The Endocytosis of Cellulose Synthase in Arabidopsis Is Dependent on μ2, a Clathrin-Mediated Endocytosis Adaptin

Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified μ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) μ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that μ2 interacts with multiple CESA proteins through the μ-homology domain of μ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, μ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of μ2 resulted in defects in bulk endocytosis. Furthermore, loss of μ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.Cellulose microfibrils, as the major load-bearing polymers in plant cell walls, are the predominant component that enforces asymmetric cell expansion (Green, 1962). In higher plants, cellulose is synthesized by multimeric rosettes, which are also referred to as cellulose synthase complexes (CSCs; Kimura et al., 1999). Genetic and coimmunoprecipitation studies have indicated that CELLULOSE SYNTHASE1 (CESA1), CESA3, and CESA6-like (CESA6, CESA2, CESA5, and CESA9) isoforms are constituents of CSCs during primary cell wall synthesis (Persson et al., 2005; Desprez et al., 2007; Persson et al., 2007; Wang et al., 2008), whereas CESA4, CESA7, and CESA8 are implicated in the cellulose synthesis of secondary cell walls (Taylor et al., 1999, 2003; Brown et al., 2005). Knowledge about cellulose synthesis has recently been enhanced by the development of a system whereby the dynamics of CESA can be imaged in living cells (Paredez et al., 2006; Desprez et al., 2007). In agreement with earlier transmission electron microscopy studies in which rosettes were visualized in Golgi cisternae, vesicles, and at the plasma membrane (Haigler and Brown, 1986), fluorescent protein tagging of CESA has identified CESA localization at the plasma membrane, in Golgi bodies, and in small intracellular compartments (Paredez et al., 2006; Desprez et al., 2007; Crowell et al., 2009; Gutierrez et al., 2009; Gu et al., 2010; Lei et al., 2012; Li et al., 2012b).Assuming that cellulose synthesis occurs solely at the plasma membrane, the trafficking of CSCs to and from the plasma membrane may act as a significant regulatory mechanism. Although the mechanistic details of CESA trafficking are lacking, live cell imaging has shown that CESA localizes to various subcellular compartments. A subset of CESAs colocalize with markers of the trans-Golgi network (TGN)/early endosome (EE), an organelle that is part of both the secretory and endocytic pathways in Arabidopsis (Arabidopsis thaliana; Dettmer et al., 2006; Lam et al., 2007; Crowell et al., 2009, 2010; Viotti et al., 2010). CESAs also localize to microtubule-associated cellulose synthase compartments (MASCs) and small CESA-containing compartments (SmaCCs). The exact function of SmaCCs/MASCs is unknown, but it has been proposed that SmaCCs/MASCs might result from the internalization of CSCs or might act in the delivery of CSCs to the plasma membrane (Crowell et al., 2009, 2010; Gutierrez et al., 2009).Clathrin-mediated endocytosis (CME) has been shown to be a major endocytic pathway in Arabidopsis (Holstein, 2002; Samaj et al., 2005; Dhonukshe et al., 2007; Kleine-Vehn and Friml, 2008; Chen et al., 2011; Beck et al., 2012; Wang et al., 2013), although there is also evidence of clathrin-independent endocytosis mechanisms (Bandmann and Homann, 2012). The function of many CME proteins has been extensively characterized in mammals (McMahon and Boucrot, 2011), and homologs of many CME components are encoded by the Arabidopsis genome, including multiple copies of clathrin H chain and clathrin light chain (CLC), all four subunits of the heterotetrameric ADAPTOR PROTEIN COMPLEX2 (AP2) complex, dynamin-related proteins, and accessory proteins such as AP180 (Holstein, 2002; Chen et al., 2011); however, many CME components have yet to be characterized in plants.It has been suggested that CME might also function in controlling cell wall metabolism. For example, dividing and growing cells internalize cross-linked cell wall pectins, which might allow for cell wall remodeling (Baluska et al., 2002, 2005; Samaj et al., 2004). Moreover, the importance of endocytosis for cell wall morphogenesis is apparent from the functional characterization of proteins involved in CME. A dynamin-related protein, DRP1A, plays a significant role in endocytosis and colocalizes with CLC (Collings et al., 2008; Konopka and Bednarek, 2008). Defective endocytosis in RADIAL SWELLING9 (rsw9) plants, which contain a mutation in DRP1A, results in cellulose deficiency and defects in cell elongation (Collings et al., 2008). A mutation in rice, brittle culm3 (bc3), was mapped to the dynamin-related gene OsDRP2A, which has been proposed to function in CME. The brittle-culm phenotype in this mutant was attributed to cellulose deficiency (Xiong et al., 2010). Although the abundance of OsCESA4 was also altered in bc3, it remains unclear whether the cellulose deficiency of either bc3 or rsw9 results directly from perturbations in CESA trafficking.To identify proteins involved in the regulation of cellulose biosynthesis, a yeast two-hybrid (Y2H) screen was performed in which the central domain of CESA6 (CESA6CD) was used as bait to screen an Arabidopsis complementary DNA library for potential interaction partners of CESA6 (Gu et al., 2010; Gu and Somerville, 2010). The Y2H screen identified μ2 as a putative interaction partner of CESA6CD. The mammalian homolog of μ2 is the medium subunit of the tetrameric AP2, which acts as the core of the CME machinery by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis (Jackson et al., 2010; McMahon and Boucrot, 2011; Cocucci et al., 2012). In this study, we provide evidence that μ2 plays a role in CME in Arabidopsis, that CESAs are a new set of CME cargo proteins, and that plant cells might regulate cellulose synthesis by controlling the abundance of CSCs at the plasma membrane through CME. To our knowledge, this study is the first to show the affect of an AP2 complex component on endocytosis in plants and the first to visualize an AP2 complex component in living plant cells. Furthermore, our data suggest that the role of AP2 in plants may differ from what has been shown in animals.     

