Anterograde and retrograde regulation of nuclear genes encoding mitochondrial proteins during growth, development, and stress.

Molecular Plant Advance Access published April 7, 2014

Anterograde and Retrograde Regulation of Nuclear Genes Encoding Mitochondrial Proteins During Growth, Development and Stress Sophia Nga,b,2, Inge De Clercqc,2, Olivier Van Akena, Simon R. Lawa, Aneta Ivanovaa, Patrick Willemsc,d, Estelle Girauda,e, Frank Van Breusegemc, James Whelanf,1 a ARC Centre of Excellence in Plant Energy Biology, University of Western b Joint Research Laboratory in Genomics and Nutriomics, College of Life Sciences, Zhejiang University, Hangzhou, 310058, P. R. China c Department of Plant Systems Biology, VIB, and Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B9052 Gent, Belgium d Department of Medical Protein Research and Department of Biochemistry, 9000 Ghent, Belgium. e Present address: Illumina, ANZ, 1 International Court, Scoresby Victoria 3179, Australia f Department of Botany, School of Life Science. La Trobe University, Bundoora 3086, Victoria, Australia 1

To

whom

correspondence

should

be

addressed.

E-mail

[email protected], tel. +61 (0)3 90327488, fax +61 (0)3 94791188 2

These authors contributed equally to this work.

Running title: Mitochondrial Signalling – Not a One Way Traffic Short Summary: Mitochondria are important organelles in plants as sensors and signaling of stress responses. Mitochondrial signaling integrates and optimises growth with energy metabolism and stress responses.

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ABSTRACT Mitochondrial biogenesis and function in plants requires the expression of over one thousand nuclear genes encoding mitochondrial proteins (NGEMPs). The expression of these genes is regulated by tissuespecific, developmental, internal and external stimuli that result in a dynamic organelle involved in both metabolic and a variety of signalling processes. Although the metabolic and biosynthetic machinery of these processes and the various signalling pathways involved are only beginning to be identified at a molecular level. The molecular components of anterograde (nuclear to mitochondrial) and retrograde (mitochondrial to nuclear) signalling pathways that regulate the expression of NGEMPs interact with chloroplast-, growth- and stress signalling pathways in the cell at a variety of levels, with common components involved in transmission and execution of these signals. This positions mitochondria as important hubs for signalling in the cell, not only in direct signalling of mitochondrial function per se, but also in sensing and/or integrating a variety of other internal and external signals. This integrates and optimises growth with energy metabolism and stress responses, which is required both in photosynthetic and non-photosynthetic cells.

Keywords: mitochondria; mitochondrial retrograde regulation (MRR); organellar crosstalk; signalling.

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mitochondria is relatively well understood, the factors that regulate

INTRODUCTION The traditional textbook view of mitochondria is that they are involved in metabolism and biosynthetic processes. With the availability of genome sequences and a broader view of the mitochondrial proteome from a combination of prediction, tagging or direct proteome studies, it is now apparent that more than a thousand proteins present in mitochondria carry out a range of additional functions. Mitochondria are not only a target for various stresses, but also play an important role in signalling and responding to of nuclear genes encoding mitochondrial proteins (NGEMPs) during growth, development and in response to adverse growth conditions, and the factors involved in defining these expression patterns will be outlined. Our knowledge of the signals that define these expression patterns and that are generated in mitochondria will also be discussed, within the concept of mitochondrial signalling. NGEMPs GENE EXPRESSION DURING GROWTH AND DEVELOPMENT Although mitochondria are essential for cell viability, they account for only a few percent of the total cell volume or protein content (Winter et al., 1994). As such, the determination of transcript abundance for mitochondrial proteins was quite limited for many years by both the lack of sequences of NGEMPs and their relatively low transcript abundancies. As a result, the initial studies examining gene expression for mitochondrial proteins were restricted to nuclear and mitochondrial encoded genes, which are highly abundant and relatively easy to detect using northern blotting. These studies, in monocots, suggested a model in which mitochondria were made early during development, and that after this burst of mitochondrial biogenesis, mitochondrial gene expression was relatively low (Ehrenshaft and Brambl, 1990; Topping and Leaver, 1990). The more recent analysis of turnover rates for transcripts of NGEMPs, confirms that many of the transcripts encoding functions of the mitochondrial metabolism have relatively long half-lives (Narsai et al., 2007). Transcript levels of the highly abundant subunits of glycine decarboxylase, an essential step in the photorespiratory pathway, are

3


stresses (Vanlerberghe, 2013). In this review, an overview of the expression

induced by light, and regulated in a similar manner to many photosynthetic compounds (McCabe et al., 2000). Transcripts encoding proteins of the respiratory chain, such as the adenine nucleotide translocator, the F1β subunit of the ATP synthase and the Rieske Iron-Sulfur Protein (RISP) of the cytochrome bc1 complex, are only abundant in specific organs, such as flowers and pollen tissue (McCabe et al., 2000). Interestingly, with many mitochondrial proteins being encoded by small gene families, often only one member was more prevalent in floral or pollen tissue (Huang et al., 1994; availability of complete genome sequences and the advent of advanced transcriptomic tools like QRT-PCR and arrays have now removed all obstacles of sensitivity when measuring transcript abundancies for NGEMPs. Seed germination is often used as a model to examine mitochondrial biogenesis, because early morphological and biochemical studies revealed that seed mitochondria contained little internal structure together with low respiratory chain capacities, but, upon imbibition, they rapidly transformed into electron dense structures, and gained high respiratory chain activity (Bewley and Black, 1994). Studies on maize and rice germination revealed populations of ‘light’ and ‘heavy’ mitochondria, proposing a maturation programme in which ‘light’ mitochondria, termed pro-mitochondria, matured into cristaecontaining and metabolising mature mitochondria, associated with sequential series of gene expression events. Surprisingly, the components of the mitochondrial electron transport chain (mtETC) are expressed only relatively late during seed germination. Biogenesis-related components, such as proteins involved in DNA or RNA metabolism (replication, transcription or translation), are expressed first (Howell et al., 2006; Howell et al., 2007b; Howell et al., 2007a; Howell et al., 2009). NGEMPs expression represents one of the earliest peaks in gene expression during germination in Arabidopsis (Figure 1) (Narsai et al., 2011; Law et al., 2012). Again, these genes do not encode components involved in metabolism, but rather in DNA replication, transcription and translation. Thus, a model for mitochondrial biogenesis during germination is that mitochondrial DNA

replication,

transcription

and

translation

represent

the

earliest 4


Grohmann et al., 1996; Zabaleta et al., 1998; Brennicke et al., 1999). The

mitochondrial events occurring prior to the expression of components involved in metabolism. This maturation model proposes an intense and early burst in mitochondrial biogenesis, that is supported by a baseline mitochondrial metabolic capacity that exists even in dry seeds, which is evident by low levels of oxygen consumption almost immediately upon imbibition (Bewley and Black, 1994; Howell et al., 2006; Howell et al., 2007b; Howell et al., 2007a). It is worth noting that the expression of the mitochondrial genome (from DNA replication, transcription, transcript processing and editing, to genes. Thus, the expression of the NGEMPs represents to a large extent the anterograde control of mitochondrial function. Especially during germination this seems to be developmentally programmed, and while the factors that control the expression of these genes essential for the expression of the mitochondrial genome is not known, this is likely to be coordinated with other cellular processes, possibly involving regulation by phytohormones. Looking beyond seed germination it is clear that expression of NGEMPs varies between tissues and during development (Figure 1; Supplemental Table 1) and that few NGEMPs display constitutive expression across all tissues or organs. Apart from the group of germination-specific genes discussed above, other notable groups of genes, that display restricted expression, are evident in mature pollen (Figure 1). NGEMPs expression is different in roots compared to shoot or rosette tissues, and small numbers of floral-specific expression patterns are also evident. Despite the current limitations in both coverage and sensitivity of proteomics, it is suggested that transcriptomic differences are also reflected at the protein level, with evident differences in abundance between floral, shoot and root tissue (Lee et al., 2012). DIURNAL REGULATION OF NGEMPs While the above datasets are comprehensive in terms of organs, tissue and development, they do not take into account that the expression of NGEMPs may show diurnal variation. While some initial reports suggested diurnal variation in transcript abundance (Escobar et al., 2004; Elhafez et al., 2006), 5


