S-Nitrosylation of Ascorbate Peroxidase Is Part of Programmed Cell Death Signaling in Tobacco Bright Yellow-2 Cells

Nitric oxide (NO) is a small redox molecule that acts as a signal in different physiological and stress-related processes in plants. Recent evidence suggests that the biological activity of NO is also mediated by S-nitrosylation, a well-known redox-based posttranslational protein modification. Here, we show that during programmed cell death (PCD), induced by both heat shock (HS) or hydrogen peroxide (H₂O₂) in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, an increase in S-nitrosylating agents occurred. NO increased in both experimentally induced PCDs, although with different intensities. In H₂O₂-treated cells, the increase in NO was lower than in cells exposed to HS. However, a simultaneous increase in S-nitrosoglutathione (GSNO), another NO source for S-nitrosylation, occurred in H₂O₂-treated cells, while a decrease in this metabolite was evident after HS. Consistently, different levels of activity and expression of GSNO reductase, the enzyme responsible for GSNO removal, were found in cells subjected to the two different PCD-inducing stimuli: low in H₂O₂-treated cells and high in the heat-shocked ones. Irrespective of the type of S-nitrosylating agent, S-nitrosylated proteins formed upon exposure to both of the PCD-inducing stimuli. Interestingly, cytosolic ascorbate peroxidase (cAPX), a key enzyme controlling H₂O₂ levels in plants, was found to be S-nitrosylated at the onset of both PCDs. In vivo and in vitro experiments showed that S-nitrosylation of cAPX was responsible for the rapid decrease in its activity. The possibility that S-nitrosylation induces cAPX ubiquitination and degradation and acts as part of the signaling pathway leading to PCD is discussed.Nitric oxide (NO) is a gaseous and diffusible redox molecule that acts as a signaling compound in both animal and plant systems (Pacher et al., 2007; Besson-Bard et al., 2008). In plants, NO has been found to play a key role in several physiological processes, such as germination, lateral root development, flowering, senescence, stomatal closure, and growth of pollen tubes (Beligni and Lamattina, 2000; Neill et al., 2002; Correa-Aragunde et al., 2004; He et al., 2004; Prado et al., 2004; Carimi et al., 2005). In addition, NO has been reported to be involved in plant responses to both biotic and abiotic stresses (Leitner et al., 2009; Siddiqui et al., 2011) and in the signaling pathways leading to programmed cell death (PCD; Delledonne et al., 1998; de Pinto et al., 2006; De Michele et al., 2009; Lin et al., 2012; Serrano et al., 2012).The cellular environment may greatly influence the chemical reactivity of NO, giving rise to different biologically active NO-derived compounds, collectively named reactive nitrogen species, which amplify and differentiate its ability to activate physiological and stress-related processes. Many of the biological properties of NO are due to its high affinity with transition metals of metalloproteins as well as its reactivity with reactive oxygen species (ROS; Hill et al., 2010). However, recent evidence suggests that protein S-nitrosylation, due to the addition of NO to reactive Cys thiols, may act as a key mechanism of NO signaling in plants (Wang et al., 2006; Astier et al., 2011). NO is also able to react with reduced glutathione (GSH), the most abundant cellular thiol, thus producing S-nitrosoglutathione (GSNO), which also acts as an endogenous trans-nitrosylating agent. GSNO is also considered as a NO store and donor and, as it is more stable than NO, acts as a long-distance NO transporter through the floematic flux (Malik et al., 2011). S-Nitrosoglutathione reductase (GSNOR), which is an enzyme conserved from