Mitochondria In Plant Cells Research Articles

Hydrogen sulfide signaling in plant response to temperature stress.

For the past 300 years, hydrogen sulfide (H2S) has been considered a toxic gas. Nowadays, it has been found to be a novel signaling molecule in plants involved in the regulation of cellular metabolism, seed germination, plant growth, development, and response to environmental stresses, including high temperature (HT) and low temperature (LT). As a signaling molecule, H2S can be actively synthesized and degraded in the cytosol, chloroplasts, and mitochondria of plant cells by enzymatic and non-enzymatic pathways to maintain homeostasis. To date, plant receptors for H2S have not been found. It usually exerts physiological functions through the persulfidation of target proteins. In the past 10 years, H2S signaling in plants has gained much attention. Therefore, in this review, based on that same attention, H2S homeostasis, protein persulfidation, and the signaling role of H2S in plant response to HT and LT stress were summarized. Also, the common mechanisms of H2S-induced HT and LT tolerance in plants were updated. These mechanisms involve restoration of biomembrane integrity, synthesis of stress proteins, enhancement of the antioxidant system and methylglyoxal (MG) detoxification system, improvement of the water homeostasis system, and reestablishment of Ca2+ homeostasis and acid-base balance. These updates lay the foundation for further understanding the physiological functions of H2S and acquiring temperature-stress-resistant crops to develop sustainable food and agriculture.

Expanding the knowledge frontier of mitoviruses in Cannabis sativa

Mitoviruses were initially known for their presence in the mitochondria of fungi and were considered exclusive to these organisms. However, recent studies have shown that they are also present in a large number of plant species. Despite the potential impact that mitoviruses might have on the mitochondria of plant cells, there is a lack of information about these ancient RNA viruses, especially within the Cannabaceae family. Cannabis sativa has been in the spotlight in recent years due to the growing industrial applications of plant derivatives, such as fiber and secondary metabolites. Given the importance of Cannabis in today's agriculture, our study aimed to expand the knowledge frontier of Mitoviruses in C. sativa by increasing the number of reference genomes of CasaMV1 available in public databases and representing a larger number of crops in countries where its industrial-scale growth is legalized. To achieve this goal, we used transcriptomics to sequence the first mitoviral genomes of Colombian crops and analyzed RNA-seq datasets available in the SRA databank. Additionally, the evolutionary analysis performed using the mitovirus genomes revealed two main lineages of CasaMV1, termed CasaMV1_L1 and CasaMV1_L2. These mitoviral lineages showed strong clustering based on the geographic location of the crops and differential expression intensities.

ENGINEERING THE N-TERMINAL SEQUENCE OF GLYCINE MAX SOYBEAN FORMATE DEHYDROGENASE

NAD(P)+ -dependent formate dehydrogenase (FDH, EC 1.2.1.2.) catalyzes the oxidation of formate ion with the coupled reduction of NAD(P)+ to NAD(P)H. Previously, in our laboratory, a genetic construct was obtained with the soyfdh2 gene encoding isoenzyme 2 of formate dehydrogenase from soybean Glycine max (SoyFDH). In this construct the nucleotide sequence encoding the signal peptide responsible for the transport of the pro-enzyme into the mitochondria of plant cells (the SoyFDH_L enzyme) was deleted. In this work, a second variant of SoyFDH_S was obtained, in which, compared to SoyFDH_L, the sequence at the N-terminus was reduced and changed to mimic the N-terminus sequence in FDH from Pseudomonas sp.101 bacteria. Next, a sequence of six histidine residues (His-tag) was added to the N-terminus of the long and short forms of SoyFDH. All four SoyFDH variants were expressed in E. coli BL21(DE3)CodonPlus, these enzymes were purified, their kinetic parameters were determined, and thermal stability was studied. In the case of SoyFDH_L, which is similar to the natural variant of the enzyme, both with and without His-tag, the expression level is two times higher compared to the truncated variant. The addition of His-tag to the N-terminus of enzymes reduces the level of expression. Changing the sequence of the N-terminus, as well as introducing the His-tag sequence to the N-terminus, does not significantly affect thermal stability of the enzymes at temperatures of 50–56 °C. However, due to the higher values of the activation enthalpy ΔH≠ of the thermal inactivation process, the shortened form at normal temperatures is 3 times more stable than the natural one. A comparison of the kinetic parameters of the two SoyFDH variants shows that the catalytic constants are the same, but the long version of SoyFDH_L has lower values KM HCOO– , and the short version has lower KM NAD+ values. The introduction of His-tag into the N-terminus of enzymes does not affect their kinetic parameters.

