Unravelling how plants benefit from ROS and NO reactions, while resisting oxidative stress

Abstract

Background and Aims Reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO), play crucial roles in the signal transduction pathways that regulate plant growth, development and defence responses, providing a nexus of reduction/oxidation (redox) control that impacts on nearly every aspect of plant biology. Here we summarize current knowledge and concepts that lay the foundations of a new vision for ROS/RNS functions – particularly through signalling hubs – for the next decade.

Scope Plants have mastered the art of redox control using ROS and RNS as secondary messengers to regulate a diverse range of protein functions through redox-based, post-translational modifications that act as regulators of molecular master-switches. Much current focus concerns the impact of this regulation on local and systemic signalling pathways, as well as understanding how such reactive molecules can be effectively used in the control of plant growth and stress responses.

Conclusions The spectre of oxidative stress still overshadows much of our current philosophy and understanding of ROS and RNS functions. While many questions remain to be addressed – for example regarding inter-organellar regulation and communication, the control of hypoxia and how ROS/RNS signalling is used in plant cells, not only to trigger acclimation responses but also to create molecular memories of stress – it is clear that ROS and RNS function as vital signals of living cells.

Plants monitor their surroundings continuously and adjust their metabolic systems accordingly to optimize productivity. Plant cells display a high degree of plasticity, enabling them to withstand or acclimate to the swathe of environmental forces they are exposed to on a daily basis because of their sedentary habit. This plasticity is reflected in the dynamics of cellular reduction/oxidation (redox) processes, particularly the regulated accumulation of reactive oxygen species (ROS), which play important roles in signalling appropriate responses to changes in parameters such as light intensity or water availability. Stress-induced metabolic imbalances can lead to the activation of NADPH oxidases and increased production of ROS from other sources such as mitochondria, chloroplasts and peroxisomes. ROS accumulation leads to oxidative signalling. Similarly, the generation of nitric oxide (NO) in plants is important in the regulation of plant growth and defence responses. NO and its derivatives (reactive nitrogen species, RNS) are also produced in cells experiencing abiotic stress. Increasing evidence suggests that NO production in plants is linked, at least in part, to nitrogen assimilation through the conversion of nitrite to NO by the enzyme nitrate reductase, and that NO functions in a feedback loop that regulates plant nitrogen fluxes.

Considerable progress has been made in our understanding of the complexities of ROS and NO interactions over the last 10 years. Accumulating evidence in the literature demonstrates that ROS and RNS play key roles as biological messengers, functioning together to facilitate appropriate responses to biotic and abiotic stresses. ROS/RNS interactions are also important in the regulation of plant development and in the regulation of a wide range of physiological responses to the environment, from germination, root development and gravitropism to stomatal closure. The scope of this volume was influenced by a workshop held at the International University of Andalusia (UNIA) in Baeza, Spain to celebrate the achievements of Professor Luis del Rio (Fig. 1), who has dedicated a lifetime of research to this crucial topic. Our aim in this volume is to present recent advances in our understanding of how plant cells manage ROS and NO signalling, particularly the interactions between these redox signals, antioxidants and proteins that function as switches to regulate gene and pathway function in response to environmental and developmental triggers. The articles provide snapshots of key areas of current endeavour, together with overviews of key topics within this ever-growing field of intensive research interest. Taken together, the findings presented in this volume demonstrate that ROS and NO impact on nearly every aspect of plant biology, making an important contribution to the cellular information exchange that governs cell function and cell fate. Several studies in this volume provide insights into the complexities of the redox regulation of cellular processes that govern plant stress responses. For example, Sandalio and Romero-Puertas (2015) provide a timely and useful overview of the central role played by peroxisomes in plant development and stress tolerance. They discuss how peroxisomes exert a strong influence on cellular redox processes through the generation of ROS and NO and associated signalling networks.

Fig. 1.

