Multiscale Metabolic Modeling: Dynamic Flux Balance Analysis on a Whole-Plant Scale

Plant metabolism is characterized by a unique complexity on the cellular, tissue, and organ levels. On a whole-plant scale, changing source and sink relations accompanying plant development add another level of complexity to metabolism. With the aim of achieving a spatiotemporal resolution of source-sink interactions in crop plant metabolism, a multiscale metabolic modeling (MMM) approach was applied that integrates static organ-specific models with a whole-plant dynamic model. Allowing for a dynamic flux balance analysis on a whole-plant scale, the MMM approach was used to decipher the metabolic behavior of source and sink organs during the generative phase of the barley (Hordeum vulgare) plant. It reveals a sink-to-source shift of the barley stem caused by the senescence-related decrease in leaf source capacity, which is not sufficient to meet the nutrient requirements of sink organs such as the growing seed. The MMM platform represents a novel approach for the in silico analysis of metabolism on a whole-plant level, allowing for a systemic, spatiotemporally resolved understanding of metabolic processes involved in carbon partitioning, thus providing a novel tool for studying yield stability and crop improvement.Plants are of vital significance as a source of food (Grusak and DellaPenna, 1999; Rogalski and Carrer, 2011), feed (Lu et al., 2011), energy (Tilman et al., 2006; Parmar et al., 2011), and feedstocks for the chemical industry (Metzger and Bornscheuer, 2006; Kinghorn et al., 2011). Given the close connection between plant metabolism and the usability of plant products, there is a growing interest in understanding and predicting the behavior and regulation of plant metabolic processes. In order to increase crop quality and yield, there is a need for methods guiding the rational redesign of the plant metabolic network (Schwender, 2009).Mathematical modeling of plant metabolism offers new approaches to understand, predict, and modify complex plant metabolic processes. In plant research, the issue of metabolic modeling is constantly gaining attention, and different modeling approaches applied to plant metabolism exist, ranging from highly detailed quantitative to less complex qualitative approaches (for review, see Giersch, 2000; Morgan and Rhodes, 2002; Poolman et al., 2004; Rios-Estepa and Lange, 2007).A widely used modeling approach is flux balance analysis (FBA), which allows the prediction of metabolic capabilities and steady-state fluxes under different environmental and genetic backgrounds using (non)linear optimization (Orth et al., 2010). Assuming steady-state conditions, FBA has the advantage of not requiring the knowledge of kinetic parameters and, therefore, can be applied to model detailed, large-scale systems. In recent years, the FBA approach has been applied to several different plant species, such as maize (Zea mays; Dal’Molin et al., 2010; Saha et al., 2011), barley (Hordeum vulgare; Grafahrend-Belau et al., 2009b; Melkus et al., 2011; Rolletschek et al., 2011), rice (Oryza sativa; Lakshmanan et al., 2013), Arabidopsis (Arabidopsis thaliana; Poolman et al., 2009; de Oliveira Dal’Molin et al., 2010; Radrich et al., 2010; Williams et al., 2010; Mintz-Oron et al., 2012; Cheung et al., 2013), and rapeseed (Brassica napus; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011), as well as algae (Boyle and Morgan, 2009; Cogne et al., 2011; Dal’Molin et al., 2011) and photoautotrophic bacteria (Knoop et al., 2010; Montagud et al., 2010; Boyle and Morgan, 2011). These models have been used to study different aspects of metabolism, including the prediction of optimal metabolic yields and energy efficiencies (Dal’Molin et al., 2010; Boyle and Morgan, 2011), changes in flux under different environmental and genetic backgrounds (Grafahrend-Belau et al., 2009b; Dal’Molin et al., 2010; Melkus et al., 2011), and nonintuitive metabolic pathways that merit subsequent experimental investigations (Poolman et al., 2009; Knoop et al., 2010; Rolletschek et al., 2011). Although FBA of plant metabolic models was shown to be capable of reproducing experimentally determined flux distributions (Williams et al., 2010; Hay and Schwender, 2011b) and generating new insights into metabolic behavior, capacities, and efficiencies (Sweetlove and Ratcliffe, 2011), challenges remain to advance the utility and predictive power of the models.Given that many plant metabolic functions are based on interactions between different subcellular compartments, cell types, tissues, and organs, the reconstruction of organ-specific models and the integration of these models into interacting multiorgan and/or whole-plant