Genetic elucidation of nitric oxide signaling in incompatible plant-pathogen interactions

Recent experiments indicate that nitric oxide (NO) plays a pivotal role in disease resistance and several other physiological processes in plants. However, most of the current information about the function of NO in plants is based on pharmacological studies, and additional approaches are therefore required to ascertain the role of NO as an important signaling molecule in plants. We have expressed a bacterial nitric oxide dioxygenase (NOD) in Arabidopsis plants and/or avirulent Pseudomonas syringae pv tomato to study incompatible plant-pathogen interactions impaired in NO signaling. NOD expression in transgenic Arabidopsis resulted in decreased NO levels in planta and attenuated a pathogen-induced NO burst. Moreover, NOD expression in plant cells had very similar effects on plant defenses compared to NOD expression in avirulent Pseudomonas. The defense responses most affected by NO reduction during the incompatible interaction were decreased H(2)O(2) levels during the oxidative burst and a blockage of Phe ammonia lyase expression, the key enzyme in the general phenylpropanoid pathway. Expression of the NOD furthermore blocked UV light-induced Phe ammonia lyase and chalcone synthase gene expression, indicating a general signaling function of NO in the activation of the phenylpropanoid pathway. NO possibly functions in incompatible plant-pathogen interactions by inhibiting the plant antioxidative machinery, and thereby ensuring locally prolonged H(2)O(2) levels. Additionally, albeit to a lesser extent, we observed decreases in salicylic acid production, a diminished development of hypersensitive cell death, and a delay in pathogenesis-related protein 1 expression during these NO-deficient plant-pathogen interactions. Therefore, this genetic approach confirms that NO is an important regulatory component in the signaling network of plant defense responses.     

Regulatory role of nitric oxide in lipopolysaccharides-triggered plant innate immunity

Recent studies have suggested that lipopolysaccharides (LPS) induce nitric oxide (NO) production and defense gene expression in plants. Our current work investigated the signaling mechanism of NO and the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) in LPS-induced innate immunity of Arabidopsis (Arabidopsis thaliana). We have provided evidence that LPS-elicited NO generation as well as increased antioxidant enzyme activities capable of maintaining the redox state could be important to protect plants against oxidative damage from pathogen attack. In addition, LPS-activated defense responses, including callose deposition and defense-related gene expression, are regulated through an NPR1-dependent signaling pathway. Our results contribute to elucidation of the signaling mechanism of NO and highlight an important role of NPR1 in modulating LPS-triggered innate immunity in plants. However, further research is necessary to clarify the cross-talk between mitochondria and NO on activating LPS-induced defense responses, and the regulatory mechanism of NO in LPS-induced innate immunity needs further improvement.     

The diversity of nitric oxide function in plant responses to metal stress

Nitric oxide (NO) emerges as signalling molecule, which is involved in diverse physiological processes in plants. High mobility metal interferes with NO signaling. The exogenous NO alleviates metal stress, whereas endogenous NO contributes to metal toxicity in plants. Owing to different cellular localization and concentration, NO may act as multifunctional regulator in plant responses to metal stress. It not only plays a crucial role in the regulation of gene expression, but serves as a long-distance signal. Through tight modulation of redox signaling, the integration among NO, reactive oxygen species and stress-related hormones in plants determines whether plants stimulate death pathway or activate survival signaling.     

Nitric oxide as a signal in plants.

Molecular, genetic and biochemical studies have identified key players in the signaling pathways regulating growth and development, as well as defense responses in plants. Recently, nitric oxide (NO) - the versatile and powerful effector of animal redox-regulated signaling and immune responses - was shown to mediate plant defense responses against pathogens. Interestingly, several key components involved in NO-mediated signaling in animals also appear to be operative in plants.     

Regulation of physiological aspects in plants by hydrogen sulfide and nitric oxide under challenging environment

Plants are exposed to a plethora of abiotic stresses such as drought, salinity, heavy metal and temperature stresses at different stages of their life cycle, from germination to seedling till the reproductive phase. As protective mechanisms, plants release signaling molecules that initiate a cascade of stress-signaling events, leading either to programmed cell death or plant acclimation. Hydrogen sulfide (H₂S) and nitric oxide (NO) are considered as new ‘gasotransmitter’ molecules that play key roles in regulating gene expression, posttranslational modification (PTM), as well as cross-talk with other hormones. Although the exact role of NO in plants remains unclear and is species dependent, various studies have suggested a positive correlation between NO accumulation and environmental stress in plants. These molecules are also involved in a large array of stress responses and act synergistically or antagonistically as signaling components, depending on their respective concentration. This study provides a comprehensive update on the signaling interplay between H₂S and NO in the regulation of various physiological processes under multiple abiotic stresses, modes of action and effects of exogenous application of these two molecules under drought, salt, heat and heavy metal stresses. However, the complete picture of the signaling cascades mediated by H₂S and NO is still elusive. Recent researches indicate that during certain plant processes, such as stomatal closure, H₂S could act upstream of NO signaling or downstream of NO in response to abiotic stresses by improving antioxidant activity in most plant species. In addition, PTMs of antioxidative pathways by these two molecules are also discussed.     

