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The plant community has a strong track record of RNA sequencing technology deployment, which combined with the recent advent of spatial platforms (e.g. 10× genomics) has resulted in an explosion of single-cell and nuclei datasets that can be positioned in an in situ context within tissues (e.g. a cell atlas). In the genomics era, application of proteomics technologies in the plant sciences has always trailed behind that of RNA sequencing technologies, largely due in part to upfront cost, ease-of-use, and access to expertise. Conversely, the use of early analytical tools for characterizing small molecules (metabolites) from plant systems predates nucleic acid sequencing and proteomics analysis, as the search for plant-based natural products has played a significant role in improving human health throughout history. As the plant sciences field now aims to fully define cell states, cell-specific regulatory networks, metabolic asymmetry and phenotypes, the measurement of proteins and metabolites at the single-cell level will be paramount. As a result of these efforts, the plant community will unlock exciting opportunities to accelerate discovery and drive toward meaningful translational outcomes., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

The calcium-dependent protein kinase CPK28 regulates several stress pathways in multiple plant species. Here, we aimed to discover CPK28-associated proteins in Arabidopsis thaliana. We used affinity-based proteomics and identified several potential CPK28 binding partners, including the C7 Raf-like kinases MRK1, RAF26, and RAF39. We used biochemistry, genetics, and physiological assays to gain insight into their function. We define redundant roles for these kinases in stomatal opening, immune-triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. We report that CPK28 associates with and trans-phosphorylates RAF26 and RAF39, and that MRK1, RAF26, and RAF39 are active kinases that localize to endomembranes. Although Raf-like kinases share some features with mitogen-activated protein kinase kinase kinases (MKKKs), we found that MRK1, RAF26, and RAF39 are unable to trans-phosphorylate any of the 10 Arabidopsis mitogen-activated protein kinase kinases (MKKs). Overall, our study suggests that C7 Raf-like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal opening and immunity, and possess substrate specificities distinct from canonical MKKKs., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Decades of research have uncovered how plants respond to two environmental variables that change across latitudes and over seasons: photoperiod and temperature. However, a third such variable, twilight length, has so far gone unstudied. Here, using controlled growth setups, we show that the duration of twilight affects growth and flowering time via the LHY/CCA1 clock genes in the model plant Arabidopsis. Using a series of progressively truncated no-twilight photoperiods, we also found that plants are more sensitive to twilight length compared to equivalent changes in solely photoperiods. Transcriptome and proteome analyses showed that twilight length affects reactive oxygen species metabolism, photosynthesis, and carbon metabolism. Genetic analyses suggested a twilight sensing pathway from the photoreceptors PHY E , PHY B , PHY D , and CRY2 through LHY/CCA1 to flowering modulation through the GI-FT pathway. Overall, our findings call for more nuanced models of day-length perception in plants and posit that twilight is an important determinant of plant growth and development.

A major limitation when undertaking quantitative proteomic time-course experimentation is the tradeoff between depth-of-analysis and speed-of-analysis. In high complexity and high dynamic range sample types, such as plant extracts, balance between resolution and time is especially apparent. To address this, we evaluate multiple compensation voltage (CV) high field asymmetric waveform ion mobility spectrometry (FAIMSpro) settings using the latest label-free single-shot Orbitrap-based DIA acquisition workflows for their ability to deeply quantify the Arabidopsis thaliana seedling proteome. Using a BoxCarDIA acquisition workflow with a -30 -50 -70 CV FAIMSpro setting, we were able to consistently quantify >5000 Arabidopsis seedling proteins over a 21-min gradient, facilitating the analysis of ∼42 samples per day. Utilizing this acquisition approach, we then quantified proteome-level changes occurring in Arabidopsis seedling shoots and roots over 24 h of salt and osmotic stress, to identify early and late stress response proteins and reveal stress response overlaps. Here, we successfully quantify >6400 shoot and >8500 root protein groups, respectively, quantifying nearly ∼9700 unique protein groups in total across the study. Collectively, we pioneer a short gradient, multi-CV FAIMSpro BoxCarDIA acquisition workflow that represents an exciting new analysis approach for undertaking quantitative proteomic time-course experimentation in plants., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Drought is a major environmental stress that affects crop productivity worldwide. Although previous research demonstrated links between strigolactones (SLs) and drought, here we used barley (Hordeum vulgare) SL-insensitive mutant hvd14 (dwarf14) to scrutinize the SL-dependent mechanisms associated with water deficit response., Results: We have employed a combination of transcriptomics, proteomics, phytohormonomics analyses, and physiological data to unravel differences between wild-type and hvd14 plants under drought. Our research revealed that drought sensitivity of hvd14 is related to weaker induction of abscisic acid-responsive genes/proteins, lower jasmonic acid content, higher reactive oxygen species content, and lower wax biosynthetic and deposition mechanisms than wild-type plants. In addition, we identified a set of transcription factors (TFs) that are exclusively drought-induced in the wild-type barley., Conclusions: Critically, we resolved a comprehensive series of interactions between the drought-induced barley transcriptome and proteome responses, allowing us to understand the profound effects of SLs in alleviating water-limiting conditions. Several new avenues have opened for developing barley more resilient to drought through the information provided. Moreover, our study contributes to a better understanding of the complex interplay between genes, proteins, and hormones in response to drought, and underscores the importance of a multidisciplinary approach to studying plant stress response mechanisms., (© 2023. BioMed Central Ltd., part of Springer Nature.)

