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Low temperatures (0–10 °C) represent a major physiological stress for plants, negatively affecting both their growth rates and overall growth. Cold stress may induce a wide range of negative physiological effects, from oxidative stress to photosynthetic damage. We investigated the effects of low temperatures in two different model plants, Arabidopsis thaliana and Hordeum vulgare. We tested whether the oxidative pentose phosphate pathway (OPPP) is involved in the increase of reductants’ levels needed to counteract oxidative stress induced by cold. The expression, occurrence, and activity of different glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) isoforms during cold stress and plant recovery from low temperatures, were measured at different growth stages from early germinated to mature pot-grown plants. Our results showed plants exhibited changes in different stress markers; ascorbate peroxidase – APX, catalase - CAT, proline, malondialdehyde, H2O2, NADPH/NADP+. We found that a major role in cold acclimation for cytosolic- and peroxisome-located G6PDHs, and different roles for plastidial/chloroplastic isoforms. This suggests that G6PDH isoforms may regulate redox homeostasis in low temperatures, in order to support the increased and continued demand of reductants during both cold stress and recovery stages. Furthermore, we found a significant involvement of (6PGDH), strengthening the idea that the contribution of the whole oxidative pentose phosphate pathway (OPPP) is required to sustain reductant supply under cold stress.

Nitrogen (N) is a crucial nutrient for plant growth and development. To optimize agricultural environments, N fertilizers represent a critical tool to regulate crop productivity. The improvement of nitrogen use efficiency (NUE) represents a promising tool that may enable cereal production to meet future food demand. Wheat reported contrasting behaviors in N utilization showing specific abilities depending on genotype. This study selected two landraces and two improved genotypes from Northern Africa to investigate grain yield (GY), grain protein content (GPC) and NUE. Plants were grown under three levels of N supply: 0, 75, 150 kg N ha−1 and for two consecutive years. Results reported a better NUE (0.40 kg kg N−1) obtained under 150 kg N ha−1, while N utilization efficiency (NUtE) showed a 13% increase using 75 kg N ha−1 compared with 150 kg N ha−1. Under low nitrogen rate (0 N), crop N supply (CNS) and N uptake efficiency (NUpE) were shown as determinant factors for improved genotypes GY (R2 = 0.72), while NUtE represented the most determinant component for GPC in landraces (R2 = 0.92). Multivariate regression models explained the dependence in GPC on NUE, NUpE, and NUtE. In conclusion, our results recognize GPC and NUtE as suitable selection traits to identify durum wheat with higher NUE.

In this study, the efficacy of Acca sellowiana fruit acetonic extract on human MDR cancer cells was tested for the first time, and it was demonstrated that the fruit extract is effective on both sensitive and resistant tumor cells. The effects of A. sellowiana extract on bacterial biofilm were also examined for the first time. By crystal violet assays and confocal microscopy analyses, it was demonstrated that the plant extract is able to strongly inhibit biofilm formation of both sensitive and resistant bacterial strains. Furthermore, antimicrobial activity assays and TEM analyses clearly demonstrated the effectiveness of plant extract on planktonic bacterial cells in both sensitive and resistant strains. Altogether, these findings intriguingly expand the panel of activities of A. sellowiana fruit extract with respect to previous reports, and open interesting perspectives to its therapeutic applications.

This work was supported by the PAI programme (Plan Andaluz de Investigacion, Grupo de Investigacion AGR282). V. P. Acknowledges Erasmus traineeship program by the University of Naples "Federico II". The results presented in this paper are based on the PhD Thesis by Paradisone ( 2018)., Zinc (Zn) deficiency causes serious issues to plant growth and development, negatively affecting crops in many world regions. On the other hand, Zn toxicity impairs plant growth, producing physiological alterations, and even cell death. In plants, two of the processes that most determine growth are nitrogen (N) metabolism and photosynthesis. In the last decades, several authors proved that silicon (Si) and calcium (Ca) mitigate the effects of various abiotic and biotic stresses in plants. The objective of this research is to study the effect of Si application to barley (Hordeum vulgare cv. Nure) plants grown under Zn deficiency and Zn toxicity. Hence, barley plants were grown in hydroponics and supplied with a low Zn dose (0.01 mu M ZnSO4) and a high Zn dose (100 mu M ZnSO4) and were supplied with CaSiO3. Parameters related to Zn accumulation, N metabolism, and photosynthesis were measured. Zn stress affected leaf Zn concentration and reduced biomass in barley plants. Both Zn toxicity and deficiency inhibited N metabolism and enhanced photorespiration, increasing stress symptoms. CaSiO3 mitigated Zn stress effects, probably regulating Zn levels in plant cells and enhancing N metabolism and photosynthesis. We conclude that CaSiO3 could be beneficial to grow barley plants in soils with high or low availability of Zn., PAI programme (Plan Andaluz de Investigacion, Grupo de Investigacion) AGR282

