The Receptor-like Pseudokinase GHR1 Is Required for Stomatal Closure.

Guard cells control the aperture of stomatal pores to balance photosynthetic carbon dioxide uptake with evaporative water loss. Stomatal closure is triggered by several stimuli that initiate complex signaling networks to govern the activity of ion channels. Activation of SLOW ANION CHANNEL1 (SLAC1) is central to the process of stomatal closure and requires the leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), among other signaling components. Here, based on functional analysis of nine Arabidopsis thaliana ghr1 mutant alleles identified in two independent forward-genetic ozone-sensitivity screens, we found that GHR1 is required for stomatal responses to apoplastic reactive oxygen species, abscisic acid, high CO2 concentrations, and diurnal light/dark transitions. Furthermore, we show that the amino acid residues of GHR1 involved in ATP binding are not required for stomatal closure in Arabidopsis or the activation of SLAC1 anion currents in Xenopus laevis oocytes and present supporting in silico and in vitro evidence suggesting that GHR1 is an inactive pseudokinase. Biochemical analyses suggested that GHR1-mediated activation of SLAC1 occurs via interacting proteins and that CALCIUM-DEPENDENT PROTEIN KINASE3 interacts with GHR1. We propose that GHR1 acts in stomatal closure as a scaffolding component.

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.

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.

Plant signalling in acute ozone exposure.

Exposure of plants to high ozone concentrations causes lesion formation in sensitive plants. Plant responses to ozone involve fast and massive changes in protein activities, gene expression and metabolism even before any tissue damage can be detected. Degradation of ozone and subsequent accumulation of reactive oxygen species (ROS) in the extracellular space activates several signalling cascades, which are integrated inside the cell into a fine-balanced network of ROS signalling. Reversible protein phosphorylation and degradation plays an important role in the regulation of signalling mechanisms in a complex crosstalk with plant hormones and calcium, an essential second messenger. In this review, we discuss the recent advances in understanding the molecular mechanisms of ozone uptake, perception and signalling pathways activated during the early steps of ozone response, and discuss the use of ozone as a tool to study the function of apoplastic ROS in signalling.

Are solar UV-B- and UV-A-dependent gene expression and metabolite accumulation in Arabidopsis mediated by the stress response regulator RADICAL-INDUCED CELL DEATH1?

Wavelengths in the ultraviolet (UV) region of the solar spectrum, UV-B (280-315 nm) and UV-A (315-400 nm), are key environmental signals modifying several aspects of plant physiology. Despite significant advances in the understanding of plant responses to UV-B and the identification of signalling components involved, there is limited information on the molecular mechanisms that control UV-B signalling in plants under natural sunlight. Here, we aimed to corroborate the previous suggested role for RADICAL-INDUCED CELL DEATH1 (RCD1) in UV-B signalling under full spectrum sunlight. Wild-type Arabidopsis thaliana and the rcd1-1 mutant were used in an experimental design outdoors where UV-B and UV-A irradiances were manipulated using plastic films, and gene expression, PYRIDOXINE BIOSYNTHESIS1 (PDX1) accumulation and metabolite profiles were analysed in the leaves. At the level of transcription, RCD1 was not directly involved in the solar UV-B regulation of genes with functions in UV acclimation, hormone signalling and stress-related markers. Furthermore, RCD1 had no role on PDX1 accumulation but modulated the UV-B induction of flavonoid accumulation in leaves of Arabidopsis exposed to solar UV. We conclude that RCD1 does not play an active role in UV-B signalling but rather modulates UV-B responses under full spectrum sunlight.

RCD1-DREB2A interaction in leaf senescence and stress responses in Arabidopsis thaliana.

Transcriptional regulation of gene expression is one major determinant of developmental control and stress adaptation in virtually all living organisms. In recent years numerous transcription factors controlling various aspects of plant life have been identified. The activity of transcription factors needs to be regulated to prevent unspecific, prolonged or inappropriate responses. The transcription factor DREB2A (DEHYDRATION-RESPONSIVE ELEMENT BINDING 2A) has been identified as one of the main regulators of drought and heat responses, and it is regulated through protein stability. In the present paper we describe evidence that the interaction with RCD1 (RADICAL-INDUCED CELL DEATH 1) contributes to the control of DREB2A under a range of conditions. The interaction is mediated by a novel protein motif in DREB2A and a splice variant of DREB2A which lacks the interaction domain accumulates during heat stress and senescence. In addition RCD1 is rapidly degraded during heat stress, thus our results suggest that removal of RCD1 protein or the loss of the interaction domain in DREB2A appears to be required for proper DREB2A function under stress conditions.

Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases.

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.

Druggable cancer phosphatases.

The phosphorylation status of oncoproteins is regulated by both kinases and phosphatases. Kinase inhibitors are rarely sufficient for successful cancer treatment, and phosphatases have been considered undruggable targets for cancer drug development. However, innovative pharmacological approaches for targeting phosphatases have recently emerged. Here, we review progress in the therapeutic targeting of oncogenic Src homology region 2 domain-containing phosphatase-2 (SHP2) and tumor suppressor protein phosphatase 2A (PP2A) and select other druggable oncogenic and tumor suppressor phosphatases. We describe the modes of action for currently available small molecules that target phosphatases, their use in drug combinations, and advances in clinical development toward future cancer therapies.

CIP2A Interacts with TopBP1 and Drives Basal-Like Breast Cancer Tumorigenesis.

Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high-proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA damage-induced initiation of mouse BLBC-like mammary tumors and for survival of HR-defective BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified, and CIP2A inhibition resulted in enhanced DNA damage-induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple-negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small-molecule reactivators of PP2A (SMAP) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibited growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNA damage-induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs. SIGNIFICANCE: These results identify CIP2A as a nongenetic driver and therapeutic target in basal-like breast cancer that regulates DNA damage-induced G2-M checkpoint and proliferative signaling.

Dynamic changes in the proteome of Synechocystis 6803 in response to CO(2) limitation revealed by quantitative proteomics.

Cyanobacteria developed efficient carbon concentrating mechanisms which significantly improve the photosynthetic performance and survival of cells under limiting CO(2) conditions. Dynamic changes of the Synechocystis proteome to CO(2) limitation were investigated using shotgun LC-MS/MS approach with isobaric tag for relative and absolute quantification (iTRAQ) technique. Synechocystis cells grown at high (3%) CO(2) were shifted to air-level CO(2) followed by protein extraction after 6, 24, and 72 h. About 19% of the cyanobacterial proteome was identified and the expression changes were quantified for 17% of theoretical ORFs. For 76 proteins, up- or down-regulation was found to be significant (more than 1.5 or less than 0.7). Major changes were observed in proteins participating in inorganic carbon uptake, CO(2) fixation, nitrogen transport and assimilation, as well as in the protection of the photosynthetic machinery from excess of light. Further, a number of hypothetical proteins with unknown functions were discovered. In general, the cells appear to acclimate to low CO(2) without a significant stress since the stress-related molecular chaperones were down-regulated and only a minor decline was detected for proteins of phycobilisomes, photosynthetic complexes, and translation machinery. The results of iTRAQ experiment were validated by the Western blot analysis for selected proteins.

Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors.

RADICAL-INDUCED CELL DEATH1 (RCD1) is an important regulator of stress and hormonal and developmental responses in Arabidopsis thaliana. Together with its closest homolog, SIMILAR TO RCD-ONE1 (SRO1), it is the only Arabidopsis protein containing the WWE domain, which is known to mediate protein-protein interactions in other organisms. Additionally, these two proteins contain the core catalytic region of poly-ADP-ribose transferases and a conserved C-terminal domain. Tissue and subcellular localization data indicate that RCD1 and SRO1 have partially overlapping functions in plant development. In contrast mutant data indicate that rcd1 has defects in plant development, whereas sro1 displays normal development. However, the rcd1 sro1 double mutant has severe growth defects, indicating that RCD1 and SRO1 exemplify an important genetic principle - unequal genetic redundancy. A large pair-wise interaction test against the REGIA transcription factor collection revealed that RCD1 interacts with a large number of transcription factors belonging to several protein families, such as AP2/ERF, NAC and basic helix-loop-helix (bHLH), and that SRO1 interacts with a smaller subset of these. Full genome array analysis indicated that in many cases targets of these transcription factors have altered expression in the rcd1 but not the sro1 mutant. Taken together RCD1 and SRO1 are required for proper plant development.

The RST and PARP-like domain containing SRO protein family: analysis of protein structure, function and conservation in land plants.