translation) is carried out completely by proteins encoded by nuclear located

regulatory mechanisms and the biological significance remained unclear. The identification of TCP transcription factors [named after the transcription factors TEOSINTE BRANCHED 1 (TB1) in maize (Zea mays), CYCLOIDEA (CYC) in Anthirrinum majus and PCF (PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR, PCNA) in rice (Oryza sativa)] as transcriptional regulators of both mitochondrial and clock components provided a mechanistic link of how NGEMPs may be regulated in a diurnal manner (Welchen and Gonzalez, 2005; Gonzalez et al., 2007; Comelli and Gonzalez, 2009; Welchen et al., identified the TCP transcription factor CCA1 HIKING EXPEDITION (CHE) as an interacting protein of TIMING OF CAB EXPRESSION1 (TOC1), an important clock component (Pruneda-Paz et al., 2009). The transcript levels of the TCP transcription factors, that can act as both positive and negative regulators of transcription, themselves displayed multi-phased diurnal patterns leading to a complex regulatory wiring mechanism. Importantly, for at least some genes, diurnal variation in transcript abundancies was also reflected in protein abundancies (Giraud et al., 2010; Lee et al., 2010). Studies have also shown that mitochondrial activity, specifically the tricarboxylic acid (TCA) cycle, is linked to the circadian clock. Thus, NGEMPs appear to be regulated diurnally, at the promoter, transcript, protein and activity level (Fukushima et al., 2009; Nakamichi et al., 2009). MATERNAL CONTROL OF GENE EXPRESSION In the simplest case when a female zygote is fertilised by a pollen cell, one would expect that gene expression in the diploid cells occurs at a similar rate from the maternal and paternal gene copies. However, for a number of genes one parental copy (usually the paternal) is silenced during early embryogenesis, so that gene expression levels are mostly maternally controlled. This phenomenon is termed gene imprinting or parent-of-origin effect (Feil and Berger, 2007), and is possibly explained by the parental conflict hypothesis which proposes that in mammals and plants where embryos can be fertilised by multiple male parents, it would be in the male parent’s favour to invest into a productive endosperm or placenta for its own 6


2009; Giraud et al., 2010). Studies on the circadian clock in Arabidopsis

progeny, hence outcompeting progeny from a different male parent. However, from the female parent’s perspective it would be advantageous to allow equal resource distribution between embryos from different fathers, to maximize the genetic variation of the mother’s progeny. This conflict of interest may be the driving force behind biased maternal gene expression during embryogenesis. Most imprinted genes encode nuclear proteins and proteins involved in plant hormone signal transduction (Ikeda, 2012). However, for most of these genes the functional contribution to endosperm and resource allocation is HAND-CONTAINING TRANSMEMBRANE-2) in Arabidopsis, which encodes a mitochondrial protein, as a maternally expressed gene is of special interest (Zhang et al., 2012). LETM proteins are evolutionary conserved and are involved in mitochondrial protein translation (Bauerschmitt et al., 2010; Zhang et al., 2012) and ion channelling (Nowikovsky et al., 2004; Jiang et al., 2009). In Arabidopsis, LETM1 and LETM2 were found to be essential proteins involved in mitochondrial protein translation. LETM1 is a highly stress inducible gene, while LETM2 is more constitutively present and maternally expressed during embryogenesis (Zhang et al., 2012). Several lines of evidence support the silencing of the paternal allele of LETM2, including reciprocal crosses and genotyping of the offspring, LETM2:GUS promoter analysis showing that GUS activity in embryo and endosperm is only present when the GUS construct comes from the female parent, and also direct sequencing of LETM2 polymorphisms between Col-0 and L.er F1 progeny. By controlling LETM2 levels during embryogenesis, the female parent gains direct control over mitochondrial translation and thus mitochondrial respiration and energy metabolism. This would provide an elegant mechanism for the female parent to direct the parental conflict in her favour by being able to provide equal resources for its entire offspring, independent of the male parent. The mechanism by which imprinting of LETM2 is controlled remains unclear, but a transposon present in the promoter of LETM2 may provide an attractive target site for RNA-directed DNA methylation (Haag and Pikaard, 2011; Zhang et al., 2012). This constitutes a first example of variable epigenetic control of NGEMPs, and it will be of interest in the future to fully 7


unclear. In this regard, the identification of LETM2 (LEUCINE ZIPPER-EF-

grasp the extent of epigenetic control over mitochondrial function. RETROGRADE CONTROL OF MITOCHONDRIAL GENE EXPRESSION The regulatory pathways described above are top down (anterograde) in nature in that they are driven from external or internal signals directly modifying the expression of NGEMPs. A bottom up pathway also exists, known as retrograde signalling, where mitochondria signal to the nuclei their functional state and steer altered expression of NGEMPs. Mitochondrial cerevisiae), by showing that perturbed respiratory function switched on a retrograde pathway that controls the expression of a variety of genes that alter metabolism (Liu and Butow, 2006), and has been since shown to be operational in all eukaryotes studied. While genome-wide transcriptomics allow a full view of NGEMPs responsiveness (Leister 2005; Schwarzländer et al., 2012 Van Aken and Whelan 2012). By far the pre-eminent model to study MRR in plants is the ALTERNATIVE OXIDASE (AOX) (Clifton et al., 2006; Rhoads and Subbaiah, 2007; Vanlerberghe, 2013). AOX catalyses cyanide insensitive oxygen consumption and oxidises its substrate without the generation of a proton motive force. It is induced by a wide variety of abiotic and biotic stresses, including photo-oxidative stress at the transcriptional, protein and activity level (Vanlerberghe and McIntosh, 1997; Millar et al., 2011). The expression characteristics of all NGEMPs in response to chemical and genetic mitochondrial perturbations were analysed. To assess the contribution of MRR signalling pathways within environmental stress responses and other intracellular signalling pathways, the MRR transcriptional landscape was overlayed with gene expression changes provoked by abiotic and biotic stress conditions, chloroplast perturbations and several oxidative stress conditions (Supplemental Table 2 for overview of experiments). Expression profiles of transcripts that were differentially expressed (p1 – see Supplemental material) in one third of the included experiments (mitochondrial, chloroplast, biotic, nutrient stress) or in one fourth (in case of abiotic stress) are displayed in Figure 2. At least two transcriptomic 8


