Plant Physiology and Biochemistry

The effect of growing pea (Pisum sativum L.) plants with CdCl 2 (0-50 mM) on different plant physiological parameters and antioxidative enzymes of leaves was studied in order to know the possible involvement of this metal in the generation of oxidative stress. In roots and leaves of pea plants Cd produced a significant inhibition of growth as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content of leaves, and an alteration in the nutrient status in both roots and leaves. The ultrastructural analysis of leaves from plants grown with 50 mM CdCl 2 , showed cell disturbances characterized by an increase of mesophyll cell size, and a reduction of intercellular spaces, as well as severe disturbances in chloroplast structure. Alterations in the activated oxygen metabolism of pea plants were also detected, as evidenced by an increase in lipid peroxidation and carbonyl-groups content, as well as a decrease in catalase, SOD and, to a lesser extent, guaiacol peroxidase activities. Glutathione reductase activity did not show significant changes as a result of Cd treatment. A strong reduction of chloroplastic and cytosolic Cu,Zn-SODs by Cd was found, and to a lesser extent of Fe-SOD, while Mn-SOD was only affected by the highest Cd concentrations. Catalase isoenzymes responded differentially, the most acidic isoforms being the most sensitive to Cd treatment. Results obtained suggest that growth of pea plants with CdCl 2 can induce a concentration-dependent oxidative stress situation in leaves, characterized by an accumulation of lipid peroxides and oxidized proteins as a result of the inhibition of the antioxidant systems. These results, together with the ultrastructural data, point to a possible induction of leaf senescence by cadmium.

The object of the work is to evaluate whether rhizobacteria populating dry salty environments can increase resistance in tomato to salt stress. Seven strains of plant growth-promoting bacteria that have 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity were isolated from soil samples taken from the Arava region of southern Israel. Following growth of these seedlings in the presence of 43 mM NaCl for 7 weeks, the bacterium that promoted growth to the greatest extent was selected for further study. DNA analysis of the 16S RNA indicated that the selected bacterium was Achromobacter piechaudii. This bacterium significantly increased the fresh and dry weights of tomato seedlings grown in the presence of up to 172 mM NaCl salt. The bacterium reduced the production of ethylene by tomato seedlings, which was otherwise stimulated when seedlings were challenged with increasing salt concentrations, but did not reduce the content of sodium. However, it slightly increased the uptake of phosphorous and potassium, which may contribute in part to activation of processes involved in the alleviation of the effect of salt. In the presence of salt the bacterium increased the water use efficiency (WUE). This may suggest that the bacterium act to alleviate the salt suppression of photosynthesis. However, the detailed mechanism was not elucidated. The work described in this report is a first step in the development of productive agricultural systems in saline environments.

Brassinosteroids are found in a wide range of organisms from lower to higher plants. They are steroidal plant hormones implicated in the promotion of plant growth and development. Brassinosteroid metabolism has long been known to be altered in plants responding to abiotic stresses and to undergo profound changes in plants interacting with bacterial, fungal and viral pathogens. This review describes the role of brassinosteroids in response to various kinds of stresses via activation of different mechanisms.

As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4 0 ,5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structureefunction studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution insights into the architecture and mechanistic underpinnings of phenylpropanoid metabolizing enzymes contribute to our understanding of the emergence and on-going evolution of specialized phenylpropanoid products, and underscore the molecular basis of metabolic biodiversity at the chemical level. Finally, the detailed knowledge of the structure, function and evolution of these enzymes of specialized metabolism provide a set of experimental templates for the enzyme and metabolic engineering of production platforms for diverse novel compounds with desirable dietary and medicinal properties.

The phytotoxicity imposed by cadmium (Cd) and its detoxifying responses of Bacopa monnieri L. have been investigated. Effect on biomass, photosynthetic pigments and protein level were evaluated as gross effect, while lipid peroxidation and electrolyte leakage reflected oxidative stress. Induction of phytochelatins and enzymatic and non-enzymatic antioxidants were monitored as plants primary and secondary metal detoxifying responses, respectively. Plants accumulated substantial amount of Cd in different plant parts (root, stem and leaf), the maximum being in roots (9240.11 μg g–1 dw after 7 d at 100 μM). Cadmium induced oxidative stress, which was indicated by increase in lipid peroxidation and electrical conductivity with increase in metal concentration and exposure duration. Photosynthetic pigments showed progressive decline while protein showed slight increase at lower concentrations. Enzymes viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) showed stimulation except catalase (CAT, EC 1.11.1.6) which showed declining trend. Initially, an enhanced level of cysteine, glutathione and non-protein thiols was observed, which depleted with increase in exposure concentration and duration. Phytochelatins induced significantly at 10 μM Cd in roots and at 50 μM Cd in leaves. The phytochelatins decreased in roots at 50 μM Cd, which may be correlated with reduced level of GSH, probably due to reduced GR activity, which exerted increased oxidative stress as also evident by the phenotypic changes in the plant like browning of roots and slight yellowing of leaves. Thus, besides synthesis of phytochelatins, availability of GSH and concerted activity of GR seem to play a central role for Bacopa plants to combat oxidative stress caused by metal and to detoxify it. Plants ability to accumulate and tolerate high amount of Cd through enhanced level of PCs and various antioxidants suggest it to be a suitable candidate for phytoremediation.

