ABSTRACT
The root cap secretes mucilage and sheds border cells (border-like cells, BLCs) in Arabidopsis (Arabidopsis thaliana). These mucilage and root cap-derived cells form a defensive barrier against soil pathogens. BEARSKIN1 (BRN1) and BRN2 are 2 homologous NAM, ATAF1/2, and CUC2 (NAC) family transcription factors of Arabidopsis, and mucilage secretion is inhibited in the brn1/2 double mutant. BRN1 and BRN2 are also involved in the expression of a pectin-digesting enzyme, POLYGALACTURONASE (RCPG), that facilitates BLC shedding. To further explore the connection between mucilage secretion and BLC shedding, we examined mucilage production in Arabidopsis lines displaying altered BLC detachment. Inactivation of BRN2 blocked mucilage synthesis and secretion, while inactivation of BRN1 and RCPG did not. Interestingly, RCPG sorted into mucilage-carrying vesicles budding from the Golgi and inhibited mucilage secretion in brn2-delayed BLC detachment. The root cap of a germinating seedling is initially covered with a cuticle, which is replaced by mucilage from BLCs as the seedling begins to shed these cells. Ectopic expression of RCPG in germinating seedlings caused early BLC formation and accelerated the cuticle-to-mucilage transition, indicating that RCPG expression and mucilage secretion are co-regulated. Furthermore, brn2 roots exhibited slower growth and increased cell death when subjected to salt or osmotic stress. Our research suggests that BRN2-mediated mucilage secretion contributes to BLC release to build an extracellular defense zone surrounding the root cap.
ABSTRACT
Rho/Rac of plant (ROP) GTPases are a plant-specific proteins that function as molecular switches, activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single copies of ROP (MpROP), GEFs (ROPGEF and SPIKE (SPK)), and GAPs (ROPGAP and ROP ENHANCER (REN)). MpROP regulates the development of various tissues and organs such as rhizoids, gemmae, and air chambers. While the ROPGEF, KARAPPO (MpKAR) is essential for gemma initiation, the functions of other ROP regulatory factors are less understood. This study focused on two GAPs: MpROPGAP and MpREN. Mpren single mutants showed defects in thallus growth, rhizoid tip growth, gemma development, and air chamber formation, whereas Mpropgap mutants showed no visible abnormalities. However, Mpropgap Mpren double mutants had more severe phenotypes than the Mpren single mutants, suggesting backup roles of MpROPGAP in MpREN-involving processes. Overexpression of MpROPGAP, MpREN resulted in similar gametophyte defects, highlighting the importance of MpROP activation/inactivation cycling (or balancing). Thus, MpREN predominantly, and MpROPGAP as a backup, regulate gametophyte development, most likely by controlling MpROP activation in M. polymorpha.
ABSTRACT
Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.
Subject(s)
Cytokinins , Gene Expression Regulation, Plant , Host Microbial Interactions , Lotus , Mesorhizobium , Plant Root Nodulation , Root Nodules, Plant , Symbiosis , Cytokinins/metabolism , Gene Expression Profiling , Lotus/genetics , Lotus/growth & development , Lotus/metabolism , Mutation , Root Nodules, Plant/growth & development , Root Nodules, Plant/microbiology , Signal Transduction , Mesorhizobium/genetics , Mesorhizobium/physiologyABSTRACT
In angiosperms, the transition from floral-organ maintenance to abscission determines reproductive success and seed dispersion. For petal abscission, cell-fate decisions specifically at the petal-cell base are more important than organ-level senescence or cell death in petals. However, how this transition is regulated remains unclear. Here, we identify a jasmonic acid (JA)-regulated chromatin-state switch at the base of Arabidopsis petals that directs local cell-fate determination via autophagy. During petal maintenance, co-repressors of JA signaling accumulate at the base of petals to block MYC activity, leading to lower levels of ROS. JA acts as an airborne signaling molecule transmitted from stamens to petals, accumulating primarily in petal bases to trigger chromatin remodeling. This allows MYC transcription factors to promote chromatin accessibility for downstream targets, including NAC DOMAIN-CONTAINING PROTEIN102 (ANAC102). ANAC102 accumulates specifically at the petal base prior to abscission and triggers ROS accumulation and cell death via AUTOPHAGY-RELATED GENEs induction. Developmentally induced autophagy at the petal base causes maturation, vacuolar delivery, and breakdown of autophagosomes for terminal cell differentiation. Dynamic changes in vesicles and cytoplasmic components in the vacuole occur in many plants, suggesting JA-NAC-mediated local cell-fate determination by autophagy may be conserved in angiosperms.
