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The synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D) functions as an agronomic weed control herbicide. High concentrations of 2,4‐D induce plant growth defects, particularly leaf epinasty and stem curvature. Although the 2,4‐D triggered reactive oxygen species (ROS) production, little is known about its signalling. In this study, by using a null mutant in peroxisomal acyl CoA oxidase 1 (acx1‐2), we identified acyl‐coenzyme A oxidase 1 (ACX1) as one of the main sources of ROS production and, in part, also causing the epinastic phenotype following 2,4‐D application. Transcriptomic analyses of wild type (WT) plants after treatment with 2,4‐D revealed a ROS‐related peroxisomal footprint in early plant responses, while other organelles, such as mitochondria and chloroplasts, are involved in later responses. Interestingly, a group of 2,4‐D‐responsive ACX1‐dependent transcripts previously associated with epinasty is related to auxin biosynthesis, metabolism, and signalling. We found that the auxin receptor auxin signalling F‐box 3 (AFB3), a component of Skp, Cullin, F‐box containing complex (SCF) (ASK‐cullin‐F‐box) E3 ubiquitin ligase complexes, which mediates auxin/indole acetic acid (AUX/IAA) degradation by the 26S proteasome, acts downstream of ACX1 and is involved in the epinastic phenotype induced by 2,4‐D. We also found that protein degradation associated with ubiquitin E3‐RING and E3‐SCF‐FBOX in ACX1‐dependent signalling in plant responses to 2,4‐D is significantly regulated over longer treatment periods. Summary Statement: 2,4‐D induced a reactive oxygen species (ROS)‐related peroxisomal footprint in early plant responses. ACX1 is one of the main sources of ROS production and associated epinasty phenotype following 2,4‐D application in plants. ACX1‐dependent signalling in plant responses to 2,4‐D point to protein degradation associated with auxin signalling at the early response and with ubiquitin E3‐RING and E3‐SCF‐FBOX/proteasome as key processes in the development of epinasty over longer treatment periods. [ABSTRACT FROM AUTHOR]

Complex signalling pathways are involved in plant protection against single and combined stresses. Plants are able to coordinate genome-wide transcriptional reprogramming and display a unique programme of transcriptional responses to a combination of stresses that differs from the response to single stresses. However, a significant overlap between pathways and some defence genes in the form of shared and general stress-responsive genes appears to be commonly involved in responses to multiple biotic and abiotic stresses. Reactive oxygen and nitrogen species, as well as redox signals, are key molecules involved at the crossroads of the perception of different stress factors and the regulation of both specific and general plant responses to biotic and abiotic stresses. In this review, we focus on crosstalk between plant responses to biotic and abiotic stresses, in addition to possible plant protection against pathogens caused by previous abiotic stress. Bioinformatic analyses of transcriptome data from cadmium- and fungal pathogen-treated plants focusing on redox gene ontology categories were carried out to gain a better understanding of common plant responses to abiotic and biotic stresses. The role of reactive oxygen and nitrogen species in the complex network involved in plant responses to changes in their environment is also discussed. [ABSTRACT FROM AUTHOR]

Highlights • Reactive oxygen and nitrogen species (ROS/RNS) have a dual role in plant responses to Cd stress. • An excess of ROS and RNS induce oxidative stress in plants under Cd treatment. • ROS and RNS in low concentrations, act as signalling molecules, capable of orchestrating plant responses to Cd stress. Abstract Although cadmium (Cd), an extremely toxic non-essential heavy metal, has no biological function, it is capable of entering plant roots. Cd not only presents a problem for plants, which have developed specific Cd detection, transport and detoxification mechanisms, but also for humans as it can enter the food chain. After entering the root, Cd can be loaded into the xylem and then into the leaves and fruits, thus constituting a major environmental and health hazard worldwide. Understanding the mechanisms involved in plant responses to Cd stress would facilitate the production of crops with a lower Cd uptake and accumulation capacity as well as plants with greater Cd uptake potential for phytoremediation. One of the most common symptoms of Cd toxicity is the induction of oxidative stress in plants, which have developed various strategies to avoid this toxicity, including the early production of reactive oxygen and nitrogen species (ROS and RNS) with signalling functions. This review focuses on the dual role of ROS and RNS in plant responses to Cd stress: in low concentrations, as signalling molecules capable of orchestrating plant responses on the one hand, and at higher concentrations, as oxidative stress inducers on the other. [ABSTRACT FROM AUTHOR]

Peroxisomes are highly dynamic and metabolically active organelles that play an important role in cellular functions, including reactive oxygen species (ROS) metabolism. Peroxisomal dynamics, such as the proliferation, movement, and production of dynamic extensions called peroxules, have been associated with ROS in plant cells. However, the function and regulation of peroxules are largely unknown. Using confocal microscopy, we have shown that treatment of Arabidopsis leaves with the heavy metal cadmium produces time course-dependent changes in peroxisomal dynamics, starting with peroxule formation, followed by peroxisome proliferation, and finally returning to the normal morphology and number. These changes during Cd treatment were regulated by NADPH oxidase (C and F)-related ROS production. Peroxule formation is a general response to stimuli such as arsenic and is regulated by peroxin 11a (PEX11a), as Arabidopsis pex11a RNAi lines are unable to produce peroxules under stress conditions. The pex11a line showed higher levels of lipid peroxidation content and lower expression of genes involved in antioxidative defenses and signaling, suggesting that these extensions are involved in regulating ROS accumulation and ROS-dependent gene expression in response to stress. Our results demonstrate that PEX11a and peroxule formation play a key role in regulating stress perception and fast cell responses to environmental cues. [ABSTRACT FROM AUTHOR]

Cellular metabolism is organized in a complex network of multiple steps called metabolic pathways to complete daily procedures and to maintain a metabolite balance in a coordinated way. As environmental conditions both inside and outside the cell are continuously changing, these pathways need to be highly regulated. Enzymes usually have different regulatory mechanisms at both the transcriptional and posttranscriptional level. A key feature of the signaling molecule nitric oxide (NO) is the direct alteration of proteins through posttranslational modifications (PTMs) such as S-nitrosylation and nitration. Certain proteins have been described as putative targets of NO-dependent PTMs and as comprising a broad range of categories, with the cellular metabolism being one of the most important. The functional effect of these NO-dependent PTMs on enzymes, the metabolic pathway flux and crosstalk between the different PTMs is still unclear. In this review, we will discuss the impact of NO-dependent PTMs on metabolic enzymes and their possible physiological effects on the regulation of the main pathways affected by these modifications. [ABSTRACT FROM AUTHOR]

