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Eukaryotic cells have developed specialized membrane structures called organelles, which compartmentalize cellular functions and chemical reactions. Recent improvements in microscopy and membrane compartment isolation techniques are now sophisticating our view. Emerging evidences support that there are distinct sub-populations or subdomains, which are spatially and/or temporally segregated within one type of organelle, contributing to specify differential sorting of various cargos to distinct destinations of the cell. In plant cells, the Golgi apparatus represents a main trafficking hub in which entry occurs through a Golgi Entry Core Compartment (GECCO), that remains to be further characterized, and sorting of cargos is mediated through multiple transport pathways with different sets of regulator proteins at the post-Golgi compartment trans -Golgi network (TGN). Both GECCO and TGN are differentiated sub-populations as compared to the rest of Golgi, and moreover, further subdomain formation within TGN is suggested to play a key role for cargo sorting. In this review, we will summarize recent findings obtained on organelle subdomains, and their relationship with cargo entry at and exit from the Golgi apparatus.

Lipids are essential components of biological membranes that present a wide diversity in eukaryotic cells. Recent impressive advances in lipid biochemistry and biophysics have enabled a refocus of our view of lipids as functional units for cellular activity. However, the gap between molecular and cellular processes remains to be bridged. Here, 2 papers meet the burden of proof that choline transporters participate in local lipid composition modifications at the trans-Golgi network, an intracellular compartment that serves as the main sorting station in the cell. Localization of choline transporters to this precise compartment could be a way for plant cells to quickly modify the membrane lipid composition and asymmetry during both the allocation of cargos and the recruitment of trafficking machineries into distinct subcellular pathways.

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.

In the plant sterol biosynthetic pathway, sterol 4α-methyl oxidase1 (SMO1) and SMO2 enzymes are involved in the removal of the first and second methyl groups at the C-4 position, respectively. SMO2s have been found to be essential for embryonic and postembryonic development, but the roles of SMO1s remain unclear. Here, we found that the three Arabidopsis ( ) genes displayed different expression patterns. Single mutants and double mutants showed no obvious phenotype, but the double mutant was embryo lethal. The embryos exhibited severe defects, including no cotyledon or shoot apical meristem formation, abnormal division of suspensor cells, and twin embryos. These defects were associated with enhanced and ectopic expression of auxin biosynthesis and response reporters. Consistently, the expression pattern and polar localization of PIN FORMED1, PIN FORMED7, and AUXIN RESISTANT1 auxin transporters were dramatically altered in embryos. Moreover, cytokinin biosynthesis and response were reduced in embryos. Tissue culture experiments further demonstrated that homeostasis between auxin and cytokinin was altered in heterozygous mutants. This disturbed balance of auxin and cytokinin in embryos was accompanied by unrestricted expression of the quiescent center marker Accordingly, exogenous application of either auxin biosynthesis inhibitor or cytokinin partially rescued the embryo lethality of Sterol analyses revealed that 4,4-dimethylsterols dramatically accumulated in heterozygous mutants. Together, these data demonstrate that SMO1s function through maintaining correct sterol composition to balance auxin and cytokinin activities during embryogenesis.

