, EC 2.5.1.10, EC 2.5.1.29) catalyses the formation of geranylgeranyl diphosphate (GGPP) from isopentenyl diphosphate and dimethylallyl diphosphate via three successive condensation reactions. A full-length nucleotide sequence of GGPS (named CrGGPS) was cloned from the medicinal plant Catharanthus roseus. The deduced polypeptide has 383 amino acids with a calculated mass of 41.6 kDa and possesses prenyltransferase signatures characteristic of plant type II GGPS. The enzyme was characterized by functional complementation in carotenoid accumulating strains of Escherichia coli. When cultures of Catharanthus cell lines were treated with methyljasmonate, no specific increase in transcript levels were observed. In plants, GGPS are encoded by a small multigene family and the isoforms have been shown to be localized in three different subcellular compartments: chloroplast, endoplasmic reticulum and mitochondria. We investigated the subcellular distribution of CrGGPS through transient transformations of C. roseus cells with a yellow fluorescent protein-fused construct. Our results clearly indicate that CrGGPS is located to plastids within stroma and stromules.

The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis.

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.

Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta.

In plants, the mevalonic acid (MVA) pathway provides precursors for the formation of triterpenes, sesquiterpenes, phytosterols and primary metabolites important for cell integrity. Here, we have cloned the cDNA encoding enzymes catalysing the final three steps of the MVA pathway from Madagascar periwinkle (Catharanthus roseus), mevalonate kinase (MVK), 5-phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MVD). These cDNA were shown to functionally complement MVA pathway deletion mutants in the yeast Saccharomyces cerevisiae. Transient transformations of C. roseus cells with yellow fluorescent protein (YFP)-fused constructs reveal that PMK and MVD are localised to the peroxisomes, while MVK was cytosolic. These compartmentalisation results were confirmed using the Arabidopsis thaliana MVK, PMK and MVD sequences fused to YFP. Based on these observations and the arguments raised here we conclude that the final steps of the plant MVA pathway are localised to the peroxisome.

Functional aspects of the photosynthetic light reactions in heat stressed Arabidopsis deficient in digalactosyl-diacylglycerol.

Plants are often submitted, in their natural environment, to various abiotic stresses such as heat stress. However, elevated temperature has a detrimental impact on overall plant growth and development. We have examined the physiological response of the dgd1-2 and dgd1-3 Arabidopsis mutants lacking 30-40% of digalactosyl-diacylglycerol (DGDG) exposed to heat constraint. These mutants, which grow similarly to wild type under normal conditions, were previously reported to be defective in basal thermotolerance as measured by cotyledon development. However their functional properties were not described. Chlorophyll fluorescence measurements and absorbance changes at 820nm were used to monitor photosystem II (PSII) and PSI activity, respectively. It was observed that both mutants have similar photosystem activities with some differences. The mutants were less able to use near saturation light energy and elicited higher rates of cyclic PSI electron flow compare to wild type. Arabidopsis leaves exposed to short-term (5min) mild (40°C) or strong (44°C) heat treatment have shown a decline in the operating effective quantum yield of PSII and in the proportion of active PSI reaction centers. However, cyclic PSI electron flow was enhanced. The establishment of the energy-dependent non-photochemical quenching of chlorophyll fluorescence was accelerated but its decline under illumination was inhibited. Furthermore, heat stress affected the process implicated in the redistribution of light excitation energy between the photosystems known as the light state transitions. All the effects of heat stress mentioned above were more intense in the mutant leaves with dgd1-3 being even more susceptible. The decreased DGDG content of the thylakoid membranes together with other lipid changes are proposed to influence the thermo-sensitivity of the light reactions of photosynthesis towards heat stress.

Abolition of photosystem I cyclic electron flow in Arabidopsis thaliana following thermal-stress.

