Phosphorylation of Merlin Regulates its Stability and Tumor Suppressive Activity

The neurofibromatosis-2 (NF2) tumor suppressor protein, merlin or schwannomin, inhibits cell proliferation by modulating the growth activities of its binding partners, including the cell surface glycoprotein CD44, membrane-cytoskeleton linker protein ezrin and PIKE (PI 3-kinase enhancer) GTPase, etc. Merlin exerts its growth suppressive activity through a folded conformation that is tightly controlled through phosphorylation by numerous protein kinases including PAK, PKA and Akt. Merlin inhibits PI 3-kinase activity through binding to PIKE-L. Now, we show that merlin is a physiological substrate of Akt, which phosphorylates merlin on both T230 and S315 residues. This phosphorylation abolishes the folded conformation of merlin and inhibits its association with PIKE-L, provoking merlin polyubiquitination and proteasome-mediated degradation. This finding demonstrates a negative feed-back loop from merlin/PIKE-L/PI 3-kinase to Akt in tumors. The proliferation repressive activity of merlin is also partially regulated by S518 phosphorylation. Thus, Akt-mediated merlin T230/S315 phosphorylation, combined with S518 phosphorylation by PAK and PKA, provides new insight into abrogating merlin function in the absence of merlin mutational inactivation.Key Words: Akt, merlin, neurofibromatosis, phosphorylation, cell invasion and migrationNeurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by bilateral occurrence of vestibular schwannomas and other brain tumors, including meningiomas and ependymomas.¹ The NF2 tumor suppressor protein merlin belongs to the band 4.1 family of cytoskeleton-associated proteins.^2,3 Merlin isoform I possesses a “closed” conformation via an NTD (N-terminal domain)/CTD (Carboxy terminal domain) intramolecular interaction. In contrast, the alternatively spliced merlin isoform II exists in an “open” conformation that cannot function as a negative growth regulator.⁴ Merlin with NF2 patient missense mutations in the NTD or CTD exhibit an “open” conformation and do not suppress cell growth.⁵ Merlin plays a key role in regulating cell proliferation and cell migration. Merlin exerts its growth suppressive activity through intramolecular folding that dictates its binding affinities to various cellular partners including HRS (hepatocyte growth factor regulated tyrosine kinase substrate), CD44 cell surface glycoprotein, schwannomin interacting protein-1 (SCHIP1), βII-spectrin or fodrin, PIKE-L GTPase and other ERM proteins.^6–10 For instance, CD44 preferentially associates with hypophosphorylated merlin, and relatively little phosphorylated merlin binds CD44. Interference with merlin binding to CD44 impairs merlin growth suppression in RT4 rat schwannoma cells.¹¹We have previously shown that the PIKE/PI 3-kinase signaling pathway is negatively regulated by protein 4.1N, a neuronal selective isoform of band 4.1 superfamily.¹² Recently, we show that PIKE-L is an important mediator of merlin growth suppression. We show that merlin blocks cell proliferation by inhibiting PI 3-kinase through binding to PIKE-L. Interestingly, wild-type merlin, but not patient-derived mutant (L64P), binds PIKE-L and inhibits PI 3-kinase activity. This suppression of PI 3-kinase activity results from merlin disrupting the binding of PIKE-L to PI 3-kinase. Mutation of PIKE-L with Proline 187 into Leucine disrupts its interaction with merlin. Accordingly, merlin suppression of PI 3-kinase activity as well as schwannoma cell growth is abrogated by a single PIKE-L point mutation (P187L).¹⁰Merlin is phosphorylated on S518 by members of the PAK family of kinases, including PAK1 and PAK2,^13–15 which mislocates merlin from the plasma membrane to the cytoplasm. A merlin mutant that mimics S518 phosphorylation (S518D) cannot suppress cell growth or motility in RT4 rat schwannoma cells, and leads to dramatic changes in cell morphology and actin cytoskeleton organization.¹⁶ S518 phosphorylation results in impaired merlin NTD/CTD folding as well as altered interactions with critical merlin associated proteins, including CD44 and HRS.¹⁷ Recently, Alfthan and colleagues demonstrated that Protein Kinase-A (PKA) induces merlin phosphorylation on both N-and C-terminal residues.¹⁸ In addition to S518 phosphorylation, PKA can phosphorylate merlin at S66 in the N-terminal domain (Fig. 1). When PAK activity is suppressed, merlin can still be phosphorylated by PKA in cells, indicating that these two kinases function independently. The N-terminus of ezrin strongly binds to a PKA-phosphorylated, but not unphosphorylated, merlin CTD. In contrast, PAK2-induced S518 phosphorylation has a minimal effect on the interaction between full-length merlin and full-length ezrin.¹⁷ Besides regulation of cell growth, merlin also mediates cell motility presumably through directly binding to actin cytoskeleton.¹⁹ Depletion of merlin in normal fibroblast results in enhanced cell invasion. Nevertheless, expression of merlin attenuates Y397 phosphorylation on FAK, an essential player in cell migration and invasion. This observation might provide a molecular mechanism accounting for merlin inhibitory activity in cell motility.²⁰Open in a separate windowFigure 1Merlin phosphorylation sites by various kinases.In addition to PAK and PKA, we show that Akt potently phosphorylates merlin at both T230 and S315 residues. Blocking one site phosphorylation abolishes the other site phosphorylation by Akt, indicating that these two phosphorylation sites are mutually regulated.²¹ The physiological significance of the tight control on merlin phosphorylation by Akt remains incompletely understood. Presumably, only when mitogenic signal or oncogenic stress is strongly enough to provoke cell proliferation or migration, does Akt simultaneously phosphorylate both sites. Akt phosphorylation of merlin attenuates the NTD/CTD interaction and inhibits its binding activity to PIKE-L, CD44 and ezrin. Further, phosphorylation mediates the biological activities of merlin, as expression of a phosphomimetic merlin mutant (T230DS315D) increases cell motility and proliferation in a rat schwannoma cell line (Fig. 2). By contrast, expression of a mutant (T230A/S315A) that was unable to undergo phosphorylation inhibited cell growth and motility. The F1 motif in FERM proteins including merlin exhibits an ubiquitin-like structure. This domain facilitates MDM2 degradation and stimulates the ubiquitination and degradation of TRBP, a double-stranded RNA binding protein. Surprisingly, inhibition of the proteasome does not affect total merlin protein levels in human glioblastoma cells, but leads to a marked increase of phospho-S315 merlin. Simultaneous treatment with MG132, which blocks proteasome-mediated degradation and PI 3-kinase inhibitor, wortmannin, which inhibits Akt phosphorylation of merlin, substantially enhances merlin levels. Coimmunoprecipitation studies demonstrate that Akt-phosphorylated merlin is rapidly ubiquitinated, presumably by spectrin, which binds to merlin and possesses ubiquitin-conjugating and ubiquitin E3 ligase function. However, S518 phosphorylation fails to trigger merlin ubiquitination, suggesting that Akt, but not PAK or PKA, phosphorylation selectively elicits merlin ubiquitination. Using a panel of human primary nervous system tumors, we found that merlin phosphorylation by Akt also mediates its degradation in primary tumors. Accordingly, tumors that possess high levels of phospho-Akt exhibited low levels of merlin. Therefore, our data suggest a novel role for Akt in promoting phosphorylation and subsequent degradation of merlin. Loss of merlin has been linked to schwannomas and other nervous system tumors, and these results indicate that inhibitors for PI 3-kinase/Akt pathway might restore merlin function in tumors.Open in a separate windowFigure 2The model for Akt interaction with merlin and its phosphorylation.     

