Nitric oxide functions in both methyl jasmonate signaling and abscisic acid signaling in Arabidopsis guard cells

Intracellular components in methyl jasmonate (MeJA) signaling remain largely unknown, to compare those in well-understood abscisic acid (ABA) signaling. We have reported that nitric oxide (NO) is a signaling component in MeJA-induced stomatal closure, as well as ABA-induced stomatal closure in the previous study. To gain further information about the role of NO in the guard cell signaling, NO production was examined in an ABA- and MeJA-insensitive Arabidopsis mutant, rcn1. Neither MeJA nor ABA induced NO production in rcn1 guard cells. Our data suggest that NO functions downstream of the branch point of MeJA and ABA signaling in Arabidopsis guard cells.Key words: abscisic acid, Arabidopsis thaliana, guard cells, methyl jasmonate, nitric oxideStomatal pores that are formed by pairs of guard cells respond to various environmental stimuli including plant hormones. Some signal components commonly function in MeJA- and ABA-induced stomatal closing signals,¹ such as cytosolic alkalization, ROS generation and cytosolic free calcium ion elevation. Recently, we demonstrated that NO functions in MeJA signaling, as well as ABA signaling in guard cells.²NO production by nitric oxide synthase (NOS) and nitrate reductase (NR) plays important roles in physiological processes in plants.^3,4 It has been shown that NO functions downstream of ROS production in ABA signaling in guard cells.⁵ NO mediates elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt), inactivation of inward-rectifying K⁺ channels and activation of S-type anion channels,⁶ which are known to be key factors in MeJA- and ABA-induced stomatal closure.^2,7–9It has been reported that ROS was not induced by MeJA and ABA in the MeJA- and ABA-insensitive mutant, rcn1 in which the regulatory subunit A of protein phosphatase 2A, RCN1, is impaired.^7,10 We examined NO production induced by MeJA and ABA in rcn1 guard cells (Fig. 1). NO production by MeJA and ABA was impaired in rcn1 mutant (p = 0.87 and 0.25 for MeJA and ABA, respectively) in contrast to wild type. On the other hand, the NO donor, SNP induced stomatal closure both in wild type and rcn1 mutant (data not shown). These results are consistent with our previous results, i.e., NO is involved in both MeJA- and ABA-induced stomatal closure and functions downstream of the branching point of MeJA and ABA signaling in Arabidopsis guard cells.⁷ Our finding implies that protein phosphatase 2A might positively regulate NO levels in guard cells (Fig. 2).Open in a separate windowFigure 1Impairment of MeJA- and ABA-induced NO production in rcn1 guard cells. (A) Effects of MeJA (n = 10) and ABA (n = 9) on NO production in wild-type guard cells. (B) Effects of MeJA (n = 7) and ABA (n = 7) on NO production in rcn1 guard cells. The vertical scale represents the percentage of diaminofluorescein-2 diacetate (DAF-2 DA) fluorescent levels when fluorescent intensities of MeJA- or ABA-treated cells are normalized to control value taken as 100% for each experiment. Each datum was obtained from at least 30 guard cells. Error bars represent standard errors. Significance of differences between data sets was assessed by Student''s t-test analysis in this paper. We regarded differences at the level of p < 0.05 as significant.Open in a separate windowFigure 2A model of signal interaction in MeJA-induced and ABA-induced stomatal closure. Neither MeJA nor ABA induces ROS production, NO production, I_Kin and stomatal closure in rcn1 mutant. These results suggest that NO functions downstream of the branch point of MeJA signaling and ABA signaling in Arabidopsis guard cells.     