The interaction between salinity and nitrogen metabolism has been investigated in two barley landraces, one tolerant (&ldquo, 100/1B&rdquo, ) and one susceptible to salinity (&ldquo, Barley medenine&rdquo, ) from the Middle East and North Africa (MENA) region. Barley plants were exposed to 50 mM NaCl for 7 days, then, salinity was increased to 150 mM NaCl in the presence (10 mM) or limitation (1 mM) of ammonium as a nitrogen source. Upon salinity, &ldquo, was shown to support N assimilation by enhancing the glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) cycle under high N, and the stimulation of the glutamate dehydrogenase (GDH) pathway under low N treatment. In &ldquo, salinity reduced the GS/GOGAT cycle, and increased GDH activity. Upon salinity, Heat Shock Proteins 70 and PEPC remained unchanged in &ldquo, while they decreased in &ldquo, The tolerance degree is a determining factor in enzymes&rsquo, occurrence and regulation: exposed to salinity, &ldquo, rapidly increased APX and PEPC activities, while this was delayed in &ldquo, Salinity increased cyt-G6PDH levels in &ldquo, while &ldquo, showed a decrease in G6PDH isoforms. Correlation analyses confirm GOGAT was related to G6PDH, GDH and APX with PEPC in &ldquo, under moderate salinity, severe salinity correlated GDH with G6PDH and PEPC. In &ldquo, under salinity, GOGAT was correlated with G6PDH, while APX showed a relation with PEPC. Therefore, specific enzymatic activities and occurrence can be used to determine stress responsiveness of different landraces. We suggest that the rapid increase in G6PDH, APX, and nitrogen assimilation enzymes activities represents an index of tolerance in &ldquo, and a stress symptom in &ldquo

Salinity is an important environmental factor that reduces plant productivity in many world regions. It affects negatively photosynthesis causing a growth reduction. Likewise, calcium (Ca2+) is crucial in plant stress response. Therefore, the modification of Ca2+ cation exchangers (CAX) transporters could be a potential strategy to increase plant tolerance to salinity. Using Targeting Induced Local Lesions in Genomes (TILLING), researchers generated three mutants of Brassica rapa CAX1a transporter: BraA.cax1a-7, BraA.cax1a-4, and BraA.cax1a-12. The aim of this study was to test the effect of those mutations on salt tolerance focusing on the response to the photosynthesis process. Thus, the three BraA.cax1a mutants and the parental line (R-o-18) were grown under salinity conditions, and parameters related to biomass, photosynthesis performance, glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), and soluble carbohydrates were measured. BraA.cax1a-4 provided higher biomass and a better photosynthetic performance manifested by higher water use efficiency (WUE), Fv/Fm, electron fluxes, and Rubisco (EC 4.1.1.39) values. In addition, BraA.cax1a-4 presented increased osmotic protection through myo-inositol accumulation. On the other hand, BraA.cax1a-7 produced some negative effects on photosynthesis performance and lower G6PDH and Rubisco accumulations. Therefore, this study points out BraA.cax1a-4 as a useful mutation to improve photosynthetic performance in plants grown under saline conditions.