bacteria to humans, has been suggested to play a role in regulating S-nitrosothiols (SNO) and the turnover of S-nitrosylated proteins in plants (Liu et al., 2001; Rusterucci et al., 2007).A number of proteins involved in metabolism, stress responses, and redox homeostasis have been identified as potential targets for S-nitrosylation in Arabidopsis (Arabidopsis thaliana; Lindermayr et al., 2005). During the hypersensitive response (HR), 16 proteins were identified to be S-nitrosylated in the seedlings of the same species (Romero-Puertas et al., 2008); in Citrus species, S-nitrosylation of about 50 proteins occurred in the NO-mediated resistance to high salinity (Tanou et al., 2009).However, while the number of candidate proteins for S-nitrosylation is increasing, the functional significance of protein S-nitrosylation has been explained only in a few cases, such as for nonsymbiotic hemoglobin (Perazzolli et al., 2004), glyceraldehyde 3-phosphate dehydrogenase (Lindermayr et al., 2005; Wawer et al., 2010), Met adenosyltransferase (Lindermayr et al., 2006), and metacaspase9 (Belenghi et al., 2007). Of particular interest are the cases in which S-nitrosylation involves enzymes controlling ROS homeostasis. For instance, it has been reported that S-nitrosylation of peroxiredoxin IIE regulates the antioxidant function of this enzyme and might contribute to the HR (Romero-Puertas et al., 2007). It has also been shown that in the immunity response, S-nitrosylation of NADPH oxidase inactivates the enzyme, thus reducing ROS production and controlling HR development (Yun et al., 2011).Recently, S-nitrosylation has also been shown to be involved in PCD of nitric oxide excess1 (noe1) rice (Oryza sativa) plants, which are mutated in the OsCATC gene coding for catalase (Lin et al., 2012). In these plants, which show PCD-like phenotypes under high-light conditions, glyceraldehyde 3-phosphate dehydrogenase and thioredoxin are S-nitrosylated. This suggests that the NO-dependent regulation of these proteins is involved in plant PCD, similar to what occurs in animal apoptosis (Sumbayev, 2003; Hara et al., 2005; Lin et al., 2012). The increase in hydrogen peroxide (H₂O₂) after exposure to high light in noe1 plants is responsible for the production of NO required for leaf cell death induction (Lin et al., 2012). There is a strict relationship between H₂O₂ and NO in PCD activation (Delledonne et al., 2001; de Pinto et al., 2002); however, the mechanism of this interplay is largely still unknown (for review, see Zaninotto et al., 2006; Zhao, 2007; Yoshioka et al., 2011). NO can induce ROS production and vice versa, and their reciprocal modulation in terms of intensity and timing seems to be crucial in determining PCD activation and in controlling HR development (Delledonne et al., 2001; Zhao, 2007; Yun et al., 2011).In previous papers, we demonstrated that heat shock (HS) at 55°C and treatment with 50 mm H₂O₂ promote PCD in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Vacca et al., 2004; de Pinto et al., 2006; Locato et al., 2008). In both experimental conditions, NO production and decrease in cytosolic ascorbate peroxidase (cAPX) were observed as early events in the PCD pathway, and cAPX decrease has been suggested to contribute to determining the redox environment required for PCD (de Pinto et al., 2006; Locato et al., 2008).In this study, the production of nitrosylating agents (NO and GSNO) in the first hours of PCD induction by HS or H₂O₂ treatment in tobacco BY-2 cells and their role in PCD were studied. The possibility that S-nitrosylation could be a first step in regulating cAPX activity and turnover as part of the signaling pathway leading to PCD was also investigated.     

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.     

