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.     

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.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

Environmental stimuli and intestinal stem cell behavior

Comment on: Wong VWY, et al. Nat Cell Biol 2012; 14:401-8.The intestine carries out important functions related to digestion and absorption. It is composed of three distinct layers, an outer muscle layer, a mesenchymal layer and the epithelial layer. The epithelial layer forms the protective barrier that faces the luminal content of the intestine. In order to maintain barrier function the epithelial layer needs constant replenishment. This is ensured by continuous cellular replication in proliferative crypt compartments. Following exit from the crypt, cells adopt fates along either secretory or absorptive lineage and will, after three to four days, be exfoliated into the lumen of the intestine from the tips of the villi. Intestinal stem cells located at the bottom of the proliferative crypt compartment ensure lifelong maintenance of the organ (Fig. 1A).Open in a separate windowFigure 1. Diagram of the intestinal stem cell niche. (A) Lgr5-expressing columnar-based crypt cells (CBCs) intercalated between Paneth cells are indicated in green. Stem cells located in position +4 are yellow. Lrig1 is expressed in a gradient along the niche axis with highest expression in the CBCs indicated with the thickness of the red line. Proliferation in the stem cell niche ensures continuous replenishment of the transit-amplifying (TA) compartment. (B) Within the stem cell niche, Lgr5-expressing CBCs are actively dividing and will give rise to both HopX-expressing +4 cells and TA cells. HopX-expressing cells, which are less mitotically active, will give rise to fewer TA cells and occasionally an Lgr5-expressing stem cell. Lrig1 expression in the stem cell niche reduces the amplitude of ErbB activation and is essential for controlling stem cell proliferation.Adult stem cell niches are far more heterogeneous than previously anticipated.¹ The intestinal stem cell niche can be subdivided by the relative position within the crypt. Stem cells located in position +4, just above secretory Paneth cells, express HopX, Bmi1 and Tert. These cells are generally less mitotically active than Lgr5-expressing stem cells located at the bottom of the proliferative crypts intercalated between Paneth cells (Fig. 1A).^2,3 It has been argued that both populations represent the most primitive stem cell; however, recent studies suggest that stem cells can interconvert between the two states (Fig. 1B).³ Fate mapping from cells in position 4 and at the bottom of the crypt supports this.^2,4 The positional cues responsible for cellular sorting into different functional stem cell compartments are poorly characterized. The only known effector of cellular positioning is Wnt (wingless-related MMTV integration site) signaling.⁵ Wnt is highly expressed by Paneth cells along with other mitotic factors, such as ErbB and Notch ligands.⁶ This could functionally account for the differences observed in proliferative potential along the stem cell axis. The discrete expression patterns of Lgr5 and HopX also support the existence of distinct microenvironments that supports cellular identities. A thorough characterization of the factors responsible for stem cell identity will help delineate and define the functional relationship between the distinct stem cell populations.Tissue homeostasis is governed by balanced loss and gain of cells. The stem cell niche supports constant proliferation via pro-mitotic stimuli. In order to control the amplitude of signaling strength, many pathways have developed negative feedback loops. Lrig1 (Leucine-rich repeats and immunoglobulin-like domains 1) is a negative feedback regulator of ErbB-mediated growth factor signaling.⁷ Lrig1 marks stem cells in various epithelial tissues including the intestinal epithelium, where it is expressed within the entire stem cell niche including the +4 and Lgr5-expressing cells (Fig. 1).^8,9 The functional relevance of Lrig1 and negative feedback regulation is clear from the pronounced expansion of the intestinal stem cell compartment observed in the Lrig1-KO mouse model.⁹ This is mediated via increased ErbB signaling and demonstrates the importance of balanced signaling strength within the stem cell niche.⁹ Moreover, an independent study reveals that Lrig1-KO animals have a higher incidence of colorectal cancer, suggesting that unbalanced stem cell proliferation increases tumor susceptibility.¹⁰ Future studies will address whether additional feedback regulators control signaling strength within the intestinal stem cell niche and how homeostasis within the stem cell compartment affects tumor susceptibility.     