Professors Francisca Sevilla Valenzuela, Luisa Maria Sandalio, Luis Del Rio and Christine Helen Foyer at the workshop titled ‘Oxygen and nitrogen reactive species: generating a new vision for 2020’, that was held at the International University of Andalusia (UNIA) in Baeza, in October 2014.

The crop varieties used in today’s agriculture have been selected largely on the basis of increased yields and not for stress-tolerance traits. It is therefore unlikely that current crop varieties will be able to sustain high yields under the increasingly stressful conditions that are predicated to occur in the future. An increase in global temperatures is considered to be one of the most important consequences of climate change. The consensus view held by climate change scientists is that the predicted increases in global temperatures that will occur over the next 50 years will have a strong negative impact on the productivity of major cereal crops such as wheat. Reductions in major crop yields of nearly 20 % have been predicted for each degree Centigrade increase in temperature during the growing season. It is therefore important to have a profound knowledge and understanding of thermotolerance mechanisms in crop plants, a topic that is reviewed by de Pinto et al. (2015), with a view to developing redox solutions with which to overcome the negative impacts of high temperatures. This article focuses on the role of ROS, redox metabolites and antioxidant enzymes in signalling pathways that activate plant cell responses. High temperatures are often associated with drought stress under field conditions. It is perhaps not surprising therefore that many parts of the world will experience drought more frequently in coming decades. Marquez-Garcia et al. (2015) highlight the role of redox signals in the source–sink relationships underpinning the symbiotic union between soybean and Bradyrhizobium species exposed to drought. Drought can undermine symbiotic nitrogen fixation; however, as the authors state, defining this system is akin to a ‘chicken and egg’ situation. By monitoring primary and nitrogen metabolism, as well as markers of redox status in the leaf and crown nodules, they show that the leaves of lowest photosynthetic capacity are sacrificed before the symbiotic nodules. Similarly, Simancas and Munne-Bosch (2015) describe a trade-off between vitamin E and phosphorus nutrition in arabidopsis plants. Strikingly, the vitamin E-deficient vte1 (tocopherol cyclase) plants display increased longevity under severe phosphorus limitation, implying a conceptual priority of vitamin E synthesis over plant longevity, i.e. a ‘vitamin E cost’ to sustained life. The authors suggest that the trade-off extends to a balance between photoprotection and jasmonate-mediated plant defence. Roach et al. (2015) investigate the relationships between the regulation of photosynthesis and ROS release into the surrounding water by several algal species. The role of the enigmatic enzyme polyphenol oxidase in photosynthesis is investigated by Boeckx et al. (2015), who provide preliminary evidence of a role for this thylakoid lumen-localized enzyme in oxidative stress tolerance.

There are clear differences in the ways that ROS and NO are produced and managed in animals and plants, not least because of the presence of chloroplasts and photosynthesis in plants but not in animals. The roles of ROS and NO in the pathways of anterograde and retrograde signalling between the organelles and the nucleus are hotbeds of current plant science research, as are the mechanisms of inter-organellar cross-talk. In this context, Ozgur et al. (2015) seek to define how the different organelles in plant cells influence the ROS-mediated unfolded protein response (UPR) in the endoplasmic reticulum (ER). While the UPR serves to facilitate protein-folding and degradation of misfolded proteins, prolonged stress exposures can lead to excessive accumulation of misfolded proteins. This triggers ER-associated degradation (ERAD), leading to the phenomenon called ER-stress. Ozgur et al. have studied the transcriptional responses of a number of UPR-hallmark genes to various concentrations of ROS-producing agents that are specific to different organelles. The findings of their study expand our understanding of ER responses to multiple oxidative signals from different organelles in plants. The alternative oxidase AOX1a has become a hallmark of oxidative stress and is known to be a key player in mitochondria–chloroplast cross-talk, as well as organelle to nucleus retrograde signalling pathways. The study described by Vishwakarma et al. (2015) suggests that lack of a functional AOX1a in arabidopsis has a negative impact on photosynthesis.