retrograde regulation (MRR) was first demonstrated in yeast (Saccharomyces

footprints are apparent from the clustering: genes that are generally upregulated and genes that are generally down-regulated under most of the conditions, respectively in the upper and lower part of the heatmap (Figure 2). A comprehensive meta-analysis of the response of nuclear genes encoding chloroplastic proteins also revealed a set of genes that were up-regulated or down-regulated in transcript abundance (Richly et al., 2003; Leister 2005). AOX1a is induced over the five categories, being activated by a wide variety of mitochondrial and chloroplast perturbations, as well as different compounds, water and oxygen availability, temperature and pathogens. Several of these stresses are known to perturb the functioning of the mitochondria. Together with AOX1a, several other genes are generally stress responsive and cluster together with AOX1a (indicated by cluster I), including several heat shock proteins and chaperones (e.g. Hsp60, sHSP23.5, sHSP23.6 and MGE1), other components of the alternative respiratory chain (NDA2, NDB2 and UCP5), transporters and several SMALL AUXIN-UP RNA (SAUR)-like proteins (Figure 2, Cluster 1). Some of these genes are part of the so-called MITOCHONDRIAL DYSFUNCTION STIMULON (MDS; De Clercq et al., 2013). Other members of the MDS are: UPOX (an universal ROS marker gene), MGE1 (At5g55200), sHsp23.5 (encoding a small heat shock protein) and BCS1 (the AAA ATPase UBIQUINOL-CYTOCHROME C REDUCTASE SYNTHESIS1) (Van Aken et al., 2009; Giraud et al., 2012; Van Aken and Whelan, 2012). They bear a common cis-regulatory element in their promoters, the mitochondrial dysfunction motif (MDM), that arbitrates the MRR-mediated induction by NAM, ATAF1/2, and CUC2 (NAC) transcription factors (De Clercq et al., 2013; Ng et al., 2013b). Moreover, this MDM is sufficient for responsiveness to various pharmacological (e.g. Antimycin A (AA), monofluoro-acetic acid (MFA), rotenone and H2O2) and genetic (e.g. prohibitin mutant atphb3; Van Aken et al., 2007) mitochondrial perturbations, indicating the integration of multiple MRR pathways at the transcriptional or more upstream level. A W-box cis-element is also present in the MDS promoters, and the W-box binding WRKY transcription factors regulate the expression of these genes, not only under mitochondrial stress conditions, but

9


environmental stress conditions, such as nutrient deficiency, phytotoxic

also during high-light exposure (Van Aken and Whelan, 2012; Van Aken et al., 2013). The MDS is also responsive to a wider range of chloroplast perturbations: by methyl viologen treatments and in the singlet oxygen producing flu mutant (Giraud et al., 2012; Van Aken and Whelan, 2012); by increased chloroplastic nitric oxide in the suppressor of salicylic acid insensitivity of npr1-5 (ssi) mutant (Mandal et al., 2012); during inhibition of photosystem II by N-octyl-3-nitro-2,4,6-trihydroxybenzamide (PNO8); and by increased chloroplastic glutathione levels in the cysteine synthase 26 mutant It was suggested that the MDS genes act as a non-specific and primary defence layer in response to a multitude of stress conditions by avoiding (through alternative respiration) and by alleviating the effects of oxidative stress, which is a common factor during different stresses (Giraud et al., 2012). However, it is currently unclear whether both mitochondria and chloroplasts induce separate signalling pathways or whether they rather signal to each other to induce a common response (Woodson and Chory, 2008). The fact that the functioning of both organelles is strongly connected through metabolism, energy and redox status (Raghavendra and Padmasree, 2003; van Lis and Atteia, 2004; Noguchi and Yoshida, 2008) makes the latter scenario an attractive hypothesis. For example, in the tobacco cytoplasmic male sterile II (CMSII) mutant, which lacks mitochondrial complex I, the rate of photosynthesis is decreased (Sabar et al., 2000). Moreover, over-expression of ANAC013 increases tolerance to chloroplast-initiated oxidative stress in Arabidopsis, likely through MRR regulation of the MDS, including the alternative respiratory enzymes, which can alleviate photo-oxidative damage in the chloroplasts by efficiently dissipating excess reducing equivalents (Yoshida et al., 2006, 2007; De Clercq et al., 2013). A second cluster (cluster II) shows a general down-regulation over the five categories. This cluster contains genes encoding photorespiratory enzymes: components of the glycine decarboxylase complex (GDCH, GLDP1, GLDP2). Due to the intimate connection between photosynthesis and photorespiration, it is not surprising that the expression of these genes is also coordinated with that of photosynthetic genes (McCabe et al., 2000; Yoshida 10


(Bermudez et al., 2010).

and Noguchi, 2009). Interesting to note is the apparent up-regulation of this cluster in gun1 (genomes uncoupled-1) and gun5 mutants. Despite the overall responsiveness of cluster 1 NGEMPs to a wide range of mitochondrial perturbations in our meta-analysis, discrete expression patterns for subsets of genes can be recognised. Whereas some are induced by the majority of chemical and genetic mutations (MDS, AOX1a, UPOX, BCS1, sHSP23.5), others are only deregulated by specific mitochondrial perturbations (Figure 2); (De Clercq et al., 2013). Different expression characteristics during revealed in other genome-wide transcriptomic studies (Rhoads and Subbaiah 2007; Schwarzländer et al., 2012a, Umbach et al., 2012) and enforce the hypothesis that multiple MRR pathways are in place. In an attempt to obtain more insight into these pathways, Ho et al. (2008) analysed promoter activities and transcript abundancies of four stress-responsive NGEMPs (AOX1a, BCS1, NDB2 and UPOX) in response to rotenone, H2O2 and salicylic acid. The kinetics and magnitude of the expression varied significantly between different treatments and between the different genes upon the same treatment, suggesting discrete regulatory mechanisms. REGULATORS OF MITOCHONDRIAL RETROGRADE SIGNALLING Members of a wide range of transcription factor classes have been identified that were shown to bind to promoters of NGEMPs, indicating that the regulation of mitochondrial function is very deeply integrated into the regulatory context of the cell (Table 1, Figure 3). In broad terms, the identified transcription factors contain regulators of oxidative phosphorylation and protein import components, or on the other hand of alternative respiration and general stress response. Transcription factors binding the promoters of one or more subunits of all five respiratory complexes have been identified (Table 1), including PSST, TYKY and 55KDa subunits (complex I), SUCCINATE DEHYDROGENASE

2-3

(SDH2-3,

complex

II),

RISP

(complex

III),

CYTOCHROME C OXIDASE 5B-1 (COX5B-1, complex IV) and ATP50 (complex V). Although a series of potential regulators has been found for respiratory complex subunits, very little information is available about the direct regulatory function of these transcription factors during plant growth and 11


