Cytochrome c levels affect the TOR pathway to regulate growth and metabolism under energy-deficient conditions.

Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.

Knockdown of double-stranded RNases (dsRNases) enhances oral RNA interference (RNAi) in the corn leafhopper, Dalbulus maidis.

The leafhopper Dalbulus maidis is a harmful pest that causes severe damage to corn crops. Conventional chemical pesticides have negative environmental impacts, emphasizing the need for alternative solutions. RNA interference (RNAi) is a more specific and environmentally friendly method for controlling pests and reducing the negative impacts of current pest management practices. Previous studies have shown that orally administered double-stranded RNA (dsRNA) is less effective than injection protocols in silencing genes. This study focuses on identifying and understanding the role of double-stranded ribonucleases (dsRNases) in limiting the efficiency of oral RNAi in D. maidis. Three dsRNases were identified and characterized, with Dmai-dsRNase-2 being highly expressed in the midgut and salivary glands. An ex vivo degradation assay revealed significant nuclease activity, resulting in high instability of dsRNA when exposed to tissue homogenates. Silencing Dmai-dsRNase-2 improved the insects' response to the dsRNA targeting the gene of interest, providing evidence of dsRNases involvement in oral RNAi efficiency. Therefore, administering both dsRNase-specific and target gene-specific-dsRNAs simultaneously is a promising approach to increase the efficiency of oral RNAi and should be considered in future control strategies.

Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity.

Hydrogen sulfide (H2S) is a gaseous signaling molecule involved in numerous physiological processes in plants, including gas exchange with the environment through the regulation of stomatal pore width. Guard cells (GCs) are pairs of specialized epidermal cells that delimit stomatal pores and have a higher mitochondrial density and metabolic activity than their neighboring cells. However, there is no clear evidence on the role of mitochondrial activity in stomatal closure induction. In this work, we showed that the mitochondrial-targeted H2S donor AP39 induces stomatal closure in a dose-dependent manner. Experiments using inhibitors of the mitochondrial electron transport chain (mETC) or insertional mutants in cytochrome c (CYTc) indicated that the activity of mitochondrial CYTc and/or complex IV are required for AP39-dependent stomatal closure. By using fluorescent probes and genetically encoded biosensors we reported that AP39 hyperpolarized the mitochondrial inner potential (Δψm) and increased cytosolic ATP, cytosolic hydrogen peroxide levels, and oxidation of the glutathione pool in GCs. These findings showed that mitochondrial-targeted H2S donors induce stomatal closure, modulate guard cell mETC activity, the cytosolic energetic and oxidative status, pointing to an interplay between mitochondrial H2S, mitochondrial activity, and stomatal closure.

Cytochrome c and the transcription factor ABI4 establish a molecular link between mitochondria and ABA-dependent seed germination.

During germination, seed reserves are mobilised to sustain the metabolic and energetic demands of plant growth. Mitochondrial respiration is presumably required to drive germination in several species, but only recently its role in this process has begun to be elucidated. Using Arabidopsis thaliana lines with changes in the levels of the respiratory chain component cytochrome c (CYTc), we investigated the role of this protein in germination and its relationship with hormonal pathways. Cytochrome c deficiency causes delayed seed germination, which correlates with decreased cyanide-sensitive respiration and ATP production at the onset of germination. In addition, CYTc affects the sensitivity of germination to abscisic acid (ABA), which negatively regulates the expression of CYTC-2, one of two CYTc-encoding genes in Arabidopsis. CYTC-2 acts downstream of the transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), which binds to a region of the CYTC-2 promoter required for repression by ABA and regulates its expression. The results show that CYTc is a main player during seed germination through its role in respiratory metabolism and energy production. In addition, the direct regulation of CYTC-2 by ABI4 and its effect on ABA-responsive germination establishes a link between mitochondrial and hormonal functions during this process.

Development of efficient RNAi methods in the corn leafhopper Dalbulus maidis, a promising application for pest control.

