Showing papers on "Arabidopsis published in 2022"

Mutation bias reflects natural selection in Arabidopsis thaliana

Abstract: Abstract Since the first half of the twentieth century, evolutionary theory has been dominated by the idea that mutations occur randomly with respect to their consequences 1 . Here we test this assumption with large surveys of de novo mutations in the plant Arabidopsis thaliana . In contrast to expectations, we find that mutations occur less often in functionally constrained regions of the genome—mutation frequency is reduced by half inside gene bodies and by two-thirds in essential genes. With independent genomic mutation datasets, including from the largest Arabidopsis mutation accumulation experiment conducted to date, we demonstrate that epigenomic and physical features explain over 90% of variance in the genome-wide pattern of mutation bias surrounding genes. Observed mutation frequencies around genes in turn accurately predict patterns of genetic polymorphisms in natural Arabidopsis accessions ( r = 0.96). That mutation bias is the primary force behind patterns of sequence evolution around genes in natural accessions is supported by analyses of allele frequencies. Finally, we find that genes subject to stronger purifying selection have a lower mutation rate. We conclude that epigenome-associated mutation bias 2 reduces the occurrence of deleterious mutations in Arabidopsis , challenging the prevailing paradigm that mutation is a directionless force in evolution.

Auxin signaling: Research advances over the past 30 years.

Abstract: Auxin, one of the first identified and most widely studied phytohormones, has been and will remain a hot topic in plant biology. After more than a century of passionate exploration, the mysteries of its synthesis, transport, signaling, and metabolism have largely been unlocked. Due to the rapid development of new technologies, new methods, and new genetic materials, the study of auxin has entered the fast lane over the past 30 years. Here, we highlight advances in understanding auxin signaling, including auxin perception, rapid auxin responses, TRANSPORT INHIBITOR RESPONSE 1 and AUXIN SIGNALING F-boxes (TIR1/AFBs)-mediated transcriptional and non-transcriptional branches, and the epigenetic regulation of auxin signaling. We also focus on feedback inhibition mechanisms that prevent the over-amplification of auxin signals. In addition, we cover the TRANSMEMBRANE KINASEs (TMKs)-mediated non-canonical signaling, which converges with TIR1/AFBs-mediated transcriptional regulation to coordinate plant growth and development. The identification of additional auxin signaling components and their regulation will continue to open new avenues of research in this field, leading to an increasingly deeper, more comprehensive understanding of how auxin signals are interpreted at the cellular level to regulate plant growth and development. This article is protected by copyright. All rights reserved.

Increasing the resilience of plant immunity to a warming climate

Abstract: Abstract Extreme weather conditions associated with climate change affect many aspects of plant and animal life, including the response to infectious diseases. Production of salicylic acid (SA), a central plant defence hormone 1–3 , is particularly vulnerable to suppression by short periods of hot weather above the normal plant growth temperature range via an unknown mechanism 4–7 . Here we show that suppression of SA production in Arabidopsis thaliana at 28 °C is independent of PHYTOCHROME B 8,9 (phyB) and EARLY FLOWERING 3 10 (ELF3), which regulate thermo-responsive plant growth and development. Instead, we found that formation of GUANYLATE BINDING PROTEIN-LIKE 3 (GBPL3) defence-activated biomolecular condensates 11 (GDACs) was reduced at the higher growth temperature. The altered GDAC formation in vivo is linked to impaired recruitment of GBPL3 and SA-associated Mediator subunits to the promoters of CBP60g and SARD1 , which encode master immune transcription factors. Unlike many other SA signalling components, including the SA receptor and biosynthetic genes, optimized CBP60g expression was sufficient to broadly restore SA production, basal immunity and effector-triggered immunity at the elevated growth temperature without significant growth trade-offs. CBP60g family transcription factors are widely conserved in plants 12 . These results have implications for safeguarding the plant immune system as well as understanding the concept of the plant–pathogen–environment disease triangle and the emergence of new disease epidemics in a warming climate.

A wheat resistosome defines common principles of immune receptor channels

Abstract: Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.