For the past two decades, the study of alternative splicing (AS) and its involvement in plant development and stress response has grown in popularity. Only recently however, has the focus shifted to the study of how AS regulation (or lack-thereof) affects downstream mRNA and protein landscapes and how these AS regulatory events impact plant development and stress tolerance. In humans, protein phosphorylation represents one of the predominant mechanisms by which AS is regulated and thus the protein kinases governing these phosphorylation events are of interest for further study. Large-scale phosphoproteomic studies in plants have consistently found that RNA splicing-related proteins are extensively phosphorylated, however, the signaling pathways involved in AS regulation have not been resolved. In this mini-review, we summarize our current knowledge of the three major splicing-related protein kinase families in plants that are suggested to mediate AS phospho-regulation and draw comparisons to their metazoan orthologs. We also summarize and contextualize the phosphorylation events identified as occurring on splicing-related protein families to illustrate the high degree to which splicing-related proteins are modified, placing a new focus on elucidating the impacts of AS at the protein and PTM-level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rodriguez Gallo and Uhrig.)

Kale is a group of diverse Brassicaceae species that are nutritious leafy greens consumed for their abundance of vitamins and micronutrients. Typified by their curly, serrated and/or wavy leaves, kale varieties have been primarily defined based on their leaf morphology and geographic origin, despite having complex genetic backgrounds. Kale is a very promising crop for vertical farming due to its high nutritional content; however, being a non-model organism, foundational, systems-level analyses of kale are lacking. Previous studies in kale have shown that time-of-day harvesting can affect its nutritional composition. Therefore, to gain a systems-level diel understanding of kale across its wide-ranging and diverse genetic landscape, we selected nine publicly available and commercially grown kale cultivars for growth under near-sunlight LED light conditions ideal for vertical farming. We then analyzed changes in morphology, growth and nutrition using a combination of plant phenotyping, proteomics and metabolomics. As the diel molecular activities of plants drive their daily growth and development, ultimately determining their productivity as a crop, we harvested kale leaf tissue at both end-of-day (ED) and end-of-night (EN) time-points for all molecular analyses. Our results reveal that diel proteome and metabolome signatures divide the selected kale cultivars into two groups defined by their amino acid and sugar content, along with significant proteome differences involving carbon and nitrogen metabolism, mRNA splicing, protein translation and light harvesting. Together, our multi-cultivar, multi-omic analysis provides new insights into the molecular underpinnings of the diel growth and development landscape of kale, advancing our fundamental understanding of this nutritious leafy green super-food for horticulture/vertical farming applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Scandola, Mehta, Castillo, Boyce and Uhrig.)

In order to mitigate the effects of a changing climate, agriculture requires more effective evaluation, selection, and production of crop cultivars in order to accelerate genotype-to-phenotype connections and the selection of beneficial traits. Critically, plant growth and development are highly dependent on sunlight, with light energy providing plants with the energy required to photosynthesize as well as a means to directly intersect with the environment in order to develop. In plant analyses, machine learning and deep learning techniques have a proven ability to learn plant growth patterns, including detection of disease, plant stress, and growth using a variety of image data. To date, however, studies have not assessed machine learning and deep learning algorithms for their ability to differentiate a large cohort of genotypes grown under several growth conditions using time-series data automatically acquired across multiple scales (daily and developmentally). Here, we extensively evaluate a wide range of machine learning and deep learning algorithms for their ability to differentiate 17 well-characterized photoreceptor deficient genotypes differing in their light detection capabilities grown under several different light conditions. Using algorithm performance measurements of precision, recall, F1-Score, and accuracy, we find that Suport Vector Machine (SVM) maintains the greatest classification accuracy, while a combined ConvLSTM2D deep learning model produces the best genotype classification results across the different growth conditions. Our successful integration of time-series growth data across multiple scales, genotypes and growth conditions sets a new foundational baseline from which more complex plant science traits can be assessed for genotype-to-phenotype connections., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors.)

