The mitochondrial copper chaperone COX11 has an additional role in cellular redox homeostasis.

Mitochondria are sites of cellular respiration, which is accompanied by the generation of dangerous reactive oxygen species (ROS). Cells have multiple mechanisms to mitigate the dangers of ROS. Here we investigate the involvement of the COX complex assembly chaperone COX11 (cytochrome c oxidase 11) in cellular redox homeostasis, using homologs from the flowering plant Arabidopsis thaliana (AtCOX11) and yeast Saccharomyces cerevisiae (ScCOX11). We found that AtCOX11 is upregulated in Arabidopsis seedlings in response to various oxidative stresses, suggesting a defensive role. In line with this, the overexpression of either AtCOX11 or ScCOX11 reduced ROS levels in yeast cells exposed to the oxidative stressor paraquat. Under normal growth conditions, both Arabidopsis and yeast COX11 overexpressing cells had the same ROS levels as the corresponding WT. In contrast, the COX11 knock-down and knock-out in Arabidopsis and yeast, respectively, significantly reduced ROS levels. In yeast cells, the ScCOX11 appears to be functionally redundant with superoxide dismutase 1 (ScSOD1), a superoxide detoxifying enzyme. The ΔSccox11ΔScsod1 mutants had dramatically reduced growth on paraquat, compared with the WT or single mutants. This growth retardation does not seem to be linked to the status of the COX complex and cellular respiration. Overexpression of putatively soluble COX11 variants substantially improved the resistance of yeast cells to the ROS inducer menadione. This shows that COX11 proteins can provide antioxidative protection likely independently from their COX assembly function. The conserved Cys219 (in AtCOX11) and Cys208 (in ScCOX11) are important for this function. Altogether, these results suggest that COX11 homologs, in addition to participating in COX complex assembly, have a distinct and evolutionary conserved role in protecting cells during heightened oxidative stress.

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.

The biochemical properties of the Arabidopsis ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 contradict a direct role in purinergic signaling.

The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three--GDP, IDP and UDP--were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca(2+), Mg(2+) and Mn(2+) were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling.

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.

Divergent functions of the Arabidopsis mitochondrial SCO proteins: HCC1 is essential for COX activity while HCC2 is involved in the UV-B stress response.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space.

BACKGROUND: The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function. RESULTS: Transgenic Arabidopsis lines expressing AtAPY1 fused to the SNAP-(O(6)-alkylguanine-DNA alkyltransferase)-tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate structures within the cell. The same signal pattern was found in seedlings stably overexpressing AtAPY1-GFP by indirect immunofluorescence and live imaging. In order to identify the nature of the AtAPY1-positive structures, AtAPY1-GFP expressing seedlings were treated with the endocytic marker stain FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)-pyridinium dibromide) and crossed with a transgenic line expressing the trans-Golgi marker Rab E1d. Neither FM4-64 nor Rab E1d co-localized with AtAPY1. However, live imaging of transgenic Arabidopsis lines expressing AtAPY1-GFP and either the fluorescent protein-tagged Golgi marker Membrin 12, Syntaxin of plants 32 or Golgi transport 1 protein homolog showed co-localization. The Golgi localization was confirmed by immunogold labeling of AtAPY1-GFP. There was no indication of extracellular AtAPY1 by indirect immunofluorescence using antibodies against SNAP and GFP, live imaging of AtAPY1-GFP and immunogold labeling of AtAPY1-GFP. Activity assays with AtAPY1-GFP revealed GDP, UDP and IDP as substrates, but neither ATP nor ADP. To determine if AtAPY1 is a soluble or membrane protein, microsomal membranes were isolated and treated with various solubilizing agents. Only SDS and urea (not alkaline or high salt conditions) were able to release the AtAPY1 protein from microsomal membranes. CONCLUSIONS: AtAPY1 is an integral Golgi protein with the substrate specificity typical for Golgi apyrases. It is therefore not likely to regulate extracellular nucleotide signals as previously thought. We propose instead that AtAPY1 exerts its growth and developmental effects by possibly regulating glycosylation reactions in the Golgi.

