Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

Expanded roles and divergent regulation of FAMA in Brachypodium and Arabidopsis stomatal development.

Stomata, cellular valves found on the surfaces of aerial plant tissues, present a paradigm for studying cell fate and patterning in plants. A highly conserved core set of related basic helix-loop-helix (bHLH) transcription factors regulates stomatal development across diverse species. We characterized BdFAMA in the temperate grass Brachypodium distachyon and found this late-acting transcription factor was necessary and sufficient for specifying stomatal guard cell fate, and unexpectedly, could also induce the recruitment of subsidiary cells in the absence of its paralogue, BdMUTE. The overlap in function is paralleled by an overlap in expression pattern and by unique regulatory relationships between BdMUTE and BdFAMA. To better appreciate the relationships among the Brachypodium stomatal bHLHs, we used in vivo proteomics in developing leaves and found evidence for multiple shared interaction partners. We reexamined the roles of these genes in Arabidopsis thaliana by testing genetic sufficiency within and across species, and found that while BdFAMA and AtFAMA can rescue stomatal production in Arabidopsis fama and mute mutants, only AtFAMA can specify Brassica-specific myrosin idioblasts. Taken together, our findings refine the current models of stomatal bHLH function and regulatory feedback among paralogues within grasses as well as across the monocot/dicot divide.

SPINDLY mediates O-fucosylation of hundreds of proteins and sugar-dependent growth in Arabidopsis.

The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates the important roles of SPY in diverse developmental and physiological processes. However, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrated that SPY mediates sugar-dependent growth in Arabidopsis (Arabidopsis thaliana). We further identified hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in our proteomic analyses were undetectable or dramatically decreased in the spy mutants, and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, phytohormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and phytohormone responses. Our study identified numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.

Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.

SECRET AGENT O-GlcNAcylates Hundreds of Proteins Involved in Diverse Cellular Processes in Arabidopsis.

O-GlcNAcylation is a critical post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. While considerable knowledge exists about over 3000 substrates in animals, our understanding of this modification in plants remains limited. Unlike animals, plants possess two putative homologs: SECRET AGENT (SEC) and SPINDLY, with SPINDLY also exhibiting O-fucosylation activity. To investigate the role of SEC as a major O-GlcNAc transferase in plants, we utilized lectin-weak affinity chromatography enrichment and stable isotope labeling in Arabidopsis labeling, quantifying at both MS1 and MS2 levels. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating the critical role of SEC in mediating O-GlcNAcylation. Through a comprehensive approach, combining higher-energy collision dissociation and electron-transfer high-energy collision dissociation fragmentation with substantial fractionations, we expanded our GlcNAc profiling, identifying 436 O-GlcNAc targets, including 227 new targets. The targets span diverse cellular processes, suggesting broad regulatory functions of O-GlcNAcylation. The expanded targets also enabled exploration of crosstalk between O-GlcNAcylation and O-fucosylation. We also examined electron-transfer high-energy collision dissociation fragmentation for site assignment. This report advances our understanding of O-GlcNAcylation in plants, facilitating further research in this field.

Uncovering the proximal proteome of CTR1 through TurboID-mediated proximity labeling.

Protein-protein interactions play a crucial role in driving cellular processes and enabling appropriate physiological responses in organisms. The plant hormone ethylene signaling pathway is complex and regulated by the spatiotemporal regulation of its signaling molecules. Constitutive Triple Response 1 (CTR1), a key negative regulator of the pathway, regulates the function of Ethylene-Insensitive 2 (EIN2), a positive regulator of ethylene signaling, at the endoplasmic reticulum (ER) through phosphorylation. Our recent study revealed that CTR1 can also translocate from the ER to the nucleus in response to ethylene and positively regulate ethylene responses by stabilizing EIN3. To gain further insights into the role of CTR1 in plants, we used TurboID-based proximity labeling and mass spectrometry to identify the proximal proteomes of CTR1 in Nicotiana benthamiana. The identified proximal proteins include known ethylene signaling components, as well as proteins involved in diverse cellular processes such as mitochondrial respiration, mRNA metabolism, and organelle biogenesis. Our study demonstrates the feasibility of proximity labeling using the N. benthamiana transient expression system and identifies the potential interactors of CTR1 in vivo, uncovering the potential roles of CTR1 in a wide range of cellular processes.

TRIPP Is a Plant-Specific Component of the Arabidopsis TRAPPII Membrane Trafficking Complex with Important Roles in Plant Development.