BACKGROUND: The SROs (SIMILAR TO RCD-ONE) are a group of plant-specific proteins which have important functions in stress adaptation and development. They contain the catalytic core of the poly(ADP-ribose) polymerase (PARP) domain and a C-terminal RST (RCD-SRO-TAF4) domain. In addition to these domains, several, but not all, SROs contain an N-terminal WWE domain. RESULTS: SROs are present in all analyzed land plants and sequence analysis differentiates between two structurally distinct groups; cryptogams and monocots possess only group I SROs whereas eudicots also contain group II. Group I SROs possess an N-terminal WWE domain (PS50918) but the WWE domain is lacking in group II SROs. Group I domain structure is widely represented in organisms as distant as humans (for example, HsPARP11). We propose a unified nomenclature for the SRO family. The SROs are able to interact with transcription factors through the C-terminal RST domain but themselves are generally not regulated at the transcriptional level. The most conserved feature of the SROs is the catalytic core of the poly(ADP-ribose) polymerase (PS51059) domain. However, bioinformatic analysis of the SRO PARP domain fold-structure and biochemical assays of AtRCD1 suggested that SROs do not possess ADP-ribosyl transferase activity. CONCLUSIONS: The SROs are a highly conserved family of plant specific proteins. Sequence analysis of the RST domain implicates a highly preserved protein structure in that region. This might have implications for functional conservation. We suggest that, despite the presence of the catalytic core of the PARP domain, the SROs do not possess ADP-ribosyl transferase activity. Nevertheless, the function of SROs is critical for plants and might be related to transcription factor regulation and complex formation.

Dissecting the interaction of photosynthetic electron transfer with mitochondrial signalling and hypoxic response in the Arabidopsis rcd1 mutant.

The Arabidopsis mutant rcd1 is tolerant to methyl viologen (MV). MV enhances the Mehler reaction, i.e. electron transfer from Photosystem I (PSI) to O2, generating reactive oxygen species (ROS) in the chloroplast. To study the MV tolerance of rcd1, we first addressed chloroplast thiol redox enzymes potentially implicated in ROS scavenging. NADPH-thioredoxin oxidoreductase type C (NTRC) was more reduced in rcd1. NTRC contributed to the photosynthetic and metabolic phenotypes of rcd1, but did not determine its MV tolerance. We next tested rcd1 for alterations in the Mehler reaction. In rcd1, but not in the wild type, the PSI-to-MV electron transfer was abolished by hypoxic atmosphere. A characteristic feature of rcd1 is constitutive expression of mitochondrial dysfunction stimulon (MDS) genes that affect mitochondrial respiration. Similarly to rcd1, in other MDS-overexpressing plants hypoxia also inhibited the PSI-to-MV electron transfer. One possible explanation is that the MDS gene products may affect the Mehler reaction by altering the availability of O2. In green tissues, this putative effect is masked by photosynthetic O2 evolution. However, O2 evolution was rapidly suppressed in MV-treated plants. Transcriptomic meta-analysis indicated that MDS gene expression is linked to hypoxic response not only under MV, but also in standard growth conditions. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Determination of in vivo protein phosphorylation in photosynthetic membranes.

Light- and redox-controlled reversible phosphorylation of thylakoid proteins regulates short- and long-term acclimation of plants to environmental cues. The major phosphoproteins in thylakoids belong to photosystem II and its light-harvesting antenna but phosphorylation of subunits of other thylakoid protein complexes has been detected as well. The detection methods include electrophoretic separation of proteins and detection of phosphoproteins with a phosphoaminoacid-specific antibody or phosphoprotein-specific dye. The use of mass spectrometry allows the identification of exact phosphorylation site(s) in the proteins. Various methods for detection of phosphoproteins in thylakoids are outlined including phosphopeptide preparation for mass spectrometric analyses and quantitative analysis of protein phosphorylation.

Interaction of methyl viologen-induced chloroplast and mitochondrial signalling in Arabidopsis.