Flavonoids are representative plant secondary products. In the model plant Arabidopsis thaliana, at least 54 flavonoid molecules (35 flavonols, 11 anthocyanins and 8 proanthocyanidins) are found. Scaffold structures of flavonoids in Arabidopsis are relatively simple. These include kaempferol, quercetin and isorhamnetin for flavonols, cyanidin for anthocyanins and epicatechin for proanthocyanidins. The chemical diversity of flavonoids increases enormously by tailoring reactions which modify these scaffolds, including glycosylation, methylation and acylation. Genes responsible for the formation of flavonoid aglycone structures and their subsequent modification reactions have been extensively characterized by functional genomic efforts-mostly the integration of transcriptomics and metabolic profiling followed by reverse genetic experimentation. This review describes the state-of-art of flavonoid biosynthetic pathway in Arabidopsis regarding both structural and genetic diversity, focusing on the genes encoding enzymes for the biosynthetic reactions and vacuole translocation.

Cakile maritima is a local oilseed halophyte exhibiting potential for secondary metabolite production. In the present study, plant growth, leaf polyphenol content and antioxidant activity were comparatively analyzed in two C. maritima Tunisian accessions (Jerba and Tabarka, respectively sampled from arid and humid bioclimatic stages) under salt constraint. Three-week-old plants were subjected to 0, 100, and 400 mM NaCl for 28 days under glasshouse conditions. A significant variability in salt response was found between both accessions: while Tabarka growth (shoot biomass, leaf expansion) was significantly restricted at 100 and 400 mM NaCl, compared to the control, Jerba growth increased at 100 mM before declining at 400 mM NaCl. The better behaviour of Jerba salt-challenged plants, compared to those of Tabarka, may be related to their higher polyphenol content (1.56- and 1.3-fold the control, at 100 and 400 mM NaCl respectively) and antioxidant activity (smaller IC50 values for both 1,1-diphenyl-2-picrylhydrazyl and superoxide scavenging), associated with lower leaf MDA accumulation (ca. −66% of the control at 100 mM NaCl). Taken together, our findings suggest that halophytes may be interesting for production of antioxidant compounds, and that the accession-dependent capacity to induce antioxidative mechanisms in response to salt, may result in a corresponding variability for growth sustainability.

Soybean plants (Glycine max [L.] Merr var Amsoy 71) were grown in growth chambers with high-phosphorus (high-P) and low-phosphorus (low-P) culture solutions. Low-P treatment reduced shoot growth significantly 7 days after treatment began. Root growth was much less affected by low-P, there being no significant reduction in root growth rate until 17 days had elapsed. The results suggest that low-P treatment decreased soybean growth primarily through an effect on the expansion of the leaf surface which was diminished by 85%, the main effect of low-P being on the rate of expansion of individual leaves. Low-P had a lesser effect on photosynthesis; light saturated photosynthetic rates at ambient and saturating CO2 levels were lowered by 55 and 45%, respectively, after 19 days of low-P treatment. Low-P treatment increased starch concentrations in mature leaves, expanding leaves and fibrous roots; sucrose concentrations, however, were reduced by low-P in leaves and increased in roots. Foliar F-2,6-BP levels were not affected by P treatment in the light but in darkness they increased with high-P and decreased with low-P. The increase in the starch/sucrose ratio in low-P leaves was correlated primarily with changes in the total activities of enzymes of starch and sucrose metabolism.

Plant resistance to pathogens requires the activation of complex metabolic pathways in the infected cells, aimed at recognizing pathogen presence and hindering its propagation within plant tissues. In spite of this both compatible and incompatible responses induce alterations in plant metabolism, only in the latter the plant is able to efficiently block pathogen penetration without suffering excessive damage. One of the most studied incompatible responses is based on the hypersensitive response (HR), in which cells surrounding the site of pathogen penetration switch on genes encoding for phytoalexin synthesis and other pathogenesis related proteins before activating programmed cell death (PCD). The production of reactive oxygen species (ROS) is a key event in HR. Several enzymatic systems have been proposed to be responsible for the oxidative burst characterizing HR. In this review, the involvement of antioxidant redox systems, in particular those related to ascorbate (ASC) and glutathione (GSH), in activating both compatible and incompatible plant responses is analysed. Increasing lines of evidence indicate that alterations in the levels and/or redox state of ASC and/or GSH, as well as in the activity of their redox enzymes, occur during the HR programme. These alterations do not seem to be a mere consequence of the oxidative stress induced by the massive ROS production, but they are induced as part of the transduction pathways triggering defence responses and PCD. The possibility that ASC and GSH systems are links in a redox signalling chain activating defence strategies is also discussed.

Nitric oxide (NO) is a bioactive molecule, which in plants was found to function as prooxidant as well as antioxidant. In the present study, we found that NO donor sodium nitroprusside (SNP) stimulates seed germination and root growth of lupin (Lupinus luteus L. cv. Ventus). Seed germination is promoted at concentrations between 0.1 and 800 µM SNP in a dose-dependent manner. The stimulation was most pronounced after 18 and 24 h and ceased after 48 h of imbibition. The promoting effect of NO on seed germination persisted even in the presence of heavy metals (Pb, Cd) and sodium chloride. Pretreatment of lupin seedlings for 24 h with 10 µM SNP resulted in efficient reduction of the detrimental effect of the abiotic stressors on root growth and morphology. The inhibitory effect of heavy metals on root growth was accompanied by increased activity of superoxide dismutase (SOD, EC 1.15.1.1.), which in roots preincubated with SNP was significantly higher. Some changes in the activity of other antioxidant enzymes, peroxidase (POX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) were also detected. Using the superoxide anion (O 2

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating CO(2) into the biosphere. At the same time Rubisco is an extremely inefficient catalyst and its carboxylase activity is compromised by an opposing oxygenase activity involving atmospheric O(2). The shortcomings of Rubisco have implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. This review uses the information provided in these structures in a structure-based sequence alignment and discusses Rubisco function in the context of structural variations at all levels--amino acid sequence, fold, tertiary and quaternary structure--with an evolutionary perspective and an emphasis on the structural features of the enzyme that may determine its function as a carboxylase.

