Search

Plants continuously respond to changing environmental conditions to prevent damage and maintain optimal performance. To regulate gas exchange with the environment and to control abiotic stress relief, plants have pores in their leaf epidermis, called stomata. Multiple environmental signals affect the opening and closing of these stomata. High temperatures promote stomatal opening (to cool down), and drought induces stomatal closing (to prevent water loss). Coinciding stress conditions may evoke conflicting stomatal responses, but the cellular mechanisms to resolve these conflicts are unknown. Here we demonstrate that the high-temperature-associated kinase TARGET OF TEMPERATURE 3 directly controls the activity of plasma membrane H + -ATPases to induce stomatal opening. OPEN STOMATA 1, which regulates stomatal closure to prevent water loss during drought stress, directly inactivates TARGET OF TEMPERATURE 3 through phosphorylation. Taken together, this signalling axis harmonizes stomatal opening and closing under high temperatures and/or drought. In the context of global climate change, understanding how different stress signals converge on stomatal regulation allows the development of climate-change-ready crops., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Signal transduction relies largely on the activity of kinases and phosphatases that control protein phosphorylation. However, we still know very little about phosphorylation-mediated signaling networks. Plant MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASEs (MAP4Ks) have recently gained more attention, given their role in a wide range of processes, including developmental processes and stress signaling. We analyzed MAP4K expression patterns and mapped protein-MAP4K interactions in Arabidopsis (Arabidopsis thaliana), revealing extensive coexpression and heterodimerization. This heterodimerization is regulated by the C-terminal, intrinsically disordered half of the MAP4K, and specifically by the coiled coil motif. The ability to heterodimerize is required for proper activity and localization of the MAP4Ks. Taken together, our results identify MAP4K-interacting proteins and emphasize the functional importance of MAP4K heterodimerization. Furthermore, we identified MAP4K4/TARGET OF TEMPERATURE3 (TOT3) and MAP4K5/TOT3-INTERACTING PROTEIN 5 (TOI5) as key regulators of the transition from cell division to elongation zones in the primary root tip., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Hypocotyl elongation is controlled by several signals and is a major characteristic of plants growing in darkness or under warm temperature. While already several molecular mechanisms associated with this process are known, protein degradation and associated E3 ligases have hardly been studied in the context of warm temperature. In a time-course phosphoproteome analysis on Arabidopsis seedlings exposed to control or warm ambient temperature, we observed reduced levels of diverse proteins over time, which could be due to transcription, translation, and/or degradation. In addition, we observed differential phosphorylation of the LRR F-box protein SLOMO MOTION (SLOMO) at two serine residues. We demonstrate that SLOMO is a negative regulator of hypocotyl growth, also under warm temperature conditions, and protein-protein interaction studies revealed possible interactors of SLOMO, such as MKK5, DWF1, and NCED4. We identified DWF1 as a likely SLOMO substrate and a regulator of warm temperature-mediated hypocotyl growth. We propose that warm temperature-mediated regulation of SLOMO activity controls the abundance of hypocotyl growth regulators, such as DWF1, through ubiquitin-mediated degradation., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Molecular biology aims to understand cellular responses and regulatory dynamics in complex biological systems. However, these studies remain challenging in non-model species due to poor functional annotation of regulatory proteins. To overcome this limitation, we develop a multi-layer neural network that determines protein functionality directly from the protein sequence. We annotate kinases and phosphatases in Glycine max. We use the functional annotations from our neural network, Bayesian inference principles, and high resolution phosphoproteomics to infer phosphorylation signaling cascades in soybean exposed to cold, and identify Glyma.10G173000 (TOI5) and Glyma.19G007300 (TOT3) as key temperature regulators. Importantly, the signaling cascade inference does not rely upon known kinase motifs or interaction data, enabling de novo identification of kinase-substrate interactions. Conclusively, our neural network shows generalization and scalability, as such we extend our predictions to Oryza sativa, Zea mays, Sorghum bicolor, and Triticum aestivum. Taken together, we develop a signaling inference approach for non-model species leveraging our predicted kinases and phosphatases., (© 2023. The Author(s).)

