Photosynth Res DOI 10.1007/s11120-015-0161-6

REGULAR PAPER

Dysfunctional chloroplasts up-regulate the expression of mitochondrial genes in Arabidopsis seedlings Jo-Chien Liao1 • Wei-Yu Hsieh1 • Ching-Chih Tseng1 • Ming-Hsiun Hsieh1

Received: 25 December 2014 / Accepted: 20 May 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Chloroplasts and mitochondria play important roles in maintaining metabolic and energy homeostasis in the plant cell. The interactions between these two organelles, especially photosynthesis and respiration, have been intensively studied. Still, little is known about the regulation of mitochondrial gene expression by chloroplasts and vice versa. The gene expression machineries in chloroplasts and mitochondria rely heavily on the nuclear genome. Thus, the interactions between nucleus and these organelles, including anterograde and retrograde regulation, have been actively investigated in the last two decades. Norflurazon (NF) and lincomycin (Lin) are two commonly used inhibitors to study chloroplast-to-nucleus retrograde signaling in plants. We used NF and Lin to block the development and functions of chloroplasts and examined their effects on mitochondrial gene expression, RNA editing and splicing. The editing of most mitochondrial transcripts was not affected, but the editing extents of nad4-107, nad6-103, and ccmFc-1172 decreased slightly in NF- and Lin-treated seedlings. While the splicing of mitochondrial transcripts was not significantly affected, steady-state mRNA levels of several mitochondrial genes increased significantly in NF- and Lintreated seedlings. Moreover, Lin seemed to have more profound effects than NF on the expression of mitochondrial Jo-Chien Liao and Wei-Yu Hsieh have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s11120-015-0161-6) contains supplementary material, which is available to authorized users. & Ming-Hsiun Hsieh [email protected] 1

Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan

genes, indicating that signals derived from these two inhibitors might be distinct. NF and Lin also significantly induced the expression of nuclear genes encoding subunits of mitochondrial electron transport chain complexes. Thus, dysfunctional chloroplasts may coordinately up-regulate the expression of nuclear and mitochondrial genes encoding subunits of respiratory complexes. Keywords Arabidopsis thaliana  Chloroplast  Mitochondrion  Retrograde regulation  Gene expression  RNA editing

Introduction Chloroplasts were once free-living cyanobacteria that became endosymbionts around 1.2 billion years ago (Timmis et al. 2004; Gould et al. 2008). Although contemporary chloroplasts still retain DNA, their genome sizes are significantly smaller than those of cyanobacteria. It is likely that most of their genes have been transferred to the nuclear genome as an endosymbiont evolves into an organelle. It has been estimated that about 3000 proteins are localized to the chloroplast (Leister 2003). However, there are only approximately 120 genes located in the chloroplast genome (Sugita and Sugiura 1996). Thus, more than 95 % of the chloroplast-localized proteins are encoded by the nuclear genes, translated in the cytosol and then transported into the chloroplast. The approximately 120 proteins encoded by the chloroplast genes are mostly subunits of protein complexes involved in energy production (e.g., photosynthesis) or gene expression machinery (Sato et al. 1999). Still, it requires many other subunits encoded by the nuclear genes to assemble into functional protein complexes. For instance,