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H₂O₂ generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H₂O₂ signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H₂O₂ generation, inhibition of inward K⁺ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H₂O₂ signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H₂O₂, is essential to plant growth and development in response to stresses,^1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.^1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,^4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H₂O₂ generation.^6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H₂O₂ signaling regardless of SA mimicking ABA in regulating stomatal closure.^2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H₂O₂ generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.^11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,^14–16 Pisum sativum,¹⁷ Medicago sativa¹⁸ and tobacco.¹⁹ The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,²⁰ tobacco tissue²¹ and suspension-cultured Oryza sativa cells.²² Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H₂O₂ signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H₂O₂ generation and decreasing K⁺ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.^23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H₂O₂ signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H₂O₂ signaling,^23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase^23,24 is not always identical to that of MEK1/2^8,25 in ABA-induced H₂O₂ signaling of Vicia guard cells. For example, H₂O₂^- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H₂O₂, the effects of both ABA- and H₂O₂-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H₂O₂-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H₂O₂-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.^8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.²⁶ Similar to this, SB203580 improves H₂O₂-inhibited inward K⁺ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H₂O₂ signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,^1,2,8,23,24 and the ABA and H₂O₂ pathways diverge further downstream in their actions on the K⁺ channels and, thus, on stomatal control. Other differences in action between ABA and H₂O₂ are known. For example, Köhler et al. (2001) reported that H₂O₂ inhibited the K⁺ outward rectifier in guard cells shows that H₂O₂ does not mimic ABA action on guard cell ion channels as it acts on the K⁺ outward rectifier in a manner entirely contrary to that of ABA.²⁷Open in a separate windowFigure 2Effect of SB203580 on ABA- and H₂O₂-inhibited inward K⁺ currents. The experimental procedure and data analysis are according to the previous publication.²⁴ SB203580 directs ABA- and H₂O₂-inactivated inward K⁺ currents across plasma membrane of Vicia guard cells. Here the inward K⁺ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H₂O₂ signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,^1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H₂O₂ signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.     

High-throughput transient transformation of Arabidopsis roots enables systematic colocalization analysis of GFP-tagged proteins

Determination of the subcellular localization of an unknown protein is a major step towards the elucidation of its function. Lately, the expression of proteins fused to fluorescent markers has been very popular and many approaches have been proposed to express these proteins. Stable transformation using Agrobacterium tumefaciens generates stable lines for downstream experiments, but is time-consuming. If only colocalization is required, transient techniques save time and effort. Several methods for transient assays have been described including protoplast transfection, biolistic bombardment, Agrobacterium tumefaciens cocultivation and infiltration. In general colocalizations are preferentially performed in intact tissues of the same species, resembling the native situation. High transformation rates were described for cotyledons of Arabidopsis, but never for roots. Here we report that it is possible to transform Arabidopsis root epidermal cells with an efficiency that is sufficient for colocalization purposes.Key words: Arabidopsis, GFP-fusions, protein localization, root, transient transformationSince the release of the Arabidopsis thaliana genome sequence plant biologists set the goal to elucidate the functions of all coded genes. Apart from the spatio-temporal expression patterns of genes, the subcellular localization of gene products can play an essential role in deciphering their function. Classical immunological approaches to localize proteins can be hindered by cross-reactivity, time-consuming generation of antibodies and the low temporal resolution. Expression of tagged proteins forms a suitable alternative. Lately, fusions with fluorescent proteins in combination with confocal (CLSM)¹ or spinning disc microscopy² allow real time protein localization and even subcellular trafficking at high resolution. An overview of fluorescent tagging approaches can be found elsewhere.³Currently several techniques to introduce the coding region for a tagged protein in a plant are available. The generation of stable lines transformed by Agrobacterium tumefaciens offers a continuous source of plant material, but it is time-consuming especially when only colocalization experiments are required. Transient assays, on the other hand, offer the advantage of being fast and amenable to high throughput strategies. Each of these techniques, however, has some limitations and drawbacks. Particle bombardment (biolistics) ^4–6 for example circumvents the host specificity of Agrobacterium strains, but requires expensive equipment. Moreover, it is rather disruptive and imposes a significant stress upon the plants, possibly influencing the results. Protoplasts lack a cell wall and protoplast transformation^7,8 is therefore not suitable for certain experiments related to cell wall proteins or when interactions between cells on tissue level might be important.