An alanine to valine mutation of glutamyl-tRNA reductase enhances 5-aminolevulinic acid synthesis in rice.

KEY MESSAGE: An alanine to valine mutation of glutamyl-tRNA reductase's 510th amino acid improves 5-aminolevulinic acid synthesis in rice. 5-aminolevulinic acid (ALA) is the common precursor of all tetrapyrroles and plays an important role in plant growth regulation. ALA is synthesized from glutamate, catalyzed by glutamyl-tRNA synthetase (GluRS), glutamyl-tRNA reductase (GluTR), and glutamate-1-semialdehyde aminotransferase (GSAT). In Arabidopsis, ALA synthesis is the rate-limiting step in tetrapyrrole production via GluTR post-translational regulations. In rice, mutations of GluTR and GSAT homologs are known to confer chlorophyll deficiency phenotypes; however, the enzymatic activity of rice GluRS, GluTR, and GSAT and the post-translational regulation of rice GluTR have not been investigated experimentally. We have demonstrated that a suppressor mutation in rice partially reverts the xantha trait. In the present study, we first determine that the suppressor mutation results from a G → A nucleotide substitution of OsGluTR (and an A → V change of its 510th amino acid). Protein homology modeling and molecular docking show that the OsGluTRA510V mutation increases its substrate binding. We then demonstrate that the OsGluTRA510V mutation increases ALA synthesis in Escherichia coli without affecting its interaction with OsFLU. We further explore homologous genes encoding GluTR across 193 plant species and find that the amino acid (A) is 100% conserved at the position, suggesting its critical role in GluTR. Thus, we demonstrate that the gain-of-function OsGluTRA510V mutation underlies suppression of the xantha trait, experimentally proves the enzymatic activity of rice GluRS, GluTR, and GSAT in ALA synthesis, and uncovers conservation of the alanine corresponding to the 510th amino acid of OsGluTR across plant species.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized ß-sheet structures that accumulate in plaques in brains of Parkinson's disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Isolation of Highly Purified, Intact, and Functional Mitochondria from Potato Tubers Using a Two-in-One Percoll Density Gradient.

The isolation of mitochondria from potato tubers (Solanum tuberosum L.) is described, but the methodology can easily be adapted to other storage tissues. After homogenization of the tissue, filtration and differential centrifugation, the key step is a Percoll density gradient centrifugation. The Percoll gradient contains two parts: a bottom part containing Percoll in 0.3 M sucrose, and a slightly less dense top part containing Percoll in 0.3 M mannitol. After centrifugation, a density gradient is formed that is almost linear in the central part, and this is where the band containing the purified intact mitochondria is formed. This method makes it possible to process large amounts of plant material (2-6 kg) and saves at least 1.5 h on the preparation time compared to methods where two consecutive purification methods are used. Nonetheless, it yields large amounts of mitochondria (50-125 mg protein) of very high purity, intactness and functionality.

Integrity Assessment of Isolated Plant Mitochondria.

The integrity of isolated mitochondria can be estimated functionally using enzymatic activities or the permeability of mitochondrial membranes to molecules of different sizes. Thus, the permeability of the outer membrane to the protein cytochrome c, the permeability of the inner membrane to protons, and the permeability of the inner membrane to NAD+, NADH and organic acids using soluble matrix dehydrogenases as markers have all been used. These assays all have limitations to how the data can be converted into a measure of integrity, are differently sensitive to artifacts and require widely variable amounts of material. Therefore, each method has a restricted utility for estimating integrity, depending on the type of mitochondria analysed. Here, we review the advantages and disadvantages of different integrity assays and present protocols for integrity assays that require relatively small amounts of mitochondria. They are based on the permeability of the outer membrane to cytochrome c, and the inner membrane to protons or NAD(H). The latter has the advantage of utilizing a membrane-bound activity (complex I) and the pore-forming peptide alamethicin to gain access to the matrix space. These methods together provide a toolbox for the determination of functionality and quality of isolated mitochondria.

Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin.

We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.

Measuring the Activity of DNA Repair Enzymes in Isolated Mitochondria.

