Maternal nitric oxide homeostasis impacts female gametophyte development under optimal and stress conditions.

In adverse environments, the number of fertilizable female gametophytes (FGs) in plants is reduced, leading to increased survival of the remaining offspring. How the maternal plant perceives internal growth cues and external stress conditions to alter FG development remains largely unknown. We report that homeostasis of the stress signaling molecule nitric oxide (NO) plays a key role in controlling FG development under both optimal and stress conditions. NO homeostasis is precisely regulated by S-nitrosoglutathione reductase (GSNOR). Prior to fertilization, GSNOR protein is exclusively accumulated in sporophytic tissues and indirectly controls FG development in Arabidopsis (Arabidopsis thaliana). In GSNOR null mutants, NO species accumulated in the degenerating sporophytic nucellus, and auxin efflux into the developing FG was restricted, which inhibited FG development, resulting in reduced fertility. Importantly, restoring GSNOR expression in maternal, but not gametophytic tissues, or increasing auxin efflux substrate significantly increased the proportion of normal FGs and fertility. Furthermore, GSNOR overexpression or added auxin efflux substrate increased fertility under drought and salt stress. These data indicate that NO homeostasis is critical to normal auxin transport and maternal control of FG development, which in turn determine seed yield. Understanding this aspect of fertility control could contribute to mediating yield loss under adverse conditions.

Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity.

Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.

Heat tolerance of a tropical-subtropical rainforest tree species Polyscias elegans: time-dependent dynamic responses of physiological thermostability and biochemistry.

Heat stress interrupts physiological thermostability and triggers biochemical responses that are essential for plant survival. However, there is limited knowledge on the speed plants adjust to heat in hours and days, and which adjustments are crucial. Tropical-subtropical rainforest tree species (Polyscias elegans) were heated at 40°C for 5 d, before returning to 25°C for 13 d of recovery. Leaf heat tolerance was quantified using the temperature at which minimal chl a fluorescence sharply rose (Tcrit ). Tcrit , metabolites, heat shock protein (HSP) abundance and membrane lipid fatty acid (FA) composition were quantified. Tcrit increased by 4°C (48-52°C) within 2 h of 40°C exposure, along with rapid accumulation of metabolites and HSPs. By contrast, it took > 2 d for FA composition to change. At least 2 d were required for Tcrit , HSP90, HSP70 and FAs to return to prestress levels. The results highlight the multi-faceted response of P. elegans to heat stress, and how this response varies over the scale of hours to days, culminating in an increased level of photosynthetic heat tolerance. These responses are important for survival of plants when confronted with heat waves amidst ongoing global climate change.

ATAD3 Proteins: Unique Mitochondrial Proteins Essential for Life in Diverse Eukaryotic Lineages.

ATAD3 proteins (ATPase family AAA domain-containing protein 3) are unique mitochondrial proteins that arose deep in the eukaryotic lineage but that are surprisingly absent from the Fungi and Amoebozoa. These ~600 amino acid proteins are anchored in the inner mitochondrial membrane and are essential in metazoans and Arabidopsis thaliana. ATAD3s comprise a C-terminal AAA+ matrix domain and an ATAD3_N domain that is located primarily in the inner membrane space but potentially extends into cytosol to interact with the ER. Sequence and structural alignments indicate ATAD3 proteins are most similar to classic chaperone unfoldases in AAA+ family, suggesting that they operate in mitochondrial protein quality control. A. thaliana has four ATAD3 genes in two distinct clades that appear first in the seed plants, and both clades are essential for viability. The four genes are generally coordinately expressed, and transcripts are highest in growing apices and imbibed seeds. Plants with disrupted ATAD3 have reduced growth, aberrant mitochondrial morphology, diffuse nucleoids and reduced oxidative phosphorylation complex I. These and other pleiotropic phenotypes are also observed in ATAD3 mutants in metazoans. Here we discuss the distribution of ATAD3 proteins as they have evolved in the plant kingdom, their unique structure, what we know about their function in plants, and the challenges in determining their essential roles in mitochondria.

