New insights into the functional roles of CrRLKs in the control of plant cell growth and development

Receptor-like kinases (RLKs) are a family of transmembrane proteins with a variable ligand-binding extracellular domain and a cytoplasmic kinase domain. In Arabidopsis, there are ∼600 RLKs believed to have diverse functions during plant growth, development and interactions with the environment. Based on the variable extracellular domain, RLKs can be classified into different subfamilies. The CrRLK subfamily contains 17 members in Arabidopsis and characterization of some of its members suggests a role for these proteins in the regulation of growth and reproduction. This review focuses on the roles of CrRLKs in the regulation of polarized growth with emphasis on the newly identified signal transduction pathways activated downstream of CrRLKs. A picture is emerging where CrRLKs are part of a conserved signal transduction cascade important for growth maintenance in different cell types.Key words: CrRLKs, FERONIA, RAC/ROP, ROS, polar growthThe ability of plants to perceive and process environmental and internal information into coordinated responses is crucial to their adaptability and survival in constantly changing environments. Most of signal perception occurs at the plasma membrane of cells where membrane-associated receptors receive signals to activate downstream signaling cascades that regulate growth and development. In plants and animals alike, receptor-like kinases (RLKs) mediate many of the signaling events at the cell surface and in the model plant Arabidopsis they comprise a monophyletic family with more than 600 members.¹ RLKs are transmembrane proteins with a variable N-terminal extracellular domain and a Ser/Thr intracellular kinase domain. The diversity of their extracellular domains suggests involvement in the transduction of a wide range of signals and allows them to be classified into different sub-families.² The CrRLK1L subfamily (from here on referred to as CrRLK) is named after the first member characterized in Catharanthus roseus cell cultures³ and contains 17 members in Arabidopsis.⁴ Several members of this family have now been implicated in growth regulatory processes.THESEUS1 (THE1) was identified through a suppressor screen of a cellulose-deficient mutant (prc1-1) which has a short hypocotyl phenotype.⁵ Loss of THE1 function resulted in reduced growth inhibition in the prc1-1 the1 double mutant. Interestingly, the the1 mutation itself has no effect in wild type background, thus leading to the suggestion that THE1 functions as a sensor of cell wall integrity in situations where the cell wall is weakened and organ elongation would be detrimental for the plant.^4,5A second CrRLK, FERONIA (FER), was first implicated in the regulation of female control of fertility. In the female gametophyte FER is involved in sensing pollen tube arrival and promoting its rupture which is necessary for double fertilization to occur.^6,7 FER is in fact involved in several processes depending on the tissue where it is expressed. In hypocotyls, FER is involved in the integration of ethylene and brassinosteroid (BR) signals to regulate hypocotyl elongation in the dark.⁸ Moreover, FER, THE1 and the closely related HERCULES1 (HERK1), were found to regulate cell elongation by interacting with BR signaling.⁹ More recently, roles for FER in the regulation of root hair development and fungal invasion have been established.^10,11 The pollen-specific ANXUR1 (ANX1) and ANXUR2 (ANX2) are closely related to FER and act redundantly to maintain pollen tube growth integrity during its journey through the style and ensure against precocious pollen tube rupture before reaching the ovule.^12,13Apparently with different biological roles, all the CrRLK members analyzed thus far have an effect on the growth of plant cells. The present review focuses on their role during cell growth with emphasis on polar cell growth and the downstream pathways activated by CrRLKs.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Mannan synthase activity in the CSLD family

