Thermoprotection by a cell membrane-localized metacaspase in a green alga.

Caspases are restricted to animals, while other organisms, including plants, possess metacaspases (MCAs), a more ancient and broader class of structurally related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis (Arabidopsis thaliana) with 9 MCAs with partially redundant activities. In contrast to streptophytes, most chlorophytes contain only 1 or 2 uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigated CrMCA-II, the single type-II MCA from the model chlorophyte Chlamydomonas (Chlamydomonas reinhardtii). Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlated with its dimerization. Most of CrMCA-II in the cell was present as a proenzyme (zymogen) attached to the plasma membrane (PM). Deletion of CrMCA-II by genome editing compromised thermotolerance, leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restored thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we connected the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

Structure-function study of a Ca2+-independent metacaspase involved in lateral root emergence.

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure-function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor.

Diverse biological effects of glycosyltransferase genes from Tartary buckwheat.

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) is an edible cereal crop whose sprouts have been marketed and commercialized for their higher levels of anti-oxidants, including rutin and anthocyanin. UDP-glucose flavonoid glycosyltransferases (UFGTs) play an important role in the biosynthesis of flavonoids in plants. So far, few studies are available on UFGT genes that may play a role in tartary buckwheat flavonoids biosynthesis. Here, we report on the identification and functional characterization of seven UFGTs from tartary buckwheat that are potentially involved in flavonoid biosynthesis (and have varying effects on plant growth and development when overexpressed in Arabidopsis thaliana.) RESULTS: Phylogenetic analysis indicated that the potential function of the seven FtUFGT proteins, FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, FtUFGT15, FtUFGT40, and FtUFGT41, could be divided into three Arabidopsis thaliana functional subgroups that are involved in flavonoid biosynthesis of and anthocyanin accumulation. A significant positive correlation between FtUFGT8 and FtUFGT15 expression and anthocyanin accumulation capacity was observed in the tartary buckwheat seedlings after cold stress. Overexpression in Arabidopsis thaliana showed that FtUFGT8, FtUFGT15, and FtUFGT41 significantly increased the anthocyanin content in transgenic plants. Unexpectedly, overexpression of FtUFGT6, while not leading to enhanced anthocyanin accumulation, significantly enhanced the growth yield of transgenic plants. When wild-type plants have only cotyledons, most of the transgenic plants of FtUFGT6 had grown true leaves. Moreover, the growth speed of the oxFtUFGT6 transgenic plant root was also significantly faster than that of the wild type. At later growth, FtUFGT6 transgenic plants showed larger leaves, earlier twitching times and more tillers than wild type, whereas FtUFGT15 showed opposite results. CONCLUSIONS: Seven FtUFGTs were isolated from tartary buckwheat. FtUFGT8, FtUFGT15, and FtUFGT41 can significantly increase the accumulation of total anthocyanins in transgenic plants. Furthermore, overexpression of FtUFGT6 increased the overall yield of Arabidopsis transgenic plants at all growth stages. However, FtUFGT15 shows the opposite trend at later growth stage and delays the growth speed of plants. These results suggested that the biological function of FtUFGT genes in tartary buckwheat is diverse.

Caught green-handed: methods for in vivo detection and visualization of protease activity.

Proteases are enzymes that cleave peptide bonds of other proteins. Their omnipresence and diverse activities make them important players in protein homeostasis and turnover of the total cell proteome as well as in signal transduction in plant stress responses and development. To understand protease function, it is of paramount importance to assess when and where a specific protease is active. Here, we review the existing methods to detect in vivo protease activity by means of imaging chemical activity-based probes and genetically encoded sensors. We focus on the diverse fluorescent and luminescent sensors at the researcher's disposal and evaluate the potential of imaging techniques to deliver in vivo spatiotemporal detail of protease activity. We predict that in the coming years, revised techniques will help to elucidate plant protease activity and functions and hence expand the current status of the field.

Extracellular peptide Kratos restricts cell death during vascular development and stress in Arabidopsis.

