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.

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.

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.

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.

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.

Deconvoluting signals downstream of growth and immune receptor kinases by phosphocodes of the BSU1 family phosphatases.

Hundreds of leucine-rich repeat receptor kinases (LRR-RKs) have evolved to control diverse processes of growth, development and immunity in plants, but the mechanisms that link LRR-RKs to distinct cellular responses are not understood. Here we show that two LRR-RKs, the brassinosteroid hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the flagellin receptor FLAGELLIN SENSING 2 (FLS2), regulate downstream glycogen synthase kinase 3 (GSK3) and mitogen-activated protein (MAP) kinases, respectively, through phosphocoding of the BRI1-SUPPRESSOR1 (BSU1) phosphatase. BSU1 was previously identified as a component that inactivates GSK3s in the BRI1 pathway. We surprisingly found that the loss of the BSU1 family phosphatases activates effector-triggered immunity and impairs flagellin-triggered MAP kinase activation and immunity. The flagellin-activated BOTRYTIS-INDUCED KINASE 1 (BIK1) phosphorylates BSU1 at serine 251. Mutation of serine 251 reduces BSU1's ability to mediate flagellin-induced MAP kinase activation and immunity, but not its abilities to suppress effector-triggered immunity and interact with GSK3, which is enhanced through the phosphorylation of BSU1 at serine 764 upon brassinosteroid signalling. These results demonstrate that BSU1 plays an essential role in immunity and transduces brassinosteroid-BRI1 and flagellin-FLS2 signals using different phosphorylation sites. Our study illustrates that phosphocoding in shared downstream components provides signalling specificities for diverse plant receptor kinases.

Application of Parallel Reaction Monitoring in 15N Labeled Samples for Quantification.

Accurate relative quantification is critical in proteomic studies. The incorporation of stable isotope 15N to plant-expressed proteins in vivo is a powerful tool for accurate quantification with a major advantage of reducing preparative and analytical variabilities. However, 15N labeling quantification has several challenges. Less identifications are often observed in the heavy-labeled samples because of incomplete labeling, resulting in missing values in reciprocal labeling experiments. Inaccurate quantification can happen when there is contamination from co-eluting peptides or chemical noise in the MS1 survey scan. These drawbacks in quantification can be more pronounced in less abundant but biologically interesting proteins, which often have very few identified peptides. Here, we demonstrate the application of parallel reaction monitoring (PRM) to 15N labeled samples on a high resolution, high mass accuracy Orbitrap mass spectrometer to achieve reliable quantification even of low abundance proteins in samples.

15N Metabolic Labeling Quantification Workflow in Arabidopsis Using Protein Prospector.

Metabolic labeling using stable isotopes is widely used for the relative quantification of proteins in proteomic studies. In plants, metabolic labeling using 15N has great potential, but the associated complexity of data analysis has limited its usage. Here, we present the 15N stable-isotope labeled protein quantification workflow utilizing open-access web-based software Protein Prospector. Further, we discuss several important features of 15N labeling required to make reliable and precise protein quantification. These features include ratio adjustment based on labeling efficiency, median and interquartile range for protein ratios, isotope cluster pattern matching to flag incorrect monoisotopic peak assignment, and caching of quantification results for fast retrieval.

Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions.

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUS's O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.

In vivo methylation of OLA1 revealed by activity-based target profiling of NTMT1.

N-Terminal methyltransferase 1 (NTMT1) catalyzes the N-terminal methylation of proteins with a specific N-terminal motif after methionine removal. Aberrant N-terminal methylation has been implicated in several cancers and developmental diseases. Together with motif sequence and signal peptide analyses, activity-based substrate profiling of NTMT1 utilizing (E)-hex-2-en-5-ynyl-S-adenosyl-l-methionine (Hey-SAM) revealed 72 potential targets, which include several previously confirmed ones and many unknowns. Target validation using normal and NTMT1 knock-out (KO) HEK293FT cells generated by CRISPR-Cas9 demonstrated that Obg-like ATPase 1 (OLA1), a protein involved in many critical cellular functions, is methylated in vivo by NTMT1. Additionally, Hey-SAM synthesis achieved ≥98% yield for SAH conversion.