Reactive oxygen species (ROS) are continuously generated as a consequence of plant metabolic processes due to incomplete reduction of O 2 . Previously considered to be only toxic by-products of metabolism, ROS are now known to act as second messengers in intracellular signalling cascades to trigger tolerance of various abiotic and biotic stresses. The accumulation of proline is frequently observed during the exposure of plants to adverse environmental conditions. Interestingly proline metabolism may also contribute to ROS formation in mitochondria, which play notably a role in hypersensitive response in plants, life-span extension in worms and tumor suppression in animals. Here we review current knowledge about the regulation of proline metabolism in response to environmental constraints and highlight the key role of ROS in the regulation of this metabolism. The impact of proline on ROS generation is also investigated. Deciphering and integrating these relationships at the whole plant level will bring new perspectives on how plants adapt to environmental stresses.

In order to study the effects of high salt stress on PS II in detached wheat (Triticum aestivum) leaves, the seedlings were grown in Knop solution and temperature was 20 AE 2 C. Detached leaves were exposed to high salt stress (0.1e0.5 M NaCl) for 1 h in dark and Chl a fluorescence induction kinetics was measured. Various parameters like Fv/Fm, ABS/RC, ETo/TRo, performance index and area over the florescence curve were measured and the energy pipeline model was deduced in response to salt stress. Our results show that the damage caused due to high salt stress is more prominent at the donor side rather than the acceptor side of PS II. Moreover the effects of high salt stress are largely reversible, as the acceptor side damage is completely recovered (w100%) while the recovery of the donor side is less than 85%. Based on our results we suggest that in response to high salt stress, the donor side of PS II is affected more as compared to the acceptor side of PS II.

Plant growth and productivity is negatively affected by soil salinity. However, it is predicted that plant growth-promoting bacterial (PGPB) endophytes that contain 1-aminocyclopropane-1-carboxylate (ACC) deaminase (E.C. 4.1.99.4) can facilitate plant growth and development in the presence of a number of different stresses. In present study, the ability of ACC deaminase containing PGPB endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6, and their ACC deaminase deficient mutants to promote tomato plant growth in the absence of salt and under two different levels of salt stress (165 mM and 185 mM) was assessed. It was evidence that wild-type bacterial endophytes (P. fluorescens YsS6 and P. migulae 8R6) promoted tomato plant growth significantly even in the absence of stress (salinity). Plants pretreated with wild-type ACC deaminase containing endophytic strains were healthier and grew to a much larger size under high salinity stress compared to plants pretreated with the ACC deaminase deficient mutants or no bacterial treatment (control). The plants pretreated with ACC deaminase containing bacterial endophytes exhibit higher fresh and dry biomass, higher chlorophyll contents, and a greater number of flowers and buds than the other treatments. Since the only difference between wildtype and mutant bacterial endophytes was ACC deaminase activity, it is concluded that this enzyme is directly responsible for the different behavior of tomato plants in response to salt stress. The use of PGPB endophytes with ACC deaminase activity has the potential to facilitate plant growth on land that is not normally suitable for the majority of crops due to their high salt contents.

Hypericum brasiliense is a medicinal herb containing several compounds with important pharmacological activity. In this study, we investigated the effects of water stress (waterlogging and drought) and temperature (low and high, constant and alternate) on the content of betulinic acid and phenolic compounds (quercetin, rutin, 1,5-dihydroxyxanthone, isouliginosin B) in this species. In general, the water stress increased the levels of all of the compounds analyzed, particularly some of the phenolic compounds. On the other hand, the responses to alternating temperatures varied according to the compound. The results for plants kept in growth chambers indicated that low light intensity might have influenced the levels of the compounds. There was also a reallocation of carbon, with water-stressed plants showing a reduction in growth while the levels of the compounds increased. In the temperature treatments, such an increase was evident only for the phenolic compounds.

The biochemical nature of the interaction between the antagonistic fungus Trichoderma harzianum strain T-203 and cucumber roots was studied during the early stages of root colonization by the fungus. Pathogenesis related (PR) proteins of the plant and enzyme activity of the fungus following the penetration and colonization of the roots by T. harzianum were explored up to 72 h post-inoculation. Scanning electron microscopy (SEM) revealed typical fungal structures previously associated with mycoparasitic interactions of T. harzianum strains during biological control. These included hyphal coiling and appressoria formation. Compared to untreated control, cucumber roots treated with T. harzianum T-203 exhibited higher activities of chitinase (EC 3.2.1.14), -1,3-glucanase (EC 3.2.1.6), cellulase (EC 3.2.1.4) and peroxidase (EC 1.11.1.7), up to 72 h post-inoculation. Plants treated with a chemical inducer of the plant defence response, 2,6-dichloroisonicotinic acid (INA) displayed responses that were similar but not identical to those of plants inoculated with T. harzianum. In vivo staining of chitinase activity in fresh root sections allowed the localization of the activity in roots treated with either T. harzianum T-203 or INA. The formation of fluorescent products mainly in intercellular spaces of the induced roots provided evidence for the involvement of the plant defence system. In addition to its well-recognized mycoparasitic nature, it is suggested that Trichoderma's association with roots reduce root disease through activation of the plant's defence response.