Crop production is facing the increase of salinity in soils and irrigation waters. This is a widespread problem that affects crops with high economic importance and reduces yields. Salinity causes oxidative stress in plants which induce complex defensive mechanisms involving the antioxidant system and osmoprotector compounds. Ca2+ plays a pivotal role in counteracting salt stress, and the modification of the Ca2+ vacuolar transporter CAX1 could represent a potential method to improve tolerance to salinity. Three new CAX1 variants in Brassica rapa (BraA.cax1a) were generated using TILLING strategy. The objective of this work is to evaluate the tolerance of these mutants to saline conditions. For this, BraA.cax1a mutants and the parental line R-o-18 were grown in pots under control and saline conditions (150 mM NaCl). Parameters of biomass, ions accumulation, antioxidant compounds concentration and antioxidant enzyme activities, heat shock protein 70 (HSP70) occurrence, and proline (Pro) levels were measured. According to the results, BraA.cax1a-4 mutation provided a higher tolerance to salinity stress. Thus, BraA.cax1a-4 plants showed higher biomass; higher Ca2+ and K+ accumulation in the shoot, a lower Na+/K+ ratio, an enhanced ROS scavenging (increased superoxide dismutase and catalase activities) and an enhanced ascorbate redox state. BraA.cax1a-4 and BraA.cax1a-12 showed higher K+ transport to the shoot which could contribute to their lower lipid peroxidation. In contrast, BraA.cax1a-7 showed a lower antioxidant response. Besides, an increased occurrence of cytosolic and chloroplastic HSP70 isoforms and Pro levels could contribute to protect these B. rapa mutants from saline stress. Therefore, this study identifies a potential useful genotype that could be used to enhance salt tolerance of crops.

Nitrogen (N) availability represents one of the most critical factors affecting cultivated crops. N is indeed a crucial macronutrient influencing major aspects, from plant development to productivity and final yield of lignocellulosic biomass, as well as content of bioactive molecules. N metabolism is fundamental as it is at the crossroad between primary and secondary metabolic pathways: Besides affecting the synthesis of fundamental macromolecules, such as nucleic acids and proteins, N is needed for other types of molecules intervening in the response to exogenous stresses, e.g. alkaloids and glucosinolates. By partaking in the synthesis of phenylalanine, N also directly impacts a central plant metabolic ‘hub’—the phenylpropanoid pathway—from which important classes of molecules are formed, notably monolignols, flavonoids and other types of polyphenols. In this review, an updated analysis is provided on the impact that N has on the multipurpose crop hemp (Cannabis sativa L.) due to its renewed interest as a multipurpose crop able to satisfy the needs of a bioeconomy. The hemp stalk provides both woody and cellulosic fibers used in construction and for biocomposites; different organs (leaves/flowers/roots) are sources of added-value secondary metabolites, namely cannabinoids, terpenes, flavonoids, and lignanamides. We survey the available literature data on the impact of N in hemp and highlight the importance of studying those genes responding to both N nutrition and abiotic stresses. Available hemp transcriptomic datasets obtained on plants subjected to salt and drought are here analyzed using Gene Ontology (GO) categories related to N metabolism. The ultimate goal is to shed light on interesting candidate genes that can be further studied in hemp varieties growing under different N feeding conditions and showing high biomass yield and secondary metabolite production, even under salinity and drought.

Nitrogen (N) availability represents one of the most critical factors affecting cultivated crops. N is indeed a crucial macronutrient influencing major aspects, from plant development to productivity and final yield of lignocellulosic biomass, as well as content of bioactive molecules. N metabolism is fundamental as it is at the crossroad between primary and secondary metabolic pathways: Besides affecting the synthesis of fundamental macromolecules, such as nucleic acids and proteins, N is needed for other types of molecules intervening in the response to exogenous stresses, e.g. alkaloids and glucosinolates. By partaking in the synthesis of phenylalanine, N also directly impacts a central plant metabolic ‘hub’—the phenylpropanoid pathway—from which important classes of molecules are formed, notably monolignols, flavonoids and other types of polyphenols. In this review, an updated analysis is provided on the impact that N has on the multipurpose crop hemp (Cannabis sativa L.) due to its renewed interest as a multipurpose crop able to satisfy the needs of a bioeconomy. The hemp stalk provides both woody and cellulosic fibers used in construction and for biocomposites; different organs (leaves/flowers/roots) are sources of added-value secondary metabolites, namely cannabinoids, terpenes, flavonoids, and lignanamides. We survey the available literature data on the impact of N in hemp and highlight the importance of studying those genes responding to both N nutrition and abiotic stresses. Available hemp transcriptomic datasets obtained on plants subjected to salt and drought are here analyzed using Gene Ontology (GO) categories related to N metabolism. The ultimate goal is to shed light on interesting candidate genes that can be further studied in hemp varieties growing under different N feeding conditions and showing high biomass yield and secondary metabolite production, even under salinity and drought.