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development

We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1.Endomembrane trafficking in plant cells is complicated such that secretory, endocytic, and recycling pathways are usually integrated with each other at the post-Golgi compartments, among which, the trans-Golgi network (TGN) and prevacuolar compartment (PVC)/multivesicular body (MVB) are best studied (Tse et al., 2004; Lam et al., 2007a, 2007b; Müller et al., 2007; Foresti and Denecke, 2008; Hwang, 2008; Otegui and Spitzer, 2008; Robinson et al., 2008; Richter et al., 2009; Ding et al., 2012; Gao et al., 2014). Following the endocytic trafficking of a lipophilic dye, FM4-64, the TGN and PVC/MVB are sequentially labeled and thus are defined as the early and late endosome, respectively, in plant cells (Lam et al., 2007a; Chow et al., 2008). While the TGN is a tubular vesicular-like structure that may include several different microdomains and fit its biological function as a sorting station (Chow et al., 2008; Kang et al., 2011), the PVC/MVB is 200 to 500 nm in size with multiple luminal vesicles of approximately 40 nm (Tse et al., 2004). Membrane cargoes destined for degradation are sequestered into these tiny luminal vesicles and delivered to the lumen of the lytic vacuole (LV) via direct fusion between the PVC/MVB and the LV (Spitzer et al., 2009; Viotti et al., 2010; Cai et al., 2012). Therefore, the PVC/MVB functions between the TGN and LV as an intermediate organelle and decides the fate of membrane cargoes in the LV.In yeast (Saccharomyces cerevisiae), carboxypeptidase S (CPS) is synthesized as a type II integral membrane protein and sorted from the Golgi to the lumen of the vacuole (Spormann et al., 1992). Genetic analyses on the trafficking of CPS have led to the identification of approximately 17 class E genes (Piper et al., 1995; Babst et al., 1997, 2002a, 2002b; Odorizzi et al., 1998; Katzmann et al., 2001) that constitute the core endosomal sorting complex required for transport (ESCRT) machinery. The evolutionarily conserved ESCRT complex consists of several functionally different subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III and the ESCRT-III-associated/Vacuolar Protein Sorting4 (VPS4) complex. Together, they form a complex protein-protein interaction network that coordinates sorting of cargoes and inward budding of the membrane on the MVB (Hurley and Hanson, 2010; Henne et al., 2011). Cargo proteins carrying ubiquitin signals are thought to be passed from one ESCRT subcomplex to the next, starting with their recognition by ESCRT-0 (Bilodeau et al., 2002, 2003; Hislop and von Zastrow, 2011; Le Bras et al., 2011; Shields and Piper, 2011; Urbé, 2011). ESCRT-0 recruits the ESCRT-I complex, a heterotetramer of VPS23, VPS28, VPS37, and MVB12, from the cytosol to the endosomal membrane (Katzmann et al., 2001, 2003). The C terminus of VPS28 interacts with the N terminus of VPS36, a member of the ESCRT-II complex (Kostelansky et al., 2006; Teo et al., 2006). Then, cargoes passed from ESCRT-I and ESCRT-II are concentrated in certain membrane domains of the endosome by ESCRT-III, which includes four coiled-coil proteins and is sufficient to induce the membrane invagination (Babst et al., 2002b; Saksena et al., 2009; Wollert et al., 2009). Finally, the ESCRT components are disassociated from the membrane by the adenosine triphosphatase (ATPase) associated with diverse cellular activities (AAA) VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1 (SKD1) before releasing the internal vesicles (Babst et al., 1997, 1998).Putative homologs of ESCRT-I–ESCRT-III and ESCRT-III-associated components have been identified in plants, except for ESCRT-0, which is only present in Opisthokonta (Winter and Hauser, 2006; Leung et al., 2008; Schellmann and Pimpl, 2009). To date, only a few plant ESCRT components have been studied in detail. The Arabidopsis (Arabidopsis thaliana) AAA ATPase SKD1 localized to the PVC/MVB and showed ATPase activity that was regulated by Lysosomal Trafficking Regulator-Interacting Protein5, a plant homolog of Vps Twenty Associated1 Protein (Haas et al., 2007). Expression of the dominant negative form of SKD1 caused an increase in the size of the MVB and a reduction in the number of internal vesicles (Haas et al., 2007). This protein also contributes to the maintenance of the central vacuole and might be associated with cell cycle regulation, as leaf trichomes expressing its dominant negative mutant form lost the central vacuole and frequently contained multiple nuclei (Shahriari et al., 2010). Double null mutants of CHARGED MULTIVESICULAR BODY PROTEIN, chmp1achmp1b, displayed severe growth defects and were seedling lethal. This may be due to the mislocalization of plasma membrane (PM) proteins, including those involved in auxin transport such as PINFORMED1, PINFORMED2, and AUXIN-RESISTANT1, from the vacuolar degradation pathway to the tonoplast of the LV (Spitzer et al., 2009).Plant ESCRT components usually contain several homologs, with the possibility of functional redundancy. Single mutants of individual ESCRT components may not result in an obvious phenotype, whereas knockout of all homologs of an ESCRT component by generating double or triple mutants may be lethal to the plant. As a first step to carry out systematic analysis on each ESCRT complex in plant cells, here, we established an efficient analysis system to monitor the localization changes of four vacuolar reporters that accumulate either in the lumen (LRR84A-GFP, EMP12-GFP, and aleurain-GFP) or on the tonoplast (GFP-VIT1) of the LV and identified several ESCRT-III dominant negative mutants. We reported that ESCRT-III subunits were involved in the release of PVC/MVB’s internal vesicles from the limiting membrane and were required for membrane protein degradation from secretory and endocytic pathways. In addition, transgenic Arabidopsis plants with induced expression of ESCRT-III dominant negative mutants showed severe cotyledon developmental defects. We also showed that membrane dissociation of ESCRT-III subunits was regulated by direct interaction with SKD1.     