Successful sexual reproduction often relies on the ability of plants to recognize self- or genetically-related pollen and prevent pollen tube growth soon after germination in order to avoid self-fertilization. Angiosperms have developed different reproductive barriers, one of the most extended being self-incompatibility (SI). With SI, pistils are able to reject self or genetically-related pollen thus promoting genetic variability. There are basically two distinct systems of SI: gametophytic (GSI) and sporophytic (SSI) based on their different molecular and genetic control mechanisms. In both types of SI, programmed cell death (PCD) has been found to play an important role in the rejection of self-incompatible pollen. Although reactive oxygen species (ROS) were initially recognized as toxic metabolic products, in recent years, a new role for ROS has become apparent: the control and regulation of biological processes such as growth, development, response to biotic and abiotic environmental stimuli, and PCD. Together with ROS, nitric oxide (NO) has become recognized as a key regulator of PCD. PCD is an important mechanism for the controlled elimination of targeted cells in both animals and plants. The major focus of this review is to discuss how ROS and NO control male-female cross-talk during fertilization in order to trigger PCD in self-incompatible pollen, providing a highly effective way to prevent self-fertilization. [ABSTRACT FROM AUTHOR]

Successful sexual reproduction often relies on the ability of plants to recognize self- or genetically-related pollen and prevent pollen tube growth soon after germination in order to avoid self-fertilization. Angiosperms have developed different reproductive barriers, one of the most extended being self-incompatibility (SI). With SI, pistils are able to reject self or genetically-related pollen thus promoting genetic variability. There are basically two distinct systems of SI: gametophytic (GSI) and sporophytic (SSI) based on their different molecular and genetic control mechanisms. In both types of SI, programmed cell death (PCD) has been found to play an important role in the rejection of selfincompatible pollen. Although reactive oxygen species (ROS) were initially recognized as toxic metabolic products, in recent years, a new role for ROS has become apparent: the control and regulation of biological processes such as growth, development, response to biotic and abiotic environmental stimuli, and PCD. Together with ROS, nitric oxide (NO) has become recognized as a key regulator of PCD. PCD is an important mechanism for the controlled elimination of targeted cells in both animals and plants. The major focus of this review is to discuss how ROS and NO control male-female cross-talk during fertilization in order to trigger PCD in self-incompatible pollen, providing a highly effective way to prevent self-fertilization. [ABSTRACT FROM AUTHOR]

Drought is one of the most limiting factors for plant survival mainly in arid areas. The predicted increase of temperature and dryness due to climate change will exacerbate this problem in many areas throughout the world. In this work, we analyze the mechanism involved in drought tolerance in a Saharan plant, Oudneya africana , by using two different irrigation regimes, a time course analysis, as well as re-watering after drought. The content of hydrogen peroxide (H 2 O 2 ), NO, lipid peroxidation, enzymatic and non-enzymatic antioxidants and NADPH sources in leaves was analyzed. Drought induces the accumulation of H 2 O 2 and NO, being the highest rate observed under the most severe conditions. The glycolate oxidase (GOX; EC 1.1.3.1) from photorespiration has been demonstrated to be one of the most important sources of H 2 O 2 . Water deficit produces disturbances in enzymatic and non enzymatic antioxidants to cope with the increase of reactive oxygen accumulation and differentially affected the pattern of superoxide dismutases (SOD; EC 1.15.1.1) and peroxidases (POX). The analysis of principal component shows a positive and significant relationship between the redox status (GSH/GSSG; ASC/DHA), glutathione reductase (GR; EC 1.6.4.2), the malate dehydrogenase (MDH; EC 1.1.1.37) and NADP-depend isocitrate dehydoregenase (NADP-ICDH; EC 1.1.1. 42) under long term treatment, suggesting that tolerance of Oudneya under these conditions is mainly due to the ASC–GSH cycle efficiency. Under medium drought stress the higher correlation was observed between DHA, ASC and glutathione S-transferase (GST; EC 2.5.1.18), while during the rehydration period catalase (CAT; EC 1.11.1.6), H 2 O 2 , GOX, NO and guayacol peroxidase (GPX; EC 1.11.1.7) show a high correlation, suggesting an important role of peroxidases and photorespiration in the adjustment of metabolic pathways during drought recovery. These results also show that longer exposure to medium stress has a stronger effect than shorter periods at higher intensity. The results obtained in this work suggest that Oudneya has the ability to activate a complex antioxidative defense system involved in the drought tolerance regulation probably involving H 2 O 2 -dependent signals. [ABSTRACT FROM AUTHOR]

Plant peroxisomes are highly dynamic organelles with regard to metabolic pathways, number and morphology and participate in different metabolic processes and cell responses to their environment. Peroxisomes from animal and plant cells house a complex system of reactive oxygen species (ROS) production associated to different metabolic pathways which are under control of an important set of enzymatic and non enzymatic antioxidative defenses. Nitric oxide (NO) and its derivate reactive nitrogen species (RNS) are also produced in these organelles. Peroxisomes can regulate ROS and NO/RNS levels to allow their role as signalling molecules. The metabolism of other reactive species such as carbonyl reactive species (CRS) and sulfur reactive species (SRS) in peroxisomes and their relationship with ROS and NO have not been explored in depth. In this review, we define a peroxisomal reactive species interactome (PRSI), including all reactive species ROS, RNS, CRS and SRS, their interaction and effect on target molecules contributing to the dynamic redox/ROS homeostasis and plasticity of peroxisomes, enabling fine-tuned regulation of signalling networks associated with peroxisome-dependent H 2 O 2. Particular attention will be paid to update the information available on H 2 O 2 -dependent peroxisomal retrograde signalling and to discuss a specific peroxisomal footprint. [Display omitted] • Peroxisomes are a multipurpose organelles necessary for both animal and plant cell functionality. • Peroxisomes participate in the perception of and cell responses to changes in their environment • Peroxisomes can be considered as a platform of signal molecules in the cell. • Peroxisomal reactive species interactome contribute to the redox/ROS/NO homeostasis and plasticity of peroxisomes. • ROS perturbations in peroxisomes trigger specific retrograde signalling. [ABSTRACT FROM AUTHOR]

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen–pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O2˙− and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O2˙− and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen–pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells. [ABSTRACT FROM PUBLISHER]