Clathrin Light Chains Regulate Clathrin-Mediated Trafficking,Auxin Signaling,and Development in Arabidopsis

Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.     

Hitching a Ride on Vesicles: Cauliflower Mosaic Virus Movement Protein Trafficking in the Endomembrane System

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) μA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.Membrane trafficking is essential in eukaryotic cells. Cellular membranes serve as a delivery system for newly synthesized proteins such as transporters and receptors exiting the endoplasmic reticulum after proper folding. They then transit through the Golgi complex, reaching the plasma membrane (PM) or the tonoplast via intermediate endomembrane compartments. Receptors and transporters returning from the PM are either recycled or targeted to the vacuole for degradation. Delivery and recycling sorting pathways overlap in the trans-Golgi network (TGN)/early endosome (EE), an intermediate compartment for both exocytosis and endocytosis (Reyes et al., 2011). In plant systems, the endoplasmic reticulum and PM provide membrane continuity between cells through the connections made by plasmodesmata (PD), cytoplasmic channels that regulate traffic in the symplasm (Maule et al., 2011).The selective transport of macromolecules between different compartments of the endomembrane system is mediated by coat proteins promoting the generation of small cargo-trafficking coated vesicles (Spang, 2008). The recognition and recruitment of cargo proteins are mediated by so-called adaptor complexes (AP complexes [AP-1–AP-4]; Robinson, 2004) one of which, AP-1, is localized on the TGN/EE and endosomes, whereas AP-2 is in the PM. The μ-subunit of AP complexes is devoted to cargo protein selection via a specific and well-characterized interaction with a Tyr-sorting signal, YXXΦ, where Φ is a bulky hydrophobic residue and X is any amino acid (Bonifacino and Dell’Angelica, 1999). YXXΦ motifs are present in the cytoplasmic tail of many proteins integral to the PM and TGN/EE and have been found in the movement proteins (MPs) of some viruses (Laporte et al., 2003; Haupt et al., 2005). Plant viruses are obligate parasites that exploit host components to move within the cell and from cell to cell into the vascular system for systemic invasion of the host. Virus movement, which requires the passage of macromolecules through PD connections, is mediated by one or more virus-encoded MPs with the help of the host cytoskeleton and/or endomembranes (Harries et al., 2010). While most MPs act to increase the size exclusion limit of PD to facilitate the passage of the viral nucleoprotein complex, other MPs are assembled in tubules that pass inside highly modified PD and transport encapsidated particles through their lumen.Here, we focus on this second group of tubule-forming MPs and examine the intracellular trafficking of cauliflower mosaic virus (CaMV) MP. The MP encoded by CaMV forms tubules guiding encapsidated virus particle cell-to-cell transport via an indirect MP-virion interaction (Stavolone et al., 2005; Sánchez-Navarro et al., 2010). However, how CaMV MP (and the other tubule-forming MPs) targets the PM and forms tubules remains to be elucidated. Tubule-forming MPs do not require an intact cytoskeleton for PM targeting (Huang et al., 2000; Pouwels et al., 2002) and/or tubule formation (Laporte et al., 2003). However, suppression of tubule formation upon treatment with brefeldin A (BFA), a specific inhibitor of secretion or endocytosis, suggests the involvement of the endomembrane system in correct functioning of some tubule-forming MPs (Huang et al., 2000; Laporte et al., 2003). In this study, we examined the three Tyr-sorting motifs in CaMV MP and show that each of the three domains interacts directly with subunit μ of an Arabidopsis (Arabidopsis thaliana) AP complex. Mutations in these domains revert in the viral context to maintain CaMV viability. MP is found in endosomal compartments labeled by AtRAB-F2b (ARA7) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). The presence of at least one functional YXXΦ domain is essential for the localization of MP to endosomes and for tubule assembly but is not required for MP targeting to the PM. We provide several lines of evidence to show CaMV MP trafficking in the endocytic pathway. Our findings are discussed in the light of the recent demonstration that the TGN/EE functions as a major hub controlling secretory and endocytic pathways in plants.     