Reversible protein phosphorylation is a post-translational modification involved in virtually all plant processes, as it mediates protein activity and signal transduction. Here, we probe dynamic protein phosphorylation during de novo shoot organogenesis in Arabidopsis thaliana . We find that application of three kinase inhibitors in various time intervals has different effects on root explants. Short exposures to the putative histidine (His) kinase inhibitor TCSA during the initial days on shoot induction medium (SIM) are detrimental for regeneration in seven natural accessions. Investigation of cytokinin signaling mutants, as well as reporter lines for hormone responses and shoot markers, suggests that TCSA impedes cytokinin signal transduction via AHK3, AHK4, AHP3, and AHP5. A mass spectrometry-based phosphoproteome analysis further reveals profound deregulation of Ser/Thr/Tyr phosphoproteins regulating protein modification, transcription, vesicle trafficking, organ morphogenesis, and cation transport. Among TCSA-responsive factors are prior candidates with a role in shoot apical meristem patterning, such as AGO1, BAM1, PLL5, FIP37, TOP1ALPHA, and RBR1, as well as proteins involved in polar auxin transport (e.g., PIN1) and brassinosteroid signaling (e.g., BIN2). Putative novel regeneration determinants regulated by TCSA include RD2, AT1G52780, PVA11, and AVT1C, while NAIP2, OPS, ARR1, QKY, and aquaporins exhibit differential phospholevels on control SIM. LC-MS/MS data are available via ProteomeXchange with identifier PXD030754., 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 Lardon, Trinh, Xu, Vu, Van De Cotte, Pernisová, Vanneste, De Smet and Geelen.)

SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies have revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different concentrations of the synthetic auxin NAA that establish growth-promoting and -repressing conditions on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data, we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results, together with previously published studies, suggest that auxin, H + -ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr 31 phosphorylation site for growth regulation in the Arabidopsis root tip.

Plants respond to mild warm temperature conditions by increased elongation growth of organs to enhance cooling capacity, in a process called thermomorphogenesis. To this date, the regulation of thermomorphogenesis has been exclusively shown to intersect with light signalling pathways. To identify regulators of thermomorphogenesis that are conserved in flowering plants, we map changes in protein phosphorylation in both dicots and monocots exposed to warm temperature. We identify MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4)/TARGET OF TEMPERATURE3 (TOT3) as a regulator of thermomorphogenesis that impinges on brassinosteroid signalling in Arabidopsis thaliana. In addition, we show that TOT3 plays a role in thermal response in wheat, a monocot crop. Altogether, the conserved thermal regulation by TOT3 expands our knowledge of thermomorphogenesis beyond the well-studied pathways and can contribute to ensuring food security under a changing climate.

The anaphase promoting complex/cyclosome (APC/C) controls unidirectional progression through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its activity is temporally regulated by the docking of different activating subunits, known in plants as CELL DIVISION PROTEIN20 (CDC20) and CELL CYCLE SWITCH52 (CCS52). Despite the importance of the APC/C during cell proliferation, the number of identified targets in the plant cell cycle is limited. Here, we used the growth and meristem phenotypes of Arabidopsis ( Arabidopsis thaliana ) CCS52A2-deficient plants in a suppressor mutagenesis screen to identify APC/C CCS52A2 substrates or regulators, resulting in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially rescues the ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhance the scored ccs52a2-1 phenotypes. Furthermore, whereas the CYCA3;4 protein is promptly broken down after prophase in wild-type plants, it remains present in later stages of mitosis in ccs52a2-1 mutant plants, marking it as a putative APC/C CCS52A2 substrate. Strikingly, increased CYCA3;4 levels result in aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with reduced RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) activity. Correspondingly, RBR1 hyperphosphorylation was observed in CYCA3;4 gain-of-function plants. Our data thus demonstrate that an inability to timely destroy CYCA3;4 contributes to disorganized formative divisions, possibly in part caused by the inactivation of RBR1., (© 2020 American Society of Plant Biologists. All rights reserved.)

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis , and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Smith et al.)

In plants, postembryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires the tight coordination of asymmetric cell division in adjacent pericycle cells. We identified EXPANSIN A1 (EXPA1) as a cell wall modifying enzyme controlling the divisions marking lateral root initiation. Loss of EXPA1 leads to defects in the first asymmetric pericycle cell divisions and the radial swelling of the pericycle during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.

Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower, and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental, and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term and mild increase in temperature on the wheat leaf and spikelet phosphoproteome. In total, 3822 (containing 5178 phosphosites) and 5581 phosphopeptides (containing 7023 phosphosites) were identified in leaf and spikelet samples, respectively. Following statistical analysis, the resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature. This community resource on the high temperature-mediated wheat phosphoproteome will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which probably impacts protein activity.

Leaf growth is a complex, quantitative trait, controlled by a plethora of regulatory mechanisms. Diverse environmental stimuli inhibit leaf growth to cope with the perceived stress. In plant research, mannitol is often used to impose osmotic stress and study the underlying growth-repressing mechanisms. In growing leaf tissue of plants briefly exposed to mannitol-induced stress, a highly interconnected gene regulatory network is induced. However, early signalling and associated protein phosphorylation events that probably precede part of these transcriptional changes and that potentially act at the onset of mannitol-induced leaf size reduction are largely unknown. Here, we performed a proteome and phosphoproteome analysis on growing leaf tissue of Arabidopsis thaliana plants exposed to mild mannitol-induced stress and captured the fast (within the first half hour) events associated with this stress. Based on this in-depth data analysis, 167 and 172 differentially regulated proteins and phosphorylated sites were found. We provide these data sets as a community resource and we flag differentially phosphorylated proteins with described growth-regulatory functions, but we also illustrate potential novel regulators of shoot growth.

The growth of plants, like that of other walled organisms, depends on the ability of the cell wall to yield without losing its integrity. In this context, plant cells can sense the perturbation of their walls and trigger adaptive modifications in cell wall polymer interactions. Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) THESEUS1 (THE1) was previously shown in Arabidopsis to trigger growth inhibition and defense responses upon perturbation of the cell wall, but so far, neither the ligand nor the role of the receptor in normal development was known. Here, we report that THE1 is a receptor for the peptide rapid alkalinization factor (RALF) 34 and that this signaling module has a role in the fine-tuning of lateral root initiation. We also show that RALF34-THE1 signaling depends, at least for some responses, on FERONIA (FER), another RALF receptor involved in a variety of processes, including immune signaling, mechanosensing, and reproduction [1]. Together, the results show that RALF34 and THE1 are part of a signaling network that integrates information on the integrity of the cell wall with the coordination of normal morphogenesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Roots explore the soil for water and nutrients through the continuous production of lateral roots. Lateral roots are formed at regular distances in a steadily elongating organ, but how future sites for lateral root formation become established is not yet understood. Here, we identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, auxin-repressed and phloem pole-expressed signal assisting in the formation of lateral roots. In addition, based on genetic and expression data, we found evidence for the involvement of its proposed receptor, XYLEM INTERMIXED WITH PHLOEM 1 (XIP1)/CEP RECEPTOR 1 (CEPR1), during the process of lateral root initiation. In conclusion, we report here on the existence of a peptide ligand-receptor kinase interaction that impacts lateral root initiation. Our results represent an important step towards the understanding of the cellular communication implicated in the early phases of lateral root formation., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In plants, many signalling molecules, such as phytohormones, miRNAs, transcription factors, and small signalling peptides, drive growth and development. However, very few small signalling peptides have been shown to be necessary for lateral root development. Here, we describe the role of the peptide RALFL34 during early events in lateral root development, and demonstrate its specific importance in orchestrating formative cell divisions in the pericycle. Our results further suggest that this small signalling peptide acts on the transcriptional cascade leading to a new lateral root upstream of GATA23, an important player in lateral root formation. In addition, we describe a role for ETHYLENE RESPONSE FACTORs (ERFs) in regulating RALFL34 expression. Taken together, we put forward RALFL34 as a new, important player in lateral root initiation., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In plants, the generation of new cell types and tissues depends on coordinated and oriented formative cell divisions. The plasma membrane-localized receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. Despite its important role in plant development, very little is known about the molecular mechanism with which ACR4 is affiliated and its network of interactions. Here, we used various complementary proteomic approaches to identify ACR4-interacting protein candidates that are likely regulators of formative cell divisions and that could pave the way to unraveling the molecular basis behind ACR4-mediated signaling. We identified PROTEIN PHOSPHATASE 2A-3 (PP2A-3), a catalytic subunit of PP2A holoenzymes, as a previously unidentified regulator of formative cell divisions and as one of the first described substrates of ACR4. Our in vitro data argue for the existence of a tight posttranslational regulation in the associated biochemical network through reciprocal regulation between ACR4 and PP2A-3 at the phosphorylation level.