Nearly 60 - 80 % of intron-containing plant genes undergo alternative splicing in response to either stress or plant developmental cues. RNA splicing is performed by a large ribonucleoprotein complex called the spliceosome in conjunction with associated subunits such as serine arginine (SR) proteins, all of which undergo extensive phosphorylation. In plants, there are three main protein kinase families suggested to phosphorylate core spliceosome subunits and related splicing factors based on orthology to human splicing-related kinases: the SERINE/ARGININE PROTEIN KINASES (SRPK), ARABIDOPSIS FUS3 COMPLEMENT (AFC), and Pre-mRNA PROCESSING FACTOR 4 (PRP4K) protein kinases. To better define the conservation and role(s) of these kinases in plants, we performed a genome-scale analysis of the three families across photosynthetic eukaryotes, followed by extensive transcriptomic and bioinformatic analysis of all Arabidopsis thaliana SRPK, AFC, and PRP4K protein kinases to elucidate their biological functions. Unexpectedly, this revealed the existence of SRPK and AFC phylogenetic groups with distinct promoter elements and patterns of transcriptional response to abiotic stress, while PRP4Ks possess no phylogenetic sub-divisions, suggestive of functional redundancy. We also reveal splicing-related kinase families are both diel and photoperiod regulated, implicating different orthologs as discrete time-of-day RNA splicing regulators. This foundational work establishes a number of new hypotheses regarding how reversible spliceosome phosphorylation contributes to both diel plant cell regulation and abiotic stress adaptation in plants., (© 2022. The Author(s).)

Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE (RVE) proteins are recently discovered members of the plant circadian circuitry that activate the evening complex and PSEUDO-RESPONSE REGULATOR genes to maintain regular circadian oscillation. The RVE8 protein and its two homologs, RVE 4 and 6 in Arabidopsis (Arabidopsis thaliana), have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass, cell size, and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we draw new connections between RVE8-like proteins and a number of core plant cell processes. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid, and lipid metabolism, including a starch excess phenotype at dawn. We further demonstrate that rve 4 6 8 plants have lower levels of 20S proteasome subunits and possess significantly reduced proteasome activity, potentially explaining the increase in cell-size observed in RVE8-like mutants. Overall, this robust, multi-omic dataset provides substantial insight into the far-reaching impact RVE8-like proteins have on the diel plant cell environment., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The Plant Cell Atlas (PCA) community hosted a virtual symposium on December 9 and 10, 2021 on single cell and spatial omics technologies. The conference gathered almost 500 academic, industry, and government leaders to identify the needs and directions of the PCA community and to explore how establishing a data synthesis center would address these needs and accelerate progress. This report details the presentations and discussions focused on the possibility of a data synthesis center for a PCA and the expected impacts of such a center on advancing science and technology globally. Community discussions focused on topics such as data analysis tools and annotation standards; computational expertise and cyber-infrastructure; modes of community organization and engagement; methods for ensuring a broad reach in the PCA community; recruitment, training, and nurturing of new talent; and the overall impact of the PCA initiative. These targeted discussions facilitated dialogue among the participants to gauge whether PCA might be a vehicle for formulating a data synthesis center. The conversations also explored how online tools can be leveraged to help broaden the reach of the PCA (i.e., online contests, virtual networking, and social media stakeholder engagement) and decrease costs of conducting research (e.g., virtual REU opportunities). Major recommendations for the future of the PCA included establishing standards, creating dashboards for easy and intuitive access to data, and engaging with a broad community of stakeholders. The discussions also identified the following as being essential to the PCA's success: identifying homologous cell-type markers and their biocuration, publishing datasets and computational pipelines, utilizing online tools for communication (such as Slack), and user-friendly data visualization and data sharing. In conclusion, the development of a data synthesis center will help the PCA community achieve these goals by providing a centralized repository for existing and new data, a platform for sharing tools, and new analytical approaches through collaborative, multidisciplinary efforts. A data synthesis center will help the PCA reach milestones, such as community-supported data evaluation metrics, accelerating plant research necessary for human and environmental health., Competing Interests: The Authors did not report any conflict of interest., (© 2022 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Clubroot of Brassicaceae , an economically important soil borne disease, is caused by Plasmodiophora brassicae Woronin, an obligate, biotrophic protist. This disease poses a serious threat to canola and related crops in Canada and around the globe causing significant losses. The pathogen is continuously evolving and new pathotypes are emerging, which necessitates the development of novel resistant canola cultivars to manage the disease. Proteins play a crucial role in many biological functions and the identification of differentially abundant proteins (DAP) using proteomics is a suitable approach to understand plant-pathogen interactions to assist in the development of gene specific markers for developing clubroot resistant (CR) cultivars. In this study, P. brassicae pathotype 3 (P3H) was used to challenge CR and clubroot susceptible (CS) canola lines. Root samples were collected at three distinct stages of pathogenesis, 7-, 14-, and 21-days post inoculation (DPI), protein samples were isolated, digested with trypsin and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. A total of 937 proteins demonstrated a significant ( q -value < 0.05) change in abundance in at least in one of the time points when compared between control and inoculated CR-parent, CR-progeny, CS-parent, CS-progeny and 784 proteins were significantly ( q < 0.05) changed in abundance in at least in one of the time points when compared between the inoculated- CR and CS root proteomes of parent and progeny across the three time points tested. Functional annotation of differentially abundant proteins (DAPs) revealed several proteins related to calcium dependent signaling pathways. In addition, proteins related to reactive oxygen species (ROS) biochemistry, dehydrins, lignin, thaumatin, and phytohormones were identified. Among the DAPs, 73 putative proteins orthologous to CR proteins and quantitative trait loci (QTL) associated with eight CR loci in different chromosomes including chromosomes A3 and A8 were identified. Proteins including BnaA02T0335400WE, BnaA03T0374600WE, BnaA03T0262200WE, and BnaA03T0464700WE are orthologous to identified CR loci with possible roles in mediating clubroot responses. In conclusion, these results have contributed to an improved understanding of the mechanisms involved in mediating response to P. brassicae in canola at the protein level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Adhikary, Mehta, Uhrig, Rahman and Kav.)