mitochondrial perturbation experiments of subsets of NGEMPs are also

development. Many of the binding sites in the target promoters have been shown to affect promoter activity in response to a variety of conditions, including light signals, diurnal cycling, sucrose, stress triggers and tissuespecific expression patterns. Perhaps the best understood function is the above described role of TCP transcription factors in controlling diurnal expression patterns, although modification of TCP levels only resulted in relatively mild expression changes of target genes, possibly due to the strong redundancy in this gene family (Welchen et al., 2009; Giraud et al., 2010). A CYTOCHROME C-2 and COX5B-1 target genes when overexpressed in plants, suggesting they are positive regulators (Welchen et al., 2009; Comelli et al., 2012). The transcription factors ABSCISIC ACID-INSENSITIVE 3 (ABI3), LEAFY COTYLEDON 2 and FUSCA 3 were shown to regulate the expression of SDH2-3 during different stages of seed maturation, while ABI3 was found to be required for normal SDH2-3 levels in different tissue types and growth stages (Roschzttardtz et al., 2009). Currently, the information on transcription factors that regulate NGEMPs related to classical cytochrome c respiration components is very scattered and focusing on only very few target genes. Therefore, there is a need for more genome-wide analyses of mutants in the different transcription factors identified, ideally over a range of developmental stages and treatments, to get a better idea of how respiration is controlled at the transcriptional level. Several transcription factors that bind promoters of alternative respiration and stress-responsive NGEMPs have been identified, mainly using AOX1a, NDB2, UPOX and AtBCS1 promoters as proxies. An increasingly detailed picture of how these transcription factors control their target genes has been obtained over the past five years. In brief, it has been shown that ABI4 keeps the AOX1a promoter constitutively repressed under normal conditions, and this repression can be lifted by external stimuli such as abscisic acid (ABA) and rotenone (Giraud et al., 2009). AtWRKY40 was shown to limit the induction of AOX1a, NDB2, UPOX and AtBCS1 when a stress such as high light and AA is applied, while AtWRKY63 was shown to positively affect their expression (Van Aken et al., 2013). A group of related 12


set of diverse transcription factors was shown to induce expression of the

NAC transcription factors that are bound to the endoplasmic reticulum (ER) membrane and released upon mitochondrial inhibition have also been shown to positively regulate the expression of AOX1a and a variety of genes encoding mitochondrial and non-mitochondrial proteins (De Clercq et al., 2013; Ng et al., 2013b). Although a basic insight into the positive and negative regulators of stress-responsive NGEMPs is established, there is a need for further exploration, because the current set of transcription factors does not seem to be responsible for responses to all known stimuli and stresses that The identification of a group of ER-bound NAC transcription factors regulating AOX1a helps to explain how signals can be transmitted from mitochondria to the nucleus. Their location in the ER, associated with actin filaments (Ng et al., 2013b), facilitates a close interaction between mitochondria and the ER. While reactive oxygen species (ROS) are well known to be involved in signalling (see below), it is unclear how these signals can be transmitted, due to the limited mobility of ROS, and also how the specificity of ROS signalling is achieved (Moller and Sweetlove, 2010). A close interaction between mitochondria and ER would allow a direct diffusion of ROS over small distances to activate the release of the membrane-bound NAC transcription factors, providing a means to relay the signal and also providing specificity in signalling. In addition to a range of transcription factors that act as both positive and negative regulators, components involved in signal transduction have also been identified (Table 1, Figure 3). Two of these components are KIN10 and CYCLIN DEPENDENT KINASE E;1 (CDKE;1) (Ng et al., 2013a). KIN10 is a catalytic subunit of the SNF1-related protein kinase (SnRK1) complex in plants, orthologous to the mammalian AMP-activated protein kinase complex (AMPK) and sucrose-non-fermenting 1 (SNF1) in yeast (Ghillebert et al., 2011) and has been proposed to be a point of integration of stress and energy signalling in plants (Arabidopsis) (Baena-Gonzalez et al., 2007; BaenaGonzalez and Sheen, 2008). CDKE;1 is a subunit of the kinase module of the plant mediator complex (Mathur et al., 2011) and has been shown to be required for the expression of AOX1a in Arabidopsis at both the transcriptional 13


induce genes such as AOX1a.

and post-transcriptional level (Ng et al., 2013a). It interacts with KIN10 in the nucleus. Furthermore, cdke;1 has been shown to display a genomes uncoupled (gun) phenotype, in response to the perturbation of the chloroplast redox, and a high-light phenotype (Blanco et al., 2014). Thus, CDKE;1 acts as an integrator of both mitochondrial and plastidic retrograde signals. Along with KIN10, which is involved in the regulation of sugar metabolism, ABI4 (Koussevitzky et al., 2007; Giraud et al., 2009) and AtWRKY40 (Van Aken et al., 2013) have been shown to be involved in both mitochondrial and retrograde signalling converges through these regulators. This molecular identification of the components integrating chloroplast and mitochondrial signaling is consistent with the frequent observations that AOX1a is responsive to a variety of treatments that affect chloroplast function (Van Aken and Whelan, 2012). While some factors identified as regulators of NGEMPs have been identified, such as ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2) (Lohrmann et al., 2001), little is known about how they may be activated. ARR2 is classified as one part of a two-component signalling system (Grefen and Harter 2004), in which one component serves as a sensing and response mechanism, via auto-phosphorylation by the histidine kinase activity of the sensor/response component. The second component, the response regulator, is phosphorylated, likely via intermediates in eukaryotes, in a phosphor relay sequence of events (Grefen and Harter 2004). Two component systems have been shown to be important in the regulation of hormone and stress signalling (Nongpiyr et al., 2012) and interaction with clock components (Mizuno and Nakamichi 2005). As yet it is unclear how ARR2 is activated to regulate the expression of NGEMPs for complex I subunits in plants, but the role of TCP transcription factors in regulating NGEMPs and their interaction with various clock components, including pseudo response regulators (PRR) (Giraud et al., 2010), provides a possible mechanism for the activation of ARR2. Also, BLAST (Basic Local Alignment Search Tool) analysis of an outer mitochondrial membrane protein (Duncan et al., 2011) reveals low sequence similarity to the Citrobacter freundii sensory histidine kinase CreC. An 14


chloroplast retrograde signaling. It is apparent that MRR and chloroplast

orthologous E. coli protein has been demonstrated to have dual functions, regulating the expression of the Cre regulon which encodes a variety of proteins including intermediary metabolic enzymes as well as cross regulating PhoB in the early response to phosphate starvation (Baek et al., 2007). CreC is the sensory subunit of a two component signalling system. Thus, it appears there may be sensory components present on the mitochondrial outer membrane that could play a role in activating ARR2, or other response regulators. how mitochondrial function is integrated with cellular function, it raises the question of what regulates or activates the regulators? As outlined above, TCP transcription factors that are implicated in the regulation of the expression of a variety of NGEMPs seemed to be regulated in a diurnal manner and interact with a variety of clock components (Giraud et al., 2010). For a variety of other components involved in regulating mitochondrial function (as listed in Table 1), few studies have been carried out directly on the regulating of these factors, especially with respect to regulating in terms of response to mitochondrial dysfunction. An analysis of the transcriptional response of these various regulators to the same stimuli as shown in Figure 2, reveal that there is no overall pattern observed, with some factors unresponsive (e.g. ANAC017, AT1G34190), while other show some response to some stresses (indicated with asterisk on Supplemental Figure 1 and in bold on Supplemental Table 3, e.g. AT1G32870, ANAC013). Overall, the most responsive set of regulators seems to be a group of WRKY transcription factors (Supplemental Figure 1). The complex redundancies observed with this large family of transcription factors and their regulation by a variety of abiotic and biotic stimuli is well documented (Rushton et al., 2010). However some details of the regulation of some of these transcription factors are known. ABI4, which acts as a repressor of AOX1a, is known to be regulated by the transcription factors SCARECROW, WRKY40 and the PHD TYPE TRANSCRIPTION FACTOR WITH TRANSMEMBRANE DOMAINS (PTM), that under various conditions relocated from the chloroplast to the nucleus to directly activate the expression of ABI4 (Leon et al., 2013). ABI4 is also known to be regulated at a post-transcriptional level mediated by the 26S 15