Mutation of the polyadenylation complex subunit CstF77 reveals that mRNA 3' end formation and HSP101 levels are critical for a robust heat stress response.

Heat shock protein 101 (HSP101) in plants, and bacterial and yeast orthologs, is essential for thermotolerance. To investigate thermotolerance mechanisms involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a missense HSP101 allele (hot1-4). hot1-4 plants are sensitive to acclimation heat treatments that are otherwise permissive for HSP101 null mutants, indicating that the hot1-4 protein is toxic. We report one suppressor (shot2, suppressor of hot1-4 2) has a missense mutation of a conserved residue in CLEAVAGE STIMULATION FACTOR77 (CstF77), a subunit of the polyadenylation complex critical for mRNA 3' end maturation. We performed ribosomal RNA depletion RNA-Seq and captured transcriptional readthrough with a custom bioinformatics pipeline. Acclimation heat treatment caused transcriptional readthrough in hot1-4 shot2, with more readthrough in heat-induced genes, reducing the levels of toxic hot1-4 protein and suppressing hot1-4 heat sensitivity. Although shot2 mutants develop like the wild type in the absence of stress and survive mild heat stress, reduction of heat-induced genes and decreased HSP accumulation makes shot2 in HSP101 null and wild-type backgrounds sensitive to severe heat stress. Our study reveals the critical function of CstF77 for 3' end formation of mRNA and the dominant role of HSP101 in dictating the outcome of severe heat stress.

Focus on Nitric Oxide Homeostasis: Direct and Indirect Enzymatic Regulation of Protein Denitrosation Reactions in Plants.

Protein cysteines (Cys) undergo a multitude of different reactive oxygen species (ROS), reactive sulfur species (RSS), and/or reactive nitrogen species (RNS)-derived modifications. S-nitrosation (also referred to as nitrosylation), the addition of a nitric oxide (NO) group to reactive Cys thiols, can alter protein stability and activity and can result in changes of protein subcellular localization. Although it is clear that this nitrosative posttranslational modification (PTM) regulates multiple signal transduction pathways in plants, the enzymatic systems that catalyze the reverse S-denitrosation reaction are poorly understood. This review provides an overview of the biochemistry and regulation of nitro-oxidative modifications of protein Cys residues with a focus on NO production and S-nitrosation. In addition, the importance and recent advances in defining enzymatic systems proposed to be involved in regulating S-denitrosation are addressed, specifically cytosolic thioredoxins (TRX) and the newly identified aldo-keto reductases (AKR).

Quantitative Proteome Profiling of a S-Nitrosoglutathione Reductase (GSNOR) Null Mutant Reveals a New Class of Enzymes Involved in Nitric Oxide Homeostasis in Plants.

Nitric oxide (NO) is a short-lived radical gas that acts as a signaling molecule in all higher organisms, and that is involved in multiple plant processes, including germination, root growth, and fertility. Regulation of NO-levels is predominantly achieved by reaction of oxidation products of NO with glutathione to form S-nitrosoglutathione (GSNO), the principal bioactive form of NO. The enzyme S-nitrosoglutathione reductase (GSNOR) is a major route of NADH-dependent GSNO catabolism and is critical to NO homeostasis. Here, we performed a proteomic analysis examining changes in the total leaf proteome of an Arabidopsis thaliana GSNOR null mutant (hot5-2/gsnor1-3). Significant increases or decreases in proteins associated with chlorophyll metabolism and with redox and stress metabolism provide insight into phenotypes observed in hot5-2/gsnor1-3 plants. Importantly, we identified a significant increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8 and 9. Because specific AKRs have been linked to NO metabolism in mammals, we expressed and purified A. thaliana AKR4C8 and 9 and close homologs AKR4C10 and 11 and determined that they have NADPH-dependent activity in GSNO and S-nitroso-coenzyme A (SNO-CoA) reduction. Further, we found an increase of NADPH-dependent GSNO reduction activity in hot5-2/gsnor1-3 mutant plants. These data uncover a new, NADPH-dependent component of NO metabolism that may be integrated with NADH-dependent GSNOR activity to control NO homeostasis in plants.

mTERF18 and ATAD3 are required for mitochondrial nucleoid structure and their disruption confers heat tolerance in Arabidopsis thaliana.