Cdk5: A regulator of epithelial cell adhesion and migration

Cell adhesion is a fundamental property of epithelial cells required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of a variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.Key words: Cdk5, Src, Rho, stress fibers, epithelial cells, cell adhesion, cell migrationAnchoring of epithelial cells to their basement membrane is essential to maintain their morphology, normal physiological function and survival. Cells attach to extracellular matrix components by means of membrane-spanning integrins, which cluster and link to the actin cytoskeleton via components of focal adhesions. At focal adhesions, actin is bundled into stress fibers, multi-protein cellular contractile machines that strengthen attachment and provide traction during migration.¹ Stress fiber contraction is generated by myosin II, a hexamer containing one pair of each non-muscle heavy chains (NMHCs), essential light chains, and myosin regulatory light chains (MRLC). Myosin motor activity is regulated by phosphorylation of MRLC at Thr18/Ser19 and is required to generate tension on actin filaments and to maintain stress fibers.¹ Although a number of kinases have been identified which phosphorylate MRLC at Thr18/Ser19, the principal kinases in most cells are myosin light chain kinase (MLCK)² and Rho-kinase (ROCK),³ a downstream effector of the small GTPas, RhoA.Rho family small GTPases play a central role in regulating many aspects of cytoskeletal organization and contraction.⁴ These GTPases are subject to both positive regulation by guanine nucleotide exchange factors (GEFs), such as GEF-H1,^5,6 and negative regulation by GTPase-activating proteins (GAPs), such as p190RhoGAP.⁷ As cells spread, the Rho-family GTPase, Cdc42, is activated at the cell periphery, leading to the formation of numerous filapodia. Focal adhesion formation is first seen at the tips of these filapodia as focal adhesion proteins such as talin and focal adhesion kinase (FAK) bind to the intracellular domains of localized integrins.⁸ Src is recruited to activated FAK at the nascent focal adhesion and generates binding sites for additional focal adhesion proteins by phosphorylating FAK and paxillin.⁹ Src activity is essential for the further maturation of the focal adhesion and for activating the Rho-family GTPase, Rac, leading to Arp2/3-dependent actin polymerization, formation of a lamellipodium and extension of the cell boundary. Simultaneously, Src inhibits RhoA by phosphorylating and activating its upstream inhibitor, p190RhoGAP. As the focal adhesion matures, Src is deactivated, allowing the Rho activation necessary for mDia-dependent actin polymerization,¹⁰ myosin-dependent cytoskeletal contraction⁵ and tight adhesion to the extracellular matrix. Since new focal adhesions continually form at the distal boundary of the spreading cell, the most mature and highly contracted stress fibers are localized at the center of the cell.Cell adhesion is an essential component of cell migration: if adhesion is too weak, cells can not generate the traction necessary for migration; if it is too strong, they are unable to overcome the forces that anchor them in place. Thus, the relationship between adhesion force and migration rate is a bell-shaped curve.¹¹ Migration rate increases as adhesive strength increases until an optimum value is reached. Thereafter, increases in adhesive strength decrease migration rate. Since the strength of adhesion depends on extracellular matrix composition as well as the types of integrin expressed in the cell, a decrease in adhesive strength may result in either faster or slower cell migration.Several lines of evidence indicate that the proline-directed serine/threonine kinase cyclin dependent kinase 5 (Cdk5) plays an integral role in regulating cell adhesion and/or migration in epithelial cells.^12–17 Cdk5 is an atypical member of cyclin dependent kinase family, which is activated by the non-cyclin proteins, p35 or p39.¹⁸ Cdk5 is most abundant in neuronal cells where it also regulates migration and cytoskeletal dynamics.¹⁹ In neurons, Cdk5 exerts its effects on migration at least in part by phosphorylating FAK,¹⁹ and the LIS1 associated protein, NDEL1.²⁰ In contrast, recent findings have revealed two novel pathways involved in Cdk5-dependent regulation of migration in epithelial cells.^16,17One of these newly discovered mechanisms links Cdk5 activation to control of stress fiber contraction.¹⁶ We have found that Cdk5 and its activator, p35, co-localize with phosphorylated myosin regulatory light chain (MRLC) on centrally located stress fibers in spreading cells.¹⁶ Moreover, Cdk5 is strongly activated in spreading cells as central stress fiber contraction becomes pronounced.²¹ Since contraction of these central stress fibers is primarily responsible for tight attachment between the cell and the extracellular matrix,⁵ the above findings suggested that Cdk5 might regulate cell adhesion by regulating MRLC phosphorylation. To test this possibility we inhibited Cdk5 activity by several independent means and found that MRLC phosphorylation was likewise inhibited. In addition, we found that inhibiting Cdk5 either prevented the formation of central stress fibers or led to their dissolution. The concave cell boundaries characteristic of contracting cells were also lost.¹⁶ Since MRLC lacks a favorable site for phosphorylation by Cdk5, we asked whether Cdk5 might affect the upstream signaling pathways that regulate MRLC phosphorylation. Experiments with specific pathway inhibitors indicated that the MRLC phosphorylation involved in stress fiber contraction in lens epithelial cells was regulated largely by Rho-kinase (ROCK). Inhibiting Cdk5 activity not only significantly reduced ROCK activity, but also blocked activation of its upstream regulator, Rho. To explore the mechanism behind the Cdk5-dependent regulation of Rho, we turned our attention to p190RhoGAP, which appears to play a major role in regulating Rho-dependent stress fiber contraction.⁷ This RhoGAP must be phosphorylated by Src to be active; as a result, Rho activity is low in the early stages of cell spreading, when Src activity is high. At later times, Src activity falls, p190RhoGAP activity is lost, and Rho-GTP is formed, enabling Rho-dependent myosin phosphorylation and stress fiber contraction.^9,10 We have found that inhibiting Cdk5 activity during this later stage of cell spreading increases Src activity and Src-dependent phosphorylation of its substrate, p190RhoGAP. This in turn leads to decreased Rho activity accompanied by loss of Rho-dependent myosin phosphorylation, dissolution of central stress fibers, and loss of cell contraction (Fig. 1). Moreover, inhibiting Src protects cells from the loss of Rho activation and dissolution of central stress fibers produced by inhibiting Cdk5.