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protein complexes in photosystem I and II, photosynthetic electron transport chain, NADH dehydrogenase, ATP synthase, and plastid ribosomes all contain proteins encoded by the plastid and nuclear genes. The CO2 fixation enzyme ribulose-bisphosphate carboxylase/oxygenase consists of the plastid-synthesized large subunit and cytoplasmically synthesized small subunit. In addition to photosynthesis and the housekeeping gene expression machinery, many important cellular reactions, such as amino acid metabolism, carbohydrate metabolism, redox regulation, etc., also occur in the chloroplast. Thus, the development and functions of chloroplasts are highly dependent on the expression of nuclear genes (Leister 2003). The coordination of nuclear and chloroplast gene expression at different levels is important for plant growth, development and in response to various environmental cues. For instance, the majority of chloroplast genes are clustered in operons and both nucleus-encoded and plastidencoded RNA polymerases (e.g., NEP and PEP) are involved in the transcription of chloroplast genes (Barkan 2011). The primary transcripts in the chloroplast have to go through a complex series of posttranscriptional processing such as endonucleolytic cleavage of di- or polycistronic transcripts, 50 and 30 end processing, intron splicing and RNA editing (Stern et al. 2010). These extensive processes require many nucleus-encoded chloroplast proteins. Nucleus-encoded proteins control most aspects of chloroplast biogenesis and function via anterograde signaling. In addition to anterograde regulation, the functional state of the chloroplast may also affect the expression of nuclear genes via retrograde regulation (Nott et al. 2006; Koussevitzky et al. 2007; Woodson and Chory 2008). The plastid-to-nucleus retrograde signaling was initially implicated in a study using barley mutants with primary defects in plastid ribosomes that failed to synthesize nucleus-encoded plastid enzymes (Bradbeer et al. 1979). Since then, a complex picture of plastid-to-nucleus retrograde regulation has emerged (Chi et al. 2013; Blanco et al. 2014). Because the development and functions of chloroplasts are highly dependent on nuclear genes, the organelles must communicate with nucleus to adjust the expression of nuclear genes in response to developmental cues and environmental conditions. It is imaginable that the chloroplast may need both anterograde and retrograde signaling pathways to allow for optimal functioning. Besides nuclear and chloroplast genomes, mitochondria also have their own genome. Similar to chloroplasts, mitochondria are semiautonomous and their development and functions also largely depend on the nucleus-encoded mitochondrial proteins. In contrast to chloroplasts, the mitochondrial genome does not contain any RNA polymerase genes. The transcription of mitochondrial genes is controlled exclusively by the nucleus-encoded RNA polymerases (Hess

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and Bo¨rner 1999). Similar to the regulation of gene expression in chloroplasts, complex post-transcriptional processes of primary transcripts including RNA cleavage, editing, and splicing also play important roles in the regulation of mitochondrial gene expression (Binder and Brennicke 2003; Falcon de Longevialle et al. 2010). Similar to the chloroplast-nucleus relationship, lots of recent studies have advanced our knowledge in the communication between the nuclear and mitochondrial genomes, e.g., anterograde and retrograde regulation of nucleus-encoded mitochondrial proteins in plants (Blanco et al. 2014; Ng et al. 2014). However, the communication between the chloroplast and mitochondrial genomes is less studied. In white leaves of barley (Hordeum vulgare cv. Haisa) albostrians mutants that lack plastid ribosomes, the expression of a select set of mitochondrial genes is up-regulated (Hedtke et al. 1999). Similarly, the expression of mitochondrial genes is also upregulated in Arabidopsis mutants defective in the methylerythritol phosphate pathway of plastid isoprenoid biosynthesis (Hsieh et al. 2008). Since little is known about the regulation of mitochondrial gene expression by the functional state of the chloroplast, we decided to examine the effects of dysfunctional chloroplasts on mitochondrial gene expression, including RNA editing and splicing in the model plant Arabidopsis thaliana. Here, we used inhibitors norflurazon (NF) and lincomycin (Lin) to block the development and functions of chloroplasts in Arabidopsis seedlings and then examined the effects of these inhibitors on the expression of mitochondrial genes. Several aspects of mitochondrial gene expression including RNA editing, splicing, and transcript abundance were examined in this study. We found that dysfunctional chloroplasts had little effects on mitochondrial RNA editing and splicing, whereas the accumulation of many mitochondrial transcripts was significantly increased in the inhibitor-treated seedlings.

Materials and methods Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia-0 was grown on half-strength Murashige and Skoog (MS) plates [MS salts (Sigma), pH adjusted to 5.7 with 1 N KOH, 0.8 % (w/v) agar] containing 2 % (w/v) sucrose in the growth chamber under a 16 h light/8 h dark cycle at 23 °C. For NF and Lin treatments, Arabidopsis seedlings were germinated and grown on ‘ MS ? 2 % (w/v) sucrose medium containing 5 lM NF or 0.5 mM Lin for 14 days. The harvested plant tissues or seedlings were frozen in liquid nitrogen and used for RNA extraction immediately or were stored at -80 °C.

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Measurements of PSII maximum quantum yield and photosynthetic pigments

quantitative RT-PCRs were performed in triplicate for each sample in three independent experiments.