Mitochondria play critical roles in generating ATP through oxidative phosphorylation (OXPHOS) and produce both damaging and signaling reactive oxygen species (ROS). They have reduced genomes that encode essential subunits of the OXPHOS machinery. Mitochondrial Transcription tERmination Factor-related (mTERF) proteins are involved in organelle gene expression, interacting with organellar DNA or RNA. We previously found that mutations in Arabidopsis thaliana mTERF18/SHOT1 enable plants to better tolerate heat and oxidative stresses, presumably due to low ROS production and reduced oxidative damage. Here we discover that shot1 mutants have greatly reduced OXPHOS complexes I and IV and reveal that suppressor of hot1-4 1 (SHOT1) binds DNA and localizes to mitochondrial nucleoids, which are disrupted in shot1. Furthermore, three homologues of animal ATPase family AAA domain-containing protein 3 (ATAD3), which is involved in mitochondrial nucleoid organization, were identified as SHOT1-interacting proteins. Importantly, disrupting ATAD3 function disrupts nucleoids, reduces accumulation of complex I, and enhances heat tolerance, as is seen in shot1 mutants. Our data link nucleoid organization to OXPHOS biogenesis and suggest that the common defects in shot1 mutants and ATAD3-disrupted plants lead to critical changes in mitochondrial metabolism and signaling that result in plant heat tolerance.

Mitochondrial ATP synthase subunit d, a component of the peripheral stalk, is essential for growth and heat stress tolerance in Arabidopsis thaliana.

As rapid changes in climate threaten global crop yields, an understanding of plant heat stress tolerance is increasingly relevant. Heat stress tolerance involves the coordinated action of many cellular processes and is particularly energy demanding. We acquired a knockout mutant and generated knockdown lines in Arabidopsis thaliana of the d subunit of mitochondrial ATP synthase (gene name: ATPQ, AT3G52300, referred to hereafter as ATPd), a subunit of the peripheral stalk, and used these to investigate the phenotypic significance of this subunit in normal growth and heat stress tolerance. Homozygous knockout mutants for ATPd could not be obtained due to gametophytic defects, while heterozygotes possess no visible phenotype. Therefore, we used RNA interference to create knockdown plant lines for further studies. Proteomic analysis and blue native gels revealed that ATPd downregulation impairs only subunits of the mitochondrial ATP synthase (complex V). Knockdown plants were more sensitive to heat stress, had abnormal leaf morphology, and were severely slow growing compared to wild type. These results indicate that ATPd plays a crucial role in proper function of the mitochondrial ATP synthase holoenzyme, which, when reduced, leads to wide-ranging defects in energy-demanding cellular processes. In knockdown plants, more hydrogen peroxide accumulated and mitochondrial dysfunction stimulon (MDS) genes were activated. These data establish the essential structural role of ATPd and support the importance of complex V in normal plant growth, and provide new information about its requirement for heat stress tolerance.

Auxin efflux controls orderly nucellar degeneration and expansion of the female gametophyte in Arabidopsis.

The nucellus tissue in flowering plants provides nutrition for the development of the female gametophyte (FG) and young embryo. The nucellus degenerates as the FG develops, but the mechanism controlling the coupled process of nucellar degeneration and FG expansion remains largely unknown. The degeneration process of the nucellus and spatiotemporal auxin distribution in the developing ovule before fertilization were investigated in Arabidopsis thaliana. Nucellar degeneration before fertilization occurs through vacuolar cell death and in an ordered degeneration fashion. This sequential nucellar degeneration is controlled by the signalling molecule auxin. Auxin efflux plays the core role in precisely controlling the spatiotemporal pattern of auxin distribution in the nucellus surrounding the FG. The auxin efflux carrier PIN1 transports maternal auxin into the nucellus while PIN3/PIN4/PIN7 further delivers auxin to degenerating nucellar cells and concurrently controls FG central vacuole expansion. Notably, auxin concentration and auxin efflux are controlled by the maternal tissues, acting as a key communication from maternal to filial tissue.