How the membrane trafficking system spatially organizes intracellular activities and intercellular signaling networks in plants is not well understood. Transport Protein Particle (TRAPP) complexes play key roles in the selective delivery of membrane vesicles to various subcellular compartments in yeast and animals but remain to be fully characterized in plants. Here, we investigated TRAPP complexes in Arabidopsis (Arabidopsis thaliana) using immunoprecipitation followed by quantitative mass spectrometry analysis of AtTRS33, a conserved core component of all TRAPP complexes. We identified 14 AtTRS33-interacting proteins, including homologs of all 13 TRAPP components in mammals and a protein that has homologs only in multicellular photosynthetic organisms and is thus named TRAPP-Interacting Plant Protein (TRIPP). TRIPP specifically associates with the TRAPPII complex through binary interactions with two TRAPPII-specific subunits. TRIPP colocalized with a subset of TRS33 compartments and trans-Golgi network markers in a TRS33-dependent manner. Loss-of-function tripp mutants exhibited dwarfism, sterility, partial photomorphogenesis in the dark, reduced polarity of the auxin transporter PIN2, incomplete cross wall formation, and altered localization of a TRAPPII-specific component. Therefore, TRIPP is a plant-specific component of the TRAPPII complex with important functions in trafficking, plant growth, and development.

Mutual Regulation of Receptor-Like Kinase SIT1 and B'κ-PP2A Shapes the Early Response of Rice to Salt Stress.

The receptor-like kinase SIT1 acts as a sensor in rice (Oryza sativa) roots, relaying salt stress signals via elevated kinase activity to enhance salt sensitivity. Here, we demonstrate that Protein Phosphatase 2A (PP2A) regulatory subunit B'κ constrains SIT1 activity under salt stress. B'κ-PP2A deactivates SIT1 directly by dephosphorylating the kinase at Thr515/516, a salt-induced phosphorylation site in the activation loop that is essential for SIT1 activity. B'κ overexpression suppresses the salt sensitivity of rice plants expressing high levels of SIT1, thereby contributing to salt tolerance. B'κ functions in a SIT1 kinase-dependent manner. During early salt stress, activated SIT1 phosphorylates B'κ; this not only enhances its binding with SIT1, it also promotes B'κ protein accumulation via Ser502 phosphorylation. Consequently, by blocking SIT1 phosphorylation, B'κ inhibits and fine-tunes SIT1 activity to balance plant growth and stress adaptation.

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis.

Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

N-Glycopeptide Profiling in Arabidopsis Inflorescence.

This study presents the first large-scale analysis of plant intact glycopeptides. Using wheat germ agglutinin lectin weak affinity chromatography to enrich modified peptides, followed by electron transfer dissociation (ETD)(1) fragmentation tandem mass spectrometry, glycan compositions on over 1100 glycopeptides from 270 proteins found in Arabidopsis inflorescence tissue were characterized. While some sites were only detected with a single glycan attached, others displayed up to 16 different glycoforms. Among the identified glycopeptides were four modified in nonconsensus glycosylation motifs. While most of the modified proteins are secreted, membrane, endoplasmic reticulum (ER), or Golgi-localized proteins, surprisingly, N-linked sugars were detected on a protein predicted to be cytosolic or nuclear.

Multisite light-induced phosphorylation of the transcription factor PIF3 is necessary for both its rapid degradation and concomitant negative feedback modulation of photoreceptor phyB levels in Arabidopsis.

Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood. Here, using mass spectrometry, we identify multiple, in vivo, light-induced Ser/Thr phosphorylation sites in PIF3. Using transgenic expression of site-directed mutants of PIF3, we provide evidence that a set of these phosphorylation events acts collectively to trigger rapid degradation of the PIF3 protein in response to initial exposure of dark-grown seedlings to light. In addition, we show that phyB-induced PIF3 phosphorylation is also required for the known negative feedback modulation of phyB levels in prolonged light, potentially through codegradation of phyB and PIF3. This mutually regulatory intermolecular transaction thus provides a mechanism with the dual capacity to promote early, graded, or threshold regulation of the primary, PIF3-controlled transcriptional network in response to initial light exposure, and later, to attenuate global sensitivity to the light signal through reductions in photoreceptor levels upon prolonged exposure.

Impact of alternative splicing on Arabidopsis proteome.

