A natural gene drive system confers reproductive isolation in rice.

Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.

Biomolecular condensates mediate bending and scission of endosome membranes.

Multivesicular bodies are key endosomal compartments implicated in cellular quality control through their degradation of membrane-bound cargo proteins1-3. The ATP-consuming ESCRT protein machinery mediates the capture and engulfment of membrane-bound cargo proteins through invagination and scission of multivesicular-body membranes to form intraluminal vesicles4,5. Here we report that the plant ESCRT component FREE16 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation. We use a minimal physical model, reconstitution experiments and in silico simulations to identify the dynamics of this process and describe intermediate morphologies of nascent intraluminal vesicles. Furthermore, we find that condensate-wetting-induced line tension forces and membrane asymmetries are sufficient to mediate scission of the membrane neck without the ESCRT protein machinery or ATP consumption. Genetic manipulation of the ESCRT pathway in several eukaryotes provides additional evidence for condensate-mediated membrane scission in vivo. We find that the interplay between condensate and machinery-mediated scission mechanisms is indispensable for osmotic stress tolerance in plants. We propose that condensate-mediated scission represents a previously undescribed scission mechanism that depends on the physicomolecular properties of the condensate and is involved in a range of trafficking processes. More generally, FREE1 condensate-mediated membrane scission in multivesicular-body biogenesis highlights the fundamental role of wetting in intracellular dynamics and organization.

Ufmylation reconciles salt stress-induced unfolded protein responses via ER-phagy in Arabidopsis.

In plants, the endomembrane system is tightly regulated in response to environmental stresses for maintaining cellular homeostasis. Autophagosomes, the double membrane organelles forming upon nutrient deprivation or stress induction, degrade bulky cytosolic materials for nutrient turnover. Though abiotic stresses have been reported to induce plant autophagy, few receptors or regulators for selective autophagy have been characterized for specific stresses. Here, we have applied immunoprecipitation followed by tandem mass spectrometry using the autophagosome marker protein ATG8 as bait and have identified the E3 ligase of the ufmylation system Ufl1 as a bona fide ATG8 interactor under salt stress. Notably, core components in the ufmylation cascade, Ufl1 and Ufm1, interact with the autophagy kinase complexes proteins ATG1 and ATG6. Cellular and genetic analysis showed that Ufl1 is important for endoplasmic reticulum (ER)-phagy under persisting salt stress. Loss-of-function mutants of Ufl1 display a salt stress hypersensitive phenotype and abnormal ER morphology. Prolonged ER stress responses are detected in ufl1 mutants that phenocopy the autophagy dysfunction atg5 mutants. Consistently, expression of ufmylation cascade components is up-regulated by salt stress. Taken together, our study demonstrates the role of ufmylation in regulating ER homeostasis under salt stress through ER-phagy.

A plant-unique protein BLISTER coordinates with core retromer to modulate endosomal sorting of plasma membrane and vacuolar proteins.

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. In this study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.

A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana.

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Recent advances in plant endomembrane research and new microscopical techniques.

The endomembrane system consists of various membrane-bound organelles including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, and the lysosome/vacuole. Membrane trafficking between distinct compartments is mainly achieved by vesicular transport. As the endomembrane compartments and the machineries regulating the membrane trafficking are largely conserved across all eukaryotes, our current knowledge on organelle biogenesis and endomembrane trafficking in plants has mainly been shaped by corresponding studies in mammals and yeast. However, unique perspectives have emerged from plant cell biology research through the characterization of plant-specific regulators as well as the development and application of the state-of-the-art microscopical techniques. In this review, we summarize our current knowledge on the plant endomembrane system, with a focus on several distinct pathways: ER-to-Golgi transport, protein sorting at the TGN, endosomal sorting on multivesicular bodies, vacuolar trafficking/vacuole biogenesis, and the autophagy pathway. We also give an update on advanced imaging techniques for the plant cell biology research.

ADP-ribosylation factor D1 modulates Golgi morphology, cell plate formation, and plant growth in Arabidopsis.