Background: Excessive rates of nitrogen (N) fertilizers may result in elevated concentrations of nitrate (NO3- ) in plants. Considering that many programs of biofortification with trace elements are being performed, it has become important to study how the application of these elements affects plant physiology and, particularly, N utilization in leaf crops. The main objective of the present study was to determine whether the NO3- accumulation and the nitrogen use efficiency was affected by the application of different doses of Zn in Lactuca sativa plants.; Results: Zn doses in the range 80-100 µmol L-1 produced an increase in Zn concentration provoking a decrease of NO3- concentration and increase of the nitrate reductase, glutamine synthetase and aspartate aminotransferase activities, as well as the photorespiration processes. As result, we observed an increase in reduced N, total N concentration and N utilization efficiency. Consequently, at a dose of 80 µmol L-1 of Zn, the amino acid concentration increased significantly.; Conclusion: Adequate Zn fertilization is an important critical player in lettuce, especially at a dose of 80 µmol L-1 of Zn, because it could result in an increase in the Zn concentration, a reduction of NO3- levels and an increase the concentration of essential amino acids, with all of them having beneficial properties for the human diet. © 2016 Society of Chemical Industry.; © 2016 Society of Chemical Industry.

Cadmium represents one of the most toxic pollutants in plant ecosystems: at high concentrations it can cause severe effects, such as plant growth inhibition, decrease in photosynthesis and changes in plant basal metabolism. Changes in pigments’ content, RubisCO large subunit, and D1 protein indicated a severe reduction in photosynthetic efficiency. Furthermore, the decrease of nitrate reductase activity and changes in free amino acids levels show a general stress condition of nitrogen assimilation. Cadmium increased the activities of ROS scavenging enzymes; among these, ascorbate peroxidase rate was the most noticeably increased. It is worth noting that glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.64), showed changes in both activities and occurrence during cadmium stress. Interestingly, our data suggest that G6PDH would modulate redox homeostasis under metal exposure, and possibly satisfy the increased request of reductants to counteract the oxidative burst induced by cadmium. Therefore, the results suggest that APX and G6PDH may play a pivotal role to counteract the oxidative stress induced by cadmium in young barley plants.

Poaceae represent the most important group of crops susceptible to abiotic stress. This large family of monocotyledonous plants, commonly known as grasses, counts several important cultivated species, namely wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays), and barley (Hordeum vulgare). These crops, notably, show different behaviors under abiotic stress conditions: wheat and rice are considered sensitive, showing serious yield reduction upon water scarcity and soil salinity, while barley presents a natural drought and salt tolerance. During the green revolution (1940–1960), cereal breeding was very successful in developing high-yield crops varieties; however, these cultivars were maximized for highest yield under optimal conditions, and did not present suitable traits for tolerance under unfavorable conditions. The improvement of crop abiotic stress tolerance requires a deep knowledge of the phenomena underlying tolerance, to devise novel approaches and decipher the key components of agricultural production systems. Approaches to improve food production combining both enhanced water use efficiency (WUE) and acceptable yields are critical to create a sustainable agriculture in the future. This paper analyzes the latest results on abiotic stress tolerance in Poaceae. In particular, the focus will be directed toward various aspects of water deprivation and salinity response efficiency in Poaceae. Aspects related to cell wall metabolism will be covered, given the importance of the plant cell wall in sensing environmental constraints and in mediating a response; the role of silicon (Si), an important element for monocots' normal growth and development, will also be discussed, since it activates a broad-spectrum response to different exogenous stresses. Perspectives valorizing studies on landraces conclude the survey, as they help identify key traits for breeding purposes.

In this work, the involvement of heat shock proteins (HSP70) in barley (Hordeum vulgare) has been studied in response to drought and salinity. Thus, 3 barley genotypes usually cultivated and/or selected in Italy, 3 Middle East/North Africa landraces and genotypes and 1 improved genotype from ICARDA have been studied to identify those varieties showing the best stress response. Preliminarily, a bioinformatic characterization of the HSP70s protein family in barley has been made by using annotated Arabidopsis protein sequences. This study identified 20 putative HSP70s orthologs in the barley genome. The construction of un-rooted phylogenetic trees showed the partition into four main branches, and multiple subcellular localizations. The enhanced HSP70s presence upon salt and drought stress was investigated by both immunoblotting and expression analyses. It is worth noting the Northern Africa landraces showed peculiar tolerance behavior versus drought and salt stresses. The drought and salinity conditions indicated the involvement of specific HSP70s to counteract abiotic stress. Particularly, the expression of cytosolic MLOC_67581, mitochondrial MLOC_50972, and encoding for HSP70 isoforms showed different expressions and occurrence upon stress. Therefore, genotypes originated in the semi-arid area of the Mediterranean area can represent an important genetic source for the improvement of commonly cultivated high-yielding varieties.