Plant roots are important for a wide range of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface with the soil environment. Several small signalling peptides and receptor kinases have been shown to affect primary root growth, but very little is known about their role in lateral root development. In this context, the CLE family, a group of small signalling peptides that has been shown to affect a wide range of developmental processes, were the focus of this study. Here, the expression pattern during lateral root initiation for several CLE family members is explored and to what extent CLE1, CLE4, CLE7, CLE26, and CLE27, which show specific expression patterns in the root, are involved in regulating root architecture in Arabidopsis thaliana is assessed. Using chemically synthesized peptide variants, it was found that CLE26 plays an important role in regulating A. thaliana root architecture and interacts with auxin signalling. In addition, through alanine scanning and in silico structural modelling, key residues in the CLE26 peptide sequence that affect its activity are pinpointed. Finally, some interesting similarities and differences regarding the role of CLE26 in regulating monocot root architecture are presented., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plants have developed a variety of mechanisms to cope with abiotic and biotic stresses. In a previous subcellular localization study of hydrogen peroxide-responsive proteins, two peptides with an unknown function (designated ARACIN1 and ARACIN2) have been identified. These peptides are structurally very similar but are transcriptionally differentially regulated during abiotic stresses during Botrytis cinerea infection or after benzothiadiazole and methyl jasmonate treatments. In Arabidopsis (Arabidopsis thaliana), these paralogous genes are positioned in tandem within a cluster of pathogen defense-related genes. Both ARACINs are small, cationic, and hydrophobic peptides, known characteristics for antimicrobial peptides. Their genes are expressed in peripheral cell layers prone to pathogen entry and are lineage specific to the Brassicaceae family. In vitro bioassays demonstrated that both ARACIN peptides have a direct antifungal effect against the agronomically and economically important necrotrophic fungi B. cinerea, Alternaria brassicicola, Fusarium graminearum, and Sclerotinia sclerotiorum and yeast (Saccharomyces cerevisiae). In addition, transgenic Arabidopsis plants that ectopically express ARACIN1 are protected better against infections with both B. cinerea and A. brassicicola. Therefore, we can conclude that both ARACINs act as antimicrobial peptides., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

The development of a multicellular embryo from a single zygote is a complex and highly organized process that is far from understood. In higher plants, apical-basal patterning mechanisms are crucial to correctly specify root and shoot stem cell niches that will sustain and drive post-embryonic plant growth and development. The auxin-responsive AtWRKY23 transcription factor is expressed from early embryogenesis onwards and the timing and localization of its expression overlaps with the root stem cell niche. Knocking down WRKY23 transcript levels or expression of a dominant-negative WRKY23 version via a translational fusion with the SRDX repressor domain affected both apical-basal axis formation as well as installation of the root stem cell niche. WRKY23 expression is affected by two well-known root stem cell specification mechanisms, that is, SHORTROOT and MONOPTEROS-BODENLOS signalling and can partially rescue the root-forming inability of mp embryos. On the basis of these results, we postulate that a tightly controlled WRKY23 expression is involved in the regulation of both auxin-dependent and auxin-independent signalling pathways towards stem cell specification.

Upon disturbance of their function by stress, mitochondria can signal to the nucleus to steer the expression of responsive genes. This mitochondria-to-nucleus communication is often referred to as mitochondrial retrograde regulation (MRR). Although reactive oxygen species and calcium are likely candidate signaling molecules for MRR, the protein signaling components in plants remain largely unknown. Through meta-analysis of transcriptome data, we detected a set of genes that are common and robust targets of MRR and used them as a bait to identify its transcriptional regulators. In the upstream regions of these mitochondrial dysfunction stimulon (MDS) genes, we found a cis-regulatory element, the mitochondrial dysfunction motif (MDM), which is necessary and sufficient for gene expression under various mitochondrial perturbation conditions. Yeast one-hybrid analysis and electrophoretic mobility shift assays revealed that the transmembrane domain-containing no apical meristem/Arabidopsis transcription activation factor/cup-shaped cotyledon transcription factors (ANAC013, ANAC016, ANAC017, ANAC053, and ANAC078) bound to the MDM cis-regulatory element. We demonstrate that ANAC013 mediates MRR-induced expression of the MDS genes by direct interaction with the MDM cis-regulatory element and triggers increased oxidative stress tolerance. In conclusion, we characterized ANAC013 as a regulator of MRR upon stress in Arabidopsis thaliana.