⁹ Moreover, protoplasts have lost their identity which might be critical for the correct functioning of certain transgenic constructs. Agrobacterium infiltration of tobacco leaves¹⁰ is regularly used and represents an efficient, fast and relatively easy transformation technique. However, tobacco leaves easily show autofluoresence due to tissue damage as a result of experimental manipulations. As it has been reported that some protein fusions expressed in an heterologous system localize to different subcellular localizations11 it is advisable not to use tobacco when localizing Arabidopsis proteins. Leaf infiltrations have been performed in Arabidopsis,¹² but apparently their leaves are much more prone to mechanical damage and the leaf developmental stage is critical, complicating this technique. Cocultivation of Agrobacterium with seedlings offers a rapid and efficient approach applicable to many mono and dicot species. It was reported to work efficiently in Arabidopsis cotyledons, but not in roots.⁹ As an alternative method, Agrobacterium infiltration of Arabidopsis seedlings¹¹ seems an efficient technique for transient expression. However, expression in root cells could not be obtained. Colocalizations are required in the native cells or tissue for the correct localization of an unknown protein or proteins that need interaction partners. As a consequence this technique can not be reliably used when root expressed gene products are studied. Here we show evidence that it is possible to use the described technique¹¹ to induce transient expression in Arabidopsis roots.We used the Agrobacterium infiltration of Arabidopsis seedlings technique¹¹ to colocalize several C-terminal (S65T)-sGFP fusions generated in the plant binary vector pGWB6.¹³ Each construct was transformed into Agrobacterium tumefaciens (C59C1Rif^R) containing the helper plasmid pMP90. Subsequently different stable marker lines, wild type Arabidopsis (Col-0) bearing mCherry fusion constructs,¹⁴ were transiently transformed.¹¹ After 2 or 3 days seedlings were studied using CLSM. Besides being expressed in cotyledons fusion proteins were clearly observed in root epidermis and root cap cells (Fig. 1A and B). As reported¹¹ the transformation efficiency in cotyledons was considerably higher than in root cells. However, in each experiment we obtained a considerable amount of transformed root epidermal cells which was more than sufficient for colocalization studies (Fig. 2). It was remarkable that transformation was repeatedly successful in groups of cells, adjacent or close to each other.Open in a separate windowFigure 1Transient transformation of Arabidopsis root cells. Expression of the protein-GFP fusion product can be seen in the epidermal (A) and root cap cells (B) on fluorescence/transmission merged images. As seen in (A) high efficiencies of root transformation can be reached.Open in a separate windowFigure 2Colocalization of mCherry and GFP constructs. Confocal image of the mCherry fluorescence (A), the GFP signal (B) and the merged image (C).In contrast to what was reported earlier we show here that the Agrobacterium infiltration technique¹¹ is perfectly capable of transiently transforming Arabidopsis root epidermal cells. It allows the transient production and study of proteins in their native environment, considerably increasing the reliability of such experiments. Additionaly the use of RFP marker constructs in colocalisation studies in the root is free of interference by the red background autofluorescence of chlorophyll.     

Biphasic ethylene production during the hypersensitive response in Arabidopsis: A window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H₂O₂, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.¹ Much work on the regulation of the HR has indicated the importance of H₂O₂,² and NO.³ A feature of H₂O₂ generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)⁴ and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.⁵Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H₂O₂ (●, Mur¹⁸); (B) nitric oxide (◇; Mur¹² (C) salicylic acid (SA, ■¹⁹) and (D) ethylene (○ Mur⁹) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H₂O₂—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.⁶ Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H₂O₂ act to initiate SA biosynthesis, where SA and NO act to initiate a “H₂O₂ biphasic switch”. This could initially suppress both SA and the H₂O₂ generation but subsequently acts to potentiate a second phase of H₂O₂ generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C₂H₄ biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.⁶ Such secondary stimuli include SA³ or PAMPs⁷ and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H₂O₂ generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.^6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.⁸We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.⁹ As with H₂O₂ generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H₂O₂ (Fig. 1A) which initiated SA biosynthesis^10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models⁵ this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H₂O₂ and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.⁹ Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H₂O₂ generation. Significantly, the second phases in both H₂O₂ and ethylene production occur exactly where SA and NO production coincides; in the case of H₂O₂ generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco^9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with ​with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.¹³ who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.¹⁴ Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.⁶Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m².sec⁻¹) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 10⁶ colony forming units.ml⁻¹) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.⁹ The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 10⁶ c.f.u.mL⁻¹ with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H₂O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.⁹A further point requires consideration; the role of ethylene as a direct contributor to plant defense.¹⁵ The contribution of ethylene to the HR has been disputed,¹⁶ but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.⁹ In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD.     