While identification of the regulators of NGEMPs provides a picture of

proteasome (Leon et al., 2013). As ABI4 has been characterised as a repressor of AOX1a, it would be interesting to elucidate how this repression is mediated, in other scenarios this is achieved by competing for binding of Gbox binding factors, thus blocking the positive action of positive regulators (Leon et al., 2013). THE NATURE OF MITOCHONDRIAL RETROGRADE SIGNAL(S) The nature of the mitochondrial retrograde signal is still yet to be retrograde signal, with some progress made with the identification of 3'phosphoadenosine 5'-phosphatehate (PAP), β-cyclocitral (β-CC), and 2-Cmethyl-D-erythritol 2, 4-cyclodiphosphate (MecPP), in addition to ROS and tetrapyrroles all implicated in chloroplast retrograde signalling (Xiao et al., 2013). While the possible nature of the mitochondrial retrograde signal has been reviewed previously (Schwarzländer and Finkemeier 2013), here the focus will be on the generation of these signals in mitochondrial processes and how the mitochondrial generation of these signals, some of which can be generated in other locations in the cell, can be specific for mitochondrial retrograde regulation, and in relation to the regulators outlined above. Reactive Oxygen Species In mitochondria, ROS are generated as part of normal metabolism in the mtETC. Once superoxide anions are formed, they can be converted into H2O2 by the mitochondrial manganese superoxide dismutase. ROS production is increased upon over-reduction of the ETC and upon perturbation of the individual respiratory complexes (Maxwell et al., 1999; Moller, 2001). For example, chemical inhibition of complex III by AA increases mtROS levels in tobacco cells (Maxwell et al., 1999; Rhoads and Subbaiah, 2007). During cold and salt stress, and phosphate starvation, mtETC impairment was associated with increased mtROS levels (Hernández et al., 1993a; Prasad et al., 1994; Parsons et al., 1999). First evidence for the signalling capacities of mtROS came from the finding that supplemental antioxidants could impair AAmediated induction of AOX transcript levels (Maxwell et al., 1999; 16


defined. Indeed there is much discussion as to what represent the chloroplast

Vanlerberghe et al., 2002). Recently, genetic evidence was provided for a key role of complex II in the formation of salicylic acid-triggered mtROS that steer plant stress gene expression. Mutations in the conserved region of the SDH-1 subunit

lowered

mtROS

production

and

downstream

stress

gene

responsiveness (Gleason et al., 2011). A comparative analysis of nuclear gene expression profiles responding to specific chemical disruptions of mitochondrial function and diverse oxidative stress treatments, consolidated that specific responses are associated with increased mtROS levels (Figure Whereas mitochondrial function perturbation is a prominent source of ROS production, increased cellular ROS levels can affect mitochondrial functions through oxidative damage. Exogenously applied H2O2 damages and degrades protein components of both the mtETC and the TCA cycle (Sweetlove et al., 2002). Hence, we can envisage a potential self-amplifying relay system that can be ignited by cellular ROS and in which mtROS can serve themselves as signalling molecules and/or generate secondary signals/triggers through the interaction with other molecules or proteins. How ROS-driven signals are transmitted to the nucleus to activate gene expression is still unclear. ROS-specific transcriptome fingerprints, however, are favouring scenarios in which local detection mechanisms are present inside or in close proximity to the mitochondria (Gadjev et al., 2006). For example, lipid peroxidation products could provoke specific signalling effects (Tang et al., 2002; Winger et al., 2005). Oxidised peptides derived from oxidatively damaged mitochondrial proteins could also act as specific ROS sensors/transducers (Sweetlove et al., 2002; Moller and Sweetlove, 2010). While peptide signalling has been shown to play an important role in plant development (Katsir et al., 2011), MRR signalling using peptides would require specificity in proteases to generate the peptides, transport across one and/or two mitochondrial membranes, and specific peptide receptors (of unknown location). Thus, while peptide signalling remains an attractive option to determine specificity of signalling, the generation, transport and detection of these signals raises many issues in signalling. Lipid peroxides and oxidised peptides in their turn might interact with proteinaceous signalling components, 17


2) (Rhoads and Subbaiah, 2007).

such as kinases/phosphatases and transcription factors to further convey the signal towards the nucleus (Tang et al., 2002; Haynes et al., 2010). Until now, no direct evidence for such protein compounds involved in MRR has been identified. Alternatively, ROS themselves could participate in the first steps of the signalling pathways. Both H2O2 and superoxide can leave the mitochondria via permeability transition pores upon mitochondrial dysfunction (Maxwell et al., 2002; Yao et al., 2002; Han et al., 2003). While it is evident that increased ROS levels will have a direct impact on the redox status (Foyer signalling in plant MRR is currently lacking. Energy and Metabolites While the concept of ROS signalling is clearly established, there may be a variety of other signals that are generated in mitochondria that could signal MRR. While there are limited studies, the energy status needs to be considered as a possible signal. In-depth analysis of a complex I mutant in Arabidopsis, i.e. nadh dehydrogenase ubiquinone iron-sulfur protein 4 (ndufs4), revealed that energy or adenylate control of metabolism was a major feature (Meyer et al., 2009). A direct RNA interference approach to reduce the amount of the mitochondrial ATP synthase via down-regulation of the δsubunit did result in a lowering of cellular ATP levels (Geisler et al., 2012). It was proposed that the resulting growth retardation, also observed with complex I mutants, was due to a metabolic re-adjustment to maintain energy homeostasis, rather than a direct consequence of an energy deficit. The combined ‘omic’ approaches of these two studies suggest that energy status does trigger cellular wide changes. The nature of the signals involved is unclear, but consequent changes in metabolite concentrations may act as signalling molecules. The original paradigm of MRR in yeast was based on the accumulation of citrate in a yeast mutant for mitochondrial citrate synthase (Liu and Butow, 2006). Based on this system, it was initially suggested that in tobacco citrate might act via a ROS independent pathway to induce AOX (Gray et al., 2004). However, later studies both in soybean (Glycine max) and Arabidopsis did not 18


and Noctor, 2003), experimental evidence for the involvement of redox

support the role of citrate as a retrograde signalling molecule for AOX1a induction. While treatment of cultures with citrate does result in changes of AOX at a transcript or protein level, these changes are small in magnitude, and the induction is only observed hours to days after treatment (Djajanegara et al., 2002; Finkemeier et al., 2013). Thus, it is difficult to envisage a signalling role for citrate in this context, but other target genes cannot be ruled out. A kinetic study using leaf slices concluded that both citrate and malate do target transcriptome changes, but notably AOX1a was only up-regulated 2induction with AA or H2O2, where peak induction is usually observed at 3 h and ranges from 10- to 50-fold (Ng et al., 2013a; Ng et al., 2013b). Thus, while citrate may have direct signalling activities, it does not seem to act as a signal for the induction of AOX. Mitochondrial Translation and Gene Expression While chloroplast gene expression has been implicated in chloroplast retrograde signalling, comparatively little is known about the signalling role of mitochondrial gene expression. In mouse, it has been proposed based on studies on mitochondrial ribosomal stalling that mitochondrial translation acts as a retrograde signal, inhibiting cell proliferation. While perturbing mitochondrial translation clearly has an effect on metabolism, it is proposed that this is due to a retrograde response, since mitochondrial stalling activates a pathway that results in the degradation of mitochondrial rRNA and mRNA pools (Richter et al., 2013). Direct studies on mutants affecting mitochondrial translation also suggest a role for mitochondrial translation as a signal in plants. A gene encoding a dual-targeted PROLYL-TRNA SYNTHETASE 1 (PRORS1) protein is targeted to both the mitochondria and chloroplasts (Jeannin et al., 1976). While knock-outs of PRORS1 are embryo lethal, leaky mutants cause a down-regulation of expression of nuclear genes encoding proteins involved in the light reaction of photosynthesis. Mutants affecting plastid (plastid ribosomal L11 protein) or mitochondrial (mitochondrial ribosomal protein L11) ribosome alone had no affect of the expression of the nuclear