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.

Environmental stresses adversely affect plant growth and development. A common theme within these adverse conditions is the perturbation of reactive oxygen species (ROS) homeostasis. Here, we demonstrate that the ROS-inducible Arabidopsis thaliana WRKY15 transcription factor (AtWRKY15) modulates plant growth and salt/osmotic stress responses. By transcriptome profiling, a divergent stress response was identified in transgenic WRKY15-overexpressing plants that linked a stimulated endoplasmic reticulum-to-nucleus communication to a disrupted mitochondrial stress response under salt-stress conditions. We show that mitochondrial calcium-flux sensing might be important for regulating an active mitochondrial retrograde signaling and launching an appropriate defense response to confer salt-stress tolerance.

Eukaryotic organisms evolved under aerobic conditions subjecting nuclear DNA to damage provoked by reactive oxygen species (ROS). Although ROS are thought to be a major cause of DNA damage, little is known about the molecular mechanisms protecting nuclear DNA from oxidative stress. Here we show that protection of nuclear DNA in plants requires a coordinated function of ROS-scavenging pathways residing in the cytosol and peroxisomes, demonstrating that nuclear ROS scavengers such as peroxiredoxin and glutathione are insufficient to safeguard DNA integrity. Both catalase (CAT2) and cytosolic ascorbate peroxidase (APX1) play a key role in protecting the plant genome against photorespiratory-dependent H(2)O(2)-induced DNA damage. In apx1/cat2 double-mutant plants, a DNA damage response is activated, suppressing growth via a WEE1 kinase-dependent cell-cycle checkpoint. This response is correlated with enhanced tolerance to oxidative stress, DNA stress-causing agents, and inhibited programmed cell death.

Reactive oxygen species and redox signaling undergo synergistic and antagonistic interactions with phytohormones to regulate protective responses of plants against biotic and abiotic stresses. However, molecular insight into the nature of this crosstalk remains scarce. We demonstrate that the hydrogen peroxide-responsive UDP-glucosyltransferase UGT74E2 of Arabidopsis thaliana is involved in the modulation of plant architecture and water stress response through its activity toward the auxin indole-3-butyric acid (IBA). Biochemical characterization of recombinant UGT74E2 demonstrated that it strongly favors IBA as a substrate. Assessment of indole-3-acetic acid (IAA), IBA, and their conjugates in transgenic plants ectopically expressing UGT74E2 indicated that the catalytic specificity was maintained in planta. In these transgenic plants, not only were IBA-Glc concentrations increased, but also free IBA levels were elevated and the conjugated IAA pattern was modified. This perturbed IBA and IAA homeostasis was associated with architectural changes, including increased shoot branching and altered rosette shape, and resulted in significantly improved survival during drought and salt stress treatments. Hence, our results reveal that IBA and IBA-Glc are important regulators of morphological and physiological stress adaptation mechanisms and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT.

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

The Arabidopsis thaliana genome expresses five evolutionarily conserved prohibitin (PHB) genes that are divided into type-I (AtPHB3 and AtPHB4) and type-II (AtPHB1, AtPHB2 and AtPHB6) classes, based on their phylogenetic relationships with yeast PHB1 and PHB2, respectively. Yeast and animal PHBs are reported to have diverse roles in the cell cycle, mitochondrial electron transport, aging and apoptosis. All transcribed Arabidopsis PHB genes are primarily expressed in both shoot and root proliferative tissues, where they are present in mitochondrial multimeric complexes. Loss of function of the type-I AtPHB4 had no phenotypic effects, while loss of function of the homologous AtPHB3 caused mitochondrial swelling, decreased meristematic cell production, increased cell division time and reduced cell expansion rates, leading to severe growth retardation. Double knockout atphb3 atphb4 plants were not viable, but transgenic lines overexpressing AtPHB3 or AtPHB4 showed leaf shape aberrations and an increased shoot branching phenotype. Genome-wide microarray analysis revealed that both knockout and overexpression perturbations of AtPHB3 and AtPHB4 provoked an altered abundance of mitochondrial and stress-related transcripts. We propose that plant type-I PHBs take part in protein complexes that are necessary for proficient mitochondrial function or biogenesis, thereby supporting cell division and differentiation in apical tissues.