Nuclear, mitochondrial and plastidic DNA is constantly exposed to conditions, such as ultraviolet radiation or reactive oxygen species, which will induce chemical modifications to the nucleotides. Unless repaired, these modifications can lead to mutations, so the nucleus, mitochondria and plastids each contains a number of DNA repair systems. We here describe assays for measuring the enzyme activities associated with the base-excision repair pathway in potato tuber mitochondria. As the name implies, this pathway involves removing a modified base and replacing it with an undamaged base. Activity of each of the enzymes involved, DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase and DNA ligase can be measured by incubating a mitochondrial extract with a specifically designed oligonucleotide. After incubation, the reaction mixture is separated on a polyacrylamide gel, and the amounts of specific products formed is estimated by autoradiography, which makes it possible to calculate the enzymatic activity.

Nitric oxide regulation of plant metabolism.

Nitric oxide (NO) has emerged as an important signal molecule in plants, having myriad roles in plant development. In addition, NO also orchestrates both biotic and abiotic stress responses, during which intensive cellular metabolic reprogramming occurs. Integral to these responses is the location of NO biosynthetic and scavenging pathways in diverse cellular compartments, enabling plants to effectively organize signal transduction pathways. NO regulates plant metabolism and, in turn, metabolic pathways reciprocally regulate NO accumulation and function. Thus, these diverse cellular processes are inextricably linked. This review addresses the numerous redox pathways, located in the various subcellular compartments that produce NO, in addition to the mechanisms underpinning NO scavenging. We focus on how this molecular dance is integrated into the metabolic state of the cell. Within this context, a reciprocal relationship between NO accumulation and metabolite production is often apparent. We also showcase cellular pathways, including those associated with nitrate reduction, that provide evidence for this integration of NO function and metabolism. Finally, we discuss the potential importance of the biochemical reactions governing NO levels in determining plant responses to a changing environment.

MULocDeep: A deep-learning framework for protein subcellular and suborganellar localization prediction with residue-level interpretation.

Prediction of protein localization plays an important role in understanding protein function and mechanisms. In this paper, we propose a general deep learning-based localization prediction framework, MULocDeep, which can predict multiple localizations of a protein at both subcellular and suborganellar levels. We collected a dataset with 44 suborganellar localization annotations in 10 major subcellular compartments-the most comprehensive suborganelle localization dataset to date. We also experimentally generated an independent dataset of mitochondrial proteins in Arabidopsis thaliana cell cultures, Solanum tuberosum tubers, and Vicia faba roots and made this dataset publicly available. Evaluations using the above datasets show that overall, MULocDeep outperforms other major methods at both subcellular and suborganellar levels. Furthermore, MULocDeep assesses each amino acid's contribution to localization, which provides insights into the mechanism of protein sorting and localization motifs. A web server can be accessed at http://mu-loc.org.

Plant mitochondria - past, present and future.

The study of plant mitochondria started in earnest around 1950 with the first isolations of mitochondria from animal and plant tissues. The first 35 years were spent establishing the basic properties of plant mitochondria and plant respiration using biochemical and physiological approaches. A number of unique properties (compared to mammalian mitochondria) were observed: (i) the ability to oxidize malate, glycine and cytosolic NAD(P)H at high rates; (ii) the partial insensitivity to rotenone, which turned out to be due to the presence of a second NADH dehydrogenase on the inner surface of the inner mitochondrial membrane in addition to the classical Complex I NADH dehydrogenase; and (iii) the partial insensitivity to cyanide, which turned out to be due to an alternative oxidase, which is also located on the inner surface of the inner mitochondrial membrane, in addition to the classical Complex IV, cytochrome oxidase. With the appearance of molecular biology methods around 1985, followed by genomics, further unique properties were discovered: (iv) plant mitochondrial DNA (mtDNA) is 10-600 times larger than the mammalian mtDNA, yet it only contains approximately 50% more genes; (v) plant mtDNA has kept the standard genetic code, and it has a low divergence rate with respect to point mutations, but a high recombinatorial activity; (vi) mitochondrial mRNA maturation includes a uniquely complex set of activities for processing, splicing and editing (at hundreds of sites); (vii) recombination in mtDNA creates novel reading frames that can produce male sterility; and (viii) plant mitochondria have a large proteome with 2000-3000 different proteins containing many unique proteins such as 200-300 pentatricopeptide repeat proteins. We describe the present and fairly detailed picture of the structure and function of plant mitochondria and how the unique properties make their metabolism more flexible allowing them to be involved in many diverse processes in the plant cell, such as photosynthesis, photorespiration, CAM and C4 metabolism, heat production, temperature control, stress resistance mechanisms, programmed cell death and genomic evolution. However, it is still a challenge to understand how the regulation of metabolism and mtDNA expression works at the cellular level and how retrograde signaling from the mitochondria coordinates all those processes.