Alternative splicing is an important regulatory process in eukaryotes. In plants, the major form of alternative splicing is intron retention. Despite its importance, the global impact of AS on the Arabidopsis proteome has not been investigated. In this study, we address this gap by performing a comprehensive integrated analysis of how changes in AS can affect the Arabidopsis proteome using mutants that disrupt ACINUS and PININ, two evolutionarily conserved alternative splicing factors. We used tandem mass tagging (TMT) with real-time search MS3 (RTS-SPS-MS3) coupled with extensive sample fractionations to achieve very high coverage and accurate protein quantification. We then integrated our proteomic data with transcriptomic data to assess how transcript changes and increased intron retention (IIR) affect the proteome. For differentially expressed transcripts, we have observed a weak to moderate correlation between transcript changes and protein changes. Our studies revealed that some IIRs have no effect on either transcript or protein levels, while some IIRs can significantly affect protein levels. Surprisingly, we found that IIRs have a much smaller effect on increasing protein diversity. Notably, the increased intron retention events detected in the double mutant are also detected in the WT under various biotic or abiotic stresses. We further investigated the characteristics of the retained introns. Our extensive proteomic data help to guide the phenotypic analysis and reveal that collective protein changes contribute to the observed phenotypes of the increased anthocyanin, pale green, reduced growth, and short root observed in the acinus pnn double mutant. Overall, our study provides insight into the intricate regulatory mechanism of intron retention and its impact on protein abundance in plants.

Spatially resolved proteomics of the Arabidopsis stomatal lineage identifies polarity complexes for cell divisions and stomatal pores.

Cell polarity is used to guide asymmetric divisions and create morphologically diverse cells. We find that two oppositely oriented cortical polarity domains present during the asymmetric divisions in the Arabidopsis stomatal lineage are reconfigured into polar domains marking ventral (pore-forming) and outward-facing domains of maturing stomatal guard cells. Proteins that define these opposing polarity domains were used as baits in miniTurboID-based proximity labeling. Among differentially enriched proteins, we find kinases, putative microtubule-interacting proteins, and polar SOSEKIs with their effector ANGUSTIFOLIA. Using AI-facilitated protein structure prediction models, we identify potential protein-protein interaction interfaces among them. Functional and localization analyses of the polarity protein OPL2 and its putative interaction partners suggest a positive interaction with mitotic microtubules and a role in cytokinesis. This combination of proteomics and structural modeling with live-cell imaging provides insights into how polarity is rewired in different cell types and cell-cycle stages.

Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade.

For over 60 years, salicylic acid (SA) has been known as a plant immune signal required for both basal and systemic acquired resistance (SAR). SA activates these immune responses by reprogramming up to 20% of the transcriptome through the function of NPR1. However, components in the NPR1-signaling hub, which appears as nuclear condensates, and the NPR1- signaling cascade remained elusive due to difficulties in studying transcriptional cofactors whose chromatin associations are often indirect and transient. To overcome this challenge, we applied TurboID to divulge the NPR1-proxiome, which detected almost all known NPR1-interactors as well as new components of transcription-related complexes. Testing of new components showed that chromatin remodeling and histone demethylation contribute to SA-induced resistance. Globally, NPR1-proxiome shares a striking similarity to GBPL3-proxiome involved in SA synthesis, except associated transcription factors (TFs), suggesting that common regulatory modules are recruited to reprogram specific transcriptomes by transcriptional cofactors, like NPR1, through binding to unique TFs. Stepwise greenCUT&RUN analyses showed that, upon SA-induction, NPR1 initiates the transcriptional cascade primarily through association with TGA TFs to induce expression of secondary TFs, predominantly WRKYs. WRKY54 and WRKY70 then play a major role in inducing immune-output genes without interacting with NPR1 at the chromatin. Moreover, a loss of NPR1 condensate formation decreases its chromatin-association and transcriptional activity, indicating the importance of condensates in organizing the NPR1- signaling hub and initiating the transcriptional cascade. This study demonstrates how combinatorial applications of TurboID and stepwise greenCUT&RUN transcend traditional genetic methods to globally map signaling hubs and transcriptional cascades.

Workflow enhancement of TurboID-mediated proximity labeling for SPY signaling network mapping.