Reactive oxygen species (ROS) are key signalling intermediates in plant metabolism, defence, and stress adaptation. In plants, both the chloroplast and mitochondria are centres of metabolic control and ROS production, which coordinate stress responses in other cell compartments. The herbicide and experimental tool, methyl viologen (MV) induces ROS generation in the chloroplast under illumination, but is also toxic in non-photosynthetic organisms. We used MV to probe plant ROS signalling in compartments other than the chloroplast. Taking a genetic approach in the model plant Arabidopsis (Arabidopsis thaliana), we used natural variation, QTL mapping, and mutant studies with MV in the light, but also under dark conditions, when the chloroplast electron transport is inactive. These studies revealed a light-independent MV-induced ROS-signalling pathway, suggesting mitochondrial involvement. Mitochondrial Mn SUPEROXIDE DISMUTASE was required for ROS-tolerance and the effect of MV was enhanced by exogenous sugar, providing further evidence for the role of mitochondria. Mutant and hormone feeding assays revealed roles for stress hormones in organellar ROS-responses. The radical-induced cell death1 mutant, which is tolerant to MV-induced ROS and exhibits altered mitochondrial signalling, was used to probe interactions between organelles. Our studies suggest that mitochondria are involved in the response to ROS induced by MV in plants.

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors.

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles.

Light regulation of CaS, a novel phosphoprotein in the thylakoid membrane of Arabidopsis thaliana.

Exposure of Arabidopsis thaliana plants to high levels of light revealed specific phosphorylation of a 40 kDa protein in photosynthetic thylakoid membranes. The protein was identified by MS as extracellular calcium-sensing receptor (CaS), previously reported to be located in the plasma membrane. By confocal laser scanning microscopy and subcellular fractionation, it was demonstrated that CaS localizes to the chloroplasts and is enriched in stroma thylakoids. The phosphorylation level of CaS responded strongly to light intensity. The light-dependent thylakoid protein kinase STN8 is required for CaS phosphorylation. The phosphorylation site was mapped to the stroma-exposed Thr380, located in a motif for interaction with 14-3-3 proteins and proteins with forkhead-associated domains, which suggests the involvement of CaS in stress responses and signaling pathways. The knockout Arabidopsis lines revealed a significant role for CaS in plant growth and development.

1H, 13C and 15N NMR chemical shift assignments of A. thaliana RCD1 RST.

The A. thaliana RCD1 (radical-induced cell death1) protein is a cellular signaling hub protein which interacts with numerous plant transcription factors from different families. It consists of three conserved domains and intervening unstructured regions, the C-terminal RST domain being responsible for the interactions with the transcription factors. It has been shown that many partner proteins interact with RCD1 RST via their intrinsically disordered regions, and that the domain is able to house partners with divergent folds. We aim to structurally characterize the RCD1 RST domain and its complexes [complex with DREB2A]. Here we report the 1H, 15N and 13C chemical shift assignments of the backbone and sidechain atoms for RCD1 (468-589) containing the RST (510-567) domain.

Plant PARPs, PARGs and PARP-like Proteins.

Poly(ADP-ribos)ylation, originally described as a mechanism of DNA break repair, is now considered as part of a complex regulatory system involved in dynamic reorganization of chromatin structure, transcriptional control of gene expression and regulation of metabolism. In plants poly(ADP-ribos)ylation has received surprisingly little attention. It has been implicated in abiotic and biotic stress responses, cell cycle control and development; however, the molecular mechanisms and proteins involved are largely unknown. In this review we summarize current knowledge on plant PARP, PARG and PARP-like domain containing proteins and discuss their possible roles in plant development, immune responses, programmed cell death and stress responses in general. The genome of the model plant Arabidopsis contains three genes encoding PARP proteins, two of which have been shown to be active PARPs, and two genes encoding PARG proteins, one of which was shown to possess enzymatic activity. In addition, SROs (Similar to RCD One) represent a plant specific family of proteins containing a PARP-like domain. Although bioinformatics and biochemical data suggest that the PARP-like domain in SRO proteins does not have PARP activity, these proteins play a significant role in stress response as revealed by mutant analyses. SRO proteins interact with transcription factors involved in various stress and developmental responses and are suggested to serve as hubs in many signaling pathways. Altogether current data imply that poly(ADP-ribos)ylation plays significant regulatory role in many aspects of plant biology.

A previously found thylakoid membrane protein of 14kDa (TMP14) is a novel subunit of plant photosystem I and is designated PSI-P.

We show that the thylakoid membrane phosphoprotein TMP14 is a novel subunit of plant photosystem I (PSI). Blue native/SDS-PAGE and sucrose gradient fractionation demonstrated the association of the protein exclusively with PSI. We designate the protein PSI-P. The presence of PSI-P subunit in Arabidopsis mutants lacking other PSI subunits was analyzed and suggested a location in the proximity of PSI-L, -H and -O subunits. The PSI-P protein was not differentially phosphorylated in state 1 and state 2.