RALF peptide signaling controls the polytubey block in Arabidopsis

Abstract: Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana, the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube–specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success. Description Block to polyspermy The next generation needs enough, but not too many, nuclear genomes. Zhong et al. show how the small mustard plant Arabidopsis both blocks polyspermy and also adds second-chance insurance when the first fertilization effort goes awry. Signals from the synergid cells in the female gametophyte invite a nearby pollen tube, which secretes peptides to block other pollen tubes from tagging along. The blockade persists as the pollen tube grows to its target. If the pollen tube successfully releases its pair of sperm (plants have a dual fertilization system), the pollen tube’s signaling system fades to silence. If the female gametophyte successfully receives the sperm nuclei, invitational signals from the synergid cells also fade to silence. If, however, fertilization fails, the persistent signal from the synergid cells continues to attract pollen tubes and, because pollen tube rupture silenced the first pollen tube’s block to polytuby, secondary pollen tubes are able to give another try at fertilization. —PJH Signaling between pollen tube and female gametophyte ensures that only one pollen tube gets through but can re-establish access in case of failure.

OUP accepted manuscript

Abstract: Abstract Galactose is an abundant and essential sugar used for the biosynthesis of many macromolecules in different organisms, including plants. Galactose metabolism is tightly and finely controlled, since excess galactose and its derivatives are inhibitory to plant growth. In Arabidopsis (Arabidopsis thaliana), root growth and pollen germination are strongly inhibited by excess galactose. However, the mechanism of galactose-induced inhibition during pollen germination remains obscure. In this study, we characterized a plasma membrane-localized transporter, Arabidopsis Sugars Will Eventually be Exported Transporter 5, that transports glucose and galactose. SWEET5 protein levels started to accumulate at the tricellular stage of pollen development and peaked in mature pollen, before rapidly declining after pollen germinated. SWEET5 levels are responsible for the dosage-dependent sensitivity to galactose, and galactokinase is essential for these inhibitory effects during pollen germination. However, sugar measurement results indicate that galactose flux dynamics and sugar metabolism, rather than the steady-state galactose level, may explain phenotypic differences between sweet5 and Col-0 in galactose inhibition of pollen germination.

DNA methylation-free Arabidopsis reveals crucial roles of DNA methylation in regulating gene expression and development

Abstract: A contribution of DNA methylation to defense against invading nucleic acids and maintenance of genome integrity is uncontested; however, our understanding of the extent of involvement of this epigenetic mark in genome-wide gene regulation and plant developmental control is incomplete. Here, we knock out all five known DNA methyltransferases in Arabidopsis, generating DNA methylation-free plants. This quintuple mutant exhibits a suite of developmental defects, unequivocally demonstrating that DNA methylation is essential for multiple aspects of plant development. We show that CG methylation and non-CG methylation are required for a plethora of biological processes, including pavement cell shape, endoreduplication, cell death, flowering, trichome morphology, vasculature and meristem development, and root cell fate determination. Moreover, we find that DNA methylation has a strong dose-dependent effect on gene expression and repression of transposable elements. Taken together, our results demonstrate that DNA methylation is dispensable for Arabidopsis survival but essential for the proper regulation of multiple biological processes.

OUP accepted manuscript

Abstract: Previously, we have shown that apoplastic wash fluid (AWF) purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs.

FIONA1 is an RNA N6-methyladenosine methyltransferase affecting Arabidopsis photomorphogenesis and flowering

