Focus Issue on the Plant Physiology of Global Change: A Nitrogen-Regulated Glutamine Amidotransferase (GAT1_2.1) Represses Shoot Branching in Arabidopsis

Shoot branching in plants is regulated by many environmental cues and by specific hormones such as strigolactone (SL). We show that the GAT1_2.1 gene (At1g15040) is repressed over 50-fold by nitrogen stress, and is also involved in branching control. At1g15040 is predicted to encode a class I glutamine amidotransferase (GAT1), a superfamily for which Arabidopsis (Arabidopsis thaliana) has 30 potential members. Most members can be categorized into known biosynthetic pathways, for the amidation of known acceptor molecules (e.g. CTP synthesis). Some members, like GAT1_2.1, are of unknown function, likely involved in amidation of unknown acceptors. A gat1_2.1 mutant exhibits a significant increase in shoot branching, similar to mutants in SL biosynthesis. The results suggest that GAT1_2.1 is not involved in SL biosynthesis since exogenously applied GR24 (a synthetic SL) does not correct the mutant phenotype. The subfamily of GATs (GATase1_2), with At1g15040 as the founding member, appears to be present in all plants (including mosses), but not other organisms. This suggests a plant-specific function such as branching control. We discuss the possibility that the GAT1_2.1 enzyme may activate SLs (e.g. GR24) by amidation, or more likely could embody a new pathway for repression of branching.Shoot branching plays an important role in establishing plant body plans during development and growth, also conferring the flexibility for plants to respond to environmental stresses. The control of bud growth/branching has been studied for many decades with much interest stemming from its value in agriculture. Indeed, many of our domesticated crops have been bred for modified branching to optimize yields. In early studies, auxin synthesized in the shoot apex was proposed to act indirectly to inhibit bud outgrowth, while cytokinin (CK) synthesized in the roots promoted bud outgrowth (Domagalska and Leyser, 2011). Studies on auxin inhibition suggested there should be another signal mediating bud growth control (Hayward et al., 2009; Stirnberg et al., 2010; Domagalska and Leyser, 2011). In the past decade, studies in Arabidopsis (Arabidopsis thaliana) and other plants have addressed this signal. Identification and characterization of mutants with increased branching in garden pea (Pisum sativum), Arabidopsis, rice (Oryza sativa), and Petunia hybrida demonstrated the existence of a long-distance signaling pathway that regulates shoot branching (Beveridge et al., 1996, 1997; Napoli, 1996; Stirnberg et al., 2002, 2007; Sorefan et al., 2003; Booker et al., 2004; Arite et al., 2007; Gomez-Roldan et al., 2008; Umehara et al., 2008, 2010; Lin et al., 2009; Liu et al., 2009, 2011; Zhang et al., 2010). Later, studies on pea (Gomez-Roldan et al., 2008) and rice (Umehara et al., 2008) demonstrated unequivocally that this hormone (or its precursor) is strigolactone (SL). Currently, it is proposed that SL acts downstream of auxin to regulate bud outgrowth (Brewer et al., 2009). It is also likely that SL and auxin have the capacity to modulate each other’s levels and distribution in a dynamic feedback loop required for the branching control (Ferguson and Beveridge, 2009; Hayward et al., 2009; Stirnberg et al., 2010). The interaction between SL and CK during bud outgrowth is less understood, although recent studies in pea indicate that SL and CK act antagonistically on bud growth (Dun et al., 2012).Branching is also modulated in response to environmental conditions, including nutrient supply. Generally, nutrient deficiency in soil causes a reduction in shoot to root ratio, resulting in decreased shoot branching (Lafever, 1981). Under nitrogen or phosphate limitation, elevated levels of SL repress shoot branching in rice, tomato (Solanum lycopersicum), and Arabidopsis (Yoneyama et al., 2007; López-Ráez et al., 2008; Umehara et al., 2008, 2010; Kohlen et al., 2011), and possibly increase lateral root formation (Ruyter-Spira et al., 2011). This makes sense physiologically, diverting resources to roots from shoots to scavenge more nutrients. The basis for modulation of SL levels or nutrient-dependent branching control is not understood.Here, we report a novel gene, GAT1_2.1 (At1g15040), predicted to encode a class I Gln amidotransferase (GAT1) in Arabidopsis, is highly repressed by long-term nitrogen stress (down 57-fold), and that mutation of this gene leads to an enhanced branching phenotype. Thus, this gene may present a link between the nitrogen stress response and branching control.     