Besides the implication of ascorbate and glutathione in the defence against oxidative stress, these two compounds are involved in plant growth and cell cycle control. Ascorbate metabolism is closely linked to the development of embryos and seedlings. Furthermore, ascorbate stimulates cell cycle activity in competent cells, while the oxidised form, dehydroascorbate, blocks normal cell cycle progression. Several possible mechanisms have been proposed to explain the effect of these compounds. The links between glutathione and the cell cycle are less clear. It has long been assumed that both compounds are closely linked by way of the Halliwell-Asada cycle. Any hypothesis concerning the pathways by which ascorbate or glutathione influence cell division, should take this connection into account. However, other mechanisms have been proposed for ascorbate-mediated cell cycle control, e.g. via the thioredoxin pathway.

To gain a better insight into long-term salt-induced oxidative stress, some physiological parameters in marigold (Calendula offıcinalis L.) under 0, 50 and 100 mM NaCl were investigated. Salinity affected most of the considered parameters. High salinity caused reduction in growth parameters, lipid peroxidation and hydrogen peroxide accumulation. Under high salinity stress, a decrease in total glutathione and an increase in total ascorbate (AsA + DHA), accompanied with enhanced glutathione reductase (GR, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) activities, were observed in leaves. In addition, salinity induced a decrease in superoxide dismutase (SOD, EC 1.15.1.1) and peroxidase (POX, EC 1.11.1.7) activities. The decrease in dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities suggests that other mechanisms play a major role in the regeneration of reduced ascorbate. The changes in catalase (CAT, EC 1.11.1.6) activities, both in roots and in leaves, may be important in H 2 O 2 homeostasis.

Abiotic stresses such as salinity, drought, clilling, heavy metal are the major limiting factors for crop productivity. These stresses induce the overproduction of reactive oxygen species (ROS) which are highly reactive and toxic, which must be minimized to protect the cell from oxidative damage. The cell organelles, particularly chloroplast and mitochondria are the major sites of ROS production in plants where excessive rate of electron flow takes place. Plant cells are well equipped to efficiently scavenge ROS and its reaction products by the coordinated and concerted action of antioxidant machinery constituted by vital enzymatic and non-enzymatic antioxidant components. Glutathione reductase (GR, EC 1.6.4.2) and tripeptide glutathione (GSH, g-Glutamyl-Cysteinyl-Glycine) are two major components of ascorbate eglutathione (AsAeGSH) pathway which play significant role in protecting cells against ROS and its reaction products-accrued potential anomalies. Both GR and GSH are physiologically linked together where, GR is a NAD(P)H-dependent enzymatic antioxidant and efficiently maintains the reduced pool of GSH e a cellular thiol. The differential modulation of both GR and GSH in plants has been widely implicated for the significance of these two enigmatic antioxidants as major components of plant defense operations. Considering recent informations gained through molecular-genetic studies, the current paper presents an overview of the structure, localization, biosynthesis (for GSH only), discusses GSH and GR significance in abiotic stress (such as salinity, drought, clilling, heavy metal)-exposed crop plants and also points out unexplored aspects in the current context for future studies.

The functioning of the photosynthetic apparatus of cotton (Gossypium hirsutum) grown during the onset of water limitation was studied by gas-exchange and chlorophyll fluorescence to better understand the adaptation mechanisms of the photosynthetic apparatus to drought conditions. For this, cotton was grown in the field in Central Asia under well-irrigated and moderately drought-stressed conditions. The light and CO 2 responses of photosynthesis (A G ), stomatal conductance ( g s ) and various chlorophyll fluorescence parameters were determined simultaneously. Furthermore, chlorophyll fluorescence images were taken from leaves to study the spatial pattern of photosystem II (PSII) efficiency and non-photochemical quenching parameters. Under low and moderate light intensity, the onset of drought stress caused an increase in the operating quantum efficiency of PSII photochemistry (f PSII ) which indicated increased photorespiration since photosynthesis was hardly affected by water limitation. The increase in f PSII was caused by an increase of the efficiency of open PSII reaction centers (F v 0 /F m 0 ) and by a decrease of the basal non-photochemical quenching (f NO ). Using a chlorophyll fluorescence imaging system a low spatial heterogeneity of f PSII was revealed under both irrigation treatments. The increased rate of photorespiration in plants during the onset of drought stress can be seen as an acclimation process to avoid an over-excitation of PSII under more severe drought conditions.

The differential pigment composition and photosynthetic activity of sun and shade leaves of deciduous (Acer pseudoplatanus, Fagus sylvatica, Tilia cordata) and coniferous (Abies alba) trees was comparatively determined by studying the photosynthetic rates via CO 2 measurements and also by imaging the Chl fluorescence decrease ratio (R Fd ), which is an in vivo indicator of the net CO 2 assimilation rates. The thicker sun leaves and needles in all tree species were characterized by a lower specific leaf area, lower water content, higher total chlorophyll (Chl) aþb and total carotenoid (Cars) content per leaf area unit, as well as higher values for the ratio Chl a/b compared to the much thinner shade leaves and needles that possess a higher Chl aþb and Cars content on a dry matter basis and higher values for the weight ratio Chls/Cars. Sun leaves and needles exhibited higher rates of maximum net photosynthetic CO 2 assimilation (P Nmax ) measured at saturating irradiance associated with higher maximum stomatal conductance for water vapor efflux. The differences in photosynthetic activity between sun and shade leaves and needles could also be sensed via imaging the Chl fluorescence decrease ratio R Fd , since it linearly correlated to the P Nmax rates at saturating irradiance. Chl fluorescence imaging not only provided the possibility to screen the differences in P N rates between sun and shade leaves, but in addition permitted detection and quantification of the large gradients in photosynthetic rates across the leaf area existing in sun and shade leaves.

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.