Cellulose Synthase Like (CSL) proteins are a group of plant glycosyltransferases that are predicted to synthesize β-1,4-linked polysaccharide backbones. CSLC, CSLF and CSLH families have been confirmed to synthesize xyloglucan and mixed linkage β-glucan, while CSLA family proteins have been shown to synthesize mannans. The polysaccharide products of the five remaining CSL families have not been determined. Five CSLD genes have been identified in Arabidopsis thaliana and a role in cell wall biosynthesis has been demonstrated by reverse genetics. We have extended past research by producing a series of double and triple Arabidopsis mutants and gathered evidence that CSLD2, CSLD3 and CSLD5 are involved in mannan synthesis and that their products are necessary for the transition between early developmental stages in Arabidopsis. Moreover, our data revealed a complex interaction between the three glycosyltransferases and brought new evidence regarding the formation of non-cellulosic polysaccharides through multimeric complexes.Key words: mannan, mannose, plant cell wall, glycosyltransferase, cellulose synthase like, CSL, biosynthesis, hemicelluloseThe plant cell wall is mainly composed of polysaccharides, which are often grouped into cellulose, hemicelluloses and pectin. Since the discovery of the first cellulose synthase (CESA) genes in cotton fibers,¹ the synthesis of cellulose has been extensively studied.² In contrast, the glycosyltransferases responsible for synthesizing hemicelluloses and pectin are still largely unidentified.^3,4,5 The CESA genes are members of a superfamily that includes genes with a high sequence similarity with CESA genes and are named Cellulose Synthase Like (CSL).⁶ The CSL genes have themselves been grouped into nine families designated CSLA, -B, -C, -D, -E, -F, -G, -H and -J (Figure 1A).^5,6 Mannan and glucomannan synthase activity has been demonstrated in the CSLA family,^7,8,9 while members of the CSLC family have been implicated in synthesis of the xyloglucan backbone.¹⁰ CSLF and CSLH, which are found only in grasses, are involved in synthesis of mixed linkage glucan.^11,12 The function of the remaining CSL families has not been determined. We have reported our research on the CSLD family in a recent publication.¹³ Of all the CSL families, CSLD possesses the most ancient intron/exon structure and is the most similar to the CESA family.⁶ CSLD genes are found in all sequenced genomes of terrestrial plants including Physcomitrella and Selaginella suggesting a highly conserved function throughout the plant kingdom (Figure 1A). Five genes (CSLD1 to CSLD5) and one apparent pseudogene (CSLD6) have been identified in Arabidopsis thaliana.¹⁴ Bernal et al.^14,15 studied knock-out mutants of the individual genes and presented evidence for a role in cell wall biosynthesis for each Arabidopsis CSLD. To elucidate the activity of the CSLD proteins and obtain further understanding of their biological role, we generated double mutants csld2/csld3, csld2/csld5, csld3/csld5 and the triple mutant csld2/csld3/csld5. Immunochemical, biochemical and complementation assays brought evidence that CSLD5 or CSLD2 in concomitance with CSLD3 act as mannan synthases.Open in a separate windowFigure 1(A) Schematic representation of the CESA superfamily phylogeny. The inset on the right is a detailed phylogenetic tree of CSLDs from Selaginella moellendorffii, Arabidopsis thaliana and Oryza sativa. The figure is modified from Ulvskov and Scheller.⁵ (B) Comparison of csld2, csld3, csld5 with Col-0 20 days after germination. The inflorescences of csld2 and csld3 were similar to Col-0 whereas csld5 had a delayed growth. Scale bar: 1 cm. (C) Col-0 and csld2/csld3/csld5 (triple mutant, TM) 40 days after germination. After 40 days, the triple mutant was barely developed and, as shown in the magnified inset, displayed purple coloration indicating accumulation of anthocyanins, a typical stress response. Scale bar: 2 mm.     

Staying in the fold: The SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins

