Cell wall integrity regulation across plant species.

Plant cell walls are highly dynamic and chemically complex structures surrounding all plant cells. They provide structural support, protection from both abiotic and biotic stress as well as ensure containment of turgor. Recently evidence has accumulated that a dedicated mechanism exists in plants, which is monitoring the functional integrity of cell walls and initiates adaptive responses to maintain integrity in case it is impaired during growth, development or exposure to biotic and abiotic stress. The available evidence indicates that detection of impairment involves mechano-perception, while reactive oxygen species and phytohormone-based signaling processes play key roles in translating signals generated and regulating adaptive responses. More recently it has also become obvious that the mechanisms mediating cell wall integrity maintenance and pattern triggered immunity are interacting with each other to modulate the adaptive responses to biotic stress and cell wall integrity impairment. Here we will review initially our current knowledge regarding the mode of action of the maintenance mechanism, discuss mechanisms mediating responses to biotic stresses and highlight how both mechanisms may modulate adaptive responses. This first part will be focused on Arabidopsis thaliana since most of the relevant knowledge derives from this model organism. We will then proceed to provide perspective to what extent the relevant molecular mechanisms are conserved in other plant species and close by discussing current knowledge of the transcriptional machinery responsible for controlling the adaptive responses using selected examples.

THESEUS1 modulates cell wall stiffness and abscisic acid production in Arabidopsis thaliana.

Plant cells can be distinguished from animal cells by their cell walls and high-turgor pressure. Although changes in turgor and the stiffness of cell walls seem coordinated, we know little about the mechanism responsible for coordination. Evidence has accumulated that plants, like yeast, have a dedicated cell wall integrity maintenance mechanism. It monitors the functional integrity of the wall and maintains integrity through adaptive responses induced by cell wall damage arising during growth, development, and interactions with the environment. These adaptive responses include osmosensitive induction of phytohormone production, defense responses, as well as changes in cell wall composition and structure. Here, we investigate how the cell wall integrity maintenance mechanism coordinates changes in cell wall stiffness and turgor in Arabidopsis thaliana We show that the production of abscisic acid (ABA), the phytohormone-modulating turgor pressure, and responses to drought depend on the presence of a functional cell wall. We find that the cell wall integrity sensor THESEUS1 modulates mechanical properties of walls, turgor loss point, ABA biosynthesis, and ABA-controlled processes. We identify RECEPTOR-LIKE PROTEIN 12 as a component of cell wall integrity maintenance-controlling, cell wall damage-induced jasmonic acid (JA) production. We propose that THE1 is responsible for coordinating changes in turgor pressure and cell wall stiffness.

TALEN-Based HvMPK3 Knock-Out Attenuates Proteome and Root Hair Phenotypic Responses to flg22 in Barley.

Mitogen activated protein kinases (MAPKs) integrate elicitor perception with both early and late responses associated with plant defense and innate immunity. Much of the existing knowledge on the role of plant MAPKs in defense mechanisms against microbes stems from extensive research in the model plant Arabidopsis thaliana. In the present study, we investigated the involvement of barley (Hordeum vulgare) MPK3 in response to flagellin peptide flg22, a well-known bacterial elicitor. Using differential proteomic analysis we show that TALEN-induced MPK3 knock-out lines of barley (HvMPK3 KO) exhibit constitutive downregulation of defense related proteins such as PR proteins belonging to thaumatin family and chitinases. Further analyses showed that the same protein families were less prone to flg22 elicitation in HvMPK3 KO plants compared to wild types. These results were supported and validated by chitinase activity analyses and immunoblotting for HSP70. In addition, differential proteomes correlated with root hair phenotypes and suggested tolerance of HvMPK3 KO lines to flg22. In conclusion, our study points to the specific role of HvMPK3 in molecular and root hair phenotypic responses of barley to flg22.

HEAT SHOCK PROTEIN 90 proteins and YODA regulate main body axis formation during early embryogenesis.