Twelve-day-old Arabidopsis seedlings grown on tissue culture medium with or without NF and Lin were used for chlorophyll fluorescence imaging and photosynthetic pigment analysis. Chlorophyll fluorescence imaging was performed with the Maxi-Imaging-PAM Chlorophyll Fluorometer (Heinz Walz GmbH, Germany). Plants were dark-adapted for 20 min before the measurement of PSII maximum quantum yield (Fv/Fm). Total chlorophylls and carotenoids were extracted and quantified as described previously (Lichtenthaler and Wellburn 1983).

RT-PCR and analysis of mitochondrial RNA editing

Transmission electron microscope analysis Leaves from two-week-old Arabidopsis seedlings treated with or without NF and Lin were used for transmission electron microscope analysis. The leaf samples were fixed in 4 % (w/v) glutaraldehyde, 100 mM sodium cacodylate, pH 7.2, for 16 h at 4 °C, and postfixed with 1 % (w/v) osmium tetroxide in the same buffer for 6 h at 4 °C. Dehydration, embedding, sectioning, staining, and observation were performed according to methods described previously (Hsieh and Goodman 2005). RNA extraction and RNA gel-blot analysis Total RNA was isolated from 2-week-old Arabidopsis seedlings as described previously (Tseng et al. 2013). 5 lg total RNA from each sample was used for RNA gel-blot analysis. Primers used to make DIG-labeled probes for RNA gel-blot analyses are listed in Supplementary Table S1. DIG probe labeling, pre-hybridization, hybridization, wash conditions and detection were performed according to Roche’s DIG Application Manual for Filter Hybridization. Quantitative RT-PCR analysis of mitochondrial transcripts The RNA was treated with DNase I prior to use in the assays. Quantitative RT-PCR analysis of mitochondrial and chloroplast transcripts was performed as described previously (Koprivova et al. 2010; Kuhn et al. 2011). The sequences of the primers used for quantitative RT-PCR analysis of mitochondrial genes are listed in Supplementary Table S2. To quantify the splicing of mitochondrial mRNAs, quantitative RT-PCR was performed using specific primers designed to intron–exon regions (unspliced forms) and exon–exon regions (spliced forms) of each gene as described previously (Falcon de Longevialle et al. 2007; Koprivova et al. 2010; Hsieh et al. 2015). All of the quantifications were normalized to the nuclear gene ACTIN 2 (ACT2). The

We used RT-PCR and bulk sequencing of the amplified cDNAs to examine the extent of mitochondrial RNA editing. The RNA samples were digested with DNase I, and reverse transcription was performed with Superscript III RT (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Random hexamers were used in reverse transcription to synthesize the first strand cDNA. Primers used for cDNA amplification of mitochondrial genes harboring more than 500 editing sites were described previously (Sung et al. 2010). As a negative control for genomic DNA contamination, we used the same PCR conditions on RNA samples that were not treated with reverse transcriptase. Bulk sequencing of the RT-PCR products with the same primers used for cDNA amplification was conducted at the DNA Sequencing Core at Academia Sinica.

Results Loss of photosynthesis in NF- and Lin-treated seedlings Arabidopsis seedlings grown on medium containing NF or Lin were albino (Fig. 1a). Measurement of photosynthetic pigments revealed that the contents of chlorophylls and carotenoids were extremely low or undetectable in these albino seedlings (Fig. 1b). Moreover, the PSII maximum efficiency in dark-adapted state (Fv/Fm) was undetectable in NF- and Lin-treated seedlings (Fig. 1c). These results suggest that the photosynthetic activity may have completely lost in the albino seedlings treated with NF or Lin. Impaired chloroplast development in NF- and Lintreated seedlings In addition to analyses of photosynthetic pigments and activity, we used transmission electron microscopy to examine the morphology of chloroplasts in NF- and Lin-treated albino seedlings. Compared with the untreated control chloroplast that had lens shape and normal grana, the NF- and Lintreated chloroplasts were oval or round shape and did not have any thylakoids (Fig. 2, top). These results suggest that treatments of NF and Lin may have blocked the development of chloroplast at a very early stage. By contrast, we did not observe significant differences in the morphology of mitochondria in NF- and Lin-treated seedlings compared with that of control (Fig. 2, bottom).