Extracellular nucleotides and apyrases regulate stomatal aperture in Arabidopsis.

This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5'-[γ-thio]triphosphate (ATPγS) or adenosine 5'-[ß-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5'-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATPγS- and adenosine 5'-[ß-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATPγS induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions.

HCC1, the Arabidopsis homologue of the yeast mitochondrial copper chaperone SCO1, is essential for embryonic development.

The Arabidopsis HCC1 gene is a homologue of the copper chaperone SCO1 from the yeast Saccharomyces cerevisiae. SCO1 (synthesis of cytochrome c oxidase 1) encodes a mitochondrial protein that is essential for the correct assembly of complex IV in the respiratory chain. GUS analyses showed HCC1 promoter activity in vascular tissue, guard cells, hydathodes, trichome support cells, and embryos. HCC1 function was studied in two hcc1 T-DNA insertion lines, hcc1-1 and hcc1-2. Gametophyte development was not affected by the disruption of HCC1, but homozygous hcc1-1 and hcc1-2 embryos became arrested at various developmental stages, mostly at the heart stage. Both the wild-type HCC1 gene and the modified gene coding for the C-terminally SNAP-tagged HCC1 were able to complement the embryo-lethal phenotype of the hcc1-1 line. Localization of the SNAP-tagged HCC1 in transgenic lines identified HCC1 as a mitochondrial protein. To determine if HCC1 is a functional homologue to Sco1p, the respiratory-deficient yeast sco1 mutant was transformed with chimeric constructs containing different combinations of HCC1 and SCO1 sequences. One of the resulting chimeric proteins restored respiration in the yeast mutant. This protein had the N-terminal mitochondrial targeting signal and the single transmembrane domain derived from Sco1p and the C-terminal half (including the copper-binding motif) derived from HCC1. Growth of the complemented yeast mutant was enhanced by the addition of copper to the medium. The data demonstrate that HCC1 is essential for embryo development in Arabidopsis, possibly due to its role in cytochrome c oxidase assembly.

Extracellular ATP: an unexpected role as a signaler in plants.

ATP and other nucleoside triphosphates not only drive energy-dependent reactions inside cells, but can also function outside the plasma membrane in the extracellular matrix, where they function as agonists that can induce diverse physiological responses without being hydrolyzed. This external role of ATP is well established in animal cells but only recently has it become apparent that extracellular ATP (eATP) can also function as a signaling agent in plants. Recent data have shown that eATP and other nucleotides can induce an increase in the cytosolic Ca(2+) concentration and diverse downstream changes that influence plant growth and defense responses. Ectoapyrase enzymes that regulate the eATP concentration also have an impact on plant growth. These results beg the question of whether there is a receptor that can bind to eATP and transduce this into signaling changes. Answering this will be key to understanding how eATP and ectoapyrases influence plant growth and development.

Developmental defects and seedling lethality in apyrase AtAPY1 and AtAPY2 double knockout mutants.

Previously it was shown that the Arabidopsis apyrase genes AtAPY1 and AtAPY2 are crucial for male fertility because mutant pollen (apy1-1; apy2-1) with T-DNA insertions in both genes could not germinate (Steinebrunner et al. (2003) Plant Physiol. 131: 1638-1647). In this study, pollen germination was restored and apyrase T-DNA double knockouts (DKO) apy1-1/apy1-1; apy2-1/apy2-1 were generated by complementation with AtAPY2 under the control of a pollen-specific promoter. The DKO phenotype displayed developmental defects including the lack of functional root and shoot meristems. In cotyledons, morphogenetic and patterning abnormalities were apparent, e.g., unlobed pavement cells and stomatal clusters. Another set of lines was created which carried either AtAPY1 or AtAPY2 under a dexamethasone-(DEX)-inducible promoter as an additional transgene to the pollen-specific gene construct. Application of DEX did not reverse the DKO phenotype to wild-type, but some inducible lines exhibited less severe defects even in the absence of the inducer, probably due to some background expression. However, even these DKO mutants were seedling-lethal and shared other defects regarding cell division, cell expansion and stomatal patterning. Taken together, the defects in the DKO mutants demonstrate that AtAPY1 and AtAPY2 are essential for normal plant development.