Abstract: Changes in the leaf antioxidant metabolism upon exposure to salinity and potassium deficiency were investigated in the annual halophyte Hordeum maritimum L. Plants were hydroponically grown either with a complete nutrient solution containing 3mM K+ without (+K/−NaCl) or with 100mM NaCl (+K/+NaCl), or in K+-free medium containing 100mM NaCl (−K/+NaCl). Malondialdehyde (MDA), carbonyl group (Cydrogen peroxide (H2O2) contents as well as antioxidant enzyme activities [superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2)] and non-enzymatic antioxidant contents (ascorbate and glutathione) were determined. Plants exposed to salinity, either alone or in combination with K+ deprivation, showed enhanced lipid peroxidation along with higher antioxidative response. This tendency was generally more marked in −K/+NaCl plants as compared to +K/+NaCl plants. H2O2 concentration was negatively correlated with the plant antioxidative capacity, either enzymatic or non-enzymatic. As a whole, these data suggest that the enhancement of the antioxidative response is of crucial significance for H. maritimum plants growing under salinity and potassium deficiency. [Copyright &y& Elsevier]

Abstract: Peroxisomes are organelles with an essentially oxidative metabolism that are involved in various metabolic pathways such as fatty acid β-oxidation, photorespiration, and metabolism of reactive oxygen species (ROS) and reactive nitrogen species. These organelles are highly dynamic but there is little information about the regulation of, and the effects of environment on, peroxisome movement. In this work a stable Arabidopsis line expressing the GFP–SKL peptide targeted to peroxisomes was characterized. Peroxisome-associated fluorescence was observed in all tissues, including leaves (mesophyll and epidermal cells, trichomes, and stomata) and roots. The dynamics of peroxisomes in epidermal cells was examined by confocal laser microscope, and various types of movement were observed. The speed of movement differed depending on the plant age. Treatment of plants with CdCl2 (100 μM) produced a significant increase in speed, which was dependent on endogenous ROS and Ca2+, but was not related to actin cytoskeleton modifications. In light of the results obtained, it is proposed that the increase in peroxisomal motility observed in Arabidopsis plants could be a cellular mechanism of protection against the Cd-imposed oxidative stress. Other possible roles for the enhanced peroxisome movement in plant cell physiology are discussed. [Copyright &y& Elsevier]

Cadmium (Cd) toxicity has been widely studied in different plant species; however, the mechanism involved in its toxicity as well as the cell response against the metal have not been well established. In this work, using pea (Pisum sativum) plants, we studied the effect of Cd on antioxidants, reactive oxygen species (ROS), and nitric oxide (NO) metabolism of leaves using different cellular, molecular, and biochemical approaches. The growth of pea plants with 50 μM CdCl2 affected differentially the expression of superoxide dismutase (SOD) isozymes at both transcriptional and posttranscriptional levels, giving rise to a SOD activity reduction. The copper/zinc-SOD down-regulation was apparently due to the calcium (Ca) deficiency induced by the heavy metal. In these circumstances, the overproduction of the ROS hydrogen peroxide and superoxide could be observed in vivo by confocal laser microscopy, mainly associated with vascular tissue, epidermis, and mesophyll cells, and the production of superoxide radicals was prevented by exogenous Ca. On the other hand, the NO synthase-dependent NO production was strongly depressed by Cd, and treatment with Ca prevented this effect. Under these conditions, the pathogen-related proteins PrP4A and chitinase and the heat shock protein 71.2, were up-regulated, probably to protect cells against damages induced by Cd. The regulation of these proteins could be mediated by jasmonic acid and ethylene, whose contents increased by Cd treatment. A model is proposed for the cellular response to long-term Cd exposure consisting of cross talk between Ca, ROS, and NO. [ABSTRACT FROM AUTHOR]

Summary: The long-term effects of 50μM CdCl2 on the enzymatic and non-enzymatic antioxidative defences of pea (Pisum sativum L.) plants was studied in terms of activity, protein content and transcripts levels. Cadmium caused a reduction of the total glutathione content (GSH+GSSG), with the reduced form of glutathione (GSH) being most affected. The content of ascorbic acid (ASC) was also decreased by the treatment. The transcript levels of catalase (CAT) and monodehydroascorbate reductase (MDHAR) showed a Cd-dependent increase, although CAT activity and its protein content were depressed, which suggests a posttranslational modification of this protein induced by cadmium. Glutathione reductase (GR), and ascorbate peroxidase (APX) did not change significantly, either in activity or accumulation of transcript. However, cadmium treatment provoked a strong reduction in mRNA, protein level and activity of CuZn-superoxide dismutase (SOD), being the most negatively affected antioxidative enzyme, and in less extent of Mn-SOD. Transcriptome analysis of the antioxidative enzymes in leaves of pea plants grown with cadmium and treated with some modulators of the signal transduction cascade suggested that at least Ca2+ channels, phosphorylation/dephosphorylation processes, nitric oxide, cGMP, salicylic acid (SA) and H2O2 were involved in some steps between the cadmium signal and transcript expression of CuZn-SOD, CAT and MDHAR. This indicated the existence of cross-talk between these elements and reactive oxygen species (ROS) metabolism during cadmium stress. [Copyright &y& Elsevier]

In this work the manganese superoxide dismutase (Mn-SOD) bound to peroxisomal membranes of watermelon cotyledons (Citrullus lanatus Schrad.) was purified to homogeneity and some of its molecular properties were determined. The stepwise purification procedure consisted of ammonium sulphate fractionation, batch anion-exchange chromatography, and anion-exchange and gel-filtration column chromatography using a fast protein liquid chromatography system. Peroxisomal membrane Mn-SOD (perMn-SOD; EC 1.15.1.1) was purified 5600-fold with a yield of 2.6 μg of enzyme g−1 of cotyledons, and had a specific activity of 480 U mg−1 of protein. The native molecular mass determined for perMn-SOD was 108 000 Da, and it was composed of four equal subunits of 27 kDa, which indicates that perMn-SOD is a homotetramer. Ultraviolet and visible absorption spectra of the enzyme showed a shoulder at 275 nm and two absorption maxima at 448 nm and 555 nm, respectively. By isoelectric focusing, a pI of 5.75 was determined for perMn-SOD. In immunoblot assays, purified perMn-SOD was recognized by a polyclonal antibody against Mn-SOD from pea leaves, and the peroxisomal enzyme rapidly dissociated in the presence of dithiothreitol and SDS. The potential binding of the Mn-SOD isozyme to the peroxisomal membrane was confirmed by immunoelectron microscopy analysis. The properties of perMn-SOD and the mitMn-SOD are compared and the possible function in peroxisomal membranes of the peripheral protein Mn-SOD is discussed. [ABSTRACT FROM PUBLISHER]