A Role for MORE AXILLARY GROWTH1 (MAX1) in Evolutionary Diversity in Strigolactone Signaling Upstream of MAX2

Strigolactones (SLs) are carotenoid-derived phytohormones with diverse roles. They are secreted from roots as attractants for arbuscular mycorrhizal fungi and have a wide range of endogenous functions, such as regulation of root and shoot system architecture. To date, six genes associated with SL synthesis and signaling have been molecularly identified using the shoot-branching mutants more axillary growth (max) of Arabidopsis (Arabidopsis thaliana) and dwarf (d) of rice (Oryza sativa). Here, we present a phylogenetic analysis of the MAX/D genes to clarify the relationships of each gene with its wider family and to allow the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, there may be a diversity of SL-like compounds, the response to which requires a D14/D14-like protein. This ancestral system could have been refined toward distinct ligand-specific pathways channeled through MAX2, the most downstream known component of SL signaling. MAX2 is tightly conserved among land plants and is more diverged from its nearest sister clade than any other SL-related gene, suggesting a pivotal role in the evolution of SL signaling. By contrast, the evidence suggests much greater flexibility upstream of MAX2. The MAX1 gene is a particularly strong candidate for contributing to diversification of inputs upstream of MAX2. Our functional analysis of the MAX1 family demonstrates the early origin of its catalytic function and both redundancy and functional diversification associated with its duplication in angiosperm lineages.Strigolactones (SLs) are carotenoid-derived terpenoid lactones, which have been identified as signaling molecules in several areas of plant biology. SLs were first identified as germination stimulants for seeds of plants in the genus Striga (Cook et al., 1966). Striga spp. and related Orobanchaceae are parasitic weeds that germinate in response to host plant root exudates and develop haustoria to penetrate the host tissue and draw nutrients. Striga spp. are major agricultural pests across much of tropical and subtropical Asia and are present in two-thirds of arable land in Africa, where they are the greatest biological cause of crop damage (Humphrey and Beale, 2006). The secretion of SLs by roots, despite its exploitation by Striga spp., has been preserved because it also serves to recruit arbuscular mycorrhizal (AM) fungi (Akiyama et al., 2005). AM fungi form symbiotic associations with most land plants, whereby the plant gains access to mineral nutrients, particularly phosphate, absorbed by the fungal hyphae, and in exchange the fungus gains fixed carbon from the plant. In several flowering plant species, SL production is correspondingly increased when phosphate availability is limiting, thereby presumably increasing fungal recruitment (Yoneyama et al., 2007, 2012).AM symbioses can be traced back to the origin of land plants, between 360 to 450 million years ago, and are thought to have facilitated plant colonization of the terrestrial environment (Simon et al., 1993). Although AM symbiosis has been lost from some lineages, such as Brassicaceae, it is still widespread, with 80% of land plants able to form associations with AM fungi (Schüssler et al., 2001). In support of a similarly ancient origin for SL secretion, the liverwort Marchantia polymorpha and the moss Physcomitrella patens, both basal land plant groups, have been shown to produce SLs (Proust et al., 2011; Delaux et al., 2012). Furthermore, the presence of SLs in charophyte algae indicates that SL production may predate the emergence of land plants (Delaux et al., 2012), and Chara corallina responds to SL treatment by producing longer rhizoids (Delaux et al., 2012). In P. patens, SLs appear to act as intercolony coordination signals, regulating colony growth and competition by controlling flexible developmental processes such as protonemal branching (Proust et al., 2011; Delaux et al., 2012). In flowering plants, SLs have also been implicated in development, including several processes regulated in response to phosphate limitation (Kohlen et al., 2011; Ruyter-Spira et al., 2011). In particular, SLs