A biosensor for the direct visualization of auxin.

One of the most important regulatory small molecules in plants is indole-3-acetic acid, also known as auxin. Its dynamic redistribution has an essential role in almost every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity1,2. So far, it has not been possible to directly determine the spatial and temporal distribution of auxin at a cellular resolution. Instead it is inferred from the visualization of irreversible processes that involve the endogenous auxin-response machinery3-7; however, such a system cannot detect transient changes. Here we report a genetically encoded biosensor for the quantitative in vivo visualization of auxin distribution. The sensor is based on the Escherichia coli tryptophan repressor8, the binding pocket of which is engineered to be specific to auxin. Coupling of the auxin-binding moiety with selected fluorescent proteins enables the use of a fluorescence resonance energy transfer signal as a readout. Unlike previous systems, this sensor enables direct monitoring of the rapid uptake and clearance of auxin by individual cells and within cell compartments in planta. By responding to the graded spatial distribution along the root axis and its perturbation by transport inhibitors-as well as the rapid and reversible redistribution of endogenous auxin in response to changes in gravity vectors-our sensor enables real-time monitoring of auxin concentrations at a (sub)cellular resolution and their spatial and temporal changes during the lifespan of a plant.

Mass-spectrometry-based draft of the Arabidopsis proteome.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities1. Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.

A rich and bountiful harvest: Key discoveries in plant cell biology.

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke's discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

A tyrosine phospho-switch within the Longin domain of VAMP721 modulates SNARE functionality.

The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.

N-terminal domain of ARF-GEF GNOM prevents heterodimerization with functionally divergent GNL1 in Arabidopsis.

Evolutionary change following gene duplication can lead to functionally divergent paralogous proteins. If comprising identical subunits their random assortment would also form potentially detrimental heteromeric proteins. In Arabidopsis, the ARF GTPase guanine-nucleotide exchange factor GNOM is essential for polar recycling of auxin-efflux transporter PIN1 from endosomes to the basal plasma membrane whereas its paralog GNL1 mediates retrograde Golgi-endoplasmic reticulum traffic. Here we show that both GNOM and GNL1 form homodimers but no heterodimers. To assess the biological significance of this, we generated transgenic plants expressing engineered heterodimer-compatible GNOM variants. Those plants showed developmental defects such as the failure to produce lateral roots. To identify mechanisms underlying heterodimer prevention, we analyzed interactions of the N-terminal dimerization and cyclophilin-binding (DCB) domain. Each DCB domain interacted with the complementary fragment (ΔDCB) both of their own and of the paralogous protein. However, only DCBGNOM interacted with itself whereas DCBGNL1 failed to interact with itself and with DCBGNOM . GNOM variants in which the DCB domain was removed or replaced by DCBGNL1 revealed a role for DCB-DCB interaction in the prevention of GNOM-GNL1 heterodimers whereas DCB-ΔDCB interaction was essential for dimer formation and GNOM function. Our data suggest a model of early DCB-DCB interaction that facilitates GNOM homodimer formation, indirectly precluding formation of detrimental heterodimers.

Specification and regulation of vascular tissue identity in the Arabidopsis embryo.

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.

Coordinated Activation of ARF1 GTPases by ARF-GEF GNOM Dimers Is Essential for Vesicle Trafficking in Arabidopsis.

Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1•GTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.

Plants have evolved a tremendous ability to respond to environmental changes by adapting their growth and development. The interaction between hormonal and developmental signals is a critical mechanism in the generation of this enormous plasticity. A good example is the response to the hormone ethylene that depends on tissue type, developmental stage, and environmental conditions. By characterizing the Arabidopsis wei8 mutant, we have found that a small family of genes mediates tissue-specific responses to ethylene. Biochemical studies revealed that WEI8 encodes a long-anticipated tryptophan aminotransferase, TAA1, in the essential, yet genetically uncharacterized, indole-3-pyruvic acid (IPA) branch of the auxin biosynthetic pathway. Analysis of TAA1 and its paralogues revealed a link between local auxin production, tissue-specific ethylene effects, and organ development. Thus, the IPA route of auxin production is key to generating robust auxin gradients in response to environmental and developmental cues.

Functional diversification of Arabidopsis SEC1-related SM proteins in cytokinetic and secretory membrane fusion.