HAPLESS13, the Arabidopsis μ1 Adaptin,Is Essential for Protein Sorting at the trans-Golgi Network/Early Endosome

In plant cells, secretory and endocytic routes intersect at the trans-Golgi network (TGN)/early endosome (EE), where cargos are further sorted correctly and in a timely manner. Cargo sorting is essential for plant survival and therefore necessitates complex molecular machinery. Adaptor proteins (APs) play key roles in this process by recruiting coat proteins and selecting cargos for different vesicle carriers. The µ1 subunit of AP-1 in Arabidopsis (Arabidopsis thaliana) was recently identified at the TGN/EE and shown to be essential for cytokinesis. However, little was known about other cellular activities affected by mutations in AP-1 or the developmental consequences of such mutations. We report here that HAPLESS13 (HAP13), the Arabidopsis µ1 adaptin, is essential for protein sorting at the TGN/EE. Functional loss of HAP13 displayed pleiotropic developmental defects, some of which were suggestive of disrupted auxin signaling. Consistent with this, the asymmetric localization of PIN-FORMED2 (PIN2), an auxin transporter, was compromised in the mutant. In addition, cell morphogenesis was disrupted. We further demonstrate that HAP13 is critical for brefeldin A-sensitive but wortmannin-insensitive post-Golgi trafficking. Our results show that HAP13 is a key link in the sophisticated trafficking network in plant cells.Plant cells contain sophisticated endomembrane compartments, including the endoplasmic reticulum, the Golgi, the trans-Golgi network (TGN)/early endosome (EE), the prevacuolar compartments/multivesicular bodies (PVC/MVB), various types of vesicles, and the plasma membrane (PM; Ebine and Ueda, 2009; Richter et al., 2009). Intracellular protein sorting between the various locations in the endomembrane system occurs in both secretory and endocytic routes (Richter et al., 2009; De Marcos Lousa et al., 2012). Vesicles in the secretory route start at the endoplasmic reticulum, passing through the Golgi before reaching the TGN/EE, while vesicles in the endocytic route start from the PM before reaching the TGN/EE (Dhonukshe et al., 2007; Viotti et al., 2010). The TGN/EE in Arabidopsis (Arabidopsis thaliana) is an independent and highly dynamic organelle transiently associated with the Golgi (Dettmer et al., 2006; Lam et al., 2007; Viotti et al., 2010), distinct from the animal TGN. Once reaching the TGN/EE, proteins delivered by their vesicle carriers are subject to further sorting, being incorporated either into vesicles that pass through the PVC/MVB before reaching the vacuole for degradation or into vesicles that enter the secretory pathway for delivery to the PM (Ebine and Ueda, 2009; Richter et al., 2009). Therefore, the TGN/EE is a critical sorting compartment that lies at the intersection of the secretory and endocytic routes.Fine-tuned control of intracellular protein sorting at the TGN/EE is essential for plant development (Geldner et al., 2003; Dhonukshe et al., 2007, 2008; Richter et al., 2007; Kitakura et al., 2011; Wang et al., 2013). An auxin gradient is crucial for pattern formation in plants, whose dynamic maintenance requires the polar localization of auxin efflux carrier PINs through endocytic recycling (Geldner et al., 2003; Blilou et al., 2005; Paciorek et al., 2005; Abas et al., 2006; Jaillais et al., 