¹⁶ Since the effects of Cdk5 on Rho-dependent cytoskeletal contraction appear to be mediated almost entirely through Cdk5-dependent regulation of Src, it will be particularly important to determine how Cdk5 limits Src activity.Open in a separate windowFigure 1Cdk5 inhibition reduces contraction of preformed stress fibers. (A) Cells were spread on fibronectin for 60 min to adhere, allowing them to form focal adhesion and stress fibers (pre-incubation) and then further incubated for 2 h in absence (control) or presence of Cdk5 inhibitor (olomoucine) and stained with phalloidin. The cells without olomoucine (control) had concave boundaries and well-formed stress fibers. Olomoucine treated cells showed loss of central stress fibers and failure to contract. Scale bar = 20 µ. (B) experimental conditions were same as shown in (A). Cdk5 inhibitor, olomoucine, was added after 1 h of spreading (indicated as t = 0) and cells were incubated for an additional 2h in absence or presence of olomoucine. Cell lysates were immunoblotted with antibodies for pMRLC (upper) and MRLC (middle). Tubulin was used as a loading control (lower). Lane 1: untreated (at 0 h); Lane 2: untreated (at 2 h); Lane 3: Cdk5 inhibitor (olomoucine) treated. (C) results of three independent experiments of the type shown in (B) were quantified by densitometry and normalized to determine the relative levels of pMRLC at each time. Statistical analysis demonstrated a significant (p < 0.05) decrease in pMRLC level in olomoucine treated cells compared to untreated cells.The central stress fibers regulated by Cdk5 play a central role in anchoring cells to the substratum, and their loss when Cdk5 is inhibited will reduce adhesion. As discussed above, reduction in cell adhesion may either increase or decrease the rate of cell migration, depending on the cell type and extracellular matrix composition. In lens and corneal epithelial cells, the reduction in adhesion produced by Cdk5 inhibition promotes cell migration.^13,15,16,22 Moreover, regulation of Rho/Rho-kinase signaling by Cdk5 seems to be a major factor in determining the migration rate, since inhibitors of Cdk5 and Rho-kinase increased lens epithelial cell migration rate equivalently and inhibiting both produced no additional effect.¹⁶Interestingly, an independent line of investigation has shown that this is not the only mechanism underlying Cdk5-dependent regulation of cell adhesion and migration. Cdk5 also localizes at focal contacts at the cell periphery and phosphorylates the focal adhesion protein talin.¹⁷ The talin phosphorylation site has been identified as S425, near the FERM domain in the talin head region. Upon focal adhesion disassembly, this region is separated from the talin rod domain by calpain-dependent cleavage.²³ Phosphorylation at S425 by Cdk5 blocks ubiquitylation and degradation of the talin head by inhibiting interaction with the E3 ligase, Smurf1. This leads, ultimately, to greater stability of lamellipodia and newly formed focal adhesions, thus strengthening adhesion to the substrate.¹⁷ Although the exact molecular events involved in this stabilization are not yet clear, it has been suggested that the talin head may “prime” integrins to bind full length talin.²⁴ One possible scenario describing how this might occur is shown in Figure 2. By permitting the isolated head region to escape degradation following calpain cleavage, Cdk5-dependent phosphorylation may stablize a pool of talin head domains to bind focal contacts within the lamellipodium. It is known that the isolated talin head region can bind and activate integrins during cell protrusion.²⁵ The resulting integrin activation would be expected to stabilize the lamellipodium by strengthening integrin-dependent adhesion. Since the head domain lacks sites for actin binding, which are located in the talin rod domain,²⁶ the bound head domain would have to be replaced by full length talin to enable focal adhesion attachment to the cytoskeleton.²⁵ The head domain might promote this replacement by recruiting the PIP-kinase needed to generate PI(4,5) P₂,²³ which facilitates binding of full length talin to integrin by exposing the auto-inhibited integrin binding sites.²⁷ The binding of full length talin and the resulting link between the integrins and the actin cytoskeleton would then further strengthen adhesion.²⁵ This model predicts that full length talin would bind poorly in the absence of Cdk5 activity, due to degradation of the talin head and the resulting limited availability of PI(4,5)P₂, and thus provides a possible explanation for the observed rapid turnover of peripheral focal adhesions.¹⁷ Clearly, other models may be proposed to explain the increase in adhesion produced by talin head phosphorylation, and deciding among them will be an active area for future investigation. Nonetheless, it is now certain that talin is a key substrate for Cdk5 at focal adhesions.Open in a separate windowFigure 2Mechanism of Cdk5-dependent regulation of cell adhesion and migration. Binding of p35 to Cdk5 forms the active Cdk5/p35 kinase, which regulates cell adhesion and migration in two distinct ways. Cdk5-dependent phosphorylation of the talin head domain at Ser425 prevents its ubiquitylation and degradation, allowing it to persist following calpain cleavage. The phosphorylated talin head may then bind to integrin at peripheral sites and recruit PIP-K, which converts PI(4)P to PI(4,5)P₂. PI(4,5)P₂ may promote replacement of the talin head by full length talin. Full length talin recruits other focal adhesion proteins to form the mature focal adhesion. The talin tail provides the site for the actin binding and polymerization. Polymerized actin is subsequently bundled into stress fibers. Cdk5/p35 also regulates the Rho-dependent myosin phosphorylation necessary for stress fiber stability and cytoskeletal contraction by limiting Src activity. This in turn decreases Src-dependent phosphorylation of p190RhoGAP, favoring Rho-GTP formation, Rho-dependent stress fiber polymerization, stabilization and contraction. Both pathways modulate cell migration by increasing adhesive strength.In summary, the presently available data indicate that Cdk5 has at least two distinct functions in cell adhesion (Fig. 2). On the one hand, it stabilizes peripheral focal adhesions and promotes their attachment to the cytoskeleton by phosphorylating the talin head. On the other hand, once the actin cytoskeleton has been organized into stress fibers, Cdk5 enhances the Rho activation essential for stability and contraction of central stress fibers by limiting Src activity. The discovery that Cdk5 is involved in two separate events required for efficient migration, suggests that it may coordinate multiple signaling pathways. The known involvement of Cdk5 and its activator, p35, in regulating microtubule stability suggests yet another mechanism by which Cdk5 activity may regulate cytoskeletal function. Microtubules are closely associated with stress fibers²⁸ and their depolymerization has been shown to release the Rho activating protein, GEF-H1, leading to Rho activation and Rho-dependent myosin contraction.⁶ Since cell adhesion and migration play an important role in the progression of many pathological conditions, Cdk5, its substrates and its downstream effectors involved in cell adhesion may provide novel targets for therapeutic intervention.^15,29     

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.     