Subject(s)
Arabidopsis , Cyclopentanes , Oxylipins , Arabidopsis/genetics , Flowers/genetics , Reactive Oxygen Species/metabolism , Autophagy , Chromatin/metabolism , Gene Expression Regulation, PlantABSTRACT
One of the fundamental questions in plant developmental biology is how cell proliferation and cell expansion coordinately determine organ growth and morphology. An amenable system to address this question is the Arabidopsis root tip, where cell proliferation and elongation occur in spatially separated domains, and cell morphologies can easily be observed using a confocal microscope. While past studies revealed numerous elements of root growth regulation including gene regulatory networks, hormone transport and signaling, cell mechanics and environmental perception, how cells divide and elongate under possible constraints from cell lineages and neighboring cell files has not been analyzed quantitatively. This is mainly due to the technical difficulties in capturing cell division and elongation dynamics at the tip of growing roots, as well as an extremely labor-intensive task of tracing the lineages of frequently dividing cells. Here, we developed a motion-tracking confocal microscope and an Artificial Intelligence (AI)-assisted image-processing pipeline that enables semi-automated quantification of cell division and elongation dynamics at the tip of vertically growing Arabidopsis roots. We also implemented a data sonification tool that facilitates human recognition of cell division synchrony. Using these tools, we revealed previously unnoted lineage-constrained dynamics of cell division and elongation, and their contribution to the root zonation boundaries.
Subject(s)
Arabidopsis , Humans , Arabidopsis/genetics , Microscopy , Plant Roots , Artificial Intelligence , Meristem , Cell DivisionABSTRACT
Lateral root (LR) formation is an important developmental event for the establishment of the root system in most vascular plants. In Arabidopsis thaliana, the fewer roots (fwr) mutation in the GNOM gene, encoding a guanine nucleotide exchange factor of ADP ribosylation factor that regulates vesicle trafficking, severely inhibits LR formation. Local accumulation of auxin response for LR initiation is severely affected in fwr. To better understand how local accumulation of auxin response for LR initiation is regulated, we identified a mutation, fewer roots suppressor1 (fsp1), that partially restores LR formation in fwr. The gene responsible for fsp1 was identified as SUPERROOT2 (SUR2), encoding CYP83B1 that positions at the metabolic branch point in the biosynthesis of auxin/indole-3-acetic acid (IAA) and indole glucosinolate. The fsp1 mutation increases both endogenous IAA levels and the number of the sites where auxin response locally accumulates prior to LR formation in fwr. SUR2 is expressed in the pericycle of the differentiation zone and in the apical meristem in roots. Time-lapse imaging of the auxin response revealed that local accumulation of auxin response is more stable in fsp1. These results suggest that SUR2/CYP83B1 affects LR founder cell formation at the xylem pole pericycle cells where auxin accumulates. Analysis of the genetic interaction between SUR2 and GNOM indicates the importance of stabilization of local auxin accumulation sites for LR initiation.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Plant Roots/metabolismABSTRACT
In land plants, sexual dimorphism can develop in both diploid sporophytes and haploid gametophytes. While developmental processes of sexual dimorphism have been extensively studied in the sporophytic reproductive organs of model flowering plants such as stamens and carpels of Arabidopsis thaliana, those occurring in gametophyte generation are less well characterized due to the lack of amenable model systems. In this study, we performed three-dimensional morphological analyses of gametophytic sexual branch differentiation in the liverwort Marchantia polymorpha, using high-depth confocal imaging and a computational cell segmentation technique. Our analysis revealed that the specification of germline precursors initiates in a very early stage of sexual branch development, where incipient branch primordia are barely recognizable in the apical notch region. Moreover, spatial distribution patterns of germline precursors differ between males and females from the initial stage of primordium development in a manner dependent on the master sexual differentiation regulator MpFGMYB. At later stages, distribution patterns of germline precursors predict the sex-specific gametangia arrangement and receptacle morphologies seen in mature sexual branches. Taken together, our data suggest a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha.
Subject(s)
Arabidopsis , Marchantia , Marchantia/genetics , Sex Characteristics , Germ Cells, PlantABSTRACT
The root cap is a multilayered tissue covering the tip of a plant root that directs root growth through its unique functions, such as gravity sensing and rhizosphere interaction. To maintain the structure and function of the root cap, its constituent cells are constantly turned over through balanced cell division and cell detachment in the inner and outer cell layers, respectively. Upon displacement toward the outermost layer, columella cells at the central root cap domain functionally transition from gravity-sensing cells to secretory cells, but the mechanisms underlying this drastic cell fate transition are largely unknown. Here, using live-cell tracking microscopy, we show that organelles in the outermost cell layer undergo dramatic rearrangements. This rearrangement depends, at least partially, on spatiotemporally regulated activation of autophagy. Notably, this root cap autophagy does not lead to immediate cell death, but is instead necessary for organized separation of living root cap cells, highlighting a previously undescribed role of developmentally regulated autophagy in plants. This article has an associated 'The people behind the papers' interview.