Four types of cDNAs corresponding to the fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) enzyme were isolated from the developing seeds of Brassica juncea, a widely cultivated species amongst the oil-seed crops. The mature polypeptides deduced from the cDNAs showed sequence identity with the FatB class of plant thioesterases. Southern hybridization revealed the presence of at least four copies of BjFatB gene in the genome of this amphidiploid species. Western blot and RT-PCR analyses showed that the BjFatB class thioesterase is expressed poorly in flowers and leaves, but significantly in seeds at the mid-maturation stage. The enzymatic activities of different BjFatB isoforms were established upon heterologous expression of the four BjFatB CDSs in Escherichia coli K27fadD88, a mutant strain of fatty acid b-oxidation pathway. The substrate specificity of each BjFatB isoform was determined in vivo by fatty acid profile analyses of the culture supernatant and membrane lipid of the recombinant K27fadD88 and E. coli DH10B (fadD þ ) clones, respectively. The BjFatB1 and BjFatB3 were predominantly active on C18:0-ACP substrate, whereas BjFatB2 and BjFatB4 were specific towards C18:0-ACP as well as C16:0-ACP. These novel FatB genes may find potential application in metabolic engineering of crop plants through their over-expression in seed tissues to generate stearate-rich vegetable fats/oils of commercial importance.

Plants store carbohydrate polymers in a number of forms. Starch is the principal form, followed by fructans and cell wall storage polysaccharides (CWSP). The latter are present mainly in seeds and consist of magnifications of one of the polysaccharides present in one of the cell wall matrices. CWSPs are classified as mannans, xyloglucans and galactans, the first being subdivided into pure mannans, galactomannans and glucomannans. The present review updates the principal discoveries concerning occurrence, structure, metabolism and eco-physiological significance of the seed storage cell wall polysaccharides with emphasis on their importance for plant metabolism and adaptation to their respective environments during evolution. The properties of CWSPs as storage polysaccharides are compared with starch and fructans and the current knowledge on the catabolism (including control) of mannan/galactomannan, xyloglucan, and galactan is reviewed. On the basis of these data, the idea that the CWSPs are multifunctional molecules is proposed and this feature is used as evidence for the hypothesis that, during evolution, the CWSPs were involved in cycles of transference of functions which led them to become storage polysaccharides, yet preserving some of their previous primary cell wall functions. © 2000 Éditions scientifiques et médicales Elsevier SAS

In the last few years programmed cell death in plants inspired many studies in development and environmental stresses. Some of these studies showed that hallmarks of animal programmed cell death were found at cellular or molecular level in plant cells in different experimental systems. Additionally the effect of over-expression in plants of animal genes implicated in programmed cell death has been tested, and some plant homologues of these genes have been found. This suggests that, despite some differences, plants and animals could share at least some common components of a core mechanism used to carry out programmed cell death in eukaryotes. In this review, we will concentrate on the last findings that suggest similarity between plant programmed cell death and its better known counterpart in animals. © 2000 Éditions scientifiques et médicales Elsevier SAS apoptosis / caspases / DNA ladder / necrosis / plants / programmed cell death / TUNEL bp, base pair / HR, hypersensitive reaction / PCD, programmed cell death / PARP, poly (ADP-ribose) polymerase / TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling * Correspondence and reprints: fax +33 4 6866 8499;

The role of the plant hormone abscisic acid (ABA) has been studied in Arabidopsis by using mutants affected in either the biosynthesis or the mode of action of this hormone. Mutants have been isolated mainly by altered germination characteristics and seedling growth. The biochemical lesions of the abal, a&2 and aba3 mutants have been identified and the zeaxanthin epoxidase gene encoded by ABA1 has been cloned by homology with a Nicotiana plumbaginifolia gene blocked at the same biosynthetic step. ABA-insensitive mutants have either a phenotype affecting several ABA processes (abil and abi2) and therefore were suggested to encode early steps in ABA signal transduction, or they affect specific steps (e.g. abi3, abi4, abi5). The ABII and ABZ2 genes encode protein phosphatase 2C enzymes and ABI3 a transcription factor with seed-specific expression. The ABA hypersensitive era1 mutant is impaired in a farnesyl transferase. The various mutants have been used to analyse the role of ABA in seed development and seed germination, stress tolerance, and stomata1 closure. 0 Elsevier, Paris.

Clonal, hydroponically grown poplar plants (Populus × canescens, a hybrid of Populus tremula × Populus alba) were exposed to Cd or H 2 O 2 to find out whether Cd-induced injury was related to the disturbance of the cellular redox control in root tips. Cd exposure resulted in an inhibition of antioxidative enzymes (superoxide dismutase, EC 1.15.1.1; catalase, EC 1.11.1.6; ascorbate peroxidase, EC 1.11.1.11; monodehydroascorbate radical reductase, EC 1.1.5.4; glutathione reductase, EC 1.6.4.2) but had fewer effects on dehydroascorbate reductase (EC 1.8.5.1) activities. Glutathione concentrations decreased, whereas ascorbate remained unaffected by Cd. Five micromoles of Cd were subinjurious in short-term experiments and stimulated root growth. Fifty micromoles of Cd retarded shoot growth faster than root growth, caused a more severe loss in antioxidative capacity than 5 µM Cd and resulted in an accumulation of H 2 O 2 in roots. Exposure to H 2 O 2 had an effect on antioxidative enzymes similar to that found under the influence of Cd, but caused GSH accumulation, and loss of ascorbate. The present data indicate that both agents acted via the disturbance of the cellular redox control.

