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.

Differential chromatin binding preference is the result of the neo-functionalization of the TB1 clade of TCP transcription factors in grasses.

The understanding of neo-functionalization of plant transcription factors (TFs) after gene duplication has been extensively focused on changes in protein-protein interactions, the expression pattern of TFs, or the variation of cis-elements bound by TFs. Yet, the main molecular role of a TF, that is, its specific chromatin binding for the direct regulation of target gene expression, continues to be mostly overlooked. Here, we studied the TB1 clade of the TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTORS (TCP) TF family within the grasses (Poaceae). We identified an Asp/Gly amino acid replacement within the TCP domain, originated within a paralog TIG1 clade exclusive for grasses. The heterologous expression of Zea mays TB1 and its two paralogs BAD1 and TIG1 in Arabidopsis mutant plants lacking the TB1 ortholog BRC1 revealed distinct functions in plant development. Notably, the Gly acquired in the TIG1 clade does not impair TF homodimerization and heterodimerization, while it modulates chromatin binding preferences. We found that in vivo TF recognition of target promoters depends on this Asp/Gly mutation and directly impacts downstream gene expression and subsequent plant development. These results provided new insights into how natural selection fine-tunes gene expression regulation after duplication of TFs to define plant architecture.

The lncRNA APOLO and the transcription factor WRKY42 target common cell wall EXTENSIN encoding genes to trigger root hair cell elongation.

Plant long noncoding RNAs (lncRNAs) are key chromatin dynamics regulators, directing the transcriptional programs driving a wide variety of developmental outputs. Recently, we uncovered how the lncRNA AUXIN REGULATED PROMOTER LOOP (APOLO) directly recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) modulating its transcriptional activation and leading to low temperature-induced RH elongation. We further demonstrated that APOLO interacts with the transcription factor WRKY42 in a novel ribonucleoprotein complex shaping RHD6 epigenetic environment and integrating signals governing RH growth and development. In this work, we expand this model showing that APOLO is able to bind and positively control the expression of several cell wall EXTENSIN (EXT) encoding genes, including EXT3, a key regulator for RH growth. Interestingly, EXT3 emerged as a novel common target of APOLO and WRKY42. Furthermore, we showed that the ROS homeostasis-related gene NADPH OXIDASE C (NOXC) is deregulated upon APOLO overexpression, likely through the RHD6-RSL4 pathway, and that NOXC is required for low temperature-dependent enhancement of RH growth. Collectively, our results uncover an intricate regulatory network involving the APOLO/WRKY42 hub in the control of master and effector genes during RH development.

The lncRNA APOLO interacts with the transcription factor WRKY42 to trigger root hair cell expansion in response to cold.

Plant long noncoding RNAs (lncRNAs) have emerged as important regulators of chromatin dynamics, impacting on transcriptional programs leading to different developmental outputs. The lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO) directly recognizes multiple independent loci across the Arabidopsis genome and modulates their three-dimensional chromatin conformation, leading to transcriptional shifts. Here, we show that APOLO recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) and controls RHD6 transcriptional activity, leading to cold-enhanced RH elongation through the consequent activation of the transcription factor gene RHD6-like RSL4. Furthermore, we demonstrate that APOLO interacts with the transcription factor WRKY42 and modulates its binding to the RHD6 promoter. WRKY42 is required for the activation of RHD6 by low temperatures and WRKY42 deregulation impairs cold-induced RH expansion. Collectively, our results indicate that a novel ribonucleoprotein complex with APOLO and WRKY42 forms a regulatory hub to activate RHD6 by shaping its epigenetic environment and integrate signals governing RH growth and development.

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.

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.

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.

The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity.

Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor.

Coordination of plant mitochondrial biogenesis: keeping pace with cellular requirements.

Plant mitochondria are complex organelles that carry out numerous metabolic processes related with the generation of energy for cellular functions and the synthesis and degradation of several compounds. Mitochondria are semiautonomous and dynamic organelles changing in shape, number, and composition depending on tissue or developmental stage. The biogenesis of functional mitochondria requires the coordination of genes present both in the nucleus and the organelle. In addition, due to their central role, all processes held inside mitochondria must be finely coordinated with those in other organelles according to cellular demands. Coordination is achieved by transcriptional control of nuclear genes encoding mitochondrial proteins by specific transcription factors that recognize conserved elements in their promoter regions. In turn, the expression of most of these transcription factors is linked to developmental and environmental cues, according to the availability of nutrients, light-dark cycles, and warning signals generated in response to stress conditions. Among the signals impacting in the expression of nuclear genes, retrograde signals that originate inside mitochondria help to adjust mitochondrial biogenesis to organelle demands. Adding more complexity, several nuclear encoded proteins are dual localized to mitochondria and either chloroplasts or the nucleus. Dual targeting might establish a crosstalk between the nucleus and cell organelles to ensure a fine coordination of cellular activities. In this article, we discuss how the different levels of coordination of mitochondrial biogenesis interconnect to optimize the function of the organelle according to both internal and external demands.