Heat tolerance of Arabidopsis thaliana (WT) and its mutants, crr2-2, lacking NADPH-dehydrogenase (Ndh-pathway), and pgr5, deficient in proton gradient regulation and/or ferredoxin-quinone-reductase (FQR-pathway), was studied from 30 to 46°C. Chlorophyll fluorescence revealed that thermal damage to photosystem II (PSII) was maximal in WT plants following short-term exposure of leaves to moderate or high temperature stress. Thermal stress impaired the photosynthetic electron flow at oxidizing and reducing sides of PSII. This was deduced from the transformation of temperature dependent OJIP to OKP patterns, changes in the relative amplitudes of K-step fluorescence rise and F(v)/F(o) ratio. The amplitude of the K-peak that corresponds to the magnitude of damage to the oxygen evolving complex (OEC) in crr2-2 mutants was about 50% of that observed in WT plants exposed to 46°C. The damage to OEC in pgr5 mutants was relatively smaller and thus their PSII complexes were more heat tolerant. P700 oxidation-reduction kinetics following heat-stress revealed that photosystem I (PSI) complexes remained oxidizable either with 10-ms multiple turn-over flashes or far-red illumination but the complementary cyclic electron flow around PSI (CEF) was abolished in both mutants. With further increase in incubation temperature, CEF was fully suppressed even in WT. Thus, P700 turn-over was not enhanced following thermal stress. Furthermore, the experimental data predicts the onset of pseudocyclic electron transport with molecular oxygen as terminal acceptor in crr2-2 and pgr5 mutants but not in wild type Arabidopsis subjected to severe thermal-stress.

Endophytic hyphal compartmentalization is required for successful symbiotic Ascomycota association with root cells.

Root endophytic fungi are seen as promising alternatives to replace chemical fertilizers and pesticides in sustainable and organic agriculture systems. Fungal endophytes structure formations play key roles in symbiotic intracellular association with plant-roots. To compare the morphologies of Ascomycete endophytic fungi in wheat, we analyzed growth morphologies during endophytic development of hyphae within the cortex of living vs. dead root cells. Confocal laser scanning microscopy (CLSM) was used to characterize fungal cell morphology within lactofuchsin-stained roots. Cell form regularity Ireg and cell growth direction Idir, indexes were used to quantify changes in fungal morphology. Endophyte fungi in living roots had a variable Ireg and Idir values, low colonization abundance and patchy colonization patterns, whereas the same endophyte species in dead (gamma-irradiated) roots had consistent form of cells and mostly grew parallel to the root axis. Knot, coil and vesicle structures dominated in living roots, as putative symbiotic functional organs. Finally, an increased hypha septation in living roots might indicate local specialization within endophytic Ascomycota. Our results suggested that the applied method could be expanded to other septate fungal symbionts (e.g. Basidiomycota). The latter is discussed in light of our results and other recent discoveries.

Biological activity evaluation of the oils from Laurus nobilis of Tunisia and Algeria extracted by supercritical carbon dioxide.

Isolation of essential oil from dried and ground leaves of Laurus nobilis L. from Algeria and Tunisia were obtained with two different methods: by means of classical hydrodistillation (HD) and by using carbon dioxide in the supercritical state (SFE). The effect of extraction pressure on the yield and composition of the L. nobilis volatile oil is examined. The best extraction conditions were as follows: pressure, 9.0 MPa; temperature, 323.15 K; 4 h of extraction and carbon dioxide flow, Phi = 1.2 kg h(-1). Waxes were entrapped in the first separator set at 9.0 MPa and 263.15 K. The oil was recovered in the second separator working at 2.0 bar and 288.15 K. GC/MS analysis of the leaves' volatile oil revealed that it mainly consisted of: 1,8-cineole, linalool, alpha-terpinyl acetate, methyl eugenol and sabinene. The comparison with the hydrodistilled oil did not reveal any big difference. The collection of samples at different extraction times during supercritical extraction allowed us to monitor the change of the oil composition. On the matrice exhaust owing to a SFE at 9.0 MPa, one further extraction at 323.15 K and 24.0 MPa was performed to obtain a mixture of compounds with higher molecular weight. Finally, the antibacterial and antifungal activity of the various extracts has been assayed.

An inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycinebetaine in response to salinity and water stress.