The involvement and the efficiency of the antioxidants scavenging system upon drought were examined by comparing traditional tomato landraces with respect to an industrial commercial genotype (Red Setter); for the first time, comprehensive analyses of physiological, biochemical and molecular parameters were investigated directly under real field conditions, in a typical agricultural environment of Southern Italy. The characterization of the responses upon drought evidenced peculiar changes in stomatal conductance, ascorbate peroxidase and catalase activities and expression in drought tolerant tomato landraces, with respect to the industrial genotype. An in silico analysis (promoter and co-expression study) coupled to a phylogenetic investigation of selected enzymes was performed, reinforcing the hypothesis of a basal activation of ROS scavenging machinery in the Mediterranean landraces. Thus our data suggest a constitutive increase in the expression and activities of specific enzymes involved in ROS detoxification that can play a pivotal role in the drought response shown by tomato landraces. Therefore, traditional landraces could represent an important source of useful genetic variability for the improvement of commercial varieties; their ROS detoxifying capabilities denote peculiar aspects worth being explored to better describe their specific stress tolerance.

Zinc (Zn) deficiency is a major problem in agricultural crops of many world regions. N metabolism plays an essential role in plants and changes in their availability and their metabolism could seriously affect crop productivity. The main objective of the present work was to perform a comparative analysis of different strategies against Zn deficiency between two plant species of great agronomic interest such as Lactuca sativa cv. Phillipus and Brassica oleracea cv. Bronco. For this, both species were grown in hydroponic culture with different Zn doses: 10μM Zn as control and 0.01μM Zn as deficiency treatment. Zn deficiency treatment decreased foliar Zn concentration, although in greater extent in B. oleracea plants, and caused similar biomass reduction in both species. Zn deficiency negatively affected NO3(-) reduction and NH4(+) assimilation and enhanced photorespiration in both species. Pro and GB concentrations were reduced in L. sativa but they were increased in B. oleracea. Finally, the AAs profile changed in both species, highlighting a great increase in glycine (Gly) concentration in L. sativa plants. We conclude that L. sativa would be more suitable than B. oleracea for growing in soils with low availability of Zn since it is able to accumulate a higher Zn concentration in leaves with similar biomass reduction. However, B. oleracea is able to accumulate N derived protective compounds to cope with Zn deficiency stress.

Glucose 6 phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is well-known as the main regulatory enzyme of the oxidative pentose phosphate pathway (OPPP) in living organisms. Namely, in Planta, different G6PDH isoforms may occur, generally localized in cytosol and plastids/chloroplasts. These enzymes are differently regulated by distinct mechanisms, still far from being defined in detail. In the last decades, a pivotal function for plant G6PDHs during the assimilation of nitrogen, providing reductants for enzymes involved in nitrate reduction and ammonium assimilation, has been described. More recently, several studies have suggested a main role of G6PDH to counteract different stress conditions, among these salinity and drought, with the involvement of an ABA depending signal. In the last few years, this recognized vision has been greatly widened, due to studies clearly showing the non-conventional subcellular localization of the different G6PDHs, and the peculiar regulation of the different isoforms. The whole body of these considerations suggests a central question: how do the plant cells distribute the reductants coming from G6PDH and balance their equilibrium? This review explores the present knowledge about these mechanisms, in order to propose a scheme of distribution of reductants produced by G6PDH during nitrogen assimilation and stress.

A cDNA coding for a plastidic P2-type G6PDH isoform from A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys175 and Cys183 (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition. Three cysteine residues (C145, C194, C242) are suggested to have a role in controlling the NADP+ access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys175 and Cys183 in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f. This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants. (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys175 and Cys183 (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition. Three cysteine residues (C145, C194, C242) are suggested to have a role in controlling the NADP+ access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys175 and Cys183 in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f. This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants.