Phragmites (Phragmites australis Cav. (Trin.) ex Steud) plants exposed to a high concentration of CdSO 4 (50 µM) for 21 d were analysed with respect to the distribution of metal, its effects on antioxidants, the antioxidant enzymes and the redox status in leaves, roots and stolons. The highest accumulation of Cd 2+ occurred in roots followed by leaves, and it was not significant in the stolons when compared with the control plants. In particular, in roots from Cd-treated plants, both the high amount of GSH and the parallel increase of glutathione-Stransferase (EC 2.5.1.18; GST) activity seemed to be associated with an induction of the detoxification processes in response to the high cadmium concentration. Superoxide dismutase (EC 1.15.1.1; SOD), ascorbate peroxidase (EC 1.11.1.11; APX), glutathione reductase (EC 1.6.4.2; GR) and catalase (EC 1.11.1.6; CAT) activities as well as reduced and oxidised glutathione contents in all samples of leaves, roots and stolons were increased in the presence of Cd 2+ when compared to control plants. Despite the fact that Cd 2+ has a redox characteristic not compatible with the Fenton-type chemistry that produces active oxygen species, the antioxidant response is widespread and generic. Increased activities of antioxidant enzymes in Cd-treated plants suggest that metal tolerance in Phragmites plants might be associated to the efficiency of these mechanisms.

This study was conducted to investigate changes of nucleic acids and protein levels in response to brassinosteroid (BR) effect in the green alga Chlorella vulgaris Beijerinck. BRs had the greatest effect on growth and metabolism of algae between 24 and 36 h after treatment in the range from 10 -12 to 10 -8 M. The highest growth of the content of nucleic acids and proteins was observed in the case of brassinolide (BL) at the concentration 10 -8 M in the 36th hour of cultivation. BL and its derivatives are biologically more active than castasterone (CS). The lowest stimulatory activity was shown by homoCS. The stimulatory effect of BRs in the algae was arranged in the following order: BL > 24-epiBL > homoBL > CS > 24-epiCS > homoCS. The relationship between the structure and the biological activity of BRs in stimulation of growth and changes in the content of DNA, RNA and proteins are discussed. © 2000 Éditions scientifiques et médicales Elsevier SAS Algae / brassinosteroids / Chlorella vulgaris / nucleic acids / phytohormones / proteins BL, brassinolide / BRs, brassinosteroids / CS, castasterone / 24-epiBL, 24-epibrassinolide / 24-epiCS, 24-epicastasterone / homoBL, homobrassinolide / homoCS, homocastasterone

The present study was undertaken to test the influence of exogenously applied phytohormones: auxins (IAA, IBA, NAA, PAA), cytokinins (BA, CPPU, DPU, 2iP, Kin, TDZ, Z), gibberellin (GA 3 ), jasmonic acid (JA) as well as polyamine -spermidine (Spd) upon the growth and metabolism of green microalga Chlorella vulgaris (Chlorophyceae) exposed to heavy metal (Cd, Cu, Pb) stress. The inhibitory effect of heavy metals on algal growth, metabolite accumulation and enzymatic as well as non-enzymatic antioxidant system was arranged in the following order: Cd > Pb > Cu. Exogenously applied phytohormones modify the phytotoxicity of heavy metals.

Soil cadmium (Cd) contamination has posed a serious problem for safe food production and become a potential agricultural and environmental hazard worldwide. Greenhouse hydroponic experiments were conducted to investigate the modulation of exogenous GSH (reduced glutathione) in antioxidant defense system against the Cd-induced toxicity in plants exposed to 5 mM Cd using two barley genotypes differing in Cd tolerance. Addition of 20 mg L À1 GSH in 5 mM Cd culture medium significantly alleviated Cd-induced growth inhibition, especially for the sensitive genotype Dong 17 and dramatically depressed O 2 À , H 2 O 2 and malondialdehyde (MDA) accumulation. GSH mediated intracellular GSH content to keep the level over the control especially in the case of Cd-induced GSH reduction. External GSH counteracted Cd-induced alterations of certain antioxidant enzymes, e.g. brought root dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione peroxidase (GPX) activities of the both genotypes down towards the control level, but elevated the depressed ascorbate peroxidase (APX) and catalase (CAT) activities in Dong 17 after 10e15 d treatment. The examination of APX and superoxide dismutase (SOD) isoenzymes revealed GSH significantly increased MnSOD, sAPX and tAPX activities in the both genotypes, and strongly stimulated Cd-induced decrease in cAPX in the sensitive genotype. Furthermore, External GSH up-regulated root cAPX and leaf cAPX, CAT1, and CAT2 expression at transcript level in Dong 17 to achieve stimulation. These data, especially from the results of depressed O 2 À , H 2 O 2 and MDA accumulation and elevated Cd-induced decrease in GSH content and APX (strongly stimulated cAPX, sAPX and tAPX) and CAT activities by GSH addition in the sensitive genotype, suggest that elevated intracellular GSH and stimulated APX (especially cAPX, sAPX and tAPX iosenzymes) and CAT activities, when concerning ROS scavenging systems, play an important role in GSH-induced alleviation of oxidative stress.

Different redox-active compounds, such as ascorbate, glutathione, NAD(P)H and proteins from the thioredoxin superfamily, contribute to the general redox homeostasis in the plant cell. The myriad of interactions between redox-active compounds, and the effect of environmental parameters on them, has been encapsulated in the concept of a cellular redox state. This concept has facilitated progress in understanding stress signalling and defence in plants. However, despite the proven usefulness of the concept of a redox state, there is no single, operational definition that allows for quantitative analysis and hypothesis testing.