ADP-ribosylation factor (ARF) family proteins, one type of small guanine-nucleotide-binding (G) proteins, play a central role in regulating vesicular traffic and organelle structures in eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains more than 21 ARF proteins, but relatively little is known about the functional heterogeneity of ARF homologs in plants. Here, we characterized the function of a unique ARF protein, ARFD1B, in Arabidopsis. ARFD1B exhibited both cytosol and punctate localization patterns, colocalizing with a Golgi marker in protoplasts and transgenic plants. Distinct from other ARF1 homologs, overexpression of a dominant-negative mutant form of ARFD1B did not alter the localization of the Golgi marker mannosidase I (ManI)-RFP in Arabidopsis cells. Interestingly, the ARFD1 artificial microRNA knockdown mutant arfd1 displayed a deleterious growth phenotype, while this phenotype was restored in complemented plants. Further, confocal imaging and transmission electron microscopy analyses of the arfd1 mutant revealed defective cell plate formation and abnormal Golgi morphology. Pull-down and liquid chromatography-tandem mass spectrometry analyses identified Coat Protein I (COPI) components as interacting partners of ARFD1B, and subsequent bimolecular fluorescence complementation, yeast (Saccharomyces cerevisiae) two-hybrid, and co-immunoprecipitation assays further confirmed these interactions. These results demonstrate that ARFD1 is required for cell plate formation, maintenance of Golgi morphology, and plant growth in Arabidopsis.

Systematic prediction of autophagy-related proteins using Arabidopsis thaliana interactome data.

Autophagy is a self-degradative process that is crucial for maintaining cellular homeostasis by removing damaged cytoplasmic components and recycling nutrients. Such an evolutionary conserved proteolysis process is regulated by the autophagy-related (Atg) proteins. The incomplete understanding of plant autophagy proteome and the importance of a proteome-wide understanding of the autophagy pathway prompted us to predict Atg proteins and regulators in Arabidopsis. Here, we developed a systems-level algorithm to identify autophagy-related modules (ARMs) based on protein subcellular localization, protein-protein interactions, and known Atg proteins. This generates a detailed landscape of the autophagic modules in Arabidopsis. We found that the newly identified genes in each ARM tend to be upregulated and coexpressed during the senescence stage of Arabidopsis. We also demonstrated that the Golgi apparatus ARM, ARM13, functions in the autophagy process by module clustering and functional analysis. To verify the in silico analysis, the Atg candidates in ARM13 that are functionally similar to the core Atg proteins were selected for experimental validation. Interestingly, two of the previously uncharacterized proteins identified from the ARM analysis, AGD1 and Sec14, exhibited bona fide association with the autophagy protein complex in plant cells, which provides evidence for a cross-talk between intracellular pathways and autophagy. Thus, the computational framework has facilitated the identification and characterization of plant-specific autophagy-related proteins and novel autophagy proteins/regulators in higher eukaryotes.

Arabidopsis ENDOMEMBRANE PROTEIN 12 contributes to the endoplasmic reticulum stress response by regulating K/HDEL receptor trafficking.

ENDOMEMBRANE PROTEIN 70 (EMP70) proteins constitute a 12-member superfamily in Arabidopsis thaliana, and are the most abundant protein species in plant Golgi proteomes. However, the physiological functions of EMPs in plants remain largely unknown. Here we have demonstrated that two AtEMP12 T-DNA insertion mutants are sensitive to ER (endoplasmic reticulum) stress as induced by tunicamycin and dithiothreitol treatments. Interestingly, the unfolded protein response (UPR) is constitutively activated in the knockout mutant emp12-1 under normal growth conditions, suggesting that the activation is a result of insufficient chaperones in the ER to aid protein folding. Indeed, we have further shown that BiP is secreted into the apoplast in emp12-1, while the K/HDEL receptor ERD2a, which regulates BiP trafficking, is exclusively localized in the ER in emp12-1, instead of its known ER-Golgi dual-localization. Given an enhanced retrograde transport of ERD2a, along with less dimerized receptor formed in the absence of EMP12, ERD2a may be prematurely returned to the ER without its bound ligands. Therefore, we propose that EMP12 may act as a novel regulator of the K/HDEL receptor to ensure an effective retrograde transport of K/HDEL ligands.

A plant-unique ESCRT component, FYVE4, regulates multivesicular endosome biogenesis and plant growth.