Most of the carbon used for starch biosynthesis in cereal endosperms is derived from ADP-glucose (ADP-Glc) synthesized by extra-plastidial AGPase activity, and imported directly across the amyloplast envelope. The properties of the wheat endosperm amyloplast ADP-Glc transporter were analysed with respect to substrate kinetics and specificities using reconstituted amyloplast envelope proteins in a proteoliposome-based assay system, as well as with isolated intact organelles. Experiments with liposomes showed that ADP-Glc transport was dependent on counter-exchange with other adenylates. Rates of ADP-Glc transport were highest with ADP and AMP as counter-exchange substrates, and kinetic analysis revealed that the transport system has a similar affinity for ADP and AMP. Measurement of ADP and AMP efflux from intact amyloplasts showed that, under conditions of ADP-Glc-dependent starch biosynthesis, ADP is exported from the plastid at a rate equal to that of ADP-Glc utilization by starch synthases. Photo-affinity labelling of amyloplast membranes with the substrate analogue 8-azido-[alpha-P-32]ADP-Glc showed that the polypeptide involved in substrate binding is an integral membrane protein of 38 kDa. This study shows that the ADP-Glc transporter in cereal endosperm amyloplasts imports ADP-Glc in exchange for ADP which is produced as a by-product of the starch synthase reaction inside the plastid.

Zinc (Zn) pollution in the soil represents a major problem for crop production worldwide. In the present work, two horticultural plants exhibiting different tolerance to Zn, Lactuca sativa cv. Phillipus and Brassica oleracea cv. Bronco, were exposed to Zn to evaluate the contribution of compatible osmolytes such as proline (Pro), glycine betaine (GB) and c-aminobutyric acid (GABA) in the mechanism(s) of tolerance to Zn stress. This study confirms the higher susceptibility of L. sativa to Zn stress: lettuce plants experienced a strong reduction in biomass, while the levels of Pro and GB increased. These results suggest that in L. sativa, the increase of Pro and GB does not represent a mechanism of resistance to toxicity, but it is likely a symptom of Zn stress. Conversely, in B. oleracea, a slight decrease in Pro levels, mainly catalysed by degradation through proline dehydrogenase, was observed; a similar behaviour affected GB levels. On the other hand, GABA synthesis was slightly, but significantly, increased. The presence of high levels of GABA in Zn-stressed B. oleracea would suggest that reactive oxygen species detoxification could be essential to improve the resistance to toxicity under metal stress conditions.

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP+/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, Km for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg2+ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots.

Summary Phosphoglucomutase (PGM) activity was measured during the development of endosperm of spring wheat ( Triticum aestivum L. cv. Axona) in crude extracts and in isolated amyloplasts. Results show a 60 % reduction in activity of plastidial PGM activity between 5 and 15 days post anthesis. Measurements of the concentrations of the substrates and co-factors of the PGM reaction in the tissue during this period indicate that the enzyme should be saturated in vivo , suggesting that the reduction in activity of PGM during endosperm development is a function of the amount of PGM protein. The implications of this for starch synthesis are discussed.

Psychrophilic organisms have evolved a number of modifications of cellular structures to survive in the cold environment; among them it is worth noting an increased efficiency of enzymes at lower temperatures. Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was purified and characterised from the psychrophilic green alga Koliella antarctica (Trebouxiophyceae, Chlorophyta) from the Ross Sea (Antarctica). It was possible to isolate a single G6PDH using biochemical strategies; its maximum activity was measured at 35 °C, and the enzyme showed an E a of 39.6 kJ mol−1. This protein reacted with antibodies raised against higher plants plastidic isoforms. KaG6PDH showed peculiar kinetic properties, with a K iNADPH value lower than $$ K_{{{\text{mNADP}}^{ + } }} $$ . Notably, catalytic activity was inactivated in vitro by DTT and chloroplastic thioredoxin f. These biochemical properties of G6PDH are discussed with respect to higher plant G6PDHs and the adaptation of K. antarctica to polar low-temperature environment.

In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms. The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coil, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction. The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies. (C) 2013 Elsevier Masson SAS. All rights reserved.

