Cold and Heat Stress Diversely Alter Both Cauliflower Respiration and Distinct Mitochondrial Proteins Including OXPHOS Components and Matrix Enzymes.

Complex proteomic and physiological approaches for studying cold and heat stress responses in plant mitochondria are still limited. Variations in the mitochondrial proteome of cauliflower (Brassica oleracea var. botrytis) curds after cold and heat and after stress recovery were assayed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) in relation to mRNA abundance and respiratory parameters. Quantitative analysis of the mitochondrial proteome revealed numerous stress-affected protein spots. In cold, major downregulations in the level of photorespiratory enzymes, porine isoforms, oxidative phosphorylation (OXPHOS) and some low-abundant proteins were observed. In contrast, carbohydrate metabolism enzymes, heat-shock proteins, translation, protein import, and OXPHOS components were involved in heat response and recovery. Several transcriptomic and metabolic regulation mechanisms are also suggested. Cauliflower plants appeared less susceptible to heat; closed stomata in heat stress resulted in moderate photosynthetic, but only minor respiratory impairments, however, photosystem II performance was unaffected. Decreased photorespiration corresponded with proteomic alterations in cold. Our results show that cold and heat stress not only operate in diverse modes (exemplified by cold-specific accumulation of some heat shock proteins), but exert some associations at molecular and physiological levels. This implies a more complex model of action of investigated stresses on plant mitochondria.

Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins.

Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation.

Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure.

The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed.

Mitochondria in COVID-19: from cellular and molecular perspective.

The rapid development of the COVID-19 pandemic resulted in a closer analysis of cell functioning during ß-coronavirus infection. This review will describe evidence for COVID-19 as a syndrome with a strong, albeit still underestimated, mitochondrial component. Due to the sensitivity of host mitochondria to coronavirus infection, SARS-CoV-2 affects mitochondrial signaling, modulates the immune response, modifies cellular energy metabolism, induces apoptosis and ageing, worsening COVID-19 symptoms which can sometimes be fatal. Various aberrations across human systems and tissues and their relationships with mitochondria were reported. In this review, particular attention is given to characterization of multiple alterations in gene expression pattern and mitochondrial metabolism in COVID-19; the complexity of interactions between SARS-CoV-2 and mitochondrial proteins is presented. The participation of mitogenome fragments in cell signaling and the occurrence of SARS-CoV-2 subgenomic RNA within membranous compartments, including mitochondria is widely discussed. As SARS-CoV-2 severely affects the quality system of mitochondria, the cellular background for aberrations in mitochondrial dynamics in COVID-19 is additionally characterized. Finally, perspectives on the mitigation of COVID-19 symptoms by affecting mitochondrial biogenesis by numerous compounds and therapeutic treatments are briefly outlined.

Novel PKD1 mutations - the effect on clinical phenotype of ADPKD patients in Lower Silesia.

INTRODUCTION: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development and progressive enlargement of cysts in the kidneys. The diagnosis of ADPKD is usually determined by criteria of renal ultrasound imaging of the development and number of cysts. However, in atypical cystic disease, for the recognition of ADPKD, DNA-based assays may be required. MATERIALS AND METHODS: In the present study PCR amplified fragments of the PKD1 gene (covering exons 15 and 43- 44) from genomic DNA of 134 Lower Silesia patients were analyzed for mutations and polymorphisms. Among them, the clinical significance of different PKD1 mutations was investigated in 81 persons. RESULTS: Eight new, previously undescribed, and 2 recurrent mutations were discovered. The presence of 3 known polymorphisms was confirmed. Seven of the 8 new discovered mutations were heterozygous. DISCUSSION: The results of the present study demonstrated that the frequency of genetic abnormalities in the analyzed fragments of the PKD1 gene in the Lower Silesian population is smaller than previously reported. Moreover, we could not detect deletions and insertions, which are often present is these regions of the PKD1 gene, which may be due to the limited number of screened patients. We conclude that none of the discovered changes in the PKD1 gene had any effect on clinical phenotype of the disease.

Plant dehydrins and dehydrin-like proteins: characterization and participation in abiotic stress response.

Abiotic stress has a significant impact on plant growth and development. It causes changes in the subcellular organelles, which, due to their stress sensitivity, can be affected. Cellular components involved in the abiotic stress response include dehydrins, widely distributed proteins forming a class II of late embryogenesis abundant protein family with characteristic properties including the presence of evolutionarily conserved sequence motifs (including lysine-rich K-segment, N-terminal Y-segment, and often phosphorylated S motif) and high hydrophilicity and disordered structure in the unbound state. Selected dehydrins and few poorly characterized dehydrin-like proteins participate in cellular stress acclimation and are also shown to interact with organelles. Through their functioning in stabilizing biological membranes and binding reactive oxygen species, dehydrins and dehydrin-like proteins contribute to the protection of fragile organellar structures under adverse conditions. Our review characterizes the participation of plant dehydrins and dehydrin-like proteins (including some organellar proteins) in plant acclimation to diverse abiotic stress conditions and summarizes recent updates on their structure (the identification of dehydrin less conserved motifs), classification (new proposed subclasses), tissue- and developmentally specific accumulation, and key cellular activities (including organellar protection under stress acclimation). Recent findings on the subcellular localization (with emphasis on the mitochondria and plastids) and prospective applications of dehydrins and dehydrin-like proteins in functional studies to alleviate the harmful stress consequences by means of plant genetic engineering and a genome editing strategy are also discussed.

Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress.

BACKGROUND: Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress. RESULTS: All the analyzed plant species showed constitutive accumulation of thermostable mitochondrial putative dehydrins ranging from 50 to 70 kDa. The mitochondrial dehydrin-like proteins observed in cauliflower and Arabidopsis ranged from 10 to 100 kDa and in lupin imbibed seeds and hypocotyls--from 20 to 90 kDa. Cold treatment increased mainly the accumulation of 10-100 kDa cauliflower and Arabidopsis dehydrin-like proteins, in the patterns different in cauliflower leaf and inflorescence mitochondria. However, in lupin mitochondria, cold affected mainly 25-50 kDa proteins and seemed to induce the appearance of some novel dehydrin-like proteins. The influence of frost stress on cauliflower leaf mitochondrial dehydrin- like proteins was less significant. The impact of heat stress was less significant in lupin and Arabidopsis than in cauliflower inflorescence mitochondria. Cauliflower mitochondrial dehydrin-like proteins are localized mostly in the mitochondrial matrix; it seems that some of them may interact with mitochondrial membranes. CONCLUSIONS: All the results reveal an unexpectedly broad spectrum of dehydrin-like proteins accumulated during some abiotic stress in the mitochondria of the plant species analyzed. They display only limited similarity in size to those reported previously in maize, wheat and rye mitochondria. Some small thermolabile dehydrin-like proteins were induced under stress conditions applied and therefore they are likely to be involved in stress response.

Proteins involved in maturation pathways of plant mitochondrial and plastid c-type cytochromes.

c-type cytochromes are characterized by the presence of two covalent bonds linking heme to apocytochrome and by the heme attachment motif in the apoprotein. Several molecular systems for the maturation of c-type cytochromes have evolved in different organisms. The best characterized are three of them: system I, system II and system III. Heme is synthesized in bacterial cytoplasm, in plastids, and in animal and fungal mitochondria. Therefore the maturation of bacterial and plastid c-type cytochromes involves the transport of heme and apocytochrome from the n-side to the p-side of the respective biological membranes and the formation of the covalent bond at the p-side. It should be underlined that the site of the c-type apocytochrome synthesis is also distinct from the site of its functioning. The aim of this review is to present the current state of knowledge concerning the structure and function of two systems - system I and system II - in the maturation of plant mitochondrial and plastid c-type cytochromes, respectively.

Plant Glycine-Rich Proteins in Stress Response: An Emerging, Still Prospective Story.

Seed plants are sessile organisms that have developed a plethora of strategies for sensing, avoiding, and responding to stress. Several proteins, including the glycine-rich protein (GRP) superfamily, are involved in cellular stress responses and signaling. GRPs are characterized by high glycine content and the presence of conserved segments including glycine-containing structural motifs composed of repetitive amino acid residues. The general structure of this superfamily facilitates division of GRPs into five main subclasses. Although the participation of GRPs in plant stress response has been indicated in numerous model and non-model plant species, relatively little is known about the key physiological processes and molecular mechanisms in which those proteins are engaged. Class I, II, and IV members are known to be involved in hormone signaling, stress acclimation, and floral development, and are crucial for regulation of plant cells growth. GRPs of class IV [RNA-binding proteins (RBPs)] are involved in alternative splicing or regulation of transcription and stomatal movement, seed, pollen, and stamen development; their accumulation is regulated by the circadian clock. Owing to the fact that the overexpression of GRPs can confer tolerance to stress (e.g., some are involved in cold acclimation and may improve growth at low temperatures), these proteins could play a promising role in agriculture through plant genetic engineering. Consequently, isolation, cloning, characterization, and functional validation of novel GRPs expressed in response to the diverse stress conditions are expected to be growing areas of research in the coming years. According to our knowledge, this is the first comprehensive review on participation of plant GRPs in the response to diverse stress stimuli.

Plant mitochondrial genes can be expressed from mRNAs lacking stop codons.

The mRNAs of the nad6 and ccmC genes of Arabidopsis and cauliflower were found to be processed upstream of the inframe stop codons. This result was confirmed by northern hybridization and by RT-PCR. There is no evidence that an alternative stop codon is created post-transcriptionally, either by RNA editing or by polyadenylation. The non-stop mRNAs are found in the high molecular weight polysomal fractions, suggesting that they are translated. Using antibodies directed against CcmC, the corresponding protein was detected in Arabidopsis mitochondrial extracts. These observations raise the question of how the plant mitochondrial translation system deals with non-stop mRNAs.