2006; Dhonukshe et al., 2007; Kleine-Vehn et al., 2008). Receptor-like kinases (RLKs) have also been recognized as major cargos undergoing endocytic trafficking, which are either recycled back to the PM or sent for vacuolar degradation (Geldner and Robatzek, 2008; Irani and Russinova, 2009). RLKs are involved in most if not all developmental processes of plants (De Smet et al., 2009).Intracellular protein sorting relies on sorting signals within cargo proteins and on the molecular machinery that recognizes sorting signals (Boehm and Bonifacino, 2001; Robinson, 2004; Dhonukshe et al., 2007). Adaptor proteins (AP) play a key role (Boehm and Bonifacino, 2001; Robinson, 2004) in the recognition of sorting signals. APs are heterotetrameric protein complexes composed of two large subunits (β and γ/α/δ/ε), a small subunit (σ), and a medium subunit (µ) that is crucial for cargo selection (Boehm and Bonifacino, 2001). APs associate with the cytoplasmic side of secretory and endocytic vesicles, recruiting coat proteins and recognizing sorting signals within cargo proteins for their incorporation into vesicle carriers (Boehm and Bonifacino, 2001). Five APs have been identified so far, classified by their components, subcellular localization, and function (Boehm and Bonifacino, 2001; Robinson, 2004; Hirst et al., 2011). Of the five APs, AP-1 associates with the TGN or recycling endosomes (RE) in yeast and mammals (Huang et al., 2001; Robinson, 2004), mediating the sorting of cargo proteins to compartments of the endosomal-lysosomal system or to the basolateral PM of polarized epithelial cells (Gonzalez and Rodriguez-Boulan, 2009). Knockouts of AP-1 components in multicellular organisms resulted in embryonic lethality (Boehm and Bonifacino, 2001; Robinson, 2004).We show here that the recently identified Arabidopsis µ1 adaptin AP1M2 (Park et al., 2013; Teh et al., 2013) is a key component in the cellular machinery mediating intracellular protein sorting at the TGN/EE. AP1M2 was previously named HAPLESS13 (HAP13), whose mutant allele hap13 showed male gametophytic lethality (Johnson et al., 2004). In recent quests for AP-1 in plants, HAP13/AP1M2 was confirmed as the Arabidopsis µ1 adaptin based on its interaction with other components of the AP-1 complex as well as its localization at the TGN (Park et al., 2013; Teh et al., 2013). A novel mutant allele of HAP13/AP1M2, ap1m2-1, was found to be defective in the intracellular distribution of KNOLLE, leading to defective cytokinesis (Park et al., 2013; Teh et al., 2013). However, it was not clear whether HAP13/AP1M2 mediated other cellular activities and their developmental consequences. Using the same mutant allele, we found that functional loss of HAP13 (hap13-1/ap1m2-1) resulted in a full spectrum of growth defects, suggestive of compromised auxin signaling and of defective RLK signaling. Cell morphogenesis was also disturbed in hap13-1. Importantly, hap13-1 was insensitive to brefeldin A (BFA) washout, indicative of defects in guanine nucleotide exchange factors for ADP-ribosylation factor (ArfGEF)-mediated post-Golgi trafficking. Furthermore, HAP13/AP1M2 showed evolutionarily conserved function during vacuolar fusion, providing additional support to its identity as a µ1 adaptin. These results demonstrate the importance of the Arabidopsis µ1 adaptin for intracellular protein sorting centered on the TGN/EE.     