Sterols are essential molecules for multiple biological processes, including embryogenesis, cell elongation, and endocytosis. The plant sterol biosynthetic pathway is unique in the involvement of two distinct sterol 4α-methyl oxidase (SMO) families, SMO1 and SMO2, which contain three and two isoforms, respectively, and are involved in sequential removal of the two methyl groups at C-4. In this study, we characterized the biological functions of members of the SMO2 gene family. SMO2-1 was strongly expressed in most tissues during Arabidopsis (Arabidopsis thaliana) development, whereas SMO2-2 showed a more specific expression pattern. Although single smo2 mutants displayed no obvious phenotype, the smo2-1 smo2-2 double mutant was embryonic lethal, and the smo2-1 smo2-2/+ mutant was dwarf, whereas the smo2-1/+ smo2-2 mutant exhibited a moderate phenotype. The phenotypes of the smo2 mutants resembled those of auxin-defective mutants. Indeed, the expression of DR5rev :GFP, an auxin-responsive reporter, was reduced and abnormal in smo2-1 smo2-2 embryos. Furthermore, the expression and subcellular localization of the PIN1 auxin efflux facilitator also were altered. Consistent with these observations, either the exogenous application of auxin or endogenous auxin overproduction (YUCCA9 overexpression) partially rescued the smo2-1 smo2-2 embryonic lethality. Surprisingly, the dwarf phenotype of smo2-1 smo2-2/+ was completely rescued by YUCCA9 overexpression. Gas chromatography-mass spectrometry analysis revealed a substantial accumulation of 4α-methylsterols, substrates of SMO2, in smo2 heterozygous double mutants. Together, our data suggest that SMO2s are important for correct sterol composition and function partially through effects on auxin accumulation, auxin response, and PIN1 expression to regulate Arabidopsis embryogenesis and postembryonic development.

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.

The secretion of proteins, lipids, and carbohydrates to the cell surface is essential for plant development and adaptation. Secreted substances synthesized at the endoplasmic reticulum pass through the Golgi apparatus and trans -Golgi network (TGN) en route to the plasma membrane via the conventional secretion pathway. The TGN is morphologically and functionally distinct from the Golgi apparatus. The TGN is located at the crossroads of many trafficking pathways and regulates a range of crucial processes including secretion to the cell surface, transport to the vacuole, and the reception of endocytic cargo. This review outlines the TGN’s central role in cargo secretion, showing that its behavior is more complex and controlled than the bulk-flow hypothesis suggests. Its formation, structure, and maintenance are discussed along with the formation and release of secretory vesicles.

Sterols are eukaryotic membrane components with crucial roles in diverse cellular processes. Elucidation of sterol function relies on development of tools for in situ sterol visualization. Here we describe protocols for in situ sterol localization in Arabidopsis thaliana root cells, using filipin as a specific probe for detection of fluorescent filipin-sterol complexes. Currently, filipin is the only established tool for sterol visualization in plants. Filipin labeling can be performed on aldehyde-fixed samples, largely preserving fluorescent proteins and being compatible with immunocytochemistry. Filipin can also be applied for probing live cells, taking into account the fact that it inhibits sterol-dependent endocytosis. The experimental procedures described are designed for fluorescence detection by confocal laser-scanning microscopy with excitation of filipin-sterol complexes at 364 nm. The protocols require 1 d for sterol covisualization with fluorescent proteins in fixed or live roots and 2 d for immunocytochemistry on whole-mount roots.

A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant’s early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.

The polarization of yeast and animal cells relies on membrane sterols for polar targeting of proteins to the plasma membrane, their polar endocytic recycling and restricted lateral diffusion 1 , 2 , 3 , 4 . However, little is known about sterol function in plant-cell polarity 5 . Directional root growth along the gravity vector requires polar transport of the plant hormone auxin. In Arabidopsis , asymmetric plasma membrane localization of the PIN–FORMED2 (PIN2) auxin transporter directs root gravitropism 6 , 7 , 8 , 9 , 10 . Although the composition of membrane sterols influences gravitropism and localization of two other PIN proteins 11 , it remains unknown how sterols contribute mechanistically to PIN polarity. Here, we show that correct membrane sterol composition is essential for the acquisition of PIN2 polarity. Polar PIN2 localization is defective in the sterol-biosynthesis mutant cyclopropylsterol isomerase1-1 ( cpi1-1 ) which displays altered sterol composition, PIN2 endocytosis, and root gravitropism. At the end of cytokinesis, PIN2 localizes initially to both newly formed membranes but subsequently disappears from one. By contrast, PIN2 frequently remains at both daughter membranes in endocytosis-defective cpi1-1 cells. Hence, sterol composition affects post-cytokinetic acquisition of PIN2 polarity by endocytosis, suggesting a mechanism for sterol action on establishment of asymmetric protein localization.