Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 32: 2905–2919 10.1038/emboj.2013.199; published online September032013Some B cells of the adaptive immune system secrete polyreactive immunoglobulin G (IgG) in the absence of immunization or infection. Owing to its limited affinity and specificity, this natural IgG is thought to play a modest protective role. In this issue, a report reveals that natural IgG binds to microbes following their opsonization by ficolin and mannan-binding lectin (MBL), two carbohydrate receptors of the innate immune system. The interaction of natural IgG with ficolins and MBL protects against pathogenic bacteria via a complement-independent mechanism that involves IgG receptor FcγRI expressing macrophages. Thus, natural IgG enhances immunity by adopting a defensive strategy that crossovers the conventional boundaries between innate and adaptive microbial recognition systems.The adaptive immune system generates protective somatically recombined antibodies through a T cell-dependent (TD) pathway that involves follicular B cells. After recognizing antigen through the B-cell receptor (BCR), follicular B cells establish a cognate interaction with CD4⁺ T follicular helper (T_FH) cells and thereafter either rapidly differentiate into short-lived IgM-secreting plasmablasts or enter the germinal centre (GC) of lymphoid follicles to complete class switch recombination (CSR) and somatic hypermutation (SHM) (Victora and Nussenzweig, 2012). CSR from IgM to IgG, IgA and IgE generates antibodies with novel effector functions, whereas SHM provides the structural correlate for the induction of affinity maturation (Victora and Nussenzweig, 2012). Eventually, this canonical TD pathway generates long-lived bone marrow plasma cells and circulating memory B cells that produce protective class-switched antibodies capable to recognize specific antigens with high affinity (Victora and Nussenzweig, 2012).In addition to post-immune monoreactive antibodies, B cells produce pre-immune polyreactive antibodies in the absence of conventional antigenic stimulation (Ehrenstein and Notley, 2010). These natural antibodies form a vast and stable repertoire that recognizes both non-protein and protein antigens with low affinity (Ehrenstein and Notley, 2010). Natural antibodies usually emerge from a T cell-independent (TI) pathway that involves innate-like B-1 and marginal zone (MZ) B cells. These are extrafollicular B-cell subsets that rapidly differentiate into short-lived antibody-secreting plasmablasts after detecting highly conserved microbial and autologus antigens through polyreactive BCRs and nonspecific germline-encoded pattern recognition receptors (Pone et al, 2012; Cerutti et al, 2013).The most studied natural antibody is IgM, a pentameric complement-activating molecule with high avidity but low affinity for antigen (Ehrenstein and Notley, 2010). In addition to promoting the initial clearance of intruding microbes, natural IgM regulates tissue homeostasis, immunological tolerance and tumour surveillance (Ochsenbein et al, 1999; Zhou et al, 2007; Ehrenstein and Notley, 2010). Besides secreting IgM, B-1 and MZ B cells produce IgG and IgA after receiving CSR-inducing signals from dendritic cells (DCs), macrophages and neutrophils of the innate immune system (Cohen and Norins, 1966; Cerutti et al, 2013). In humans, certain natural IgG and IgA are moderately mutated and show some specificity, which may reflect the ability of human MZ B cells to undergo SHM (Cerutti et al, 2013). Yet, natural IgG and IgA are generally perceived as functionally quiescent.In this issue, Panda et al show that natural IgG bound to a broad spectrum of bacteria with high affinity by cooperating with ficolin and MBL (Panda et al, 2013), two ancestral soluble lectins of the innate immune system (Holmskov et al, 2003). This binding involved some degree of specificity, because it required the presence of ficolin or MBL on the microbial surface as well as lower pH and decreased calcium concentration in the extracellular environment as a result of infection or inflammation (see Figure 1).Open in a separate windowFigure 1Ficolins and MBL are produced by hepatocytes and various cells of the innate immune system and opsonize bacteria after recognizing conserved carbohydrates. Low pH and calcium concentrations present under infection-inflammation conditions promote the interaction of ficolin or MBL with natural IgG on the surface of bacteria. The resulting immunocomplex is efficiently phagocytosed by macrophages through FcγR1 independently of the complement protein C3, leading to the clearance of bacteria.Ficolins and MBL are soluble pattern recognition receptors that opsonize microbes after binding to glycoconjugates through distinct carbohydrate recognition domain (CRD) structures (Holmskov et al, 2003). While ficolins use a fibrinogen domain, MBL and other members of the collectin family use a C-type lectin domain attached to a collagen-like region (Holmskov et al, 2003). Similar to pentraxins, ficolins and MBL are released by innate effector cells and hepatocytes, and thus may have served as ancestral antibody-like molecules prior to the inception of the adaptive immune system (Holmskov et al, 2003; Bottazzi et al, 2010). Of note, MBL and the MBL-like complement protein C1q are recruited by natural IgM to mediate complement-dependent clearance of autologous apoptotic cells and microbes (Holmskov et al, 2003; Ehrenstein and Notley, 2010). Panda et al found that a similar lectin-dependent co-optation strategy enhances the protective properties of natural IgG (Panda et al, 2013).By using bacteria and the bacterial glycan N-acetylglicosamine, Panda et al show that natural IgG isolated from human serum or T cell-deficient mice interacted with the fibrinogen domain of microbe-associated ficolins (Panda et al, 2013). The resulting immunocomplex was phagocytosed by macrophages via the IgG receptor FcγRI in a complement-independent manner (Panda et al, 2013). The additional involvement of MBL was demonstrated by experiments showing that natural IgG retained some bacteria-binding activity in the absence of ficolins (Panda et al, 2013).Surface plasmon resonance provided some clues regarding the molecular requirements of the ficolin–IgG interaction (Panda et al, 2013), but the conformational changes required by ficolin to interact with natural IgG remain to be addressed. In particular, it is unclear what segment of the effector Fc domain of natural IgG binds to ficolins and whether Fc-associated glycans are involved in this binding. Specific glycans have been recently shown to mitigate the inflammatory properties of IgG emerging from TI responses (Hess et al, 2013) and this process could implicate ficolins and MBL. Moreover, it would be important to elucidate whether and how the antigen-binding Fab portion of natural IgG regulates its interaction with ficolins and MBL.The in vivo protective role of natural IgG was elegantly demonstrated by showing that reconstitution of IgG-deficient mice lacking the CSR-enzyme activation-induced cytidine deaminase with natural IgG from T cell-insufficient animals enhanced resistance to pathogenic Pseudomonas aeruginosa (Panda et al, 2013). This protective effect was associated with reduced production of proinflammatory cytokines, occurred independently of the complement protein C3 and was impaired by peptides capable to inhibit the binding of natural IgG to ficolin (Panda et al, 2013). Additional in vivo studies will be needed to determine whether natural IgG exerts protective activity in mice lacking ficolin, MBL or FcγRI, and to ascertain whether these molecules also enhance the protective properties of canonical or natural IgG and IgA released by bone marrow plasma cells and mucosal plasma cells, respectively.In conclusion, the findings by Panda et al show that natural IgG adopts ‘crossover'' defensive strategies that blur the conventional boundaries between the innate and adaptive immune systems. The sophisticated integration of somatically recombined and germline-encoded antigen recognition systems described in this new study shall stimulate immunologists to further explore the often underestimated protective virtues of our vast natural antibody repertoire. This effort may lead to the development of novel therapies against infections.     