In Arabidopsis, a model genus missing a functional ornithine decarboxylase pathway, most of the key genes involved in polyamine biosynthesis are duplicated. This gene redundancy has been related to the involvement of certain gene isoforms in the response to specific environmental stimuli. We have previously shown that drought stress induces Arginine decarboxlase 2 expression, while transcript levels for Arginine decarboxlase 1 remain constant. Accumulation of putrescine and increased arginine decarboxlase activity (EC 4.1.1.19) levels in response to different abiotic stresses have been reported in many different plant systems, but the biological meaning of this increase remains unclear. To get a new insight into these questions, we have studied the response to drought of transgenic Arabidopsis thaliana lines constitutively expressing the homologous Arginine decarboxlase 2 gene. These lines contain high levels of putrescine with no changes in spermidine and spermine content even under drought stress. Drought tolerance experiments indicate that the different degree of resistance to dehydration correlates with Put content. Although no significant differences were observed in the number of stomata between wild-type and transgenic plants, a reduction in transpiration rate and stomata conductance was observed in the ADC2 over-expressor lines. These results indicate that one of the mechanisms involved in the drought tolerance of transgenic plants over-producing Put is related to a reduction of water loss by transpiration.

Cadmium (Cd 2þ ) is a widespread heavy metal pollutant in the environment with a long biological half-life, originating mainly from industrial processes and phosphate fertilizers. It is easily taken up by plants, resulting in toxicity symptoms, such as chlorosis, wilting, growth reduction, and cell death. This cellular toxicity might result from interactions with vital metabolic pathways, carboxyl or thiol groups of proteins and reactive oxygen species (ROS) burst in plants. Plant exposure even to low concentrations of Cd may lead to cell death but the mechanism of its toxicity is still debatable. Therefore, exploring various ways to improve crop productivity and/or alleviate Cd stress effects is one of the major areas of concern. Nitric oxide (NO) is a hydrophobic gaseous molecule involved in various physiological processes such as germination, root growth, stomatal closure, control of the flowering timing etc. NO also functions as cell signaling molecule in plants and play important roles in the regulation of plant responses to both abiotic and biotic stress conditions. At the molecular level, NO signaling includes protein modification by binding to critical cysteine residues, heme or ironesulfur centers and tyrosine residue nitration via peroxynitrite formation (ONOO À ), mobilization of secondary messengers (Ca 2þ , cyclic GMP and cyclic ADP-Rib) and modulation of protein kinase activities. Significant research had been done to understand the NO biosynthesis and signaling in plants under stress, but several questions still need to be answered. The present review is focused specifically on the importance of NO as Cd stress modulator in crop plants.

Rice, a first cereal crop whose draft genome sequence from two subspecies (japonica-type cv. Nipponbare and indica-type 93-11) was available in 2002, along with its almost complete genome sequence in 2005, has drawn the attention of researchers worldwide because of its immense impact on human existence. One of the most critical research areas in rice is to discern the self-defense mechanism(s), an innate property of all living organisms. The last few decades have seen scattered research into rice responses to diverse environmental stimuli and stress factors. Our understanding on rice self-defense mechanism has increased considerably with accelerated research during recent years mainly due to identification and characterization of several defense/stress-related components, genes, proteins and secondary metabolites. As these identified components have been used to study the defense/stress pathways, their compilation in this review will undoubtedly help rice (and others) researchers to effectively use them as a potential marker for better understanding, and ultimately, in defining rice (and plant) self-defense response pathways.

Effects of hydrogen sulfide (H 2 S) on plant physiology have been previously studied, but such studies have relied on the use of NaSH as a method for supplying H 2 S to tissues. Now new compounds which give a less severe H 2 S shock and a more prolonged exposure to H 2 S have been developed. Here the effects of one such compound, GYY4137, has been investigated to determine its effects on stomatal closure in Arabidopsis thaliana. It was found that both NaSH and GYY4137 caused stomatal opening in the light and prevented stomatal closure in the dark. Nitric oxide (NO) has been well established as a mediator of stomatal movements and here it was found that both NaSH and GYY4137 reduced the accumulation of NO in guard cells, perhaps suggesting a mode of action for H 2 S in this system. GYY4137, and future related compounds, will be important tools to unravel the effects of plant exposure to H 2 S and to determine how H 2 S may fit into plant cell signalling pathways.

Salicylic acid (SA) inhibited mycelial growth of Eutypa lata (Pers. Fr.)Tul. in a solid as well as in a liquid culture medium, in a concentration-dependent manner, the threshold value being 0.1 mM. In conditions mimicking the plant environment (in particular, a pH near the apoplastic value, i.e. 5.5), 1 mM SA showed only fungistatic properties. However, modifications were observed in the structural organization of the mycelium at various levels (wall, mitochondria, vacuole and nucleus). A fungicidal effect was obtained at 2 mM or higher concentrations and following this treatment, fungal filaments appeared empty. Antifungal efficiency of the molecule was increased when the experimental pH was brought to more acidic values (pH 4). This observation was correlated with the increased capacity of compound uptake by the fungus. The in vitro antifungal effects of 57 benzoic acid derivatives were investigated on E. lata, in order to define a possible structure-activity relationship. A strategy leading to the synthesis of particular benzoic conjugates and integrating the data obtained here is proposed to treat in planta against E. lata, the causal agent of eutypa dieback, a severe disease of the grapevine and other woody plants resulting from an infection in the xylem of the host plant.