Abiotic stresses such as drought result in large annual economic losses around the world. As sessile organisms, plants cannot escape the environmental stresses they encounter but instead must adapt to survive. Studies investigating plant responses to osmotic and/or salt stress have largely focused on short-term systemic responses, leaving our understanding of intermediate to longer-term adaptation (24 h to d) lacking. In addition to protein abundance and phosphorylation changes, evidence suggests reversible lysine acetylation may also be important for abiotic stress responses. Therefore, to characterize the protein-level effects of osmotic and salt stress, we undertook a label-free proteomic analysis of Arabidopsis thaliana roots exposed to 300 mM mannitol and 150 mM NaCl for 24 h. We assessed protein phosphorylation, lysine acetylation and changes in protein abundance, detecting significant changes in 245, 35 and 107 total proteins, respectively. Comparison with available transcriptome data indicates that transcriptome- and proteome-level changes occur in parallel, while post-translational modifications (PTMs) do not. Further, we find significant changes in PTMs, and protein abundance involve different proteins from the same networks, indicating a multifaceted regulatory approach to prolonged osmotic and salt stress. In particular, we find extensive protein-level changes involving sulfur metabolism under both osmotic and salt conditions as well as changes in protein kinases and transcription factors that may represent new targets for drought stress signaling. Collectively, we find that protein-level changes continue to occur in plant roots 24 h from the onset of osmotic and salt stress and that these changes differ across multiple proteome levels., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them., Competing Interests: SJ, AB, BC, NF, AF, SH, PK, ML, NP, SR, MS, PA, AA, CA, SB, JB, PD, LD, ME, SG, FG, KK, DK, SK, AM, NN, TO, MO, GP, EQ, RS, RU, JW, KV, RW, DE, KB, SR No competing interests declared, (© 2021, Plant Cell Atlas Consortium et al.)