Amphisomes: out of the autophagosome shadow?

EMBO J (2013) 32: 3130–3144 doi: 10.1038/emboj.2013.233; published online November012013Amphisomes are intermediate organelles, formed during autophagy through the fusion between autophagosomes and endosomes. Complex multivesicular vacuoles that resemble amphisomes have been observed in various cell types, but whether they have cellular roles other than being a precursor structure is still enigmatic. While autophagy-related (ATG) proteins interact with the endocytic pathways in other processes different from autophagy, Patel and colleagues now report that these factors come together to generate amphisome-like compartments that regulate mucin secretion in goblet cells.ATG and endosomal proteins have been linked to secretion, and the specific loss of them impairs the function of different secretory cell types (Jung et al, 2008; DeSelm et al, 2011; Ushio et al, 2011; Sasidharan et al, 2012). ATG proteins have also been shown to interact with the endocytic pathway in few situations that do not involve autophagy. For example in phagocytic cells, the surface of bacteria-containing phagosomes acquires LC3/Atg8 through the concerted action of a subpopulation of ATG proteins. This process, which has been termed LC3-associated phagocytosis (LAP), promotes the fusion of phagosomes with lysosomes (Sanjuan et al, 2007). Something similar occurs during entotic cell death, an engulfment programme leading to the elimination of cells into lysosomes. The entotic vacuole membranes surrounding the internalized cells also recruit LC3 through a mechanism that depends on several ATG proteins, but not on autophagosome formation (Florey et al, 2011).In their work aimed to understand the function of ATG proteins in goblet cells, Patel et al (2013) show that the autophagy and endocytic machinery converge at the amphisomes to promote the secretion of mucins. In the gastrointestinal tract, secretory cells have a crucial role in providing the mucus barrier that protects against intestinal pathogens. Mucins, the main components of the mucus, are produced in goblet cells where large polymers of these highly glycosylated proteins are packed into secretory granules that accumulate at the apical surface. The release of these mucin granules relies on a series of cellular events that are tightly coordinated. Patel et al (2013) show that knockout mice lacking ATG5 in the intestinal epithelium, that is, Atg5VC mice, exhibit both a dramatic accumulation of mucin granules in goblet cells and a diminished mucus secretion. Taking advantage of a newly developed in vitro system to culture and differentiate intestinal epithelial stem cells into secretory goblet cells, the authors also demonstrate that the ablation of other ATG proteins causes the same phenotype showing that the autophagy machinery is required for mucin secretion in these specialized cells (Patel et al, 2013). Interestingly, ATG proteins affect the functionality of another gastrointestinal secretory lineage, the Paneth cells. Paneth cells homozygous for the atg16L1 risk allele, associated with Crohn disease, produce less secretory granules than in controls (Cadwell et al, 2008). This suggests that although ATG proteins regulate secretion in the two most abundant secretory lineages in the intestinal tract, two different mechanisms are probably involved.A microarray analysis of mRNA from Atg5VC mouse colonic epithelial cells revealed a possible alteration in the endocytic pathway. Indeed, blocking endocytosis also provoked an accumulation of mucin granules. While LC3B has been previously found on the surface of secretory granules (Ushio et al, 2011; Ishibashi et al, 2012), immuno-electron microscopy of wild-type mouse intestinal tissue revealed a distribution of LC3B not on mucin granules, but on multivesicular vacuoles positive for several endosomal proteins (Patel et al, 2013). Because of the morphological and molecular characteristics of these compartments, it appears that the ATG proteins together with the endocytic pathway regulate secretion in goblet cells by converging in what could be a new amphisome-like organelle (Figure 1).Open in a separate windowFigure 1Schematic representation for the regulated secretion of mucin granules by amphisome-like structures in goblet cells. ROS generated by NADPH oxidases promote the fusion of LC3-positive vesicles with endosomes marked by Rab5 and containing the NADPH oxidase subunit p22phox. The resulting amphisomes-like organelles are decorated with LC3, endosomal proteins (Rab5, Rab7 and EEA1) and p22phox and localize near the mucin granules. The formation of these copartments probably prolong and/or enhance the production of ROS by the NADPH oxidase, which in turn increases the levels of cytoplasmic calcium through an unknown mechanism leading to the release of the mucin granules.NADPH oxidases are known to be present in endosomes, and NADPH oxidase-generated reactive oxygen species (ROS) are necessary for LC3 recruitment to phagosomes.(Huang et al, 2009). Patel et al (2013) thus explored whether these enzymes played a role in mucin granule secretion in goblet cells. Indeed, expression of a mutant form of p22phox, a transmembrane subunit of several NADPH oxidase complexes, altered the exocytosis of these carriers. Moreover, p22phox was found to localize to Rab5-positive endosomes and also with the observed amphisome-like structures (Figure 1). Because a mutant form of p22phox also caused a misslocalization of both LC3 and the early-endosomal marker protein EEA1, the obvious conclusion was that ROS production by endosomes is necessary to trigger the formation of the amphisome-like organelles via the acquisition of the ATG machinery (Figure 1). Interestingly, addition of H₂O₂ that mimics ROS generation was able to induce mucin granule exocytosis in the p22phox mutant cells, showing that ROS was also required to regulate secretion in goblet cells (Patel et al, 2013). Furthermore, H₂O₂ bypassed as well the mucin granule secretion defect in autophagy and endocytosis-deficient goblet cells through an increase of cytosolic calcium levels (Patel et al, 2013). This, together with the observation that the loss of ATG5 and the block of the endocytic pathway impair the production of ROS has led Patel et al (2013) to propose that amphisome-like organelles are a signalling platform, where NADPH oxidase-driven ROS production promotes the release of the mucin granules.Amphisomes have been characterized and defined as autophagic vacuoles formed upon fusion between autophagosomes and endosomes. Given that ATG and endosomal proteins converge in multivesicular and/or vacuolar compartments resembling amphisomes in cellular processes independent of autophagy, one could consider to use the term amphisomes to describe a more heterogenous and ampler population of unnamed compartments where part of the autophagy and endosomal machineries co-localize. Based on this consideration, the study by Patel et al (2013) has identified an amphisome-like structure where molecular events interconnect to trigger granule secretion. While their work adds to the still limited number of non-degradative roles of the autophagic pathway, which include unconventional secretion (Subramani and Malhotra, 2013), it is one of the first reports highlighting that amphisomes (or any autophagosomal intermediate structure) could be more than just a transport intermediate, and at least in goblet cells, they could act as a platform where signals integrating some aspects of the cell physiology are elicited.Though it remains to be establish whether the organelles described by Patel et al (2013) are indeed amphisomes, especially as they are formed by fusion of endosomes with LC3-positive single-membrane vesicles rather than LC3-positive double-membrane autophagosomes, their study raises some intriguing questions. Are these compartments persistent or will they eventually fuse with lysosomes? Why has the cell opted to signal from amphisomes and not from endosomes, where the NADPH oxidases are normally present? Maybe the answer to these questions is hidden in the transient life of amphisomes. In the most classical signalling pathways, the transduction cascade amplifies the initial cue but it also turn it off subsequently through negative feedback loops. This permits to precisely modulate the signal output temporally (and locally). The amphisome-like structures observed in goblet cells could also act as the molecular switch for the signal-stimulating mucin granule secretion. The ROS generated initially from endosomes would trigger the recruitment of LC3 through vesicle fusion events, and the production of this second messenger will be prolonged and/or enhanced in the resulting amphisomes-like structure, leading to a stimulation of mucin granule exocytosis (Figure 1). The subsequent fusion of the amphisomes with lysosomes could lead to the termination of the signal. Other scenarios, however, cannot be excluded like, for example, the delivery of a protein enhancing the NADPH oxidase activity to the endosomes by the LC3-positive vesicles.While these are just hypotheses, it is clear that Patel et al (2013) have opened a window on a new and unexplored area of the autophagy field. Future investigations will tell us whether what observed in goblet cells is a unique situation or the intermediate organelles characterizing autophagy can carry out cellular functions different from the one delivering unwanted structures into the lysosome interior for degradation, including to serve as signalling platforms.     