Abstract: N6-methyladenosine (m6A) mRNA modification is essential for mammalian and plant viability. The U6 m6A methyltransferases in other species regulate S-adenosylmethionine (SAM) homeostasis through installing m6A in pre-mRNAs of SAM synthetases. However, U6 m6A methyltransferase has not been characterized in Arabidopsis and little is known about its role in regulating photomorphogenesis and flowering.Here we characterize that FIONA1 is an Arabidopsis U6 m6A methyltransferase that installs m6A in U6 snRNA and a small subset of poly(A)+ RNA. Disruption of FIONA1 leads to phytochrome signaling-dependent hypocotyl elongation and photoperiod-independent early flowering. Distinct from mammalian METTL16 and worm METT-10, FIONA1 neither installs m6A in the mRNAs of Arabidopsis SAM synthetases nor affects their transcript expression levels under normal or high SAM conditions. We confirm that FIONA1 can methylate plant mRNA m6A motifs in vitro and in vivo. We further show that FIONA1 installs m6A in several phenotypic related transcripts, thereby affecting downstream mRNA stability and regulating phytochrome signaling and floral transition.FIONA1 is functional as a U6 m6A methyltransferase in Arabidopsis, distinct from mammalian METTL16 and worm METT-10. Our results demonstrate that FIONA1-mediated m6A post-transcriptional regulation is an autonomous regulator for flowering and phytochrome signaling-dependent photomorphogenesis.

A dirigent family protein confers variation of Casparian strip thickness and salt tolerance in maize

Abstract: Abstract Plant salt-stress response involves complex physiological processes. Previous studies have shown that some factors promote salt tolerance only under high transpiring condition, thus mediating transpiration-dependent salt tolerance (TDST). However, the mechanism underlying crop TDST remains largely unknown. Here, we report that ZmSTL1 ( Salt-Tolerant Locus 1 ) confers natural variation of TDST in maize. ZmSTL1 encodes a dirigent protein (termed ZmESBL) localized to the Casparian strip (CS) domain. Mutants lacking ZmESBL display impaired lignin deposition at endodermal CS domain which leads to a defective CS barrier. Under salt condition, mutation of ZmESBL increases the apoplastic transport of Na + across the endodermis, and then increases the root-to-shoot delivery of Na + via transpiration flow, thereby leading to a transpiration-dependent salt hypersensitivity. Moreover, we show that the ortholog of ZmESBL also mediates CS development and TDST in Arabidopsis. Our study suggests that modification of CS barrier may provide an approach for developing salt-tolerant crops.

Plant immunity suppression via PHR1‐RALF‐FERONIA shapes the root microbiome to alleviate phosphate starvation

Abstract: The microbiome plays an important role in shaping plant growth and immunity, but few plant genes and pathways impacting plant microbiome composition have been reported. In Arabidopsis thaliana, the phosphate starvation response (PSR) was recently found to modulate the root microbiome upon phosphate (Pi) starvation through the transcriptional regulator PHR1. Here, we report that A. thaliana PHR1 directly binds to the promoters of rapid alkalinization factor (RALF) genes, and activates their expression under phosphate‐starvation conditions. RALFs in turn suppress complex formation of pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) receptor through FERONIA, a previously‐identified PTI modulator that increases resistance to certain detrimental microorganisms. Suppression of immunity via the PHR1‐RALF‐FERONIA axis allows colonization by specialized root microbiota that help to alleviate phosphate starvation by upregulating the expression of PSR genes. These findings provide a new paradigm for coordination of host‐microbe homeostasis through modulating plant innate immunity after environmental perturbations.

FIONA1 is an RNA N6-methyladenosine methyltransferase affecting Arabidopsis photomorphogenesis and flowering

Abstract: N6-methyladenosine (m6A) mRNA modification is essential for mammalian and plant viability. The U6 m6A methyltransferases in other species regulate S-adenosylmethionine (SAM) homeostasis through installing m6A in pre-mRNAs of SAM synthetases. However, U6 m6A methyltransferase has not been characterized in Arabidopsis and little is known about its role in regulating photomorphogenesis and flowering.Here we characterize that FIONA1 is an Arabidopsis U6 m6A methyltransferase that installs m6A in U6 snRNA and a small subset of poly(A)+ RNA. Disruption of FIONA1 leads to phytochrome signaling-dependent hypocotyl elongation and photoperiod-independent early flowering. Distinct from mammalian METTL16 and worm METT-10, FIONA1 neither installs m6A in the mRNAs of Arabidopsis SAM synthetases nor affects their transcript expression levels under normal or high SAM conditions. We confirm that FIONA1 can methylate plant mRNA m6A motifs in vitro and in vivo. We further show that FIONA1 installs m6A in several phenotypic related transcripts, thereby affecting downstream mRNA stability and regulating phytochrome signaling and floral transition.FIONA1 is functional as a U6 m6A methyltransferase in Arabidopsis, distinct from mammalian METTL16 and worm METT-10. Our results demonstrate that FIONA1-mediated m6A post-transcriptional regulation is an autonomous regulator for flowering and phytochrome signaling-dependent photomorphogenesis.