Enantioselective Synthesis of Dilignol Model Compounds and Their Stereodiscrimination Study with a Dye-Decolorizing Peroxidase.

A four-step enantioselective approach was developed to synthesize anti (1R,2S)-1a and (1S,2R)-1b containing a ß-O-4 linkage in good yields. A significant difference was observed for the apparent binding affinities of four stereospecific lignin model compounds with TcDyP by surface plasmon resonance, which was not translated into a significant difference in enzyme activities. The discrepancy may be attributed to the conformational change involving a loop widely present in DyPs upon H2O2 binding.

Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects.

Dye-decolorizing peroxidases (DyPs) are a family of H2O2-dependent heme peroxidases, which have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been uncovered for the B-class ElDyP from Enterobacter lignolyticus. Its structure revealed that a water molecule acts as the sixth axial ligand with two channels at diameters of ~3.0 and 8.0 Å leading to the heme center. A conformational change of ERS* to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs' bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type ElDyP with D2O2 at pH 3.5 suggested that cmpd 0 deprotonation by the distal aspartate is rate-limiting in the formation of cmpd I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with cmpd I instead of the free enzyme. The significant inverse sKIEs of kcat/KM and kERS* suggested that the aquo release in DyPs is mechanistically important and may explain the enzyme's adoption of two-electron reduction for cmpd I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining DyPs' acidic pH optimum. The kinetic mechanism of D143H-ElDyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs' lignolytic activity.

Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from Thermomonospora curvata.

Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases, in which a catalytic distal aspartate is involved in H2O2 activation to catalyze oxidations in acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial TcDyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds (J. Biol. Chem.2015, 290, 23447). Based on the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II-like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the TcDyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in TcDyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the crosslinking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class TcDyP is minimal possibly due to its extremely small solvent accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.

Characterization of Dye-decolorizing Peroxidase (DyP) from Thermomonospora curvata Reveals Unique Catalytic Properties of A-type DyPs.

Dye-decolorizing peroxidases (DyPs) comprise a new family of heme peroxidases, which has received much attention due to their potential applications in lignin degradation. A new DyP from Thermomonospora curvata (TcDyP) was identified and characterized. Unlike other A-type enzymes, TcDyP is highly active toward a wide range of substrates including model lignin compounds, in which the catalytic efficiency with ABTS (kcat(app)/Km(app) = (1.7 × 10(7)) m(-1) s(-1)) is close to that of fungal DyPs. Stopped-flow spectroscopy was employed to elucidate the transient intermediates as well as the catalytic cycle involving wild-type (wt) and mutant TcDyPs. Although residues Asp(220) and Arg(327) are found necessary for compound I formation, His(312) is proposed to play roles in compound II reduction. Transient kinetics of hydroquinone (HQ) oxidation by wt-TcDyP showed that conversion of the compound II to resting state is a rate-limiting step, which will explain the contradictory observation made with the aspartate mutants of A-type DyPs. Moreover, replacement of His(312) and Arg(327) has significant effects on the oligomerization and redox potential (E°') of the enzyme. Both mutants were found to promote the formation of dimeric state and to shift E°' to a more negative potential. Not only do these results reveal the unique catalytic property of the A-type DyPs, but they will also facilitate the development of these enzymes as lignin degraders.

Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum.

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model.

Mechanistic insight with HBCH2CoA as a probe to polyhydroxybutyrate (PHB) synthases.

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1-2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 µM, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [(3)H]-saturated trimer-CoA ([(3)H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [(3)H]-sTCoA ([(3)H]-sT-CH2-CoA), saturated dimer-([(3)H]-sD-CO2H), and trimer-acid ([(3)H]-sT-CO2H), distinct from the expected methylene analogue of [(3)H]-saturated tetramer-CoA ([(3)H]-sTet-CH2-CoA). Detection of [(3)H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.