Transgenic plants with reduced poly(ADP-ribose) polymerase (PARP) levels have broad-spectrum stress-resistant phenotypes. Both Arabidopsis thaliana and oilseed rape (Brassica napus) lines overexpressing RNA interference-PARP constructs were more resistant to various abiotic stress treatments in laboratory and greenhouse experiments without negative effects on growth, development, and seed production. This outperforming stress tolerance was initially attributed solely to a maintained energy homeostasis due to reduced NAD(+) consumption. We show that in PARP2-deficient Arabidopsis plants, the observed abiotic stress resistance can also be explained by alterations in abscisic acid levels that facilitate the induction of a wide set of defense-related genes.

Metacaspases are distant relatives of animal caspases found in plants, fungi and protozoa. We demonstrated previously that two type II metacaspases of Arabidopsis thaliana, AtMC4 and AtMC9 are Arg/Lys-specific cysteine-dependent proteases. We screened a combinatorial tetrapeptide library of 130,321 substrates with AtMC9. Here, we show that AtMC9 is a strict Arg/Lys-specific protease. Based on the position-specific scoring matrix derived from the substrate library results, the tetrapeptide Val-Arg-Pro-Arg was identified as an optimized substrate. AtMC9 had a kcat/KM of 4.6x10(5) M-1 s-1 for Ac-Val-Arg-Pro-Arg-amido-4-methyl-coumarin, representing a more than 10-fold improvement over existing fluorogenic substrates. A yeast two-hybrid screen with catalytically inactive AtMC9 as bait identified a serine protease inhibitor, designated AtSerpin1, which was found to be a potent inhibitor of AtMC9 activity in vitro through cleavage of its reactive center loop and covalent binding to AtMC9. On the basis of the substrate profiling of AtMC9 and confirmation through site-directed mutagenesis, the inhibitory P4-P1 cleavage site of AtSerpin1 was determined to be Ile-Lys-Leu-Arg351. Further mutagenesis of the AtSerpin1 inhibitory cleavage site modulated AtMC9 inhibition positively or negatively. Both AtMC9 and AtSerpin1 were localized in the extracellular space, suggesting an in vivo interaction as well. To our knowledge, this is the first report of plant protease inhibition by a plant serpin.

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H(2)O(2)-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H(2)O(2) plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H(2)O(2) and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H(2)O(2) and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.

Nine potential caspase counterparts, designated metacaspases, were identified in the Arabidopsis thaliana genome. Sequence analysis revealed two types of metacaspases, one with (type I) and one without (type II) a proline- or glutamine-rich N-terminal extension, possibly representing a prodomain. Production of recombinant Arabidopsis type II metacaspases in Escherichia coli resulted in cysteine-dependent autocatalytic processing of the proform into large and small subunits, in analogy to animal caspases. A detailed biochemical characterization with a broad range of synthetic oligopeptides and several protease inhibitors of purified recombinant proteins of both metacaspase 4 and 9 showed that both metacaspases are arginine/lysine-specific cysteine proteases and did not cleave caspase-specific synthetic substrates. These findings suggest that type II metacaspases are not directly responsible for earlier reported caspase-like activities in plants.

In transgenic tobacco plants with reduced catalase activity, high levels of hydrogen peroxide (H2O2) can accumulate under photorespiratory conditions. Such a perturbation in H2O2 homeostasis induced cell death in clusters of palisade parenchyma cells, primarily along the veins. Ultrastructural alterations, such as chromatin condensation and disruption of mitochondrial integrity, took place before cell death. Furthermore, enhanced transcript levels of mitochondrial defense genes accompanied these mitochondrial changes. Pharmacological data indicated that the initiation and execution of cell death require de novo protein synthesis and that the signal transduction pathway leading to cell death involved changes in ion homeostasis, (de)phosphorylation events and an oxidative burst, as observed during hypersensitive responses. This oxidase-dependent oxidative burst is essential for cell death, but it is not required for the accumulation of defense proteins, suggesting a more prominent role for the oxidative burst in abiotic stress-induced cell death.

