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.     

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

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

Mannan synthase activity in the CSLD family

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

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.     

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

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

Protein Misfolding and the Serpinopathies

The serpins are the largest superfamily of protease inhibitors. They are found in almost all branches of life including viruses, prokaryotes and eukaryotes. They inhibit their target protease by a unique mechanism that involves a large conformational transition and the translocation of the enzyme from the upper to the lower pole of the protein. This complex mechanism, and the involvement of serpins in important biological regulatory processes, makes them prone to mutation-related diseases. For example the polymerization of mutant α₁-antitrypsin leads to the accumulation of ordered polymers within the endoplasmic reticulum of hepatocytes in association with cirrhosis. An identical process in the neuron specific serpin, neuroserpin, results in the accumulation of polymers in neurons and the dementia FENIB. In both cases there is a clear correlation between the molecular instability, the rate of polymer formation and the severity of disease. A similar process underlies the hepatic retention and plasma deficiency of antithrombin, C1 inhibitor, α₁-antichymotrypsin and heparin co-factor II. The common mechanism of polymerization has allowed us to group these conditions together as a novel class of disease, the serpinopathies.Key Words: serpins, α1-antitrypsin, neuroserpin, polymerization, dementia, conformational disease, serpinopathiesSerpins (or serine protease inhibitors) are the largest family of protease inhibitors. They have been found in all major branches of life including viruses, prokaryotes and eukaryotes.^1–3 Despite their name there is increasing evidence that serpins can also inhibit other classes of proteases as demonstrated by the viral serpin CrmA and recently by a plant serpin, serpin1.^4,5 They can even play a non-inhibitory role in events as diverse as blood pressure regulation (angiotensinogen), chromatin condensation (MENT), tumor progression (maspin), protein folding (hsp47) and hormone transport (cortisol and thyroxine binding globulin).⁶One of the most important roles of serpins is the regulation of enzymes involved in proteolytic cascades. Among these serpins are α₁-antitrypsin, α₁-antichymotrypsin, C1 inhibitor, antithrombin and plasminogen activator inhibitor-1, which play an important role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways, respectively.^1,3 The serpin superfamily is characterised by more than 30% homology with the archetypal serpin α₁-antitrypsin and conservation of tertiary structure.^7,8 Serpins adopt a metastable conformation composed in most cases of 9 α-helices, three β-sheet (A to C) and an exposed mobile reactive centre loop (RCL). This flexible RCL typically contains 20 residues that act as a pseudo substrate for the target protease (Fig. 1A).^9–15 After formation of a Michaelis complex^16,17 the enzyme cleaves the P1-P1′ bond of the serpin, releasing the P1'' residue and forming an ester bond between the protease and the serpin.^18,19 This is then followed by a dramatic conformational transition from a stressed to relaxed conformation with the enzyme being pulled from the upper to the lower pole of the serpin and the insertion of the reactive loop as an extra strand in β-sheet A.^20–25 As a consequence of this conformational change the thermal stability of the serpin is greatly enhanced. Whereas a typical serpin in its native state exhibits a midpoint of thermal denaturation of around 50–60°C, a cleaved serpin with its RCL fully incorporated into β-sheet A denatures at temperatures >120°C.^9,26,27 Another consequence is the inactivation of the enzyme, stabilised at the acyl-intermediate and unable to proceed further to deacylation of the complex.^24,28 This serpin-protease complex then binds to members of the lipoprotein receptor family and is cleared from the circulation.^29–31Open in a separate windowFigure 1Inhibition of neutrophil elastase by α₁-antitrypsin and the structural basis of polymerization. (A) After docking (left) the neutrophil elastase (grey) is inactivated by movement from the upper to the lower pole of the protein (right). This is associated with the insertion of the RCL (red) as an extra strand into β-sheet A (green). (B) The structure of α₁-antitrypsin is centred on β-sheet A (green) and the mobile reactive centre loop (red). Polymer formation results from the Z variant of α₁-antitrypsin (Glu342Lys at P17; indicated by arrow) or mutations in the shutter domain (blue circle) that open β-sheet A to favour partial loop insertion and the formation of an unstable intermediate (M*). The patent β-sheet A then accepts the loop of another molecule to form a dimer (D), which then extends into polymers (P). The individual molecules of α₁-antitrypsin within the polymer, although identical, are coloured red, yellow and blue for clarity. Figure reproduced with permission from Lomas et al.⁹⁷Despite the evolutionary advantage conferred upon serpins by the remarkable mobility of the native state, their complexity is also their weak point.^19,32 Mutations affecting the serpins can lead to a variety of diseases, resulting from either a gain or loss of function.^6,19 For example mutations can cause aberrant conformational transitions that result in the retention of the serpin within the cell of synthesis. This will lead to either protein overload and death of the cell in which the serpin is synthesised, or disease as a consequence of the resulting plasma deficiency. Such a mechanism underlies diseases as diverse as cirrhosis, thrombosis, angio-oedema, emphysema and dementia. We review here the common mechanism underlying these diseases that we have grouped together as the serpinopathies.^33–35 The aggregation and accumulation of conformationally destabilized proteins is an important feature of many neurodegenerative diseases, including Alzheimer''s and Parkinson''s disease and the spongiform encephalopathies. Indeed we have used the serpinopathies as a paradigm for these other ‘conformational diseases’.³⁶     