Arabidopsis glutathione reductase 1 is dually targeted to peroxisomes and the cytosol

We recently established a proteome methodology for Arabidopsis leaf peroxisomes and identified more than 90 putative novel proteins of the organelle. These proteins included glutathione reductase isoform 1 (GR1), a major enzyme of the antioxidative defense system that was previously reported to be cytosolic. In this follow-up study, we validated the proteome data by analyzing the in vivo subcellular targeting of GR1 and the function of its C-terminal tripeptide, TNL>, as a putative novel peroxisome targeting signal type 1 (PTS1). The full-length protein was targeted to peroxisomes in onion epidermal cells when fused N-terminally with the reporter protein. The efficiency of peroxisome targeting, however, was weak upon expression from a strong promoter, consistent with the idea that the enzyme is dually targeted to peroxisomes and the cytosol in vivo. The reporter protein that was extended C-terminally by 10 amino acid residues of GR1 was directed to peroxisomes, characterizing TNL> as a novel PTS1. The data thus identify plant peroxisomal GR at the molecular level in the first plant species and complete the plant peroxisomal ascorbate-glutathione cycle. Moreover, GR1 is the first plant protein that is dually targeted to peroxisomes and the cytosol. The evolutionary origin and regulatory mechanisms of dual targeting are discussed.Key words: ascorbate-glutathione cycle, dual targeting, proteome analyses, reactive oxygen species, targeting signalsMassive amounts of hydrogen peroxide (H₂O₂) are produced during photosynthesis in peroxisomes by glycolate oxidase activity as part of the photorespiratory cycle.¹ Next to catalase, the ascorbate-glutathione cycle is the secondary scavenging system for H₂O₂ detoxification.^2–4 The cycle comprises four enzymes, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and NADPH-dependent glutathione reductase (GR). GR plays a major physiological role in maintaining and regenerating reduced glutathione in response to biotic and abiotic stresses in plants.⁵ Jiminez et al. (1997) provided biochemical evidence for the presence of the antioxidants ascorbate and glutathione and the enzymes of the ascorbate-glutathione cycle in pea peroxisomes.^6–8 While Arabidopsis APX3, MDAR1 and MDAR4 have been characterized as peroxisomal isoforms,^9–11 the molecular identity of plant peroxisomal GR and DHAR have not been determined in any plant species to date.⁵ Arabidopsis encodes two GR and five DHAR isoforms that are either shown to be or predicted to be cytosolic, mitochondrial or plastidic.¹² We recently identified specific isoforms of GR (GR1, At3g24170) and DHAR (DHAR1, At1g19570) as being peroxisome-associated by proteome analysis of Arabidopsis leaf peroxisomes.^13,14 Both isoforms were previously reported to be or predicted to be cytosolic.¹⁵Arabidopsis GR1 terminates with TNL>, which is related to functional plant PTS1 tripeptides such as SNL> and ANL>.^16,17 Threonine (T), however, has not yet been described as an allowed residue at position −3 of PTS1s in any plant peroxisomal protein.¹⁶ Analysis of homologous plant proteins and expressed sequence tags (ESTs) shows that TNL> is generally highly conserved in putative plant GR1 orthologs (Fig. 1). A few other sequences terminate with related tripeptides, such TSL>, TTL>, NNL> and TKL>. Only a single EST (Picrorhiza kurrooa) carries the canonical PTS1, SKI> (Fig. 1). The data provide only weak additional support for peroxisome targeting of plant GR1 orthologs. However, GR homologs from green algae (chlorophyta) carry canonical PTS1 tripeptides, such as SKL> (Chlamydomonas, Volvox) and AKM> (Micromonas, Fig. 1, Suppl. Fig. 1).Open in a separate windowFigure 1Analysis of PTS1 conservation in plant GR1 homologs. Sequences of full-length protein (FLP) plant GR1 homologs or ESTs (“EST”) were identified by BLAST and phylogenetic analysis, aligned by ClustalX, and conserved residues were shaded by Genedoc. In addition to spermatophyta, homologs from bryophyta and chlorophyta were analyzed for PTS1 conservation. For a phylogenetic analysis of the full-length proteins, see also Supplementary Figure 1. The species abbreviations are as follows: Aa, Artemisia annua; At, Arabidopsis thaliana; Bn, Brassica napus; Br, Brassica rapa; Ci, Cichorium intybus; Cr, Chlamydomonas reinhardtii; Cs, Cynara scolymus; Fv, Fragaria vesca; Ha, Helianthus annuus; Msp, Micromonas sp. RCC 299; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, Oryza sativa; Pk, Picrorhiza kurrooa; Ppat, Physcomitrella patens subsp. patens; Ps, Pisum sativum; Ptri, Populus trichocarpa; Rc, Ricinus communis; Rs, Raphanus sativus; Tp, Trifolium pratense; Tpus, Triphysaria pusilla; Vc, Volvox carteri f. nagariensis; Vv, Vitis vinifera; Zm, Zea mays.     

