Biology subjects

Vierling, E.

Publications and source records attributed to Vierling, E..

6 recordsLinked to original sources

bioRxiv2024

Disrupted Nitric Oxide Homeostasis Impacts Fertility Through Multiple Processes Including Protein Quality Control

Plant fertility is fundamental to plant survival and requires the coordinated interaction of developmental pathways and signaling molecules. Nitric oxide (NO) is a small gaseous signaling molecule that plays crucial roles in plant fertility as well as other developmental processes and stress responses. NO influences biological processes through S-nitrosation, the posttranslational modification of protein cysteines to S-nitrosocysteine (R-SNO). NO homeostasis is controlled by S-nitrosoglutathione reductase (GSNOR), which reduces S-nitrosoglutathione (GSNO), the major form of NO in cells. GSNOR mutants (hot5-2/gsnor1) have defects in female gametophyte development along with elevated levels of reactive nitrogen species and R-SNOs. To better understand the fertility defects in hot5-2 we investigated the in vivo nitrosoproteome of floral tissues coupled with quantitative proteomics of pistils. To identify protein-SNOs we employed, for the first time in plants, an organomercury-based method that involves direct reaction with S-nitrosocysteine, enabling specific identification of S-nitrosocysteine-containing peptides and S-nitrosated proteins. We identified 1102 endogenously S-nitrosated proteins in floral tissues, of which 1049 were unique to hot5-2. Among the identified proteins, 728 were novel S-nitrosation targets. Notably, specific UGT-glycosyltransferases and argonaute proteins are S-nitrosated in floral tissues and differentially regulated in pistils. We also discovered S-nitrosation of proteins of the 26S proteasome together with increased abundance of proteasomal components and enhanced trypsin-like proteasomal activity in hot5-2 pistils. Our data establish a new method for nitrosoprotein detection in plants, expand knowledge of the plant S-nitrosoproteome, and suggest that nitro-oxidative modification and NO homeostasis are critical to protein quality control in reproductive tissues.

plant biology↗

bioRxiv2024

Mitochondria affect photosynthesis through altered tissue levels of oxygen

Interactions between plant energy organelles, the chloroplasts and the mitochondria, are crucial for plant development and acclimation. These interactions occur at different levels including exchange of metabolites and reducing power, organelle signaling pathways and intracellular gas exchange. Mitochondrial retrograde stress signaling activates expression of nuclear genes encoding mitochondrial components, including alternative oxidases. High abundances of these respiratory enzymes coincide not only with the changes in plant respiration but also with alterations in the chloroplast. For example, plants that overexpress alternative oxidases are tolerant to methyl viologen, a redox-active compound that catalyzes transfer of electrons from Photosystem I to molecular oxygen. The mechanism of this inter-organelle interaction is unclear but could be related to diminished availability of tissue oxygen. Here we assessed respiration, photosynthesis and in vivo levels of oxygen in a set of Arabidopsis lines with perturbations in diverse mitochondrial functions, including defects in respiratory complex I, mitochondrial protein processing, transcription, nucleoid organization, altered fission and architecture or suppressed ATP synthase activity. In these lines, the increased abundance and activity of alternative oxidases strongly correlated with higher oxygen consumption in darkness, lower oxygen re-accumulation in light, and diminished effects of methyl viologen in chloroplasts. These results support the hypothesis that increased mitochondrial oxygen sink capacity affects photosynthesis by decreasing oxygen levels in tissues. This phenomenon can be one of the reasons for the impact that stressed mitochondria have on chloroplasts and photosynthesis. It contributes to our understanding of the mechanisms of hypoxia establishment and acclimation in plants.

plant biology↗

bioRxiv2021

A temperature sensitive mutation in the CstF77 subunit of the polyadenylation complex reveals the critical function of mRNA 3' end formation for a robust heat stress response in plants

BackgroundHeat Shock Protein 101 (HSP101) in plants and orthologs in bacteria (Caseinolytic peptidase B, ClpB) and yeast (Hsp104) are essential for thermotolerance. To investigate molecular mechanisms of thermotolerance involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a semi-dominant, missense HSP101 allele, hot1-4 (A499T). Plants carrying the hot1-4 mutation are more heat-sensitive than an HSP101 null mutant (hot1-3), indicating the toxicity of hot1-4 allele. ResultsWe report that one suppressor (shot2, suppressor of hot1-4 2) has a temperature-sensitive, missense mutation (E170K) in the CstF77 (Cleavage stimulation factor 77) subunit of the polyadenylation complex, which is critical for 3 end maturation of pre-mRNA. RNA-Seq analysis of total RNA depleted of ribosomes reveals that heat treatment causes transcriptional readthrough events in shot2, specifically in highly heat-induced genes, including the toxic hot1-4 gene. In addition, failure of correct transcript processing leads to reduced accumulation of many HSP RNAs and proteins, suppressing heat sensitivity of the hot1-4 mutant, due to reduction of the toxic mutant HSP101 protein. Notably, the shot2 mutation makes plants more sensitive to heat stress in the HSP101 null (hot1-3) and wild-type backgrounds correlated with the reduced expression of other heat-inducible genes required for thermotolerance. ConclusionsOur study reveals the critical function of CstF77 for 3 end formation of mRNA during heat stress, as well as the dominant role of HSP101 in dictating the outcome of severe heat stress in plants.