Participation of non-coding RNAs in plant organelle biogenesis.

The biogenesis of plant mitochondria and plastids is a multistep process that depends on the expression of both, organellar and nuclear genes. A growing body of evidence suggests that the indispensable coordination of different steps in this process may be gained by participation of the non-coding RNAs. A plethora of non-coding RNAs of diverse length, both intraorganellar ones, as well as encoded by the nuclear genome (including microRNAs and short interfering RNAs), were also suggested to play a role in the stress response by regulating the expression levels of targeted genes important for organelle biogenesis. Selected points of current interest regarding the regulation of plant mitochondrial and plastid gene expression by diverse non-coding RNAs, also discussed in the aspect of abiotic stress conditions, are highlighted here.

Lupin nad9 and nad6 genes and their expression: 5' termini of the nad9 gene transcripts differentiate lupin species.

The mitochondrial nad9 and nad6 genes were analyzed in four lupin species: Lupinus luteus, Lupinus angustifolius, Lupinus albus and Lupinus mutabilis. The nucleotide sequence of these genes confirmed their high conservation, however, higher number of nucleotide substitution was observed in the L. albus genes. Southern hybridizations confirmed the presence of single copy number of these genes in L. luteus, L. albus and L. angustifolius. The expression of nad9 and nad6 genes was analyzed by Northern in different tissue types of analyzed lupin species. Transcription analyses of the two nad genes displayed single predominant mRNA species of about 0.6 kb in L. luteus and L. angustifolius. The L. albus transcripts were larger in size. The nad9 and nad6 transcripts were modified by RNA editing at 8 and 11 positions, in L. luteus and L. angustifolius, respectively. The gene order, rps3-rpl16-nad9, found in Arabidopsis thaliana is also conserved in L. luteus and L. angustifolius mitochondria. L. luteus and L. angustifolius showed some variability in the sequence of the nad9 promoter region. The last feature along with the differences observed in nad9 mRNA 5' termini of two lupins differentiate L. luteus and L. angustifolius species.

Plant mitochondria under a variety of temperature stress conditions.

The biogenesis of plant mitochondria is a multistep process that depends on a concerted expression of mitochondrial and nuclear genes. The balance between different steps of this process, embracing various fluctuations in mitochondrial transcriptome and proteome, may be affected by diverse temperature treatments. A plethora of genes with altered expression during the acting of these stimuli were identified and their expression characterized, including those encoding for classical components of energy dissipating system. Selected aspects of current interest, regarding the functioning of plant mitochondria under cold and heat stresses, are highlighted.

Application of DNA markers linked to the potato H1 gene conferring resistance to pathotype Ro1 of Globodera rostochiensis.

Ninety-one potato genotypes (cultivars and breeding lines) selected as resistant or susceptible to pathotype Ro1 of Globodera rostochiensis were screened for the presence of two PCR markers, 0.14 and 0.76 kb in length. Both PCR markers were linked with the H1 gene, located at the distal end of the long arm of chromosome V, and were present in 88 to 100% of the resistant cultivars and breeding lines. The 0.76 kb PCR marker was detected in all resistant genotypes and in approximately 86% of susceptible breeding lines as well as in all susceptible cultivars. The 0.14 kb marker was detected in 88% of resistant breeding lines and in 94% of resistant cultivars. Most of the susceptible genotypes tested (91% of cultivars, but only 50% of breeding lines) did not show the presence of the 0.14 kb marker. We conclude that the 0.14 kb H1 marker is likely to be useful for the proper selection of potato genotypes resistant to the Ro1 pathotype of G. rostochiensis.

PPR336 is associated with polysomes in plant mitochondria.

The function of pentatricopeptide repeat (PPR) proteins has been associated with various post-transcriptional steps of organelle gene expression. Among them, translation and its regulation are essential processes. However, in plant mitochondria, they are also the steps of gene expression that are the least understood. In this study, PPR336 was identified as part of a high-molecular-weight complex in Arabidopsis mitochondria. PPR336 is an unusual representative of the large PPR family because it is relatively short and is characterised by a high expression level compared with other PPR proteins. PPR336 defines a small subgroup of eight class P PPR proteins that are similar in terms of motif organization. Among them, PPR336-like is the closest homolog of PPR336. Biochemical analysis has indicated that PPR336 is a strictly mitochondrial protein, extrinsically attached to the inner mitochondrial membrane and part of an RNase-sensitive complex. Sucrose gradients and polysome destabilisation experiments show that PPR336 is associated with ribosomes in plant mitochondria. Moreover, in Ppr336/336-like mutants, mitochondrial polysomes of lower molecular weight accumulate compared with wild-type plants. Polysome association and these unusual features suggest that PPR336 could be involved in a distinctive process, possibly translation in plant mitochondria.