Compact Parkin only: insights into the structure of an autoinhibited ubiquitin ligase

EMBO J 32 15, 2099–2112 doi:10.1038/emboj.2013.125; published online May312013Mutations in Parkin represent ∼50% of disease-causing defects in autosomal recessive-juvenile onset Parkinson''s disease (AR-JP). Recently, there have been four structural reports of autoinhibited forms of this RING-IBR-RING (RBR) ubiquitin ligase (E3) by the Gehring, Komander, Johnston and Shaw groups. The important advances from these studies set the stage for the next steps in understanding the molecular basis for Parkinson''s disease (PD).Regulated protein degradation requires that E3s and their access to substrates be exquisitely controlled. RBR family E3s provide striking examples of this regulation. The complex and compact structures of Parkin (Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) as well as another RBR E3, human homologue of Ariadne (HHARI) (Duda et al, 2013), demonstrate extraordinarily intricate inter-domain arrangements. These autoinhibited structures ensure that their functions are restricted until activated.Until recently, RBR E3s were believed to be a subclass of RING E3s, which allosterically activate E2 conjugated with ubiquitin (E2∼Ub). However, Wenzel et al (2011) determined that they are actually hybrid E3s, containing an E2 binding site in RING1 and a catalytic cysteine residue in the domain designated as RING2. The catalytic cysteine is an acceptor for an ubiquitin from RING1-bound E2∼Ub forming an intermediate (E3∼Ub) that leads to substrate or autoubiquitination. In this way, RBRs resemble HECT E3s, which also form catalytic intermediates in ubiquitination. There are 13 human RBR family E3s. Besides Parkin, two notable RBRs are HOIL-1 and HOIP, which form part of a complex integral to NF-κB activation (Wenzel and Klevit, 2012).In addition to causal roles in AR-JP, single allele mutations of Parkin are found in some sporadic cases of PD (references in Wauer and Komander, 2013). Mutations in the Parkin-associated kinase PINK1, which is upstream of Parkin, also account for a significant number of AR-JP cases (Hardy et al, 2009; Narendra et al, 2012; Lazarou et al, 2013). A number of diverse Parkin substrates have been postulated to be associated with PD. There is substantial evidence that one role for Parkin is at mitochondria. Once activated and recruited to damaged/depolarized mitochondria by PINK1, it ubiquitinates exposed mitochondrial proteins leading to both proteasomal degradation and mitophagy (Narendra et al, 2012; Sarraf et al, 2013). Parkin has also been implicated in cell surface signalling and as a tumour suppressor (see references in Wauer and Komander, 2013).Parkin encodes five structured domains, beginning with an N-terminal ubiquitin-like domain (UbLD) and followed by four domains that each bind two zinc (Zn) atoms (Figure 1A). The most N-terminal of the Zn-binding domains is RING0. C-terminal to this is the RBR, consisting of RING1, the IBR and RING2. The crystal structures of inactive Parkin from Riley et al (2013), Trempe et al (2013) and Wauer and Komander (2013) show remarkable congruity. Spatially, the IBR is at the complete opposite end of the molecule from RING2, to which it is connected by a partially unstructured ∼37 residue linker. This linker includes a two-turn helix, referred to as the repressor element of Parkin (REP) or tether, which binds and occludes the E2 binding face of RING1. RING1 occupies the central position in these structures, and RING0 separates RING1 from RING2 (Figure 1B and C). The latter contains the residue identified by Wenzel et al (2011), and confirmed by all three groups, to be the catalytic cysteine, C431. A lower resolution structure also includes the UbLD and places this domain adjacent to RING1 (Trempe et al, 2013). A second unstructured linker connects the UbLD and RING0. UbLDs are involved in a number of protein–protein interactions and small angle X-ray scattering confirms that this domain is integral to the core structure of Parkin (Spratt et al, 2013; Trempe et al, 2013). Biophysical characterization of Parkin and HHARI suggests that each is a monomer in solution.Open in a separate windowFigure 1Schematic and spatial representation of Parkin. (A) Primary structure and domain designations of Parkin, including the REP sequence within the otherwise unstructured IBR-RING2 linker. (B) Structural representation of full-length Parkin (PDB 4K95) highlighting the complex domain interactions in the three-dimensional structure, the catalytic C431 residue, and residue W403 within the REP, which plays a role in stabilizing the autoinhibited form of Parkin. (C) A model of Parkin with the E2 UbcH5B/Ube2D2 bound (devised using PDB 4K95 and PDB 4AP4 to mimic the position of an E2 bound to RING1) to illustrate the required displacement of UbLD and REP and the large distance between the E2∼Ub attachment site of the E2 and the catalytic active site of Parkin. Note that in this conformation the catalytic Cys within RING2 (C431) remains buried by RING0.RING1 is the only bona fide RING domain. All NMR and crystal structures of IBR domains from Parkin, HHARI and HOIP (PDB ID: 2CT7) are in good agreement. The Parkin and HHARI RING2s are structurally highly homologous and share a common Zn-coordinating arrangement with IBR domains. In contrast to the IBR and RING2, RING0 has a distinct arrangement of Zn-coordinating residues (Beasley et al, 2007; Duda et al, 2013; Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) (see Figure 1F of Trempe et al (2013) for the various Zn coordination arrangements).All of the Parkin crystal structures represent inactive forms of the E3. This is imposed by the quaternary positioning of the domains, which precludes activity in multiple ways. RING0 plays two obvious roles to maintain Parkin in an inactive state. RING0 shares an interface with RING2 and buries C431, making it unavailable as an ubiquitin acceptor. Moreover, RING0 intervenes between RING1 and RING2, creating an insurmountable separation of >50 Å between the active site Cys of an E2 bound to RING1 and C431 (Figure 1B and C). Thus, RING0 must be displaced for ubiquitin transfer to occur. Accordingly, deletion of RING0 results in a marked increase in Parkin autoubiquitination and in C431 reactivity (Riley et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013). In HHARI, these two inhibitory functions are fulfilled by the C-terminal Ariadne domain, which similarly interposes between RING1 and RING2 (Duda et al, 2013).Additional inhibition is provided by the REP, which binds to RING1 at the canonical RING-E2 binding site and prevents E2 binding. This provides at least a partial explanation for the impaired ability of Parkin to bind E2 when compared to HHARI, which lacks this element (Duda et al, 2013). A disease-associated REP mutant (A398T) at the RING1 interface increases autoubiquitination (Wauer and Komander, 2013). The significance of inhibition by REP-RING1 binding was verified by mutating a critical RING1-interacting REP residue (W403A). This increased autoubiquitination and E2 binding (Trempe et al, 2013). Consistent with the requirement for charging C431 with ubiquitin in mitochondrial translocation (Lazarou et al, 2013), Parkin association with depolarized mitochondria is accelerated with this mutation (Trempe et al, 2013). Interestingly, W403 also interacts with the C-terminal Val of Parkin within RING2, and could therefore potentially further stabilize the autoinhibited form of the protein (Riley et al, 2013), consistent with previous observations (Henn et al, 2005).The quaternary structure of full-length Parkin also suggests that displacement of its N-terminal UbLD must occur for full