BACKGROUND: The corn leafhopper Dalbulus maidis is the main vector of important stunting pathogens that affect maize production. Currently, there are no effective methods available to manage this pest without adverse impact on the environment. In this context, genomic-based technologies such as RNA interference (RNAi) provide a more environmentally friendly pest control strategy. Therefore, we aimed to assess the application of RNAi in D. maidis and determine the function of a candidate gene related to insect reproduction and propagation. RESULTS: We have characterized the core RNAi genes and evaluated the functionality of the RNAi machinery. We assessed the potential of RNAi technology in D. maidis via injection or ingestion of double-stranded RNA (dsRNA) to adult females. We chose Bicaudal C (BicC) as a target gene due to its important role during insect oogenesis. Administration of dsRNABicC caused significant reductions in the transcript levels (fold changes up to 170 times) and ovipositions. Phenotypic analysis of the ovaries revealed alterations in oocyte development, providing additional confirmation for our results and supporting the idea that Dmai-BicC is a key player of D. maidis oogenesis. CONCLUSION: This is, to our knowledge, the first report of efficient RNAi in D. maidis. We believe our findings provide a starting point for future control strategies against one of the most important maize pests in the Americas. © 2022 Society of Chemical Industry.

Breaking boundaries: exploring short- and long-distance mitochondrial signalling in plants.

Communication of mitochondria with other cell compartments is essential for the coordination of cellular functions. Mitochondria send retrograde signals through metabolites, redox changes, direct organelle contacts and protein trafficking. Accumulating evidence indicates that, in animal systems, changes in mitochondrial function also trigger responses in other, either neighbouring or distantly located, cells. Although not clearly established, there are indications that this type of communication may also be operative in plants. Grafting experiments suggested that the translocation of entire mitochondria or submitochondrial vesicles between neighbouring cells is possible in plants, as already documented in animals. Changes in mitochondrial function also regulate cell-to-cell communication via plasmodesmata and may be transmitted over long distances through plant hormones acting as mitokines to relay mitochondrial signals to distant tissues. Long-distance movement of transcripts encoding mitochondrial proteins involved in crucial aspects of metabolism and retrograde signalling was also described. Finally, changes in mitochondrial reactive species (ROS) production may affect the 'ROS wave' that triggers systemic acquired acclimation throughout the plant. In this review, we summarise available evidence suggesting that mitochondria establish sophisticated communications not only within the cell but also with neighbouring cells and distant tissues to coordinate plant growth and stress responses in a cell nonautonomous manner.

Cytochrome c Deficiency Differentially Affects the In Vivo Mitochondrial Electron Partitioning and Primary Metabolism Depending on the Photoperiod.

Plant respiration provides metabolic flexibility under changing environmental conditions by modulating the activity of the nonphosphorylating alternative pathways from the mitochondrial electron transport chain, which bypass the main energy-producing components of the cytochrome oxidase pathway (COP). While adjustments in leaf primary metabolism induced by changes in day length are well studied, possible differences in the in vivo contribution of the COP and the alternative oxidase pathway (AOP) between different photoperiods remain unknown. In our study, in vivo electron partitioning between AOP and COP and expression analysis of respiratory components, photosynthesis, and the levels of primary metabolites were studied in leaves of wild-type (WT) plants and cytochrome c (CYTc) mutants, with reduced levels of COP components, under short- and long-day photoperiods. Our results clearly show that differences in AOP and COP in vivo activities between WT and cytc mutants depend on the photoperiod likely due to energy and stress signaling constraints. Parallel responses observed between in vivo respiratory activities, TCA cycle intermediates, amino acids, and stress signaling metabolites indicate the coordination of different pathways of primary metabolism to support growth adaptation under different photoperiods.

The tip of the iceberg: emerging roles of TORC1, and its regulatory functions in plant cells.

