Proteome-wide lysine acetylation profiling to investigate the involvement of histone deacetylase HDA5 in the salt stress response of Arabidopsis leaves.

Post-translational modifications (PTMs) of proteins play important roles in the acclimation of plants to environmental stress. Lysine acetylation is a dynamic and reversible PTM, which can be removed by histone deacetylases. Here we investigated the role of lysine acetylation in the response of Arabidopsis leaves to 1 week of salt stress. A quantitative mass spectrometry analysis revealed an increase in lysine acetylation of several proteins from cytosol and plastids, which was accompanied by altered histone deacetylase activities in the salt-treated leaves. While activities of HDA14 and HDA15 were decreased upon salt stress, HDA5 showed a mild and HDA19 a strong increase in activity. Since HDA5 is a cytosolic-nuclear enzyme from the class II histone deacetylase family with yet unknown protein substrates, we performed a lysine acetylome analysis on hda5 mutants and characterized its substrate proteins. Next to histone H2B, the salt stress-responsive transcription factor GT2L and the dehydration-related protein ERD7 were identified as HDA5 substrates. In addition, in protein-protein interaction studies, HDA18 was discovered, among other interacting proteins, to work in a complex together with HDA5. Altogether, this study revealed the substrate proteins of HDA5 and identified new lysine acetylation sites which are hyperacetylated upon salt stress. The identification of specific histone deacetylase substrate proteins, apart from histones, will be important to unravel the acclimation response of Arabidopsis to salt stress and their role in plant physiology.

ABI5 binding protein2 inhibits ABA responses during germination without ABA-INSENSITIVE5 degradation.

Overexpression of ABA-INSENSITIVE5 binding proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. We tested the hypothesis that AFPs promote germination in Arabidopsis (Arabidopsis thaliana) by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING, were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although weak direct interactions were detected between AFPs and E3 ligases, loss of function for these E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP-AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 overexpression reduces ABA sensitivity, thereby permitting germination prior to ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance. Our quantitative proteomics analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of wild-type or dwa1 dwa2 mutants with or without AFP2 overexpression allowed us to separate and refine the changes in protein accumulation patterns associated with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

A New Role for Plastid Thioredoxins in Seed Physiology in Relation to Hormone Regulation.

In Arabidopsis seeds, ROS have been shown to be enabling actors of cellular signaling pathways promoting germination, but their accumulation under stress conditions or during aging leads to a decrease in the ability to germinate. Previous biochemical work revealed that a specific class of plastid thioredoxins (Trxs), the y-type Trxs, can fulfill antioxidant functions. Among the ten plastidial Trx isoforms identified in Arabidopsis, Trx y1 mRNA is the most abundant in dry seeds. We hypothesized that Trx y1 and Trx y2 would play an important role in seed physiology as antioxidants. Using reverse genetics, we found important changes in the corresponding Arabidopsis mutant seeds. They display remarkable traits such as increased longevity and higher and faster germination in conditions of reduced water availability or oxidative stress. These phenotypes suggest that Trxs y do not play an antioxidant role in seeds, as further evidenced by no changes in global ROS contents and protein redox status found in the corresponding mutant seeds. Instead, we provide evidence that marker genes of ABA and GAs pathways are perturbed in mutant seeds, together with their sensitivity to specific hormone inhibitors. Altogether, our results suggest that Trxs y function in Arabidopsis seeds is not linked to their previously identified antioxidant roles and reveal a new role for plastid Trxs linked to hormone regulation.

Sequence Polymorphisms at the REDUCED DORMANCY5 Pseudophosphatase Underlie Natural Variation in Arabidopsis Dormancy.

Seed dormancy controls the timing of germination, which regulates the adaptation of plants to their environment and influences agricultural production. The time of germination is under strong natural selection and shows variation within species due to local adaptation. The identification of genes underlying dormancy quantitative trait loci is a major scientific challenge, which is relevant for agricultural and ecological goals. In this study, we describe the identification of the DELAY OF GERMINATION18 (DOG18) quantitative trait locus, which was identified as a factor in natural variation for seed dormancy in Arabidopsis (Arabidopsis thaliana). DOG18 encodes a member of the clade A of the type 2C protein phosphatases family, which we previously identified as the REDUCED DORMANCY5 (RDO5) gene. DOG18/RDO5 shows a relatively high frequency of loss-of-function alleles in natural accessions restricted to northwestern Europe. The loss of dormancy in these loss-of-function alleles can be compensated for by genetic factors like DOG1 and DOG6, and by environmental factors such as low temperature. RDO5 does not have detectable phosphatase activity. Analysis of the phosphoproteome in dry and imbibed seeds revealed a general decrease in protein phosphorylation during seed imbibition that is enhanced in the rdo5 mutant. We conclude that RDO5 acts as a pseudophosphatase that inhibits dephosphorylation during seed imbibition.