activation (Trempe et al, 2013). The positioning of the UbLD adjacent to RING1 indicates that it would provide a steric impediment to E2∼Ub binding (Figure 1B and C). Additionally, displacement of the UbLD could be important to relieve interactions with the IBR-RING2 linker, which, as suggested in a previous study (Chaugule et al, 2011), might help to maintain Parkin in an inactive state. Finally, the crystal structure of the full-length Parkin indicates that the UbLD is not available for interactions with other proteins. This would limit Parkin''s range of intermolecular interactions.RBR E3s have at least two domains critical for sequential ubiquitin transfer and full activity, RING1 and RING2. The RING1 of Parkin, as well as all other RBR E3s, is notable in lacking the basic residue in the second Zn coordinating loop (or its equivalent in U-box proteins), which has recently been implicated in RING-mediated transfer of Ub from E2∼Ub (Metzger et al, 2013). This suggests that other factors play compensatory roles in positioning ubiquitin for transfer from E2∼Ub to C431. A non-mutually exclusive possibility is that the lack of this basic residue in RING1 limits unwanted attack on the E2∼Ub linkage, thereby minimizing the unregulated ubiquitination. Turning to RING2, the area surrounding the active site C431 of Parkin is notable in that it includes a sequence recognizable as a catalytic triad, similar to that in deubiquitinating enzymes. The Cys-His-Glu grouping, found in Parkin and other RBR E3s, contributes to in vitro activity (Riley et al, 2013; Wauer and Komander, 2013). Interestingly, however, the Glu was dispensable in a cellular assay (Riley et al, 2013). This triad is conserved in HHARI, where an Asn between the Cys and His residues (found in a number of RBRs but not conserved in Parkin), was found to be important for catalysis (Duda et al, 2013).The advances made in these studies impart significant information about an important and clinically relevant E3. However, Parkin, as well as HHARI, has been captured in their inactive, unmodified forms. One obvious question is how does Parkin transition between inactive and active states. PINK1 is implicated in phosphorylating Parkin on its UbLD and potentially other sites, with evidence that phosphorylation contributes to Parkin activation (Narendra et al, 2012). How phosphorylation could contribute to protein interactions that might facilitate Parkin activation, potentially including Parkin oligomerization (Lazarou et al, 2013), is unknown. Regardless, it is evident that considerable unwinding of its quaternary structure must take place.While there is much work ahead to understand these processes, one important interface that must be disrupted for activation is that between the REP and RING1. It is intriguing to consider that such interruption might be associated with other alterations in the IBR-RING2 linker, potentially facilitating the movement of the UbLD from RING1 and contributing to activation. Related to activation is the all-important question of how Parkin recognizes and targets specific substrates. While the UbLD represents a potential site of interaction, most purported substrates are not known to have UbLD-interaction domains. Although interactions involving the UbLD could occur indirectly, through bridging molecules, there is also evidence that other regions of Parkin, including the RBR region, might recognize substrates either directly or indirectly (Tsai et al, 2003) and that some substrates may be phosphorylated by PINK1 (Narendra et al, 2012). Conformational changes induced by substrate interactions, particularly in the IBR RING2 linker, could, as above, represent an important aspect of activation.There are over 75 missense mutations of Parkin associated with AR-JP, most of these inactivate the protein, but there are also some that are activating (Wauer and Komander, 2013). Activating mutations presumably result in pathology at least partially as a consequence of increased autoubiquitination and degradation (e.g., A398T). The current studies help to provide a classification of missense mutations into those that affect (i) folding or stability, (ii) catalytic mechanism, and (iii) interactions between domains. Interdomain mutations might inactivate or contribute to constitutive activation leading to autoubiquitination and degradation.Finally, we know little about how the autosomal recessive and the much more prevalent sporadic forms of PD overlap in their molecular pathology. However, mitochondrial dysfunction is increasingly a common theme. Thus, with the structure of the inactive protein in hand, there is hope that we can begin to consider ways in which domain interactions might be altered in a controlled manner to activate, but not hyperactivate, this critical E3 and lessen the progression of PD.     

FIBRILLIN4 Is Required for Plastoglobule Development and Stress Resistance in Apple and Arabidopsis

The fibrillins are a large family of chloroplast proteins that have been linked with stress tolerance and disease resistance. FIBRILLIN4 (FIB4) is found associated with the photosystem II light-harvesting complex, thylakoids, and plastoglobules, which are chloroplast compartments rich in lipophilic antioxidants. For this study, FIB4 expression was knocked down in apple (Malus 3 domestica) using RNA interference. Plastoglobule osmiophilicity was decreased in fib4 knockdown (fib4 KD) tree chloroplasts compared with the wild type, while total plastoglobule number was unchanged. Compared with the wild type, net photosynthetic CO₂ fixation in fib4 KD trees was decreased at high light intensity but was increased at low light intensity. Furthermore, fib4 KD trees produced more anthocyanins than the wild type when transferred from low to high light intensity, indicating greater sensitivity to high light stress. Relative to the wild type, fib4 KD apples were more sensitive to methyl viologen and had higher superoxide levels during methyl viologen treatment. Arabidopsis (Arabidopsis thaliana) fib4 mutants and fib4 KD apples were more susceptible than their wild-type counterparts to the bacterial pathogens Pseudomonas syringae pathovar tomato and Erwinia amylovora, respectively, and were more sensitive to ozone-induced tissue damage. Following ozone stress, plastoglobule osmiophilicity decreased in wild-type apple and remained low in fib4 KD trees; total plastoglobule number increased in fib4 KD apples but not in the wild type. These results indicate that FIB4 is required for plastoglobule development and resistance to multiple stresses. This study suggests that FIB4 is involved in regulating plastoglobule content and that defective regulation of plastoglobule content leads to broad stress sensitivity and altered photosynthetic activity.Increased production of reactive oxygen species (ROS) is among the first biochemical responses of plants when challenged by pathogens and harsh environmental conditions (Mehdy, 1994; Lamb and Dixon, 1997; Joo et al., 2005). ROS are implicated in tissue damage during environmental stress and in the promotion of disease development by necrotrophic and hemibiotrophic pathogens (Venisse et al., 2001; Apel and Hirt, 2004; Shetty et al., 2008). For example, ROS production is critical for host colonization and pathogenesis by the bacterium Erwinia amylovora, which causes fire blight disease in rosaceous plants such as apple (Malus 3 domestica) and pear (Pyrus communis; Venisse et al., 2001).The chloroplast is a site of ROS production during biotic and abiotic stress (Joo et al., 2005; Liu et al., 2007). The chloroplast has a battery of enzymes such as superoxide dismutase and ascorbate peroxidase, and antioxidants such as ascorbate, glutathione, and