Plant Low-Temperature Stress: Signaling and Response

Abstract: Cold stress has always been a significant limitation for plant development and causes substantial decreases in crop yield. Some temperate plants, such as Arabidopsis, have the ability to carry out internal adjustment, which maintains and checks the metabolic machinery during cold temperatures. This cold acclimation process requires prior exposure to low, chilling temperatures to prevent damage during subsequent freezing stress and maintain the overall wellbeing of the plant despite the low-temperature conditions. In comparison, plants of tropical and subtropical origins, such as rice, are sensitive to chilling stress and respond differently to low-temperature stress. Plants have evolved various physiological, biochemical, and molecular mechanisms to sense and respond to low-temperature stress, including membrane modifications and cytoskeletal rearrangement. Moreover, the transient increase in cytosolic calcium level leads to the activation of many calcium-binding proteins and calcium-dependent protein kinases during low-temperature stress. Recently, mitogen-activated protein kinases have been found to regulate low-temperature signaling through ICE1. Besides, epigenetic control plays a crucial role during the cold stress response. This review primarily focuses on low-temperature stress experienced by plants and their strategies to overcome it. We have also reviewed recent progress and previous knowledge for a better understanding of plant cold stress response.

OUP accepted manuscript

Abstract: Flowering is a critical agricultural trait that substantially affects tomato fruit yield. Although drought stress influences flowering time, the molecular mechanism underlying drought-regulated flowering in tomato remains elusive. In this study, we demonstrated that loss of function of tomato OPEN STOMATA 1 (SlOST1), a protein kinase essential for abscisic acid (ABA) signaling and abiotic stress responses, lowers the tolerance of tomato plants to drought stress. slost1 mutants also exhibited a late flowering phenotype under both normal and drought stress conditions. We also established that SlOST1 directly interacts with and phosphorylates the NAC (NAM, ATAF and CUC)-type transcription factor VASCULAR PLANT ONE-ZINC FINGER 1 (SlVOZ1), at residue serine 67, thereby enhancing its stability and nuclear translocation in an ABA-dependent manner. Moreover, we uncovered several SlVOZ1 binding motifs from DNA affinity purification sequencing analyses and revealed that SlVOZ1 can directly bind to the promoter of the major flowering-integrator gene SINGLE FLOWER TRUSS to promote tomato flowering transition in response to drought. Collectively, our data uncover the essential role of the SlOST1-SlVOZ1 module in regulating flowering in response to drought stress in tomato and offer insights into a novel strategy to balance drought stress response and flowering.

ARFs are keys to the many auxin doors.

Abstract: In plants, most developmental programs depend on auxin action. The best described model of auxin signaling pathway that explains most, but not all, auxin transcriptional responses relies on a derepression mechanism. Repressors called Aux/IAAs (Auxin/Indole-3-Acetic Acid) interact with ARFs (Auxin Response Factors), the transcription factors of the auxin signaling pathway, leading to a repression of ARF-controlled genes. Auxin induces Aux/IAA degradation, releases ARFs and activates transcription. However, this elegant model is not suitable for all ARFs. Indeed, in Arabidopsis thaliana which has 22 ARFs, only 5 of them fit into the model since they are the ones able to interact with Aux/IAAs. The 17 left have a limited capacity to interact with the repressors and their mechanism is still unclear. ARF-Aux/IAA differential interaction is one of the many examples of ARFs biochemical and structural diversification that affects ARFs action and therefore, auxin transcriptional responses. A deeper understanding of the structural properties of ARFs is fundamental to better explain the action of auxin in plants.