During evolution, land plants generated unique proteins that participate in endosomal sorting and multivesicular endosome (MVE) biogenesis, many of them with specific phosphoinositide-binding capabilities. Nonetheless, the function of most plant phosphoinositide-binding proteins in endosomal trafficking remains elusive. Here, we analysed several Arabidopsis mutants lacking predicted phosphoinositide-binding proteins and first identified fyve4-1 as a mutant with a hypersensitive response to high-boron conditions and defects in degradative vacuolar sorting of membrane proteins such as the borate exporter BOR1-GFP. FYVE4 encodes a plant-unique, FYVE domain-containing protein that interacts with SNF7, a core component of ESCRT-III (Endosomal Sorting Complex Required for Transport III). FYVE4 affects the membrane association of the late-acting ESCRT components SNF7 and VPS4, and modulates the formation of intraluminal vesicles (ILVs) inside MVEs. The critical function of FYVE4 in the ESCRT pathway was further demonstrated by the strong genetic interactions with SNF7B and LIP5. Although the fyve4-1, snf7b and lip5 single mutants were viable, the fyve4-1 snf7b and fyve4-1 lip5 double mutants were seedling lethal, with strong defects in MVE biogenesis and vacuolar sorting of ubiquitinated membrane proteins. Taken together, we identified FYVE4 as a novel plant endosomal regulator, which functions in ESCRTing pathway to regulate MVE biogenesis.

Signal motif-dependent ER export of the Qc-SNARE BET12 interacts with MEMB12 and affects PR1 trafficking in Arabidopsis.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are well-known for their role in controlling membrane fusion, the final, but crucial step, in vesicular transport in eukaryotes. SNARE proteins contribute to various biological processes including pathogen defense and channel activity regulation, as well as plant growth and development. Precise targeting of SNARE proteins to destined compartments is a prerequisite for their proper functioning. However, the underlying mechanism(s) for SNARE targeting in plants remains obscure. Here, we investigate the targeting mechanism of the Arabidopsis thaliana Qc-SNARE BET12, which is involved in protein trafficking in the early secretory pathway. Two distinct signal motifs that are required for efficient BET12 ER export were identified. Pulldown assays and in vivo imaging implicated that both the COPI and COPII pathways were required for BET12 targeting. Further studies using an ER-export-defective form of BET12 revealed that the Golgi-localized Qb-SNARE MEMB12, a negative regulator of pathogenesis-related protein 1 (PR1; At2g14610) secretion, was its interacting partner. Ectopic expression of BET12 caused no inhibition in the general ER-Golgi anterograde transport but caused intracellular accumulation of PR1, suggesting that BET12 has a regulatory role in PR1 trafficking in A. thaliana.

The roles of endomembrane trafficking in plant abiotic stress responses.

Endomembrane trafficking is a fundamental cellular process in all eukaryotic cells and its regulatory mechanisms have been extensively studied. In plants, the endomembrane trafficking system needs to be constantly adjusted to adapt to the ever-changing environment. Evidence has accumulated supporting the idea that endomembrane trafficking is tightly linked to stress signaling pathways to meet the demands of rapid changes in cellular processes and to ensure the correct delivery of stress-related cargo molecules. However, the underlying mechanisms remain unknown. In this review, we summarize the recent findings on the functional roles of both secretory trafficking and endocytic trafficking in different types of abiotic stresses. We also highlight and discuss the unique properties of specific regulatory molecules beyond their conventional functions in endosomal trafficking during plant growth under stress conditions.

Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation.

Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 domain-containing protein2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.

Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana.

Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.

How vacuolar sorting receptor proteins interact with their cargo proteins: crystal structures of apo and cargo-bound forms of the protease-associated domain from an Arabidopsis vacuolar sorting receptor.

In plant cells, soluble proteins are directed to vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptors (VSR). To understand how a VSR recognizes its cargo, we present the crystal structures of the protease-associated domain of VSR isoform 1 from Arabidopsis thaliana (VSR1PA) alone and complexed with a cognate peptide containing the barley (Hordeum vulgare) aleurain VSD sequence of 1ADSNPIRPVT10. The crystal structures show that VSR1PA binds the sequence, Ala-Asp-Ser, preceding the NPIR motif. A conserved cargo binding loop, with a consensus sequence of 95RGxCxF100, forms a cradle that accommodates the cargo-peptide. In particular, Arg-95 forms a hydrogen bond to the Ser-3 position of the VSD, and the essential role of Arg-95 and Ser-3 in receptor-cargo interaction was supported by a mutagenesis study. Cargo binding induces conformational changes that are propagated from the cargo binding loop to the C terminus via conserved residues in switch I-IV regions. The resulting 180° swivel motion of the C-terminal tail is stabilized by a hydrogen bond between Glu-24 and His-181. A mutagenesis study showed that these two residues are essential for cargo interaction and trafficking. Based on our structural and functional studies, we present a model of how VSRs recognize their cargos.