tocopherols, for protection against ROS (Noctor and Foyer, 1998; Asada, 2006). Plastoglobules are lipoprotein bodies attached to the thylakoids (Austin et al., 2006) that store lipids, including antioxidants such as tocopherols, carotenes, and plastoquinones (Steinmüller and Tevini, 1985; Tevini and Steinmüller, 1985). In addition to antioxidants, plastoglobules contain tocopherol cyclase, which is involved in γ-tocopherol synthesis (Austin et al., 2006; Vidi et al., 2006). The antioxidant content of plastoglobules and their apparent involvement in tocopherol biosynthesis imply that they could play a role in plant responses to oxidative stress.Plastoglobules contain fibrillins, which were initially described as protein components of chromoplast fibrils with a molecular mass of approximately 30 kD (Winkenbach et al., 1976; Knoth et al., 1986; Emter et al., 1990; Deruère et al., 1994). Fibrillins are ubiquitous proteins present from cyanobacteria to plants (Laizet et al., 2004). Fibrillins maintain plastoglobule structural integrity (Deruère et al., 1994; Pozueta-Romero et al., 1997; Langenkämper et al., 2001; Vidi et al., 2006; Bréhélin et al., 2007) and stabilize the photosynthetic apparatus during photooxidative stress (Gillet et al., 1998; Yang et al., 2006; Youssef et al., 2010), osmotic stress (Gillet et al., 1998), drought (Pruvot et al., 1996; Rey et al., 2000), and low temperature (Rorat et al., 2001). Fibrillins are involved in abscisic acid-mediated protection from photoinhibition (Yang et al., 2006), and a subfamily of Arabidopsis (Arabidopsis thaliana) fibrillins (FIB1a, -1b, and -2) conditions jasmonate production during low-temperature, photooxidative stress (Youssef et al., 2010). Arabidopsis plants lacking one fibrillin (At4g22240) and tomato (Solanum lycopersicum) plants with suppressed expression of a fibrillin (LeCHRC) are susceptible to Pseudomonas syringae and Botrytis cinerea, respectively (Cooper et al., 2003; Leitner-Dagan et al., 2006), indicating that fibrillins play a role in disease resistance.The Arabidopsis fibrillin encoded by At3g23400 has received various appellations, including FIBRILLIN4 (FIB4; Laizet et al., 2004), Harpin-Binding Protein1 (Song et al., 2002), AtPGL 30.4 (Vidi et al., 2006), and Fibrillin6 (Galetskiy et al., 2008); here, it will be referred to by its earliest published name, FIB4. FIB4 is found associated with the PSII light-harvesting complex (Galetskiy et al., 2008). FIB4 has also been detected in plastoglobules (Vidi et al., 2006; Ytterberg et al., 2006) and thylakoids (Friso et al., 2004; Peltier et al., 2004). However, the specific function of FIB4 is unknown. Several lines of evidence suggest that FIB4 may be involved in plant disease resistance responses: pathogen-associated molecular patterns trigger its phosphorylation (Jones et al., 2006); pathogen-associated molecular patterns stimulate the expression of its ortholog in tobacco (Nicotiana tabacum; Jones et al., 2006; Sanabria and Dubery, 2006); and it can physically interact with the HrpN (harpin) virulence protein of the fire blight pathogen E. amylovora in a yeast two-hybrid assay, suggesting that it could be a receptor or target of HrpN (Song et al., 2002). In addition, it is thought that FIB4 may be involved in the transport of small, hydrophobic molecules because it contains a conserved lipocalin signature (Jones et al., 2006). Here, we report a genetic analysis of FIB4 function in apple and Arabidopsis in terms of its role in plastoglobule development and plant resistance to biotic and abiotic stresses.     

Overexpression of Flavodoxin in Bacteroids Induces Changes in Antioxidant Metabolism Leading to Delayed Senescence and Starch Accumulation in Alfalfa Root Nodules

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.Symbiotic nodules have a limited functional life that varies among different legume species. Nodule senescence is the sequence of structural, molecular, biochemical, and physiological events taking place in the process that a mature and functional nodule undergoes leading to the loss of the nitrogen-fixing activity and culminating in cell death of symbiotic tissue (Swaraj and Bishnoi, 1996; Puppo et al., 2005; Van de Velde et al., 2006).Various models have been proposed to explain the mechanisms that trigger the process of natural or stress-induced nodule senescence. However, it is generally accepted that a senescence-inducing signal from the plant causes a decrease in antioxidant levels and thus an increase in reactive oxygen species (ROS) up to a point of no return. Numerous studies have shown that ROS and antioxidant systems are involved in natural (Lucas et al., 1998; Evans et al., 1999; Hernández-Jiménez et al., 2002; Puppo et al., 2005) as well as induced (Dalton et al., 1993; Becana et al., 2000; Hernández-Jiménez et al., 2002; Matamoros et al., 2003) nodule senescence. Nitrogen fixation is very sensitive to ROS, and nitrogenase activity drastically decreases during nodule senescence (Dalton et al., 1986).Antioxidant systems that protect cells from oxidative damage have been described in symbiotic nodules (Dalton et al., 1986, 1993; Evans et al., 1999; Becana et al., 2000; Matamoros et al., 2003; Puppo et al., 2005). These include the enzymes superoxide dismutase (SOD), catalase, and peroxidase. Another enzymatic system associated with ROS detoxification is the ascorbate-glutathione pathway, which includes ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR; Dalton et al., 1986, 1992; Noctor and Foyer 1998; Becana et al., 2000). Ascorbate and reduced glutathione (GSH) in this pathway can also scavenge superoxide and hydrogen peroxide.During nodule senescence, several ultrastructural alterations in the nodule tissues and cells have been observed (Lucas et al., 1998; Hernández-Jiménez et al., 2002; Puppo et al., 2005, and refs. therein; Van de Velde et al., 2006). Cytosol becomes electron dense, altered vesicles proliferate, and eventually the cytosol undergoes lysis. The number of peroxisomes increases, mitochondria form complex elongated structures, and symbiosomes change in size and shape and fuse during natural and induced senescence of nodules (Hernández-Jiménez et al., 2002). Damage of the symbiosome membrane is also detected (Puppo et al., 2005; Van de Velde et al., 2006).A strategy of delayed nodule senescence could lead to increased nitrogen fixation and legume productivity. Delayed nodule senescence together with enhanced sustainability under field conditions are among the key aims of legume improvement programs (Puppo et al., 2005). An interesting approach proposed to achieve delayed senescence is to induce nodulation in legumes using rhizobial strains with modified redox capacity (Zahran, 2001).The protein flavodoxin contains a FMN group acting as a redox center transferring electrons at low potentials (Pueyo et al., 1991; Pueyo and Gómez-Moreno, 1991). The FMN cofactor of flavodoxin can exist in three different redox states: oxidized, one-electron-reduced semiquinone, and two-electron-reduced hydroquinone. This property confers high versatility to flavodoxins in electron transport systems (Simondsen and Tollin, 1980; McIver et al., 1998). To date, flavodoxin has not been described in plants, as flavodoxin-encoding genes were lost during the transition of algae to plants (Zurbriggen et al., 2007) and, consequently, no homologs have been identified in the sequenced genome of Arabidopsis (Arabidopsis thaliana; Arabidopsis Genome Initiative, 2000). Flavodoxin is present as a constitutive or inducible protein in different microorganisms (Klugkist et al., 1986). In the nitrogen-fixing cyanobacterium Anabaena variabilis PCC 7119, flavodoxin is expressed under conditions of limited iron