An ancestral function of strigolactones as symbiotic rhizosphere signals

Abstract: Abstract In flowering plants, strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), an SL from the bryophyte Marchantia paleacea . BSB is also found in vascular plants, indicating its origin in the common ancestor of land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

HY5: A Pivotal Regulator of Light-Dependent Development in Higher Plants

Abstract: ELONGATED HYPOCOTYL5 (HY5), a bZIP-type transcription factor, acts as a master regulator that regulates various physiological and biological processes in plants such as photomorphogenesis, root growth, flavonoid biosynthesis and accumulation, nutrient acquisition, and response to abiotic stresses. HY5 is evolutionally conserved in function among various plant species. HY5 acts as a master regulator of light-mediated transcriptional regulatory hub that directly or indirectly controls the transcription of approximately one-third of genes at the whole genome level. The transcription, protein abundance, and activity of HY5 are tightly modulated by a variety of factors through distinct regulatory mechanisms. This review primarily summarizes recent advances on HY5-mediated molecular and physiological processes and regulatory mechanisms on HY5 in the model plant Arabidopsis as well as in crops.

OUP accepted manuscript

Abstract: The bacterial pathogen Ralstonia solanacearum causes wilt disease on Arabidopsis thaliana and tomato (Solanum lycopersicum). This pathogen uses type III effectors to inhibit the plant immune system; however, how individual effectors interfere with plant immune responses, including transcriptional reprograming, remain elusive. Here, we show that the type III effector RipAB targets Arabidopsis TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors, the central regulators of plant immune gene regulation, via physical interaction in the nucleus to dampen immune responses. RipAB was required for R. solanacearum virulence on wild-type tomato and Arabidopsis but not Arabidopsis tga1 tga4 and tga2 tga5 tga6 mutants. Stable expression of RipAB in Arabidopsis suppressed the pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) burst and immune gene induction as well as salicylic acid (SA) regulons including RBOHD and RBOHF, responsible for ROS production, all of which were phenocopied by the tga1 tga4 and tga2 tga5 tga6 mutants. We found that TGAs directly activate RBOHD and RBOHF expression and that RipAB inhibits this through interfering with the recruitment of RNA polymerase II. These results suggest that TGAs are the bona fide and major virulence targets of RipAB, which disrupts SA signaling by inhibiting TGA activity to achieve successful infection.

Mechanosensory trichome cells evoke a mechanical stimuli–induced immune response in Arabidopsis thaliana

Abstract: Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces, including raindrops, to anticipate pathogen infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) and mitogen-activated protein kinases. We propose an early layer of plant immunity in which trichomes function as mechanosensory cells that detect potential risks.

The tomato OST1-VOZ1 module regulates drought-mediated flowering.

Abstract: Flowering is a critical agricultural trait that substantially affects tomato fruit yield. Although drought stress influences flowering time, the molecular mechanism underlying drought-regulated flowering in tomato remains elusive. In this study, we demonstrated that loss of function of tomato OPEN STOMATA 1 (SlOST1), a protein kinase essential for abscisic acid (ABA) signaling and abiotic stress responses, lowers the tolerance of tomato plants to drought stress. slost1 mutants also exhibited a late flowering phenotype under both normal and drought stress conditions. We also established that SlOST1 directly interacts with and phosphorylates the NAC-type transcription factor VASCULAR PLANT ONE-ZINC FINGER 1 (SlVOZ1), at residue serine 67, thereby enhancing its stability and nuclear translocation in an ABA-dependent manner. Moreover, we uncovered several SlVOZ1 binding motifs from DNA affinity purification sequencing analyses and revealed that SlVOZ1 can directly bind to the promoter of the major flowering-integrator gene SINGLE FLOWER TRUSS (SFT) to promote tomato flowering transition in response to drought. Collectively, our data uncover the essential role of the SlOST1-SlVOZ1 module in regulating flowering in response to drought stress in tomato and offer insights into a novel strategy to balance drought stress response and flowering.

Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components

Abstract: Abstract In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.