From leaves to roots: Biophysical models of transport of substances in plants.

The transport processes of substances in various plant tissues are extremely diverse. However, models aimed at elucidating the mechanisms of such processes are almost absent in the literature. A unified view of all these transport processes is necessary, considering the laws of statistical physics and thermodynamics. A model of active ion transport was constructed based on the laws of statistical physics. Using this model, we traced the entire pathway of substances and energy in a plant. The pathway included aspects of the production of energy in the process of photosynthesis, consumption of energy to obtain nutrients from the soil, transport of such substances to the main organelles of all types of plant cells, the rise of water with dissolved substances along the trunk to the leaves, and the evaporation of water, accompanied by a change in the percentage of isotopes caused by different rates of evaporation. Models of ion transport in the chloroplasts and mitochondria of plant cells have been constructed. A generalized model comprising plant cells and their vacuoles was analyzed. A model of the transport of substances in the roots of plants was also developed. Based on this model, the problem of transport of substances in tall trees has been considered. The calculated concentrations of ions in the vacuoles of cells and resting potentials agreed well with the experimental data.

Synthetic Mitochondria-Targeting Peptides Incorporating α-Aminoisobutyric Acid with a Stable Amphiphilic Helix Conformation in Plant Cells.

In the genetic modification of plant cells, the mitochondrion is an important target in addition to the nucleus and plastid. However, gene delivery into the mitochondria of plant cells has yet to be established by conventional methods, such as particle bombardment, because of the small size and high mobility of mitochondria. To develop an efficient mitochondria-targeting signal (MTS) that functions in plant cells, we designed the artificial peptide (LURL)3 and its analogues, which periodically feature hydrophobic α-aminoisobutyric acid (Aib, U) and cationic arginine (R), considering the consensus motif recognized by the mitochondrial import receptor Tom20. Circular dichroism measurements and molecular dynamics simulation studies revealed that (LURL)3 had a propensity to form a stable α-helix in 0.1 M phosphate buffer solution containing 1.0 wt % sodium dodecyl sulfate. After internalization into plant cells via particle bombardment, (LURL)3 revealed highly selective accumulation in the mitochondria, whereas its analogue (LARL)3 was predominantly located in the vacuoles in addition to mitochondria. The high selectivity of (LURL)3 can be attributed to the incorporation of Aib, which promotes the hydrophobic interaction between the MTS and Tom20 by increasing the hydrophobicity and helicity of (LURL)3. The present study provided a prospective mitochondrial targeting system using the simple design of artificial peptides.

Molecular Mechanism of the Specificity of Protein Import into Chloroplasts and Mitochondria in Plant Cells

Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.

Metal tolerance protein MTP6 affects mitochondrial iron and manganese homeostasis in cucumber.

Members of the cation diffusion facilitator (CDF) family have been identified in all kingdoms of life. They have been divided into three subgroups, namely Zn-CDF, Fe/Zn-CDF, and Mn-CDF, based on their putative specificity to transported metal ions. The plant metal tolerance protein 6 (MTP6) proteins fall into the Fe/Zn-CDF subgroup; however, their function in iron/zinc transport has not yet been confirmed. Here, we characterized the MTP6 protein from cucumber, Cucumis sativus. When expressed in yeast and in protoplasts isolated from Arabidopsis cells, CsMTP6 localized in mitochondria and contributed to the efflux of Fe and Mn from these organelles. Immunolocalization of CsMTP6 in cucumber membranes confirmed this association with mitochondria. Root expression and protein levels of CsMTP6 were significantly up-regulated in conditions of Fe deficiency and excess, but were not affected by Mn availability. These results indicate that MTP6 proteins contribute to the distribution of Fe and Mn between the cytosol and mitochondria of plant cells, and are regulated by Fe to maintain mitochondrial and cytosolic iron homeostasis under varying conditions of Fe availability.

Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana

In higher plants, biosynthesis of cysteine is catalysed by OAS-TL [O-acetylserine(thiol)lyase], which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. The sole knockout of mitochondrial OAS-TL activity (oastlC) leads to significant reduction of growth in Arabidopsis thaliana. The reason for this phenotype is still enigmatic, since mitochondrial OAS-TL accounts only for approximately 5% of total OAS-TL activity. In the present study we demonstrate that sulfide specifically intoxicates Complex IV activity, but not electron transport through Complexes II and III in isolated mitochondria of oastlC plants. Loss of mitochondrial OAS-TL activity resulted in significant inhibition of dark respiration under certain developmental conditions. The abundance of mitochondrially encoded proteins and Fe-S cluster-containing proteins was not affected in oastlC. Furthermore, oastlC seedlings were insensitive to cyanide, which is detoxified by β-cyano-alanine synthase in mitochondria at the expense of cysteine. These results indicate that in situ biosynthesis of cysteine in mitochondria is not mandatory for translation, Fe-S cluster assembly and cyanide detoxification. Finally, we uncover an OAS-TL-independent detoxification system for sulfide in mitochondria of Arabidopsis that allows oastlC plants to cope with high sulfide levels caused by abiotic stresses.

Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria

With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and driven by a transfer RNA-like (tRNA-like) moiety are taken up by mitochondria in plant cells. The process appears to follow the natural tRNA import specificity, suggesting that translocation indeed occurs through the regular tRNA uptake pathway. Upon validation of the strategy with a reporter sequence, we developed a chimeric catalytic RNA composed of a specially designed trans-cleaving hammerhead ribozyme and a tRNA mimic. Organelle import of the chimeric ribozyme and specific target cleavage within mitochondria were demonstrated in transgenic tobacco cell cultures and Arabidopsis thaliana plants, providing the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. Further observations point to mitochondrial messenger RNA control mechanisms related to the plant developmental stage and culture conditions. Transformation of mitochondria is only accessible in yeast and in the unicellular alga Chlamydomonas. Based on the widespread tRNA import pathway, our data thus make a breakthrough for direct investigation and manipulation of mitochondrial genetics.

Effect of Mitochondrial Dysfunction on Carbon Metabolism and Gene Expression in Flower Tissues of Arabidopsis thaliana

We characterized the transcriptomic response of transgenic plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase subunit 9. The u-ATP9 transgene driven by A9 and APETALA3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and adenine nucleotides (ATP, ADP) levels without changes in the ATP/ADP ratio. Furthermore, we measured an increase in ROS accumulation and a decrease in glutathione and ascorbate levels with a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by qRT-PCR and enzymatic or functional tests, showed dramatic changes in u-ATP9 plants. Both lines display a modification in the expression of various genes involved in carbon, lipid, and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial respiration, protein synthesis, and degradation are affected. Moreover, the levels of several mRNAs encoding for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone responses, suggesting that several signaling pathways overlap. Indeed, the transcriptome data revealed that the mitochondrial dysfunction dramatically alters the expression of genes involved in signaling pathways, including those related to ethylene, absicic acid, and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this report may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells.

Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization

Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.

Arabidopsis thaliana Mitochondrial Glyoxalase 2-1 Exhibits β-Lactamase Activity

In an effort to determine the physiological role of Arabidopsis thaliana Glx2-1, we attempted to uncover a substrate for the enzyme. Glx2-1 did not effectively process 192 diverse substrates found in a commercial screen used for microorganism identification or exhibit arylsulfatase, lactonase, or phosphotriesterase activities. However, Glx2-1 does exhibit beta-lactamase activity with k(cat)/KM values from 10(3) to 10(5) M(-1) s(-1). Glx2-1 can hydrolyze cephalosporins and carbapenems, albeit with rate constants slower than those of most metallo-beta-lactamases. The potential role of a beta-lactamase in the mitochondria of plant cells is briefly discussed.

Light-Dependent Intracellular Positioning of Mitochondria in Arabidopsis thaliana Mesophyll Cells

Mitochondria, the power house of the cell, are one of the most dynamic cell organelles. Although there are several reports on actin- or microtubule-dependent movement of mitochondria in plant cells, intracellular positioning and motility of mitochondria under different light conditions remain open questions. Mitochondria were visualized in living Arabidopsis thaliana leaf cells using green fluorescent protein fused to a mitochondrion-targeting signal. In darkness, mitochondria were distributed randomly in palisade cells. In contrast, mitochondria accumulated along the periclinal walls, similar to the accumulation response of chloroplasts, when treated with weak blue light (470 nm, 4 micromol m(-2) s(-1)). Under strong blue light (100 micromol m(-2) s(-1)), mitochondria occupied the anticlinal positions similar to the avoidance response of chloroplasts and nuclei. While strong red light (660 nm, 100 micromol m(-2) s(-1)) induced the accumulation of mitochondria along the inner periclinal walls, green light exhibited little effect on the distribution of mitochondria. In addition, the mode of movement of individual mitochondria along the outer periclinal walls under different light conditions was precisely analyzed by time-lapse fluorescence microscopy. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts with a time period of blue light illumination clearly demonstrated the accumulation response of mitochondria. Light-induced co-localization of mitochondria with chloroplasts strongly suggested their mutual metabolic interactions. This is the first characterization of the light-dependent redistribution of mitochondria in plant cells.