models is a prerequisite to get insight into complex plant metabolic processes organized on a whole-plant scale (e.g. source-sink interactions). Almost all FBA models of plant metabolism are restricted to one cell type (Boyle and Morgan, 2009; Knoop et al., 2010; Montagud et al., 2010; Cogne et al., 2011; Dal’Molin et al., 2011), one tissue or one organ (Grafahrend-Belau et al., 2009b; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011; Mintz-Oron et al., 2012), and only one model exists taking into account the interaction between two cell types by specifying the interaction between mesophyll and bundle sheath cells in C4 photosynthesis (Dal’Molin et al., 2010). So far, no model representing metabolism at the whole-plant scale exists.Considering whole-plant metabolism raises the problem of taking into account temporal and environmental changes in metabolism during plant development and growth. Although classical static FBA is unable to predict the dynamics of metabolic processes, as the network analysis is based on steady-state solutions, time-dependent processes can be taken into account by extending the classical static FBA to a dynamic flux balance analysis (dFBA), as proposed by Mahadevan et al. (2002). The static (SOA) and dynamic optimization approaches introduced in this work provide a framework for analyzing the transience of metabolism by integrating kinetic expressions to dynamically constrain exchange fluxes. Due to the requirement of knowing or estimating a large number of kinetic parameters, so far dFBA has only been applied to a plant metabolic model once, to study the photosynthetic metabolism in the chloroplasts of C3 plants by a simplified model of five biochemical reactions (Luo et al., 2009). Integrating a dynamic model into a static FBA model is an alternative approach to perform dFBA.In this study, a multiscale metabolic modeling (MMM) approach was applied with the aim of achieving a spatiotemporal resolution of cereal crop plant metabolism. To provide a framework for the in silico analysis of the metabolic dynamics of barley on a whole-plant scale, the MMM approach integrates a static multiorgan FBA model and a dynamic whole-plant multiscale functional plant model (FPM) to perform dFBA. The performance of the novel whole-plant MMM approach was tested by studying source-sink interactions during the seed developmental phase of barley plants.     

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.     

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.     

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the “membrane raft” hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment.The adaptive capacity of biological membranes is a primary determinant of cell survival in fluctuating conditions. In particular, membrane physical properties are adjusted in the perception of and response to environmental modifications (including temperature, mechanical, and osmotic stresses) in various organisms (Los and Murata, 2004; Vígh et al., 2007; Verstraeten et al., 2010), including plants (Vaultier et al., 2006; Königshofer et al., 2008). Moreover, it has been shown that modifications of plasma membrane (PM) physical properties induced by pharmacological treatments can trigger signaling events in tobacco (Nicotiana tabacum) suspension cells (Bonneau et al., 2010). This reinforces the need to analyze the relationships between membrane organization and signaling in greater detail.Fluidity, a physical property of the PM, is a measure of the rotational and translational motions of molecules within the membrane, and consequently this reflects the level of lipid order in the bilayer. Lipid order is comprised of structure, microviscosity, and membrane phase; the latter feature includes lipid shape, packing, and curvature (Rilfors et al., 1984; van der Meer et al., 1984; Bloom et al., 1991). Lipid self-association induces a physical segregation into lipid bilayers, wherein a liquid-ordered (Lo) phase coexists with a liquid-disordered (Ld) phase (Veatch and Keller, 2005; Gaus et al., 2006; Klymchenko et al., 2009; Heberle et al., 2010). The Lo phase couples a high rotational mobility with a high conformational order in the lipid acyl chain, two physical properties that could be spatially resolved by fluorescence microscopy (Kubiak et al., 2011). Moreover, some observations indicate that Lo size or proportion could be controlled by temperature or cholesterol content (Roche et al., 2008; Orth et al., 2011).This preferential association of some lipids in complex mixtures has resulted in the “membrane raft” hypothesis within the cell biology field. This theory postulates the existence of small (20–200 nm), short-lived, sterol-, and sphingolipid-enriched Lo