Growth of pea ( Pisum sativum L.) plants with 50 µm CdCl2 for 15 d produced a reduction in the number and length of lateral roots, and changes in structure of the principal roots affecting the xylem vessels. Cadmium induced a reduction in glutathione (GSH) and ascorbate (ASC) contents, and catalase (CAT), GSH reductase (GR) and guaiacol peroxidase (GPX) activities. CuZn-superoxide dismutase (SOD) activity was also diminished by the Cd treatment, although Mn-SOD was slightly increased. CAT and CuZn-SOD were down-regulated at transcriptional level, while Mn-SOD, Fe-SOD and GR were up-regulated. Analysis of reactive oxygen species (ROS) and nitric oxide (NO) levels by fluorescence and confocal laser microscopy (CLM) showed an over-accumulation of O2.− and H2O2, and a reduction in the NO content in lateral and principal roots. ROS overproduction was dependent on changes in intracellular Ca+2 content, and peroxidases and NADPH oxidases were involved. Cadmium also produced an increase in salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) contents. The rise of ET and ROS, and the NO decrease are in accordance with senescence processes induced by Cd, and the increase of JA and SA could regulate the cellular response to cope with damages imposed by cadmium. [ABSTRACT FROM AUTHOR]

• The glutathione reductase (GR; EC 1.6.4.2) isozyme present in peroxisomes has been purified for the first time, and its unequivocal localization in these organelles, by immunogold electron microscopy, is reported. • The enzyme was purified c. 21-fold with a specific activity of 9523 units mg−1 protein, and a yield of 44 µg protein kg−1 leaves was obtained. The subunit size of the peroxisomal GR was 56 kDa and the isoelectric point was 5.4. The enzyme was recognized by a polyclonal antibody raised against total GR from pea ( Pisum sativum) leaves. • The localization of GR in peroxisomes adds to chloroplasts and mitochondria where GR isozymes are also present, and suggests a multiple targeting of this enzyme to distinct cell compartments depending on the metabolism of each organelle under the plant growth conditions. • The expression level of GR in several organs of pea plants and under different stress conditions was investigated. The possible role of peroxisomal GR under abiotic stress conditions, such as cadmium toxicity, high light, darkness, high temperature, wounding and low temperature, is discussed. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a highly reactive signalling molecule that has numerous targets in plants. Both enzymatic and non-enzymatic synthesis of NO has been detected in several plant species, and NO functions have been characterized during diverse physiological processes such as plant growth, development, and resistance to biotic and abiotic stresses. This wide variety of effects reflects the basic signalling mechanisms that are utilized by virtually all mammalian and plant cells and suggests the necessity of detoxification mechanisms to control the level and functions of NO. During the last two years an increasing number of reports have implicated non-symbiotic haemoglobins as the key enzymatic system for NO scavenging in plants, indicating that the primordial function of haemoglobins may well be to protect against nitrosative stress and to modulate NO signalling functions. The biological relevance of plant haemoglobins during specific conditions of plant growth and stress, and the existence of further enzymatic and non-enzymatic NO scavenging systems, suggest the existence of precise NO modulation mechanisms in plants, as observed for different NO sources. [ABSTRACT FROM PUBLISHER]

Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. During the last few years NO has been detected in several plant species, and the increasing number of reports on its function in plants have implicated NO as a key molecular signal that participates in the regulation of several physiological processes; in particular, it has a significant role in plant resistance to pathogens by triggering resistance-associated cell death and by contributing to the local and systemic induction of defence genes. NO stimulates signal transduction pathways through protein kinases, cytosolic Ca2+ mobilization and protein modification (i.e. nitrosylation and nitration). In this review we will examine the synthesis of NO, its effects, functions and signalling giving rise to the hypersensitive response and systemic acquired resistance during plant–pathogen interactions. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO), first characterized as an endothelium-derived relaxation factor, is involved in diverse cellular processes including neuronal signaling, blood pressure homeostasis, and immune response. Recent studies have also revealed a role for NO as a signaling molecule in plants. As a developmental regulator, NO promotes germination, leaf extension and root growth, and delays leaf senescence and fruit maturation. Moreover, NO acts as a key signal in plant resistance to incompatible pathogens by triggering resistance-associated hypersensitive cell death. In addition, NO activates the expression of several defense genes (e.g. pathogenesisrelated genes, phenylalanine ammonialyase, chalcone synthase) and could play a role in pathways leading to systemic acquired resistance. [ABSTRACT FROM AUTHOR]

Arsenic in groundwater constitutes an agronomic problem due to its potential accumulation in the food chain. Among the agro‐sustainable tools to reduce metal(oid)s toxicity, the use of plant growth‐promoting bacteria (PGPB) becomes important. For that, and based on previous results in which significant differences of As translocation were observed when inoculating maize plants with Az39 or CD Azospirillum strains, we decided to decipher the redox metabolism changes and the antioxidant system response of maize plants inoculated when exposed to a realistic arsenate (AsV) dose. Results showed that AsV caused morphological changes in the root exodermis. Photosynthetic pigments decreased only in CD inoculated plants, while oxidative stress evidence was detected throughout the plant, regardless of the assayed strain. The antioxidant response was strain‐differential since only CD inoculated plants showed an increase in superoxide dismutase, glutathione S‐transferase (GST), and glutathione reductase (GR) activities while other enzymes showed the same behavior irrespective of the inoculated strain. Gene expression assays reported that only GST23 transcript level was upregulated by arsenate, regardless of the inoculated strain. AsV diminished the glutathione (GSH) content of roots inoculated with the Az39 strain, and CD inoculated plants showed a decrease of oxidized GSH (GSSG) levels. We suggest a model in which the antioxidant response of the maize‐diazotrophs system is modulated by the strain and that GSH plays a central role acting mainly as a substrate for GST. These findings generate knowledge for a suitable PGPB selection, and its scaling to an effective bioinoculant formulation for maize crops exposed to adverse environmental conditions. [ABSTRACT FROM AUTHOR]

Deterioration and impoverishment of soil, caused by environmental pollution and climate change, result in reduced crop productivity. To adapt to hostile soils, plants have developed a complex network of factors involved in stress sensing, signal transduction, and adaptive responses. The chemical properties of reactive oxygen species (ROS) and reactive nitrogen species (RNS) allow them to participate in integrating the perception of external signals by fine-tuning protein redox regulation and signal transduction, triggering specific gene expression. Here, we update and summarize progress in understanding the mechanistic basis of ROS and RNS production at the subcellular level in plants and their role in the regulation of ion channels/transporters at both transcriptional and post-translational levels. We have also carried out an in silico analysis of different redox-dependent modifications of ion channels/transporters and identified cysteine and tyrosine targets of nitric oxide in metal transporters. Further, we summarize possible ROS- and RNS-dependent sensors involved in metal stress sensing, such as kinases and phosphatases, as well as some ROS/RNS-regulated transcription factors that could be involved in metal homeostasis. Understanding ROS- and RNS-dependent signaling events is crucial to designing new strategies to fortify crops and improve plant tolerance of nutritional imbalance and metal toxicity. [ABSTRACT FROM AUTHOR]