The conserved eukaryotic protein SGT1 (suppressor of G₂ allele of skp1) participates in diverse physiological processes such as cell cycle progression in yeast, plant immunity against pathogens and plant hormone signalling. Recent genetic and biochemical studies suggest that SGT1 functions as a novel co-chaperone for cytosolic/nuclear HSP90 and HSP70 molecular chaperones in the folding and maturation of substrate proteins. Since proteins containing the leucine-rich repeat (LRR) protein-protein interaction motif are overrepresented in SGT1-dependent phenomena, we consider whether LRR-containing proteins are preferential substrates of an SGT1/HSP70/HSP90 complex. Such a chaperone organisation is reminiscent of the HOP/HSP70/HSP90 machinery which controls maturation and activation of glucocorticoid receptors in animals. Drawing on this parallel, we discuss the possible contribution of an SGT1-chaperone complex in the folding and maturation of LRR-containing proteins and its evolutionary consequences for the emergence of novel LRR interaction surfaces.Key words: heat shock protein, SGT1, co-chaperone, HSP90, HSP70, leucine-rich repeat, LRR, resistance, SCF, ubiquitinThe proper folding and maturation of proteins is essential for cell viability during de novo protein synthesis, translocation, complex assembly or under denaturing stress conditions. A complex machinery composed of molecular chaperones (heat-shock proteins, HSPs) and their modulators known as co-chaperones, catalyzes these protein folding events.^1,2 In animals, defects in the chaperone machinery is implicated in an increasing number of diseases such as cancers, susceptibility to viruses, neurodegenerative disease and cystic fibrosis, and thus it has become a major pharmacological target.^3,4 In plants, molecular genetic studies have identified chaperones and co-chaperones as components of various physiological responses and are now starting to yield important information on how chaperones work. Notably, processes in plant innate immunity rely on the HSP70 and HSP90^5–7 chaperones as well as two recently characterised co-chaperones, RAR1 (required for Mla12 resistance) and SGT1 (suppressor of G₂ allele of skp1).^8–11SGT1 is a highly conserved and essential co-chaperone in eukaryotes and is organized into three structural domains: a tetratricopeptide repeat (TPR), a CHORD/SGT1 (CS) and an SGT1-specific (SGS) domain (Fig. 1A). SGT1 is involved in a number of apparently unrelated physiological responses ranging from cell cycle progression and adenylyl cyclase activity in yeast to plant immunity against pathogens, heat shock tolerance and plant hormone (auxin and jasmonic acid) signalling.^7–9,12,13 Because the SGT1 TPR domain is able to interact with Skp1, SGT1 was initially believed to be a component of SCF (Skp1/Cullin/F-box) E3 ubiquitin ligases that are important for auxin/JA signalling in plants and cell cycle progression in yeast.^13,14 However, mutagenesis of SGT1 revealed that the TPR domain is dispensable for plant immunity and auxin signalling.¹⁵ Also, SGT1-Skp1 interaction was not observed in Arabidopsis.¹³ More relevant to SGT1 functions appear to be the CS and SGS domains.¹⁶ The former is necessary and sufficient for RAR1 and HSP90 binding. The latter is the most conserved of all SGT1 domains and the site of numerous disabling mutations.^14,16,17Open in a separate windowFigure 1Model for SGT1/chaperone complex functions in the folding of LRR-containing proteins. (A) The structural domains of SGT1, their sites of action (above) and respective binding partners (below) are shown. N- and C-termini are indicated. TPR, tetratricopeptide repeat; CS, CHORD/SGT1; SGS, SGT1-specific. (B) Conceptual analogy between steroid receptor folding by the HOP/chaperone machinery and LRR protein folding by the SGT1/chaperone machinery. LRR motifs are overrepresented in processes requiring SGT1 such as plant immune receptor signalling, yeast adenylyl cyclase activity and plant or yeast SCF (Skp1/Cullin/F-box) E3 ubiquitin ligase activities. (C) Opposite forces drive LRR evolution. Structure of LRRs 16 to 18 of the F-box auxin receptor TIR1 is displayed as an illustration of the LRR folds.³⁰ Leucine/isoleucine residues (side chain displayed in yellow) are under strong purifying selection and build the hydrophobic LRR backbone (Left). By contrast, solvent-exposed residues of the β-strands define a polymorphic and hydrophilic binding surface conferring substrate specificity to the LRR (Right) and are often under diversifying selection.We recently demonstrated that Arabidopsis SGT1 interacts stably through its SGS domain with cytosolic/nuclear HSP70 chaperones.⁷ The SGS domain was both necessary and sufficient for HSP70 binding and mutations affecting SGT1-HSP70 interaction compromised JA/auxin signalling and immune responses. An independent in vitro study also found interaction between human SGT1 and HSP70.