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The trans-Golgi network (TGN), a key compartment in endomembrane trafficking, participates in both secretion to and endocytosis from the plasma membrane. Consequently, the TGN plays a key role in plant growth and development. Understanding how proteins are sorted for secretion or endocytic recycling at the TGN is critical for elucidating mechanisms of plant development. We previously showed that the protein ECHIDNA is essential for phytohormonal control of hypocotyl bending because it mediates secretion of cell wall components and the auxin influx carrier AUXIN RESISTANT 1 (AUX1) from the TGN. Despite the critical role of ECHIDNA in TGN-mediated trafficking, its mode of action remains unknown in Arabidopsis (Arabidopsis thaliana). We therefore performed a suppressor screen on the ech mutant. Here, we report the identification of TGN-localized TYPHON 1 (TPN1) and TPN2 proteins. A single amino acid change in either TPN protein causes dominant suppression of the ech mutant's defects in growth and AUX1 secretion, while also restoring wild-type-like ethylene-responsive hypocotyl bending. Importantly, genetic and cell biological evidence shows that TPN1 acts through RAS-ASSOCIATED BINDING H1b (RABH1b), a TGN localized RAB-GTPase. These results provide insights into ECHIDNA-mediated secretory trafficking of cell wall and auxin carriers at the TGN, as well as its role in controlling plant growth., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Phosphatidic acid (PA) and lysophosphatidic acid acyltransferases (LPAATs) might be critical for the secretory pathway. Four extra-plastidial LPAATs (LPAAT2, 3, 4, and 5) were identified in Arabidopsis thaliana. These AtLPAATs display a specific enzymatic activity converting lysophosphatidic acid to PA and are located in the endomembrane system. We investigate a putative role for AtLPAATs 3, 4, and 5 in the secretory pathway of root cells through genetical (knockout mutants), biochemical (activity inhibitor, lipid analyses), and imaging (live and immuno-confocal microscopy) approaches. Treating a lpaat4;lpaat5 double mutant with the LPAAT inhibitor CI976 produced a significant decrease in primary root growth. The trafficking of the auxin transporter PIN2 was disturbed in this lpaat4;lpaat5 double mutant treated with CI976, whereas trafficking of H+-ATPases was unaffected. The lpaat4;lpaat5 double mutant is sensitive to salt stress, and the trafficking of the aquaporin PIP2;7 to the plasma membrane in the lpaat4;lpaat5 double mutant treated with CI976 was reduced. We measured the amounts of neo-synthesized PA in roots, and found a decrease in PA only in the lpaat4;lpaat5 double mutant treated with CI976, suggesting that the protein trafficking impairment was due to a critical PA concentration threshold., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

During early seedling development, the shoot apical meristem is protected from damage as the seedling emerges from soil by the formation of apical hook. Hook formation requires differential growth across the epidermis below the meristem in the hypocotyl. The plant hormones ethylene and auxin play key roles during apical hook development by controlling differential growth. We provide genetic and cell biological evidence for the role of ADP-ribosylation factor 1 (ARF1)-GTPase and its effector ARF-guanine-exchange factors (GEFs) of the Brefeldin A-inhibited GEF (BIG) family and GNOM in ethylene- and auxin-mediated control of hook development. We show that ARF-GEF GNOM acts early, whereas BIG ARF-GEFs act at a later stage of apical hook development. We show that the localization of ARF1 and BIG4 at the trans -Golgi network (TGN) depends on ECHIDNA (ECH), a plant homolog of yeast Triacylglycerol lipase (TLG2/SYP4) interacting protein Tgl2-Vesicle Protein 23 (TVP23). BIGs together with ECH and ARF1 mediate the secretion of AUX1 influx carrier to the plasma membrane from the TGN during hook development and defects in BIG or ARF1 result in insensitivity to ethylene. Thus, our data indicate a division of labor within the ARF-GEF family in mediating differential growth with GNOM acting during the formation phase whereas BIGs act during the hook maintenance phase downstream of plant hormone ethylene., (© 2017 American Society of Plant Biologists. All rights reserved.)

SNARE proteins are central elements of the machinery involved in membrane fusion of eukaryotic cells. In animals and plants, SNAREs have diversified to sustain a variety of specific functions. In animals, R-SNARE proteins called brevins have diversified; in contrast, in plants, the R-SNARE proteins named longins have diversified. Recently, a new subfamily of four longins named 'phytolongins' (Phyl) was discovered. One intriguing aspect of Phyl proteins is the lack of the typical SNARE motif, which is replaced by another domain termed the 'Phyl domain'. Phytolongins have a rather ubiquitous tissue expression in Arabidopsis but still await intracellular characterization. In this study, we found that the four phytolongins are distributed along the secretory pathway. While Phyl2.1 and Phyl2.2 are strictly located at the endoplasmic reticulum network, Phyl1.2 associates with the Golgi bodies, and Phyl1.1 locates mainly at the plasma membrane and partially in the Golgi bodies and post-Golgi compartments. Our results show that export of Phyl1.1 from the endoplasmic reticulum depends on the GTPase Sar1, the Sar1 guanine nucleotide exchange factor Sec12, and the SNAREs Sec22 and Memb11. In addition, we have identified the Y48F49 motif as being critical for the exit of Phyl1.1 from the endoplasmic reticulum. Our results provide the first characterization of the subcellular localization of the phytolongins, and we discuss their potential role in regulating the secretory pathway., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical for the function of the secretory pathway. The SNARE Memb11 is involved in membrane trafficking at the ER-Golgi interface. The aim of the work was to decipher molecular mechanisms acting in Memb11-mediated ER-Golgi traffic. In mammalian cells, the orthologue of Memb11 (membrin) is potentially involved in the recruitment of the GTPase Arf1 at the Golgi membrane. However molecular mechanisms associated to Memb11 remain unknown in plants. Memb11 was detected mainly at the cis-Golgi and co-immunoprecipitated with Arf1, suggesting that Arf1 may interact with Memb11. This interaction of Memb11 with Arf1 at the Golgi was confirmed by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was found to be specific to Memb11 as compared to either Memb12 or Sec22. Using a structural bioinformatic approach, several sequences in the N-ter part of Memb11 were hypothesized to be critical for this interaction and were tested by BiFC on corresponding mutants. Finally, by using both in vitro and in vivo approaches, we determined that only the GDP-bound form of Arf1 interacts with Memb11. Together, our results indicate that Memb11 interacts with the GDP-bound form of Arf1 in the Golgi apparatus., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes., (© 2015 American Society of Plant Biologists. All rights reserved.)

Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H(+)-pyrophosphatase and the vacuolar H(+)-adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)-Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.