Plant small heat shock proteins - evolutionary and functional diversity.

Small heat shock proteins (sHSPs) are an ubiquitous protein family found in archaea, bacteria and eukaryotes. In plants, as in other organisms, sHSPs are upregulated by stress and are proposed to act as molecular chaperones to protect other proteins from stress-induced damage. sHSPs share an 'α-crystallin domain' with a ß-sandwich structure and a diverse N-terminal domain. Although sHSPs are 12-25 kDa polypeptides, most assemble into oligomers with ≥ 12 subunits. Plant sHSPs are particularly diverse and numerous; some species have as many as 40 sHSPs. In angiosperms this diversity comprises ≥ 11 sHSP classes encoding proteins targeted to the cytosol, nucleus, endoplasmic reticulum, chloroplasts, mitochondria and peroxisomes. The sHSPs underwent a lineage-specific gene expansion, diversifying early in land plant evolution, potentially in response to stress in the terrestrial environment, and expanded again in seed plants and again in angiosperms. Understanding the structure and evolution of plant sHSPs has progressed, and a model for their chaperone activity has been proposed. However, how the chaperone model applies to diverse sHSPs and what processes sHSPs protect are far from understood. As more plant genomes and transcriptomes become available, it will be possible to explore theories of the evolutionary pressures driving sHSP diversification.

HSP101 Interacts with the Proteasome and Promotes the Clearance of Ubiquitylated Protein Aggregates.

Stressful environments often lead to protein unfolding and the formation of cytotoxic aggregates that can compromise cell survival. The molecular chaperone heat shock protein (HSP) 101 is a protein disaggregase that co-operates with the small HSP (sHSP) and HSP70 chaperones to facilitate removal of such aggregates and is essential for surviving severe heat stress. To better define how HSP101 protects plants, we investigated the localization and targets of this chaperone in Arabidopsis (Arabidopsis thaliana). By following HSP101 tagged with GFP, we discovered that its intracellular distribution is highly dynamic and includes a robust, reversible sequestration into cytoplasmic foci that vary in number and size among cell types and are potentially enriched in aggregated proteins. Affinity isolation of HSP101 recovered multiple proteasome subunits, suggesting a functional interaction. Consistent with this, the GFP-tagged 26S proteasome regulatory particle non-ATPase (RPN) 1a transiently colocalized with HSP101 in cytoplasmic foci during recovery. In addition, analysis of aggregated (insoluble) proteins showed they are extensively ubiquitylated during heat stress, especially in plants deficient in HSP101 or class I sHSPs, implying that protein disaggregation is important for optimal proteasomal degradation. Many potential HSP101 clients, identified by mass spectrometry of insoluble proteins, overlapped with known stress granule constituents and sHSP-interacting proteins, confirming a role for HSP101 in stress granule function. Connections between HSP101, stress granules, proteasomes, and ubiquitylation imply that dynamic coordination between protein disaggregation and proteolysis is required to survive proteotoxic stress caused by protein aggregation at high temperatures.

Small heat shock proteins: multifaceted proteins with important implications for life.

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world's experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26-29 August 2018.

Metabolic adaptation of wheat grain contributes to a stable filling rate under heat stress.