Determining the Subcellular Location of Synthesis and Assembly of the Cell Wall Polysaccharide (1,3; 1,4)-β-d-Glucan in Grasses

The current dogma for cell wall polysaccharide biosynthesis is that cellulose (and callose) is synthesized at the plasma membrane (PM), whereas matrix phase polysaccharides are assembled in the Golgi apparatus. We provide evidence that (1,3;1,4)-β-d-glucan (mixed-linkage glucan [MLG]) does not conform to this paradigm. We show in various grass (Poaceae) species that MLG-specific antibody labeling is present in the wall but absent over Golgi, suggesting it is assembled at the PM. Antibodies to the MLG synthases, cellulose synthase-like F6 (CSLF6) and CSLH1, located CSLF6 to the endoplasmic reticulum, Golgi, secretory vesicles, and the PM and CSLH1 to the same locations apart from the PM. This pattern was recreated upon expression of VENUS-tagged barley (Hordeum vulgare) CSLF6 and CSLH1 in Nicotiana benthamiana leaves and, consistent with our biochemical analyses of native grass tissues, shown to be catalytically active with CSLF6 and CSLH1 in PM-enriched and PM-depleted membrane fractions, respectively. These data support a PM location for the synthesis of MLG by CSLF6, the predominant enzymatically active isoform. A model is proposed to guide future experimental approaches to dissect the molecular mechanism(s) of MLG assembly.     

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 Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1

Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1–AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.Membrane trafficking in plants shares many fundamental features with those in yeast and animals (Bassham et al., 2008). In general, vacuolar proteins are synthesized on the rough endoplasmic reticulum and then transported to vacuoles via the Golgi apparatus (Xiang et al., 2013; Robinson and Pimpl, 2014). The vacuolar trafficking in plants has been studied by monitoring the transport of reporter proteins to lytic vacuoles in vegetative cells and tissues (Jin et al., 2001; Pimpl et al., 2003; Miao et al., 2008; Niemes et al., 2010). Recently, seed storage proteins became a model cargo for monitoring the transport of endogenous vacuolar proteins in plants (Shimada et al., 2003a; Sanmartín et al., 2007; Isono et al., 2010; Pourcher et al., 2010; Uemura et al., 2012; Shirakawa et al., 2014). During seed maturation, a large amount of storage proteins are synthesized and sorted to specialized vacuoles, the protein storage vacuoles (PSVs). To properly deliver vacuolar proteins, sorting receptors play a critical role in recognizing the vacuole-targeting signal of the proteins. VACUOLAR PROTEIN SORTING10 and Man-6-P receptor function as sorting receptors for vacuolar/lysosomal proteins in the trans-Golgi network (TGN) of yeast and mammals, respectively. The best-characterized sorting receptors in plants are VACUOLAR SORTING RECEPTOR (VSR) family proteins (De Marcos Lousa et al., 2012). VSRs have been shown to function in sorting both storage proteins to PSVs (Shimada et al., 2003a; Fuji et al., 2007) and lytic cargos to lytic vacuoles (Zouhar et al., 2010).To sort the receptors in the TGN into vacuoles/lysosomes, the adaptor protein (AP) complex binds the cytosolic domain of the receptors. The AP complexes form evolutionarily conserved machinery that mediates the post-Golgi trafficking in eukaryotic cells (Robinson, 2004). There are five types of AP complexes, AP-1 to AP-5. The functions of AP-1, AP-2, and AP-3 have been established. AP-1 appears to be involved in trafficking between the TGN and endosomes (Hirst et al., 2012), AP-2 is involved in clathrin-mediated endocytosis (McMahon and Boucrot, 2011), and AP-3 is involved in protein trafficking from the TGN/endosomes to the vacuole/lysosomes (Dell’Angelica, 2009). However, little is known about AP-4 and AP-5. Mammalian AP-4 may be involved in basolateral sorting in polarized cells and in the transport of specific cargo proteins, such as the amyloid precursor protein APP, from the TGN to endosomes (Burgos et al., 2010). The fifth AP complex, AP-5, was recently identified, and its orthologs are widely conserved in the eukaryotic genomes (Hirst et al., 2011). The AP complexes exist as heterotetrameric proteins that consist of two large subunits (β1-5 and one each of ɣ/α/δ/ε/ζ), one medium subunit (µ1-5), and one small subunit (σ1-5). The sorting mechanism is best characterized for the medium (µ) subunit, which is known to recognize the Tyr-based YXXФ motif (where Ф represents Leu, Ile, Phe, Met, or Val) that is present in the cytosolic domains of cargo proteins (Ohno et al., 1995). Mutations of the YXXФ motif abolish the interaction with µ and alter the subcellular localization of the cargo proteins.The genome of Arabidopsis (Arabidopsis thaliana) contains all five sets of putative AP genes (Bassham et al., 2008; Hirst et al., 2011). The function of AP-4 in membrane trafficking and its physiological roles in plants are largely unknown. In this study, we identified and characterized the AP-4 complex in Arabidopsis. Mutants lacking the AP-4 subunits exhibited defects in VSR1-mediated vacuolar sorting of storage proteins in seeds. Our results provide new insights into the receptor-mediated vacuolar trafficking in post-Golgi pathways.     

Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.     

Functional Analysis of the Hydrophilic Loop in Intracellular Trafficking of Arabidopsis PIN-FORMED Proteins

Different PIN-FORMED proteins (PINs) contribute to intercellular and intracellular auxin transport, depending on their distinctive subcellular localizations. Arabidopsis thaliana PINs with a long hydrophilic loop (HL) (PIN1 to PIN4 and PIN7; long PINs) localize predominantly to the plasma membrane (PM), whereas short PINs (PIN5 and PIN8) localize predominantly to internal compartments. However, the subcellular localization of the short PINs has been observed mostly for PINs ectopically expressed in different cell types, and the role of the HL in PIN trafficking remains unclear. Here, we tested whether a long PIN-HL can provide its original molecular cues to a short PIN by transplanting the HL. The transplanted long PIN2-HL was sufficient for phosphorylation and PM trafficking of the chimeric PIN5:PIN2-HL but failed to provide the characteristic polarity of PIN2. Unlike previous observations, PIN5 showed clear PM localization in diverse cell types where PIN5 is natively or ectopically expressed and even polar PM localization in one cell type. Furthermore, in the root epidermis, the subcellular localization of PIN5 switched from PM to internal compartments according to the developmental stage. Our results suggest that the long PIN-HL is partially modular for the trafficking behavior of PINs and that the intracellular trafficking of PIN is plastic depending on cell type and developmental stage.     