genes

encoding

protein

involved

in

the

light

reaction

of 19


fold after 8 h of citrate treatment (Finkemeier et al., 2013), in contrast to

photosynthesis, but the double mutants had a similar response to the prors1 leaky mutants (Pesaresi et al., 2006). Thus, it is the combined translational stress in mitochondria and chloroplasts that results in a retrograde signal that alters gene expression. Furthermore, in the letm mutant lines outlined above, it has been shown that in lines that contain just a single functional allele for LETM2 (letm1 -/-; LETM2 +/-), organelle translation is reduced, resulting in MRR and alteration in the global transcriptome (Zhang et al., 2012). The majority of mitochondrial-encoded proteins are subunits of the components into the membrane to form intermediate assembly subcomplexes may also trigger a variety of signals that can be relayed to activate nuclear gene expression (Law et al., 2012). The recent report of a dual located protein, B14.7, located in the respiratory complex I and in the TRANSLOCASE OF THE INNER MEMBRANE 17:23 (TIM17:23), provides an example of how this may function. Alteration in the amount of either of these complexes results in a feedback on the other complex, so all complex I mutants have elevated levels of TIM17:23, and display higher import rates and also elevated levels of in organelle translation (Wang et al., 2012). Notably, in humans, the Tim21 subunit of TIM17:23 associates with newly imported proteins and regulates mitochondrial protein synthesis (Mick et al., 2012). A variety of other proteins are also dual functioning and/or dual located, such as the mitochondrial processing peptidase subunits of the cytochrome bc1 complex (Braun and Schmitz, 1997; Glaser and Dessi, 1999), and TIM21, that links TIM17:23 to the cytochrome bc1 complex (van der Laan et al., 2006; Murcha et al., [Submitted]). Some of these proteins may have signalling functions in addition to their biochemical role in a protein complex. Dual-Targeted Proteins It has been proposed that dual-targeted proteins, which can be targeted to mitochondria and the nucleus, could act as retrograde signalling molecules (Duchene and Giege 2012), e.g. PNM1 (a pentatricopeptide protein located in nuclei and mitochondria) (Hammani et al., 2011). The re-location of factors bound to the chloroplast membrane seems to be an emerging theme

20


multi-subunit complexes of the ETC. Production and insertion of these

in chloroplast retrograde signalling (Sun et al., 2011; Shang et al. 2010). However, while proteins may be located in two locations, direct re-location under specific signalling needs to be shown before a role in retrograde regulation can be concluded. Dual-targeted protein may also play a role in organelle-to-organelle signalling. Over 100 proteins have been shown to be dual-targeted in Arabidopsis (Carrie and Whelan 2013), and also dual targeting seems to have arisen and to be largely conserved early in land plant evolution (Xu et al., organelle, activating gene expression for genes encoding dual targeted proteins, and modulate the activity in another organelle where the protein is also targeted. As many dual targeted protein to mitochondria and chloroplasts are involved in organelle gene expression or various reactions associated with redox balance, signals from one organelle can modify these activities in another organelle. Thus for enzymes involved in the ascorbate-glutathione cycle in plants that are dual targeted, the expression can be affected by chloroplast or mitochondrial perturbation (Chew et al., 2003). Known Signalling Molecules Mitochondrial functions that are integrated and coordinated with other cellular functions are necessary for growth and development. Thus, signalling molecules characterised in other systems can also function in mitochondria. PAP, a by-product of sulfur assimilation has been identified as a chloroplast retrograde signal during drought and high light in Arabidopsis (Estavillo et al., 2011). The protein that regulates the level of PAP in cells, SAL1, a phosphatase converting PAP to AMP, is also located in mitochondria and chloroplasts. While its physiological function is not clear, since PAP is a mobile signal, the location of the protein that controls its levels in multiple compartments, suggests that PAP may also be a mitochondrial retrograde signal or that mitochondria affect the retrograde signals from chloroplasts. In a previous meta-analysis of transcriptomes of mitochondrial or chloroplast perturbation, it was noted that the alterations of the transcriptome in the sal1 mutant clearly clustered with mutants and treatments affecting mitochondrial function (Van Aken and Whelan, 2012). The fact that one of the most notable 21


2013). These proteins may coordinate organelle activity, as signals from one

phenotypes for aox1a mutants in Arabidopsis is observed with a combined high-light and drought treatment, the same treatments that elicit chloroplast PAP retrograde signalling, suggests that there is at least some integration of both mitochondrial and chloroplast retrograde signalling via PAP. Calcium Ions The uptake and release of calcium ions (Ca2+) in mitochondria has been the subject of extensive studies in the animal field, where it is with the adjacent ER to produce Ca2+ microdomains and affect Ca2+ spikes. In animals, the ER releases Ca2+ via the inositol-1,4,5-triphosphate (IP3) and ryanodine receptors (RyR) at contact sites with the mitochondria, stimulating mitochondrial Ca2+ uptake (de Brito and Scorrano, 2010). Increased Ca2+ concentration inside mitochondria also has positive effects on ATP synthesis through directly or indirectly (via Ca2+-dependent dephosphorylation) stimulating the activity of TCA cycle enzymes, such as alpha-ketoglutarate, isocitrate and pyruvate dehydrogenases (Bernardi, 1999), as well as mitochondrial metabolite transporters (Lasorsa et al., 2003) and manganese superoxide dismutase (Hopper et al., 2006). Paradoxically, Ca2+ import into the mitochondrial matrix is energetically demanding and can result in loss of inner mitochondrial transmembrane potential (Duchen, 2000). Furthermore, excessive Ca2+ in the mitochondrial matrix can lead to opening of the permeability transition pore, which allows ions and small molecules to diffuse, leading to a reduced mitochondrial membrane potential and even cell death (Bernardi, 1999; Hajnoczky et al., 2006). Despite the multitude in information on mitochondrial Ca2+ metabolism in animals and yeast, little is known in plants. It has been described that Ca2+ and calmodulin – a Ca2+ binding protein – can promote mitochondrial protein import (Kuhn et al., 2009), while calmodulin inhibitors impair pyruvate dehydrogenase activity (Miernyk and Randall, 1987). Ca2+ was also shown to induce mitochondrial swelling and cytochrome c release in wheat (Virolainen et al., 2002) and to affect fluctuations in the mitochondrial transmembrane potential in Arabidopsis (Schwarzländer et al., 2012b). Very little is also 22


established that mitochondria act as calcium stores that can exchange Ca2+

known about plant counterparts of the Ca2+ transporters. Homologues of the low-affinity mitochondrial calcium uniporter (Baughman et al., 2011; De Stefani et al., 2011), its regulatory protein mitochondrial calcium uptake 1 (MICU1) (Perocchi et al., 2010), and the mitochondrial Ca2+/H+ antiporter LETM1 (Jiang et al., 2009) are conserved in plants (Stael et al., 2012), but remain

poorly

characterised.