¹⁸ The finding that SGT1 protein interacts directly with two chaperones (HSP90/70) and one co-chaperone (RAR1) reinforces the notion that SGT1 behaves as a co-chaperone, nucleating a larger chaperone complex that is essential for eukaryotic physiology. A future challenge will be to dissect the chaperone network at the molecular and subcellular levels. In plant cells, SGT1 localization appears to be highly dynamic with conditional nuclear localization⁷ and its association with HSP90 was recently shown to be modulated in vitro by RAR1.¹⁶A co-chaperone function suits SGT1 diverse physiological roles better than a specific contribution to SCF ubiquitin E3 ligases. Because SGT1 does not affect HSP90 ATPase activity, SGT1 was proposed rather as a scaffold protein.^16,19 In the light of our findings and earlier studies,²⁰ SGT1 is reminiscent of HOP (Hsp70/Hsp90 organizing protein) which links HSP90 and HSP70 activities and mediates optimal substrate channelling between the two chaperones (Fig. 1B).²¹ While the contribution of the HSP70/HOP/HSP90 to the maturation of glucocorticoid receptors is well established,²¹ direct substrates of an HSP70/SGT1/HSP90 complex remain elusive.It is interesting that SGT1 appears to share a functional link with leucine-rich repeat- (LRR) containing proteins although LRR domains are not so widespread in eukaryotes. For example, plant SGT1 affects the activities of the SCF^TIR1 and SCF^COI1 E3 ligase complexes whose F-box proteins contain LRRs.¹³ Moreover, plant intracellular immune receptors comprise a large group of LRR proteins that recruit SGT1.^8,9 LRRs are also found in yeast adenylyl cyclase Cyr1p and the F-box protein Grr1p which is required for SGT1-dependent cyclin destruction during G₁/S transition.^12,14 Yeast 2-hybrid interaction assays also revealed that yeast and plant SGT1 tend to associate directly or indirectly with LRR proteins.^12,22,23 We speculate that SGT1 bridges the HSP90-HSC70 chaperone machinery with LRR proteins during complex maturation and/or activation. The only other structural motif linked to SGT1 are WD40 domains found in yeast Cdc4p F-box protein and SGT1 interactors identified in yeast two-hybrid screens.¹²What mechanisms underlie a preferential SGT1-LRR interaction? HSP70/SGT1/HSP90 may have co-evolved to assist specifically in folding and maturation of LRR proteins. Alternatively, LRR structures may have an intrinsically greater need for chaperoning activity to fold compared to other motifs. These two scenarios are not mutually exclusive. The LRR domain contains multiple 20 to 29 amino acid repeats, forming an α/β horseshoe fold.²⁴ Each repeat is rich in hydrophobic leucine/isoleucine residues which are buried inside the structure and form the structural backbone of the motif (Fig. 1C, left). Such residues are under strong purifying selection to preserve structure. These hydrophobic residues would render the LRR a possible HSP70 substrate.²⁵ By contrast, hydrophilic solvent- exposed residues of the β strands build a surface which confers ligand recognition specificity of the LRRs (Fig. 1C). In many plant immune receptors for instance, these residues are under diversifying selection that is likely to favour the emergence of novel pathogen recognition specificities in response to pathogen evolution.²⁶ The LRR domain of such a protein has to survive such antagonist selection forces and yet remain functional. Under strong selection pressure, LRR proteins might need to accommodate less stable LRRs because their recognition specificities are advantageous. This could be the point at which LRRs benefit most from a chaperoning machinery such as the HSP90/SGT1/HSP70 complex. This picture is reminiscent of the genetic buffering that HSP90 exerts on many traits to mask mutations that would normally be deleterious to protein folding and/or function, as revealed in Drosophila and Arabidopsis.²⁷ It will be interesting to test whether the HSP90/SGT1/HSP70 complex acts as a buffer for genetic variation, favouring the emergence of novel LRR recognition surfaces in, for example, highly co-evolved plant-pathogen interactions.^28,29     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

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.     