TurboID-based proximity labeling coupled to mass spectrometry (PL-MS) has emerged as a powerful tool for mapping protein-protein interactions in both plant and animal systems. Despite advances in sensitivity, PL-MS studies can still suffer from false negatives, especially when dealing with low abundance bait proteins and their transient interactors. Protein-level enrichment for biotinylated proteins is well developed and popular, but direct detection of biotinylated proteins by peptide-level enrichment and the difference in results between direct and indirect detection remain underexplored. To address this gap, we compared and improved enrichment and data analysis methods using TurboID fused to SPY, a low-abundance O-fucose transferase, using an AAL-enriched SPY target library for cross-referencing. Our results showed that MyOne and M280 streptavidin beads significantly outperformed antibody beads for peptide-level enrichment, with M280 performing best. In addition, while a biotin concentration ≤ 50 µM is recommended for protein-level enrichment in plants, higher biotin concentrations can be used for peptide-level enrichment, allowing us to improve detection and data quality. FragPipe's MSFragger protein identification and quantification software outperformed Maxquant and Protein Prospector for SPY interactome enrichment due to its superior detection of biotinylated peptides. Our improved washing protocols for protein-level enrichment mitigated bead collapse issues, improving data quality, and reducing experimental time. We found that the two enrichment methods provided complementary results and identified a total of 160 SPY-TurboID-enriched interactors, including 60 previously identified in the AAL-enriched SPY target list and 100 additional novel interactors. SILIA quantitative proteomics comparing WT and spy-4 mutants showed that SPY affects the protein levels of some of the identified interactors, such as nucleoporin proteins. We expect that our improvement will extend beyond TurboID to benefit other PL systems and hold promise for broader applications in biological research.

Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis.

Oxygen (O2), a dominant element in the atmosphere and essential for most life on Earth, is produced by the photosynthetic oxidation of water. However, metabolic activity can cause accumulation of reactive O2 species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O2 sensitivity screen on a genome-wide insertional mutant library of the unicellular alga Chlamydomonas reinhardtii. This screen led to identification of a gene encoding a protein designated Rubisco methyltransferase 2 (RMT2). Although homologous to methyltransferases, RMT2 has not been experimentally demonstrated to have methyltransferase activity. Furthermore, the rmt2 mutant was not compromised for Rubisco (first enzyme of Calvin-Benson Cycle) levels but did exhibit a marked decrease in accumulation/activity of photosystem I (PSI), which causes light sensitivity, with much less of an impact on other photosynthetic complexes. This mutant also shows increased accumulation of Ycf3 and Ycf4, proteins critical for PSI assembly. Rescue of the mutant phenotype with a wild-type (WT) copy of RMT2 fused to the mNeonGreen fluorophore indicates that the protein localizes to the chloroplast and appears to be enriched in/around the pyrenoid, an intrachloroplast compartment present in many algae that is packed with Rubisco and potentially hypoxic. These results indicate that RMT2 serves an important role in PSI biogenesis which, although still speculative, may be enriched around or within the pyrenoid.

Proximity Labeling in Plants.

Proteins are workhorses in the cell; they form stable and more often dynamic, transient protein-protein interactions, assemblies, and networks and have an intimate interplay with DNA and RNA. These network interactions underlie fundamental biological processes and play essential roles in cellular function. The proximity-dependent biotinylation labeling approach combined with mass spectrometry (PL-MS) has recently emerged as a powerful technique to dissect the complex cellular network at the molecular level. In PL-MS, by fusing a genetically encoded proximity-labeling (PL) enzyme to a protein or a localization signal peptide, the enzyme is targeted to a protein complex of interest or to an organelle, allowing labeling of proximity proteins within a zoom radius. These biotinylated proteins can then be captured by streptavidin beads and identified and quantified by mass spectrometry. Recently engineered PL enzymes such as TurboID have a much-improved enzymatic activity, enabling spatiotemporal mapping with a dramatically increased signal-to-noise ratio. PL-MS has revolutionized the way we perform proteomics by overcoming several hurdles imposed by traditional technology, such as biochemical fractionation and affinity purification mass spectrometry. In this review, we focus on biotin ligase-based PL-MS applications that have been, or are likely to be, adopted by the plant field. We discuss the experimental designs and review the different choices for engineered biotin ligases, enrichment, and quantification strategies. Lastly, we review the validation and discuss future perspectives.

UPL3 Promotes BZR1 Degradation, Growth Arrest, and Seedling Survival under Starvation Stress in Arabidopsis.