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Fig. 1 Effects of norflurazon (NF) and lincomycin (Lin) on Arabidopsis seedlings. a 7-day-old Arabidopsis seedlings grown on tissue culture medium containing NF or Lin. b Contents of chlorophylls and carotenoids in NF- or Lin-treated seedlings compared with those of control (Col). c Light (top) and chlorophyll fluorescence (bottom)

Fig. 2 Transmission electron micrographs of Arabidopsis leave chloroplasts (top) and mitochondria (bottom). Scale bars are 500 nm (top) and 200 nM (bottom), respectively. Col control; NF norflurazon; Lin lincomycin

Up-regulation of mitochondrial genes in NFand Lin-treated seedlings At the molecular level, the expression of the nuclear gene LHCB1.3 that encodes a chlorophyll a/b-binding protein was also dramatically reduced in NF- and Lin-treated seedlings compared with that of control (Fig. 3a). This is consistent with the notion that the development and functions of the chloroplast are dramatically impaired in NF- and Lin-treated albino seedlings. By contrast, the expression of the nuclear

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images of control (Col) and NF- or Lin-treated 12-day-old Arabidopsis seedlings. Imaging chlorophyll fluorometer was used to measure Photosystem-II activity (Fv/Fm), and the values are indicated by pseudo-color images at the bottom

gene AOX1a that encodes an alternative oxidase, and other nuclear genes encoding 40 kD subunit of complex I, succinate dehydrogenase (SDH2) of complex II, 14 kD subunit of complex III, coxVc subunit of complex IV, and Atpd’ subunit of complex V was significantly increased in NF- and Lintreated seedlings compared with that of control (Fig. 3a). In addition to nuclear genes, we also used RNA gel-blot analysis to examine the effects of NF and Lin on the expression of a select set of mitochondrial genes encoding subunits of NADH dehydrogenase (complex I), cytochrome bc1 complex (complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V). These genes are located in the mitochondrial genome. The expression of nad2 and nad4 was slightly induced in Lin-treated seedlings, whereas steady-state levels of cob mRNA were increased in both NFand Lin-treated seedlings compared with those of control (Fig. 3b). By contrast, the expression of cox1 was induced in NF-treated seedlings, and steady-state levels of cox2 and cox3 transcripts were similar between control and inhibitortreated seedlings (Fig. 3b). The expression of atp8 was induced in both NF- and Lin-treated seedlings as compared with the control (Fig. 3b). In contrast to mitochondrial genes, the expression of PEP-transcribed psaA, psbA, and psbE genes was significantly repressed in NF- and Lin-treated seedlings (Fig. 3c). Similar to the expression of LHCB1.3 (Fig. 3a), Lin showed a stronger effect than NF on the repression of psaA, psbA, and psbE (Fig. 3c). By contrast, Lin, but not NF, significantly induced the expression of NEP-transcribed rpoA gene as compared with that of the untreated control (Fig. 3c). In addition to RNA gel-blot analysis, we also used a more sensitive quantitative RT-PCR assay to compare the abundance of mitochondrial transcripts in the untreated

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Fig. 3 RNA gel-blot analysis of nuclear, mitochondrial, and chloroplastic genes. a Treatments of norflurazon (NF) and lincomycin (Lin) increased the expression of nuclear genes encoding mitochondrionlocalized proteins. Steady-state mRNA levels of LHCB1.3 decreased dramatically, whereas the expression of AOX1a increased significantly in NF- and Lin-treated Arabidopsis seedlings. b Steady-state levels of mitochondrial transcripts were increased or not affected in NF- and Lin-treated Arabidopsis seedlings. 40 kD SU, nad2, and nad4 are complex I genes. SDH2 is a complex II gene. 14 kD SU and cob are

complex III genes. coxVc, cox1, cox2, and cox3 are complex IV genes. atpd0 and atp8 are complex V genes. c Steady-state levels of plastid-encoded RNA polymerase-dependent psaA, psbA, and psbE transcripts were down-regulated in NF- and Lin-treated Arabidopsis seedlings. Lin but not NF induced the expression of nucleus-encoded RNA polymerase-dependent rpoA gene. A representative ethidium bromide-stained agarose gel of the same samples is shown at the bottom of each panel

control and NF- and Lin-treated seedlings. We did not detect significant transcript reduction in any of the mitochondrial genes examined. By contrast, the abundance of several mitochondrial transcripts such as nad1, nad3, nad7, nad9, rps3, rps4, rps12, rpl16, and ccmFn1 showed significant increases (e.g., greater than 2-fold) in Lin-treated seedlings, whereas only the expression of cox1 in NFtreated seedlings had more than 2-fold increase as compared with the control (Fig. 4).

treated seedlings as compared with those of the untreated control (Fig. 5).