During plant vascular development, xylem tracheary elements (TEs) form water-conducting, empty pipes by genetically regulated cell death. Cell death is prevented from spreading to non-TEs by unidentified intercellular mechanisms, downstream of METACASPASE9 (MC9)-mediated regulation of autophagy in TEs. Here, we identified differentially abundant extracellular peptides in vascular-differentiating wild-type and MC9-down-regulated Arabidopsis cell suspensions. A peptide named Kratos rescued the abnormally high ectopic non-TE death resulting from either MC9 knockout or TE-specific overexpression of the ATG5 autophagy protein during experimentally induced vascular differentiation in Arabidopsis cotyledons. Kratos also reduced cell death following mechanical damage and extracellular ROS production in Arabidopsis leaves. Stress-induced but not vascular non-TE cell death was enhanced by another identified peptide, named Bia. Bia is therefore reminiscent of several known plant cell death-inducing peptides acting as damage-associated molecular patterns. In contrast, Kratos plays a novel extracellular cell survival role in the context of development and during stress response.

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes.

The function of two type II metacaspases in woody tissues of Populus trees.

Metacaspases (MCs) are cysteine proteases that are implicated in programmed cell death of plants. AtMC9 (Arabidopsis thaliana Metacaspase9) is a member of the Arabidopsis MC family that controls the rapid autolysis of the xylem vessel elements, but its downstream targets in xylem remain uncharacterized. PttMC13 and PttMC14 were identified as AtMC9 homologs in hybrid aspen (Populus tremula × tremuloides). A proteomic analysis was conducted in xylem tissues of transgenic hybrid aspen trees which carried either an overexpression or an RNA interference construct for PttMC13 and PttMC14. The proteomic analysis revealed modulation of levels of both previously known targets of metacaspases, such as Tudor staphylococcal nuclease, heat shock proteins and 14-3-3 proteins, as well as novel proteins, such as homologs of the PUTATIVE ASPARTIC PROTEASE3 (PASPA3) and the cysteine protease RD21 by PttMC13 and PttMC14. We identified here the pathways and processes that are modulated by PttMC13 and PttMC14 in xylem tissues. In particular, the results indicate involvement of PttMC13 and/or PttMC14 in downstream proteolytic processes and cell death of xylem elements. This work provides a valuable reference dataset on xylem-specific metacaspase functions for future functional and biochemical analyses.

The ROS Wheel: Refining ROS Transcriptional Footprints.

In the last decade, microarray studies have delivered extensive inventories of transcriptome-wide changes in messenger RNA levels provoked by various types of oxidative stress in Arabidopsis (Arabidopsis thaliana). Previous cross-study comparisons indicated how different types of reactive oxygen species (ROS) and their subcellular accumulation sites are able to reshape the transcriptome in specific manners. However, these analyses often employed simplistic statistical frameworks that are not compatible with large-scale analyses. Here, we reanalyzed a total of 79 Affymetrix ATH1 microarray studies of redox homeostasis perturbation experiments. To create hierarchy in such a high number of transcriptomic data sets, all transcriptional profiles were clustered on the overlap extent of their differentially expressed transcripts. Subsequently, meta-analysis determined a single magnitude of differential expression across studies and identified common transcriptional footprints per cluster. The resulting transcriptional footprints revealed the regulation of various metabolic pathways and gene families. The RESPIRATORY BURST OXIDASE HOMOLOG F-mediated respiratory burst had a major impact and was a converging point among several studies. Conversely, the timing of the oxidative stress response was a determining factor in shaping different transcriptome footprints. Our study emphasizes the need to interpret transcriptomic data sets in a systematic context, where initial, specific stress triggers can converge to common, aspecific transcriptional changes. We believe that these refined transcriptional footprints provide a valuable resource for assessing the involvement of ROS in biological processes in plants.

The Arabidopsis metacaspase9 degradome.