The AGC Kinase MtIRE: A Link to Phospholipid Signaling During Nodulation?

The development of nitrogen fixing root nodules is complex and involves an interplay of signaling processes. During maturation of plant host cells and their endocytosed rhizobia in symbiosomes, host cells and symbiosomes expand. This expansion is accompanied by a large quantity of membrane biogenesis. We recently characterized an AGC kinase gene, MtIRE, that could play a role in this expansion. MtIRE''s expression coincides with host cell and symbiosome expansion in the proximal side of the invasion zone in developing Medicago truncatula nodules. MtIRE''s closest homolog is the Arabidopsis AGC kinase family IRE gene, which regulates root hair elongation. AGC kinases are regulated by phospholipid signaling in animals and fungi as well as in the several instances where they have been studied in plants. Here we suggest that a phospholipid signaling pathway may also activate MtIRE activity and propose possible upstream activators of MtIRE protein''s presumed AGC kinase activity.Key Words: AGC kinase, nitrogen fixation, nodulation, Medicago truncatula, Sinorhizobium meliloti, infection zone, 3-phosphoinositide-dependent kinase, root hair elongationDuring symbiotic nitrogen-fixing nodule development, both plant cells and rhizobia undergo cell division and expansion.^1–3 In legume roots, nodule organogenesis is triggered by rhizobial Nod factor at the emerging root hair zone. In the indeterminate Medicago-Sinorhizobium symbiosis, inner cortical cell divisions form nodule primordia which emerge from the root and differentiate into complex nodule structures. Rhizobia enter the nodules through plant derived conduits, the infection threads (ITs). ITs begin in curled root hairs, grow through several cell layers and end at nodule primordia where rhizobia are deposited into host cell symbiosomes.² In mature nodules, the meristematic zone I at the nodule apex contains dividing cells. Rhizobia from ITs infect these cells as they exit zone I and enter the infection zone, zone II. The newly released rhizobia, now termed bacteroids, are rod-shaped. In the distal part of zone II, bacteroids divide along with the symbiosome membrane (also called the peribacteroid membrane) that contains them.⁴ As the plant cells with their internalized bacteroids progress toward the proximal end of zone II, bacteroid division ceases. Bacteroid elongation and expansion of the surrounding symbiosome space and membrane is a feature of the proximal side of zone II.⁴ Enormous membrane biogenesis accompanies progression through zone II. As the cells exit zone II, both host cells and bacteroids stop expanding. Interzone II-III is characterized by starch accumulation and zone III is where nitrogen fixation takes place.Members of the protein kinase AGC (for cAMP dependent, cGMP dependent, and protein kinase C) family have been shown to be important in yeast and mammalian signal transduction. The interaction of growth factors with their receptors leads to the activation of phosphatidylinositol (PtdIns) 3-kinase and the phosphorylation of PtdIns species.⁵ These then activate PDK1 enzymes, 3-phosphoinositide-dependent kinases, also AGC kinases,⁵ which then phosphorylate and activate downstream AGC kinases. Several plant AGC kinases have important roles in development and defense,^6–8 although most plant AGC kinases'' functions are still to be discovered.⁹ Two Arabidopsis AGC kinases, IRE and AGC2-1 have been shown to have roles regulating root hair elongation.^10,11We recently cloned and characterized a Medicago IRE-like AGC kinase gene MtIRE,¹² possibly orthologous to the Arabidopsis IRE gene, AtIRE.¹⁰ Because of MtIRE''s homology to AtIRE we thought it might function during infection, because infection threads can be viewed as inward root hair growth. However, MtIRE''s expression is novel. It is expressed only in nodules and flowers and not in roots or root hairs. During nodule development, its initial expression correlates with the onset of host cell and symbiosome expansion. Expression studies with nodulation mutants demonstrate that MtIRE expression correlates with mutant nodules'' abilities to support host cell and symbiosome expansion.