Focus on Weed Control: Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.Glyphosate [N-(phosphonomethyl) Gly] is the most widely used agricultural pesticide globally (Duke and Powles, 2008). Originally, being a nonselective herbicide, its use was limited to vegetation management in noncrop areas; however, introduction of glyphosate-resistant (GR) crops in the late 1990s, coupled with their exceptional adoption, led to accelerated use totaling approximately 128 million ha worldwide in 2012 (James, 2012). GR crop technology has made a significant contribution to global agriculture and the environment, as it not only increased farm income by $32.2 billion (Brookes and Barfoot, 2013), but also moderated the negative environmental impacts of mechanical weed management practices (Gardner and Nelson, 2008; Bonny, 2011). Glyphosate offers a simple, effective, and economic weed management option in GR crops. In addition, it provides immense value in no-till crop production systems by enabling soil and moisture conservation. However, due to intensive glyphosate selection pressure, several weed populations globally have evolved resistance through a variety of mechanisms. Globally, herbicide resistance, in particular the recent proliferation of glyphosate resistance in weed species, is a major crop protection threat; nearly two dozen GR weed species have been reported in the last 15 years (Heap, 2014).Glyphosate, an aminophosphonic analog of the natural amino acid Gly, nonselectively inhibits 5-Enolpyruvylshikimate-3-Phosphate synthase (EPSPS) in plants, preventing the biosynthesis of the aromatic amino acids Phe, Tyr, and Trp (Steinrücken and Amrhein, 1980), resulting in the death of glyphosate-sensitive individuals. In plants, EPSPS is one of the key enzymes in the shikimate pathway (Herrmann and Weaver, 1999), and glyphosate inhibits EPSPS by binding to EPSPS-shikimate-3-P binary complex forming an EPSPS-shikimate-3-P-glyphosate complex (Alibhai and Stallings, 2001). Bradshaw et al. (1997) hypothesized against the likelihood of weeds evolving resistance to glyphosate, primarily because of its complex biochemical interactions in the shikimate pathway and also due to the absence of known glyphosate metabolism in plants. Nonetheless, several cases of glyphosate resistance, as a result of difference in glyphosate translocation (Preston and Wakelin, 2008) or mutations in the EPSPS, were confirmed (Baerson et al., 2002). More importantly, duplication/amplification of the EPSPS appears to be the basis for glyphosate resistance in several weeds (Sammons and Gaines, 2014). Here, we use duplication to refer to the formation of first repetition of a chromosomal segment and amplification to refer to increase in number of the repetitions (more than two repetitions of a chromosomal segment) under positive selection. The first case of EPSPS amplification as a basis for glyphosate resistance was reported in an Amaranthus palmeri population from GA (Gaines et al., 2010). In this A. palmeri population, there is a massive increase (>100-fold relative to glyphosate-susceptible [GS] plants) in EPSPS copies, and these copies are dispersed throughout the genome (Gaines et al., 2010).Field-evolved GR Kochia scoparia populations were first reported in western Kansas in 2007 (Heap, 2014). We previously determined that evolution of GR populations of K. scoparia in the U.S. Great Plains is also due to amplification of the EPSPS (A. Wiersma and P. Westra, unpublished data). Unlike in GR A. palmeri, we found relative EPSPS:acetolactate synthase (ALS) copies ranging from three to nine in GR K. scoparia populations. While it quickly became widespread in the region, its presence was reported in another five Great Plains states by 2013 (Heap, 2014). GR K. scoparia populations we tested were 3- to 11-times resistant (population level) to glyphosate compared with a GS population (Godar, 2014), and EPSPS expression positively correlated with genomic EPSPS copy number (A. Wiersma and P. Westra, unpublished data). Here, we reveal the genomic organization of the amplified EPSPS copies in two GR K. scoparia populations, an alternative mechanism of gene amplification to that reported in GR A. palmeri.     

Focus on Weed Control: In Vivo 31P-Nuclear Magnetic Resonance Studies of Glyphosate Uptake,Vacuolar Sequestration,and Tonoplast Pump Activity in Glyphosate-Resistant Horseweed