assemblies within the membrane. An important feature is that these aggregations are believed to coalesce, upon a biological stimulus, into larger structures whose dynamics can regulate many cellular processes (Simons and Ikonen, 1997; Pike, 2006; Lingwood and Simons, 2010; Simons and Gerl, 2010). An increased resistance to solubilization by detergents of Lo versus Ld phases has led researchers to consider that membrane fractions insoluble to nonionic detergents at low temperatures could contain the putative “raft” fractions. One caveat of this theory is that recovered detergent-insoluble membrane fractions (DIMs) only exist after detergent treatment and do not correspond to the native membrane structure (Lichtenberg et al., 2005). Nevertheless, their significant enrichment in sterols, sphingolipids, and specific subsets of proteins, some of which displaying a clustered distribution within the PM (Simons and Gerl, 2010), has encouraged their use as a biochemical counterpart of Lo microdomains existing in biological membranes.Plant DIMs with a lipid content similar to animal DIMs have been isolated from several species, including tobacco cells, and are enriched in proteins involved in signaling and stress responses (Mongrand et al., 2004; Borner et al., 2005; Morel et al., 2006; Lefebvre et al., 2007; Kierszniowska et al., 2009). Moreover, immunoelectron microscopy experiments have revealed that lateral segregation of lipids and proteins occurs at the nanoscale level at the tobacco PM, thus correlating detergent insolubility with membrane domain localization of presumptive raft proteins (Raffaele et al., 2009; Furt et al., 2010; Demir et al., 2013). Together, these data point to the existence of specialized lipid domains in plants. Concomitantly, the presence of sterol-rich Lo membrane domains was observed in vivo at the tip of the growing pollen tube in Picea meyeri, using both filipin and the fluorescent probe 1-[2-hydroxy-3-(N,N-dimethyl-N-hydroxyethyl)ammoniopropyl]-4-[β-[2-(di-n-butylamino)-6-napthyl]vinyl] pyridinium dibromide (di-4-ANEPPDHQ; Liu et al., 2009). This observation argues in favor of a sterol-dependent organization of ordered domains at the plant PM surface. In addition, the combined use of fluorescent lipid analogs and the environmental dye laurdan has revealed different lipid phases that emerge in the PM of Arabidopsis (Arabidopsis thaliana) protoplasts during restoration of the cell wall (Blachutzik et al., 2012). Despite these details, necessary data concerning the presence and in vivo characterization of Lo domains at a micrometer to nanometer scale are still lacking.The importance of a more refined resolution for observing Lo domains was proposed in several recent reviews (Bagatolli, 2006; Duggan et al., 2008; García-Sáez and Schwille, 2010; Owen et al., 2010a; Stöckl and Herrmann, 2010; Klenerman et al., 2011). Although the physical properties of biological membranes have been studied in situ by various techniques, including two-channel ratiometric microscopy (Owen et al., 2010c) and microscopy imaging of partitioning of fluorescent lipids and proteins (Rosetti et al., 2010) or environmentally sensitive probes (Parasassi et al., 1990; Jin et al., 2006), membrane segregation into microscopic Lo- and Ld-like phases has been difficult to observe in living cells. Furthermore, only a few studies have demonstrated that a microscopic phase separation involving an ordered phase similar to the Lo domain of model membranes could occur in biomembranes using PM giant vesicles (Baumgart et al., 2007; Lingwood et al., 2008; Sengupta et al., 2008). A potentially powerful approach for imaging small ordered membrane domains relies on environment-sensitive probes coupled with fluorescence spectroscopy (Gaus et al., 2003, 2006; Oncul et al., 2010). In particular, analysis of the fluorescence of the di-4-ANEPPDHQ probe, which exhibits an emission shift independent of local chemical composition under different lipid packing conditions (Jin et al., 2005; Demchenko et al., 2009; Dinic et al., 2011), recently enabled the imaging of plant membrane domains at the micrometer scale (Liu et al., 2009). The relevance of this approach has been confirmed by mapping membrane domains using generalized anisotropy-based images of di-4-ANEPPDHQ-stained T cell immunological synapses (Owen et al., 2010c), together with the characterization of membrane organization of nonadherent cells (such as living zebrafish embryo tissues) labeled with this dye (Owen et al., 2012a).The function of dynamic PM compartmentalization in the detection and transduction of environmental signals in plant cells has only recently begun to emerge, along with a crucial role for sterols in this organization (for