Redox regulation of chloroplastic G6PDH activity by thioredoxin occurs through structural changes modifying substrate accessibility and cofactor binding.

In chloroplasts, redox regulation of enzyme activities by TRXs (thioredoxins) allows the co-ordination of light/dark metabolisms such as the reductive (so-called Calvin-Benson) pathway and the OPPP (oxidative pentose phosphate pathway). Although the molecular mechanisms underlying the redox regulation of several TRX-regulated enzymes have been investigated in detail, only partial information was available for plastidial G6PDH (glucose-6-phosphate dehydrogenase) catalysing the first and rate-limiting step of the OPPP. In the present study, we investigated changes in catalytic and structural properties undergone by G6PDH1 from Arabidopsis thaliana upon treatment with TRX f1, the most efficient regulator of the enzyme that did not show a stable interaction with its target. We found that the formation of the regulatory disulfide bridge that leads to activation of the enzyme allows better substrate accessibility to the active site and strongly modifies the cofactor-binding properties. Structural modelling and data from biochemical and biophysical studies of site-directed mutant proteins support a mechanism in which the positioning/function of the highly conserved Arg(131) in the cofactor-binding site can be directly influenced by the redox state of the adjacent regulatory disulfide bridge. These findings constitute another example of modifications to catalytic properties of a chloroplastic enzyme upon redox regulation, but by a mechanism unique to G6PDH.

ABI5 binding proteins: key players in coordinating plant growth and development.

During the course of terrestrial evolution, plants have developed complex networks that involve the coordination of phytohormone signalling pathways in order to adapt to an ever-changing environment. Transcription factors coordinate these responses by engaging in different protein complexes and exerting both positive and negative effects. ABA INSENSITIVE 5 (ABI5) binding proteins (AFPs), which are closely related to NOVEL INTERACTOR OF JAZ (NINJA)-like proteins, are known for their fundamental role in plants' morphological and physiological growth. Recent studies have shown that AFPs regulate several hormone-signalling pathways, including abscisic acid (ABA) and gibberellic acid (GA). Here, we review the genetic control of AFPs and their crosstalk with plant hormone signalling, and discuss the contributions of AFPs to plants' growth and development.

Dry side of the core: a meta-analysis addressing the original nature of the ABA signalosome at the onset of seed imbibition.

The timing of seedling emergence is a major agricultural and ecological fitness trait, and seed germination is controlled by a complex molecular network including phytohormone signalling. One such phytohormone, abscisic acid (ABA), controls a large array of stress and developmental processes, and researchers have long known it plays a crucial role in repressing germination. Although the main molecular components of the ABA signalling pathway have now been identified, the molecular mechanisms through which ABA elicits specific responses in distinct organs is still enigmatic. To address the fundamental characteristics of ABA signalling during germination, we performed a meta-analysis focusing on the Arabidopsis dry seed proteome as a reflexion basis. We combined cutting-edge proteome studies, comparative functional analyses, and protein interaction information with genetic and physiological data to redefine the singular composition and operation of the ABA core signalosome from the onset of seed imbibition. In addition, we performed a literature survey to integrate peripheral regulators present in seeds that directly regulate core component function. Although this may only be the tip of the iceberg, this extended model of ABA signalling in seeds already depicts a highly flexible system able to integrate a multitude of information to fine-tune the progression of germination.

Thioredoxins m regulate plastid glucose-6-phosphate dehydrogenase activity in Arabidopsis roots under salt stress.