Plant survival depends on seed germination and progression through post-germinative developmental checkpoints. These processes are controlled by the stress phytohormone abscisic acid (ABA). ABA regulates the basic leucine zipper transcriptional factor ABI5, a central hub of growth repression, while the reactive nitrogen molecule nitric oxide (NO) counteracts ABA during seed germination. However, the molecular mechanisms by which seeds sense more favourable conditions and start germinating have remained elusive. Here we show that ABI5 promotes growth via NO, and that ABI5 accumulation is altered in genetic backgrounds with impaired NO homeostasis. S-nitrosylation of ABI5 at cysteine-153 facilitates its degradation through CULLIN4-based and KEEP ON GOING E3 ligases, and promotes seed germination. Conversely, mutation of ABI5 at cysteine-153 deregulates protein stability and inhibition of seed germination by NO depletion. These findings suggest an inverse molecular link between NO and ABA hormone signalling through distinct posttranslational modifications of ABI5 during early seedling development. [ABSTRACT FROM AUTHOR]

Leaf epinasty involves the downward bending of leaves as a result of disturbances in their growth, with a greater expansion in adaxial cells as compared to abaxial surface cells. The co-ordinated anisotropy of growth in epidermal, palisade mesophyll and vascular tissues contributes to epinasty. This phenotype, which is regulated by auxin (indole-3-acetic acid, IAA), controls plant cell division and elongation by regulating the expression of a vast number of genes. Other plant hormones, such as ethylene, abscisic acid and brassinosteroids, also regulate epinasty and hyponasty. Reactive oxygen species (ROS) accumulation induced by auxins and 2,4-dichlorophenoxyacetic acid (2,4-D) triggers epinasty. The role of ROS and nitric oxide (NO) in the regulation of epinasty has recently been established. Thus, treatment with synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) induces disturbances in the actin cytoskeleton through ROS and NO-dependent post-translational modifications in actin by carbonylation and S -nitrosylation, which cause a reduction in the actin filament. Reorientation of microtubules has become a major feature of the response to auxin. The cytoskeleton is therefore a key player in epinastic development. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is one of the most toxic heavy metals for plants and humans. Reactive oxygen species (ROS) are some of the primary signaling molecules produced after Cd treatment in plants but the contribution of different organelles and specific cell types, together with the impact of light is unknown. We used Arabidopsis lines expressing GRX1-roGFP2 (glutaredoxin1-roGFP) targeted to different cell compartments and analysed changes in redox state over 24 h light/dark cycle in Cd-treated leaf discs. We imaged redox state changes in peroxisomes and chloroplasts in leaf tissue. Chloroplasts and peroxisomes were the most affected organelles in the dark and blocking the photosynthetic electron transport chain (pETC) by DCMU (3-(3,4-dichlorophenyl)−1,1-dimethylurea) promotes higher Cd-dependent oxidation in all organelles. Peroxisomes underwent the most rapid changes in redox state in response to Cd and DCMU and silencing chloroplastic NTRC (NADPH thioredoxin reductase C) considerably increases peroxisome oxidation. Total NAD(P)H and cytosolic NADH decreased during exposure to Cd, while Ca+2 content in chloroplasts and cytosol increased in the dark period. Our results demonstrate a Cd-, time- and light-dependent increase of oxidation of all organelles analysed, that could be in part triggered by disturbances in pETC and photorespiration, the decrease of NAD(P)H availability, and differential antioxidants expression at subcellular level. [Display omitted] • Cadmium promotes time- and light-dependent oxidation of peroxisomes, chloroplasts, cytosol and mitochondria. • Photosynthetic electron transport chain regulates light- and Cd-dependent oxidation of chloroplasts, peroxisomes and cytosol. • Peroxisomes and cytosol are the earliest cell compartments perceiving Cd. • Photorespiration contributes to cell redox homeostasis in response to Cd. • Cd-dependent NAD(P)H availability and differential antioxidant genes expression regulate subcellular redox homeostasis. [ABSTRACT FROM AUTHOR]

ABSTRACT In this work the differential response of adult and young leaves from pea ( Pisum sativum L.) plants to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (23 m m) applied by foliar spraying was investigated. The concentration of 2,4-D (23 m m) and the time of treatment (72 h) were previously optimized in order to visualize its toxic effects on pea plants. Under these conditions, the herbicide induced severe disturbances in mesophyll cells structure and proliferation of vascular tissue in young leaves and increased acyl-CoA oxidase (ACX), xanthine oxidase (XOD) and lipoxygenase (LOX) activities in young leaves, and only ACX and LOX in adult leaves. This situation produced reactive oxygen species (ROS) over-accumulation favoured by the absence of significant changes in the enzymatic antioxidants, giving rise to oxidative damages to proteins and membrane lipids. An increase of ethylene took place in both young and adult leaves and the induction of genes encoding the stress proteins, PRP4A and HSP 71,2, was observed mainly in young leaves. These results suggest that ROS overproduction is a key factor in the effect of high concentrations of 2,4-D, and ROS can trigger a differential response in young and adult leaves, either epinasty development in young leaves or senescence processes in adult tissues. [ABSTRACT FROM AUTHOR]

Volatile compounds (VCs) of Trichoderma fungi trigger induced systemic resistance (ISR) in Arabidopsis that is effective against a broad spectrum of pathogens. The root-specific transcription factor MYB72 is an early regulator of ISR and also controls the activation of iron-deficiency responses. Nitric oxide (NO) is involved in the regulation of MYB72-dependent iron-deficiency responses in Arabidopsis roots, but the role of NO in the regulation of MYB72 and ISR by Trichoderma VCs remains unexplored. Using in vitro bioassays, we applied Trichoderma VCs to Arabidopsis seedlings. Plant perception of Trichoderma VCs triggered a burst of NO in Arabidopsis roots. By suppressing this burst using an NO scavenger, we show the involvement of NO in Trichoderma VCs-mediated regulation of MYB72 expression. Using an NO scavenger and the Arabidopsis lines myb72 and nia1nia2 in in planta bioassays, we demonstrate that NO signalling is required in the roots for activation of Trichoderma VCs-mediated ISR against the leaf pathogen Botrytis cinerea. Analysis of the defence-related genes PR1 and PDF1.2 points to the involvement of root NO in priming leaves for enhanced defence. Our results support a key role of root NO signalling in the regulation of MYB72 expression during the activation of ISR by Trichoderma VCs. Nitric oxide signalling in roots is required for induced systemic resistance by Trichoderma volatile compounds in leaves by regulating the root-specific transcription factor MYB72 in Arabidopsis. [ABSTRACT FROM AUTHOR]

