Reactive oxygen species- and nitric oxide-dependent regulation of ion and metal homeostasis in plants.

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.

Insights into ROS-dependent signalling underlying transcriptomic plant responses to the herbicide 2,4-D.

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.

Histochemical Techniques in Plant Science: More Than Meets the Eye.

Histochemistry is an essential analytical tool interfacing extensively with plant science. The literature is indeed constellated with examples showing its use to decipher specific physiological and developmental processes, as well as to study plant cell structures. Plant cell structures are translucent unless they are stained. Histochemistry allows the identification and localization, at the cellular level, of biomolecules and organelles in different types of cells and tissues, based on the use of specific staining reactions and imaging. Histochemical techniques are also widely used for the in vivo localization of promoters in specific tissues, as well as to identify specific cell wall components such as lignin and polysaccharides. Histochemistry also enables the study of plant reactions to environmental constraints, e.g. the production of reactive oxygen species (ROS) can be traced by applying histochemical staining techniques. The possibility of detecting ROS and localizing them at the cellular level is vital in establishing the mechanisms involved in the sensitivity and tolerance to different stress conditions in plants. This review comprehensively highlights the additional value of histochemistry as a complementary technique to high-throughput approaches for the study of the plant response to environmental constraints. Moreover, here we have provided an extensive survey of the available plant histochemical staining methods used for the localization of metals, minerals, secondary metabolites, cell wall components, and the detection of ROS production in plant cells. The use of recent technological advances like CRISPR/Cas9-based genome-editing for histological application is also addressed. This review also surveys the available literature data on histochemical techniques used to study the response of plants to abiotic stresses and to identify the effects at the tissue and cell levels.

An update on redox signals in plant responses to biotic and abiotic stress crosstalk: insights from cadmium and fungal pathogen interactions.

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.

Role of potassium transporter KUP8 in plant responses to heavy metals.

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.

Characterization of the biochemical basis for copper homeostasis and tolerance in Biscutella auriculata L.

Biscutella auriculata L. is a plant that belongs to the Brassicaceae family and it has been found growing in a metal-contaminated area of the San Quíntín mine (Ciudad Real, Spain). The purpose of this work was to evaluate the mechanisms that allow this plant to tolerate high concentrations of copper. Seedlings were grown in a semi-hydroponic system for 15 days under 125 µM of Cu (NO3 )2 . Exposure to copper resulted in growth inhibition and reduction in the photosynthetic parameters. Copper was mainly accumulated in vascular tissue and vacuoles of the roots and only a minor proportion was transferred to the shoot. Biothiol analysis showed a greater enhancement of reduced glutathione in leaves and increases of phytochelatins (PC2 and PC3) in both leaves and roots. Copper treatment induced oxidative stress, which triggered a response of the enzymatic and non-enzymatic antioxidant mechanisms. The results show that B. auriculata is able to tolerate high metal levels through the activation of specific mechanisms to neutralize the oxidative stress produced and also by metal sequestration through phytochelatins. The preferential accumulation of copper in roots provides clues for further studies on the use of this plant for phytostabilization and environmental recovery purposes in Cu-contaminated areas.

Nitric oxide is essential for cadmium-induced peroxule formation and peroxisome proliferation.

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.

Cadmium induces reactive oxygen species-dependent pexophagy in Arabidopsis leaves.

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.

Role of nitric oxide in plant responses to heavy metal stress: exogenous application versus endogenous production.

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.

Multilevel Regulation of Peroxisomal Proteome by Post-Translational Modifications.

Peroxisomes, which are ubiquitous organelles in all eukaryotes, are highly dynamic organelles that are essential for development and stress responses. Plant peroxisomes are involved in major metabolic pathways, such as fatty acid ß-oxidation, photorespiration, ureide and polyamine metabolism, in the biosynthesis of jasmonic, indolacetic, and salicylic acid hormones, as well as in signaling molecules such as reactive oxygen and nitrogen species (ROS/RNS). Peroxisomes are involved in the perception of environmental changes, which is a complex process involving the regulation of gene expression and protein functionality by protein post-translational modifications (PTMs). Although there has been a growing interest in individual PTMs in peroxisomes over the last ten years, their role and cross-talk in the whole peroxisomal proteome remain unclear. This review provides up-to-date information on the function and crosstalk of the main peroxisomal PTMs. Analysis of whole peroxisomal proteomes shows that a very large number of peroxisomal proteins are targeted by multiple PTMs, which affect redox balance, photorespiration, the glyoxylate cycle, and lipid metabolism. This multilevel PTM regulation could boost the plasticity of peroxisomes and their capacity to regulate metabolism in response to environmental changes.

Autophagy-related approaches for improving nutrient use efficiency and crop yield protection.

Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.

Peroxisomes Extend Peroxules in a Fast Response to Stress via a Reactive Oxygen Species-Mediated Induction of the Peroxin PEX11a.

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.

The role of reactive oxygen species and nitric oxide in programmed cell death associated with self-incompatibility.

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.

Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana.

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.

Cadmium induces two waves of reactive oxygen species in Glycine max (L.) roots.

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 (Glycine 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 H(2)O(2) and NO, and to a lesser extent O(2)(·-) 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 O(2)(·-) 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.

Role of peroxisomes as a source of reactive oxygen species (ROS) signaling molecules.

Peroxisomes are very dynamic and metabolically active organelles and are a very important source of reactive oxygen species (ROS), H2O2, O2 (.-) and · OH, which are mainly produced in different metabolic pathways, including fatty acid ß-oxidation, photorespiration, nucleic acid and polyamine catabolism, ureide metabolism, etc. ROS were originally associated to oxygen toxicity; however, these reactive species also play a central role in the signaling network regulating essential processes in the cell. Peroxisomes have the capacity to rapidly produce and scavenge H2O2 and O2 (.-) which allows to regulate dynamic changes in ROS levels. This fact and the plasticity of these organelles, which allows adjusting their metabolism depending on different developmental and environmental cues, makes these organelles play a central role in cellular signal transduction. The use of catalase and glycolate oxidase loss-of-function mutants has allowed to study the consequences of changes in the levels of endogenous H2O2 in peroxisomes and has improved our knowledge of the transcriptomic profile of genes regulated by peroxisomal ROS. It is now known that peroxisomal ROS participate in more complex signaling networks involving calcium, hormones, and redox homeostasis which finally determine the response of plants to their environment.

Deciphering peroxisomal reactive species interactome and redox signalling networks.

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 H2O2. Particular attention will be paid to update the information available on H2O2-dependent peroxisomal retrograde signalling and to discuss a specific peroxisomal footprint.