Target of Rapamycin (TOR) is an evolutionarily conserved protein kinase that plays a central role in coordinating cell growth with light availability, the diurnal cycle, energy availability, and hormonal pathways. TOR Complex 1 (TORC1) controls cell proliferation, growth, metabolism, and defense in plants. Sugar availability is the main signal for activation of TOR in plants, as it also is in mammals and yeast. Specific regulators of the TOR kinase pathway in plants are inorganic compounds in the form of major nutrients in the soils, and light inputs via their impact on autotrophic metabolism. The lack of TOR is embryo-lethal in plants, whilst dysregulation of TOR signaling causes major alterations in growth and development. TOR exerts control as a regulator of protein translation via the action of proteins such as S6K, RPS6, and TAP46. Phytohormones are central players in the downstream systemic physiological TOR effects. TOR has recently been attributed to have roles in the control of DNA methylation, in the abundance of mRNA splicing variants, and in the variety of regulatory lncRNAs and miRNAs. In this review, we summarize recent discoveries in the plant TOR signaling pathway in the context of our current knowledge of mammalian and yeast cells, and highlight the most important gaps in our understanding of plants that need to be addressed in the future.

Cross-talk between mitochondrial function, growth, and stress signalling pathways in plants.

Plant mitochondria harbour complex metabolic routes that are interconnected with those of other cell compartments, and changes in mitochondrial function remotely influence processes in different parts of the cell. This implies the existence of signals that convey information about mitochondrial function to the rest of the cell. Increasing evidence indicates that metabolic and redox signals are important for this process, but changes in ion fluxes, protein relocalization, and physical contacts with other organelles are probably also involved. Besides possible direct effects of these signalling molecules on cellular functions, changes in mitochondrial physiology also affect the activity of different signalling pathways that modulate plant growth and stress responses. As a consequence, mitochondria influence the responses to internal and external factors that modify the activity of these pathways and associated biological processes. Acting through the activity of hormonal signalling pathways, mitochondria may also exert remote control over distant organs or plant tissues. In addition, an intimate cross-talk of mitochondria with energy signalling pathways, such as those represented by TARGET OF RAPAMYCIN and SUCROSE NON-FERMENTING1-RELATED PROTEIN KINASE 1, can be envisaged. This review discusses available evidence on the role of mitochondria in shaping plant growth and stress responses through various signalling pathways.

The sunflower TLDc-containing protein HaOXR2 confers tolerance to oxidative stress and waterlogging when expressed in maize plants.

The sunflower (Helianthus annuus L.) genome encodes six proteins containing a TLDc domain, typical of the eukaryotic OXidation Resistance (OXR) protein family. Expression of sunflower HaOXR2 in Arabidopsis generated plants with increased rosette diameter, higher number of leaves and increased seed production. Maize inbred lines expressing HaOXR2 also showed increased total leaf area per plant. In addition, heterologous expression of HaOXR2 induced an increase in the oxidative stress tolerance in Arabidopsis and maize. Maize transgenic plants expressing HaOXR2 experienced less oxidative damage and exhibited increased photosynthetic performance and efficiency than non-transgenic segregant plants after treatment of leaves with the reactive oxygen species generating compound Paraquat. Expression of HaOXR2 in maize also improved tolerance to waterlogging. The number of expanded leaves, aerial biomass, and stem height and cross-section area were less affected by waterlogging in HaOXR2 expressing plants, which also displayed less aerial tissue damage under these conditions. Transgenic plants also showed an increased production of roots, a typical adaptive stress response. The results show the existence of functional conservation of OXR proteins in dicot and monocot plants and indicate that HaOXR2 could be useful to improve plant performance under conditions that increase oxidative stress.

OXR2 Increases Plant Defense against a Hemibiotrophic Pathogen via the Salicylic Acid Pathway.