Effects of NF and Lin on mitochondrial RNA splicing In Arabidopsis mitochondria, 23 introns, including 4 in each of nad1, nad2, nad5, and nad7, 3 in nad4, and 1 in each of cox2, ccmFc, rpl2, and rps3, have been described (Bonen 2008). To quantify the splicing of mitochondrial mRNAs, quantitative RT-PCR was performed using specific primers designed to exon–exon regions (spliced forms) and intron–exon regions (unspliced forms) of each gene. The ratios of spliced to unspliced RNA for each mitochondrial intron in NF- and Lin-treated seedlings as compared with those of control were shown in Fig. 5. An Arabidopsis mutant, slo3, which is specifically impaired in the splicing of mitochondrial nad7 intron 2 was also included as a control (Hsieh et al. 2015). The results indicated that the splicing efficiencies of mitochondrial transcripts were not significantly affected in NF- and Lin-

Effects of NF and Lin on mitochondrial RNA editing In addition to the abundance of mitochondrial transcripts and RNA splicing, we also examined the effects of NF and Lin on mitochondrial RNA editing. More than 500 RNA editing sites have been identified in Arabidopsis. We used RT-PCR and bulk sequencing to compare the editing of mitochondrial transcripts in NF-, Lin-treated, and the untreated control seedlings. The primer sets that we used for RT-PCR cover most of the mitochondrial RNA editing sites (Sung et al. 2010). Most of the mitochondrial RNA editing sites we examined did not show significant difference between control and inhibitor-treated seedlings (data not shown). Nevertheless, compared with those of control, the C-to-U editing extents of nad4-107, nad6-103, and ccmFc-1172 were slightly decreased in NF- and Lintreated seedlings (Fig. 6). The nad4-107 is a highly edited site as the nucleotide C was hardly detectable in the control. Treatments of NF and Lin slightly decreased the editing of nad4-107 as indicated by the appearance of C peaks at this position (Fig. 6, nad4-107). By contrast, nad6-103 and ccmFc-1172 are partially edited in the control seedlings. Although both Lin and NF treatments would reduce the editing extents of these two sites, Lin seemed to have a more severe effect on the editing of nad6-103 and

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Fig. 4 Quantitative RT-PCR analysis of mitochondrial transcripts. The histogram shows the relative RNA abundance in norflurazon (NF)- and lincomycin (Lin)-treated Arabidopsis seedlings as compared with that of the untreated control

Fig. 5 Quantitative RT-PCR analysis of intron-containing mitochondrial transcripts. The histogram shows the log2 ratio of spliced to unspliced RNA in norflurazon (NF)- and lincomycin (Lin)-treated Arabidopsis seedlings as compared with the corresponding control (Col). Arabidopsis slo3 mutant that is impaired in the splicing of nad7

intron 2 was included as a control. The splicing efficiency of nad7 intron 2 was significantly reduced in slo3 as compared with that of wild type (WT). Treatments of NF and Lin did not significantly affect the splicing of mitochondrial transcripts

ccmFc-1172. The relative editing extents (T/C ? T) of these two sites were lower in Lin-treated seedlings as compared with those of NF-treated seedlings (Fig. 6, nad6103 and ccmFc-1172).

interactions between photosynthesizing chloroplasts and oxidative-respiring mitochondria have been intensively studied for decades (Noctor et al. 2007; Noguchi and Yoshida 2008). By contrast, little is known about the interactions between chloroplasts and mitochondria at the molecular levels. In addition to anterograde and retrograde responses between nucleus and chloroplasts, it is possible that the functional state of chloroplasts may also regulate the expression of mitochondrial genes. NF and Lin are commonly used inhibitors, which will block the development and functions of chloroplast (Sullivan and Gray 1999; Beisel et al. 2011; Jung 2004; Guseinova et al. 2005). NF is an inhibitor of phytoene desaturase, whereas Lin blocks the translation of plastid proteins. Despite the underlying mechanisms are different, plants treated with these inhibitors usually are similar in appearance. It is well known that the functional state of chloroplasts will affect the expression of nuclear genes. For instance, the expression of LHCB1.3, a nuclear gene