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.     

Significance of microtubule catastrophes at focal adhesion sites

Directional cell migration requires cell polarization and asymmetric distribution of cell signaling. Focal adhesions and microtubules are two systems which are essential for these. It was shown that these two systems closely interact with each other. It is known that microtubule targeting stimulates focal adhesion dissociation. Our recent study shows that focal adhesions, in turn, specifically induce microtubule catastrophe via a biochemical mechanism. We were able to track down one of the focal adhesion proteins paxillin which is involved in this process. Paxillin phosphorylation was previously shown to be the key component in the regulation of focal adhesion assembly or disassembly. Since microtubule catastrophe dynamic differs at the leading edge and cell rear, similar to paxillin phosphorylation levels, we suggest a model connecting asymmetric distribution of focal adhesions and asymmetric distribution of microtubule catastrophes at adhesion sites as a feedback loop.Key words: microtubule catastrophe, focal adhesion, microtubules, paxillin, cell motilityCell migration is important for many biological processes. It requires organized asymmetric dynamics of focal adhesions (FAs), sites where cells interact with extra cellular matrix. FAs appear at the leading edge as small transient dot-like structures termed focal complexes (FXs).^1,2 FX assembly and disassembly is regulated by phosphorilation status of paxillin a major FX protein.^3,4 Most of FXs form and disassemble rapidly. However, some adhesions mature in a force-dependent manner, into larger late adhesions. This process, involves both an increase in size and change in molecular composition^3,5 and is accompanied by a reduction in local paxillin phosphorylation.⁴ Late adhesions are more stable, immobile and undergo forced disassembly by multiple microtubule targeting events⁶ only underneath the approaching cell body or transform into fibrillar adhesions by a Src-dependent mechanism.⁷Similarly to the leading edge, proper adhesion patterns at the cell rear are also essential. Most trailing adhesions are initiated in protrusions at the rear and flanks of the cell as FX rapidly mature in response to tension and transform into sliding trailing adhesions.⁸ The process of sliding is complex. While adhesion proteins coupled with the actin cytoskeleton can be translocated relative to substratum, those that are associated with the membrane are thought to undergo treadmilling within the adhesion site.^9,10 Treadmilling, which includes disassembly of adhesion proteins at the distal end and reassembly at the proximal end,¹⁰ is accompanied by fusion with new adhesions formed in front of the sliding one.⁶ Thus, despite a protein composition similar to late adhesions, sliding adhesions are more dynamic. Not surprisingly, sliding adhesions have high paxillin phosphorylation at the distal end of the adhesion site, indicating very dynamic assembly/disassembly rates.⁴Several mechanisms have been proposed for the regulation of adhesion turnover (reviewed in ref. ¹¹). However, these have not accounted for the observed asymmetry of adhesion turnover. Understanding this requires examining the connection with another asymmetric intracellular system, the microtubule network. This dynamic network closely interacts with FAs. Microtubules play an essential role in cell migration and polarized distribution of signals within the cell. Multiple microtubule targeting to FA leads to their disassembly both at the leading edge and at the cell rear.⁶Unlike microtubule growth in other cell regions, growth at its leading edge is persistent, characterized by short periods of shrinkage.⁸ Simultaneous observation of microtubules and FAs show that microtubules specifically target adhesion sites.¹² More detailed analysis of microtubule dynamics reveals that FAs are preferable sites for microtubule catastrophes.¹³ Although FAs cover only about 5% of cell area more than 40% of catastrophes occur at these sites. The likelihood of microtubule catastrophe is seven times higher when a microtubule grows through a FA rather than through an adhesion-free area¹³ and about 90% of microtubules approaching adhesion sites undergo catastrophe. Although most of the catastrophes occur at late adhesions, due to their increased stability and lifespan, there is no difference in efficiency of catastrophe induction between small focal complexes and large rigid late adhesions.¹³ As FX do not have dense adhesion or actin plaque, it is likely that microtubule catastrophe is triggered by a biochemical mechanism rather than mechanical rigidity. This is also supported by the fact that mechanical obstacles in a cell do not necessarily cause microtubule catastrophe.¹³At the cell rear, microtubule dynamics differ from those at the leading edge. Microtubules spend less time in a growing phase and more time in pauses and shrinkage.⁸ Polymerization and depolymerization occur within a very limited area close to the cell edge.⁸ Live-cell imaging of cells expressing both microtubule and focal adhesion markers show that this complex dynamic sequence often happens within a single sliding adhesion. Microtubules that are captured at the proximal end of adhesion undergo multiple repetitive catastrophes at the distal end (Fig. 1) accompanied by rescue at the capture site. Thus, the capture mechanism significantly increases the lifetime of a microtubule and ensures that repetitive catastrophes occur at the single adhesion. This scenario leads to high catastrophe frequency at the cell rear, resulting in intensive catastrophe-dependent regulation in this cell region.Open in a separate windowFigure 1Multiple microtubule catastrophes at the sliding adhesion. (A) Frame from TIRF video sequence of a fish fibroblast cell (CAR) co-transfected with GFP-tubulin (green) to visualize microtubules and Cherry-Zyxin (red) to mark focal adhesions. The boxed region is presented in the kymograph in (B). Bar, 10 µm. (B) Kymograph of microtubule dynamics at a trailing end focal adhesion. Top panel shows microtubule (MT) only. Bottom panel shows life history plot of MT (green line shows movement of MT end) in relation to focal adhesions (red). Arrows show catastrophes at the distal end of adhesion, arrowheads show capture at the proximal end of adhesion.Detailed analysis of microtubule catastrophe localization shows that they occur at the areas of FAs where paxillin is enriched and highly phosphorylated.^4,13 Paxillin was shown to interact with microtubules through its Lim2/Lim3 domain.¹⁴ Purified GST-Lim2/Lim3 fragment injected into the cell localizes to FAs, displacing endogenous paxillin.¹³ This leads to a 40% decrease in the number of microtubule catastrophe events at adhesion sites,¹³ indicating that paxillin is needed for catastrophe initiation.In summary, we conclude that microtubule catastrophes at focal adhesions are specific events that are triggered by a biochemical mechanism. This process involves the focal adhesion protein paxillin, which may serve as a docking site for microtubules and/or microtubule catastrophe factors. The nature of catastrophe factors remains to be clarified. Possible mechanisms include molecules which induce microtubule catastrophe directly, such as stathmin,¹⁵ or molecules which regulate catastrophe-inducing factors activity. Alternatively, catastrophe factors at adhesion sites could act by removing stabilizing factors from microtubule tips. Thus, allowing already active catastrophe-inducing molecules such as kinesin-13 family member MCAK^16,17 to complete their function. Furthermore, microtubule catastrophe at paxillin-enriched areas, followed by release of microtubule-associated factors, may be involved in paxillin phosphorylation. This local regulation of adhesion disassembly would close the feed-back loop to microtubule regulation of FA turnover.In this model, asymmetric distribution of microtubule catastrophes is tightly linked to asymmetric regulation of FA. Since asymmetric FA dynamics in a cell are critical for organization of the actin cytoskeleton, tensile force distribution and directional cell migration, we conclude that microtubule catastrophes serve as important regulatory events for asymmetric signaling and dynamics of the whole cell (Fig. 2).Open in a separate windowFigure 2Model for asymmetric focal adhesion and microtubule dynamics. Focal complexes at the leading edge either disassemble or mature in response to tension. Microtubules undergo catastrophe both at focal complexes and late adhesions. Late adhesions disassemble in response to multiple microtubule targeting. At the cell rear a microtubule is captured at the proximal end of sliding adhesion and undergoes multiple catastrophes at its distal end, supporting disassembly of this region.     