The regulation of genetically programmed cell death events and related mechanisms are another widely studied topic within the context of plant responses to the environment. Ortiz-Espín et al. (2015) examine the potential of the mitochondrial thioredoxin TRXO1 to mitigate ROS-induced programmed cell death in tobacco BY-2 cells. Over-expression of TRXO1 was shown to decrease markers of oxidative stress, and lower the cellular H₂O₂ and NO levels as well as delaying cell death. These features are linked to an increase in catalase activity and a greater capacity to maintain the cellular glutathione pool in a highly reduced state. Within this context, Pu et al. (2015) provide a useful and insightful description of the critical importance of the mitochondrial electron transport proteins such as AOX in the control of cellular redox homeostasis, and Jozefczak et al. (2015) examine the effects of low antioxidant status on plant responses to the heavy metal cadmium. Using a range of arabidopsis mutants that have low glutathione contents (cad2-1), low ascorbate (vtc1-1) or both (cad2-1 vtc1-1), Jozefczak et al. present data showing that the cad2-1 mutants have an increased sensitivity to cadmium, but the vtc1-1 mutants have a lower sensitivity. These differences are explained in terms of low phytochelatin production in the cad2-1 and cad2-1 vtc1-1 lines, while the vtc2-1 lines have a higher capacity for phytochelatin synthesis. Returning to crop species, Feigl et al. (2015) examine markers of oxidative and nitrosative stress to evaluate the relative sensitivities of Brassica napus (oilseed rape) and B. juncea (Indian mustard) to zinc toxicity. Declines in the primary root length, total root fresh weight and root meristem cell viability of B. napus show it is substantially more sensitive to increasing zinc concentrations (50–300 µm) than B. juncea. This sensitivity of B. napus is underscored by considerable lipid peroxidation in the roots as well as accumulation of superoxide, which is not seen in B. juncea. By contrast, markers of nitrosative stress do not discriminate the sensitivities of the two species.

Jose Palma and colleagues (Palma et al., 2015) provide an overview of our current knowledge of the fruit ripening process using capsicum as a model system for climacteric fruit. They highlight the roles of ascorbate and pyridine nucleotides in developmental and environmental stress conditions in their review of this important topic. Evidence of a role for protein tyrosine nitration in the control of pepper fruit ripening is presented by Chaki et al. (2015). Tanou et al. (2015) describe the respective functions of oxidative processes caused by ozone exposure and NO signalling imposed by application of SNP on kiwifruit ripening, with a particular focus on changes to the fruit proteome. Data are presented to show that ozone-induced inhibition of fruit ripening could be prevented by NO, highlighting the interactions between these signalling pathways.

Crop yield is dependent on seed quality. ROS are closely associated with different aspects of seed physiology from birth to death that regulate dormancy and play a role in combatting pathogen infection, among others. The dual role of ROS in seed germination and in cell death is discussed by Jeevan Kumar et al. (2015). New data concerning the role of the cellular redox environment in seed dormancy, ageing and deterioration are presented by Morscher et al. (2015), who show that high oxygen concentrations favour seed dormancy and that breaking dormancy and loss of viability are associated with oxidation of proteins in the cytosol rather than oxidation of membrane lipids. Following seed germination, Airaki et al. (2015) describe the spatial and temporal production of ROS and RNS in pepper, linking these changes to seedling establishment. Protein S-nitrosylation can regulate the activity, localization or conformation of target proteins. The role of protein denitrosylation in auxin-mediated root development is explored by Correa-Aragunde et al. (2015), who demonstrate that NADPH-dependent thioredoxin reductase is a key player in this process and suggest that a feedback mechanism exists to control unregulated protein S-nitrosylation.

Like seed germination, the transition from quiescence to a metabolically active state during bud burst requires the management of a transition from hypoxia to atmospheric oxygen levels, together with the co-ordination of intercellular signalling networks including the cellular redox hub. Meitha et al. (2015) provide new information concerning the regulation of hypoxia release in ecodormant buds, showing that the transition to atmospheric oxygen levels upon budburst is accompanied by ROS accumulation in the developing vascular tissues of heterotrophic grapevine buds.