The prediction of functional domains is typically among the first steps towards understanding the function of new proteins and protein families. There are numerous databases of annotated protein domains that permit researchers to identify domains on individual proteins of interest. However, it is necessary to perform high-throughput domain searches to gain evolutionary insight into the functions of proteins and protein families. Unfortunately, at present, it is difficult to search for, and visualize domain conservation across multiple proteins and/or multiple groups of proteins in an intuitive manner. Here we present DomainViz, a new web-server that streamlines the identification and visualization of domains across multiple protein sequences. Currently, DomainViz uses the well-established PFAM and Prosite databases for domain searching and assembles intuitive, publication-ready 'monument valley' plots (mv-plots) that display the extent of domain conservation along two dimensions: positionality and frequency of occurrence in the input protein sequences. In addition, DomainViz produces a conventional domain-ordering figure. DomainViz can be used to explore the conservation of domains within a single protein family, across multiple families, and across families from different species to support studies into protein function and evolution. The web-server is publicly available at: https://uhrigprotools.biology.ualberta.ca/domainviz., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Our modern understanding of diel cell regulation in plants stems from foundational work in the late 1990s that analysed the dynamics of selected genes and mutants in Arabidopsis thaliana. The subsequent rise of transcriptomics technologies such as microarrays and RNA sequencing has substantially increased our understanding of anticipatory (circadian) and reactive (light- or dark-triggered) diel events in plants. However, it is also becoming clear that gene expression data fail to capture critical events in diel regulation that can only be explained by studying protein-level dynamics. Over the past decade, mass spectrometry technologies and quantitative proteomic workflows have significantly advanced, finally allowing scientists to characterise diel protein regulation at high throughput. Initial proteomic investigations suggest that the diel transcriptome and proteome generally lack synchrony and that the timing of daily regulatory events in plants is impacted by multiple levels of protein regulation (e.g., post-translational modifications [PTMs] and protein-protein interactions [PPIs]). Here, we highlight and summarise how the use of quantitative proteomics to elucidate diel plant cell regulation has advanced our understanding of these processes. We argue that this new understanding, coupled with the extraordinary developments in mass spectrometry technologies, demands greater focus on protein-level regulation of, and by, the circadian clock. This includes hitherto unexplored diel dynamics of protein turnover, PTMs, protein subcellular localisation and PPIs that can be masked by simple transcript- and protein-level changes. Finally, we propose new directions for how the latest advancements in quantitative proteomics can be utilised to answer outstanding questions in plant chronobiology., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Phosphorus absorbed in the form of phosphate (H 2 PO 4 - ) is an essential but limiting macronutrient for plant growth and agricultural productivity. A comprehensive understanding of how plants respond to phosphate starvation is essential for the development of more phosphate-efficient crops. Here we employed label-free proteomics and phosphoproteomics to quantify protein-level responses to 48 h of phosphate versus phosphite (H 2 PO 3 - ) resupply to phosphate-deprived Arabidopsis thaliana suspension cells. Phosphite is similarly sensed, taken up and transported by plant cells as phosphate, but cannot be metabolized or used as a nutrient. Phosphite is thus a useful tool for differentiating between non-specific processes related to phosphate sensing and transport and specific responses to phosphorus nutrition. We found that responses to phosphate versus phosphite resupply occurred mainly at the level of protein phosphorylation, complemented by limited changes in protein abundance, primarily in protein translation, phosphate transport and scavenging, and central metabolism proteins. Altered phosphorylation of proteins involved in core processes such as translation, RNA splicing and kinase signaling was especially important. We also found differential phosphorylation in response to phosphate and phosphite in 69 proteins, including splicing factors, translation factors, the PHT1;4 phosphate transporter and the HAT1 histone acetyltransferase - potential phospho-switches signaling changes in phosphorus nutrition. Our study illuminates several new aspects of the phosphate starvation response and identifies important targets for further investigation and potential crop improvement., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Protein phosphorylation and acetylation are the two most abundant post-translational modifications (PTMs) that regulate protein functions in eukaryotes. In plants, these PTMs have been investigated individually; however, their co-occurrence and dynamics on proteins is currently unknown. Using Arabidopsis thaliana, we quantified changes in protein phosphorylation, acetylation and protein abundance in leaf rosettes, roots, flowers, siliques and seedlings at the end of day (ED) and at the end of night (EN). This identified 2549 phosphorylated and 909 acetylated proteins, of which 1724 phosphorylated and 536 acetylated proteins were also quantified for changes in PTM abundance between ED and EN. Using a sequential dual-PTM workflow, we identified significant PTM changes and intersections in these organs and plant developmental stages. In particular, cellular process-, pathway- and protein-level analyses reveal that the phosphoproteome and acetylome predominantly intersect at the pathway- and cellular process-level at ED versus EN. We found 134 proteins involved in core plant cell processes, such as light harvesting and photosynthesis, translation, metabolism and cellular transport, that were both phosphorylated and acetylated. Our results establish connections between PTM motifs, PTM catalyzing enzymes and putative substrate networks. We also identified PTM motifs for further characterization of the regulatory mechanisms that control cellular processes during the diurnal cycle in different Arabidopsis organs and seedlings. The sequential dual-PTM analysis expands our understanding of diurnal plant cell regulation by PTMs and provides a useful resource for future analyses, while emphasizing the importance of analyzing multiple PTMs simultaneously to elucidate when, where and how they are involved in plant cell regulation., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Background: Reversible protein acetylation occurring on Lys-N e has emerged as a key regulatory post-translational modification in eukaryotes. It is mediated by two groups of enzymes: lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) that catalyze the addition and removal of acetyl groups from target proteins. Estimates indicate that protein acetylation is second to protein phosphorylation in abundance, with thousands of acetylated sites now identified in different subcellular compartments. Considering the important regulatory role of protein phosphorylation, elucidating the diversity of KATs and KDACs across photosynthetic eukaryotes is essential in furthering our understanding of the impact of reversible protein acetylation on plant cell processes., Results: We report a genome-scale analysis of lysine acetyltransferase (KAT)- and lysine deacetylase (KDAC)-families from 53 photosynthetic eukaryotes. KAT and KDAC orthologs were identified in sequenced genomes ranging from glaucophytes and algae to land plants and then analyzed for evolutionary relationships. Based on consensus molecular phylogenetic and subcellular localization data we found new sub-classes of enzymes in established KAT- and KDAC-families. Specifically, we identified a non-photosynthetic origin of the HD-tuin family KDACs, a new monocot-specific Class I HDA-family sub-class, and a phylogenetically distinct Class II algal/heterokont sub-class which maintains an ankyrin domain not conserved in land plant Class II KDACs. Protein structure analysis showed that HDA- and SRT-KDACs exist as bare catalytic subunits with highly conserved median protein length, while all KATs maintained auxiliary domains, with CBP- and TAF II 250-KATs displaying protein domain gain and loss over the course of photosynthetic eukaryote evolution in addition to variable protein length. Lastly, promoter element enrichment analyses across species revealed conserved cis-regulatory sequences that support KAT and KDAC involvement in the regulation of plant development, cold/drought stress response, as well as cellular processes such as the circadian clock., Conclusions: Our results reveal new evolutionary, structural, and biological insights into the KAT- and KDAC-families of photosynthetic eukaryotes, including evolutionary parallels to protein kinases and protein phosphatases. Further, we provide a comprehensive annotation framework through our extensive phylogenetic analysis, from which future research investigating aspects of protein acetylation in plants can use to position new findings in a broader context.