Reactive oxygen species in aerobic methane formation from vegetation

The first report of aerobic methane emissions from vegetation by an unknown mechanism¹ suggested that this potential new source may make a significant contribution to global methane emissions. We recently investigated possible mechanisms and reported^2,3 experiments in which UV-irradiation caused methane emissions from pectin, a major plant cell wall polysaccharide. Our findings also suggest that UV-generated reactive oxygen species (ROS) release methane from pectin. This has implications for all other, UV-independent processes which may generate ROS in or close to the plant cell wall and suggests a need to evaluate additional systems for ROS-generated methane emissions in leaves.Key words: methane, hydroxyl radicals, reactive oxygen species, UV, methyl esters, pectinUntil recently, the global methane budget was thought to be well understood, the only natural process for methane generation being an anaerobic microbial mechanism.⁴ However, observations by Keppler et al.¹ of aerobic methane emissions from vegetation caused controversy and called for a re-assessment of the natural sources of methane. While no mechanism was originally suggested, a putative source, the methyl ester groups of pectin, was proposed based on carbon isotope analyses.¹ We tested this hypothesis directly and reported that UV light could drive methane emissions from pectin in vitro under aerobic conditions.² While UV light was necessary for generation of methane from pectin, it is not tenable that UV was directly attacking pectic methyl ester groups since these do not absorb UV of the wavelengths used (280–400 nm). Instead, we proposed that the energy from the UV light was being absorbed by compounds such as phenolics, and that a reactive intermediary would be formed in the process. Importantly, our process had to be non-enzymic since no enzymes were present in either experimental system.^1,2 Following this hypothesis, we tested the effect of reactive oxygen species (ROS) on pectin in vitro and discovered that certain ROS cause production of methane: hydroxyl radicals (^•OH) and singlet oxygen were effective, but hydrogen peroxide and superoxide were not.³ Also, the addition of ROS-specific scavengers to pectin sheets stopped or severely reduced UV-induced methane emissions from pectin, suggesting that ROS are the intermediary in the mechanism of aerobic methane formation from pectin (Fig. 1). De-esterified pectin was produced by saponification and emitted only trace amounts of methane upon UV-irradiation, clearly establishing ester groups as the source of methane^2,3 and confirming findings of other research groups.^5,6 However, we also found that acetyl ester groups may contribute to methane emissions from pectin and should therefore be considered in future experiments attempting to identify methane sources. Interestingly, we also observed, for the first time, ethylene, ethane and CO₂ emissions from pectin upon UV-irradiation,² which corroborates the ROS hypothesis since ROS attack of methyl esters is likely to form methyl radicals, which can then either form methane or dimerise to form ethylene or ethane.Open in a separate windowFigure 1Proposed pathway for ^•OH-driven methane generation from pectin upon UV irradiation. The compound illustrated here, l-tryptophan, is merely an example of a possible photosensitiser. Hydroxyl radicals (^•OH) are shown to attack a methyl galacturonate residue of the homogalacturonan component of the pectin molecule since this is likely to be the most abundant source of methane, but the methyl esters found in xylogalacturonan domains and the acetyl esters found in homogalacturonan and rhamnogalacturonan domains are also possible methane sources. Note that only ∼70% of all galacturonic acid residues of the pectin backbone are methyl-esterified. Inset photograph shows experimental set-up during UV-irradiation of pectin.ROS are produced and destroyed constantly throughout the lifetime of plants. The generation of ROS in vivo can generally be linked to two sources: (i) a response to an external stimulus which may be perceived as a threat or (ii) a signaling process in the cell which may happen during growth, hormone action or programmed cell death.⁷ Our experiments showed that ROS could lead to methane formation from methyl ester groups; however, the origin of the ROS may not be important, only their nature. Indeed, hydrogen peroxide and superoxide, widely reported to be formed during an oxidative burst following a biotic stress,⁸ did not generate methane from pectin in vitro, and are therefore unlikely to do so in vivo. Only the hydroxyl radical (^•OH) and singlet oxygen generation led to methane formation, and therefore any process which generates them could also trigger UV-independent methane production. Abiotic stresses, such as drought, heat or salinity, which have been shown to lead to the production of ^•OH in vegetation,⁹ could therefore be processes leading to aerobic methane formation, as could exposure to elevated ozone concentrations.¹⁰ Indeed, physical injury (by cutting) of plant material has recently been demonstrated to cause methane emissions.¹¹The origin of the ROS may not be important, as long as their generation is in or close to the pectin of the plant cell wall, since ^•OH cannot travel far within a cell. Indeed, it is estimated that ^•OH typically reacts with organic matter within ∼1 nm of the site of radical production.¹² Processes such as growth^13,14 and calcium signaling,¹⁵ which both involve ROS production as an intermediary in the mechanism but are not necessarily due to external stress, may therefore have the potential to generate methane aerobically. Any process involved in the complicated pathways of ROS-regulation, for which 152 genes are responsible in Arabidopsis thaliana,¹⁶ could be involved in methane emission if the ROS generation is localised close to pectin or other potential substrates.In addition, hydrogen peroxide, which is generated in the cell walls of healthy plants,¹⁷ can be converted in the cell wall into ^•OH by processes such as the Fenton reaction,^18,19 especially in the presence of apoplastic ascorbate.^20,21 A complete analysis of the potential for ^•OH and singlet oxygen to be present in the plant cell wall is therefore necessary for a proper understanding of the different mechanisms that may drive aerobic methane generation. Further experiments into the effects of abiotic stresses other than UV on aerobic methane production from different types of vegetation are necessary in order that future in-vitro studies under simulated natural conditions can be carried out correctly. This type of study, in conjunction with direct in-vivo field studies and satellite observations, are essential to allow global estimates to be made accurately in the future and help us understand the significance of ROS-driven methane emission.