Arabidopsis (Arabidopsis thaliana) OXIDATION RESISTANCE2 (AtOXR2) is a mitochondrial protein belonging to the Oxidation Resistance (OXR) protein family, recently described in plants. We analyzed the impact of AtOXR2 in Arabidopsis defense mechanisms against the hemibiotrophic bacterial pathogen Pseudomonas syringae oxr2 mutant plants are more susceptible to infection by the pathogen and, conversely, plants overexpressing AtOXR2 (oeOXR2 plants) show enhanced disease resistance. Resistance in these plants is accompanied by higher expression of WRKY transcription factors, induction of genes involved in salicylic acid (SA) synthesis, accumulation of free SA, and overall activation of the SA signaling pathway. Accordingly, defense phenotypes are dependent on SA synthesis and SA perception pathways, since they are lost in isochorismate synthase1/salicylic acid induction deficient2 and nonexpressor of pathogenesis-related genes1 (npr1) mutant backgrounds. Overexpression of AtOXR2 leads to faster and stronger oxidative burst in response to the bacterial flagellin peptide flg22 Moreover, AtOXR2 affects the nuclear localization of the transcriptional coactivator NPR1, a master regulator of SA signaling. oeOXR2 plants have increased levels of total glutathione and a more oxidized cytosolic redox cellular environment under normal growth conditions. Therefore, AtOXR2 contributes to establishing plant protection against infection by P. syringae acting on the activity of the SA pathway.

Arabidopsis thaliana SURFEIT1-like genes link mitochondrial function to early plant development and hormonal growth responses.

Mutations in SURFEIT1 (SURF1) genes affect cytochrome c oxidase (COX) levels in different prokaryotic and eukaryotic organisms. In this work, we report that Arabidopsis thaliana has two genes that potentially encode SURF1 proteins, as a result of a duplication that took place in Brassicaceae. Both genes encode mitochondrial proteins and mutation in AtSURF1a causes embryonic lethality. Mutation in AtSURF1b, instead, causes defects in hypocotyl elongation under growth-stimulating conditions, such as low light intensity, increased ambient temperature and incubation with glucose. Mutants in AtSURF1b show reduced expression of the auxin reporter DR5:GUS and increased levels of the gibberellin reporter GFP-RGA, suggesting that auxin and gibberellin homeostasis are affected. In agreement, growth defects caused by AtSURF1b mutation can be overcome by treatment with indole-3-acetic acid and gibberellin A3 , and also by increasing expression of the auxin biosynthesis gene YUC8 or the transcription factor PIF4, which shows lower abundance in AtSURF1b-deficient plants. Mutants in AtSURF1b display lower COX levels, higher alternative oxidase and superoxide levels, and increased expression of genes that respond to mitochondrial dysfunction. Decreased hypocotyl growth and DR5:GUS expression can be reversed by treatment with reduced glutathione, suggesting that redox changes, probably related to mitochondrial dysfunction, are responsible for the effect of AtSURF1b deficiency on hormone responses. The results indicate that changes in AtSURF1b affect mitochondrial function and the production of reactive oxygen species, which, in turn, impinges on a growth regulatory circuit that involves auxin, gibberellins and the transcription factor PIF4.

Arabidopsis SCO Proteins Oppositely Influence Cytochrome c Oxidase Levels and Gene Expression during Salinity Stress.

SCO (synthesis of cytochrome c oxidase) proteins are involved in the insertion of copper during the assembly of cytochrome c oxidase (COX), the final enzyme of the mitochondrial respiratory chain. Two SCO proteins, namely, homolog of copper chaperone 1 and 2 (HCC1 and HCC2) are present in seed plants, but HCC2 lacks the residues involved in copper binding, leading to uncertainties about its function. In this study, we performed a transcriptomic and phenotypic analysis of Arabidopsis thaliana plants with reduced expression of HCC1 or HCC2. We observed that a deficiency in HCC1 causes a decrease in the expression of several stress-responsive genes, both under basal growth conditions and after applying a short-term high salinity treatment. In addition, HCC1 deficient plants show a faster decrease in chlorophyll content, photosystem II quantum efficiency and COX levels after salinity stress, as well as a faster increase in alternative oxidase capacity. Notably, HCC2 deficiency causes opposite changes in most of these parameters. Bimolecular fluorescence complementation analysis indicated that both proteins are able to interact. We postulate that HCC1 is a limiting factor for COX assembly during high salinity conditions and that HCC2 probably acts as a negative modulator of HCC1 activity through protein-protein interactions. In addition, a direct or indirect role of HCC1 and HCC2 in the gene expression response to stress is proposed.