availability, replacing ferredoxin in the photosynthetic electron transport from PSI to NADP⁺ and in nitrogenase reduction (Sandmann et al., 1990). Reversible electron transfer from flavodoxin to NADP⁺ is catalyzed by ferredoxin NADP⁺ reductase in different pathways of oxidative metabolism (Arakaki et al., 1997). In its reduced state, flavodoxin might be able to react with ROS and revert to its original redox state in the presence of an appropriate electron source. This could probably occur without the associated molecular damage that metallic complexes in catalases or SODs suffer (Keyer et al., 1995). The presence of flavodoxin has not been documented to date in the symbiotic bacterium Sinorhizobium meliloti. In Escherichia coli, however, flavodoxin induction is linked to the oxidative stress-responsive regulon soxRS (Zheng et al., 1999). It has been suggested that flavodoxin and ferredoxin (flavodoxin) NADP⁺ reductase might be induced and have a role in reestablishing the cell redox balance under oxidative stress conditions (Liochev et al., 1994). The properties of flavodoxin suggest that its presence in the cell may have a facilitating effect on ROS detoxification. In fact, an increase in the amount of flavodoxin has been observed in some bacterial species subjected to oxidative stress (Zheng et al., 1999; Yousef et al., 2003; Singh et al., 2004), and transgenic tobacco (Nicotiana tabacum) plants expressing flavodoxin in chloroplasts show enhanced tolerance to a broad range of stresses related to oxidative damage (Tognetti et al., 2006, 2007a, 2007b).In this work, Sinorhizobium meliloti was transformed with the A. variabilis flavodoxin gene and used to nodulate alfalfa (Medicago sativa) plants. The effects of flavodoxin expression on nodulation dynamics, on nodule development and senescence processes, and on nitrogen-fixing activity were analyzed. Mechanistic insights suggesting putative roles for flavodoxin in protection from ROS and the induced delay of nodule senescence are likewise discussed.     

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.The classical heterotrimeric G protein consists of a GDP/GTP-binding Gα subunit with GTPase activity bound to an obligate dimer formed by Gβ and Gγ subunits. In the signaling paradigm largely elucidated from mammalian systems, the plasma membrane-associated heterotrimer contains Gα in its GDP-bound form. Upon receiving a molecular signal, typically transduced by a transmembrane protein (e.g. a G protein-coupled receptor), Gα exchanges GDP for GTP and dissociates from the Gβγ dimer. Both Gα and Gβγ interact with intracellular effectors to initiate downstream signaling cascades. The intrinsic GTPase activity of Gα restores Gα to the GDP-bound form, which binds Gβγ, thereby reconstituting the heterotrimer (McCudden et al., 2005; Oldham and Hamm, 2008).Signal transduction through a heterotrimeric G protein complex is an evolutionarily conserved eukaryotic mechanism common to metazoa and plants, although there are distinct differences in the functional intricacies between the evolutionary branches (Jones et al., 2011a, 2011b; Bradford et al., 2013). The numbers of each subunit encoded within genomes, and therefore the potential for combinatorial complexity within the heterotrimer, is one of the most striking differences between plants and animals. For example, the human genome encodes 23 Gα (encoded by 16 genes), five Gβ, and 12 Gγ subunits (Hurowitz et al., 2000; McCudden et al., 2005; Birnbaumer, 2007). The Arabidopsis (Arabidopsis thaliana) genome, however, only encodes one canonical Gα (GPA1; Ma et al., 1990), one Gβ (AGB1; Weiss et al., 1994), and three Gγ (AGG1, AGG2, and AGG3) subunits (Mason and Botella, 2000, 2001; Chakravorty et al., 2011), while the rice (Oryza sativa) genome encodes one Gα (Ishikawa et al., 1995), one Gβ (Ishikawa et al., 1996), and either four or five Gγ subunits (Kato et al., 2004; Chakravorty et al., 2011; Botella, 2012). As expected, genomes of polyploid plants have more copies due to genome duplication, with the soybean (Glycine max) genome encoding four Gα, four Gβ (Bisht et al., 2011), and 10 Gγ subunits (Choudhury et al., 2011). However, Arabidopsis heterotrimeric G proteins have been implicated in a surprisingly large number of phenotypes, which is seemingly contradictory given the relative scarcity of subunits. Arabidopsis G proteins have been implicated in cell division (Ullah et al., 2001; Chen et al., 2006) and morphological development in various tissues, including hypocotyls (Ullah et al., 2001, 2003), roots (Ullah et al., 2003; Chen et al., 2006; Li et al., 2012), leaves (Lease et al., 2001; Ullah et al., 2001), inflorescences (Ullah et al., 2003), and flowers and siliques (Lease et al., 2001), as well as in pathogen responses (Llorente et al., 2005; Trusov et al., 2006; Cheng et al., 2015), regulation of stomatal movement (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2008) and development (Zhang et al., 2008; Nilson and Assmann, 2010), cell wall composition (Delgado-Cerezo et al., 2012), responses to various light stimuli (Warpeha et al., 2007; Botto et al., 2009), responses to multiple abiotic stimuli (Huang et al., 2006; Pandey et al., 2006; Trusov et al., 2007; Zhang et al., 2008; Colaneri et al., 2014), responses to various hormones during germination (Ullah et al., 2002), and postgermination development (Ullah et al., 2002; Pandey et al., 2006; Trusov et al., 2007). Since the Gγ subunit appeared to be the only subunit that provides diversity in heterotrimer composition in Arabidopsis, it was proposed that all functional specificity in heterotrimeric G protein signaling was provided by the Gγ subunit (Trusov et al., 2007; Chakravorty et al., 2011; Thung et al., 2012, 2013). This allowed for only three heterotrimer combinations to account for the wide range of G protein-associated phenotypes.In addition to the above typical G protein subunits, the plant kingdom contains a conserved protein family of extra-large GTP-binding proteins (XLGs). XLGs differ from typical Gα subunits in that they possess a long N-terminal extension of unknown function, but they are similar in that they all have a typical C-terminal Gα-like region, with five semiconserved G-box (G1–G5) motifs. The XLGs also possess the two sequence features that differentiate heterotrimeric G protein Gα subunits from monomeric G proteins: a helical region between the G1 and G2 motifs and an Asp/Glu-rich loop between the G3 and G4 motifs (Lee and Assmann, 1999; Ding et al., 2008; Heo et al., 2012). The Arabidopsis XLG family comprises XLG1, XLG2, and XLG3, and all three have demonstrated GTP-binding and GTPase activities, although they differ from GPA1 in exhibiting a much slower rate of GTP hydrolysis, with a Ca²⁺ cofactor requirement instead of an Mg²⁺ requirement, as for canonical Gα proteins (Heo et al., 2012). All three Arabidopsis XLGs were observed to be nuclear localized (Ding et al., 2008). Although much less is known about XLGs than canonical Gα subunits, XLG2 positively regulates resistance to the bacterial pathogen Pseudomonas syringae and was immunoprecipitated with AGB1 from tissue infected with P. syringae (Zhu et al., 2009). xlg3 mutants, like agb1 mutants, are impaired in root-waving and root-skewing responses (Pandey et al., 2008). During the preparation of this report, Maruta et al. (2015) further investigated XLG2, particularly focusing on the link between XLG2 and Gβγ in pathogen responses. Based on symptom progression in xlg mutants, they found that XLG2 is a positive regulator of resistance to both bacterial and fungal pathogens, with a minor contribution from XLG3 in resistance to Fusarium oxysporum. XLG2 and XLG3 are also positive regulators of reactive