The mixed effects of nitrogen nutrition and sulphate assimilation were investigated in barley plants (Hordeum vulgare var. Alfeo) that were subjected to long-term sulphur and/or nitrogen starvation, by measuring the O-acetylserine(thio)lyase (OASTL–EC 4.2.99.8) activity, changes in –SH compounds and amino acid levels. The growth of barley plants cultured in the hydroponic vessels was severely affected by altered nutrient levels. The barley plants grown in medium deprived of nitrogen and/or sulphur sources for 21 days showed increase in both root length and weight. In contrast, the shoot growth was reduced in nitrogen-starved plants and was unaffected by sulphur deprivation. Sulphur starvation affected the level of proteins in barley plants more than nitrogen deprivation. The decline in the protein levels observed under sulphur-deficient conditions was coupled with the accumulation of glutamine, asparagine and serine, mainly in the roots; additionally, a nitrogen deficiency in the roots promoted a decrease in both glutathione and cysteine levels. The simultaneous deprivation of nitrogen and sulphur in plants leads to an alteration in their metabolism; high levels of glutathione (GSH) in the shoots could signify the induction of a mechanism intended for coping with stressful conditions. Sulphate deprivation enhanced OASTL activity, mainly in the roots; on the other hand, OASTL increases observed under S deprivation were clearly dependent on the nitrogen availability in the culture medium. In fact, the nitrate supply to the N and S starved plants that showed OASTL activity very low, rapidly recovered the OASTL activities to the levels typical of control plants. Nevertheless, the ammonium supply had negligible effects on the OASTL activity only observed after three days in the roots. Our results support the hypothesis that in barley plants, a portion of S assimilation (up to cysteine biosynthesis) occurs in the roots, and a reciprocal influence of nitrogen assimilation on cysteine synthesis occurs.

Plants respond to changes of nutrient availability in the soil by modulating their root system developmental plan. This response is mediated by systemic changes of the nutritional status and/or by local perception of specific signals. The effect of nitrate on Arabidopsis (Arabidopsis thaliana) root development represents a paradigm of these responses, and nitrate transporters are involved both in local and systemic control. Ammonium (NH4 +) represents an important nitrogen (N) source for plants, although toxicity symptoms are often associated with high NH4 + concentration when this is present as the only N source. The reason for these effects is still controversial, and mechanisms associating ammonium supply and plant developmental programs are completely unknown. We determined in Lotus japonicus the range of ammonium concentration that significantly inhibits the elongation of primary and lateral roots without affecting the biomass of the shoot. The comparison of the growth phenotypes in different N conditions indicated the specificity of the ammonium effect, suggesting that this was not mediated by assimilatory negative feedback mechanisms. In the range of inhibitory NH4 + conditions, only the LjAMT1;3 gene, among the members of the LjAMT1 family, showed a strong increased transcription that was reflected by an enlarged topology of expression. Remarkably, the short-root phenotype was phenocopied in transgenic lines by LjAMT1;3 overexpression independently of ammonium supply, and the same phenotype was not induced by another AMT1 member. These data describe a new plant mechanism to cope with environmental changes, giving preliminary information on putative actors involved in this specific ammonium-induced response.

The effects of nitrogen and sulphur deprivation on photosynthetic apparatus were investigated in barley grown in hydroponic culture: no difference was observed in AN, qP, and ETR/AN among control (C), nitrogen (-N), and sulphur (-S) deprivation. Conversely, plants grown in nitrogen and sulphur deprivation (-N-S) exhibited a reduction of AN and qP, a significant increase of NPQ and ETR/AN, as well as higher levels of glutathione and cysteine. The lowest total protein content was found in -N and -N-S plants. Data indicate that the simultaneous starvation of N and S leads to an alteration of plant metabolism that affects negatively photosynthesis. However, since no difference in Fv/Fm was found, it may be hypothesized that in -N-S seedlings processes other than photosynthesis and thermal dissipation are effective in photoprotection at low light. In -N-S plants, the highest glutathione content could represent a mean to face oxidative stress triggered within photosynthetic membranes by carbon fixation decline.

In Chlorella sorokiniana (211/8k), glucose-6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) activity is similar in both N-starved cells and nitrate-grown algae when expressed on a PCV basis. A single G6PDH isoform was purified from Chlorella cells grown under different nutrient conditions; the presence of a single G6PDH was confirmed by native gels stained for enzyme activity and by Western blots. The algal G6PDH is recognised only by antibodies raised against higher plants plastidic protein, but not by chloroplastic and cytosolic isoform-specific antisera. Purified G6PDH showed kinetic parameters similar to plastidic isoforms of higher plants, suggesting a different biochemical structure which would confer peculiar regulative properties to the algal G6PDH with respect to higher plants enzymes. The most remarkable property of algal G6PDH is represented by the response to NADPH inhibition. The algal enzyme is less sensitive to NADPH effects compared to higher plants G6PDH: Ki(NADPH) is 103 microM for G6PDH from nitrogen-starved C. sorokiniana, similarly to root plastidic P2-G6PDH. In nitrate-grown C. sorokiniana the Ki(NADPH) decreased to 48 microM, whereas other kinetic parameters remained unchanged. These results will allow further investigations in order to rule out possible modifications of the enzyme, and/or the expression of a different G6PDH isoform during nitrate assimilation.