Sorting out the role of reactive oxygen species during plant programmed cell death induced by ultraviolet-C overexposure

Previous studies have reported that light is required for activating Arabidopsis programmed cell death (PCD) induced by ultraviolet-C (UV-C) overexposure, and a caspase-like protease cleaving the caspase-3 substrate Asp-Glu-Val-Asp (DEVDase activity) is induced during this process. Our recent report has suggested that a quick burst of reactive oxygen species (ROS), which is mainly derived from mitochondria and chloroplasts, is induced in a light dependent manner during the early stages of UV-induced plant PCD. Concomitantly, the mitochondria undergo serious dysfunction including the MTP loss and the changes in distribution and mobility, which ultimately lead to apoptotic-cell death. Though some of signaling molecules have been elucidated in this type of plant cell death, the molecular mechanism about UV-induce Arabidopsis PCD is still poorly understood when comparing with the study of signaling pathways involved in animal cell apoptosis induced by UV. By using the Arabidopsis mesophyll protoplasts as a reference model, we have begun to shed light on the complexity of signaling pathway in UV-induced plant PCD. Recently we have tried to real-time detect the presence of caspase-like proteolytic activation, and to sort out the key role of ROS as well as to further assess the relationship between the ROS production and caspase-like activation in this type of plant apoptotic cell death.Key words: caspase-like activation, FRET, programmed cell death, reactive oxygen species, ultraviolet-CUltraviolet-C has been shown to be a very convenient trigger to induce PCD in plants and protoplasts.^1,2 Others have shown that UV induction of plant PCD requires light and that caspase-like proteolytic activation is induced in this process.¹ Our recent works have shown that ROS mainly localizing in mitochondria and chloroplasts are produced in a light dependent manner during the early stages of UV stress, and that ROS production and mitochondrial dysfunction play important roles during UV-induced Arabidopsis PCD (Fig. 1).² We also found that if the Arabidopsis plants, which were kept at light for 1 h after UV irradiation then were moved to the dark and kept for 60 h, showed no evident plant death phenomena (unpublished data), though burst of ROS has appeared after UV exposure and subsequent 1 h light irradiation.² In contrast, seedlings developed an obvious bleaching when kept in light for 60 h after UV treatment. These findings prompt us to carry out further investigations to dig out the role of ROS in the execution of this type of cell death, and to ask whether the produced ROS in the early stages is involved in the activation of caspase-like protease.Open in a separate windowFigure 1Hypothetical model of the signal transduction pathways in the plant programmed cell death induced by UV-C overexposure. After UV and light treatment a quick burst of ROS appear in the region of mitochondria and chloroplasts, then the mitochondria undergo functional dysfunction, which ultimately leads to cell death. Caspase-like activation and nucleus damage are also involved in the control of this type cell death. Solid line means the issues have been detected. Dotted line and question marks indicate events that have not been detected in this process. For detailed explanation, see the text.It has been reported that ROS is required for the release of cytochrome c (cyt c) and subsequent activation of caspase-like proteases during heat-shock induced plant PCD, and the addition of caspase inhibitors (zVAD-fmk or AC-DEVD-CHO) can prevent the degradation of cyt c and protect the plant cells from cell death.