The YODA (YDA) kinase pathway is intimately associated with the control of Arabidopsis (Arabidopsis thaliana) embryo development, but little is known regarding its regulators. Using genetic analysis, HEAT SHOCK PROTEIN 90 (HSP90) proteins emerge as potent regulators of YDA in the process of embryo development and patterning. This study is focused on the characterization and quantification of early embryonal traits of single and double hsp90 and yda mutants. HSP90s genetic interactions with YDA affected the downstream signaling pathway to control the development of both basal and apical cell lineage of embryo. Our results demonstrate that the spatiotemporal expression of WUSCHEL-RELATED HOMEOBOX 8 (WOX8) and WOX2 is changed when function of HSP90s or YDA is impaired, suggesting their essential role in the cell fate determination and possible link to auxin signaling during early embryo development. Hence, HSP90s together with YDA signaling cascade affect transcriptional networks shaping the early embryo development.

HSP90 chaperones regulate stomatal differentiation under normal and heat stress conditions.

Stomatal development is tightly connected with the overall plant growth, while changes in environmental conditions, like elevated temperature, affect negatively stomatal formation. Stomatal ontogenesis follows a well-defined series of cell developmental transitions in the cotyledon and leaf epidermis that finally lead to the production of mature stomata. YODA signaling cascade regulates stomatal development mainly through the phosphorylation and inactivation of SPEECHLESS (SPCH) transcription factor, while HSP90 chaperones have a central role in the regulation of YODA cascade. Here, we report that acute heat stress affects negatively stomatal differentiation, leads to high phosphorylation levels of MPK3 and MPK6, and alters the expression of SPCH and MUTE transcription factors. Genetic depletion of HSP90 leads to decreased stomatal differentiation rates. Thus, HSP90 chaperones safeguard the completion of distinct stomatal differentiation steps depending on these two transcription factors under normal and heat stress conditions.

Multifaceted roles of HEAT SHOCK PROTEIN 90 molecular chaperones in plant development.

HEAT SHOCK PROTEINS 90 (HSP90s) are molecular chaperones that mediate correct folding and stability of many client proteins. These chaperones act as master molecular hubs involved in multiple aspects of cellular and developmental signalling in diverse organisms. Moreover, environmental and genetic perturbations affect both HSP90s and their clients, leading to alterations of molecular networks determining respectively plant phenotypes and genotypes and contributing to a broad phenotypic plasticity. Although HSP90 interaction networks affecting the genetic basis of phenotypic variation and diversity have been thoroughly studied in animals, such studies are just starting to emerge in plants. Here, we summarize current knowledge and discuss HSP90 network functions in plant development and cellular homeostasis.

YODA-HSP90 Module Regulates Phosphorylation-Dependent Inactivation of SPEECHLESS to Control Stomatal Development under Acute Heat Stress in Arabidopsis.

Stomatal ontogenesis, patterning, and function are hallmarks of environmental plant adaptation, especially to conditions limiting plant growth, such as elevated temperatures and reduced water availability. The specification and distribution of a stomatal cell lineage and its terminal differentiation into guard cells require a master regulatory protein phosphorylation cascade involving the YODA mitogen-activated protein kinase kinase kinase. YODA signaling results in the activation of MITOGEN-ACTIVATED PROTEIN KINASEs (MPK3 and MPK6), which regulate transcription factors, including SPEECHLESS (SPCH). Here, we report that acute heat stress affects the phosphorylation and deactivation of SPCH and modulates stomatal density. By using complementary molecular, genetic, biochemical, and cell biology approaches, we provide solid evidence that HEAT SHOCK PROTEINS 90 (HSP90s) play a crucial role in transducing heat-stress response through the YODA cascade. Genetic studies revealed that YODA and HSP90.1 are epistatic, and they likely function linearly in the same developmental pathway regulating stomata formation. HSP90s interact with YODA, affect its cellular polarization, and modulate the phosphorylation of downstream targets, such as MPK6 and SPCH, under both normal and heat-stress conditions. Thus, HSP90-mediated specification and differentiation of the stomatal cell lineage couples stomatal development to environmental cues, providing an adaptive heat stress response mechanism in plants.

Measurement of S-Nitrosoglutathione Reductase Activity in Plants.

S-nitrosation as a redox-based posttranslational modification of protein cysteine has emerged as an integral part of signaling pathways of nitric oxide across all types of organisms. Protein S-nitrosation status is controlled by two key mechanisms: by direct denitrosation performed by the thioredoxin/thioredoxin reductase system, and in an indirect way mediated by S-nitrosoglutathione reductase (GSNOR). GSNOR, which has been identified as a key component of S-nitrosothiols catabolism, catalyzes an irreversible decomposition of abundant intracellular S-nitrosothiol, S-nitrosoglutathione (GSNO) to oxidized glutathione using reduced NADH cofactor. In plants, GSNOR has been shown to play important roles in plant growth and development and plant responses to abiotic and biotic stress stimuli. In this chapter, optimized protocols of spectrophotometric measurement of GSNOR enzymatic activity and activity staining in native polyacrylamide gels in plant GSNOR are presented.