Shewanella-like PPP family phosphatases (SLPs) are a unique lineage of eukaryote PPP-family phosphatases of bacterial origin which are not found in metazoans. 1,2 Their absence in metazoans is marked by their ancient bacterial origins and presence in plants. 1 Recently, we found that the SLP2 phosphatase ortholog of Arabidopsis thaliana localized to the mitochondrial intermembrane space (IMS) where it was determined to be activated by mitochondrial intermembrane space protein 40 (MIA40) to regulate seed germination. 3 Through examination of atslp2 knockout (accelerated germination) and 35S::AtSLP2 over-expressing (delayed germination) plants it was found that AtSLP2 influences Arabidopsis thaliana germination rates via gibberellic acid (GA) biosynthesis. 3 However, the exact mechanism by which this occurs remains unresolved. To identify potential partners of AtSLP2 in regulating germination through GA, we undertook a gene co-expression network analysis using RNA-sequencing data available through Genevestigator ( https://genevestigator.com/gv/ ).

The circadian clock regulates physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have integrated RNA-sequencing and protein mass spectrometry data to comparatively analyse the lhycca1 , prr7prr9 , gi and toc1 circadian clock mutant rosette at the end of day and end of night. Each mutant affects specific sets of genes and proteins, suggesting that the circadian clock regulation is modular. Furthermore, each circadian clock mutant maintains its own dynamically fluctuating transcriptome and proteome profile specific to subcellular compartments. Most of the measured protein levels do not correlate with changes in their corresponding transcripts. Transcripts and proteins that have coordinated changes in abundance are enriched for carbohydrate- and cold-responsive genes. Transcriptome changes in all four circadian clock mutants also affect genes encoding starch degradation enzymes, transcription factors and protein kinases. The comprehensive transcriptome and proteome datasets demonstrate that future system-driven research of the circadian clock requires multi-level experimental approaches. Our work also shows that further work is needed to elucidate the roles of post-translational modifications and protein degradation in the regulation of clock-related processes., (© 2017 The Authors.)