NO and ROS implications in the organization of root system architecture

Over the past decades the role of nitric oxide (NO) and reactive oxygen species (ROS) in signaling and cellular responses to stress has witnessed an exponential trend line. Despite advances in the subject, our knowledge of the role of NO and ROS as regulators of stress and plant growth and their implication in signaling pathways is still partial. The crosstalk between NO and ROS during root formation offers new domains to be explored, as it regulates several plant functions. Previous findings indicate that plants utilize these signaling molecules for regulating physiological responses and development. Depending upon cellular concentration, NO either can stimulate or impede root system architecture (RSA) by modulating enzymes through post-translational modifications. Similarly, the ROS signaling molecule network, in association with other hormonal signaling pathways, control the RSA. The spatial regulation of ROS controls cell growth and ROS determine primary root and act in concert with NO to promote lateral root primordia. NO and ROS are two central messenger molecules which act differentially to upregulate or downregulate the expression of genes pertaining to auxin synthesis and to the configuration of root architecture. The investigation concerning the contribution of donors and inhibitors of NO and ROS can further aid in deciphering their role in root development. With this background, this review provides comprehensive details about the effect and function of NO and ROS in the development of RSA.     

Brassinosteroid-Mediated Stress Responses

Brassinosteroids (BRs) are a group of naturally occurring plant steroidal compounds with wide-ranging biological activity that offer the unique possibility of increasing crop yields through both changing plant metabolism and protecting plants from environmental stresses. In recent years, genetic and biochemical studies have established an essential role for BRs in plant development, and on this basis BRs have been given the stature of a phytohormone. A remarkable feature of BRs is their potential to increase resistance in plants to a wide spectrum of stresses, such as low and high temperatures, drought, high salt, and pathogen attack. Despite this, only a few studies aimed at understanding the mechanism by which BRs promote stress resistance have been undertaken. Studies of the BR signaling pathway and BR gene-regulating properties indicate that there is cross-talk between BRs and other hormones, including those with established roles in plant defense responses such as abscisic acid, jasmonic acid, and ethylene. Recent studies aimed at understanding how BRs modulate stress responses suggest that complex molecular changes underlie BR-induced stress tolerance in plants. Analyses of these changes should generate exciting results in the future and clarify whether the ability of BRs to increase plant resistance to a range of stresses lies in the complex interactions of BRs with other hormones. Future studies should also elucidate if BRI1, an essential component of the BR receptor, directly participates in stress response signaling through interactions with ligands and proteins involved in plant defense responses.     

Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.     

Functional role of nitric oxide in plants

The review considers involvement of nitric oxide (NO) in regulation of basic physiological processes underlying growth, development, and senescence in plants. The NO sources in plants, as well as direct and indirect NO signaling mechanisms are also reviewed. Particular attention is paid to the role of this secondary messenger in plant responses to various abiotic stresses, such as mechanical injury, salinity, drought, UV irradiation, high and low temperatures, ozonation, hypoxia, the impacts of heavy metals and herbicides. The role of NO in the hypersensitive response and in a systemic response upon plant infection with invasive pathogens is described.     

Nitric oxide synthesis and signalling in plants

As with all organisms, plants must respond to a plethora of external environmental cues. Individual plant cells must also perceive and respond to a wide range of internal signals. It is now well-accepted that nitric oxide (NO) is a component of the repertoire of signals that a plant uses to both thrive and survive. Recent experimental data have shown, or at least implicated, the involvement of NO in reproductive processes, control of development and in the regulation of physiological responses such as stomatal closure. However, although studies concerning NO synthesis and signalling in animals are well-advanced, in plants there are still fundamental questions concerning how NO is produced and used that need to be answered. For example, there is a range of potential NO-generating enzymes in plants, but no obvious plant nitric oxide synthase (NOS) homolog has yet been identified. Some studies have shown the importance of NOS-like enzymes in mediating NO responses in plants, while other studies suggest that the enzyme nitrate reductase (NR) is more important. Still, more published work suggests the involvement of completely different enzymes in plant NO synthesis. Similarly, it is not always clear how NO mediates its responses. Although it appears that in plants, as in animals, NO can lead to an increase in the signal cGMP which leads to altered ion channel activity and gene expression, it is not understood how this actually occurs.
NO is a relatively reactive compound, and it is not always easy to study. Furthermore, its biological activity needs to be considered in conjunction with that of other compounds such as reactive oxygen species (ROS) which can have a profound effect on both its accumulation and function. In this paper, we will review the present understanding of how NO is produced in plants, how it is removed when its signal is no longer required and how it may be both perceived and acted upon.     