Mutations of the Genomes Uncoupled 4 Gene Cause ROS Accumulation and Repress Expression of Peroxidase Genes in Rice.

The Genomes Uncoupled 4 (GUN4) is one of the retrograde signaling genes in Arabidopsis and its orthologs have been identified in oxygenic phototrophic organisms from cyanobacterium to higher plants. GUN4 is involved in tetrapyrrole biosynthesis and its mutation often causes chlorophyll-deficient phenotypes with increased levels of reactive oxygen species (ROS), hence it has been speculated that GUN4 may also play a role in photoprotection. However, the biological mechanism leading to the increased ROS accumulation in gun4 mutants remains largely unknown. In our previous studies, we generated an epi-mutant allele of OsGUN4 (gun4 epi ), which downregulated its expression to ∼0.5% that of its wild-type (WT), and a complete knockout allele gun4-1 due to abolishment of its translation start site. In the present study, three types of F2 plant derived from a gun4-1/gun4 epi cross, i.e., gun4-1/gun4-1, gun4-1/gun4 epi and gun4 epi /gun4 epi were developed and used for further investigation by growing them under photoperiodic condition (16 h/8 h light/dark) with low light (LL, 100 µmol photons m-2 s-1) or high light (HL, 1000 µmol photons m-2 s-1). The expression of OsGUN4 was light responsive and had two peaks in the daytime. gun4-1/gun4-1-F2 seeds showed defective germination and died within 7 days. Significantly higher levels of ROS accumulated in all types of OsGUN4 mutants than in WT plants under both the LL and HL conditions. A comparative RNA-seq analysis of WT variety LTB and its gun4 epi mutant HYB led to the identification of eight peroxidase (PRX)-encoding genes that were significantly downregulated in HYB. The transcription of these eight PRX genes was restored in transgenic HYB protoplasts overexpressing OsGUN4, while their expression was repressed in LTB protoplasts transformed with an OsGUN4 silencing vector. We conclude that OsGUN4 is indispensable for rice, its expression is light- and oxidative-stress responsive, and it plays a role in ROS accumulation via its involvement in the transcriptional regulation of PRX genes.

Insights into triterpene synthesis and unsaturated fatty-acid accumulation provided by chromosomal-level genome analysis of Akebia trifoliata subsp. australis.

Akebia trifoliata subsp. australis is a well-known medicinal and potential woody oil plant in China. The limited genetic information available for A. trifoliata subsp. australis has hindered its exploitation. Here, a high-quality chromosome-level genome sequence of A. trifoliata subsp. australis is reported. The de novo genome assembly of 682.14 Mb was generated with a scaffold N50 of 43.11 Mb. The genome includes 25,598 protein-coding genes, and 71.18% (485.55 Mb) of the assembled sequences were identified as repetitive sequences. An ongoing massive burst of long terminal repeat (LTR) insertions, which occurred ~1.0 million years ago, has contributed a large proportion of LTRs in the genome of A. trifoliata subsp. australis. Phylogenetic analysis shows that A. trifoliata subsp. australis is closely related to Aquilegia coerulea and forms a clade with Papaver somniferum and Nelumbo nucifera, which supports the well-established hypothesis of a close relationship between basal eudicot species. The expansion of UDP-glucoronosyl and UDP-glucosyl transferase gene families and ß-amyrin synthase-like genes and the exclusive contraction of terpene synthase gene families may be responsible for the abundant oleanane-type triterpenoids in A. trifoliata subsp. australis. Furthermore, the acyl-ACP desaturase gene family, including 12 stearoyl-acyl-carrier protein desaturase (SAD) genes, has expanded exclusively. A combined transcriptome and fatty-acid analysis of seeds at five developmental stages revealed that homologs of SADs, acyl-lipid desaturase omega fatty acid desaturases (FADs), and oleosins were highly expressed, consistent with the rapid increase in the content of fatty acids, especially unsaturated fatty acids. The genomic sequences of A. trifoliata subsp. australis will be a valuable resource for comparative genomic analyses and molecular breeding.