oxygen species (ROS) production in response to pathogen-associated molecular pattern elicitors. The resistance and pathogen-associated molecular pattern-induced ROS phenotypes of the agg1 agg2 and xlg2 xlg3 double mutants were not additive in an agg1 agg2 xlg2 xlg3 quadruple mutant, indicating that these two XLGs and the two Gγ subunits function in the same, rather than parallel, pathways. Unfortunately, the close proximity of XLG2 and AGB1 on chromosome 4 precluded the generation of an agb1 xlg2 double mutant; therefore, direct genetic evidence of XLG2 and AGB1 interaction is still lacking, but physical interactions between XLG2 and the Gβγ dimers were shown by yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescence complementation (BiFC) assays (Maruta et al., 2015). Localization of all three XLGs was also reexamined, indicating that XLGs are capable of localizing to the plasma membrane in addition to the nucleus (Maruta et al., 2015).Interestingly, several other plant G protein-related phenotypes, in addition to pathogen resistance, have been observed only in Gβ and Gγ mutants, with opposite phenotypes observed in Gα (gpa1) mutants. Traditionally, the observation of opposite phenotypes in Gα versus Gβγ mutants in plants and other organisms has mechanistically been attributed to signaling mediated by free Gβγ, which increases in abundance in the absence of Gα. However, an intriguing alternative is that XLG proteins fulfill a Gα-like role in forming heterotrimeric complexes with Gβγ and function in non-GPA1-based G protein signaling processes. If XLGs function like Gα subunits, the corresponding increase in subunit diversity could potentially account for the diversity of G protein phenotypes. In light of this possibility, we assessed the heterotrimerization potential of all possible XLG and Gβγ dimer combinations, XLG localization and its regulation by Gβγ, and the effect of xlg mutation on selected known phenotypes associated with heterotrimeric G proteins. Our results provide compelling evidence for the formation of XLG-Gβγ heterotrimers and reveal that plant G protein signaling is substantially more complex than previously thought.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

The Involvement of Lipid Peroxide-Derived Aldehydes in Aluminum Toxicity of Tobacco Roots

Oxidative injury of the root elongation zone is a primary event in aluminum (Al) toxicity in plants, but the injuring species remain unidentified. We verified the hypothesis that lipid peroxide-derived aldehydes, especially highly electrophilic α,β-unsaturated aldehydes (2-alkenals), participate in Al toxicity. Transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis (Arabidopsis thaliana) 2-alkenal reductase (AER-OE plants), wild-type SR1, and an empty vector-transformed control line (SR-Vec) were exposed to AlCl₃ on their roots. Compared with the two controls, AER-OE plants suffered less retardation of root elongation under AlCl₃ treatment and showed more rapid regrowth of roots upon Al removal. Under AlCl₃ treatment, the roots of AER-OE plants accumulated Al and H₂O₂ to the same levels as did the sensitive controls, while they accumulated lower levels of aldehydes and suffered less cell death than SR1 and SR-Vec roots. In SR1 roots, AlCl₃ treatment markedly increased the contents of the highly reactive 2-alkenals acrolein, 4-hydroxy-(E)-2-hexenal, and 4-hydroxy-(E)-2-nonenal and other aldehydes such as malondialdehyde and formaldehyde. In AER-OE roots, accumulation of these aldehydes was significantly less. Growth of the roots exposed to 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal were retarded more in SR1 than in AER-OE plants. Thus, the lipid peroxide-derived aldehydes, formed downstream of reactive oxygen species, injured root cells directly. Their suppression by AER provides a new defense mechanism against Al toxicity.Aluminum (Al) is the most abundant metal in the earth''s crust and is a major factor limiting plant growth and productivity in acid soils, which cover about 50% of the world''s potentially arable land surface (Kochian, 1995; Kochian et al., 2004). The primary site of Al accumulation and toxicity is the root meristem, and inhibition of root elongation is the most notable symptom of Al toxicity (Delhaize and Ryan, 1995; Yamamoto et al., 2003). Al causes various adverse effects, such as disruption of signal transduction pathways, inhibition of cell division and ion fluxes, disruption of cytoskeletal dynamics, induced generation of reactive oxygen species (ROS), and disturbance of plasma membrane stability and function (Jones and Kochian, 1995; Blancaflor et al., 1998; Yamamoto et al., 2001, 2002; Kochian et al., 2004; Ma et al., 2007). Of all these toxic effects, the generation of ROS is observed rapidly and sustainably in roots after Al exposure. Al-induced generation of ROS has been shown in maize (Zea mays) and Allium cepa roots (Jones et al., 2006; Achary et al., 2008). Tahara et al. (2008) showed that ROS generated to a greater degree in Al-sensitive species than in Al-tolerant species. Yamamoto et al. (2002, 2003) have shown a correlation between ROS level and inhibition of growth capacity in cultured tobacco (Nicotiana tabacum) cells. Furthermore, ROS generation increases with increasing Al concentration (Achary et al., 2008; Xue et al., 2008). Generation of ROS appears to be a cause, rather than a result, of Al-induced cell injury, because high ROS scavenging ability resulted in enhanced Al tolerance (Devi et al., 2003; Ezaki et al., 2008). In addition, overexpression of genes encoding antioxidant enzymes (peroxidase and superoxide dismutase) conferred Al tolerance to the transgenic plants (Ezaki et al., 2000; Basu et al., 2001). Thus, ROS appears to be the primary factors that cause growth inhibition in Al-stressed roots.Downstream of ROS generation, lipid peroxidation is a common symptom of Al toxicity (Yamamoto et al., 2001), and it increases with increasing Al concentration (Achary et al., 2008). From animal cell studies, it is now recognized that the toxicity of lipid peroxide (LOOH) is largely ascribable to LOOH-derived aldehydes. In particular, α,β-unsaturated aldehydes, such as 4-hydroxy-(E)-2-nonenal (HNE) and acrolein, are strong electrophiles and readily modify proteins and nucleic acids (Esterbauer et al., 1991; Taylor et al., 2002; O''Brien et al., 2005; Møller et al., 2007). HNE causes depletion of glutathione, a decrease in protein thiols, disturbance of calcium homeostasis, inhibition of DNA, RNA, and protein synthesis, lactate release, morphological changes of cells, and finally leading to cell death (Esterbauer et al., 1991; Burcham, 1998). Increase of HNE has been observed in a wide range of human diseases, including Alzheimer''s disease, Parkinson''s disease, and mitochondrial complex 1 deficiency (Poli and Schaur, 2000).In plants, too, a close correlation between the level of LOOH-derived aldehydes (determined as thiobarbituric acid-reactive substances [TBARS]) and cellular damage has been shown under environmental stresses caused by heat, chilling, UV-B radiation, salinity, heavy metals, and Al (Ma et al., 2007; Ezaki et al., 2008). Their involvement in cellular damage has been demonstrated by the protective effects of the aldehyde-scavenging enzymes aldehyde dehydrogenase (Sunkar et al., 2003; Kotchoni et al., 2006) and aldehyde reductase (Oberschall et al., 2000; Hideg et al., 2003; Hegedüs et al., 2004) to confer tolerance against various environmental stresses when they were overexpressed in plants. In barley (Hordeum vulgare) roots, the formation of HNE in association with Al treatment was observed (Sakihama and Yamasaki, 2002). Occurrence of HNE in Arabidopsis (Arabidopsis thaliana) leaves