³ Thus these findings suggest that ROS can trigger the release of cyt c, but do not cause cell death, which requires caspase-like activation.³ Conversely, caspase inhibitors have also shown to effectively block the oxidative burst and the plant cell death induced by camptothecin incubation.⁴ These studies suggest the complex relationship between ROS production and caspase activation during execution of plant PCD event. The ROS production and the mitochondrial dysfunction during UV-induced plant PCD have been illustrated in our research. We have suggested the occurrence of MTP disruption during UV stress; however, whether cyt c is released from mitochondria has not been assessed (Fig. 1). The important roles of cyt c release and subsequent caspase activation have been suggested in various types of programmed cell death including mammal and plant cells.^3,5,6 It will be a very challenging work to detect whether cyt c is released from mitochondria and is involved in the caspase-like proteolytic activation, and to further elucidate the relationship between ROS production and caspase-like activation in UV-induced plant PCD (Fig. 1).The involvement of caspase-like proteases in the control of cell death activation in plants has been shown in various forms of plant PCD.⁷ Using synthetic fluorogenic caspase-3 substrate, DEVD cleavage activity was detected during UV or heat shock-induced apoptosis of plant cells, and caspase inhibitors were able to suppress these types of cell death.^1,3 Caspase-like activities have also been detected in plant hypersensitive response (HR) triggered by tobacco mosaic virus (TMV), or plant PCD induced by chemicals like camptothecin.^8,9 All these experiments suggest the existence of functional caspase proteolytic activity in plant cells undergoing PCD. However, most of these results are from in vitro analysis using synthetic fluorogenic substrates or synthetic peptide inhibitor to caspases, this demand us to further dig out the plant caspase encoding gene and to real-time detect the caspase-like activity in vivo.Another of our ongoing work is aiming to detect the caspase-3-like proteolytic activation in living plant cells during UV-induced plant PCD, which is achieved by using the fluorescence resonance energy transfer (FRET) technique. FRET is the phenomenon whereby a fluorescent molecule—the donor—transfers energy by a nonradiative (through space) mechanism to a neighboring chromophore - the acceptor.¹⁰ FRET as a powerful technique to monitor compartmentation and subcellular targeting as well as to visualize protein-protein interactions and proteases activity in living cells has gained increasing importance for biotechnological applications during the last few years.¹¹ During the past few years FRET technique has been successfully used to monitor interactions and distances between molecules in living plant cells.^12–14 Presently, we have constructed a recombinant caspase substrate to monitor caspase-3-like protease activation in single living plant protoplast in real time. This recombinant is composed of enhanced cyan fluorescence protein (ECFP) as the FRET donor and enhanced yellow fluorescence protein (EYFP) as the acceptor, linked by peptides containing the caspase-3 cleavage sequence, DEVD (ECFP-DEVD-EYFP) as the papers demonstrated. 15 Arabidopsis mesophyll protoplasts have been successfully transiently transfected with our recombinant plasmid for expression of ECFP-DEVD-EYFP fusion proteins under control of the CaMV 35S promoter according to a modified procedure (as described previously, ref. ¹⁶). Preliminary experimental results have proved the feasibility of this method to real-time detect the caspase-like activation in living plant cells during UV-induced plant PCD.Using this FRET probe, we may real-time detect the caspase-like activation during UV-induced plant PCD, and elucidate the relationship between ROS production and caspase-like activation as well as verify our hypothesis that whether ROS is necessary for the activation of caspase-like proteases during this process. So the role of ROS in the execution of this type cell death can be further investigated. These subsequent researches will certainly increase our knowledge about the signal transduction pathways in UV-induced Arabidopsis PCD.     