Heavy metal concentrations, which have been increasing over the last 200 years, affect soil quality and crop yields. These elements are difficult to eliminate from soils and may constitute a human health hazard by entering the food chain. Recently, we obtained a selection of mutants with different degrees of tolerance to a mixture of heavy metals (HMmix) in order to gain a deeper insight into the underlying mechanism regulating plant responses to these elements. In this study, we characterized the mutant obtained Atkup8 (in this work, Atkup8‐2), which showed one of the most resistant phenotypes, as determined by seedling root length. Atkup8‐2 is affected in the potassium transporter KUP8, a member of the high‐affinity K+ uptake family KUP/HAK/KT. Atkup8‐2 mutants, which are less affected as measured by seedling root length under HMmix conditions, showed a resistant phenotype with respect to WT seedlings which, despite their delayed growth, are able to develop true leaves at levels similar to those under control conditions. Adult Atkup8‐2 plants had a higher fresh weight than WT plants, a resistant phenotype under HMmix stress conditions and lower levels of oxidative damage. KUP8 did not appear to be involved in heavy metal or macro‐ and micro‐nutrient uptake and translocation from roots to leaves, as total concentrations of these elements were similar in both Atkup8‐2 and WT plants. However, alterations in cellular K+ homeostasis in this mutant cannot be ruled out. [ABSTRACT FROM AUTHOR]

Arsenic (As) can be present naturally in groundwater from peanut fields, constituting a serious problem, as roots can accumulate and mobilize the metalloid to their edible parts. Understanding the redox changes in the legume exposed to As may help to detect potential risks to human health and recognize tolerance mechanisms. Thirty-days old peanut plants inoculated with Bradyrhizobium sp. strains (SEMIA6144 or C-145) were exposed to a realistic arsenate concentration, in order to unravel the redox response and characterize the oxidative stress indexes. Thus, root anatomy, reactive oxygen species detection by fluorescence microscopy and, ROS histochemical staining along with the NADPH oxidase activity were analyzed. Besides, photosynthetic pigments and damage to lipids and proteins were determined as oxidative stress indicators. Results showed that at 3 μM AsV, the cross-section areas of peanut roots were augmented; NADPH oxidase activity was significantly increased and O 2 ˙¯and H 2 O 2 accumulated in leaves and roots. Likewise, an increase in the lipid peroxidation and protein carbonyls was also observed throughout the plant regardless the inoculated strain, while chlorophylls and carotenes were increased only in those inoculated with Bradyrhizobium sp. C-145. Interestingly, the oxidative burst, mainly induced by the NADPH oxidase activity, and the consequent oxidative stress was strain-dependent and organ-differential. Additionally, As modifies the root anatomy, acting as a possibly first defense mechanism against the metalloid entry. All these findings allowed us to conclude that the redox response of peanut is conditioned by the rhizobial strain, which contributes to the importance of effectively formulating bioinoculants for this crop. Graphical Abstract. A proposed model to explain the impact of a realistic arsenate concentration on the peanut- Bradyrhizobium sp. (strains SEMIA6144 or C-145) symbiotic interaction. Plant maintains or increase their photosynthetic pigment content as a possible strategy to sustain carbon fixation. Additionally, the main root increases its cross section area as a potential first restriction mechanism against metalloid entry. In leaves and roots, NADPH oxidase activity is increased along with the accumulation of superoxide anion (O 2 ˙¯) and hydrogen peroxide (H 2 O 2). Consequently, the oxidative burst can be associated with lipid and protein damages, as revealed by the increase in TBARS and protein carbonyls content, respectively. The extent of oxidative stress is modulated by the tolerance to As of the rhizobial strain. Image 1 • The redox response of the peanut-bradyrhizobia symbiosis exposed to AsV is unveiled. • Root anatomy changes are the first defense mechanism against As entry. • The NADPH activity in peanut organs is modulated by the rhizobial tolerance. • Oxidative stress is Bradyrhizobium sp. strain-dependent and organ-differential. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) and nitrosylated derivatives are produced in peroxisomes, but the impact of NO metabolism on organelle functions remains largely uncharacterised. Double and triple NO‐related mutants expressing cyan florescent protein (CFP)‐SKL (nox1 × px‐ck and nia1 nia2 × px‐ck) were generated to determine whether NO regulates peroxisomal dynamics in response to cadmium (Cd) stress using confocal microscopy. Peroxule production was compromised in the nia1 nia2 mutants, which had lower NO levels than the wild‐type plants. These findings show that NO is produced early in the response to Cd stress and was involved in peroxule production. Cd‐induced peroxisomal proliferation was analysed using electron microscopy and by the accumulation of the peroxisomal marker PEX14. Peroxisomal proliferation was inhibited in the nia1 nia2 mutants. However, the phenotype was recovered by exogenous NO treatment. The number of peroxisomes and oxidative metabolism were changed in the NO‐related mutant cells. Furthermore, the pattern of oxidative modification and S‐nitrosylation of the catalase (CAT) protein was changed in the NO‐related mutants in both the absence and presence of Cd stress. Peroxisome‐dependent signalling was also affected in the NO‐related mutants. Taken together, these results show that NO metabolism plays an important role in peroxisome functions and signalling. [ABSTRACT FROM AUTHOR]

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves. To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course‐dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly up‐regulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14. Arabidopsis lines expressing GFP‐ATG8a and CFP‐SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd‐dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)‐related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron‐dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium. The molecular mechanisms governing pexophagy have been elusive. The data presented in this study show that pexophagy takes place in response to Cd toxicity to regulate peroxisomal populations and quality and is regulated by H2O2 and peroxisomal oxidation. NBR1 may be an autophagic receptor for peroxisome, and cathepsin B, legumain, and caspase 6 could be involved in the regulation of pexophagy. [ABSTRACT FROM AUTHOR]

Anthropogenic activities, such as industrial processes, mining, and agriculture, lead to an increase in heavy metal concentrations in soil, water, and air. Given their stability in the environment, heavy metals are difficult to eliminate and can constitute a human health risk by entering the food chain through uptake by crop plants. An excess of heavy metals is toxic for plants, which have various mechanisms to prevent their accumulation. However, once metals enter the plant, oxidative damage sometimes occurs, which can lead to plant death. Initial production of nitric oxide (NO), which may play a role in plant perception, signalling, and stress acclimation, has been shown to protect against heavy metals. Very little is known about NO-dependent mechanisms downstream from signalling pathways in plant responses to heavy metal stress. In this review, using bioinformatic techniques, we analyse studies of the involvement of NO in plant responses to heavy metal stress, its possible role as a cytoprotective molecule, and its relationship with reactive oxygen species. Some conclusions are drawn and future research perspectives are outlined to further elucidate the signalling mechanisms underlying the role of NO in plant responses to heavy metal stress. [ABSTRACT FROM AUTHOR]