Trans-Golgi Network-Located AP1 Gamma Adaptins Mediate Dileucine Motif-Directed Vacuolar Targeting in Arabidopsis

Membrane proteins on the tonoplast are indispensible for vacuolar functions in plants. However, how these proteins are transported to the vacuole and how they become separated from plasma membrane proteins remain largely unknown. In this study, we used Arabidopsis thaliana vacuolar ion transporter1 (VIT1) as a reporter to study the mechanisms of tonoplast targeting. We showed that VIT1 reached the tonoplast through a pathway involving the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN), prevacuolar compartment, and tonoplast. VIT1 contains a putative N-terminal dihydrophobic type ER export signal, and its N terminus has a conserved dileucine motif (EKQTLL), which is responsible for tonoplast targeting. In vitro peptide binding assays with synthetic VIT1 N terminus identified adaptor protein complex-1 (AP1) subunits that interacted with the dileucine motif. A deficiency of AP1 gamma adaptins in Arabidopsis cells caused relocation of tonoplast proteins containing the dileucine motif, such as VIT1 and inositol transporter1, to the plasma membrane. The dileucine motif also effectively rerouted the plasma membrane protein SCAMP1 to the tonoplast. Together with subcellular localization studies showing that AP1 gamma adaptins localize to the TGN, we propose that the AP1 complex on the TGN mediates tonoplast targeting of membrane proteins with the dileucine motif.     

CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.     

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.     

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H⁺-ATPases (PM H⁺-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H⁺-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H⁺-ATPase function. We find that SAUR19 stimulates PM H⁺-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H⁺-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H⁺-ATPases, and negatively regulate PM H⁺-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion.     

Glycosylphosphatidylinositol (GPI) Modification Serves as a Primary Plasmodesmal Sorting Signal

