Abstract
Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.
Citation: Umbach AL, Zarkovic J, Yu J, Ruckle ME, McIntosh L, et al. (2012) Comparison of Intact Arabidopsis thaliana Leaf Transcript Profiles during Treatment with Inhibitors of Mitochondrial Electron Transport and TCA Cycle. PLoS ONE 7(9): e44339. doi:10.1371/journal.pone.0044339 Editor: Zhengguang Zhang, Nanjing Agricultural University, China Received May 17, 2011; Accepted August 2, 2012; Published September 18, 2012 Copyright: ?2012 Umbach et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Science Foundation (www.nsf.gov; IBN0110768 to J.Y. and L.M. and IOB0344497 and IOS0822521 to D.M.R.) and the United States Department of Energy (www.energy.gov; DEFG0291ER20021 to L.M.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. �a Current address: National Renewable Energy Laboratory, Golden, Colorado, United States of America ???�b Current address: Department of Biology, Eidgenossische Technische Hochschule Zurich, Zurich, Switzerland �c Current address: Department of Fisheries Services, University of Washington, Seattle, Washington, United States of America �d Current address: Valent BioSciences, Long Grove, Illinois, United States of America
Introduction
Plant mitochondria and chloroplasts communicate with the cell nucleus to modify nuclear gene expression so that organelle and cell properties can be adjusted as metabolism and the environment change. For mitochondria, this signaling is termed mitochondrial retrograde regulation (MRR) [1], [2]. Reactive oxygen species (ROS) are generated by mitochondria (mtROS) as part of normal metabolism [3?] and mtROS appear to be signaling intermediates in MRR when mitochondrial function is perturbed [4], [7], [8]. MRR could be involved in plant response to stress because increases in mtROS have been associated with various biotic and abiotic stresses in plants [4], [9]. In addition to mtROS, mitochondrial calcium has been identified as a likely MRR signaling component [10]. Whether mtROS, calcium and/or
other molecules are necessary for all MRR, which nuclear genes are affected by MRR, and how much MRR contributes to the response of plants to environmental stresses are subjects of ongoing study. Plants with mutations in genes encoding mitochondrial electron transport chain (mtETC) components demonstrate the importance of mitochondria for many processes. Different Complex I mutations alone affect chloroplasts [11], cold acclimation [12], and development and stress resistance [13?5]. Large scale disruption of the mitochondrial genome can also make plants more heat tolerant [16]. However, for these and most other stable mutations causing mitochondrial dysfunction, whether an observed effect results directly from altered MRR or indirectly from compensatory mechanisms or metabolic limitations is difficult to discern because the mutant plants are in a steady state [17].
In one approach to the analysis of MRR, chemicals applied to leaves or suspension culture cells have been assessed for their ability to alter transcription of nuclear genes. Most work has focused on nuclear genes encoding mitochondrial proteins (NEMP genes), particularly genes for alternative NAD(P)H dehydrogenases (NDHs) and for alternative oxidases (AOXs). Together, NDH and AOX make a non-phosphorylating bypass pathway for the cytochrome pathway of the mtETC [18], and, accordingly, specific genes for AOXs and NDHs are often induced coordinately [19?2]. Two exogenous chemical treatments that may mimic MRR signals are H2O2, representing increased mtROS production, and organic acids that are part of the TCA cycle, including citrate and malate. These treatments all induce AOX [8], [19], [22?5] and NDH genes [19], [22]. The organic acids can induce AOX genes without a marked increase in cellular ROS (tobacco, Nicotiana tabacum [24]; soybean, Glycine max [23]), indicating that ROSindependent pathways inducing AOX and NDH, as well as ROSdependent ones, operate in cells. These results with organic acids have been interpreted more specifically as indicating that mtROSindependent MRR pathways also exist.