plant biology↗

bioRxiv2021

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 RNAi 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 of the electron transport chain). 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 provide new information about complex V assembly and quality control, as well as support the importance of mitochondrial respiration in normal plant growth and heat stress tolerance. SIGNIFICANCE STATEMENTThe energy converter, mitochondrial ATP synthase, is critical for all organisms, but the functional importance of ATP synthase subunit d remains largely unknown in plants. We demonstrate the contributions of subunit d to plant growth, development, and heat stress tolerance, as well as to ATP synthase stability, ROS signaling and mitochondrial dysfunction regulation.

plant biology↗

bioRxiv2020

Mitochondrial nucleoid organization and biogenesis of complex I require mTERF18/SHOT1 and ATAD3 in Arabidopsis thaliana.

Mitochondria play critical roles in eukaryotes in ATP generation through oxidative phosphorylation (OXPHOS) and also produce both damaging and signaling reactive oxygen species (ROS). Originating from endosymbiosis, mitochondria have their own reduced genomes that encode essential subunits of the OXPHOS machinery. MTERF (Mitochondrial Transcription tERmination Factor-related) proteins have been shown to be involved in organelle gene expression by interacting with organellar DNA or RNA in multicellular eukaryotes. We previously identified mutations in Arabidopsis thaliana MTERF18/SHOT1 that enable plants to better tolerate heat and oxidative stresses, presumably due to low ROS and reduced oxidative damage. To understand molecular mechanisms leading to shot1 phenotypes, we investigated mitochondrial defects of shot1 mutants and targets of the SHOT1 protein. shot1 mutants have problems accumulating OXPHOS complexes that contain mitochondria-encoded subunits, with complex I and complex IV most affected. SHOT1 binds specific mitochondrial DNA sequences and localizes to mitochondrial nucleoids, which are diffuse in shot1 mutants. Furthermore, three homologues of mammalian ATAD3A proteins, which are suggested to be involved in mitochondrial nucleoid organization, were identified as SHOT1-interacting proteins (designated SHOT1 BINDING ATPASES (SBA)1, 2 and 3). Importantly, disrupting SBA function also disrupts nucleoids, compromises accumulation of complex I and enhances heat tolerance. We conclude that proper nucleoid organization is critical for correct expression and accumulation of complex I, and propose that nucleoid disruption results in unique changes in mitochondrial metabolism and signaling that lead to heat tolerance. SignificanceIn all eukaryotes, mitochondria are critical organelles that supply chemical energy for life, which is produced by the oxidative phosphorylation (OXPHOS) machinery on the inner mitochondrial membrane. The OXPHOS machinery comprises multiple protein complexes with subunits encoded by both nuclear and mitochondrial genes. Nuclear-encoded mTERF proteins are important for expression of mitochondrial genes, interacting with mitochondrial DNA or RNA. Our study reveals that the Arabidopsis mTERF18/SHOT1 protein interacts with mtDNA and homologs of human ATAD3A proteins, and that both proteins are critical for mitochondrial nucleoid organization and accumulation of OXPHOS Complex I. Further, the data indicate nucleoid disruption leads to unique mitochondrial and cellular responses such that mutant plants have enhanced heat tolerance.

plant biology↗

bioRxiv2018

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 their 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 that substructural dimers of 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 non-dimeric, to restrict how their assemblies can dissociate. When disulfide bonded at the non-dimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive, but when reduced had wild-type-like chaperone activity, demonstrating that dissociation at non-dimeric interfaces is essential for activity. In addition, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different than the Ta16.9 x-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) showed greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Size and secondary structure changes with temperature revealed that folded sHsp dimers interact with substrate, and support dimer stability as a determinant of chaperone efficiency. These data yield a model in which sHsp dimers capture substrate prior to assembly into larger, heterogeneous sHSP-substrate complexes for subsequent substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

molecular biology↗