Soil salinity and drought compromise water uptake and lead to osmotic adjustment in xero-halophyte plant species. These important environmental constraints may also have specific effects on plant physiology. Stress-induced accumulation of osmocompatible solutes was analysed in two Tunisian populations of the Mediteranean shrub Atriplex halimus L.-plants originating from a salt-affected coastal site (Monastir) or from a non-saline semi-arid area (Sbikha)-were exposed to nutrient solution containing either low (40 mM) or high (160 mM) doses of NaCl or 15% polyethylene glycol. The low NaCl dose stimulated plant growth in both populations. Plants from Monastir were more resistant to high salinity and exhibited a greater ability to produce glycinebetaine in response to salt stress. Conversely, plants from Sbikha were more resistant to water stress and displayed a higher rate of proline accumulation. Proline accumulated as early as 24 h after stress imposition and such accumulation was reversible. By contrast, glycinebetaine concentration culminated after 10 d of stress and did not decrease after the stress relief. The highest salt resistance of Monastir plants was not due to a lower rate of Na(+) absorption; plants from this population exhibited a higher stomatal conductance and a prodigal water-use strategy leading to lower water-use efficiency than plants from Sbikha. Exogenous application of proline (1 mM) improved the level of drought resistance in Monastir plants through a decrease in oxidative stress quantified by the malondialdehyde concentration, while the exogenous application of glycinebetaine improved the salinity resistance of Sbikha plants through a positive effect on photosystem II efficiency.

Sensitivity of two wheat species's seeds (Triticum durum, variety Karim and Triticum aestivum, variety Salambô) to heat constraint during germination.

The aim of this study is determine the effect of different temperature regimes on germination of wheat seeds and early germination events. Germination is very sensitive to environment conditions, particularly the temperature. Physiological and biochemical responses of wheat seed germination during time at various temperatures (5, 15, 25, 35 and 45 degrees C) have shown that optimal temperature (25 degrees C) favorites a good aptitude to germinate, whereas low (5 degrees C) and high temperature (45 degrees C) were extend the delay of germination. Residuary dry matter's mass of germinating, both Karim and Salambô wheat species, Seeds at 5 and 45 degrees C decreased lightly. This show a less mobilization of reserves to embryo. The activity of totals solubles peroxidases changes with temperature of wheat seed germination. At low (5 degrees C) and high (45 degrees C) temperatures, for both two cultivars, peroxidase activity decreases during germination. With exception, during imbibition (2 h), only 45 degrees C involves a high stimulation of this activity for Karim cultivar. This increase is considered as a biochemical response to high temperature. In control temperature (25 degrees C), we have obtained a light increase in peroxidase activity in comparison with that of dry seeds for both these two wheat species. At the end of this study, we have studied the effect of heat stress on totals proteins content. Nevertheless, both for these two wheat cultivars, at 5 degrees C there is no a markedly change in proteins amount during germination. In control condition (25 degrees C) of germination, there is a slight decrease in this content. Germination under high temperature, for Karim variety, induces a rapid synthesis of Heat Shock Proteins (HSPs) and concomitantly a weak degradation of normal proteins. The opposite phenomenon was observed for Salambô variety (weak synthesis of HSPs and important degradation of normal proteins).

Iron-superoxide dismutase and monodehydroascorbate reductase transcripts accumulate in response to internode rubbing in tomato.

A cDNA encoding an iron-superoxide dismutase (Fe-SOD) was isolated by RACE-PCR from a Lycopersicon esculentum cDNA library. The Fe-SOD cDNA consists of a 746-bp open reading frame and is predicted to encode a protein of 249 amino acids with a calculated molecular mass of 27.9 kDa. The deduced amino acid sequence was very similar to other plant Fe-SODs and a potential chloroplastic targeting was found. To study the induction of oxidative burst in response to mechanical stimulation, the accumulation of Fe-SOD and monodehydroascorbate reductase (MDHAR) mRNAs was analysed in response to young growing internode rubbing in tomato plants. Northern analyses show that Fe-SOD mRNA and MDHAR mRNA accumulated in tomato internodes 10 min after the mechanical stimulation. These results suggest that reactive oxygen species are early involved in the response of a plant to a mechanical stimulation, such as rubbing. The nucleotide sequence data reported in this paper will appear in the NCBI Nucleotide Sequence Databases under the accession number AY262025.