Nonhost resistance to Magnaporthe oryzae in Arabidopsis thaliana

Rice blast, caused by Magnaporthe oryzae, is a devastating disease of rice (Oryza sativa). The mechanisms involved in resistance of rice to blast have been studied extensively and the rice—M. oryzae pathosystem has become a model for plant—microbe interaction studies. However, the mechanisms involved in nonhost resistance (NHR) of other plants to rice blast are still poorly understood. We have recently demonstrated that AGB1 and PMR5 contribute to PEN2-mediated preinvasion resistance to M. oryzae in Arabidopsis thaliana, suggesting a complex genetic network regulating the resistance. To determine whether other defense factors: RAR1, SGT1 and NHO1, affected the A. thaliana-M. oryzae interactions, double mutants were generated between pen2 and these defense-related mutants. All these double mutants exhibited a level of penetration resistance similar to that of the pen2 mutant, suggesting that none of these mutants significantly compromised resistance to M. oryzae in a pen2 background.Key words: nonhost resistance, PEN2, RAR1, SGT1, NHO1Plants face microbial attacks and have evolved innate immunity systems to defend against these threats. The initial step of the immunity signaling pathway is recognition of intra- or extracellular pathogen-derived molecules. Externally oriented transmembrane-type proteins containing leucine-rich repeat (LRR) domains detect extracellular molecules, whereas cytoplasmic sensors possess nucleotide-binding (NB) and LRR domains (NLR).^1,2 The LRR domain serves as a pattern-recognition receptor to detect pathogen-derived molecules or host proteins that are targeted by pathogen peptides that have entered the cell, effectors.³ NLR-type sensors are the substrates of a structurally and functionally conserved chaperone complex that consists of HEAT SHOCK PROTEIN 90 (HSP90) and its cochaperone SUPPRESSOR OF THE G2 ALLELE OF SKP1 (SGT1). REQUIRED FOR MLA12 RESISTANCE 1 (RAR1) regulated the HSP90-SGT1 complex, resulting in the stabilization of NLR proteins. Thus, SGT1 and RAR1 are required for the function of multiple and distinct R genes that encode NLR immune sensors in plants.⁴ Experiments in RAR1-silenced transgenic rice lines showed that RAR1 is not essential for Pib, which encodes an NLR against rice blast fungus.⁵ In contrast, basal resistance to normally virulent races of rice blast fungus or bacterial blight is significantly reduced in RAR1-silenced lines. This result is consistent with earlier reports that RAR1 is involved in basal resistance to virulent Pseudomonas bacteria in Arabidopsis or blast fungus in barley.^6,7 The requirement of SGT1 for immunity in plants is shown mostly by transient silencing of a number of NLR proteins.^8,9 In addition, SGT1 is also required for immune responses triggered by non-NLR-type sensors.¹⁰ This requirement indicates that either SGT1 function is not limited to the NLR sensors, or some unknown SGT1-dependent NLR proteins also operate downstream of non NLR-type sensors. Furthermore, SGT1 is involved in nonhost resistance, indicating that SGT1 may be a general factor of disease resistance.¹⁰ An Arabidopsis mutant, nho1 (nonhost resistance 1), has been isolated on which Pseudomonas syringae pv. phaseolicola grows and causes disease symptoms.^11,12 It is significant that this mutant is also compromised in R-gene-mediated resistance to P. syringae.¹¹ Although NHO1 is the flagellin-induced glycerol kinase, whose exact function in NHR remains elusive.^12,13 A possible explanation might be that altered plant glycerol pools either directly or indirectly affect nutrient availability for P. syringae. NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance.¹² However, these contributions to NHR to M. oryzae in A. thaliana have not been understood.To determine whether these factors were necessary for the resistance to M. oryzae in A. thaliana, the following A. thaliana mutants were inoculated with M. oryzae and monitored by microscopy: rar1-21;¹⁴ edm1-1;¹⁵ nho1-1,¹¹ (all Col-0 background). All these mutants exhibited a level of penetration resistance similar to that of the wild-type plants (data not shown), suggesting that none of these mutants significantly compromised resistance to M. oryzae. We have recently shown that among the penetration (pen) mutants, only the pen2,¹⁶ mutant allowed increased penetration into epidermal cells by M. oryzae.¹⁷ Thus, double mutants were generated between pen2 and these mutants to determine whether these factors were necessary for the resistance to M. oryzae in a pen2 background: pen2 rar1-21; pen2 edm1-1; pen2 nho1-1. All these double mutants exhibited a level of penetration resistance similar to that of the pen2 mutant (Fig. 1), suggesting that none of these mutants significantly compromised resistance to M. oryzae in a pen2 background. This might indicate that NHR against M. oryzae may not be conferred by RAR1- and SGT1-dependent NLR immune sensors. Alternatively, since there has been no report that RAR1 is required for any known transmembrane sensors, such as FLS2, EFR or Xa21, RAR1- and SGT1-independent transmembrane-type immune sensors may be required for NHR against M. oryzae. Future studies will be required to reveal the genetic and mechanistic requirements for NHR in A. thaliana-M. oryzae interactions.Open in a separate windowFigure 1Double mutant analysis to evaluate the role of the defense related genes on resistance to Magnaporthe oryzae in Arabidopsis thaliana. The frequency of M. oryzae penetration on double mutants at 3 days post-inoculation was expressed as a percentage of total appressoria. Data were collected from six independent plants per line. A minimum of 100 infection sites was inspected per leaf. Results represent mean ± standard error of three independent experiments.