under oxidative stress has been also deduced by detection of modified proteins in the mitochondria (Winger et al., 2007). HNE rapidly inhibited respiration in isolated potato (Solanum tuberosum) mitochondria by inactivating pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, NAD-malic enzyme (Millar and Leaver, 2000), and alternative oxidase (Winger et al., 2005). HNE and other 2-alkenals also inactivated photosynthesis in isolated chloroplasts (Mano et al., 2009). Arabidopsis contains 2-alkenal reductase (AER; E.C. 1.3.1.74) that catalyzes the reduction of the α,β-unsaturated bond of 2-alkenals to produce n-alkanals (Mano et al., 2002). Overexpression of AER in tobacco (Mano et al., 2005) and in Arabidopsis (Papdi et al., 2008) improved the tolerance to photooxidative stress and NaCl stress, respectively. Thus, accumulated observation indicates that LOOH-derived aldehydes, especially 2-alkenals, are commonly involved in oxidative damage in plant cells. Considering the critical importance of ROS in Al toxicity to roots, it is expected that 2-alkenals are produced and mediate damage in the stressed root cells.To evaluate the roles of LOOH-derived aldehydes in root injury under Al stress, we employed transgenic tobacco plants that overexpress the AER gene (AER-OE plants; Mano et al., 2005). With Al treatment, the roots of AER-OE accumulated Al and H₂O₂ to the same levels as those of the wild type, but they showed resistance to inhibition of elongation. Aldehyde analysis revealed that the Al treatment increased the contents of several toxic aldehydes, including HNE and acrolein in wild-type plants, but these aldehydes were significantly suppressed in the AER-OE plants. On the basis of these results, we propose that the inhibition of root growth by Al ions is induced by toxic aldehydes generated with ROS.     

Split decisions: oesophageal progenitor cell behaviour

Science advance online publication July192012; doi:10.1126/science.1218835The maintenance and regeneration of continually shedding epithelial tissues that make up the linings and barriers of our bodies requires rapid and continual input of proliferative progenitor cells for tissue homeostasis. The mechanisms by which epithelial progenitors cells maintain tissues remain controversial. In a recent Science paper, Doupé et al (2012) demonstrate that a population of equivalent progenitor cells support tissue homeostasis of the oesophagus without the need for slow cycling cells as described in other rapidly dividing epithelia.In tissues such as blood and skin in which differentiated cells constantly turnover, proliferative progenitor populations are required to continually produce lost differentiated cells. Several models have been proposed to explain mechanisms by which progenitor cells contribute to tissue maintenance (Figure 1). A hierarchical model has been suggested in which longer lived stem cells, which may also cycle slowly, produce highly proliferative cells with less self-renewal potential that differentiate into a restricted number of cells. Following proliferative cells in pulse-chase experiments and genetic lineage tracing has supported a hierarchical model in the blood, epidermis and intestine (Fuchs, 2009). Alternatively, an equivalency model has been proposed in which all proliferative progenitor cells are equally able to produce proliferative and differentiated progeny in a stochastic manner. Analysis of labelled clones has supported an equivalency model for progenitors in the interfollicular epidermis and intestine (Clayton et al, 2007; Doupé et al, 2010; Snippert et al, 2010).Open in a separate windowFigure 1Two types of models have been put forward to describe the pattern of progenitor behaviour within mammalian tissues. In the hierarchical model, a stem cell can produce proliferative progenitors with less self-renewal potential that differentiate into lineage-specific cells. Alternatively, an equivalency model has been proposed that assumes equal behaviour of progenitor cells to maintain tissue homeostasis.An elevated interest in understanding the dynamics of oesophageal epithelium has resulted, in part, from the rapid increase in the incidence of oesophageal adenocarcinoma (Devesa et al, 1998). The oesophagus is a stratified epithelium that lacks any appendages or glands, and thus consists of a basal layer of proliferative keratinocytes and several suprabasal layers of differentiated cells, which are continually shed. Previously, labelling of proliferative cells with DNA analogues has demonstrated that proliferation is restricted to the basal cells, which all proliferate in 5 days seemingly stochastically, supporting an equivalency model (Marques-Periera and Leblond, 1965). In contrast, studies using chimeric mice have suggested that proliferation of labelled progenitor cells occurs in a hierarchical manner (Thomas et al, 1988; Croagh et al, 2008).To address this controversy, a recent study in Science uses several genetic mouse models to define the contribution of proliferative basal cells to oesophageal homeostasis (Doupé et al, 2012). In one mouse model, the authors utilized a genetic pulse-chase system based on the tetracycline-regulated expression of the histone H2B-GFP (Tumbar et al, 2004). They find that the rapidly dividing epithelial cells of the oesophagus lose H2B-GFP expression after 4 weeks. These data suggest that either H2B-GFP is degraded (Waghmare et al, 2008) or oesophageal progenitor cells proliferate faster than their counterparts in skin epithelial appendages or blood lineages, which retain H2B-GFP after 4 weeks (Tumbar et al, 2004; Foudi et al, 2009).To analyse the properties of oesophageal progenitor cells in more detail, the authors label single cells using an inducible cre-lox genetic system and followed clones for a year. Similar to their results with this system in the tail and ear epidermis (Clayton et al, 2007; Doupé et al, 2010), the authors find that the size of the persistent clones is linear with time. Statistical analysis of the clone size data supports the ability of the cells to contribute to proliferative and non-proliferative (i.e., differentiated) progeny with equal probability. Thus, these data support a model in which all of the labelled cells are equivalent.In addition to homeostasis, the authors explore how proliferative progenitors contribute to alterations in tissue homeostasis. After inflicting wounds by biopsy, marked clones span both proliferative and non-proliferative zones of the healing oesophageal epithelium, suggesting that they maintain a progenitor fate with distinct phenotypes. With atRA treatment, the authors show that suprabasal cell formation increases, which is consistent with the known effect of atRA on the oesophagus (Lasnitzki, 1963). Statistical analysis reveals that the probability of forming basal and suprabasal cells was not altered with atRA administration. However, since proliferative cells exist in suprabasal layers during epithelial hyperplasia, additional analyses of cell state are required to determine if atRA maintains stochastic fate decisions of progenitor cells. Furthermore, the progenitor response to atRA treatment might be limited by niche space along the basement membrane like in intestinal crypt progenitor cells (Snippert et al, 2010).In summary, this study together with the authors'' previous work provides additional support for the existence of equivalent progenitor cells within stratified epithelium in several tissues. Additional studies revealing how epithelial progenitor cells behave when proliferation and differentiation are altered in the oesophagus could shed light on mechanisms for the pathogenesis of oesophageal tumours or diseases such as Barrett''s oesophagus.