review, see Zappel and Panstruga, 2008; Mongrand et al., 2010; Simon-Plas et al., 2011). These observations make it indispensable to align how the surface membrane of living cells might reorganize during signaling with the membrane raft hypothesis. To investigate possible modifications of membrane organization during the initial steps of plant defense signaling, tobacco cells were treated with two well-described elicitors of defense reaction, cryptogein, a small protein able to trigger an hypersensitive reaction (HR) and an acquired resistance in tobacco plants (Ponchet et al., 1999; Garcia Brugger et al., 2006) together with a widely described signaling cascade in tobacco suspension cells, and flg22 (a 22-amino acid peptide corresponding to a conserved domain of bacterial flagellin). The latter peptide is also a potent elicitor in plants, yet it does not induce an HR type of necrosis (Gomez-Gomez and Boller, 2002; Chinchilla et al., 2007). The study of cryptogein response reveals that the earliest steps of the signal transduction pathway mainly involve PM activities (Ponchet et al., 1999; Garcia-Brugger et al., 2006). How the PM is laterally organized and possibly reorganized in response to this stress so it can efficiently trigger a signaling cascade remains unknown.Here, we have developed a confocal multispectral microscopy approach to generate in vivo ratiometric pictures of large areas of the tobacco cell PM labeled with di-4-ANEPPDHQ, allowing the in vivo characterization of the global level of order of this membrane. Although an increase in the proportion of ordered phase within the membrane transiently occurred in the early steps of the cryptogein and flg22 signaling cascades, the fluorescence recovery after photobleaching (FRAP) technique revealed an increase in PM fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of Lo phases on the membrane of living plant cells and monitored the variations induced by cryptogein elicitation. The results are discussed within the framework of the “membrane raft” hypothesis, in which we propose a new mechanism of signaling platform formation in the context of plant defense.     

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.     

The Role of a Potassium Transporter OsHAK5 in Potassium Acquisition and Transport from Roots to Shoots in Rice at Low Potassium Supply Levels

In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A β-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants.Potassium (K) is one of the three most important macronutrients and the most abundant cation in plants. As a major osmoticum in the vacuole, K drives the generation of turgor pressure, enabling cell expansion. In the vascular tissue, K is an important participant in the generation of root pressure (for review, see Wegner, 2014 [including his new hypothesis]). In the phloem, K is critical for the transport of photoassimilates from source to sink (Marschner, 1996; Deeken et al., 2002; Gajdanowicz et al., 2011). In addition, enhancing K absorption and decreasing sodium (Na) accumulation is a major strategy of glycophytes in salt stress tolerance (Maathuis and Amtmann, 1999; Munns and Tester, 2008; Shabala and Cuin, 2008).Plants acquire K through K-permeable proteins at the root surface. Since available K concentration in the soil may vary by 100-fold, plants have developed multiple K uptake systems for adapting to this variability (Epstein et al., 1963; Grabov, 2007; Maathuis, 2009). In a classic K uptake experiment in barley (Hordeum vulgare), root K absorption has been described as a high-affinity and low-affinity biphasic transport process (Epstein et al., 1963). It is generally assumed that the low-affinity transport system (LATS) in the roots mediates K uptake in the millimolar range and that the activity of this system is insensitive to external K concentration (Maathuis and Sanders, 1997; Chérel et al., 2014). In contrast, the high-affinity transport system (HATS) was rapidly up-regulated when the supply of exogenous K was halted (Glass, 1976; Glass and Dunlop, 1978).The membrane transporters for K flux identified in plants are generally classified into three channels and three transporter families based on phylogenetic analysis (Mäser et al., 2001; Véry and Sentenac, 2003; Lebaudy et al., 2007; Alemán et al., 2011). For K uptake, it was predicted that, under most circumstances, K transporters function as HATS, while K-permeable channels mediate LATS (Maathuis and Sanders, 1997). However, a root-expressed K channel in Arabidopsis (Arabidopsis thaliana), Arabidopsis K Transporter1 (AKT1), mediates K absorption over a wide range of external K concentrations (Sentenac et al., 1992; Lagarde et al., 1996; Hirsch et al., 1998; Spalding et al., 