Dynamic protein trafficking to the cell wall

Recently we have studied the secretion pattern of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2) in tobacco protoplast using the protein fusions, secGFP-PMEI1 and PGIP2-GFP. Both chimeras reach the cell wall by passing through the endomembrane system but using distinct mechanisms and through a pathway distinguishable from the default sorting of a secreted GFP. After reaching the apoplast, sec-GFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP undergoes endocytic trafficking. Here we describe the final localization of PGIP2-GFP in the vacuole, evidenced by co-localization with the marker Aleu-RFP, and show a graphic elaboration of its sorting pattern. A working model taking into consideration the presence of a regulated apoplast-targeted secretion pathway is proposed.Key words: cell wall trafficking, endocytosis, GPI-anchor, PGIP2, PMEI1, secretion pathway, vacuole fluorescent markerCell wall biogenesis, growth, differentiation and remodeling, as well as wall-related signaling and defense responses depend on the functionality of the secretory pathway. Matrix polysaccharides, synthesized in the Golgi stacks, and cell wall proteins, synthesized in the ER, are packaged into secretory vesicles that fuse with the plasma membrane (PM) releasing their cargo into the cell wall. Also the synthesis and deposition of cellulose itself are driven by the endomembrane system which controls the assembly, within the Golgi, and the export to the plasma membrane of rosette complexes of cellulose synthase.¹ Secretion to the cell wall has always been considered a default pathway² but recent studies have evidenced a complex regulation of wall component trafficking that does not seem to follow the default secretion model. Recent evidence that several cell wall proteins are retained in the Golgi stacks until specific signals at the N-terminal domain are proteolitically removed is a case in point.^3–5 Moreover, it has previously been reported that secretion of exogenous marker proteins (secGFP and secRGUS) and cell wall polysaccharides reach the PM through different pathways.⁶ More recently, we have reported that cell wall protein trafficking also occurs through mechanisms distinguishable from that of a secreted GFP suggesting that more complex events than the mechanisms of bulk flow control cell wall growth and differentiation.⁷ To follow cell wall protein trafficking we used a Phaseolus vulgaris polygalacturonase inhibitor protein (PGIP2) and an Arabidopsis pectin methylesterase inhibitor protein (PMEI1) fused to GFP (PGIP2-GFP and secGFP-PMEI1). Both apoplastic proteins are involved in the remodeling of pectin network with different mechanisms. PGIP2 specifically inhibits exogenous fungal polygalacturonases (PGs) and is involved in the plant defense mechanisms against pathogenic fungi.^8,9 PMEI1 counteracts endogenous PME and takes part in the physiological synthesis and remodeling of the cell wall during growth and differentiation.^10,11 The specific functions of the two apoplastic proteins seem to be strictly related to the distinct mechanisms that control their secretion and stability in the cell wall. In fact, while secGFP-PMEI1 moves through ER and Golgi stacks linked to a glycosyl phosphatidylinositol (GPI)-anchor, PGIP2-GFP moves as a cargo soluble protein. Furthermore, secGFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP, over the time, is internalized into endosomes and targeted to vacuole, likely for degradation. After reaching the cell wall, the different fate of the two proteins seems to be strictly related to the presence/absence of their physiological counteractors. PMEI regulates the demethylesterification of homogalacturonan by inhibiting pectin methyl esterase (PME) activity through the formation of a reversible 1:1 complex which is stable in the acidic cell wall environment.¹² Stable wall localization of PMEI1 is likely related to its interaction with endogenous PME, always present in the wall. Unlike PMEs, fungal polygalacturonases (PGs), the physiological interactors of PGIP2, are present in the cell wall only during a pathogen attack. The absence of PGs may determine PGIP2 internalization. Internalization events have been already reported for PM proteins,^13–16 while cell wall protein internalization is surely a less well-known event. To date, only internalization of an Arabidopsis pollen-specific PME^4,5,17 and PGIP2 ⁷ has been reported.To further confirm the internalization of PGIP2-GFP and its final localization into the vacuole, we constructed a red fluorescent variant (RFP) of the green fluorescent marker protein that accumulates in lytic or acidic vacuole because of the barley aleurain sorting determinants (Aleu-RFP).¹⁸ The localization of PGIP2-GFP was compared to that of Aleu-RFP by confocal microscopy in tobacco protoplasts transiently expressing both fusions. Sixty hours after transformation, PGIP2-GFP labeled the central vacuole as indicated by complete co-localization with the vacuolar marker (Fig. 1A–D). Instead, at the same time point, secGFP-PMEI1 still labeled the cell wall (Fig. 1E–H) and never reached the vacuolar compartment. To summarize PGIP2-GFP secretion pattern, a graphic elaboration of confocal images is reported describing the sorting of PGIP2GFP in tobacco protoplast (Fig. 1I). The protein transits through the endomembrane system (green) and reaches the cell wall which is rapidly regenerating as evidenced by immunostaining with the red monoclonal antibody JIM7 that binds to methylesterified pectins.¹⁹ PGIP2-GFP is then internalized in endosomes, labeled in yellow because of the co-localization with the styryl dye FM4-64, a red marker of the endocytic pathway.Open in a separate windowFigure 1PGIP2-GFP, but not secGFP-PMEI1, is internalized and reaches the vacuole in tobacco leaf protoplasts. (A) Approximately 60 h after transformation, PGIP2-GFP labeled the central vacuole as indicated by co-localization with the vacuole marker Aleu-RFP (B). (C) Merged image of (A and B). (D) Differential interference contrast (DIC) image of (A–C). On the contrary, secGFP-PMEI1 still labeled cell wall (E). (F) No co-localization is present in the vacuole labeled by Aleu-RFP. (G) Merged image of (E and F). (H) DIC image of (E–G). (I) Graphic elaboration of confocal images describing the sorting of PGIP2. The protein is sorted by the endomembrane system (green) to the cell wall (red) that is regenerated by the protoplast. Lacking the specific ligand, it is then internalized in endosome (yellow). Details are reported in the text.In Figure 2 we propose a model of the mechanism of secGFP-PMEI1 and PGIP2-GFP secretion derived from the different lines of evidence previously reported in reference ⁷. SecGFPPMEI1 (Fig. 2-1), but not PGIP2-GFP (Fig. 2-2), carries a GPI-anchor, required for its secretion to the cell wall. When the anchorage of GPI is inhibited by mannosamine (Fig. 2-a) or by the fusion of GFP to the C-terminus of PMEI1 (Fig. 2-b), the two non-anchored proteins accumulate in the Golgi stacks. Evidence of retention in Golgi stacks has already been reported for other two cell wall proteins.^3–5 Unlike secGFP-PMEI1, PGIP2-GFP is not stably accumulated in the cell wall and undergoes endocytic trafficking (Fig. 2-3). PGIP2-GFP internalization, likely due to the absence of PGs, might also be related with its ability to interact with homogalacturonan and oligogalacturonides,²⁰ which have been reported to internalize^21,22 (Fig. 2-4). Since SYP 121, a Qa-SNARE, is involved in the default secretion of secGFP,²³ but not in secretion of PGIP2-GFP and secGFP-PMEI1, trafficking mechanisms underlying secretion into the apoplast are likely different from those underlying the default route (Figs. 2-5). Taken as a whole, evidence suggests the existence of currently undefined signals that control apoplast-targeted secretion.Open in a separate windowFigure 2Schematic illustration for secGFP-PMEI1 and PGIP2-GFP trafficking. See text for details.     