play important roles in the regulation of shoot branching in higher plants (Gomez-Roldan et al., 2008; Umehara et al., 2008). It is through work on their effects on shoot branching that some of the genes in the SL pathway were first identified.Arabidopsis (Arabidopsis thaliana) MORE AXILLARY GROWTH (MAX) mutants show increased branching and reduced stature relative to wild-type plants, and analogous phenotypes have been identified in pea (Pisum sativum; RAMOSUS [RMS]), petunia (Petunia hybrida; DECREASED APICAL DOMINANCE [DAD]), and rice (Oryza sativa; DWARF [D] or HIGH TILLERING DWARF) mutants. So far, six MAX/RMS/DAD/D genes have been identified, with roles in SL biosynthesis or signaling. MAX3/RMS5/HIGH TILLERING DWARF1/D17 (Booker et al., 2004; Johnson et al., 2006; Zou et al., 2006) and MAX4/RMS1/DAD1/D10 (Sorefan et al., 2003; Snowden et al., 2005; Arite et al., 2007) encode carotenoid cleavage dioxygenases (CCD7 and CCD8, respectively). These enzymes are capable of sequentially cleaving the carotenoid 9-cis-β-carotene to produce a novel compound, carlactone, a putative strigolactone intermediate (Alder et al., 2012). Another biosynthetic gene, D27, was originally mutationally defined in rice (Lin et al., 2009), and reverse genetic approaches in Arabidopsis indicate a similar function in this species (Waters et al., 2012a). D27 is an iron-containing protein with isomerase activity that can produce the 9-cis-β-carotene substrate for MAX3 from all-trans-β-carotene (Alder et al., 2012). The fourth gene known to be involved in SL biosynthesis, MAX1, encodes a cytochrome p450 monooxygenase belonging to the CYP711 clan (Booker et al., 2005). Mutant phenotypes associated with this gene have so far only been identified in one species, Arabidopsis, although the gene is present in all tracheophytes (Nelson et al., 2008). The excessive-branching phenotypes associated with mutations in all of these genes can be rescued by exogenous application of SL, while mutants in the two remaining genes in the pathway are SL insensitive. D14 encodes an α/β hydrolase, which is proposed to act in signaling or in the hydrolysis of SLs to an active compound and provides specificity to signaling via MAX2/RMS4/D3, an F-box protein that mediates both SL signaling and signaling of karrikins (Stirnberg et al., 2002, 2007; Ishikawa et al., 2005; Johnson et al., 2006; Arite et al., 2009; Hamiaux et al., 2012; Waters et al., 2012b). Karrikins are compounds structurally related to SLs that are found in smoke and act as germination stimulants for plants that colonize ground cleared by forest fires (Nelson et al., 2010; Waters et al., 2012b).Homology searches described in the original publications for each of the MAX/D genes revealed two general patterns. MAX1, MAX3, and MAX4 are members of widespread gene families and are more closely related to nonplant sequences than to other plant genes (Sorefan et al., 2003; Booker et al., 2005). By contrast, MAX2, D14, and D27 are members of plant-specific gene families (Stirnberg et al., 2002; Arite et al., 2009; Lin et al., 2009). These contrasting patterns of SL pathway gene ancestry and the diverse biological roles of SLs present interesting evolutionary questions. The identification of SLs and SL responses in charophyte algae demonstrate their early evolution, but these species lack many of the genes required for SL synthesis and signaling in angiosperms. In an attempt to trace the evolution of the angiosperm SL pathway, we conducted a phylogenetic analysis of the known SL biosynthesis and signaling genes, allowing the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, the angiosperm pathway seems to have been defined by the rapid evolution of MAX2 in early land plants. Upstream of MAX2, there appears to be much greater flexibility, especially in the requirements for the synthesis of SLs. We present evidence for the contribution of MAX1 to this flexibility. Our functional analysis of MAX1 orthologs from phylogenetically diverse species demonstrates the early origin of its catalytic activity and both redundancy and functional diversification associated with its duplication in angiosperm lineages.     

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.