A Comparative Study of the Involvement of 17 Arabidopsis Myosin Family Members on the Motility of Golgi and Other Organelles

Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.

Microcalorimetric Study of the Effect of CeIII on Metabolic Activity of Mitochondria Isolated from Indice Rice 9311

Rare earth elements (REEs) have beneficial influence on plant growth and are widely used in agriculture practice, but little is known about behavior of the REEs on mitochondria in plant cells. Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from indice rice 9311 (Oryza sativa L.), and the effect of Ce(III) on mitochondrial metabolism was investigated. By analyzing the obtained heat flux curves, the crucial parameters such as activity recovery rate constant (k) and maximum heat power (P(m)) were investigated. Application of Ce3+ in concentrations ranging from 0 to 120 microg/ml significantly increased k and P(m) values, with the maximum reaching 261 and 180% of the control, respectively. Concentrations from 140 to 150 microg/ml had the opposite effect. These results were consistent with previous reports on the effects of REEs on plant growth. It was concluded that the Ce(III)-induced change of mitochondrial metabolic activity is a possible mechanism by which Ce(III) influenced indice rice 9311 growth.

Unique properties of NADP-thioredoxin reductase C in legumes

NADP-thioredoxin reductases (NTRs) reduce thioredoxins (Trxs), using NADPH as a reductant, together constituting complete redox systems (NTS). Beside NTRA and NTRB targeted to both cytosol and mitochondria of plant cells, there is in chloroplasts an unusual NTR (NTRC) harbouring a Trx domain in a C-terminal extension, as recently reported in Oryza sativa. Although NTRC may constitute a complete NTS, it was described as a bifunctional enzyme. Because the gene is only present in photosynthetic organisms and the protein in green tissues, NTRC was thought to have a role restricted to photosynthetic cells. To determine whether NTRC from dicot plants is a bifunctional enzyme or a complete NTS, as well as to identify its putative target, NTRC from Medicago truncatula was cloned and NTRA was cloned for comparison. Here evidence is presented that MtNTRC (i) acts as an NTS and reduces dithiobisnitrobenzoate (DTNB) with a turnover (0.62 s(-1)) similar to that measured with MtNTRA in the presence of a Trxh (0.81 s(-1)); (ii) is able to use both NADPH (k(M)=2.4 microM) and NADH (k(M)=11 microM) as cofactors; (iii) efficiently reduces BAS1, a plastidial peroxiredoxin; and (iv) is expressed in both leaves and stems but unexpectedly is even more abundant in cotyledons from dry and germinating seeds. Because BAS1 is also present in both green tissues and seeds, NTRC/BAS1 may be involved in the scavenging of peroxides produced in green tissues during the day or the night and in seeds during germination. These results suggest different roles for NTRC in monocot and dicot plants.

Improved staining of F‐actin and co‐localization of mitochondria in plant cells

Permeating the cell membrane with glycerol instead of detergents (e.g. NP‐40) and/or organic solvents (e.g. DMSO) significantly improves the visualization of F‐actin with rhodamine phalloidin. In particular, the thinnest bundles are more intensely stained, better delineated and observation time is prolonged dramatically. Furthermore, the method is gentle to membranous organelles, which makes, for example, co‐localization of mitochondria and actin possible.

Mitochondrial localization of a NADP-dependent [corrected] isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker.

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5' region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.

Rhodamine 123 as a vital stain for mitochondria of plant cells

Rhodamine 123 as a vital stain for mitochondria of plant cells

Localization of mitochondria in plant cells by vital staining with rhodamine 123

The positively-charged fluorescent dye rhodamine 123 (r-123) specifically stains mitochondria in living plant protoplasts, suspensionculture cells, and root hairs. This dye functions as a vital stain and permits visualization of the localization, distribution and movement of the mitochondria. Dehydration of root hairs caused mitochondria to aggregate into clumps. Mitochondria were either homogenous or heterogeneous and were frequently seen to accumulate in the perinuclear regions of suspension-culture cells but not in those of protoplasts or root-hair cells. Dinitrophenol and high concentrations of ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and KCl immediately eliminated fluorescence in r-123-stained mitochondria whereas ionomycin enhanced it. Treatment of seedlings with r-123 resulted in differential brightness of fluorescence in different tissues. Meristematic tissues, such as root and shoot tips, exhibited the brightest fluorescence. The cytotoxicity of r-123 in both germinating seedlings and suspension-culture cells was low. The specificity, sensitivity and low toxicity of r-123 should make it a useful tool in experiments designed to examine agents and conditions which affect the location, the physiological status or the viability of mitochondria.