Anticipating future conditions via trajectory sensitivity

Plants are known to be highly responsive to environmental heterogeneity and normally allocate more biomass to organs that grow in richer patches. However, recent evidence demonstrates that plants can discriminately allocate more resources to roots that develop in patches with increasing nutrient levels, even when their other roots develop in richer patches. Responsiveness to the direction and steepness of spatial and temporal trajectories of environmental variables might enable plants to increase their performance by improving their readiness to anticipated resource availabilities in their immediate proximity. Exploring the ecological implications and mechanisms of trajectory-sensitivity in plants is expected to shed new light on the ways plants learn their environment and anticipate its future challenges and opportunities.Key words: Gradient perception, phenotypic plasticity, anticipatory responses, plant behavior, plant learningNatural environments present organisms with myriad challenges of surviving and reproducing under changing conditions.¹ Depending on its extent, predictability and costs, environmental heterogeneity may select for various combinations of genetic differentiation and phenotypic plasticity.^2–6 However, phenotypic plasticity is both limited and costly.⁷ One of the main limitations of phenotypic plasticity is the lag between the perception of the environment and the time the products of the plastic responses are fully operational.⁷ For instance, the developmental time of leaves may significantly limit the adaptive value of their plastic modification due to mismatches between the radiation levels and temperatures prevailing during their development and when mature and fully functional.^8,9 Accordingly, selection is expected to promote responsiveness to cues that bear information regarding the probable future environment.^9,10Indeed, anticipatory responses are highly prevalent, if not universal, amongst living organisms. Whether through intricate cerebral processes, such as in vertebrates, nervous coordination, as in Echinoderms,¹¹ or by relatively rudimentary non-neural processes, such as in plants¹² and bacteria,¹³ accumulating examples suggest that virtually all known life forms are able to not only sense and plastically respond to their immediate environment but also anticipate probable future conditions via environmental correlations.¹⁰Perhaps the best known example of plants'' ability to anticipate future conditions is their responsiveness to spectral red/far-red cues, which is commonly tightly correlated with future probability of light competition.¹⁴ Among others, plants have been shown to respond to cues related to anticipated herbivory^15,16 and nitrogen availability.¹⁷ Imminent stress is commonly anticipated by the perception of a prevailing stress. For example, adaptation to anticipated severe stress was demonstrated to be inducted by early priming by sub-acute drought,¹⁸ root competition¹⁹ and salinity.²⁰Future conditions can also be anticipated by gradient perception: because resource and stress levels are often changing along predictable spatial and temporal trajectories, spatio-temporal dynamics of environmental variables might convey information regarding anticipated growth conditions (Fig. 1). For example, the order of changes in day length, rather than day length itself, are known to assist plants in differentiating fall from spring and thus avoid blooming in the wrong season.²¹ In addition, responsiveness to environmental gradients as such, i.e., sensitivity to the direction and steepness of environmental trajectories, independently from the stationary levels of the same factors, has been demonstrated in higher organisms, such as the perception of acceleration in contrast to velocity;²² and the dynamics of skin temperature in contrast to stationary skin temperature;²³ where the adaptive value of the second-order derivatives of environmental factors is paramount. Similar perception capabilities have also been demonstrated in rudimentary life forms such as bacteria (reviewed in refs. ¹³ and ²⁴) and plants.^25,26 Specifically, perception of environmental trajectories might assist organisms to both anticipate future conditions and better utilize the more promising patches in their immediate environment.^27,28Open in a separate windowFigure 1Trajectory sensitivity in plants. The hypothetical curves depict examples of spatio-temporal trajectories of resource availability, which might be utilized by plants to increase foraging efficiency in newly-encountered patches. When young or early-in-the-season (segment 1–2), plants are expected to allocate more resources to roots that experience the most promising (steepest increases or shallowest decreases) resource availabilities (e.g., allocating more resources to organs in INC-1 than INC-2). In addition, plants are predicted to avoid allocation to roots experiencing decreasing trajectories (DEC, segment 1–2); although temporarily more abundant with resources, such DEC patches are expected to become poorer than alternative patches in the longer run (segment 2–3).²⁹ However, responsiveness to environmental trajectories is only predicted where the expected period of resource uptake is relatively long, e.g., when plants are still active in segment 2–3, a stipulation which might not be fulfilled in e.g., short-living annuals with life span shorter than segment 1–2.In a recent study, Pisum plants have been demonstrated to be sensitive to temporal changes in nutrient availabilities. Specifically, plants allocated greater biomass to roots growing under dynamically-improving nutrient levels than to roots that grew under continuously higher, yet stationary or deteriorating, nutrient availabilities.²⁹ Allocation to roots in poorer patches might seem maladaptive if only stationary nutrient levels are accounted for, and indeed-almost invariably, plants are known to allocate more resources to organs that experience higher (non-toxic) resource levels (reviewed in ref. ³³). Accordingly, the new findings suggest that rather than merely responding to the prevailing nutrient availabilities, root growth and allocation are also responsive to trajectories of nutrient availabilities (Fig. 1).¹⁰Although Shemesh et al.²⁹ demonstrated trajectory-sensitivity of individual roots to temporal gradient of nutrient availabilities, it is likely that this sensitivity helps plants sense spatial gradients, whereby root tips perceive changes in growth conditions as they move through space.³⁴ Interestingly, because the trajectory-sensitivity was observed when whole roots were subjected to changing nutrient levels, it is likely that trajectory sensitivity in roots is based on the integration of sensory inputs perceived by yet-to-be-determined parts of the root over time, i.e., temporal sensitivity/memory (e.g. reviewed in ref. ³⁵), rather than on the integration of sensory inputs at different locations on the same individual roots (i.e., spatial sensitivity).Besides the direction of change, it is hypothesized that plants are also sensitive to the steepness of environmental trajectories (Fig. 1). This might be especially crucial in short-living annuals, which are expected to only be responsive to trajectories steep enough to be indicative of changes in growth conditions before the expected termination of the growth season (Fig. 1).Studying responsiveness to environmental variability is pivotal for understanding the ecology and evolution of any living organism. However, until recently most attention has been given to the study of responses to stationary spatial and temporal heterogeneities in growth conditions. Exploring the ecological implications and mechanisms of trajectory sensitivity in plants is expected to shed new light on the ways plants learn their immediate environment and anticipate its future challenges and opportunities.     