The mitochondrial oxidation resistance protein AtOXR2 increases plant biomass and tolerance to oxidative stress.

This study demonstrates the existence of the oxidation resistance (OXR) protein family in plants. There are six OXR members in Arabidopsis that contain the highly conserved TLDc domain that is characteristic of this eukaryotic protein family. AtOXR2 is a mitochondrial protein able to alleviate the stress sensitivity of a yeast oxr1 mutant. It was induced by oxidative stress and its overexpression in Arabidopsis (oeOXR2) increased leaf ascorbate, photosynthesis, biomass, and seed production, as well as conferring tolerance to methyl viologen, antimycin A, and high light intensities. The oeOXR2 plants also showed higher ABA content, changes in ABA sensitivity, and modified expression of ABA- and stress-regulated genes. While the oxr2 mutants had a similar shoot phenotype to the wild-type, they exhibited increased sensitivity to stress. We propose that by influencing the levels of reactive oxygen species (ROS), AtOXR2 improves the efficiency of photosynthesis and elicits basal tolerance to environmental challenges that increase oxidative stress, allowing improved plant growth and biomass production.

Assembly of the Complexes of the Oxidative Phosphorylation System in Land Plant Mitochondria.

Plant mitochondria play a major role during respiration by producing the ATP required for metabolism and growth. ATP is produced during oxidative phosphorylation (OXPHOS), a metabolic pathway coupling electron transfer with ADP phosphorylation via the formation and release of a proton gradient across the inner mitochondrial membrane. The OXPHOS system is composed of large, multiprotein complexes coordinating metal-containing cofactors for the transfer of electrons. In this review, we summarize the current state of knowledge about assembly of the OXPHOS complexes in land plants. We present the different steps involved in the formation of functional complexes and the regulatory mechanisms controlling the assembly pathways. Because several assembly steps have been found to be ancestral in plants-compared with those described in fungal and animal models-we discuss the evolutionary dynamics that lead to the conservation of ancestral pathways in land plant mitochondria.

The mitochondrial copper chaperone COX19 influences copper and iron homeostasis in arabidopsis.

KEY MESSAGE: The mitochondrial metallochaperone COX19 influences iron and copper responses highlighting a role of mitochondria in modulating metal homeostasis in Arabidopsis. The mitochondrial copper chaperone COX19 participates in the biogenesis of cytochrome c oxidase (COX) in yeast and humans. In this work, we studied the function of COX19 in Arabidopsis thaliana, using plants with either decreased or increased COX19 levels. A fusion of COX19 to the red fluorescent protein localized to mitochondria in vivo, suggesting that Arabidopsis COX19 is a mitochondrial protein. Silencing of COX19 using an artificial miRNA did not cause changes in COX activity levels or respiration in plants grown under standard conditions. These amiCOX19 plants, however, showed decreased expression of the low-copper responsive miRNA gene MIR398b and an induction of the miR398 target CSD1 relative to wild-type plants. Plants with increased COX19 levels, instead, showed induction of MIR398b and other low-copper responsive genes. In addition, global transcriptional changes in rosettes of amiCOX19 plants resembled those observed under iron deficiency. Phenotypic analysis indicated that the roots of amiCOX19 plants show altered growth responses to copper excess and iron deficiency. COX activity levels and COX-dependent respiration were lower in amiCOX19 plants than in wild-type plants under iron deficiency conditions, suggesting that COX19 function is particularly important for COX assembly under iron deficiency. The results indicate that the mitochondrial copper chaperone COX19 has a role in regulating copper and iron homeostasis and responses in plants.