NdhV Is a Subunit of NADPH Dehydrogenase Essential for Cyclic Electron Transport in Synechocystis sp. Strain PCC 6803

Two mutants sensitive to heat stress for growth and impaired in NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET) were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in the same sll0272 gene, encoding a protein highly homologous to NdhV identified in Arabidopsis (Arabidopsis thaliana). Deletion of the sll0272 gene (ndhV) did not influence the assembly of NDH-1 complexes and the activities of CO₂ uptake and respiration but reduced the activity of NDH-CET. NdhV interacted with NdhS, a ferredoxin-binding subunit of cyanobacterial NDH-1 complex. Deletion of NdhS completely abolished NdhV, but deletion of NdhV had no effect on the amount of NdhS. Reduction of NDH-CET activity was more significant in ΔndhS than in ΔndhV. We therefore propose that NdhV cooperates with NdhS to accept electrons from reduced ferredoxin.Cyanobacterial NADPH dehydrogenase (NDH-1) complexes are localized in the thylakoid membrane (Ohkawa et al., 2001, 2002; Zhang et al., 2004; Xu et al., 2008; Battchikova et al., 2011b) and participate in a variety of bioenergetic reactions, such as respiration, cyclic electron transport around photosystem I (NDH-CET), and CO₂ uptake (Ogawa, 1991; Mi et al., 1992; Ohkawa et al., 2000). Structurally, the cyanobacterial NDH-1 complexes closely resemble energy-converting complex I in eubacteria and the mitochondrial respiratory chain regardless of the absence of homologs of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I (Friedrich et al., 1995; Friedrich and Scheide, 2000; Arteni et al., 2006). Over the past decade, new subunits of NDH-1 complexes specific to oxygenic photosynthesis have been identified in several cyanobacterial strains. They are NdhM to NdhQ and NdhS (Prommeenate et al., 2004; Battchikova et al., 2005, 2011b; Nowaczyk et al., 2011; Wulfhorst et al., 2014; Zhang et al., 2014; Zhao et al., 2014b, 2015), in addition to NdhL first identified in the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) about 20 years ago (Ogawa, 1992). Among them, NdhS possesses a ferredoxin (Fd)-binding motif and was shown to bind Fd, which suggested that Fd is one of the electron donors to NDH-1 complexes (Mi et al., 1995; Battchikova et al., 2011b; Ma and Ogawa, 2015). Deletion of NdhS strongly reduced the activity of NDH-CET but had no effect on respiration and CO₂ uptake (Battchikova et al., 2011b; Ma and Ogawa, 2015). The NDH-CET plays an important role in coping with various environmental stresses regardless of its elusive mechanism. For example, this function can greatly alleviate heat-sensitive growth phenotypes (Wang et al., 2006a; Zhao et al., 2014a). Thus, heat treatment strategy can help in identifying the proteins essential to NDH-CET.Here, a new oxygenic photosynthesis-specific (OPS) subunit NdhV was identified in Synechocystis 6803 with the help of heat treatment strategy, and its deletion did not influence the assembly of NDH-1L and NDH-1MS complexes and the activities of CO₂ uptake and respiration but impaired the NDH-CET activity. We give evidence that NdhV interacts with NdhS and is another component of Fd-binding domain of cyanobacterial NDH-1 complex. A possible role of NdhV on the NDH-CET activity is discussed.     