Proteomic and Bioinformatic Profiling of Transporters in Higher Plant Mitochondria.

To function as a metabolic hub, plant mitochondria have to exchange a wide variety of metabolic intermediates as well as inorganic ions with the cytosol. As identified by proteomic profiling or as predicted by MU-LOC, a newly developed bioinformatics tool, Arabidopsis thaliana mitochondria contain 128 or 143 different transporters, respectively. The largest group is the mitochondrial carrier family, which consists of symporters and antiporters catalyzing secondary active transport of organic acids, amino acids, and nucleotides across the inner mitochondrial membrane. An impressive 97% (58 out of 60) of all the known mitochondrial carrier family members in Arabidopsis have been experimentally identified in isolated mitochondria. In addition to many other secondary transporters, Arabidopsis mitochondria contain the ATP synthase transporters, the mitochondria protein translocase complexes (responsible for protein uptake across the outer and inner membrane), ATP-binding cassette (ABC) transporters, and a number of transporters and channels responsible for allowing water and inorganic ions to move across the inner membrane driven by their transmembrane electrochemical gradient. A few mitochondrial transporters are tissue-specific, development-specific, or stress-response specific, but this is a relatively unexplored area in proteomics that merits much more attention.

Detoxification of toxin A and toxin B by copper ion-catalyzed oxidation in production of a toxoid-based vaccine against Clostridioides difficile.

Clostridioides difficile infections (CDI) has emerged worldwide as a serious antimicrobial-resistant healthcare-associated disease resulting in diarrhea and pseudomembranous colitis. The two cytotoxic proteins, toxin A (TcdA) and toxin B (TcdB) are the major virulence factor responsible for the disease symptoms. We examined time-dependent oxidative detoxification of TcdA and TcdB using different molar ratios of protein:Cu2+:H2O2. The metal-catalyzed oxidation (MCO) reaction in molar ratios of 1:60:1000 for protein:Cu2+:H2O2 at pH 4.5 resulted in a significant 6 log10 fold reduction in cytotoxicity after 120-min incubation at 37 °C. Circular dichroism revealed that MCO-detoxified TcdA and TcdB had secondary and tertiary structural folds similar to the native proteins. The conservation of immunogenic epitopes of both proteins was tested using monoclonal antibodies in an ELISA, comparing our MCO-detoxification approach to a conventional formaldehyde-detoxification method. The oxidative detoxification of TcdA and TcdB led to an average 2-fold reduction in antibody binding relative to native proteins, whereas formaldehyde cross-linking resulted in 3-fold and 5-fold reductions, respectively. Finally, we show that mice immunized with a vaccine consisting of MCO-detoxified TcdA and TcdB were fully protected against disease symptoms and death following a C. difficile infection and elicited substantial serum IgG responses against both TcdA and TcdB. The results of this study present copper ion-catalyzed oxidative detoxification of toxic proteins as a method highly suitable for the rapid production of safe, immunogenic and irreversible toxoid antigens for future vaccine development and may have the potential for replacing cross-linking reagents like formaldehyde.

Mitochondria in parasitic plants.

Plant mitochondrial genomes are renowned for their structural complexity, extreme variation in size and mutation rates, and ability to incorporate foreign DNA. Parasitic flowering plants are no exception, and the close association between parasite and host may even enhance the likelihood of horizontal gene transfer (HGT) between them. Recent studies on mistletoes (Viscum) have revealed that these parasites have lost an exceptional number of mitochondrial genes, including all complex I genes of the respiratory chain. At the same time, an altered respiratory pathway has been demonstrated. Here we review the current understanding of mitochondrial evolution in parasitic plants with a special emphasis on HGT to and from parasite mitochondrial genomes, as well as the uniquely altered mitochondria in Viscum and related plants.

The Hypoxic Proteome and Metabolome of Barley (Hordeum vulgare L.) with and without Phytoglobin Priming.

