Antisense Expression of Mitochondrial ATP Synthase Subunits OSCP (ATP5) and (ATP3) Alters Leaf Morphology, Metabolism and Gene Expression in Arabidopsis

Department of Plant Agriculture, Bovey Bldg, University of Guelph, Guelph, ON N1G 2W1, Canada.
Plant and Cell Physiology (Impact Factor: 4.93). 09/2009; 50(10):1840-50. DOI: 10.1093/pcp/pcp125
Source: PubMed


Determination of the role of mitochondrial (mt) ATP synthesis in plant metabolism is complicated by chloroplastic ATP synthesis. To differentiate ATP synthesis from these two organelles, we created transgenic Arabidopsis plants in which two different subunits of the mt ATP synthase, the oligomycin sensitivity-conferring protein (OSCP) (=delta) (ATP5) and the gamma (ATP3) subunit, were expressed individually in antisense orientation under the control of a dexamethasone-inducible promoter. The phenotypic effects of antisense expression were identical for both atp5 and atp3. Seedling lethality resulted from induction during germination in the light, demonstrating the essentiality of both gene products. Reduced expression of either gene resulted in stunting of dark-grown (etiolated) seedlings, downward curling or wavy-edged leaf margins of light-grown plants and ball-shaped unexpanded flowers. Antisense induction reduced total ATP levels in dark-grown (etiolated) seedlings germinated on media lacking sucrose, but increased total ATP levels in induced light-grown plants and in induced dark-grown seedlings germinated on media containing sucrose. Induction reduced transcript levels for two transcription factors (TCP3 and TCP4) whose decreased expression is associated with a similar wavy-edged leaf phenotype in Arabidopsis, and increased transcript levels for dynamin-related proteins whose increased expression is associated with increased mt division. Reduced expression of these subunits of the mt ATP synthase is proposed to disturb cellular redox states, which ultimately manifest downstream as diverse and seemingly unrelated phenotypes.

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    • "Inducible knock down mutants for two ATP Synthase subunits have been constructed. Upon induction, ATP levels are decreased but no effect on pollen fitness was observed (Robison et al., 2009). In light of these data, the " ATP hypothesis " appears unlikely to explain the male-sterile phenotype of CMS lines; thus another mitochondrial function should be affected in CMS plants. "
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    ABSTRACT: Cytoplasmic male sterility (CMS) is a common feature encountered in plant species. It is the result of a genomic conflict between the mitochondrial and the nuclear genomes. CMS is caused by mitochondrial encoded factors which can be counteracted by nuclear encoded factors restoring male fertility. Despite extensive work, the molecular mechanism of male sterility still remains unknown. Several studies have suggested the involvement of respiration on the disruption of pollen production through an energy deficiency. By comparing recent works on CMS and respiratory mutants, we suggest that the "ATP hypothesis" might not be as obvious as previously suggested.
    Mitochondrion 04/2014; DOI:10.1016/j.mito.2014.04.009 · 3.25 Impact Factor
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    • "TCPs were shown to regulate nuclear-encoded mitochondrial genes and contribute in this way to the control of mitochondrial function, in particular TCA cycle function and core energy metabolism/amino acid metabolism (Giraud et al., 2010). Perhaps related to this, and in accordance with our results, TCPs were shown to be downregulated in mutants of the mitochondrial ATP synthase, suggesting that they could be controlled by ATP generation by mitochondria (Robison et al., 2009). Finally, numerous signaling components with known or potential connection to the SnRK1 pathway, such as AKINβ1, SnRK2.10, or SnRK3.10, were also uncovered as candidate miRNA targets. "
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    ABSTRACT: The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24 nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response.
    Frontiers in Plant Science 06/2013; 4:197. DOI:10.3389/fpls.2013.00197 · 3.95 Impact Factor
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    • "Again, similar results are seen in CMSII mutants where leaf extracts and protoplasts contained even higher total ATP levels compared with the wild type at the end of the dark period (Szal et al., 2008). Higher ATP concentrations were also measured in slow growing plants carrying antisense constructs for the ATP synthase subunits gamma and ATP5 (Robison et al., 2009). ATP and ADP levels in dRNAi lines were largely unchanged in our study and resulted in an unchanged ATP/ADP ratio. "
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    ABSTRACT: The mitochondrial ATP synthase (F(1)F(o) complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the δ-subunit links the membrane-embedded F(o) part to the matrix-facing central stalk of F(1). We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the δ-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the δ-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development.
    The Plant Cell 07/2012; 24(7):2792-811. DOI:10.1105/tpc.112.099424 · 9.34 Impact Factor
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