Subject(s)
Arabidopsis , Arabidopsis/metabolism , Autophagy , Cell Separation , Humans , Organelles , Plant Root Cap , Plant Roots/metabolismABSTRACT
Organ morphologies are diverse but also conserved under shared developmental constraints among species. Any geometrical similarities in the shape behind diversity and the underlying developmental constraints remain unclear. Plant root tip outlines commonly exhibit a dome shape, which likely performs physiological functions, despite the diversity in size and cellular organization among distinct root classes and/or species. We carried out morphometric analysis of the primary roots of ten angiosperm species and of the lateral roots (LRs) of Arabidopsis, and found that each root outline was isometrically scaled onto a parameter-free catenary curve, a stable structure adopted for arch bridges. Using the physical model for bridges, we analogized that localized and spatially uniform occurrence of oriented cell division and expansion force the LR primordia (LRP) tip to form a catenary curve. These growth rules for the catenary curve were verified by tissue growth simulation of developing LRP development based on time-lapse imaging. Consistently, LRP outlines of mutants compromised in these rules were found to deviate from catenary curves. Our analyses demonstrate that physics-inspired growth rules constrain plant root tips to form isometrically scalable catenary curves.
Subject(s)
Plant Development/physiology , Plant Roots/growth & development , Arabidopsis/growth & development , Cell Division , Meristem/anatomy & histology , Meristem/cytology , Meristem/growth & development , Plant Roots/anatomy & histology , Plant Roots/cytologyABSTRACT
In Arabidopsis thaliana, PROPEPs and their derived elicitor-active Pep epitopes provide damage-associated molecular patterns (DAMPs), which trigger defence responses through cell-surface receptors PEPR1 and PEPR2. In addition, Pep peptides induce root growth inhibition and root hair formation, however their relationships and coordinating mechanisms are poorly understood. Here, we reveal that Pep1-mediated root hair formation requires PEPR-associated kinases BAK1/BKK1 and BIK1/PBL1, ethylene, auxin and root hair differentiation regulators, in addition to PEPR2. Our analysis on 69 accessions unravels intraspecies variations in Pep1-induced root hair formation and growth inhibition. The absence of a positive correlation between the two traits suggests their separate regulation and diversification in natural populations of A. thaliana. Restricted PEPR2 expression to certain root tissues is sufficient to induce root hair formation and growth inhibition in response to Pep1, indicating the capacity of non-cell-autonomous receptor signalling in different root tissues. Of particular note, root hair cell-specific PEPR2 expression uncouples defence activation from root growth inhibition and root hair formation, suggesting a unique property of root hairs in root defence activation following Pep1 recognition.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Indoleacetic Acids , Peptides , Plant Roots , Protein Serine-Threonine Kinases , Receptors, Cell SurfaceABSTRACT
Plant shoots can bend upward against gravity, a behavior known as shoot gravitropism. The conventional quantification of shoot bending has been restricted to measurements of shoot tip angle, which cannot fully describe the spatio-temporal bending process. Recently, however, advanced imaging analyses have been developed to quantify in detail the spatio-temporal changes in inclination angle and curvature of the shoot. We used one such method (KymoRod) to analyze the gravitropism of the Arabidopsis thaliana inflorescence stem, and successfully extracted characteristics that capture when and where bending occurs. Furthermore, we implemented an elastic spring theoretical model and successfully determined best fitted parameters that may explain typical bending behaviors of the inflorescence stem. Overall, we propose a data-model combined framework to quantitatively investigate shoot gravitropism in plants.