BRASSINAZONE RESISTANT 1 (BZR1) is a key transcription factor of the brassinosteroid signaling pathway but also a signaling hub that integrates diverse signals that modulate plant growth. Previous studies have shown that starvation causes BZR1 degradation, but the underlying mechanisms are not understood. Here we performed quantitative proteomic analysis of BZR1 interactome under starvation conditions and identified two BZR1-interacting ubiquitin ligases, BAF1 and UPL3. Compared to the wild type, the upl3 mutants show long hypocotyl and increased BZR1 levels when grown under sugar starvation conditions but not when grown on sugar-containing media, indicating a role of UPL3 in BZR1 degradation specifically under starvation conditions. The upl3 mutants showed a reduced survival rate after starvation treatment, supporting the importance of UPL3-mediated BZR1 degradation and growth arrest for starvation survival. Treatments with inhibitors of TARGET of RAPAMYCIN (TOR) and autophagy altered BZR1 level in the wild type but were less effective in upl3 , suggesting that UPL3 mediates the TOR-regulated and autophagy-dependent degradation of BZR1. Further, the UPL3 protein level is increased posttranscriptionally by starvation but decreased by sugar treatment. Our study identifies UPL3 as a key component that mediates sugar regulation of hormone signaling pathways, important for optimal growth and survival in plants. IN A NUTSHELL: Background: The coordination between signaling pathways that monitor the levels of photosynthate and growth hormones is crucial for optimizing growth and survival, but the underlying mechanisms are not fully understood. When the sugar level is low, the BZR1 transcription factor of the brassinosteroid (BR) signaling pathway is degraded, and hence growth is attenuated to prevent starvation and enhance survival. When sugar is sufficient, sugar signaling inhibits BZR1 degradation and enables BR promotion of plant growth. The key component that mediates starvation-induced BZR1 degradation remains unknown.Question: What proteins interact with BZR1 and mediate its degradation under sugar starvation?Finding: We performed immunoprecipitation mass spectrometry analysis of BZR1 in starvation-treated Arabidopsis and identified many BZR1-interacting proteins, including two E3 ligases UPL3 and BAF1. Genetic analysis showed that UPL3 plays a specific and prominent role in promoting autophagy-dependent BZR1 degradation and plant survival under sugar-starvation conditions.Next step: How sugar-TOR signaling regulates UPL3 level remains to be studied in the future.

Cell Fate Programming by Transcription Factors and Epigenetic Machinery in Stomatal Development.

The development of multi-cellular organisms requires coordinated changes in gene expression that are often mediated by the interaction between transcription factors (TFs) and their corresponding cis-regulatory elements (CREs). During development and differentiation, the accessibility of CREs is dynamically modulated by the epigenome. How the epigenome, CREs and TFs together exert control over cell fate commitment remains to be fully understood. In the Arabidopsis leaf epidermis, meristemoids undergo a series of stereotyped cell divisions, then switch fate to commit to stomatal differentiation. Newly created or reanalyzed scRNA-seq and ChIP-seq data confirm that stomatal development involves distinctive phases of transcriptional regulation and that differentially regulated genes are bound by the stomatal basic-helix-loop-helix (bHLH) TFs. Targets of the bHLHs often reside in repressive chromatin before activation. MNase-seq evidence further suggests that the repressive state can be overcome and remodeled upon activation by specific stomatal bHLHs. We propose that chromatin remodeling is mediated through the recruitment of a set of physical interactors that we identified through proximity labeling - the ATPase-dependent chromatin remodeling SWI/SNF complex and the histone acetyltransferase HAC1. The bHLHs and chromatin remodelers localize to overlapping genomic regions in a hierarchical order. Furthermore, plants with stage-specific knock-down of the SWI/SNF components or HAC1 fail to activate specific bHLH targets and display stomatal development defects. Together these data converge on a model for how stomatal TFs and epigenetic machinery cooperatively regulate transcription and chromatin remodeling during progressive fate specification.

Proteomic analysis defines the interactome of telomerase in the protozoan parasite, Trypanosoma brucei.

Telomerase is a ribonucleoprotein enzyme responsible for maintaining the telomeric end of the chromosome. The telomerase enzyme requires two main components to function: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), which provides the template for telomeric DNA synthesis. TR is a long non-coding RNA, which forms the basis of a large structural scaffold upon which many accessory proteins can bind and form the complete telomerase holoenzyme. These accessory protein interactions are required for telomerase activity and regulation inside cells. The interacting partners of TERT have been well studied in yeast, human, and Tetrahymena models, but not in parasitic protozoa, including clinically relevant human parasites. Here, using the protozoan parasite, Trypanosoma brucei (T. brucei) as a model, we have identified the interactome of T. brucei TERT (TbTERT) using a mass spectrometry-based approach. We identified previously known and unknown interacting factors of TbTERT, highlighting unique features of T. brucei telomerase biology. These unique interactions with TbTERT, suggest mechanistic differences in telomere maintenance between T. brucei and other eukaryotes.