Sec1/Munc18 (SM) proteins contribute to membrane fusion by interacting with Qa-SNAREs or nascent trans-SNARE complexes. Gymnosperms and the basal angiosperm Amborella have only a single SEC1 gene related to the KEULE gene in Arabidopsis However, the genomes of most angiosperms including Arabidopsis encode three SEC1-related SM proteins of which only KEULE has been functionally characterized as interacting with the cytokinesis-specific Qa-SNARE KNOLLE during cell-plate formation. Here we analyze the closest paralog of KEULE named SEC1B. In contrast to the cytokinesis defects of keule mutants, sec1b mutants are homozygous viable. However, the keule sec1b double mutant was nearly gametophytically lethal, displaying collapsed pollen grains, which suggests substantial overlap between SEC1B and KEULE functions in secretion-dependent growth. SEC1B had a strong preference for interaction with the evolutionarily ancient Qa-SNARE SYP132 involved in secretion and cytokinesis, whereas KEULE interacted with both KNOLLE and SYP132. This differential interaction with Qa-SNAREs is likely conferred by domains 1 and 2a of the two SM proteins. Comparative analysis of all four possible combinations of the relevant SEC1 Qa-SNARE double mutants revealed that in cytokinesis, the interaction of SEC1B with KNOLLE plays no role, whereas the interaction of KEULE with KNOLLE is prevalent and functionally as important as the interactions of both SEC1B and KEU with SYP132 together. Our results suggest that functional diversification of the two SEC1-related SM proteins during angiosperm evolution resulted in enhanced interaction of SEC1B with Qa-SNARE SYP132, and thus a predominant role of SEC1B in secretion.

A single class of ARF GTPase activated by several pathway-specific ARF-GEFs regulates essential membrane traffic in Arabidopsis.

In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.

Specificity of plant membrane trafficking - ARFs, regulators and coat proteins.

Approximately one-third of all eukaryotic proteins are delivered to their destination by trafficking within the endomembrane system. Such cargo proteins are incorporated into forming membrane vesicles on donor compartments and delivered to acceptor compartments by vesicle fusion. How cargo proteins are sorted into forming vesicles is still largely unknown. Here we review the roles of small GTPases of the ARF/SAR1 family, their regulators designated ARF guanine-nucleotide exchange factors (ARF-GEFs) and ARF GTPase-activating proteins (ARF-GAPs) as well as coat protein complexes during membrane vesicle formation. Although conserved across eukaryotes, these four functional groups of proteins display plant-specific modifications in composition, structure and function.

VPS9a Activates the Rab5 GTPase ARA7 to Confer Distinct Pre- and Postinvasive Plant Innate Immunity.

Plant innate immunity can effectively prevent the proliferation of filamentous pathogens. Papilla formation at the site of attack is essential for preinvasive immunity; in postinvasive immunity, the encasement of pathogen structures inside host cells can hamper disease. Whereas papillae are highly dependent on transcytosis of premade material, little is known about encasement formation. Here, we show that endosome-associated VPS9a, the conserved guanine-nucleotide exchange factor activating Rab5 GTPases, is required for both pre- and postinvasive immunity against a nonadapted powdery mildew fungus (Blumeria graminis f. sp hordei) in Arabidopsis thaliana Surprisingly, VPS9a acts in addition to two previously well-described innate immunity components and thus represents an additional step in the regulation of how plants resist pathogens. We found VPS9a to be important for delivering membrane material to the encasement and VPS9a also plays a predominant role in postinvasive immunity. GTP-bound Rab5 GTPases accumulate in the encasement, but not the papillae, suggesting that two independent pathways form these defense structures. VPS9a also mediates defense to an adapted powdery mildew fungus, thus regulating a durable type of defense that works in both host and nonhost resistance. We propose that VPS9a plays a conserved role in organizing cellular endomembrane trafficking, required for delivery of defense components in response to powdery mildew fungi.

Dynamic PIN-FORMED auxin efflux carrier phosphorylation at the plasma membrane controls auxin efflux-dependent growth.