AtLETM1

and

AtLETM2

have

been

experimentally confirmed to be localised in the mitochondria (Van Aken et al., 2009; Zhang et al., 2012), and were shown to be essential for viability via proteins contain a putative Ca2+ binding EF-hand motif, a direct role in Ca2+ transport has not been reported. Pharmacological inhibition of mitochondrial Ca2+-flux sensing reduced AOX1a expression during salt stress, indicating that mitochondrial Ca2+-flux sensing is at least necessary to induce salt-stress triggered MRR (Vanderauwera et al., 2012). A significant research effort is therefore required to obtain further insight into the role of mitochondrial Ca2+ homeostasis in various aspects of mitochondrial function and signalling. The physical interaction between mitochondria and the ER in yeast and animal systems (Copeland and Dalton, 1959; Achleitner et al., 1999) facilitates Ca2+ transport between both organelles (Hayashi et al., 2009; Elbaz and Schuldiner, 2011). It was shown that Ca2+-mediated ER-mitochondria crosstalk is important for AOX1a induction in Arabidopsis during salt stress conditions, indicating it might be part of plant MRR (Vanderauwera et al., 2012). Moreover, the ER also signals to the nucleus during the so-called unfolded protein response (UPR). The UPR is an evolutionarily conserved transcriptional response to preserve ER homeostasis upon accumulation of unfolded proteins (Urade, 2007). The UPR might be involved in the Ca2+mediated AOX1a induction by regulating the activity of an ER Ca2+ pump, indicative for a tight link between ER-to-nucleus communication and MRR. FUTURE OUTLOOK While the understanding of the molecular components that control the expression of NGEMPs has progressed in the last decade, still only a handful of these regulators are known (Table 1, Figure 3), and importantly the 23


positively affecting mitochondrial protein translation. Although AtLETM

regulatory targets of these transcription factors are not fully elucidated. The latter is important to give the context in which NGEMPs are regulated. Largescale efforts that map the binding sites of all transcription factors will be useful to understand the regulatory context of NGEMPs. While AOX has served as a useful model for MRR, approaches using a variety of resources that are now available need to be undertaken, such as using other promoters and stress inducible genes that encode mitochondrial proteins. Furthermore, while signalling pathways from the mitochondria and chloroplasts to the nucleus chloroplasts (plastids) communicate at a signalling level, if they indeed do so. While there is clearly traffic of metabolites between these two organelles, and they also share many proteins, i.e. dual-targeted protein, the question if they signal each other via other means still remains unanswered. Currently, studies analysing organelle retrograde signalling rely on treatments that may not have a signal target, or T-DNA insertional knock-outs, in which the initial signalling events may be long missed. Thus, inducible genetic interventions would be informative to analyse the initial stages of organelle signalling. Furthermore, the emerging areas of cell specificity and epigenetic regulation need to be explored. Retrograde signalling is likely to differ between shoots and roots, but a finer cellular control may also exist. Finally, all our knowledge on the regulation of the expression of NGEMPs comes almost exclusively from Arabidopsis. Moving to other plant models that may be more agronomically relevant may be informative. In this respect rice is an attractive target, an important model and crop monocot species. SUPPLEMENTAL DATA Supplemental Data are available at Molecular Plant Online. FUNDING This work was supported by grants to J.W. from the Australian Research Council (CEO561495; CE140100008). O.v.A. is funded by an Australian Research Council Australian Postdoctoral fellowship (DP110102868). This work was also supported by grants to F.V.B. from Ghent University (Multidisciplinary Research Partnership “Biotechnology for a Sustainable 24


have been characterised, an important question is how mitochondria and

Economy”

[Grant

01MRB510W]

and

special

research

fund

project

[01J11311]), and from the Interuniversity Attraction Poles Programme (IUAP P7/29 “MARS”), initiated by the Belgian Science Policy Office. I.D.C. is indebted to the Agency for Innovation by Science and Technology for a predoctoral fellowship. ACKNOWLEDGMENTS We thank Szymon Kubiszewski-Jakubiak for help with Figure 3 and Annick Downloaded from http://mplant.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014

Bleys for help in preparing the manuscript. No conflict of interest declared.

25

FIGURE LEGENDS Figure 1. Transcript Abundance of Mitochondrial Genes During Development in Arabidopsis thaliana. A list of 980 mitochondrial genes was curated as outlined in Law et al., 2012 (Supplemental Table 1) and referenced against the publically available AtGenExpress developmental set (Schmid et al., 2005), which sampled wild-type tissues collected throughout

seedling, flower, leaf, root and pollen. In addition, the germination time course outlined in Narsai et al. (2011) was utilised to give an in-depth view of germination. The resulting data set was hierarchically clustered based on Euclidean distance as outlined in Law et al. (2012).

Figure 2. Transcript Abundance of the Stress-Responsive NGEMPs During Environmental Stress and Organellar Dysfunction Conditions. Hierarchical clustering of the expression profiles of the 118 stress-responsive NGEMPs (p< 0.01, |log2-fold change| >1, in one third of the included experiments (mitochondrion, chloroplast, biotic, nutrient) or in one fourth (in case of abiotic stress). Colour codes represent the actual log2-fold changes in transgenic or treated plants compared with wild-type or untreated plants, respectively. See supplemental material for details of clustering.

Figure 3. The Expression of Nuclear Genes Encoding Mitochondrial Proteins Is Regulated by a Complex Network of Signals That Operate Under Growth Permitting and Stress Conditions. Signals coming from outside the cell or from different intracellular locations are transmitted by a variety of messenger molecules, leading to the (de)activation of transcription 26


an exhaustive developmental time course from Arabidopsis; including seed,

factors. Abbreviations: PAP: 3'-phosphoadenosine 5'-phosphatehate; TCA: tricarboxylic acid; LeuZip: leucine zipper; ROS: reactive oxygen species.


27

Table 1. Transcription Factors Known to Bind Promoters of Nuclear Genes Encoding Mitochondrial Proteins. Transcription factors that were previously described as binding NGEMPs are listed by transcription factor class, name and AGI identifier. The name and AGI of the bound NGEMP target gene is shown as well as the sequence and position of the binding site relative to the transcriptional start site. Transcription factor class

Transcription factor name

AGI

Target gene(s)

AGI

Binding site(s)

ERF/AP2

ABI4

At2g40220

AOX1a

At3g22370

CCAC (-1580)

" Comelli et al., 2012

NAM

TCP

ESE1

At3g23220

COX5B-1

At3g15640

CACCG (-1593) CCACTTG (-117 and -99)

AtWRKY9

At1g68150

AOX1a

At3g22370

ATTGACA (-411)

Van Aken et al., 2013

AtWRKY13

At4g39410

TTTGACC (-290)

"

AtWRKY15

At2g23320

NDB2

At4g05020

TTTGACT (-213)

"

AtWRKY27

At5g52830

AtBCS1

At3g50930

CTTGACG (-321)

"

AtWRKY30

At5g24110

"

AtWRKY33

At2g38470

"

AtWRKY40

At1g80840

"

AtWRKY42

At4g04450

"

AtWRKY45

At3g01970

"

AtWRKY57

At1g69310

"

AtWRKY63

At1g66600

"

AtWRKY75

At5g13080

"

ANAC017

At1g34190

ANAC013

At1g32870

ANAC016

At1g34180

ANAC053

At3g10500

ANAC078

At5g04410

TCP20

At3g27010

AOX1a

AOX1a

UPOX

At3g22370

At3g22370

At2g21640

ACACG (-308)