ABSTRACT
Lateral roots (LRs) are crucial for increasing the surface area of root systems to explore heterogeneous soil environments. Major advances have recently been made in the model plant arabidopsis (Arabidopsis thaliana) to elucidate the cellular basis of LR development and the underlying gene regulatory networks (GRNs) that control the morphogenesis of the new root organ. This has provided a foundation for understanding the sophisticated adaptive mechanisms that regulate how plants pattern their root branching to match the spatial availability of resources such as water and nutrients in their external environment. We review new insights into the molecular, cellular, and environmental regulation of LR development in arabidopsis.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Gene Expression Regulation, Plant , Gene Regulatory Networks , Indoleacetic Acids , Plant RootsABSTRACT
How plant cells re-establish differential growth to initiate organs is poorly understood. Morphogenesis of lateral roots relies on the asymmetric cell division of initially symmetric founder cells. This division is preceded by the tightly controlled asymmetric radial expansion of these cells. The cellular mechanisms that license and ensure the coordination of these events are unknown. Here, we quantitatively analyze microtubule and F-actin dynamics during lateral root initiation. Using mutants and pharmacological and tissue-specific genetic perturbations, we show that dynamic reorganization of both microtubule and F-actin networks is necessary for the asymmetric expansion of the founder cells. This cytoskeleton remodeling intertwines with auxin signaling in the pericycle and endodermis in order for founder cells to acquire a basic polarity required for initiating lateral root development. Our results reveal the conservation of cell remodeling and polarization strategies between the Arabidopsis zygote and lateral root founder cells. We propose that coordinated, auxin-driven reorganization of the cytoskeleton licenses asymmetric cell growth and divisions during embryonic and post-embryonic organogenesis.
Subject(s)
Actins/metabolism , Arabidopsis/growth & development , Microtubules/metabolism , Plant Proteins/metabolism , Plant Roots/growth & development , Arabidopsis/metabolism , Cytoskeleton/metabolism , Plant Roots/metabolismABSTRACT
Lateral root (LR) formation in Arabidopsis thaliana is initiated by asymmetric division of founder cells, followed by coordinated cell proliferation and differentiation for patterning new primordia. The sequential developmental processes of LR formation are triggered by a localized auxin response. LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), an auxin-inducible transcription factor, is one of the key regulators linking auxin response in LR founder cells to LR initiation. We identified key genes for LR formation that are activated by LBD16 in an auxin-dependent manner. LBD16 targets identified include the transcription factor gene PUCHI, which is required for LR primordium patterning. We demonstrate that LBD16 activity is required for the auxin-inducible expression of PUCHI. We show that PUCHI expression is initiated after the first round of asymmetric cell division of LR founder cells and that premature induction of PUCHI during the preinitiation phase disrupts LR primordium formation. Our results indicate that LR initiation requires the sequential induction of transcription factors LBD16 and PUCHI.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Arabidopsis/genetics , Gene Expression Regulation, Plant/physiology , Transcription Factors/metabolism , Arabidopsis Proteins/genetics , Gene Expression Regulation, Developmental/physiology , Indoleacetic Acids/metabolism , Plant Roots/growth & development , Transcription Factors/geneticsABSTRACT
Lateral root organogenesis plays an essential role in elaborating plant root system architecture. In Arabidopsis, the AP2 family transcription factor PUCHI controls cell proliferation in lateral root primordia. To identify potential targets of PUCHI, we analyzed a time course transcriptomic dataset of lateral root formation. We report that multiple genes coding for very long chain fatty acid (VLCFA) biosynthesis enzymes are induced during lateral root development in a PUCHI-dependent manner. Significantly, several mutants perturbed in VLCFA biosynthesis show similar lateral root developmental defects as puchi-1 Moreover, puchi-1 roots display the same disorganized callus formation phenotype as VLCFA biosynthesis-deficient mutants when grown on auxin-rich callus-inducing medium. Lipidomic profiling of puchi-1 roots revealed reduced VLCFA content compared with WT. We conclude that PUCHI-regulated VLCFA biosynthesis is part of a pathway controlling cell proliferation during lateral root and callus formation.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Bony Callus/growth & development , Plant Roots/growth & development , Transcription Factors/genetics , Arabidopsis/growth & development , Bony Callus/metabolism , Cell Proliferation/genetics , Fatty Acids/biosynthesis , Fatty Acids/genetics , Indoleacetic Acids/metabolism , Plant Development/genetics , Plant Roots/geneticsABSTRACT
In plants, postembryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires the tight coordination of asymmetric cell division in adjacent pericycle cells. We identified EXPANSIN A1 (EXPA1) as a cell wall modifying enzyme controlling the divisions marking lateral root initiation. Loss of EXPA1 leads to defects in the first asymmetric pericycle cell divisions and the radial swelling of the pericycle during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Cell Division , Plant Roots , Arabidopsis/cytology , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , Cell Division/genetics , Cell Division/physiology , Cell Wall/genetics , Cell Wall/physiology , Plant Roots/cytology , Plant Roots/growth & development , Plant Roots/physiology , TranscriptomeABSTRACT