The directional distribution of the phytohormone auxin is essential for plant development. Directional auxin transport is mediated by the polarly distributed PIN-FORMED (PIN) auxin efflux carriers. We have previously shown that efficient PIN1-mediated auxin efflux requires activation through phosphorylation at the four serines S1-S4 in Arabidopsis thaliana The Brefeldin A (BFA)-sensitive D6 PROTEIN KINASE (D6PK) and the BFA-insensitive PINOID (PID) phosphorylate and activate PIN1 through phosphorylation at all four phosphosites. PID, but not D6PK, can also induce PIN1 polarity shifts, seemingly through phosphorylation at S1-S3. The differential effects of D6PK and PID on PIN1 polarity had so far been attributed to their differential phosphosite preference for the four PIN1 phosphosites. We have mapped PIN1 phosphorylation at S1-S4 in situ using phosphosite-specific antibodies. We detected phosphorylation at PIN1 phosphosites at the basal (rootward) as well as the apical (shootward) plasma membrane in different root cell types, in embryos, and shoot apical meristems. Thereby, PIN1 phosphorylation at all phosphosites generally followed the predominant PIN1 distribution but was not restricted to specific polar sides of the cells. PIN1 phosphorylation at the basal and apical plasma membrane was differentially sensitive to BFA treatments, suggesting the involvement of different protein kinases or trafficking mechanisms in PIN1 phosphorylation control. We conclude that phosphosite preferences are not sufficient to explain the differential effects of D6PK and PID on PIN1 polarity, and suggest that a more complex model is needed to explain the effects of PID.

Plant cytokinesis-No ring, no constriction but centrifugal construction of the partitioning membrane.

Plants have evolved a unique way of partitioning the cytoplasm of dividing cells: Instead of forming a contractile ring that constricts the plasma membrane, plant cells target membrane vesicles to the plane of division where the vesicles fuse with one another to form the partitioning membrane. Plant cytokinesis starts in the centre and progresses towards the periphery, culminating in the fusion of the partitioning membrane with the parental plasma membrane. This membrane dynamics is orchestrated by a specific cytoskeletal array named phragmoplast that originates from interzone spindle remnants. Here we review the properties of the process as well as molecules that play specific roles in that process.

Evolutionarily diverse SYP1 Qa-SNAREs jointly sustain pollen tube growth in Arabidopsis.

Intracellular membrane fusion is effected by SNARE proteins that reside on adjacent membranes and form bridging trans-SNARE complexes. Qa-SNARE members of the Arabidopsis SYP1 family are involved in membrane fusion at the plasma membrane or during cell plate formation. Three SYP1 family members have been classified as pollen-specific as inferred from gene expression profiling studies, and two of them, SYP124 and SYP125, are confined to angiosperms. The SYP124 gene appears genetically unstable, whereas its sister gene SYP125 shows essentially no variation among Arabidopsis accessions. The third pollen-specific member SYP131 is sister to SYP132, which appears evolutionarily conserved in the plant lineage. Although evolutionarily diverse, the three SYP1 proteins are functionally overlapping in that only the triple mutant syp124 syp125 syp131 shows a specific and severe male gametophytic defect. While pollen development and germination appear normal, pollen tube growth is arrested during passage through the style. Our results suggest that angiosperm pollen tubes employ a combination of ancient and modern Qa-SNARE proteins to sustain their growth-promoting membrane dynamics during the reproductive process.

Auxin responsiveness of the MONOPTEROS-BODENLOS module in primary root initiation critically depends on the nuclear import kinetics of the Aux/IAA inhibitor BODENLOS.

Primary root formation in early embryogenesis of Arabidopsis thaliana is initiated with the specification of a single cell called hypophysis. This initial step requires the auxin-dependent release of the transcription factor MONOPTEROS (MP, also known as ARF5) from its inhibition by the Aux/IAA protein BODENLOS (BDL, also known as IAA12). Auxin-insensitive bdl mutant embryos and mp loss-of-function embryos fail to specify the hypophysis, giving rise to rootless seedlings. A suppressor screen of rootless bdl mutant seedlings yielded a mutation in the nuclear import receptor IMPORTIN-ALPHA 6 (IMPα6) that promoted primary root formation through rescue of the embryonic hypophysis defects, without causing additional phenotypic changes. Aux/IAA proteins are continually synthesized and degraded, which is essential for rapid transcriptional responses to changing auxin concentrations. Nuclear translocation of bdl:3×GFP was slowed down in impα6 mutants as measured by fluorescence recovery after photobleaching (FRAP) analysis, which correlated with the reduced inhibition of MP by bdl in transient expression assays in impα6 knock-down protoplasts. The MP-BDL module acts like an auxin-triggered genetic switch because MP activates its own expression as well as the expression of its inhibitor BDL. Using an established simulation model, we determined that the reduced nuclear translocation rate of BDL in impα6 mutant embryos rendered the auxin-triggered switch unstable, impairing the fast response to changes in auxin concentration. Our results suggest that the instability of the inhibitor BDL necessitates a fast nuclear uptake in order to reach the critical threshold level required for auxin responsiveness of the MP-BDL module in primary root initiation.

Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo.

In multicellular organisms, cellular differences in gene activity are a prerequisite for differentiation and establishment of cell types. In order to study transcriptome profiles, specific cell types have to be isolated from a given tissue or even the whole organism. However, whole-transcriptome analysis of early embryos in flowering plants has been hampered by their size and inaccessibility. Here, we describe the purification of nuclear RNA from early stage Arabidopsis thaliana embryos using fluorescence-activated nuclear sorting (FANS) to generate expression profiles of early stages of the whole embryo, the proembryo and the suspensor. We validated our datasets of differentially expressed candidate genes by promoter-reporter gene fusions and in situ hybridization. Our study revealed that different classes of genes with respect to biological processes and molecular functions are preferentially expressed either in the proembryo or in the suspensor. This method can be used especially for tissues with a limited cell population and inaccessible tissue types. Furthermore, we provide a valuable resource for research on Arabidopsis early embryogenesis.

MONOPTEROS controls embryonic root initiation by regulating a mobile transcription factor.

Acquisition of cell identity in plants relies strongly on positional information, hence cell-cell communication and inductive signalling are instrumental for developmental patterning. During Arabidopsis embryogenesis, an extra-embryonic cell is specified to become the founder cell of the primary root meristem, hypophysis, in response to signals from adjacent embryonic cells. The auxin-dependent transcription factor MONOPTEROS (MP) drives hypophysis specification by promoting transport of the hormone auxin from the embryo to the hypophysis precursor. However, auxin accumulation is not sufficient for hypophysis specification, indicating that additional MP-dependent signals are required. Here we describe the microarray-based isolation of MP target genes that mediate signalling from embryo to hypophysis. Of three direct transcriptional target genes, TARGET OF MP 5 (TMO5) and TMO7 encode basic helix-loop-helix (bHLH) transcription factors that are expressed in the hypophysis-adjacent embryo cells, and are required and partially sufficient for MP-dependent root initiation. Importantly, the small TMO7 transcription factor moves from its site of synthesis in the embryo to the hypophysis precursor, thus representing a novel MP-dependent intercellular signal in embryonic root specification.

Arabidopsis µ-adaptin subunit AP1M of adaptor protein complex 1 mediates late secretory and vacuolar traffic and is required for growth.

Adaptor protein (AP) complexes are the predominant coat proteins of membrane vesicles in post-Golgi trafficking of mammalian cells. Each AP complex contains a specific medium subunit, µ-adaptin, that selects cargo proteins bearing sequence-specific sorting motifs. Much less is known about the AP complexes and their µ subunits in plants. Because of uncertain homology, the µ-adaptins of Arabidopsis have been designated muA through muD [Happel et al. (2004) Plant J 37(5):678-693]. Furthermore, only muD has been assigned to a specific AP complex, AP-3, involved in Golgi-vacuolar trafficking [Niihama et al. (2009) Plant Cell Physiol 50(12):2057-2068, Zwiewka et al. (2011) Cell Res 21(12):1711-1722, and Wolfenstetter et al. (2012) Plant Cell 24(1):215-232]. In contrast, the µ subunit of neither the post-Golgi trafficking AP-1 complex nor the endocytic AP-2 complex has been identified. Here, we report the functional analysis of redundant AP-1 µ-adaptins AP1M1 (also known as muB1) and AP1M2 (also known as muB2). Coimmunoprecipitation revealed that both AP1M2 and its less strongly expressed isoform AP1M1 are complexed with the large subunit γ-adaptin of AP-1. In addition, AP1M2 was localized at or near the trans-Golgi network. Knockout mutations of AP1M2 impaired pollen function and arrested plant growth whereas the ap1m1 ap1m2 double mutant was nearly pollen-lethal. At the cellular level, the absence of AP1M2 entailed inhibition of multiple trafficking pathways from the trans-Golgi network to the vacuole and to the plasma membrane in interphase and to the plane of cell division in cytokinesis. Thus, AP-1 is crucial in post-Golgi trafficking in plant cells and required for cell division and plant growth.