1999), while evidence is accumulating that many K transporters, including members of the K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) family, are low-affinity K transporters (Quintero and Blatt, 1997; Senn et al., 2001), implying that functions of plant K channels and transporters overlap at different K concentration ranges.Out of the three families of K transporters, cation proton antiporter (CPA), high affinity K/Na transporter (HKT), and KT/HAK/KUP, CPA was characterized as a K⁺(Na⁺)/H⁺ antiporter, HKT may cotransport Na and K or transport Na only (Rubio et al., 1995; Uozumi et al., 2000), while KT/HAK/KUP were predicted to be H⁺-coupled K⁺ symporters (Mäser et al., 2001; Lebaudy et al., 2007). KT/HAK/KUP were named by different researchers who first identified and cloned them (Quintero and Blatt, 1997; Santa-María et al., 1997). In plants, the KT/HAK/KUP family is the largest K transporter family, including 13 members in Arabidopsis and 27 members in the rice (Oryza sativa) genome (Rubio et al., 2000; Mäser et al., 2001; Bañuelos et al., 2002; Gupta et al., 2008). Sequence alignments show that genes of this family share relatively low homology to each other. The KT/HAK/KUP family was divided into four major clusters (Rubio et al., 2000; Gupta et al., 2008), and in cluster I and II, they were further separated into A and B groups. Genes of cluster I or II likely exist in all plants, cluster III is composed of genes from both Arabidopsis and rice, while cluster IV includes only four rice genes (Grabov, 2007; Gupta et al., 2008).The functions of KT/HAK/KUP were studied mostly in heterologous expression systems. Transporters of cluster I, such as AtHAK5, HvHAK1, OsHAK1, and OsHAK5, are localized in the plasma membrane (Kim et al., 1998; Bañuelos et al., 2002; Gierth et al., 2005) and exhibit high-affinity K uptake in the yeast Saccharomyces cerevisiae (Santa-María et al., 1997; Fu and Luan, 1998; Rubio et al., 2000) and in Escherichia coli (Horie et al., 2011). Transporters of cluster II, like AtKUP4 (TINY ROOT HAIRS1, TRH1), HvHAK2, OsHAK2, OsHAK7, and OsHAK10, could not complement the K uptake-deficient yeast (Saccharomyces cerevisiae) but were able to mediate K fluxes in a bacterial mutant; they might be tonoplast transporters (Senn et al., 2001; Bañuelos et al., 2002; Rodríguez-Navarro and Rubio, 2006). The function of transporters in clusters III and IV is even less known (Grabov, 2007).Existing data suggest that some KT/HAK/KUP transporters also may respond to salinity stress (Maathuis, 2009). The cluster I transporters of HvHAK1 mediate Na influx (Santa-María et al., 1997), while AtHAK5 expression is inhibited by Na (Rubio et al., 2000; Nieves-Cordones et al., 2010). Expression of OsHAK5 in tobacco (Nicotiana tabacum) BY2 cells enhanced the salt tolerance of these cells by accumulating more K without affecting their Na content (Horie et al., 2011).There are only scarce reports on the physiological function of KT/HAK/KUP in planta. In Arabidopsis, mutation of AtKUP2 (SHORT HYPOCOTYL3) resulted in a short hypocotyl, small leaves, and a short flowering stem (Elumalai et al., 2002), while a loss-of-function mutation of AtKUP4 (TRH1) resulted in short root hairs and a loss of gravity response in the root (Rigas et al., 2001; Desbrosses et al., 2003; Ahn et al., 2004). AtHAK5 is the only system currently known to mediate K uptake at concentrations below 0.01 mm (Rubio et al., 2010) and provides a cesium uptake pathway (Qi et al., 2008). AtHAK5 and AtAKT1 are the two major physiologically relevant molecular entities mediating K uptake into roots in the range between 0.01 and 0.05 mm (Pyo et al., 2010; Rubio et al., 2010). AtAKT1 may contribute to K uptake within the K concentrations that belong to the high-affinity system described by Epstein et al. (1963).Among all 27 members of the KT/HAK/KUP family in rice, OsHAK1, OsHAK5, OsHAK19, and OsHAK20 were grouped in cluster IB (Gupta et al., 2008). These four rice HAK members share 50.9% to 53.4% amino acid identity with AtHAK5. OsHAK1 was expressed in the whole plant, with maximum expression in roots, and was up-regulated by K deficiency; it mediated high-affinity K uptake in yeast (Bañuelos et al., 2002). In this study, we examined the tissue-specific localization and the physiological functions of OsHAK5 in response to variation in K supply and to salt stress in rice. By comparing K uptake and translocation in OsHAK5 knockout (KO) mutants and in OsHAK5-overexpressing lines with those in their respective wild-type lines supplied with different K concentrations, we found that OsHAK5 not only mediates high-affinity K acquisition but also participates in root-to-shoot K transport as well as in K-regulated salt tolerance.