In plants, the position of lateral roots (LRs) depends on initiation sites induced by auxin. The domain of high auxin response responsible for LR initiation stretches over several cells, but only a pair of pericycle cells (LR founder cells) will develop into LRs. In this work, we identified a signaling cascade controlling LR formation through lateral inhibition. It comprises a peptide hormone TARGET OF LBD SIXTEEN 2 (TOLS2), its receptor RLK7, and a downstream transcription factor PUCHI. TOLS2 is expressed at the LR founder cells and inhibits LR initiation. Time-lapse imaging of auxin-responsive DR5:LUCIFERASE reporter expression revealed that occasionally two pairs of LR founder cells are specified in close proximity even in wild-type and that one of them exists only transiently and disappears in an RLK7-dependent manner. We propose that the selection of LR founder cells by the peptide hormone-receptor cascade ensures proper LR spacing.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Gene Expression Regulation, Plant/physiology , Plant Roots/metabolism , Cell Communication/physiology , Cell Differentiation/physiology , Cell Division/physiology , Indoleacetic Acids/metabolism , Organogenesis, Plant/physiology , Plants, Genetically Modified/metabolismABSTRACT
Lateral roots comprise the majority of the branching root system and are important for acquiring nutrients and water from soil in addition to providing anchorage. Lateral roots develop post-embryonically from existing root parts and originate from a subset of specified pericycle cells (lateral root founder cells) located deep inside roots. Small numbers of these specified pericycle cells undergo several rounds of cell division to create a dome-shaped primordium, which eventually organizes a meristem, an essential region for plant growth with active cell division, and emerges from its parental root as a lateral root. Observing cellular and molecular processes for an extended time at various scales are crucial for understanding biological processes during organogenesis. Lateral root formation is an example of the successful application of live-cell imaging approaches to understand various aspects of developmental events in plants, including cell fate determination, cell proliferation, cell-to-cell interaction and cell wall modification. Here I review the recent progress in understanding the molecular mechanisms of lateral root formation and the contribution of live-cell imaging approaches.
Subject(s)
Arabidopsis/growth & development , Meristem/growth & development , Organogenesis, Plant/physiology , Plant Roots/growth & development , Arabidopsis/anatomy & histology , Indoleacetic Acids/metabolism , Time-Lapse ImagingABSTRACT
Plants adapt to heterogeneous soil conditions by altering their root architecture. For example, roots branch when in contact with water by using the hydropatterning response. We report that hydropatterning is dependent on auxin response factor ARF7. This transcription factor induces asymmetric expression of its target gene LBD16 in lateral root founder cells. This differential expression pattern is regulated by posttranslational modification of ARF7 with the small ubiquitin-like modifier (SUMO) protein. SUMOylation negatively regulates ARF7 DNA binding activity. ARF7 SUMOylation is required to recruit the Aux/IAA (indole-3-acetic acid) repressor protein IAA3. Blocking ARF7 SUMOylation disrupts IAA3 recruitment and hydropatterning. We conclude that SUMO-dependent regulation of auxin response controls root branching pattern in response to water availability.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Plant Roots/growth & development , Sumoylation , Transcription Factors/metabolism , Water/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , DNA, Plant/metabolism , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , Nuclear Proteins/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Protein Binding , SUMO-1 Protein/metabolismABSTRACT
Membrane trafficking plays pivotal roles in various cellular activities and higher-order functions of eukaryotes and requires tethering factors to mediate contact between transport intermediates and target membranes. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are known to act in endosomal/vacuolar transport in yeast and animals. Both complexes share a core subcomplex consisting of Vps11, Vps18, Vps16, and Vps33, and in addition to this core, HOPS contains Vps39 and Vps41, whereas CORVET contains Vps3 and Vps8. HOPS and CORVET subunits are also conserved in the model plant Arabidopsis. However, vacuolar trafficking in plants occurs through multiple unique transport pathways, and how these conserved tethering complexes mediate endosomal/vacuolar transport in plants has remained elusive. In this study, we investigated the functions of VPS18, VPS3, and VPS39, which are core complex, CORVET-specific, and HOPS-specific subunits, respectively. Impairment of these tethering proteins resulted in embryonic lethality, distinctly altering vacuolar morphology and perturbing transport of a vacuolar membrane protein. CORVET interacted with canonical RAB5 and a plant-specific R-soluble NSF attachment protein receptor (SNARE), VAMP727, which mediates fusion between endosomes and the vacuole, whereas HOPS interacted with RAB7 and another R-SNARE, VAMP713, which likely mediates homotypic vacuolar fusion. These results indicate that CORVET and HOPS act in distinct vacuolar trafficking pathways in plant cells, unlike those of nonplant systems that involve sequential action of these tethering complexes during vacuolar/lysosomal trafficking. These results highlight a unique diversification of vacuolar/lysosomal transport that arose during plant evolution, using evolutionarily conserved tethering components.