Role of nitric oxide and reactive oxide species in disease resistance to necrotrophic pathogens

Nitric oxide (NO) and reactive oxygen species (ROS) are important signaling molecules in plant immunity. However, roles of NO and ROS in disease resistance to necrotrophic pathogens are not fully understood. We have recently demonstrated that NO plays a pivotal role in basal defense against Botrytis cinerea and the expression of the salicylic acid (SA)-responsive gene PR-1 in Nicotiana benthamiana. By contrast, ROS function negatively in resistance or positively in expansion of disease lesions during B. cinerea-N. benthamiana interaction. Here, analysis in NahG-transgenic N. benthamiana showed that SA signaling is not involved in resistance to B. cinerea in N. benthamiana. We discuss how NO and ROS participate in disease resistance to necrotrophic pathogens on the basis of recent reports.Key words: NO burst, oxidative burst, necrotrophic pathogen, salicylic acid, plant immunity, MAPKNecrotrophs are pathogens that kill host cells by means of toxic molecules and lytic enzymes, and they feed on the remains for their own growth. If the toxic molecule shows differential activity to one or a few plant species, the pathogen has a limited host range and the metabolite is referred to as a host-selective toxin (HST).¹ Several well-studied necrotrophs, in particular Cochliobolus and Alternaria spp., produce HSTs required for the pathogenicity. There are also necrotrophic fungal pathogens with a broad host range, particularly those in the order of Helotiales, including Sclerotinia sclerotiorum and Botrytis cinerea.Rapid production of nitric oxide (NO) and reactive oxygen species (ROS), called NO burst and oxidative burst, respectively, is one of the earliest responses of plants to pathogen attacks. Our recent study showed that NO and oxidative bursts accompanied by activation of the mitogen-activated protein kinase (MAPK)² are induced after inoculation with B. cinerea, and that NO plays a key role, but ROS have an opposite effect in basal defense against B. cinerea in Nicotiana benthamiana.³ NO and ROS are believed to play key roles independently or coordinately in plant innate immunity.^4,5 NO signaling comprises complex processes including increases in cytosolic Ca²⁺ concentration, cyclic GMP (cGMP), cyclic ADP ribose and activation of protein kinases. NO also modulates protein activities directly by cysteine S-nitrosylation.⁶ In addition, NO appears to act as an antioxidant of ROS, because NO can react quickly with superoxide (O₂⁻) to form peroxynitrite (ONOO⁻) and then, reduces the amount of endogenous ROS. Actually, treatment with a mammalian NO synthase inhibitor and silencing NbNOA1 decreased endogenous NO levels and increased the levels of ROS after inoculation with B. cinerea.³ The suppression of NO burst induced high susceptibility to B. cinerea, and depletion of oxidative burst by an NADPH oxidase inhibitor or silencing NbRBOHB led to reduction in disease lesions by B. cinerea,³ suggesting that the growth of B. cinerea might be determined by endogenous levels of ROS which is an important component of virulence.⁷ However, depletion of both NO and oxidative bursts by double silencing NbNOA1/NbRBOHB resulted in expansion of disease lesions compared with reduction of oxidative burst alone by silencing NbRBOHB.³ Similarly, our most recent study showed that silencing NbRibA which compromises production of both NO and ROS do not affect basal resistance against B. cinerea.⁸ These findings suggest that NO positively functions in resistance to necrotrophic pathogens in the manner other than as an antioxidant of ROS.The relationship between NO and salicylic acid (SA) has been studied.⁹ SA signaling-deficient mutants of Arabidopsis thaliana show high susceptibility to B. cinerea.