¹² An MtIRE promoter-gusA reporter construct (Fig. 1A) shows expression in the proximal part of zone II, the site of continued host cell expansion and bacteroid and symbiosome elongation. RNA interference experiments were unfortunately inconclusive,¹² probably because of closely related more ubiquitously expressed IRE homologs.Open in a separate windowFigure 1(A) Localization of pMtIRE-gusA expression in wild-type nodulated roots. Composite M. truncatula plants with transgenic roots were grown in the presence of S. meliloti and stained with X-Gluc (blue) for the localization of MtIRE promoter activity. The arrow points to the X-Gluc staining in the proximal side of zone II in a 15 dpi nodule. The arrowhead points to root hairs in which no staining was observed. Bar = 100 µM. (B) Phospholipid signaling pathway that may activate MtIRE protein''s presumed kinase activity.We predict that MtIRE is part of a signal pathway regulating an aspect of host cell expansion or symbiosome elongation, or both. The CCS52A gene has a demonstrated role in host cell expansion, mediating endoreduplication.¹³ In contrast to MtIRE, its expression is found throughout zone II, as well as zone I, where it acts in cell division. One might expect other genes that regulate host cell expansion to also be expressed throughout zone II, which MtIRE is not. A unique feature of the region expressing MtIRE is symbiosome elongation.⁴ Because of MtIRE''s temporal and spatial expression patterns, we favor it having a role in symbiosome expansion, although we cannot rule out a role in the latter stages of host cell expansion.Signaling pathway for MtIRE activation is speculative (Fig. 1B) and based on AGC kinase signaling in other systems. AGC kinases are activated by phosphorylation by phosphoinositide-dependent kinase (PDK1) enzymes, also AGC kinases.⁹ We found 4 tentative consensus sequences (TCs) in the DFCI index (compbio.dfci.harvard.edu) that correspond to PDK1 genes of which 3, TC107355, TC94724 and TC94899, were isolated from expression libraries from roots with developing or mature nodules. PDKs are activated by interaction with lipids. The Arabidopsis PDK1 binds to several signaling lipids, including phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidic acid (PA).¹⁴ Phosphatidylinositol 3-kinase (PI3K) activity produces PtdIns3P and PI3K genes have been observed to be induced during nodule organogenesis in soybean¹⁵ and in M. truncatula.¹⁶ In soybean, two PI3K genes were identified with one specifically expressed during the early stages of nodulation when membrane biogenesis takes place. This gene''s predicted protein has potential phosphorylation sites for cAMP dependent kinases and Ca/calmodulin-dependent kinases.¹⁵ In soybean, PI3K enzymatic activity correlated with membrane proliferation during nodulation.¹⁵ More generally, PI3Ks are implicated in vesicular trafficking and cytoskeletal organization;¹⁷ both are required for host cell and symbiosome elongation. We suggest a model where MtIRE kinase activity is activated by PDK1, which is itself regulated by PI3K through the production of PtdIns3P. More speculatively, PI3K could be under the control of the Nod factor signaling pathway Ca/calmodulin-dependent kinase DMI3.^18,19 DMI3 is induced during nodulation, with highest expression levels found in the distal side of the infection zone,²⁰ before expression of MtIRE. Expression could persist to the proximal side of this zone, similar to the expression of another Nod factor signaling component, DMI2.²¹ Alternatively, MtIRE could be activated by PA in a PDK1-dependent manner similar to Arabidopsis AGC2-1.¹¹ PA can be produced by phospholipase C (PLC) or phospholipase D (PLD) pathways, both of which have been implicated in transducing Nod factor signals.^22–26 Either of these models includes Nod factor signaling in proximal zone II, which has not been well-studied. Expression of rhizobial nod genes has been observed in zone II,²⁷ making Nod factor signaling in this zone plausible. Further examination of zone II and predicted upstream regulators of MtIRE will address this model.