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pd-associated β-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd. Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd. Moreover, the GPI modification signal from both Pd- and non-Pd GPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd. As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-Pd GPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domain-swap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd- and non-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.Plant cells are interconnected with cross-wall membranous channels called plasmodesmata (Pd). Recent studies have shown that the region of the plasma membrane (PM) lining the Pd channel is a specialized membrane microdomain whose lipid and protein composition differs from the rest of the PM (Tilsner et al., 2011, 2016; Bayer et al., 2014; González-Solís et al., 2014; Grison et al., 2015). In a similar manner, the cell wall domain surrounding the Pd channel is specialized and, unlike the rest of the cell wall, is devoid of cellulose, rich in pectin, and contains callose (an insoluble β-1,3-glucan; Zavaliev et al., 2011; Knox and Benitez-Alfonso, 2014). In response to physiological signals, callose can be transiently deposited and degraded at Pd, which provides a mechanism for controlling the Pd aperture in diverse developmental and stress-related processes (Zavaliev et al., 2011). Control of Pd functioning is mediated by proteins that are specifically targeted to Pd. Plasmodesmal proteins localized to the PM domain of Pd can be integral transmembrane proteins, such as Pd-localized proteins (Thomas et al., 2008), the receptor kinase ARABIDOPSIS CRINKLY4 (Stahl et al., 2013), and callose synthases (Vatén et al., 2011). Alternatively, Pd proteins can associate with the membrane through a lipid modification like myristoylation (e.g. remorins; Raffaele et al., 2009) or be attached by a glycosylphosphatidylinositol (GPI) anchor (e.g. Pd-associated β-1,3-glucanases [BG_pap]; Levy et al., 2007; Rinne et al., 2011; Benitez-Alfonso et al., 2013), Pd-associated callose-binding proteins (PDCBs; Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING PROTEIN2 (LYM2; Faulkner et al., 2013).Among the known Pd proteins involved in Pd-specific callose degradation is BG_pap, a cell wall enzyme carrying a glycosyl hydrolase family 17 (GH17) module as its functional domain (Levy et al., 2007). Another group of proteins controlling callose dynamics at Pd are PDCBs that harbor a callose-binding domain termed carbohydrate-binding module 43 (CBM43), implicated in stabilizing callose at Pd (Simpson et al., 2009). Some β-1,3-glucanases may combine the two callose-modifying activities by harboring both GH17 and CBM43 functional domains, and several such proteins were shown to localize to Pd (Rinne et al., 2011; Benitez-Alfonso et al., 2013; Gaudioso-Pedraza and Benitez-Alfonso, 2014). A distinct feature of BG_pap and PDCBs is that both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs). The GPI anchor is a form of posttranslational modification common to many cell surface proteins in all eukaryotes. GPI-APs are covalently attached to the outer leaflet of the PM through the GPI anchor. The basic structure of the anchor consists of ethanolamine phosphate, followed by a glycan chain of three Man residues and glucosamine, followed by phosphatidylinositol lipid moiety (EtNP-6Manα1-2Manα1-6Manα1-4GlcNα1-6myoinositol-1-P-lipid; Ferguson et al., 2009). All predicted GPI-APs carry an N-terminal secretion signal peptide (SP) similar to other secreted proteins. Distinctly, GPI-APs also carry a structurally conserved 25- to 30-residue C-terminal GPI attachment signal, which typically begins with a small amino acid (e.g. Ala, Asn, Asp, Cys, Gly, or Ser) termed omega, followed by a spacer region of five to 10 polar residues, and ending with a transmembrane segment of 15 to 20 hydrophobic residues (Ferguson et al., 2009). The entire region between the N-terminal and the C-terminal signals of a GPI-AP is termed the ectodomain and carries the protein’s functional domain(s). The GPI modification process takes place in the lumenal face of the endoplasmic reticulum (ER) in a cotranslational manner. Upon translocation into the ER, a GPI-AP is stabilized in the ER membrane by its C-terminal signal, which is concurrently cleaved after the omega amino acid, and a preassembled GPI anchor is covalently attached to the C terminus of the omega amino acid. After attachment to a protein, the GPI anchor undergoes a series of modifications (remodeling), both at the glucan chain and at the lipid moiety. Such remodeling is crucial for the sorting of GPI-APs in the secretory pathway and the subsequent lateral heterogeneity at the PM (Kinoshita, 2015). In particular, the addition of saturated fatty acid chains to the lipid moiety of the anchor leads to the enriched accumulation of GPI-APs in the PM microdomains, also termed lipid rafts (Muñiz and Zurzolo, 2014). In Arabidopsis (Arabidopsis thaliana), GPI modification has been predicted for 210 proteins of diverse functions at the PM or the cell wall or both (Borner et al., 2002). Despite extensive research on the GPI modification pathway and the function of GPI-APs in mammalian and yeast cells, such knowledge in plant systems is scarce. In particular, despite an emerging role of GPI-APs in the regulation of the cell wall domain of Pd, their subcellular targeting and compartmentalization mechanism have not been studied. In addition, it is not known how the targeting mechanism of Pd-resident GPI-APs is different from that of other classes of GPI-APs, which are not localized to Pd.In this study, we investigated the subcellular targeting mechanism of Pd-associated callose-modifying GPI-APs, BG_pap and PDCB1, and compared it with that of two unrelated non-Pd GPI-APs, ARABINOGALACTAN PROTEIN4 (AGP4) and LIPID TRANSFER PROTEIN1 (LTPG1). Using sequential fluorescent labeling of protein domains, we found that the C-terminal GPI modification signal present in both Pd- and non-Pd GPI-APs can function as a primary signal in targeting proteins to the Pd-enriched PM domain. Moreover, we show that while the GPI signal is sufficient for Pd targeting, the ectodomains in BG_pap and PDCB1 further enhance their accumulation at Pd. In contrast, the ectodomains in non-Pd GPI-APs mediate exclusion of the proteins from the Pd-enriched targeting pathway. The Pd exclusion effect was found to be dominant over the Pd-targeting function of the GPI signal and the Pd-enhancing function of the Pd ectodomain, and it possibly occurs prior to PM localization. Our findings thus uncover a novel Pd-targeting signal and provide, to our knowledge, the first evidence of the cellular mechanism that regulates the sorting of GPI-APs in plants.     