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.

The effects of ammonium and glutamine supply on amino acid levels and the activity of glucose-6P dehydrogenase (G6PDH EC 1.1.1.49), the main regulated enzyme of the oxidative pentose phosphate pathway, were investigated in barley roots (Hordeum vulgare cv. Alfeo). Feeding ammonium to barley plants increased the contents of glutamine, asparagine and G6PDH in roots. These effects were abolished by using inhibitors of glutamine synthetase. Glutamine-fed barley roots showed a similar increase in G6PDH activities to ammonium-fed plants. Two G6PDH enzymes (G6PDH 1 and 2) were partially purified and characterized from ammonium-fed and glutamine-fed roots. The isozymes had different pH optima and apparent Km values for glucose-6P. G6PDH 2 showed similar kinetic parameters to the G6PDH present in root extracts of barley grown without any nitrogen source, while G6PDH 1 exhibited different kinetic parameters, suggesting the appearance of a second G6PDH isoform in response to ammonium. Western blot analysis demonstrated the existence of two G6PDH subunits of different molecular mass in barley roots grown in the presence of ammonium or glutamine, while only one isoform could be detected in roots grown without any nitrogen source. The results suggest a primary role of ammonium and/or glutamine in the appearance of a novel G6PDH isoform; this enzyme (G6PDH 1) shows kinetic parameters similar to those measured previously for chloroplastic and plastidic isoforms and seems to be induced by changes in glutamine content or a related compound(s) in the roots.

Barley plants (Hordeum vutgare L.) grown for 10 d in nitrogen-free hydroponic culture, after a rapid initial phase absorbed supplied NH4 (+) at a constant rate of 15.1 ±1.2 μ mol h(-1) g(-1) f. wt in the light, arid at a rate of 13.81 ± 1.6 μ mol h(-1) g(-1) f. wt in darkness. Ammonium-grown plants assimilated NH4 (+) at a rate of 7.5 ± 0.33 μmol h-1 g(-1) f. wt and at a 50% lower rate in darkness. Nitrogen-free grown plants showed low concentrations of free amino acids in both root and shoot tissues. Supplying NH4 (+) caused an immediate increase in the concentration of free amino in the root tissues of both illuminated and darkened plants over a 120 mm period. The increase in concentration of glutamine then exhibited a lag period of 120 min, after which it resumed, but to a very small extent. Glutamine also accumulated in shoot tissue of illuminated plants at increasing rates, attaining a concentration which, 8 h after NH4 (+) supply, was 1.61-fold greater than that attained in the roots. In shoots of darkened plants, by contrast, the concentration of glutamine increased slowly and was always smaller than that in the root tissue. Overall formation of glutamine (in shoots and roots) occurred at decreasing rates during the first 4 h, and then at increasing rates. The increase was more pronounced in illuminated plants than in darkened plants, liven 24 h after NH4 (+) was supplied, glutamine content in root tissue was lower than that in shoot tissue. However, 48 h later, the concentrations of glutamine in root and shoot were similar, attaining values that were almost 47-fold (in root) and 134-fold (in shoot) greater than initial values. Significant levels of asparagine were detected in the root and in the shoot 24 h after adding NH4 (+) . These increased further during the succeeding period. Ammonium supply caused a transitory drop in the concentration of ATP in root tissue, along with noticeable transitory variations in glucose-6-P concentration. A permanent decrease in free glucose concentration was also detected. Addition of NH4 (+) caused 2- and 1.43-fold increases in respiratory oxygen consumption by roots of illuminated and darkened plants, respectively. Both in the light and in the dark, the root tissue accumulated methylammonium up to a concentration of 55-67 μmol h(-1) g(-1) f. wt. Methylammonium was never found in shoot tissue of either illuminated or darkened plants. Methylammonium stimulated respiration of root barley plants by a factor of 1.2. Regulatory aspects of NH4 (+) metabolism are discussed.