It is now more than 80 years since boron was convincingly demonstrated to be essential for normal growth of higher plants. However, its biochemical role is not well understood at the moment. Several recent reviews propose that B is implicated in three main processes: keeping cell wall structure, maintaining membrane function, and supporting metabolic activities. However, in the absence of conclusive evidence, the primary role of boron in plants remains elusive. Besides plants, growth of specific bacteria, such as heterocystous cyanobacteria and the recently reported actinomycetes of the genus Frankia, requires B, particularly for the stability of the envelopes that control the access of the nitrogenase-poisoning oxygen when they grow under N 2 -fixing conditions. Likewise, a role for B for animal embryogenesis and other developmental processes is being established. Finally, a new feature of the role of boron comes from signaling mechanisms for communication among bacteria and among legumes and rhizobia leading to N 2 -fixing symbiosis, and it is possible that new roles for B, based on its special chemistry and its interaction with Ca would appear in the world of signal transduction pathways. In conclusion, the diversity of roles played by B might indicate that either the micronutrient is involved in numerous processes or that its deficiency has a pleiotropic effect. The arising question is why such an element? Since all of the roles clearly established for B are related to its capacity to form diester bridges between cis-hydroxyl-containing molecules, we propose that the main reason for B essentiality is the stabilization of molecules with cis-diol groups turning them effective, irrespectively of their function.

Using the perspective of full scale application of phytoremediation techniques, research is focusing on the optimization of agronomic practices. Two annual high biomass yield crops, Sorghum bicolor and Helianthus annuus, were grown in a polymetallic soil. The experimental site, polluted by pyrite cinders, is located in an industrial site that has been listed in the clean-up national priority list since 2001. Specific aims of this work were to observe the concentration of metals in plants during the crop cycle and to establish the amount of metal removed by the crops. The field trial, arranged in a randomized block design, started in 2005. The concentrations of heavy metals in the soil were: As 309, Cd 4.29, Co 50.9, Cu 1527 and Zn 980 mg kg À1 . The crops grown on the polluted soil received mineral fertilization (Fert) and organic amendment (Org), while plants in control soil (Ctrl) did not receive anything. The plots were watered during the crop cycle during two drought periods, using a sprinkler irrigation system. The phytoextraction potential of crops was estimated during the whole growth cycle and the plant biomass that was collected in each sampling date was ICP-analyzed. Plantebiomass growth curves were obtained. The concentrations of the metals in the shoots and in the total plant biomass were recorded. Finally, the metal removal was calculated for the harvestable parts of the crops. The amelioration of the nutritive status of the substrate that resulted, was highly effective for the biomass yield. However, fertilization and soil amendment did not heighten the concentration of metals in the harvestable tissue of the plants during the crop cycle. In some cases, organic matter appeared to bind the elements making them less available for the plants. The evaluation of the potential of phytoremediation of our plants compared to other crops in terms of metal removal was positive. Our results of metal removal are consistent with the results from other in situ experiments. The Zn removal by S. bicolor and H. annuus reached about 2000 g ha À1 and 1000 g ha À1 , respectively.

Twenty-two components of human interferon-a (IFN-a) derived from Sendai virus-induced Namalwa cells were purified by sequential immunoadsorbent affinity chromatography using four monoclonal antibody affinity columns followed by ultrafiltration and reversed-phase high-performance liquid chromatography. The specific activity ranged from 0.2 to 2.6 x lo8 IU/mg protein on Madin-Darby bovine kidney cells, 0.3 to 4.6 X lo* IU/mg protein on human WISH cells, and lo4 to 7 x lo5 units/mg protein on mouse L929 cells. The apparent molecular weights of the components ranged from 17,500 to 23,300 using nonreducing sodium dodecyl polyacrylamide-gel electrophoresis and 17,500 to 27,600 using reducing sodium dodecyl polyacylamide-gel electrophoresis. The amino-terminal amino acid sequences were similar among the components as well as to those reported for the cloned human IFN-a genes (Pestka, S. (1986) Methods Enzymol. 119, 3-14). However, four components, f, i, 1, and m, have amino-terminal amino acid sequences which appear to be unique when compared to those predicted from the cDNA clones. One component, prea, has a potential N-linked glycosylation site on the Asn of residues 2 through 4, Asn-Leu-Ser.

Amine oxidases (AOs) catalyse the oxidative de-amination of polyamines, ubiquitous polycationic compounds involved in important events of cell life. They include the copper-containing amine oxidases (CuAOs; EC 1.4.3.6) and the flavin-containing polyamine oxidases (PAOs; EC 1.5.3.11). The main physiological role of these moonlighting proteins has been linked to compartment-specific H 2 O 2 synthesis in different phases of development and differentiation as well as in the course of defence mechanisms against pathogens and abiotic stress. Moreover, several studies evidenced a correlation of AO expression levels with physiological stages characterized by intense metabolism, such as cell division or organ formation, thus leaving open the hypothesis that AOs may have also a role in the regulation of cell cycle through the modulation of polyamine cellular content. This update will deal with recent reports on the involvement of CuAOs and PAOs in abiotic (salt) stress, wound-healing and hostepathogen interactions.

To have better understanding of the processes that occur in Withania somnifera L. Dunal, proteome analyses were initiated on two tissues (seeds & leaves) of this plant. Protein extracts were separated by twodimensional gel electrophoresis (2-DE) across a broad 3.0-10.0 immobilized pH gradient (IPG) strip that yielded 434 protein spots. A total of 167 individual spots (82 from seeds and 85 from leaves) were excised from the gel and were characterized by peptide mass fingerprinting. From these analyses, 70 individual proteins from seeds and 74 from leaves were identified by protein sequence database interrogation and were catalogued accordingly to different protein functions. A comparative analysis of the two tissues indicated that some enzymes/proteins involved in housekeeping pathways were common to both, whereas some were exclusively tissue specific with specialized metabolic complement. The knowledge gained by this study towards the tissue specific protein expression in W. somnifera would form the basis for our future endeavor of characterization of proteins to understand the physiology and the associated complex metabolic network during its ontogenetic development.