Pro dehydrogenase (PDH) catalyzes the first and rate-limiting step in the Pro catabolic pathway. In Arabidopsis, this enzyme is encoded by At-PDH. To investigate the role of Pro catabolism in plants, we generated transgenic Arabidopsis plants with altered levels of PDH by sense (PDH-S plants) and antisense (PDH-AS plants) strategies. Free Pro levels were reduced by up to 50% in PDH-S plants under stress and recovery conditions and enhanced by a maximum of 25% in PDH-AS plants, despite large modifications of the At-PDH transcript and At-PDH protein levels. A similar trend in free Pro levels was observed in the PDH-S and PDH-AS seeds without visible effects on germination or growth. Under stress conditions, PDH transgenic plants showed no signs of change in osmotolerance. However, addition of exogenous Pro increased survival rates of salt-stressed PDH-S plants by 30%. Isotope-labeling studies showed that the conversion of [14C]Pro to Glu was reduced in PDH-AS plants and increased in PDH-S plants, especially under stress conditions. Furthermore, PDH-AS plants were hypersensitive to exogenous Pro, whereas PDH-S plants were sensitive to Pro analogs. These findings demonstrate that altered At-PDH levels lead to weakly modified free Pro accumulation with a limited impact on plant development and growth, suggesting a tight control of Pro homeostasis and/or gene redundancy.

Owing to their sessile lifestyle, it is crucial for plants to acquire stress tolerance. The function of heat-shock proteins, including small heat-shock proteins (smHSPs), in stress tolerance is not fully explored. To gain further knowledge about the smHSPs, the gene that encoded the cytosolic class II smHSP in Arabidopsis thaliana (At-HSP17.6A) was characterized. The At-HSP17.6A expression was induced by heat and osmotic stress, as well as during seed development. Accumulation of At-HSP17.6A proteins could be detected with heat and at a late stage of seed development, but not with osmotic stress, suggesting stress-induced post-transcriptional regulation of At-HSP17.6A expression. Overproduction of At-HSP17.6A could increase salt and drought tolerance in Arabidopsis. The chaperone activity of At-HSP17.6A was demonstrated in vitro.

The steady-state level of transcripts coding for the pyrroline-5-carboxylate reductase of Arabidopsis (At-P5R) increased under salt and heat stress, mainly because of an enhanced mRNA stability. However, the At-P5R protein level was not induced, and its translation was inhibited at initiation stage and probably also at later stages. Replacement of the 5' untranslated region (5'UTR) and beta-glucuronidase (gus) fusion analysis revealed that the first 92 bp region of the At-P5R 5'UTR was sufficient to mediate transcript stabilization and translation inhibition during salt and heat stresses. Furthermore, the first 92 bp region of the At-P5R 5'UTR was also involved in transcription efficiency in a promoter-dependent manner. The results demonstrated that the stress regulation of At-P5R is complex and involves the 5'UTR which acts at three levels, partly in opposing directions.

Hyperosmotic stress severely affects plant growth and development. To examine the effect of salt stress on cell cycle activity in Arabidopsis thaliana (L.) Heynh., the transcriptional regulation of a cyclin-dependent kinase, CDC2aAt, and two mitotic cyclins, Arath;CycB1;1 and Arath;CycA2;1, was studied by using the beta-glucuronidase (gus) reporter gene. Moreover, the mRNA abundance of these cell cycle genes as well as CDC2bAt were monitored during salt stress. Upon NaCl treatment, the promoter activities and transcript levels of all cell cycle genes diminished initially in the shoot apex and were subsequently induced during salt-stress adaptation. Additionally, the promoter activities of CDC2aAt and CycA2;1 decreased in the vascular cylinder of the root in correlation with reduced lateral root formation. In the root tips, a regression of CDC2aAt, CycA2;1, and CycB1;1:gus expression was observed, concomitant with a shrinkage of the root meristem and inhibition of root growth. Our data indicate that salt stress interferes with cell cycle regulation at the transcriptional level, resulting in an adaptive growth response.

The Arabidopsis thaliana gene that encodes pyrroline-5-carboxylate reductase (At-P5R), the last enzyme in proline biosynthesis in A. thaliana, is developmentally regulated and is highly expressed in cells that divide rapidly or undergo changes in osmotic potential. A 69 bp region (P69; -120 to -51) has previously been identified in a 5' deletion analysis of the At-P5R promoter to be necessary for the basal expression. Here, the essential role of P69 for tissue-specific expression of At-P5R is demonstrated by loss- and gain-of-function experiments.