Reversible protein phosphorylation catalyzed by protein kinases and phosphatases represents the most prolific and well-characterized posttranslational modification known. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) Shewanella-like protein phosphatase 2 (AtSLP2) is a bona fide Ser/Thr protein phosphatase that is targeted to the mitochondrial intermembrane space (IMS) where it interacts with the mitochondrial oxidoreductase import and assembly protein 40 (AtMIA40), forming a protein complex. Interaction with AtMIA40 is necessary for the phosphatase activity of AtSLP2 and is dependent on the formation of disulfide bridges on AtSLP2. Furthermore, by utilizing atslp2 null mutant, AtSLP2 complemented and AtSLP2 overexpressing plants, we identify a function for the AtSLP2-AtMIA40 complex in negatively regulating gibberellic acid-related processes during seed germination. Results presented here characterize a mitochondrial IMS-localized protein phosphatase identified in photosynthetic eukaryotes as well as a protein phosphatase target of the highly conserved eukaryotic MIA40 IMS oxidoreductase., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mg+2/Mn+2-dependent type 2C protein phosphatases (PP2Cs) are ubiquitous in eukaryotes, mediating diverse cellular signaling processes through metal ion catalyzed dephosphorylation of target proteins. We have identified a distinct PP2C sequence class ("PP2C7s") which is nearly universally distributed in Eukaryotes, and therefore apparently ancient. PP2C7s are by far most prominent and diverse in plants and green algae. Combining phylogenetic analysis, subcellular localization predictions, and a distillation of publically available gene expression data, we have traced the evolutionary trajectory of this gene family in photosynthetic eukaryotes, demonstrating two major sequence assemblages featuring a succession of increasingly derived sub-clades. These display predominant expression moving from an ancestral pattern in photosynthetic tissues toward non-photosynthetic, specialized and reproductive structures. Gene co-expression network composition strongly suggests a shifting pattern of PP2C7 gene functions, including possible regulation of starch metabolism for one homologue set in Arabidopsis and rice. Distinct plant PP2C7 sub-clades demonstrate novel amino terminal protein sequences upon motif analysis, consistent with a shifting pattern of regulation of protein function. More broadly, neither the major events in PP2C sequence evolution, nor the origin of the diversity of metal binding characteristics currently observed in different PP2C lineages, are clearly understood. Identification of the PP2C7 sequence clade has allowed us to provide a better understanding of both of these issues. Phylogenetic analysis and sequence comparisons using Hidden Markov Models strongly suggest that PP2Cs originated in Bacteria (Group II PP2C sequences), entered Eukaryotes through the ancestral mitochondrial endosymbiosis, elaborated in Eukaryotes, then re-entered Bacteria through an inter-domain gene transfer, ultimately producing bacterial Group I PP2C sequences. A key evolutionary event, occurring first in ancient Eukaryotes, was the acquisition of a conserved aspartate in classic Motif 5. This has been inherited subsequently by PP2C7s, eukaryotic PP2Cs and bacterial Group I PP2Cs, where it is crucial to the formation of a third metal binding pocket, and catalysis.

Protein phosphorylation is a reversible regulatory process catalyzed by the opposing reactions of protein kinases and phosphatases, which are central to the proper functioning of the cell. Dysfunction of members in either the protein kinase or phosphatase family can have wide-ranging deleterious effects in both metazoans and plants alike. Previously, three bacterial-like phosphoprotein phosphatase classes were uncovered in eukaryotes and named according to the bacterial sequences with which they have the greatest similarity: Shewanella-like (SLP), Rhizobiales-like (RLPH), and ApaH-like (ALPH) phosphatases. Utilizing the wealth of data resulting from recently sequenced complete eukaryotic genomes, we conducted database searching by hidden Markov models, multiple sequence alignment, and phylogenetic tree inference with Bayesian and maximum likelihood methods to elucidate the pattern of evolution of eukaryotic bacterial-like phosphoprotein phosphatase sequences, which are predominantly distributed in photosynthetic eukaryotes. We uncovered a pattern of ancestral mitochondrial (SLP and RLPH) or archaeal (ALPH) gene entry into eukaryotes, supplemented by possible instances of lateral gene transfer between bacteria and eukaryotes. In addition to the previously known green algal and plant SLP1 and SLP2 protein forms, a more ancestral third form (SLP3) was found in green algae. Data from in silico subcellular localization predictions revealed class-specific differences in plants likely to result in distinct functions, and for SLP sequences, distinctive and possibly functionally significant differences between plants and nonphotosynthetic eukaryotes. Conserved carboxyl-terminal sequence motifs with class-specific patterns of residue substitutions, most prominent in photosynthetic organisms, raise the possibility of complex interactions with regulatory proteins.

Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, "bacterial-like" enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the "eukaryotic-like" phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.

Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature.