Metacaspases are distant relatives of the metazoan caspases, found in plants, fungi, and protists. However, in contrast with caspases, information about the physiological substrates of metacaspases is still scarce. By means of N-terminal combined fractional diagonal chromatography, the physiological substrates of metacaspase9 (MC9; AT5G04200) were identified in young seedlings of Arabidopsis thaliana on the proteome-wide level, providing additional insight into MC9 cleavage specificity and revealing a previously unknown preference for acidic residues at the substrate prime site position P1'. The functionalities of the identified MC9 substrates hinted at metacaspase functions other than those related to cell death. These results allowed us to resolve the substrate specificity of MC9 in more detail and indicated that the activity of phosphoenolpyruvate carboxykinase 1 (AT4G37870), a key enzyme in gluconeogenesis, is enhanced upon MC9-dependent proteolysis.

Phosphorylation of Arabidopsis transketolase at Ser428 provides a potential paradigm for the metabolic control of chloroplast carbon metabolism.

Calcium is an important second messenger in eukaryotic cells that regulates many different cellular processes. To elucidate calcium regulation in chloroplasts, we identified the targets of calcium-dependent phosphorylation within the stromal proteome. A 73 kDa protein was identified as one of the most dominant proteins undergoing phosphorylation in a calcium-dependent manner in the stromal extracts of both Arabidopsis and Pisum. It was identified as TKL (transketolase), an essential enzyme of both the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate pathway. Calcium-dependent phosphorylation of both Arabidopsis isoforms (AtTKL1 and AtTKL2) could be confirmed in vitro using recombinant proteins. The phosphorylation is catalysed by a stroma-localized protein kinase, which cannot utilize GTP. Phosphorylation of AtTKL1, the dominant isoform in most tissues, occurs at a serine residue that is conserved in TKLs of vascular plants. By contrast, an aspartate residue is present in this position in cyanobacteria, algae and mosses. Characterization of a phosphomimetic mutant (S428D) indicated that Ser428 phosphorylation exerts significant effects on the enzyme's substrate saturation kinetics at specific physiological pH values. The results of the present study point to a role for TKL phosphorylation in the regulation of carbon allocation.

Post mortem function of AtMC9 in xylem vessel elements.

Cell death of xylem elements is manifested by rupture of the tonoplast and subsequent autolysis of the cellular contents. Metacaspases have been implicated in various forms of plant cell death but regulation and execution of xylem cell death by metacaspases remains unknown. Analysis of the type II metacaspase gene family in Arabidopsis thaliana supported the function of METACASPASE 9 (AtMC9) in xylem cell death. Progression of xylem cell death was analysed in protoxylem vessel elements of 3-d-old atmc9 mutant roots using reporter gene analysis and electron microscopy. Protoxylem cell death was normally initiated in atmc9 mutant lines, but detailed electron microscopic analyses revealed a role for AtMC9 in clearance of the cell contents post mortem, that is after tonoplast rupture. Subcellular localization of fluorescent AtMC9 reporter fusions supported a post mortem role for AtMC9. Further, probe-based activity profiling suggested a function of AtMC9 on activities of papain-like cysteine proteases. Our data demonstrate that the function of AtMC9 in xylem cell death is to degrade vessel cell contents after vacuolar rupture. We further provide evidence on a proteolytic cascade in post mortem autolysis of xylem vessel elements and suggest that AtMC9 is part of this cascade.

Mechanisms controlling plant proteases and their substrates.

In plants, proteolysis is emerging as an important field of study due to a growing understanding of the critical involvement of proteases in plant cell death, disease and development. Because proteases irreversibly modify the structure and function of their target substrates, proteolytic activities are stringently regulated at multiple levels. Most proteases are produced as dormant isoforms and only activated in specific conditions such as altered ion fluxes or by post-translational modifications. Some of the regulatory mechanisms initiating and modulating proteolytic activities are restricted in time and space, thereby ensuring precision activity, and minimizing unwanted side effects. Currently, the activation mechanisms and the substrates of only a few plant proteases have been studied in detail. Most studies focus on the role of proteases in pathogen perception and subsequent modulation of the plant reactions, including the hypersensitive response (HR). Proteases are also required for the maturation of coexpressed peptide hormones that lead essential processes within the immune response and development. Here, we review the known mechanisms for the activation of plant proteases, including post-translational modifications, together with the effects of proteinaceous inhibitors.

Detection and Quantification of Protein Aggregates in Plants.