Nitric oxide participates in cold-responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana

Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated. The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation. NO production was detected after 1-4h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO. Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction.     

Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F-box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to induce auxin-regulated responses. A broad spectrum of NO-mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin-dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1-Aux/IAA interaction as evidenced by pull-down and two-hybrid assays. In addition, we provide evidence for NO-mediated modulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor. S-nitrosylation of cysteine is a redox-based post-translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1-Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S-nitrosylation enhances TIR1-Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.     

植物细胞一氧化氮信号转导研究进展

一氧化氮(nitric oxide, NO)作为重要的信号分子, 调控植物的种子萌发、根形态建成和花器官发生等许多生长发育过程, 并参与气孔运动的调节以及植物对多种非生物胁迫和病原体侵染的应答过程。已经知道, 精氨酸依赖的NOS途径和亚硝酸盐依赖的NR途径是植物细胞NO产生的主要酶促合成途径。NO及其衍生物能够直接修饰底物蛋白的金属基团、半胱氨酸和酪氨酸残基, 通过金属亚硝基化、巯基亚硝基化和Tyr-硝基化等化学修饰方式, 调节靶蛋白的活性, 并影响cGMP和Ca²⁺信使系统等下游信号途径, 调控相应的生理过程。最新的一些研究结果也显示, MAPK级联系统与NO信号转导途径之间存在复杂的交叉调控。此外, 作为活跃的小分子信号, NO和活性氧相互依赖并相互影响, 共同介导了植物的胁迫应答和激素响应过程。文章综述了植物NO信号转导研究领域中一些新的研究进展, 对NO与活性氧信号途径间的交叉作用等也作了简要介绍。     

Protein S-nitrosylation: What's going on in plants?

Nitric oxide (NO) is now recognized as a key regulator of plant physiological processes. Understanding the mechanisms by which NO exerts its biological functions has been the subject of extensive research. Several components of the signaling pathways relaying NO effects in plants, including second messengers, protein kinases, phytohormones, and target genes, have been characterized. In addition, there is now compelling experimental evidence that NO partly operates through posttranslational modification of proteins, notably via S-nitrosylation and tyrosine nitration. Recently, proteome-wide scale analyses led to the identification of numerous protein candidates for S-nitrosylation in plants. Subsequent biochemical and in silico structural studies revealed certain mechanisms through which S-nitrosylation impacts their functions. Furthermore, first insights into the physiological relevance of S-nitrosylation, particularly in controlling plant immune responses, have been recently reported. Collectively, these discoveries greatly extend our knowledge of NO functions and of the molecular processes inherent to signal transduction in plants.     

Nitric oxide signaling in plant-pathogen interactions

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. pathogenesis-related genes, phenylalanine ammonialyase, chalcone synthase) and could play a role in pathways leading to systemic acquired resistance.     

Two-component elements mediate interactions between cytokinin and salicylic acid in plant immunity

Recent studies have revealed an important role for hormones in plant immunity. We are now beginning to understand the contribution of crosstalk among different hormone signaling networks to the outcome of plant-pathogen interactions. Cytokinins are plant hormones that regulate development and responses to the environment. Cytokinin signaling involves a phosphorelay circuitry similar to two-component systems used by bacteria and fungi to perceive and react to various environmental stimuli. In this study, we asked whether cytokinin and components of cytokinin signaling contribute to plant immunity. We demonstrate that cytokinin levels in Arabidopsis are important in determining the amplitude of immune responses, ultimately influencing the outcome of plant-pathogen interactions. We show that high concentrations of cytokinin lead to increased defense responses to a virulent oomycete pathogen, through a process that is dependent on salicylic acid (SA) accumulation and activation of defense gene expression. Surprisingly, treatment with lower concentrations of cytokinin results in increased susceptibility. These functions for cytokinin in plant immunity require a host phosphorelay system and are mediated in part by type-A response regulators, which act as negative regulators of basal and pathogen-induced SA-dependent gene expression. Our results support a model in which cytokinin up-regulates plant immunity via an elevation of SA-dependent defense responses and in which SA in turn feedback-inhibits cytokinin signaling. The crosstalk between cytokinin and SA signaling networks may help plants fine-tune defense responses against pathogens.