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.

Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis.

Rab GTPases serve as multifaceted organizers during vesicle trafficking. Rab7, a member of the Rab GTPase family, has been shown to perform various essential functions in endosome trafficking and in endosome-to-lysosome trafficking in mammalian systems. The Arabidopsis thaliana genome encodes eight putative Rab7 homologs; however, the detailed function and activation mechanism of Rab7 in plants remain unknown. Here, we demonstrate that Arabidopsis RABG3f, a member of the plant Rab7 small GTPase family, localizes to prevacuolar compartments (PVCs) and the tonoplast. The proper activation of Rab7 is essential for both PVC-to-vacuole trafficking and vacuole biogenesis. Expression of a dominant-negative Rab7 mutant (RABG3fT22N) induces the formation of enlarged PVCs and affects vacuole morphology in plant cells. We also identify Arabidopsis MON1 (MONENSIN SENSITIVITY1) and CCZ1 (CALCIUM CAFFEINE ZINC SENSITIVITY1) proteins as a dimeric complex that functions as the Rab7 guanine nucleotide exchange factor. The MON1-CCZ1 complex also serves as the Rab5 effector to mediate Rab5-to-Rab7 conversion on PVCs. Loss of functional MON1 causes the formation of enlarged Rab5-positive PVCs that are separated from Rab7-positive endosomes. Similar to the dominant-negative Rab7 mutant, the mon1 mutants show pleiotropic growth defects, fragmented vacuoles, and altered vacuolar trafficking. Thus, Rab7 activation by the MON1-CCZ1 complex is critical for vacuolar trafficking, vacuole biogenesis, and plant growth.

ClpP-deletion impairs the virulence of Legionella pneumophila and the optimal translocation of effector proteins.

BACKGROUND: The opportunistic bacterial pathogen Legionella pneumophila uses substrate effectors of Dot/Icm type IVB secretion system (T4BSS) to accomplish survival and replication in amoebae cells and mammalian alveolar macrophages. During the conversion between its highly resistant, infectious dormant form and vigorously growing, uninfectious replicative form, L. pneumophila utilizes a complicated regulatory network in which proteolysis may play a significant role. As a highly conserved core protease, ClpP is involved in various cellular processes as well as virulence in bacteria, and has been proved to be required for the expression of transmission traits and cell division of L. pneumophila. RESULTS: The clpP-deficient L. pneumophila strain failed to replicate and was digested in the first 3 h post-infection in mammalian cells J774A.1. Further investigation demonstrates that the clpP deficient mutant strain was unable to escape the endosome-lysosomal pathway in host cells. We also found that the clpP deficient mutant strain still expresses T4BSS components, induces contact-dependent cytotoxicity and translocate effector proteins RalF and LegK2, indicating that its T4BSS was overall functional. Interestingly, we further found that the translocation of several effector proteins is significantly reduced without ClpP. CONCLUSIONS: The data indicate that ClpP plays an important role in regulating the virulence and effector translocation of Legionella pneumophila.

Protein S-ACYL Transferase10 is critical for development and salt tolerance in Arabidopsis.

Protein S-acylation, commonly known as palmitoylation, is a reversible posttranslational modification that catalyzes the addition of a saturated lipid group, often palmitate, to the sulfhydryl group of a Cys. Palmitoylation regulates enzyme activity, protein stability, subcellular localization, and intracellular sorting. Many plant proteins are palmitoylated. However, little is known about protein S-acyl transferases (PATs), which catalyze palmitoylation. Here, we report that the tonoplast-localized PAT10 is critical for development and salt tolerance in Arabidopsis thaliana. PAT10 loss of function resulted in pleiotropic growth defects, including smaller leaves, dwarfism, and sterility. In addition, pat10 mutants are hypersensitive to salt stresses. We further show that PAT10 regulates the tonoplast localization of several calcineurin B-like proteins (CBLs), including CBL2, CBL3, and CBL6, whose membrane association also depends on palmitoylation. Introducing a C192S mutation within the highly conserved catalytic motif of PAT10 failed to complement pat10 mutants, indicating that PAT10 functions through protein palmitoylation. We propose that PAT10-mediated palmitoylation is critical for vacuolar function by regulating membrane association or the activities of tonoplast proteins.