The Complexity of Mitochondrial Complex IV: An Update of Cytochrome c Oxidase Biogenesis in Plants.

Mitochondrial respiration is an energy producing process that involves the coordinated action of several protein complexes embedded in the inner membrane to finally produce ATP. Complex IV or Cytochrome c Oxidase (COX) is the last electron acceptor of the respiratory chain, involved in the reduction of O2 to H2O. COX is a multimeric complex formed by multiple structural subunits encoded in two different genomes, prosthetic groups (heme a and heme a3), and metallic centers (CuA and CuB). Tens of accessory proteins are required for mitochondrial RNA processing, synthesis and delivery of prosthetic groups and metallic centers, and for the final assembly of subunits to build a functional complex. In this review, we perform a comparative analysis of COX composition and biogenesis factors in yeast, mammals and plants. We also describe possible external and internal factors controlling the expression of structural proteins and assembly factors at the transcriptional and post-translational levels, and the effect of deficiencies in different steps of COX biogenesis to infer the role of COX in different aspects of plant development. We conclude that COX assembly in plants has conserved and specific features, probably due to the incorporation of a different set of subunits during evolution.

Interplay between cytochrome c and gibberellins during Arabidopsis vegetative development.

We studied the effect of reducing the levels of the mitochondrial electron carrier cytochrome c (CYTc) in Arabidopsis thaliana. Plants with CYTc deficiency have delayed growth and development, and reach flowering several days later than the wild-type but with the same number of leaves. CYTc-deficient plants accumulate starch and glucose during the day, and contain lower levels of active gibberellins (GA) and higher levels of DELLA proteins, involved in GA signaling. GA treatment abolishes the developmental delay and reduces glucose accumulation in CYTc-deficient plants, which also show a lower raise in ATP levels in response to glucose. Treatment of wild-type plants with inhibitors of mitochondrial energy production limits plant growth and increases the levels of DELLA proteins, thus mimicking the effects of CYTc deficiency. In addition, an increase in the amount of CYTc decreases DELLA protein levels and expedites growth, and this depends on active GA synthesis. We conclude that CYTc levels impinge on the activity of the GA pathway, most likely through changes in mitochondrial energy production. In this way, hormone-dependent growth would be coupled to the activity of components of the mitochondrial respiratory chain.

d-Lactate Dehydrogenase Links Methylglyoxal Degradation and Electron Transport through Cytochrome c.

Glycolysis generates methylglyoxal (MGO) as an unavoidable, cytotoxic by-product in plant cells. MGO scavenging is performed by the glyoxalase system, which produces d-lactate as an end product. d-Lactate dehydrogenase (d-LDH) is encoded by a single gene in Arabidopsis (Arabidopsis thaliana; At5g06580). It catalyzes in vitro the oxidation of d-lactate to pyruvate using flavin adenine dinucleotide as a cofactor; knowledge of its function in the context of the plant cell remains sketchy. Blue native-polyacrylamide gel electrophoresis of mitochondrial extracts combined with in gel activity assays using different substrates and tandem mass spectrometry allowed us to definitely show that d-LDH acts specifically on d-lactate, is active as a dimer, and does not associate with respiratory supercomplexes of the inner mitochondrial membrane. The combined use of cytochrome c (CYTc) loss-of-function mutants and respiratory complex III inhibitors showed that CYTc acts as the in vivo electron acceptor of d-LDH. CYTc loss-of-function mutants, as well as the d-LDH mutants, were more sensitive to d-lactate and MGO, indicating that they function in the same pathway. In addition, overexpression of d-LDH and CYTc increased tolerance to d-lactate and MGO Together with fine-localization of d-LDH, the functional interaction with CYTc in vivo strongly suggests that d-lactate oxidation takes place in the mitochondrial intermembrane space, delivering electrons to the respiratory chain through CYTc These results provide a comprehensive picture of the organization and function of d-LDH in the plant cell and exemplify how the plant mitochondrial respiratory chain can act as a multifunctional electron sink for reductant from cytosolic pathways.