Wheat (Triticum aestivum) is particularly vulnerable to heat stress during the grain filling stage, and this can adversely affect the final yield. However, the underlying physiological and molecular mechanisms are largely unknown. In this study, the effects of heat stress on grain filling were investigated using wheat varieties with different levels of thermotolerance. Decreased grain weights and filling durations, increased protein contents, and stable filling rates across diverse varieties under different heat regimes suggested a general mechanism for heat adaptation. Proteomic analysis identified 309 heat-responsive proteins (HRPs), and revealed a general decrease in protein synthesis components and metabolic proteins, but a significant increase in stress-response proteins and storage proteins. Metabolomic analysis identified 98 metabolites specifically changed by heat stress, and suggested a global decrease in the content of carbohydrate metabolites, an increased content of amino acids, and stable levels of starch synthesis precursors. The energy-consuming HRPs suggested that less energy was channelled into metabolism and protein synthesis, whereas more energy was allocated to the stress response under elevated heat conditions. Collectively, the data demonstrated a widely distributed mechanism for heat adaptation of metabolism, in which the assimilation and energy required for metabolism and protein synthesis are reallocated to heat protection and deposition of reserves, resulting in increased storage protein accumulation and a stable filling rate.

It takes a dimer to tango: Oligomeric small heat shock proteins dissociate to capture substrate.

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that substructural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide-bonded at the nondimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive but, when reduced, had WT-like chaperone activity, demonstrating that dissociation at nondimeric interfaces is essential for sHsp activity. Moreover, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHsp-substrate complexes for substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

Correction to: Structural and functional aspects of the interaction partners of the small heat-shock protein in Synechocystis.

Table 1 in the original publication has been corrected.

Direct Measurement of S-Nitrosothiols with an Orbitrap Fusion Mass Spectrometer: S-Nitrosoglutathione Reductase as a Model Protein.

Recent studies suggest cysteine S-nitrosation of S-nitrosoglutathione reductase (GSNOR) could regulate protein redox homeostasis. "Switch" assays enable discovery of putatively S-nitrosated proteins. However, with few exceptions, researchers have not examined the kinetics and biophysical consequences of S-nitrosation. Methods to quantify protein S-nitrosothiol (SNO) abundance and formation kinetics would bridge this mechanistic gap and allow interpretation of the consequences of specific modifications, as well as facilitate development of specific S-nitrosation inhibitors. Here, we describe a rapid assay to estimate protein SNO abundance with intact protein electrospray ionization mass spectrometry. Originally designed using recombinant GSNOR, these methods are applicable to any purified protein to test for or further study nitrosatable cysteines.

Structural and functional aspects of the interaction partners of the small heat-shock protein in Synechocystis.

The canonical function of small heat-shock proteins (sHSPs) is to interact with proteins destabilized under conditions of cellular stress. While the breadth of interactions made by many sHSPs is well-known, there is currently little knowledge about what structural features of the interactors form the basis for their recognition. Here, we have identified 83 in vivo interactors of the sole sHSP in the cyanobacterium Synechocystis sp. PCC 6803, HSP16.6, reflective of stable associations with soluble proteins made under heat-shock conditions. By performing bioinformatic analyses on these interactors, we identify primary and secondary structural elements that are enriched relative to expectations from the cyanobacterial genome. In addition, by examining the Synechocystis interactors and comparing them with those identified to bind sHSPs in other prokaryotes, we show that sHSPs associate with specific proteins and biological processes. Our data are therefore consistent with a picture of sHSPs being broadly specific molecular chaperones that act to protect multiple cellular pathways.

Structural principles that enable oligomeric small heat-shock protein paralogs to evolve distinct functions.

Oligomeric proteins assemble with exceptional selectivity, even in the presence of closely related proteins, to perform their cellular roles. We show that most proteins related by gene duplication of an oligomeric ancestor have evolved to avoid hetero-oligomerization and that this correlates with their acquisition of distinct functions. We report how coassembly is avoided by two oligomeric small heat-shock protein paralogs. A hierarchy of assembly, involving intermediates that are populated only fleetingly at equilibrium, ensures selective oligomerization. Conformational flexibility at noninterfacial regions in the monomers prevents coassembly, allowing interfaces to remain largely conserved. Homomeric oligomers must overcome the entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer subunits than homomers that have no paralogs.