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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

The cytochrome c oxidase biogenesis factor AtCOX17 modulates stress responses in Arabidopsis.

COX17 is a soluble protein from the mitochondrial intermembrane space that participates in the transfer of copper for cytochrome c oxidase (COX) assembly in eukaryotic organisms. In this work, we studied the function of both Arabidopsis thaliana AtCOX17 genes using plants with altered expression levels of these genes. Silencing of AtCOX17-1 in a cox17-2 knockout background generates plants with smaller rosettes and decreased expression of genes involved in the response of plants to different stress conditions, including several genes that are induced by mitochondrial dysfunctions. Silencing of either of the AtCOX17 genes does not affect plant development or COX activity but causes a decrease in the response of genes to salt stress. In addition, these plants contain higher reactive oxygen and lipid peroxidation levels after irrigation with high NaCl concentrations and are less sensitive to abscisic acid. In agreement with a role of AtCOX17 in stress and abscisic acid responses, both AtCOX17 genes are induced by several stress conditions, abscisic acid and mutation of the transcription factor ABI4. The results indicate that AtCOX17 is required for optimal expression of a group of stress-responsive genes, probably as a component of signalling pathways that link stress conditions to gene expression responses.

Cytochrome c, a hub linking energy, redox, stress and signaling pathways in mitochondria and other cell compartments.

Cytochrome c (CYTc) is a soluble redox-active heme protein that transfers electrons from complex III to complex IV in the cyanide-sensitive mitochondrial respiratory pathway. CYTc biogenesis is a complex process that requires multiple steps until the mature active protein is obtained. CYTc levels and activity are finely regulated, revealing the importance of this protein not only as electron carrier but also in many other processes. In this article, we describe the role of CYTc in mitochondrial respiration, from its canonical role as electron carrier for ATP production to its involvement in protein import and the stabilization of respiratory complexes and supercomplexes. In plants, CYTc is connected to the synthesis of the antioxidant ascorbate and the detoxification of toxic compounds. Finally, CYTc is also a multi-functional signaling molecule that influences the balance between life and death, acting in energy provision for cellular functions or triggering programmed cell death. The confluence of several metabolic routes into a single protein that links redox reactions with energy producing pathways seems logical from the point of view of cellular economy, control and organization.

AtCOX10, a protein involved in haem o synthesis during cytochrome c oxidase biogenesis, is essential for plant embryogenesis and modulates the progression of senescence.

Cytochrome c oxidase (CcO) biogenesis requires several accessory proteins implicated, among other processes, in copper and haem a insertion. In yeast, the farnesyltransferase Cox10p that catalyses the conversion of haem b to haem o is the limiting factor in haem a biosynthesis and is essential for haem a insertion in CcO. In this work, we characterized AtCOX10, a putative Cox10p homologue from Arabidopsis thaliana. AtCOX10 was localized in mitochondria and was able to restore growth of a yeast Δcox10 null mutant on non-fermentable carbon sources, suggesting that it also participates in haem o synthesis. Plants with T-DNA insertions in the coding region of both copies of AtCOX10 could not be recovered, and heterozygous mutant plants showed seeds with embryos arrested at early developmental stages that lacked CcO activity. Heterozygous mutant plants exhibited lower levels of CcO activity and cyanide-sensitive respiration but normal levels of total respiration at the expense of an increase in alternative respiration. AtCOX10 seems to be implicated in the onset and progression of senescence, since heterozygous mutant plants showed a faster decrease in chlorophyll content and photosynthetic performance than wild-type plants after natural and dark-induced senescence. Furthermore, complementation of mutants by expressing AtCOX10 under its own promoter allowed us to obtain plants with T-DNA insertions in both AtCOX10 copies, which showed phenotypic characteristics comparable to those of wild type. Our results highlight the relevance of haem o synthesis in plants and suggest that this process is a limiting factor that influences CcO activity levels, mitochondrial respiration, and plant senescence.

Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III.

We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.

Delta subclass HD-Zip proteins and a B-3 AP2/ERF transcription factor interact with promoter elements required for expression of the Arabidopsis cytochrome c oxidase 5b-1 gene.

We have identified transcription factors that interact with a promoter region involved in expression of the Arabidopsis thaliana COX5b-1 gene, which encodes an isoform of the cytochrome c oxidase zinc binding subunit. Elements with the core sequence ATCATT, involved in induction by sugars, are recognized both in vitro and in one-hybrid assays in yeast by HD-Zip proteins from the delta subclass and, though less efficiently, by the trihelix transcription factor GT-3b. DistalB-like elements (CCACTTG), required for induction by abscisic acid (ABA), interact with ESE1, a member of the B-3 subgroup of AP2/ERF transcription factors. The HD-Zip protein Athb-21 and ESE1 are able to interact in yeast two-hybrid assays with the ABA responsive element binding factor AREB2/ABF4, which binds to a G-box absolutely required for expression of the COX5b-1 gene. Overexpression of the identified transcription factors in plants produces an increase in COX5b-1 transcript levels. Moreover, these factors are able to induce the expression of a reporter gene located in plants under the control of the relevant COX5b-1 promoter regions required for expression. Analysis of promoter regions of COX5b genes from different plant species suggests that the identified transcription factors were recruited for the regulation of COX5b gene expression at different stages during the evolution of dicot plants.