Focus Issue on Calcium Signaling: Calcium-Dependent Protein Kinase CPK6 Positively Functions in Induction by Yeast Elicitor of Stomatal Closure and Inhibition by Yeast Elicitor of Light-Induced Stomatal Opening in Arabidopsis

Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca²⁺-dependent signaling pathway. A Ca²⁺-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca²⁺-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca²⁺ concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K⁺ channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress.Stomata, formed by pairs of guard cells, play a critical role in regulation of plant CO₂ uptake and water loss, thus critically influencing plant growth and water stress responsiveness. Guard cells respond to a variety of abiotic and biotic stimuli, such as light, drought, and pathogen attack (Israelsson et al., 2006; Shimazaki et al., 2007; Melotto et al., 2008).Elicitors derived from microbial surface mimic pathogen attack and induce stomatal closure in various plant species such as Solanum lycopersicum (Lee et al., 1999), Commelina communis (Lee et al., 1999), Hordeum vulgare (Koers et al., 2011), and Arabidopsis (Arabidopsis thaliana; Melotto et al., 2006; Khokon et al., 2010). Yeast elicitor (YEL) induces stomatal closure in Arabidopsis (Klüsener et al., 2002; Khokon et al., 2010; Salam et al., 2013). Our recent studies showed that YEL inhibits light-induced stomatal opening and that protein phosphorylation is involved in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening (Salam et al., 2013).Cytosolic Ca²⁺ has long been recognized as a conserved second messenger in stomatal movement (Shimazaki et al., 2007; Roelfsema and Hedrich 2010; Hubbard et al., 2012). Elevation of cytosolic free Ca²⁺ concentration ([Ca2+]_cyt) is triggered by influx of Ca²⁺ from apoplast and release of Ca²⁺ from intracellular stores in guard cell signaling (Leckie et al., 1998; Hamilton et al., 2000; Pei et al., 2000; Garcia-Mata et al., 2003; Lemtiri-Chlieh et al., 2003). The influx of Ca²⁺ is carried by nonselective Ca²⁺-permeable cation (I_Ca) channels that are activated by plasma membrane hyperpolarization and H₂O₂ (Pei et al., 2000; Murata et al., 2001; Kwak et al., 2003). Elevation of [Ca2+]_cyt activates slow anion (S-type) channels and down-regulates inward-rectifying potassium (K_in) channels in guard cells (Schroeder and Hagiwara, 1989; Grabov and Blatt, 1999). The activation of S-type channels is a hallmark of stomatal closure, and the suppression of K_in channels is favorable to stomatal closure but not to stomatal opening (Pei et al., 1997; Kwak et al., 2001; Xue et al., 2011; Uraji et al., 2012).YEL induces stomatal closure with extracellular H₂O₂ production, intracellular H₂O₂ accumulation, activation of I_Ca channels, and transient [Ca2+]_cyt elevations (Klüsener et al., 2002; Khokon et al., 2010). However, it remains to be clarified whether YEL activates S-type channels and inhibits K_in channels in guard cells.Calcium-dependent protein kinases (CDPKs) are regulators in Ca²⁺-dependent guard cell signaling (Mori et al., 2006; Zhu et al., 2007; Geiger et al., 2010, 2011; Zou et al., 2010; Munemasa et al., 2011; Brandt et al., 2012; Scherzer et al., 2012). In guard cells, CDPKs regulate activation of S-type and I_Ca channels and inhibition of K_in channels (Mori et al., 2006; Zou et al., 2010; Munemasa et al., 2011). A CDPK, CPK6, positively regulates activation of S-type channels and I_Ca channels without affecting H₂O₂ production in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure (Mori et al., 2006; Munemasa et al., 2011). CPK6 phosphorylates and activates SLOW ANION CHANNEL-ASSOCIATED1 expressed in Xenopus spp. oocyte (Brandt et al., 2012; Scherzer et al., 2012). These findings underline the role of CPK6 in regulation of ion channel activation and stomatal movement, leading us to test whether CPK6 regulates the induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening.In this study, we investigated activation of S-type channels and inhibition of K_in channels by YEL and roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. For this purpose, we examined the effects of mutation of CPK6 on induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening, activation of I_Ca channels, transient [Ca2+]_cyt elevations, activation of S-type channels, inhibition of K_in channels, H₂O₂ production in leaves, and H₂O₂ accumulation in leaves and guard cells.     

Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins

Spatial segregation of metabolism, such as cellular-localized CO₂ fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are self-assembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic carboxysomes for plant engineering.A key enzyme in photosynthesis is the CO₂ fixation enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco not only fixes CO₂, resulting in carbon assimilation, but it can also fix O₂, leading to photorespiration. Suppressing the unwanted oxygenase activity of Rubisco by sequestering Rubisco with a source of CO₂ is Nature’s solution to this substrate discrimination problem. While C4 plants compartmentalize CO₂ fixation in specific cells (Hibberd et al., 2008; Parry et al., 2011), cyanobacteria have evolved a specialized organelle composed entirely of protein to encapsulate Rubisco—the carboxysome.The carboxysome is just one type of bacterial microcompartment (BMC), widespread, functionally diverse bacterial organelles (Axen et al., 2014). All BMCs consist of an enzymatic core surrounded by a selectively permeable protein shell (Kerfeld et al., 2005; Tanaka et al., 2008; Chowdhury et al., 2014; Kerfeld and Erbilgin, 2015). While the encapsulated enzymes differ among functionally distinct BMCs, they share an architecturally similar shell composed of three types of proteins: BMC-H, BMC-T, and BMC-P forming hexamers, pseudohexamers, and pentamers, respectively (Kerfeld and Erbilgin, 2015). These constitute the building blocks of a self-assembling, apparently icosahedral shell with a diameter ranging from 40 to 400 nm (Shively et al., 1973a,b, 1998; Price and Badger, 1991; Bobik et al., 1999; Iancu et al., 2007, 2010; Petit et al., 2013; Erbilgin et al., 2014). Recent studies have also shown that in the biogenesis of BMCs an encapsulation peptide (EP) (Fan and Bobik, 2011; Kinney et al., 2012; Aussignargues et al., 2015; Jakobson et al., 2015), a short (approximately 18 residues) amphipathic α-helix mediates interactions between a subset of core protein and the shell (Fan and Bobik, 2011; Choudhary et al., 2012; Kinney et al., 2012; Lawrence et al., 2014; Lin et al., 2014; Aussignargues et al., 2015). Indeed, because they are self-assembling organelles composed entirely of protein, BMCs hold great promise for diverse applications in bioengineering and development of bionanomaterials (Frank et al., 2013; Chowdhury et al., 2014; Chessher et al., 2015; Kerfeld and Erbilgin, 2015); the key features of BMCs include selective permeability, spatial colocalization of enzymes, the establishment of private cofactor pools, and the potentially beneficial effects of confinement on protein stability. For example, introducing carboxysomes into plants could provide a saltational enhancement of crop photosynthesis (Price et al., 2013; Zarzycki et al., 2013; Lin et al., 2014; McGrath and Long, 2014).The β-carboxysome, which sequesters form 1B Rubisco, has been an important model system for the study of the structural basis of carboxysome function, assembly, and engineering (Kerfeld et al., 2005; Tanaka et al., 2008; Cameron et al., 2013; Aussignargues et al., 2015; Cai et al., 2015). Beta-carboxysomes assemble from the inside out (Cameron et al., 2013; Gonzalez-Esquer et al., 2015). Two proteins that are absolutely conserved and unique to β-carboxysomes, CcmM and CcmN, play essential roles in this process: CcmM crosslinks Rubisco through its C-terminal Rubisco small subunit-like domains (SSLDs; pfam00101); CcmM and CcmN interact through their N-terminal domains; and C-terminal EP of CcmN interacts with the carboxysome shell.Here we describe a system for producing synthetic β-carboxysome shells and encapsulating nonnative cargo. We constructed a synthetic operon composed of ccmK1, ccmK2, ccmL, and ccmO, genes encoding, respectively, two BMC-H proteins, a BMC-P protein, and a BMC-T protein of the carboxysome shell of the halotolerant cyanobacterium, Halothece sp. PCC 7418 (Halo hereafter). Recombinant shells composed of all four proteins were produced and purified. We also demonstrated that the terminal α-helices of CcmK1 and CcmK2 are not, as had been proposed (Samborska and Kimber, 2012), required for the shell formation, and that the synthetic shell is a single-layered protein membrane. Cargo could be targeted to the interior of the synthetic shells using either the EP of CcmN or the N-terminal domain of CcmM; the latter observation provides new insight into the organization of the β-carboxysome. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly but also provide a platform to study carboxysome shell permeability. These results will be useful in guiding the design and optimization of carboxysomes and other BMCs for introduction into plants.