Horseweed (Conyza canadensis) is considered a significant glyphosate-resistant (GR) weed in agriculture, spreading to 21 states in the United States and now found globally on five continents. This laboratory previously reported rapid vacuolar sequestration of glyphosate as the mechanism of resistance in GR horseweed. The observation of vacuole sequestration is consistent with the existence of a tonoplast-bound transporter. ³¹P-Nuclear magnetic resonance experiments performed in vivo with GR horseweed leaf tissue show that glyphosate entry into the plant cell (cytosolic compartment) is (1) first order in extracellular glyphosate concentration, independent of pH and dependent upon ATP; (2) competitively inhibited by alternative substrates (aminomethyl phosphonate [AMPA] and N-methyl glyphosate [NMG]), which themselves enter the plant cell; and (3) blocked by vanadate, a known inhibitor/blocker of ATP-dependent transporters. Vacuole sequestration of glyphosate is (1) first order in cytosolic glyphosate concentration and dependent upon ATP; (2) competitively inhibited by alternative substrates (AMPA and NMG), which themselves enter the plant vacuole; and (3) saturable. ³¹P-Nuclear magnetic resonance findings with GR horseweed are consistent with the active transport of glyphosate and alternative substrates (AMPA and NMG) across the plasma membrane and tonoplast in a manner characteristic of ATP-binding cassette transporters, similar to those that have been identified in mammalian cells.Glyphosate is arguably the world’s most important herbicide (Duke and Powles, 2008). Environmental factors affecting its uptake and translocation in higher plants have been widely studied (Kells and Rieck, 1979; Coupland, 1983; Devine et al., 1983; Masiunas and Weller, 1988; Zhou et al., 2007). Notably, the role of light is important for effective uptake and translocation, suggesting that metabolic energy plays a role in this process (Simarmata et al., 2003; Shaner et al., 2005). Death of the whole plant requires effective glyphosate translocation from source to sink tissue, a process requiring ATP to maintain Suc gradients, which drive phloem movement (Bromilow et al., 1990; Dill et al., 2010).Weed species have developed glyphosate-resistant (GR) biotypes during the past decade (Heap, 2014). This has spurred interest in factors that may contribute to resistant attribute(s) as well as methods that can be used to screen plants for herbicide toxicity (Shaner, 2010). Resistance mechanisms have been reported for horseweed (Conyza canadensis; Feng et al., 2004; Zelaya et al., 2004; Ge et al., 2010, 2011), Palmer amaranth (Amaranthus palmeri; Gaines et al., 2010), and ryegrass (Lolium rigidum and Lolium multiflorum; Powles et al., 1998; Perez et al., 2004; Ge et al., 2012).Since glyphosate is foliar applied, glyphosate toxicity involves a multistep delivery process. Glyphosate must traverse the nonliving structures of the leaf cuticle and the cell walls of the epidermis, apoplast, and mesophyll prior to accessing the phloem for transport to sink tissues (Bromilow et al., 1990; Bromilow and Chamberlain, 2000). Indeed, restriction of glyphosate delivery to the plant cell cytoplasm (and chloroplast) by any means is, in itself, a resistance mechanism (Shaner, 2009; Ge et al., 2013). Elucidation of key factors governing delivery to the intracellular milieu of plant source leaves is critical for developing a complete understanding of the mechanism(s) of resistance to glyphosate.Glyphosate’s phosphono group offers the opportunity to employ in vivo ³¹P-NMR spectroscopy to track glyphosate movement and metabolism, additionally including monitoring of cellular pH, and gradients therein, and ATP levels, both indicators of tissue viability (Roberts, 1984). This laboratory has extended the ³¹P-NMR approach initially used by Gout et al. (1992) with suspension-cultured sycamore (Acer pseudoplatanus) cells. The initial findings, that source and sink leaf tissue from GR horseweed rapidly and avidly sequestered glyphosate within the vacuole compartment and that leaf tissue from glyphosate-sensitive (GS) horseweed did not, led to the hypothesis that vacuole sequestration was a key, perhaps the dominant, component of the resistance mechanism (Ge et al., 2010). It was then shown that GR horseweed acclimated and maintained at cold temperature (approximately 10°C–12°C) did not rapidly and avidly sequester glyphosate within the vacuole. Importantly, under such conditions, GR horseweed succumbed to the toxic effects of glyphosate. In short, by preventing glyphosate sequestration, GR horseweed became glyphosate sensitive, a laboratory finding confirmed in the field (Ge et al., 2011).The proposition that, by limiting the herbicide available for translocation, glyphosate vacuole sequestration could serve as an important if not dominant resistance mechanism was further strengthened by experiments that showed vacuolar glyphosate sequestration correlated with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America, and Europe (Ge et al., 2012). However, ³¹P-NMR studies of other weeds revealed that in some species, for example, Palmer amaranth, waterhemp (Amaranthus tuberculatus), and johnsongrass (Sorghum halepense), resistance correlated strongly with a lack of glyphosate uptake into the plant cell, a frontline resistance mechanism (Ge et al., 2013).Throughout these previous ³¹P-NMR studies, the finding that plants could regulate the compartmental access of glyphosate led us to speculate that the apoplast, tonoplast, and perhaps chloroplast possessed glyphosate-active transporters whose up-regulation or down-regulation and/or expression would confer resistance (Ge et al., 2010, 2011, 2012, 2013). This hypothesis motivated additional in vivo ³¹P-NMR experiments to further describe the determinants of glyphosate delivery in horseweed leaf tissue. Specifically, experiments with GR horseweed were designed with the goal of probing the transporter hypothesis.Findings from these experiments are reported herein and are consistent with the existence of a tonoplast transporter that is responsible for glyphosate resistance via vacuole sequestration. As described here, vacuole sequestration requires ATP, is active for multiple substrates, and shows substrate competition. Furthermore, glyphosate entry into the cell can be markedly inhibited by vanadate pretreatment. These characteristics are similar to those of ATP-binding cassette transporters in plants (Hetherington et al., 1998; Rea, 2007; Verrier et al., 2008; Prosecka et al., 2009; Conte and Lloyd, 2011) and mammalian cells (van de Ven et al., 2009; Ernst et al., 2010).     

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.     

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.     

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.