^10,11 We have suggested that reduced basal defense against B. cinerea in N. benthamiana resulting from compromised endogenous NO production may be due to depletion of SA signaling, because NbNOA1-silenced plants showed suppression of the SA-responsive gene NbPR-1 expression induced by inoculation with B. cinerea.³ To confirm the possibility, we used N. benthamiana expressing NahG that converts all SA to catechol. NahG and non-NahG (WT) leaves were inoculated with B. cinerea. NahG plants showed similar susceptibility to B. cinerea compared with WT plants (Fig. 1). We also evaluated effects of silencing NbNOA1 and NbRBOHB in NahG plants on susceptibility to B. cinerea. Like NbNOA1-silenced WT plants shown previusly,³ NbNOA1-silenced NahG leaves showed high susceptibility to B. cinerea. On the other hand, NbRBOHB-silenced NahG leaves showed marked reduction of disease lesions compared with silencing-control NahG leaves. NbNOA1/NbRBOHB-silenced NahG leaves showed expansion of disease lesions compared with NbRBOHB-silenced NahG leaves (Fig. 2). These results suggest that NO-mediated basal defense against B. cinerea is not due to SA signaling, and effects of ROS on disease lesions may not depend on SA in N. benthamiana.Open in a separate windowFigure 1Effects of NahG transgene on susceptibility to B. cinerea. NahG and non-NahG (WT) leaves were inoculated with B. cinerea conidial suspension (1 × 10⁵ conidia/ml). (A) Inoculated leaves were photographed at 4 days postinoculation (dpi). (B) Average diameter of lesions formed on the leaves at 3 and 4 dpi. Data are means ± SD from fourteen experiments.Open in a separate windowFigure 2Effects of silencing NbNOA1 (N), NbRBOHB (B) or NbNOA1/NbRBOHB (N/B) in NahG plants on susceptibility to B. cinerea. Silenced NahG leaves were inoculated with B. cinerea conidial suspension (1 × 10⁵ conidia/ml). (A) Inoculated leaves were photographed at 4 dpi. (B) Average diameter of lesions formed on the leaves at 3 and 4 dpi. Data are means ± SD from four experiments. Data were subjected to Student''s t-test. *p < 0.05 versus silencing-control plants (TRV). **p < 0.05 versus NbRBOHB-silenced plants.Recently, it has been reported that NO and ROS are involved in HSTs responses.^12–15 Victorin, an HST produced by Cochliobolus victoriae, elicits generation of NO and ROS in victorin-sensitive oat leaves.¹² Cell death induced by victorin is suppressed by treatment with ROS scavengers.¹³ Similarly, treatment with ToxA, an HST produced by Pyrenophora triticirepentis, induces oxidative burst, and scavenging ROS compromises ToxA-inducible cell death in ToxA-sensitive wheatleaves.^14,15 SA-induced MAPK, which regulates both NO and ROS production,² is activated by AAL-toxin produced by Alternaria alternata f. sp. lycopersici in AAL-toxin-sensitive tobacco (Mizuno et al. unpublished data). These findings indicate requirement of ROS for the HST-inducible cell death and participation of NO in HST responses.In conclusion, NO and ROS appear to play a contrasting role in disease resistance to necrotrophic pathogens as shown in Figure 3. However, how NO signaling participates in defense responses against necrotrophic pathogens has yet to be elucidated. Recently, several targets of protein S-nitrosylation during hypersensitive response have been characterized in A. thaliana.¹⁶ Evidence is also accumulating for cGMP as an important component of NO-related signal transduction.¹⁷ Further investigations of NO signaling will lead to our understanding of interactions between plants and necrotrophic pathogens.Open in a separate windowFigure 3Model showing role of NO and oxidative bursts in disease resistance to necrotrophic pathogens. After recognition of necrotrophs, plants immediately provoke activation of MAPK which could regulate production of both NO and ROS,² and then NO and oxidative bursts. NO burst plays an important role in disease resistance to necrotrophic pathogens, whereas oxidative burst has a negative role in resistance or has a positive role in expansion of disease lesions by necrotrophs.