Apyrases (nucleoside triphosphate-diphosphohydrolases) play a key role in growth control in Arabidopsis.

Expression of two Arabidopsis (Arabidopsis thaliana) apyrase (nucleoside triphosphate-diphosphohydrolase) genes with high similarity, APY1 and APY2, was analyzed during seedling development and under different light treatments using beta-glucuronidase fusion constructs with the promoters of both genes. As evaluated by beta-glucuronidase staining and independently confirmed by other methods, the highest expression of both apyrases was in rapidly growing tissues and/or tissues that accumulate high auxin levels. Red-light treatment of etiolated seedlings suppressed the protein and message level of both apyrases at least as rapidly as it inhibited hypocotyl growth. Adult apy1 and apy2 single mutants had near-normal growth, but apy1apy2 double-knockout plants were dwarf, due primarily to reduced cell elongation. Pollen tubes and etiolated hypocotyls overexpressing an apyrase had faster growth rates than wild-type plants. Growing pollen tubes released ATP into the growth medium and suppression of apyrase activity by antiapyrase antibodies or by inhibitors simultaneously increased medium ATP levels and inhibited pollen tube growth. These results imply that APY1 and APY2, like their homologs in animals, act to reduce the concentration of extracellular nucleotides, and that this function is important for the regulation of growth in Arabidopsis.

Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis.

Extracellular ATP can serve as a signaling agent in animal cells, and, as suggested by recent reports, may also do so in plant cells. In animal cells it induces the production of reactive oxygen species through the mediation of NADPH oxidase. Similarly, here we report that in leaves of Arabidopsis (Arabidopsis thaliana), applied ATP, but not AMP or phosphate, induces the accumulation of superoxide (O2-) in a biphasic, dose-dependent manner, with a threshold at 500 nm ATP. This effect did not require ATP hydrolysis for it was mimicked by ATPgammaS. ATP also induced increased levels of Arabidopsis respiratory burst oxidase homolog D (AtrbohD) mRNA, but ATP-treated plants that had disrupted AtrbohD and AtrbohF genes did not accumulate O2-, indicating that NADPH oxidases are responsible for the induced O2- accumulation. Inhibitors of mammalian P2-type ATP receptors abolished ATP-induced O2- production, suggesting that the ATP effects may be mediated through P2-like receptors in plants. Cytosolic Ca2+ and calmodulin are likely to help transduce the ATP responses, as they do in animal cells, because a Ca2+ channel blocker, a Ca2+ chelator, and calmodulin antagonist all reduced ATP-induced O2- accumulation. Furthermore, ATP treatment enhanced the expression of genes that are induced by wounds and other stresses. The ATP measured at wound sites averaged 40 microm, well above the level needed to induce O2- accumulation and gene expression changes. Transgenic plants overexpressing an apyrase gene had reduced O2- production in response to applied ATP and wounding. Together, these data suggest a possible role for extracellular ATP as a signal potentially in wound and stress responses.

Disruption of apyrases inhibits pollen germination in Arabidopsis.

In Arabidopsis, we previously identified two highly similar apyrases, AtAPY1 and AtAPY2. Here, T-DNA knockout (KO) mutations of each gene were isolated in a reverse genetic approach. The single KO mutants lacked a discernible phenotype. The double KO mutants, however, exhibited a complete inhibition of pollen germination, and this correlated with positive beta-glucuronidase staining in the pollen of apyrase promoter:beta-glucuronidase fusion transgenic lines. The vast majority of the pollen grains of these mutants were identical to wild type in size, shape, and nuclear state and were viable as assayed by metabolic activity and plasma membrane integrity. Complementation with either AtAPY1 or AtAPY2 cDNA rescued pollen germination, confirming that the phenotype was apyrase specific. Despite the redundancy of the two apyrases in rescue potential, transmission analyses suggested a greater role for AtAPY2 in male gamete success. The effect of mutant apyrase on the transmission through the female gametophyte was only marginal, and embryo development appeared normal in the absence of apyrases. The male-specific double KO mutation is fully penetrant and shows that apyrases play a crucial role in pollen germination.