Ng et al., 2013

CACGCA (-261)

"

CGTGT (-91)

"

CTTGgcgacCACG (-262) CTTGgagagCAAG (-235) CTTGctctcCAAG (-646)

De Clercq et al., 2013 " " "

Cytc-2

At4g10040

AGCCCA (-257)

Welchen et al., 2009

AGCCCA (-269) TCP1-24 (except TCP8, TCP16 and TCP22)

3x TGGGCC/T

Giraud et al., 2010

RISP

At5g13440

ATP50

At5g47030

"

MCP4

At5g27520

"

SCO1

At3g08950

"

TOM20-2

At3g27080

"

MCP3

At2g30160

"

28


WRKY

Reference Giraud et al., 2009

Transcription factor class

Transcription factor name

AGI

Target gene(s)

AGI

Binding site(s)

Leucine zipper

GBF1

At4g36730

Cytc-2

At4g10040

-189 to -139

Reference Welchen et al., 2009

GBF3

At2g43270

Cytc-2

At4g10040

-189 to -139

"

GBF-Like

At1g32150

Cytc-2

At4g10040

-189 to -139

"

ABF4

At3g19290

Cytc-2

At4g10040

-189 to -139

Athb-6

At2g22430

Cox5-b1

At3g15640

−169 to −95

" Comelli et al., 2012

Athb-21

At2g18550

Cox5-b1

At3g15640

ATCATT (-127)

Athb-40

At4g36740

Cox5-b1

At3g15640

−169 to −95

bZIP53

At3g62420

SDH2-3

At5g65165

CACGTA (-100)

" Roschzttardtz et al., 2009

bZIP10

At4g02640

SDH2-3

At5g65165

CACGTA (-100)

"

bZIP25

At3g54620

SDH2-3

At5g65165

CACGTA (-100)

"

PHD

AL5

At5g20510

Cox5-b1

At3g15640

−169 to −95

Comelli et al., 2012

bHLH

bHLH80

At1g35460

Cytc-2

At4g10040

-189 to -139

Welchen et al., 2009

bHLH81

At4g09180

Cytc-2

At4g10040

-189 to -139

"

Trihelix

GT-3b

At2g38250

Cox5b-1

At3g15640

ATCATT (-127)

Response regulator

ARR2

At4g16110

PSST

At5g11770

-215 to -171

Lohrmann et al., 2001

"

TYKY

At1g16700

-144 to -100

"

"

55/51kDA

At5g08530

-176 to -132

"

AOX1a

At3g22370

CGTGAT (-1589)

SDH2-3

At5g65165

CATGCA (-45)

MYB

Comelli et al., 2012

FUS3

At3g26790

SDH2-3

At5g65165

CATGCA (-45)

Giraud et al., 2009 Roschzttardtz et al., 2009 Roschzttardtz et al., 2009

MYB80

At5g56110

UNDEAD

At4g12920

AAACCA (-254 &104)

Phan et al., 2011

CTAACCT (-159)

"

ABI3

At3g24650

29


B3 domain

"

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Mitochondrial genes

Seed

Figure 1 Transcript abundance of mitochondrial genes during Arabidopsis thaliana development. A list of mitochondrial genes was curated using a combination of localisation data and and over 40 relevant publications (as outlined in Law et al., 2012). This list was referenced against the publically available AtGenExpress developmental set (Schmid et al., 2005), which sampled wild type Arabidopsis tissues collected throughout an exhaustive developmental time course; including seed, seedling, flower, leaf, root and pollen. In addition, the germination time course outlined in Narsai et al., 2011 was utilised to increase the coverage of this analysis. The resulting data set was hierarchically clustered based on Euclidean distance.


Relative expression level


Figure 2. Transcript Abundance of the Stress-Responsive NGEMPs During Environmental Stress and Organellar Dysfunction Conditions. Hierarchical clustering of the expression profiles of the 118 stress-responsive NGEMPs (p< 0.01, |log2-fold change| >1, in one third of the included experiments (mitochondrion, chloroplast, biotic, nutrient) or in one fourth (in case of abiotic stress). Colour codes represent the actual log2-fold changes in transgenic or treated plants compared with wild-type or untreated plants, respectively. See supplemental material for details of clustering.

Growth stimuli hυ

Germination & Growth

CHLOROPLASTS Diurnal regulation

TCP ABI3 FUS3 LeuZip ARR2

High light stress

PAP KIN10 MITOCHONDRIA ATP:ADP

Translation

ROS Ca2+ TCA intermediates Oxidised peptides & lipids


NUCLEUS

Stress response MEDIATOR COMPLEX

CDKE ABI4 NAC WRKY15/40/63

mitochondrial stress response genes

Figure 3. The Expression of nuclear genes encoding mitochondrial proteins Is regulated by a complex network of signals that operate under growth permitting and stress conditions. Signals coming from outside the cell or from different intracellular locations are transmitted by a variety of messenger molecules, leading to the (de)activation of transcription factors. Abbreviations: PAP: phosphoadenosine phosphate; TCA: tricarboxylic acid; LeuZip: leucine zipper; ROS: reactive oxygen species.

NAC

Catalase

H2O2

PEROXISOMES ENDOPLASMIC RETICULUM

mitochondrial biogenesis & respiration genes

Regulation of nuclear genes encoding chloroplast proteins in transgenic plants.

Transcriptional regulation of genes encoding ABA metabolism enzymes during the fruit development and dehydration stress of pear 'Gold Nijisseiki'.

Expression of nuclear and plastid genes for photosynthesis-specific proteins during tomato fruit development and ripening.

Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development.

Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae.

Regulation by thyroid hormone of nuclear and mitochondrial genes encoding subunits of cytochrome-c oxidase in rat liver and skeletal muscle.

Genes Encoding Cucumber Full-Size ABCG Proteins Show Different Responses to Plant Growth Regulators and Sclareolide.

The hippocampal-mamillary system: anterograde and retrograde amnesia.

Characterization and mapping of human genes encoding zinc finger proteins.

Expression of nuclear and mitochondrial genes encoding ATP synthase is synchronized by disassembly of a multisynthetase complex.

Transcriptional regulation of genes encoding proteins involved in biogenesis of peroxisomes in Saccharomyces cerevisiae.

Opposing roles of mitochondrial and nuclear PARP1 in the regulation of mitochondrial and nuclear DNA integrity: implications for the regulation of mitochondrial function.

Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

Regulation and expression of a growth arrest-specific gene (gas5) during growth, differentiation, and development.

Analysis of the polyadenylation consensus sequence context in the genes of nuclear encoded mitochondrial proteins.

Genes encoding aromatases in teleosts: evolution and expression regulation.

Nuclear-mitochondrial proteins: too much to process?

Complex formation between nuclear RNA and mitochondrial proteins.

His-Purkinje conduction during retrograde stress.

Down-regulation of GIGANTEA-like genes increases plant growth and salt stress tolerance in poplar.

Differential expression of nuclear- and organelle-encoded genes during tomato fruit development.

Chloroplast Retrograde Regulation of Heat Stress Responses in Plants.

The phosphorylation-dependent regulation of nuclear SREBP1 during mitosis links lipid metabolism and cell growth.

Different tissue distribution and hormonal regulation of messenger RNAs encoding rat insulin-like growth factor-binding proteins-1 and -2.