Immunodetection of S-Nitrosoglutathione Reductase Protein in Plant Samples.

S-nitrosation, the attachment of a nitroso group to cysteine thiols, has been recognized as an important posttranslational modification of proteins by nitric oxide and related reactive nitrogen species. Mechanisms and significance of S-nitrosation in the regulation of the structure and activity of proteins have been extensively studied in animal and plant systems. In plants, protein S-nitrosation is involved in signaling pathways of plant hormones and regulators during plant growth and development and in responses to abiotic and biotic stress stimuli. S-nitrosoglutathione reductase (GSNOR) has been identified as a key enzyme controlling the intracellular level of S-nitrosothiols. GSNOR irreversibly degrades S-nitrosoglutathione (GSNO), the major low molecular weight S-nitrosothiol involved in the formation of protein S-nitrosothiols through transnitrosylation. GSNOR level and activity in plant cells are modulated during plant development and in response to external stimuli such as pathogen infection. In this chapter, we give a detailed description of the immunochemical detection of the GSNOR protein in plant samples.

Involvement of S-nitrosothiols modulation by S-nitrosoglutathione reductase in defence responses of lettuce and wild Lactuca spp. to biotrophic mildews.

MAIN CONCLUSION: Resistant Lactuca spp. genotypes can efficiently modulate levels of S-nitrosothiols as reactive nitrogen species derived from nitric oxide in their defence mechanism against invading biotrophic pathogens including lettuce downy mildew. S-Nitrosylation belongs to principal signalling pathways of nitric oxide in plant development and stress responses. Protein S-nitrosylation is regulated by S-nitrosoglutathione reductase (GSNOR) as a key catabolic enzyme of S-nitrosoglutathione (GSNO), the major intracellular S-nitrosothiol. GSNOR expression, level and activity were studied in leaves of selected genotypes of lettuce (Lactuca sativa) and wild Lactuca spp. during interactions with biotrophic mildews, Bremia lactucae (lettuce downy mildew), Golovinomyces cichoracearum (lettuce powdery mildew) and non-pathogen Pseudoidium neolycopersici (tomato powdery mildew) during 168 h post inoculation (hpi). GSNOR expression was increased in all genotypes both in the early phase at 6 hpi and later phase at 72 hpi, with a high increase observed in L. sativa UCDM2 responses to all three pathogens. GSNOR protein also showed two-phase increase, with highest changes in L. virosa-B. lactucae and L. sativa cv. UCDM2-G. cichoracearum pathosystems, whereas P. neolycopersici induced GSNOR protein at 72 hpi in all genotypes. Similarly, a general pattern of modulated GSNOR activities in response to biotrophic mildews involves a two-phase increase at 6 and 72 hpi. Lettuce downy mildew infection caused GSNOR activity slightly increased only in resistant L. saligna and L. virosa genotypes; however, all genotypes showed increased GSNOR activity both at 6 and 72 hpi by lettuce powdery mildew. We observed GSNOR-mediated decrease of S-nitrosothiols as a general feature of Lactuca spp. response to mildew infection, which was also confirmed by immunohistochemical detection of GSNOR and GSNO in infected plant tissues. Our results demonstrate that GSNOR is differentially modulated in interactions of susceptible and resistant Lactuca spp. genotypes with fungal mildews and uncover the role of S-nitrosylation in molecular mechanisms of plant responses to biotrophic pathogens.

Redox regulation of plant S-nitrosoglutathione reductase activity through post-translational modifications of cysteine residues.