Plants contain several NADPH-producing enzymes including glucose-6-phosphate dehydrogenases (G6PDH) with different sub-cellular localizations. The activity of plastidial G6PDHs is redox-regulated by thioredoxins (TRX). Although specific TRXs are known to regulate chloroplastic isoforms of G6PDH, little information is available for plastidic isoforms found in heterotrophic organs or tissues. Here, we investigated TRX regulation of the two G6PDH plastidic isoforms of Arabidopsis roots during exposure to a mild salt stress. We report that in vitro m-type TRXs are the most efficient regulators of the G6PDH2 and G6PDH3 mainly found in Arabidopsis roots. While expression of the corresponding G6PD and plastidic TRX genes was marginally affected by salt, it impaired root growth of several of the corresponding mutant lines. Using an in situ assay for G6PDH, G6PDH2 was found to be the major contributor to salt-induced increases in activity, while data from ROS assays further provide in vivo evidence that TRX m acts in redox regulation during salt stress. Taken together, our data suggest that regulation of plastid G6PDH activity by TRX m may be an important player regulating NADPH production in Arabidopsis roots undergoing salt stress.

Meta-analysis of the diagnostic performance of stress perfusion cardiovascular magnetic resonance for detection of coronary artery disease.

AIM: Evaluation of the diagnostic accuracy of stress perfusion cardiovascular magnetic resonance for the diagnosis of significant obstructive coronary artery disease (CAD) through meta-analysis of the available data. METHODOLOGY: Original articles in any language published before July 2009 were selected from available databases (MEDLINE, Cochrane Library and BioMedCentral) using the combined search terms of magnetic resonance, perfusion, and coronary angiography; with the exploded term coronary artery disease. Statistical analysis was only performed on studies that: (1) used a [greater than or equal to] 1.5 Tesla MR scanner; (2) employed invasive coronary angiography as the reference standard for diagnosing significant obstructive CAD, defined as a [greater than or equal to] 50% diameter stenosis; and (3) provided sufficient data to permit analysis. RESULTS: From the 263 citations identified, 55 relevant original articles were selected. Only 35 fulfilled all of the inclusion criteria, and of these 26 presented data on patient-based analysis. The overall patient-based analysis demonstrated a sensitivity of 89% (95% CI: 88-91%), and a specificity of 80% (95% CI: 78-83%). Adenosine stress perfusion CMR had better sensitivity than with dipyridamole (90% (88-92%) versus 86% (80-90%), P = 0.022), and a tendency to a better specificity (81% (78-84%) versus 77% (71-82%), P = 0.065). CONCLUSION: Stress perfusion CMR is highly sensitive for detection of CAD but its specificity remains moderate.

A Versatile Workflow for the Identification of Protein-Protein Interactions Using GFP-Trap Beads and Mass Spectrometry-Based Label-Free Quantification.

Protein functions often rely on protein-protein interactions. Hence, knowledge about the protein interaction network is essential for an understanding of protein functions and plant physiology. A major challenge of the postgenomic era is the mapping of protein-protein interaction networks. This chapter describes a mass spectrometry-based label-free quantification approach to identify in vivo protein interaction networks. The procedure starts with the extraction of intact protein complexes from transgenic plants expressing the protein of interest fused to a GFP-Tag (bait-GFP), as well as plants expressing a free GFP as background control. Enrichment of the GFP-tagged protein together with its interaction partners, as well as the free GFP, is performed by immunoaffinity purification. The pull-down quality can be evaluated by simple gel-based techniques. In parallel, the captured proteins are trypsin-digested and relatively quantified by label-free mass spectrometry-based quantification. The relative quantification approach largely relies on the normalization of protein abundances of background-binding proteins, which occur in both bait-GFP and free GFP pull-downs. Therefore, relative quantification of the protein pull-down is superior over methods that solely rely on protein identifications and removal of often copurified high-abundance proteins from the bait-GFP pull-downs, which might remove real interaction partners. A further strength of this method is that it can be applied to any soluble GFP-tagged protein.

Functional Divergence of the Arabidopsis Florigen-Interacting bZIP Transcription Factors FD and FDP.