Overexpression of phytoglobins (formerly plant hemoglobins) increases the survival rate of plant tissues under hypoxia stress by the following two known mechanisms: (1) scavenging of nitric oxide (NO) in the phytoglobin/NO cycle and (2) mimicking ethylene priming to hypoxia when NO scavenging activates transcription factors that are regulated by levels of NO and O2 in the N-end rule pathway. To map the cellular and metabolic effects of hypoxia in barley (Hordeum vulgare L., cv. Golden Promise), with or without priming to hypoxia, we studied the proteome and metabolome of wild type (WT) and hemoglobin overexpressing (HO) plants in normoxia and after 24 h hypoxia (WT24, HO24). The WT plants were more susceptible to hypoxia than HO plants. The chlorophyll a + b content was lowered by 50% and biomass by 30% in WT24 compared to WT, while HO plants were unaffected. We observed an increase in ROS production during hypoxia treatment in WT seedlings that was not observed in HO seedlings. We identified and quantified 9694 proteins out of which 1107 changed significantly in abundance. Many proteins, such as ion transporters, Ca2+-signal transduction, and proteins related to protein degradation were downregulated in HO plants during hypoxia, but not in WT plants. Changes in the levels of histones indicates that chromatin restructuring plays a role in the priming of hypoxia. We also identified and quantified 1470 metabolites, of which the abundance of >500 changed significantly. In summary the data confirm known mechanisms of hypoxia priming by ethylene priming and N-end rule activation; however, the data also indicate the existence of other mechanisms for hypoxia priming in plants.

Matrix Redox Physiology Governs the Regulation of Plant Mitochondrial Metabolism through Posttranslational Protein Modifications.

Mitochondria function as hubs of plant metabolism. Oxidative phosphorylation produces ATP, but it is also a central high-capacity electron sink required by many metabolic pathways that must be flexibly coordinated and integrated. Here, we review the crucial roles of redox-associated posttranslational protein modifications (PTMs) in mitochondrial metabolic regulation. We discuss several major concepts. First, the major redox couples in the mitochondrial matrix (NAD, NADP, thioredoxin, glutathione, and ascorbate) are in kinetic steady state rather than thermodynamic equilibrium. Second, targeted proteomics have produced long lists of proteins potentially regulated by Cys oxidation/thioredoxin, Met-SO formation, phosphorylation, or Lys acetylation, but we currently only understand the functional importance of a few of these PTMs. Some site modifications may represent molecular noise caused by spurious reactions. Third, different PTMs on the same protein or on different proteins in the same metabolic pathway can interact to fine-tune metabolic regulation. Fourth, PTMs take part in the repair of stress-induced damage (e.g., by reducing Met and Cys oxidation products) as well as adjusting metabolic functions in response to environmental variation, such as changes in light irradiance or oxygen availability. Finally, PTMs form a multidimensional regulatory system that provides the speed and flexibility needed for mitochondrial coordination far beyond that provided by changes in nuclear gene expression alone.

The Mitogenome of Norway Spruce and a Reappraisal of Mitochondrial Recombination in Plants.

Plant mitogenomes can be difficult to assemble because they are structurally dynamic and prone to intergenomic DNA transfers, leading to the unusual situation where an organelle genome is far outnumbered by its nuclear counterparts. As a result, comparative mitogenome studies are in their infancy and some key aspects of genome evolution are still known mainly from pregenomic, qualitative methods. To help address these limitations, we combined machine learning and in silico enrichment of mitochondrial-like long reads to assemble the bacterial-sized mitogenome of Norway spruce (Pinaceae: Picea abies). We conducted comparative analyses of repeat abundance, intergenomic transfers, substitution and rearrangement rates, and estimated repeat-by-repeat homologous recombination rates. Prompted by our discovery of highly recombinogenic small repeats in P. abies, we assessed the genomic support for the prevailing hypothesis that intramolecular recombination is predominantly driven by repeat length, with larger repeats facilitating DNA exchange more readily. Overall, we found mixed support for this view: Recombination dynamics were heterogeneous across vascular plants and highly active small repeats (ca. 200 bp) were present in about one-third of studied mitogenomes. As in previous studies, we did not observe any robust relationships among commonly studied genome attributes, but we identify variation in recombination rates as a underinvestigated source of plant mitogenome diversity.

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.