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.Brassinosteroids (BRs) are ubiquitous plant hormones that promote plant growth by regulating cell elongation and division (Clouse, 1996; Clouse et al., 1996). BRs have other diverse roles, including enhancing tracheary element differentiation, stimulating ATPase activity, controlling microtubule orientation, and controlling flowering time, fertility, and leaf development (Iwasaki and Shibaoka, 1991; Clouse et al., 1996; Li et al., 1996; Schumacher et al., 1999; Catterou et al., 2001; Oh et al., 2011). BRs also function in tolerance to both biotic and abiotic stresses such as extreme temperatures, drought, and pathogens (Krishna, 2003).Deficiencies in BR biosynthesis or signaling produce characteristic dwarf plant phenotypes (Clouse et al., 1996; Szekeres et al., 1996; Fujioka et al., 1997). Plant height is an important agricultural trait, as seen in the Green Revolution, where semidwarf mutants contributed to increased yields in small-grain crops (Salas Fernandez et al., 2009). BR-deficient dwarf rice (Oryza sativa) produced increased grain and biomass yields because the erect leaf habit allowed higher planting densities under field conditions (Sakamoto et al., 2006). In fact, Green Revolution Uzu barley (Hordeum vulgare) is based on a mutation of the UZU1 gene, which encodes a homolog of BRASSINOSTEROID INSENSITIVE1 (BRI1), a BR receptor (Chono et al., 2003).Genes functioning in BR pathways have been identified by the analysis of dwarf mutants in several species, including Arabidopsis (Arabidopsis thaliana) and rice. Arabidopsis bri1 mutants are shortened, have reduced apical dominance, and are male sterile (Clouse et al., 1996). BRI1 encodes a Leu-rich repeat (LRR) receptor-like kinase that is located in the plasma membrane and contains an extracellular domain responsible for BR binding, a transmembrane sequence, and a cytoplasmic protein kinase domain (Li and Chory, 1997; Vert et al., 2005; Belkhadir and Chory, 2006). The island domain and subsequent LRR 22 are critical for BR binding (Kinoshita et al., 2005; Hothorn et al., 2011; She et al., 2011). Phosphorylation of the conserved residues Ser-1044 and Thr-1049 in the kinase activation loop activates the BRI1 kinase (Wang et al., 2005), while dephosphorylation of BRI1 by PROTEIN PHOSPHATASE2A inhibits its function (Wu et al., 2011).BRI1 is partially redundant in BR signaling with related BRASSINOSTEROID INSENSITIVE1-LIKE RECEPTOR KINASE (BRL) paralogs, both in Arabidopsis and rice. In Arabidopsis, even though null alleles of brl1 or brl3 did not show obvious phenotypic defects in shoots, they enhanced the developmental defects of a weak bri1-5 mutant. In contrast to ubiquitously expressed BRI1, BRL1, BRL2, and BRL3 are tissue specific, mostly expressed in vascular tissues, while BRL1 and BRL3 are also expressed in root apices (Caño-Delgado et al., 2004; Zhou et al., 2004; Fàbregas et al., 2013). Both BRL1 and BRL3 can bind brassinolide (BL; Caño-Delgado et al., 2004). In rice, OsBRI1 is similar to the Arabidopsis BRI1 gene, and phenotypes of OsBRI1 rice mutants include dwarf plants with shortened internodes, erect leaves that are twisted and dark green, and photomorphogenesis in the dark (Yamamuro et al., 2000). There are three BR receptors in rice as well, and while OsBRI1 is universally expressed in all organs, OsBRL1 and OsBRL3 are expressed mostly in roots (Nakamura et al., 2006).To date, two mutant genes of the BR biosynthetic pathway have been reported in maize (Zea mays). A classic dwarf mutant, nana plant1 (na1), has a mutation in a DE-ETIOLATED2 homologous gene, which encodes a 5α-reductase enzyme in the BR biosynthesis pathway (Hartwig et al., 2011), while the brassinosteroid-dependent1 (brd1) gene encodes brassinosteroid C-6 oxidase (Makarevitch et al., 2012). The maize BR-deficient mutants have shortened internodes, twisted, dark green, erect leaves, and feminized male flowers (Hartwig et al., 2011; Makarevitch et al., 2012). However, no genes in BR signaling have yet been reported in maize. Understanding BR signaling in maize might help improve this important crop for the production of biofuels, biomass, and grain yield. Here, we took a transgenic RNA interference (RNAi) approach to generate maize plants partially deficient for BRI1. These knockdown lines demonstrate that BRI1 functions are generally conserved in maize compared with other plant species, but they also exhibit unique phenotypes, suggesting either that maize possesses novel BR-regulated developmental processes or that aspects of maize morphology reveal processes not evident in other plants.