Flowering of many plant species depends on interactions between basic leucine zipper (bZIP) transcription factors and systemically transported florigen proteins. Members of the genus Arabidopsis contain two of these bZIPs, FD and FDP, which we show have largely complementary expression patterns in shoot apices before and during flowering. CRISPR-Cas9-induced null mutants for FDP flower slightly earlier than wild-type, whereas fd mutants are late flowering. Identical G-box sequences are enriched at FD and FDP binding sites, but only FD binds to genes involved in flowering and only fd alters their transcription. However, both proteins bind to genes involved in responses to the phytohormone abscisic acid (ABA), which controls developmental and stress responses. Many of these genes are differentially expressed in both fd and fdp mutant seedlings, which also show reduced ABA sensitivity. Thus, florigen-interacting bZIPs have distinct functions in flowering dependent on their expression patterns and, at earlier stages in development, play common roles in phytohormone signaling.

Beyond Histones: New Substrate Proteins of Lysine Deacetylases in Arabidopsis Nuclei.

The reversible acetylation of lysine residues is catalyzed by the antagonistic action of lysine acetyltransferases and deacetylases, which can be considered as master regulators of their substrate proteins. Lysine deacetylases, historically referred to as histone deacetylases, have profound functions in regulating stress defenses and development in plants. Lysine acetylation of the N-terminal histone tails promotes gene transcription and decondensation of chromatin, rendering the DNA more accessible to the transcription machinery. In plants, the classical lysine deacetylases from the RPD3/HDA1-family have thus far mainly been studied in the context of their deacetylating activities on histones, and their versatility in molecular activities is still largely unexplored. Here we discuss the potential impact of lysine acetylation on the recently identified nuclear substrate proteins of lysine deacetylases from the Arabidopsis RPD3/HDA1-family. Among the deacetylase substrate proteins, many interesting candidates involved in nuclear protein import, transcriptional regulation, and chromatin remodeling have been identified. These candidate proteins represent key starting points for unraveling new molecular functions of the Arabidopsis lysine deacetylases. Site-directed engineering of lysine acetylation sites on these target proteins might even represent a new approach for optimizing plant growth under climate change conditions.

The release of dormancy, a wake-up call for seeds to germinate.

Seed dormancy determines the timing of germination, thereby contributing to successful seedling establishment and plant fitness. The induction and release of dormancy are controlled by various regulators like plant hormones and dormancy proteins. The relative strengths of these regulators are influenced by environmental factors during seed maturation and storage. In the last few years additional processes have been identified to be involved in the release of dormancy during seed storage with an important role for non-enzymatic oxidative reactions. However, the relations between the different dormancy regulators are not fully understood yet. Finally, all accumulated information will be processed in the seed during early seed imbibition and lead to the decision to germinate or not.

DELAY OF GERMINATION1 requires PP2C phosphatases of the ABA signalling pathway to control seed dormancy.

The time of seed germination is a major decision point in the life of plants determining future growth and development. This timing is controlled by seed dormancy, which prevents germination under favourable conditions. The plant hormone abscisic acid (ABA) and the protein DELAY OF GERMINATION 1 (DOG1) are essential regulators of dormancy. The function of ABA in dormancy is rather well understood, but the role of DOG1 is still unknown. Here, we describe four phosphatases that interact with DOG1 in seeds. Two of them belong to clade A of type 2C protein phosphatases: ABA-HYPERSENSITIVE GERMINATION 1 (AHG1) and AHG3. These phosphatases have redundant but essential roles in the release of seed dormancy epistatic to DOG1. We propose that the ABA and DOG1 dormancy pathways converge at clade A of type 2C protein phosphatases.The DOG1 protein is a major regulator of seed dormancy in Arabidopsis. Here, Née et al. provide evidence that DOG1 can interact with the type 2C protein phosphatases AHG1 and AHG3 and that this represents the convergence point of the DOG1-regulated dormancy pathway and signalling by the plant hormone abscisic acid.

Redox regulation of chloroplastic glucose-6-phosphate dehydrogenase: a new role for f-type thioredoxin.

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme of the oxidative pentose phosphate pathway supplying reducing power (as NADPH) in non-photosynthesizing cells. We have examined in detail the redox regulation of the plastidial isoform predominantly present in Arabidopsis green tissues (AtG6PDH1) and found that its oxidative activation is strictly dependent on plastidial thioredoxins (Trxs) that show differential efficiencies. Light/dark modulation of AtG6PDH1 was reproduced in vitro in a reconstituted ferredoxin/Trx system using f-type Trx allowing to propose a new function for this Trx isoform co-ordinating both reductive (Calvin cycle) and oxidative pentose phosphate pathways.