2-Cys peroxiredoxin A plays a dual role as both a peroxidase and a chaperone depending on its conformation and metabolic functions. The mechanisms involved in this functional switch are investigated by Lee et al. (2015), who provide evidence that substitution of Ser¹⁵⁰ with Cys¹⁵⁰ leads to a remarkable increase in chaperone and peroxidase activity. These changes are associated with structural alterations in the α2 helix, the fully folded α2 helix being associated with high peroxidase activity.

Research in the popular and ever-growing field of redox biology continues to be exciting and challenging. Slowly but surely we are gaining a better understanding of the roles of ROS and NO in the signalling networks that link cellular responses to changing environmental and developmental stimuli. In addition, NO readily reacts with glutathione to form S-nitrosoglutathione (GSNO), an important molecule that functions as a natural NO donor. Within this context ROS, NO and GSNO interact as control hubs that include master-switches regulating the genetic and epigenetic controls of plant growth and stress tolerance (Fig. 2). There are a large number of redox-based, post-translational modifications that act as regulators of the master-switches. This includes the addition of a glutathione to a protein cysteine thiol in a glutathionylation reaction or a NO moiety to form a S-nitrosothiol (SNO). The latter mechanism represents a key mechanism for the transfer of NO bioactivity. The size of the GSNO pool is regulated by GSNO reductase 1, which reduces GSNO to oxidized glutathione (GSSG) and ammonium. Despite this extensive and increasing knowledge, many questions remain unanswered. For example, further research is required to understand how plant cells use these mechanisms to transmit information in order to regulate gene expression. In addition, while local and systemic signalling is thought to involve waves of ROS, Ca²⁺ or NO production (Fig. 2), little is known about how plant cells perceive and distinguish between different stimuli. The characterization of new signalling molecules, such as oxidized or aggregated proteins is an important new field that is as yet relatively unexplored. Little is known about the functional dynamics of ROS/NO interactions or how ROS and NO regulate cross-talk between organelles. Deciphering the mechanisms by which ROS, NO and Ca²⁺ interact with phytohormones to create molecular memories of stress is an exciting new topic for future exploration. Answering these questions requires multidisciplinary approaches using physiological, biochemical, proteomic, molecular and cellular techniques. These, together with an armoury of more traditional whole-plant physiology, genetics, breeding and crop science approaches, and newer nanotechnological tools will enable us to dissect signalling networks at multiple levels of complexity from the cell, to the tissue to the whole plant. Such information is essential if we are to design improved biotechnology-driven strategies for sustained or enhanced productivity together with better plant resistance to biotic and abiotic stress conditions. Finally, it is worth remembering that in nature plants are often exposed to multiple stresses simultaneously. Regular exposures to changing mild stresses results in acclimation of metabolic functions, together with molecular and physiological adjustments that include increased resistance to oxidative stress, which translate into plant growth and development that are optimized to prevailing environmental conditions. Without regular stress exposures that entrain genetic and epigenetic cross-tolerance responses, it is likely that sustainability and vigour in natural environments will be compromised because plants will be poorly adapted to stress, and hence less able to recover. At present, we understand relatively little about the mechanisms that create short- and long-lasting molecular memories of stress. Hence, there are many new avenues and developing topics for researchers in the field of plant redox biology to explore.

Fig. 2.

A schematic diagram showing the central roles of reactive oxygen species (ROS) and nitric oxide (NO) in the signal transduction pathways that facilitate acclimation to stress allowing high crop productivity over a wide range of environmental conditions. ETC, respiratory electron transport chain; NOX, NADPH oxidases; Mit ETC, respiratory electron transport chain; Chl ETC, photosynthetic electron transport chain; Perox, peoxisomes; GOX, glycollate oxidase; NR, nitrate reductase; NOS-like, nitric oxide synthase-like enzymes; GSNO, S-Nitrosoglutathione.