Protein quality control is an important aspect of stress recovery. It maintains protein homeostasis through a machinery of regulatory proteins such as chaperones and proteases. When the system recognizes accumulation of misfolded or aggregated proteins, the cell recruits a set of regulatory proteins to initiate protein quality control. To understand the dynamics of stress-mediated aggregate protein formation and recovery in plants, robust methods aimed at detecting and measuring such protein aggregates are needed. This will help us to deepen our understanding of protein quality control mechanisms in plants.

Chloroplast-localized protein kinases: a step forward towards a complete inventory.

In addition to redox regulation, protein phosphorylation has gained increasing importance as a regulatory principle in chloroplasts in recent years. However, only very few chloroplast-localized protein kinases have been identified to date. Protein phosphorylation regulates important chloroplast processes such as photosynthesis or transcription. In order to better understand chloroplast function, it is therefore crucial to obtain a complete picture of the chloroplast kinome, which is currently constrained by two effects: first, recent observations showed that the bioinformatics-based prediction of chloroplast-localized protein kinases from available sequence data is strongly biased; and, secondly, protein kinases are of very low abundance, which makes their identification by proteomics approaches extremely difficult. Therefore, the aim of this study was to obtain a complete list of chloroplast-localized protein kinases from different species. Evaluation of protein kinases which were either highly predicted to be chloroplast localized or have been identified in different chloroplast proteomic studies resulted in the confirmation of only three new kinases. Considering also all reports of experimentally verified chloroplast protein kinases to date, compelling evidence was found for a total set of 15 chloroplast-localized protein kinases in different species. This is in contrast to a much higher number that would be expected based on targeting prediction or on the general abundance of protein kinases in relation to the entire proteome. Moreover, it is shown that unusual protein kinases with differing ATP-binding sites or catalytic centres seem to occur frequently within the chloroplast kinome, thus making their identification by mass spectrometry-based approaches even more difficult due to a different annotation.

Plant organellar calcium signalling: an emerging field.

This review provides a comprehensive overview of the established and emerging roles that organelles play in calcium signalling. The function of calcium as a secondary messenger in signal transduction networks is well documented in all eukaryotic organisms, but so far existing reviews have hardly addressed the role of organelles in calcium signalling, except for the nucleus. Therefore, a brief overview on the main calcium stores in plants-the vacuole, the endoplasmic reticulum, and the apoplast-is provided and knowledge on the regulation of calcium concentrations in different cellular compartments is summarized. The main focus of the review will be the calcium handling properties of chloroplasts, mitochondria, and peroxisomes. Recently, it became clear that these organelles not only undergo calcium regulation themselves, but are able to influence the Ca(2+) signalling pathways of the cytoplasm and the entire cell. Furthermore, the relevance of recent discoveries in the animal field for the regulation of organellar calcium signals will be discussed and conclusions will be drawn regarding potential homologous mechanisms in plant cells. Finally, a short overview on bacterial calcium signalling is included to provide some ideas on the question where this typically eukaryotic signalling mechanism could have originated from during evolution.

Cross-talk between calcium signalling and protein phosphorylation at the thylakoid.

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.

Chemical Perturbation of Chloroplast Ca2+ Dynamics in Arabidopsis thaliana Suspension Cell Cultures and Seedlings.

Ca2+ signaling is part of universal signal transduction pathways to respond to external and internal stimuli or stress and in plants plays a central role in chloroplasts, such as in the regulation of photosynthetic enzymes or the transition from light to dark. Only recently, the underlying molecular machinery, e.g., transporters and channels that enable chloroplast Ca2+ fluxes, has started to be elucidated. However, chemical tools to specifically perturb these chloroplast Ca2+ fluxes are largely lacking. Here, we describe an efficient aequorin-based system in Arabidopsis thaliana suspension cell cultures to screen for chemicals that alter light-to-dark-induced chloroplast stroma Ca2+ signals. Subsequently, the effect of the hits on chloroplast Ca2+ signals is validated in Arabidopsis seedlings. The research lays a foundation for the identification of novel proteins involved in Ca2+ transport in chloroplast stroma under light-to-dark transition and for investigating the interaction of chloroplast Ca2+ signaling with photosynthesis in general.