Leaf carbohydrate metabolism during defense: Intracellular sucrose-cleaving enzymes do not compensate repression of cell wall invertase

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild type (wt) tobacco Nicotiana tabacum L. Samsun NN (SNN) with plants with RNA interference-mediated repression of cwINV (SNN::cwINV) during the interaction with the oomycetic phytopathogen Phytophthora nicotianae. We have previously shown that the transgenic plants developed normally under standard growth conditions, but exhibited weaker defense reactions in infected source leaves and were less tolerant to the pathogen. Here, we show that repression of cwINV was not accompanied by any compensatory activities of intracellular sucrose-cleaving enzymes such as vacuolar and alkaline/neutral invertases or sucrose synthase (SUSY), neither in uninfected controls nor during infection. In wt source leaves vacuolar invertase did not respond to infection, and the activity of alkaline/neutral invertases increased only slightly. SUSY however, was distinctly stimulated, in parallel to enhanced cwINV. In SNN::cwINV SUSY-activation was largely repressed upon infection. SUSY may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions. Its activity, however, seems to be directly affected by cwINV and the related reflux of carbohydrates from the apoplast into the mesophyll cells.Key words: cell wall invertase, apoplastic invertase, alkaline invertase, neutral invertase, sucrose synthase, plant defense, Nicotiana tabacum, Phytophthora nicotianaePlant defense against pathogens is costly in terms of energy and carbohydrates.^1,2 Sucrose (Suc) and its cleavage products glucose and fructose are central molecules for metabolism and sensing in higher plants (reviewed in refs. ³ and ⁴). Rapid mobilization of these carbohydrates seems to be an important factor determining the outcome of plant-pathogen interactions. In particular in source cells reprogramming of the carbon flow from Suc to hexoses may be a crucial process during defense.^1,2There are two alternative routes of sucrolytic carbohydrate mobilization. One route is reversible and involves an uridine 5′-diphosphate (UDP)-dependent cleavage catalyzed by sucrose synthase (SUSY). Its activity is limited by the concentrations of Suc and UDP in the cytosol, as the affinity of the enzyme to its substrate is relatively low (K_m for Suc 40–200 mM). The other route is the irreversible, hydrolytic cleavage by invertases (INVs), which exhibit high affinity to Suc (K_m 7–15 mM).⁵Plants possess three different types of INV isoenzymes, which can be distinguished by their solubility, subcellular localization, pH-optima and isoelectric point. Usually, they are subdivided into cell wall (cwINV), vacuolar (vacINV), and alkaline/neutral (a/nINVs) INVs.cwINV, also referred to as extracellular or apoplastic INV, is characterized by a low pH-optimum (pH 3.5–5.0) and usually ionically bound to the cell wall. It is the key enzyme of the apoplastic phloem unloading pathway and plays a crucial role in the regulation of source/sink relations (reviewed in refs. ^{3, 6–8}). A specific role during plant defense has been suggested, based on observations that cwINV is often induced during various plant-pathogen interactions, and the finding that overexpression of a yeast INV in the apoplast increases plant resistance.^6,8–10 It was shown, that a rapid induction of cwINV is, indeed, one of the early defense-related reactions in resistant tobacco source leaves after infection with Phytophthora nicotianae (P. nicotianae).¹¹ Finally, the whole infection area in wt leaves was covered with hypersensitive lesions, indicating that all cells had undergone hypersensitive cell death (Fig. 1A).^1,11 When the activity of cwINV was repressed by an RNAi construct, defense-related processes were impaired, and the infection site exhibited only small spots of hypersensitive lesions. Finally, the pathogen was able to sporulate, indicating a reduced resistance of these transgenic plants (Fig. 1A).¹Open in a separate windowFigure 1Defense-induced changes in the activity of intracellular sucrose-cleaving enzymes and their contribution to defense. (A) The repression of cwINV in source leaves of tobacco leads to impaired pathogen resistance and can not be compensated by other sucrose-cleaving enzymes. The intensity of defense reactions is amongst others indicated by the extent of hypersensitive lesions. (B and C) Absolute activity of vacuolar (B) and alkaline/neutral (C) INVs at the infection site (white symbols, control; black symbols, infection site). (D) Increase in SUSY activity at the infection site. All data points taken from noninfected control parts of the plants in each individual experiment and each point along the time scale of an experiment are set as 0%. At least three independent infections are averaged and their means are presented as percentage changes ± SE (circles, SNN; triangles, SNN::cwINV). Insets show the means of the absolute amount of activities (white symbols, control; black symbols, infection site). Material and methods according to Essmann, et al.¹vacINV, also labeled as soluble acidic INV, is characterized by a pH optimum between pH 5.0–5.5. Among others it determines the level of Suc stored in the vacuole and generates hexose-based sugar signals (reviewed in refs. ³ and ¹²). Yet, no specific role of vacINV during pathogen response has been reported. Although vacINV and cwINV are glycoproteins with similar enzymatic and biochemical properties and share a high degree of overall sequence homology and two conserved amino acid motifs,⁴ the activity of vacINV in tobacco source leaves was not changed due to the repression of the cwINV (Fig. 1B).¹ After infection with P. nicotianae the activity of vacINV in wt SNN did not respond under conditions where cwINV was stimulated.¹ There was also no significant change in the transgenic SNN::cwINV (Fig. 1B). This suggests that during biotic stress, there is no crosstalk between the regulation of cwINV and vacINV.a/nINVs exhibit activity maxima between pH 6.5 and 8.0, are not glycosylated and thought to be exclusively localized in the cytosol. But recent reports also point to a subcellular location in mitochondria and chloroplasts.^13,14 Only a few a/nINVs have been cloned and characterized, and not much is known about their physiological functions (reviewed in refs. ^{4, 14} and ¹⁵). Among other things they seem to be involved in osmotic or low-temperature stress response.^14,15 During the interaction between tobacco and P. nicotianae the activity of a/nINVs rose on average 17% in the resistant wt SNN between 1 to 9 hours post infection (Fig. 1C). By contrast, in SNN::cwINV the a/nINVs activities remained unchanged in control leaves and even after infection (Fig. 1C). This suggests that the defense related stimulation in a/nINVs activities is rather a secondary phenomenon, possibly in response to the enhanced cwINV activity and the related carbohydrate availability in the cytosol.SUSY can be found as a soluble enzyme in the cytosol, bound to the inner side of the plasma membrane or the outer membrane of mitochondria, depending on the phosphorylation status. It channels hexoses into polysaccharide biosynthesis (i.e., starch, cellulose and callose) and respiration.^12,16 There is also evidence that SUSY improves the metabolic performance at low internal oxygen levels¹⁷ but little is known about its role during plant defense. Callose formation is presumably one of the strongest sink reactions in plant cells.^1,18 Defense-related SUSY activity may serve to allocate Suc into callose deposition and other carbohydrate-consuming defense reactions. In fact, in the resistant wt the activity of SUSY increased upon interaction with P. nicotianae in a biphasic manner (Fig. 1D). The time course is comparable to that of cwINV activity and correlates with callose deposition and enhanced respiration.^1,11 However, repression of cwINV leads in general to a reduction of SUSY activity in source leaves of tobacco.¹ After infection the activation of SUSY was also significantly impaired (Fig. 1D). At the same time, the early defense-related callose deposition in infected mesophyll cells of SNN::cwINV plants is substantially delayed.¹ It is known that expression of SUSY isoforms is differentially controlled by sugars,¹² and there is evidence that hexoses generated by the defense-induced cwINV activity deliver sugar signals to the infected cells.¹ In this sense, the reduction of defense-related, cwINV-generated sugar signals could be responsible for the repression of SUSY activity in SNN::cwINV plants after infection with P. nicotianae.Only limited hexoses or hexose-based sugar signals could be generated by cytoplasmic Suc cleavage.¹² The reduction of soluble carbohydrates for sugar signaling and also as fuel for metabolic pathways that support defense reactions could be responsible for the impaired resistance in SNN::cwINV plants (Fig. 1A).Obviously, neither intracellular INV isoforms, nor SUSY can compensate for the reduced carbohydrate availability due to cwINV repression during plant defense. The data also suggest that the activity of SUSY is affected by cwINV and related reflux of carbohydrates. It is known that SUSY activity can be controlled, e.g., by sugar-mediated phosphorylation¹² and one may speculate that posttranslational modulation of the protein is affected by the defense-related carbohydrate status of the cell.     

Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants

Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulinregulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.Key words: calcium, calmodulin, cold stress, MAPK, Arabidopsis, protein phosphorylationCalcium, a universal second messenger in eukaryotic cells, mediates changes in external and internal signals leading to the physiological responses.^1–4 Calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinases (CaMKs) are very important players in calcium/calmodulin mediated signaling in mammalian cells.⁵ In plants, Ca²⁺/CaM-dependent protein phosphorylation was observed more than 25 years ago.⁶ Several calmodulin-regulated protein kinases have been identified and characterized.^7,8 For example, plants have a unique chimeric Ca²⁺/CaM-dependent protein kinase (CCaMK), which exhibits Ca²⁺-dependent autophosphorylation and Ca²⁺/CaM-dependent substrate phosphorylation.⁹ CCaMK is required for bacterial and fungal symbioses in plants.^10–12 Recently, we characterized a novel plant-specific calcium/CaM-regulated receptor-like kinase, CRLK1.¹³ Ca²⁺/CaM binds to CRLK1 and stimulates its kinase activity. Functional studies with CRLK1 indicate that CRLK1 acts as a positive regulator in plant response to chilling and freezing temperatures. To further define the CRLK1-mediated signal pathway, we isolated CRLK1 interacting proteins by co-immunoprecipitation using an anti-CRLK1 antibody. Since cold increases the amount of CRLK1 protein, wildtype plants (WT) were treated at 4°C for 1 hr before co-immunoprecipitation. The resulting CRLK1 immunocomplex was separated by SDS-PAGE. We observed several bands of different sizes only in the wild-type but not in the crlk1 knockout mutant plants (Fig. 1A). Furthermore, the intensity of these bands increased upon cold treatment, suggesting that they are the putative partners or associated proteins of the CRLK1 immunocomplex.Open in a separate windowFigure 1CRLK1 Interacts with MEKK1. (A) One-dimension SDS-PAGE of anti-CRLK1 immunocomplexes from 3-week-old WT or crlk1 plants with or without cold treatment. One mg of total protein was used for immunoprecipitation. (B) A list of putative CRLK1-interacting proteins determined by MALDI-TOF-MS analysis. (C) CRLK1 interacts with MEKK1 as shown by GST pull-down assay. (D) BiFC analysis show that CR LK associates with MEKK1 in vivo. Upper row shows that CRLK and MEKK1 associate both on cell membrane and in endosomes. The middle and last rows are controls. Bar = 10 µm.To determine the identities of these proteins, mass spectrometric analysis was performed with the total immunocomplex.¹⁴ In addition to CRLK1, there were 12 other proteins which matched the Arabidopsis database. Several of them appeared in the pull-down complex from WT, but not from crlk1 mutants. These putative interacting proteins included MEKK1, another unknown protein kinase, a type 2C phosphatase and CaM (Fig. 1B). MEKK1 is one of the 60 putative MAPKKKs in the Arabidopsis genome, and sits on the top of mitogen-activated protein kinase (MAPK) cascade. The MAPK signaling consists of a cascade of three consecutively acting protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and a MAP kinase (MAPK). Plants possess multiple MAPKKKs, MAPKKs and MAPKs, which respond to different upstream signals and activate distinct downstream pathways.^15–17 The specific MAPK module responding to lower temperature has been determined in Arabidopsis.^18,19 MEKK1, a member of MAPKKKs, specifically interacts and phosphorylates MKK2 and regulates COR genes expression in response to cold stress.¹⁹ MEKK1 has been shown to play a role in mediating reactive oxygen species homeostasis.^20,21 Therefore we selected MEKK1 from the putative CRLK1 partners for further studies.