Whilst many interactions with fungi are detrimental for plants, others are beneficial and result in improved growth and stress tolerance. Thus, plants have evolved sophisticated mechanisms to restrict pathogenic interactions while promoting mutualistic relationships. Numerous studies have demonstrated the importance of nitric oxide (NO) in the regulation of plant defence against fungal pathogens. NO triggers a reprograming of defence-related gene expression, the production of secondary metabolites with antimicrobial properties, and the hypersensitive response. More recent studies have shown a regulatory role of NO during the establishment of plant–fungal mutualistic associations from the early stages of the interaction. Indeed, NO has been recently shown to be produced by the plant after the recognition of root fungal symbionts, and to be required for the optimal control of mycorrhizal symbiosis. Although studies dealing with the function of NO in plant–fungal mutualistic associations are still scarce, experimental data indicate that different regulation patterns and functions for NO exist between plant interactions with pathogenic and mutualistic fungi. Here, we review recent progress in determining the functions of NO in plant–fungal interactions, and try to identify common and differential patterns related to pathogenic and mutualistic associations, and their impacts on plant health. [ABSTRACT FROM AUTHOR]

Summary: The regulatory role of nitric oxide (NO) and phytoglobins in plant response to pathogenic and mutualistic microbes has been evidenced. However, little is known about their function in the arbuscular mycorrhizal (AM) symbiosis. We investigated whether NO and phytoglobin PHYTOGB1 are regulatory components in the AM symbiosis.Rhizophagus irregularis in vitro‐grown cultures and tomato plants were used to monitor AM‐associated NO‐related root responses as compared to responses triggered by the pathogen Fusarium oxysporum. A genetic approach was conducted to understand the role of PHYTOGB1 on NO signaling during both interactions.After a common early peak in NO levels in response to both fungi, a specific NO accumulation pattern was triggered in tomato roots during the onset of the AM interaction. PHYTOGB1 was upregulated by the AM interaction. By contrast, the pathogen triggered a continuous NO accumulation and a strong downregulation of PHYTOGB1. Manipulation of PHYTOGB1 levels in overexpressing and silenced roots led to a deregulation of NO levels and altered mycorrhization and pathogen infection.We demonstrate that the onset of the AM symbiosis is associated with a specific NO‐related signature in the host root. We propose that NO regulation by PHYTOGB1 is a regulatory component of the AM symbiosis. [ABSTRACT FROM AUTHOR]

Emissions of heavy metals have risen over the past 200 years and significantly exceed those from natural sources. Phytoremediation strategies may be able to recover soil productivity in self-sustaining ecosystems; however, our knowledge of the molecular mechanisms involved in plant heavy-metal perception and signalling is scarce. The aim of this study was to assemble a “molecular tool box” of genes useful for phytoremediation. To identify mutants with different heavy-metal-tolerance, we first selected a medium from mixtures containing three metals based on their presence in two Spanish mining areas and then screened about 7000 lines of Arabidopsis T-DNA mutants and found 74 lines more resistant and 56 more susceptible than the wild type (WT). Classification of the genes showed that they were mainly linked to transport, protein modification and signalling, with RNA metabolism being the most representative category in the resistant phenotypes and protein metabolism in the sensitive ones. We have characterized one resistant mutant, Athpp9 and one sensitive, Atala4 . These mutants showed differences in growth and metal translocation. Additionally, we found that these mutants keep their phenotype in amended former soils, suggesting that these genes may be useful for phytoremediation and the recovery of contaminated soils. [ABSTRACT FROM AUTHOR]

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin-Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) have emerged as important factors linking reactive oxygen species (ROS) metabolism to redox-dependent signaling events. Together with ROS, nitric oxide (NO) is a free radical product of the cell metabolism that is essential in the signal transduction. S -Nitrosylation is emerging as a fundamental protein modification for the transduction of NO bioactivity. Using recombinant pea mitochondrial PsPrxII F (PrxII F), the effect of S -nitrosoglutathione (GSNO) and sodium nitroprusside dehydrate (SNP), which are known to mediate protein S -nitrosylation processes, was studied. S -Nitrosylation of the PrxII F was demonstrated using the biotin switch method and LC ESI-QTOF tandem MS analysis. S -nitrosylated PrxII F decreased its peroxidase activity and acquired a new transnitrosylase activity, preventing the thermal aggregation of citrate synthase (CS). For the first time, we demonstrate the dual function for PrxII F as peroxidase and transnitrosylase. This switch was accompanied by a conformational change of the protein that could favor the protein–protein interaction CS-PrxII F. The observed in vivo S -nitrosylation of PrxII F could probably function as a protective mechanism under oxidative and nitrosative stress, such as occurs under salinity. We conclude that we are dealing with a novel regulatory mechanism for this protein by NO. Biological significance S -Nitrosylation is a post-translational modification that is increasingly viewed as fundamental for the signal transduction role of NO in plants. This study demonstrates that S -nitrosylation of the mitochondrial peroxiredoxin PrxII F induces a conformational change in the protein and provokes a reduction in its peroxidase activity, while acquiring a novel function as transnitrosylase. The implication of this mechanism will increase our understanding of the role of posttranslational modifications in the protein function in plants under stress situations such as salinity, in which NO could act as signaling molecule. [ABSTRACT FROM AUTHOR]

Cadmium ( Cd) is a non-essential heavy metal that may be toxic or even lethal to plants as it can be easily taken up by the roots and loaded into the xylem to the leaves. Using soybean roots ( G lycine max L.) DM 4800, we have analysed various parameters related to reactive oxygen metabolism and nitric oxide ( NO) during a 6 day Cd exposure. A rise in H2O2 and NO, and to a lesser extent O2·− content was observed after 6 h exposure with a concomitant increase in lipid peroxidation and carbonyl group content. Both oxidative markers were significantly reduced after 24 h. A second, higher wave of O2·− production was also observed after 72 h of exposure followed by a reduction until the end of the treatment. NOX and glicolate oxidase activity might be involved in the initial Cd-induced reactive oxygen species (ROS) production and it appears that other sources may also participate. The analysis of antioxidative enzymes showed an increase in glutathione- S-transferase activity and in transcript levels and activity of enzymes involved in the ascorbate-glutathione cycle and the NADPH-generating enzymes. These results suggest that soybean is able to respond rapidly to oxidative stress imposed by Cd by improving the availability of NADPH necessary for the ascorbate-glutathione cycle. [ABSTRACT FROM AUTHOR]

