| Abstract: | Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF–defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)–Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.Due to their sessile lifestyle, the development of plants is characterized by continuous growth, generating the capacity to adapt to environmental conditions. Such flexibility has been made possible by a set of morphological adjustments that are accomplished through altered growth regulation of different plant organs, such as leaves or roots. Most aspects of plant development are regulated by the differential distribution of the plant hormone indole-3-acetic acid (IAA, or auxin) between cells or tissues (reviewed by ref. 1). The formation of auxin maxima is generated concomitantly by local auxin biosynthesis, metabolism, and directional transport (2–8).Polar auxin transport occurs in a cell-to-cell manner and is dependent on plasma membrane-localized auxin influx and efflux carriers (reviewed by ref. 9). Among them, the PIN-FORMED (PIN) auxin efflux carriers are essential for plant development, and single or multiple pin mutants display phenotypes typical for auxin transport defects, such as tropism, embryo development, organogenesis, and root meristem patterning defects (6, 7, 10–14). A polar subcellular localization has been shown for most of the plasma membrane-localized auxin transporters, in particular for the PIN proteins (PIN1-4 and PIN7) and, to some extent, also for the ATP-BINDING CASSETTE SUBFAMILY B proteins (ABCBs) and AUXIN RESISTANT 1 (AUX1) (11–13, 15–20). The PIN proteins are known to be essential for targeting and redirecting auxin flux, which modulates the spatial pattern of expression of auxin response markers (21). PINs can be targeted toward the apical (shootward), basal (rootward), or lateral plasma membrane depending upon the PIN protein identity, the cell type, and the developmental context (reviewed by ref. 22). In the root, PIN1 is localized basally toward the root tip in stele provascular cells (12). PIN2 is also localized basally in young cortex cells close to the root meristem but is localized apically in mature cortex cells, epidermal cells, and the lateral root cap (16, 22, 23).Until now, it has been unclear whether newly synthesized PIN proteins are initially secreted to the plasma membrane in a polar or apolar manner. In Arabidopsis thaliana, the current model for PIN polar localization establishment and maintenance at the plasma membrane is based on endocytosis, polar recycling, and restriction of lateral diffusion (reviewed by ref. 24). PIN proteins are internalized via clathrin-mediated endocytosis (25, 26) and can cycle back to plasma membrane domains via distinct trafficking routes. Recycling and endocytosis of PIN1 depend on the endosome-localized fraction of the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM (27, 28), which is sensitive to the fungal toxin brefeldin A (BFA) (29). ARF-GEFs are essential regulators of vesicle formation and, among the eight ARF-GEFs in Arabidopsis, GNOM is the only one reported as being essential specifically for basal PIN recycling, whereas apical PIN and AUX1 localization and dynamics are not affected in gnom mutants (30, 31). Additionally, although apical targeting of AUX1 is resistant to BFA, subcellular AUX1 trafficking is BFA-sensitive, suggesting that trafficking of apical proteins may require both BFA-sensitive and -insensitive, GNOM-independent, ARF-GEF–mediated pathways (30, 32).In addition to GNOM, other Arabidopsis ARF-GEFs have been characterized, including GNOM-LIKE 1 (GNL1), which localizes to Golgi stacks and is BFA-resistant (33, 34). GNL1 acts in the early secretory pathway where it regulates COPI-mediated recycling of endoplasmic reticulum (ER)–resident proteins from the Golgi back to the ER (33, 34). Moreover, GNOM has recently been shown to predominantly localize to Golgi stacks (35) where it plays a minor but redundant function to GNL1 in ER-Golgi trafficking (33). The other Arabidopsis ARF-GEFs include GNL2, which is expressed specifically in pollen (36), and the five BIG ARF-GEFs, BIG1 to -5. BIG5, which is BFA-sensitive, has been described under the name BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE 1 (BEN1) as mediating early endosomal trafficking (37). BIG1 to -4, of which BIG3 is BFA-resistant whereas BIG1, -2, and -4 are BFA-sensitive, have recently been described as acting redundantly in the late secretory pathway from the trans Golgi network (TGN) to the plasma membrane, as well as in late vacuolar trafficking (38).Endosomal PIN homeostasis is tightly controlled by the retromer complex through the regulation of PIN protein trafficking to the vacuole, thus controlling polar PIN abundance within the cell (39–43). Additionally, a large amount of data has demonstrated that not only trafficking routes per se are essential to determine the polar localization of PIN proteins but also internal protein signals such as posttranslational phosphorylation via the protein kinase PINOID (PID) and the protein phosphatase 2A (PP2A) (44–46). Despite recent progress, our understanding of the mechanisms establishing basal polarity remains limited. In the present work, we aimed to unravel the details of PIN basal polarity establishment by identifying selective inhibitors of this process.A number of genetic screens have been successfully used to discover new components of the endomembrane system (for examples, see refs. 34, 37, and 47–51). However, most of the molecular actors regulating endomembrane trafficking are either essential to plant survival or belong to large protein families, leading to lethality of knock-out mutants or lack of a phenotype due to redundancy. The use of fast-acting molecules suitable for the highly dynamic nature of the endomembrane system circumvents these problems and has deepened our understanding of interconnected networks of trafficking routes (52–58). While BFA has expanded our knowledge of the GNOM-dependent recycling pathway (27), other small compounds can be used to dissect different trafficking routes. In recent studies, automated screening of small molecules based on inhibition of tobacco pollen tube growth led to the isolation of a set of compounds interfering with the endomembrane system (52). Through the screening of 46,418 diverse molecules, 360 were identified as inhibitors of pollen germination (53). To dissect the trafficking routes of plasma membrane proteins specifically, a secondary screen was established based on confocal laser-scanning microscopy, leading to the identification of 123 compounds named plasma membrane recycling compound set A (PMRA), which induce mislocalization of plasma membrane markers in the Arabidopsis root meristem (53).In the present study, we reasoned that using the PMRA endomembrane trafficking modulators in combination with BFA could unravel trafficking routes regulating basal plasma membrane targeting. We designed a chemical screen to identify PMRA molecules that modulated the accumulation of PIN1 in BFA-induced agglomerations. We subsequently identified the endosidin 8 (ES8) compounds, including the original compound ES8.0 and its more potent analog ES8.1, which selectively modify PIN1 basal plasma membrane targeting in Arabidopsis with minimal effects on apical plasma membrane proteins. Using this pharmacological approach, we herein confirm that GNOM plays a role in ER-Golgi trafficking independently of its role in recycling and reveal that a GNL1/GNOM-dependent early secretory pathway is essential for targeting PIN1 toward the basal plasma membrane. Furthermore, we demonstrate that this pathway is specific for basal polarity establishment, revealing an essential and previously unknown regulatory mechanism for establishing cell polarity and regulating auxin transport and plant development. |