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/β-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Reversible protein phosphorylation is of central importance to the proper cellular functioning of all living organisms. Catalyzed by the opposing reactions of protein kinases and phosphatases, dysfunction in reversible protein phosphorylation can result in a wide variety of cellular aberrations. In eukaryotic organisms there exists four classes of protein phosphatases, of which the PPP-family protein phosphatases have documented susceptibility to a range of protein and small molecule inhibitors. These inhibitors have been of great importance to the biochemical characterization of PPP-family protein phosphatases since their discovery, but also maintain in natura biological significance with their endogenous regulatory properties (protein inhibitors) and toxicity (small molecule inhibitors). Recently, two unique PPP-family protein phosphatases, named the Shewanella-like protein phosphatases (SLP phosphatases), from Arabidopsis thaliana were characterized and found to be phylogenetically similar to the PPP-family protein phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), while completely lacking sensitivity to the classic PPP-family phosphatase small molecule inhibitors okadaic acid and microcystin-LR. SLP phosphatases were also found to be absent in metazoans, but present in a wide range of bacteria, fungi and protozoa responsible for human disease. The unique biochemical properties and evolutionary heritage of SLP phosphatases suggests they could not only be potential biotechnology targets for agriculture, but may also prove to be of interest for future therapeutic drug development., (© 2011 Landes Bioscience)

Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme situated at the core of plant C-metabolism. Although its anaplerotic role and control by allosteric effectors, reversible phosphorylation, and oligomerization have been well documented in the endosperm of developing castor oil seeds (COS), relatively little is known about PEPC in germinating COS. The initial phase of COS germination was accompanied by elevated PEPC activity and accumulation of comparable amounts of pre-existing 107-kDa and inducible 110-kDa immunoreactive PEPC polypeptides (p107 and p110, respectively). A 440-kDa PEPC heterotetramer composed of an equivalent ratio of non-phosphorylated p110 and p107 subunits was purified from germinated COS. N-terminal microsequencing, mass spectrometry, and immunoblotting revealed that both subunits arose from the same gene (RcPpc3) that encodes the p107 subunit of a phosphorylated 410-kDa PEPC homotetramer in developing COS but that p110 is a monoubiquitinated form of p107. Tandem mass spectrometry sequencing of a diglycinated tryptic peptide identified Lys-628 as p110's monoubiquitination site. This residue is conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Incubation with a human deubiquitinating enzyme (USP-2 core) converted the p110:p107 PEPC heterotetramer into a p107 homotetramer while significantly reducing the enzyme's K(m)(PEP) and sensitivity to allosteric activators (hexose-Ps, glycerol-3-P) and inhibitors (malate, aspartate). Monoubiquitination is a non-destructive and reversible post-translational modification involved in the control of diverse processes such as transcription, endocytosis, and signal transduction. The current study demonstrates that tissue-specific monoubiquitination of a metabolic enzyme can also occur and that this modification influences its kinetic and regulatory properties.

The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDC(pl)) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDC(pl) could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO(2) from PDC(pl) to PEPC.

Two classes of phosphoenolpyruvate carboxylase (PEPC) sharing the same 107-kDa catalytic subunit (p107) were previously purified from developing castor oil seed (COS) endosperm. The association of p107 with an immunologically unrelated 64-kDa polypeptide (p64) causes pronounced physical and kinetic differences between the Class-1 PEPC p107 homotetramer and Class-2 PEPC p107/p64 hetero-octamer. Tryptic peptide sequencing matched p64 to the deduced C-terminal half of several bacterial-type PEPCs (BTPCs) of vascular plants. Immunoblots probed with anti-(COS p64 peptide or p107)-IgG established that: (i) BTPC exists in vivo as an approximately 118-kDa polypeptide (p118) that is rapidly truncated to p64 by an endogenous cysteine endopeptidase during incubation of COS extracts on ice, and (ii) mature and germinated COS contain Class-1 PEPC and p107, but no detectable Class-2 PEPC nor p118. Non-denaturing PAGE, in-gel PEPC activity staining and immunoblotting of developing COS extracts demonstrated that p118 and p107 are subunits of the non-proteolysed approximately 910-kDa Class-2 PEPC complex. As total PEPC activity of clarified COS extracts was unaffected following p118 truncation to p64, the BTPC p118 may function as a regulatory rather than catalytic subunit of the Class-2 PEPC. Moreover, recombinant AtPPC3 and AtPPC4 (Arabidopsis orthologs of COS p107 and p118) expressed as active and inactive PEPCs, respectively. Cloning of cDNAs encoding p118 (RcPpc4) and p107 (RcPpc3) confirmed their respective designation as bacterial- and plant-type PEPCs. Levels of RcPpc3 and RcPpc4 transcripts generally mirrored the respective amounts of p107 and p118. The collective findings provide insights into the molecular features and functional significance of vascular plant BTPCs.