NADPH oxidase, an enzyme associated with the plasma membrane, constitutes one of the main sources of reactive oxygen species (ROS) which regulate different developmental and adaptive responses in plants. In this work, the involvement of NADPH oxidases in the regulation of photosynthesis and cell ionic homeostasis in response to short cadmium exposure was compared between wild type (WT) and three RBOHs (Respiratory Burst Oxidase Homologues) Arabidopsis mutants (AtrbohC , AtrbohD , and AtrbohF). Plants were grown under hydroponic conditions and supplemented with 50 µM CdCl 2 for 24 h. Cadmium treatment differentially affected photosynthesis, stomatal conductance, transpiration, and antioxidative responses in WT and Atrboh s mutants. The loss of function of RBOH isoforms resulted in higher Cd2+ influx, mainly in the elongation zone of roots, which was more evident in AtrbohD and AtrbohF mutants. In the mature zone, the highest Cd2+ influx was observed in rbohC mutant. The lack of functional RBOH isoforms also resulted in altered patterns of net K+ transport across cellular membranes, both in the root epidermis and leaf mesophyll. The analysis of expression of metal transporters by qPCR demonstrated that a loss of functional RBOH isoforms has altered transcript levels for metal NRAMP3 , NRAMP6 and IRT1 and the K+ transporters outward-rectifying K+ efflux GORK channel, while RBOHD specifically regulated transcripts for high-affinity K+ transporters KUP8 and HAK5 , and IRT1 and RBOHD and F regulated the transcription factors TGA3 and TGA10. It is concluded that RBOH-dependent H 2 O 2 regulation of ion homeostasis and Cd is a highly complex process involving multilevel regulation from transpirational water flow to transcriptional and posttranslational modifications of K/metals transporters. [Display omitted] • RBOHs C, D and F differentially regulate photosynthesis and transpiration. • The lack of any RBOHs produced an increase in Cd2+ influx and changes in net K+ fluxes in roots, mainly in the elongation zone. • RBOHs differentially regulate the transporters IRT1 , NRAMP3 and NRAMP6 , KUP8 , HAK5 and GORK, and the transcription factors TGA3 and TGA10. [ABSTRACT FROM AUTHOR]

Peroxisomes, single-membrane-bounded organelles with essentially oxidative metabolism, are key in plant responses to abiotic and biotic stresses. Recently, the presence of nitric oxide (NO) described in peroxisomes opened the possibility of new cellular functions, as NO regulates diverse biological processes by directly modifying proteins. However, this mechanism has not yet been analysed in peroxisomes. This study assessed the presence of S-nitrosylation in pea-leaf peroxisomes, purified S-nitrosylated peroxisome proteins by immunoprecipitation, and identified the purified proteins by two different mass-spectrometry techniques (matrix-assisted laser desorption/ionization tandem time-of-flight and two-dimensional nano-liquid chromatography coupled to ion-trap tandem mass spectrometry). Six peroxisomal proteins were identified as putative targets of S-nitrosylation involved in photorespiration, β-oxidation, and reactive oxygen species detoxification. The activity of three of these proteins (catalase, glycolate oxidase, and malate dehydrogenase) is inhibited by NO donors. NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D). Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes. However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H2O2 level under abiotic stress. [ABSTRACT FROM PUBLISHER]

S-nitrosoglutathione (GSNO) is considered a natural nitric oxide (NO·) reservoir and a reactive nitrogen intermediate in animal cells, but little is known about this molecule and its metabolism in plant systems. In this work, using pea plants as a model system, the presence of GSNO in collenchyma cells was demonstrated by an immunohistochemical method. When pea plants were grown with a toxic Cd concentration (50 μM) the content of GSNO in collenchyma cells was drastically reduced. Determination of the nitric oxide (NO·) and gluthathione contents in leaves by confocal laser scanning microscopy and HPLC, respectively, showed a marked decrease of both compounds in plants treated with cadmium. The analysis of the S-nitrosoglutathione reductase (GSNOR) activity and its transcript expression in leaves showed a reduction of 31% by cadmium. These results indicate that GSNO is associated with a specific plant cell type, and this metabolite and its related catabolic activity, GSNOR, are both down-regulated under Cd stress. [ABSTRACT FROM PUBLISHER]

The effect of growing five different cultivars of pepper plants (Capsicum annuum L.) with CdCl2 concentrations ranging from 0.125 to 0.5 mM on different physiological parameters, and antioxidative enzyme activities of leaves was studied. On the basis of growth parameters, pepper plants were relatively tolerant to cadmium, although metal concentrations higher than 0.125 mM produced a significant inhibition of growth, net photosynthesis, and water use efficiency. Different sensitivities to Cd++ ions were observed among cultivars, Abdera being the most resistant to cadmium stress, while Mondo and Herminio were the most sensitive cultivars. Cadmium concentrations of 0.5 mM produced an increase in the activity of glutathione reductase, and guaiacol peroxidase in most cultivars, while catalase and superoxide dismutase (SOD) were slightly depressed. The analysis of the SOD activity pattern by native-PAGE showed the presence in most cultivars of four SODs which were identified as Mn–SOD, Fe–SOD, CuZn–SOD I and CuZn–SOD II. However, the two CuZn–SODs were absent in the Cd-sensitive cv. Herminio. The growth of pepper plants with 0.5 mM cadmium inhibited the activity of CuZn–SODs in all cultivars, while the activity of Mn- and Fe–SOD was enhanced. The activity of NADPH-dehydrogenases (glucose-6-P-dehydrogenase, 6-phosphogluconate dehydrogenase, NADP–isocitrate dehydrogenase and malic enzyme) showed a Cd-dependent enhancement in most cultivars, the highest increase being observed in the tolerant cv. Abdera. These results suggest that in pepper plants the tolerance to Cd toxicity is more dependent on the availability of NADPH than on its antioxidant capacity. [Copyright &y& Elsevier]

Growth and development in all organisms occur as a result of an overall balance between synthesis and proteolysis. In plants, protein degradation is a crucial mechanism in some developmental stages such as germination, morphogenesis and cell biogenesis, senescence, and programmed cell death. In this work, the main proteases that take part in these processes are reviewed. Proteolysis is also an important component together with protein oxidation in oxidative stress situations induced by senescence and heavy metals. The presence of exo- and endoproteolytic activity in plant peroxisomes is analyzed, and the role of peroxisomal proteases in different physiological events that take place under oxidative stress situations is discussed. [Copyright &y& Elsevier]