Nitric oxide (NO) is considered as a signalling molecule involved in a variety of important physiological and pathological processes in plant and animal systems. The major pathway of NO reactions in vivo represents S-nitrosation of thiols to form S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is the key enzyme in the degradation pathway of S-nitrosoglutathione (GSNO), a low-molecular weight adduct of NO and glutathione. GSNOR indirectly regulates the level of protein S-nitrosothiol in the cells. This study was focused on the dynamic regulation of the activity of plant GSNORs through reversible S-nitrosation and/or oxidative modifications of target cysteine residues. Pre-incubation with NO/NO- donors or hydrogen peroxide resulted in a decreased reductase and dehydrogenase activity of all studied plant GSNORs. Incubation with thiol reducing agent completely reversed inhibitory effects of nitrosative modifications and partially also oxidative inhibition. In biotin-labelled samples, S-nitrosation of plant GSNORs was confirmed after immunodetection and using mass spectrometry S-nitrosation of conserved Cys271 was identified in tomato GSNOR. Negative regulation of constitutive GSNOR activity in vivo by nitrosative or oxidative modifications might present an important mechanism to control GSNO levels, a critical mediator of the downstream signalling effects of NO, as well as for formaldehyde detoxification in dehydrogenase reaction mode.

Characterization of S-nitrosoglutathione reductase from Brassica and Lactuca spp. and its modulation during plant development.

Cellular homeostasis of S-nitrosoglutathione (GSNO), a major cache of nitric oxide bioactivity in plants, is controlled by the NADH-dependent S-nitrosoglutathione reductase (GSNOR) belonging to the family of class III alcohol dehydrogenases (EC 1.1.1.1). GSNOR is a key regulator of S-nitrosothiol metabolism and is involved in plant responses to abiotic and biotic stresses. This study was focused on GSNOR from two important crop plants, cauliflower (Brassica oleracea var. botrytis, BoGSNOR) and lettuce (Lactuca sativa, LsGSNOR). Both purified recombinant GSNORs were characterized in vitro and found to exists as dimers, exhibit high thermal stability and substrate preference towards GSNO, although both enzymes have dehydrogenase activity with a broad range of long-chain alcohols and ω-hydroxy fatty acids in presence of NAD+. Data on enzyme affinities to their cofactors NADH and NAD+ obtained by isothermal titration calorimetry suggest the high affinity to NADH might underline the GSNOR capacity to function in the intracellular environment. GSNOR activity and gene expression peak during early developmental stages of lettuce and cauliflower at 20 and 30 days after germination, respectively. GSNOR activity was also measured in four other Lactuca spp. genotypes with different degree of resistance to biotrophic pathogen Bremia lactucae. Higher GSNOR activities were found in non-infected plants of susceptible genotypes L. sativa UCDM2 and L. serriola as compared to resistant genotypes. GSNOR and GSNO were localized by confocal laser scanning microscopy in vascular bundles and in epidermal and parenchymal cells of leaf cross-sections. The presented results bring new insight in the role of GSNOR in the regulation of S-nitrosothiol levels in plant growth and development.

Detection of S-Nitrosoglutathione Reductase Activity in Plants.

S-nitrosoglutathione reductase (GSNOR) is considered a key enzyme in the regulation of intracellular levels of S-nitrosoglutathione and protein S-nitrosylation. As a part of nitric oxide catabolism, GSNOR catalyzes the irreversible decomposition of GSNO to oxidized glutathione. GSNOR is involved in the regulation of plant growth and development, mediated by NO-dependent signaling mechanisms, and is known to play important roles in plant responses to various abiotic and biotic stress conditions. Here we present optimized protocols to determine GSNOR enzyme activities in plant samples by spectrophotometric measurements and by activity staining after the native polyacrylamide gel electrophoresis.

Effect of abiotic stress stimuli on S-nitrosoglutathione reductase in plants.

S-nitrosylation of protein cysteine thiol groups has recently emerged as a widespread and important reversible post-translational protein modification, involved in redox signalling pathways of nitric oxide and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR), member of class III alcohol dehydrogenase family (EC 1.1.1.1), is considered the key enzyme in the catabolism of major low molecular S-nitrosothiol, S-nitrosoglutathione, and hence to control the level of protein S-nitrosylation. Changes of GSNOR activity after exposure to different abiotic stress conditions, including low and high temperature, continuous dark and de-etiolation, and mechanical injury, were investigated in important agricultural plants. Significantly higher GSNOR activity was found under normal conditions in leaves of Cucumis spp. genotype sensitive to biotrophic pathogen Golovinomyces cichoracearum. GSNOR activity was generally increased in all studied plants by all types of stress conditions. Strong down-regulation of GSNOR was observed in hypocotyls of etiolated pea plants, which did not recover to values of green plants even 168 h after the transfer of etiolated plants to normal light regime. These results point to important role of GSNOR during normal plant development and in plant responses to several types of abiotic stress conditions.