Discussion There are three genomes, e.g., nuclear, chloroplastic, and mitochondrial genomes, in the plant cell. Chloroplasts and mitochondria are considered to be semiautonomous organelles as their development and functions mostly depend on genes located in the nuclear genome. In addition to the interactions with nucleus, chloroplasts and mitochondria are highly interdependent on each other. For instance, the mutually beneficial interactions between mitochondrial metabolism and photosynthetic carbon assimilation are important for plant growth and development (Raghavendra and Padmasree 2003). It is not surprising that metabolic

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Fig. 6 Effects of norflurazon (NF) and lincomycin (Lin) on the editing of mitochondrial transcripts. The editing extents of nad4-107, nad6-103, and ccmFc-1172 decreased slightly in NF- and Lin-treated seedlings as compared with those of control (Col)

encoding chlorophyll a/b-binding protein, was strongly repressed in NF- and Lin-treated plants (Fig. 3). Therefore, NF and Lin have been broadly used to dissect the retrograde signaling pathways in plants (Nott et al. 2006). In addition to their effects on nuclear genes, we previously showed that NF and Lin would differentially affect the expression of chloroplast genes and RNA editing in Arabidopsis seedlings (Tseng et al. 2013). In general, the expression of PEP-transcribed genes is down-regulated and the expression of NEP-dependent genes is up-regulated or not affected in NF- and Lin-treated seedlings. Compared with that of control, Lin has a more severe effect than NF on the editing efficiency of chloroplast transcripts (Tseng et al. 2013). Here, we extended to study the effects of NF and Lin on mitochondrial gene expression, RNA editing and splicing. The mitochondrial gene products include ribosomal proteins, subunits of electron transport chain protein complexes (e.g., Complex I–V), and cytochrome c biogenesis. The formation of these protein complexes also requires subunits encoded by the nuclear genes. Thus, coordinated regulation of nuclear and mitochondrial genes is important for many cellular activities (Gonzalez et al. 2007). In NFand Lin-treated Arabidopsis seedlings, the expression of nucleus-encoded LHCB1.3 gene was dramatically reduced, whereas the expression of nuclear genes encoding subunits of respiratory complex I–V was significantly induced (Fig. 3a). These results suggest that the retrograde regulation of nuclear genes by the functional state of chloroplasts is not limited to nuclear genes encoding chloroplast-localized proteins.

It is interesting that signals derived from dysfunctional chloroplasts in NF- and Lin-treated seedlings would simultaneously induce the expression of nuclear and mitochondrial genes encoding subunits of complex I–V (Fig. 3). Compared with those of control and NF-treated seedlings, more profound effects on the expression of mitochondrial genes were observed in Lin-treated seedlings. Lin seemed to induce more mitochondrial genes than NF. Among those genes that were commonly induced by NF and Lin, the expression levels, in general, were higher in Lin-treated seedlings (Fig. 4). These results suggest that the specific signals or the underlying molecular mechanisms are not exactly the same between NF and Lin. Indeed, several distinct retrograde signaling pathways such as the plastid gene expression (PGE), tetrapyrrole biosynthesis, chloroplast-generated reactive oxygen species (ROS), and redox signals from the photosynthetic electron transport chain have been characterized in chloroplasts (Woodson and Chory 2008). Treatments of NF and Lin will trigger ROS and PGE pathways, respectively. These differences may be accountable for the distinct molecular phenotypes observed in the NF- and Lin-treated Arabidopsis seedlings. In addition to NF and Lin, high light treatment will also result in impaired chloroplasts and trigger retrograde signals (Szechyn´ska-Hebda and Karpin´ski 2013; Dietz 2015). Previous studies revealed that high light would induce the expression of Arabidopsis nuclear genes encoding mitochondrial substrate carrier family proteins and mitochondrion-localized antioxidant enzymes (Rossel et al. 2007; Szechyn´ska-Hebda and Karpin´ski 2013). It is not clear if the expression of mitochondrial genes is also induced by high light. Further studies on the regulation of mitochondrial gene expression by various chloroplast retrograde signals may facilitate our understanding of the molecular interactions between chloroplasts and mitochondria. Chloroplasts and mitochondria are the major organelles to produce energy in the plant cell. The coordinated regulation of nuclear and mitochondrial genes by dysfunctional chloroplasts may allow mitochondria to optimize their functions. In NF- and Lin-treated Arabidopsis seedlings, photosynthesis is completely abolished. The induction of nuclear and mitochondrial genes involved in electron transport chain may enhance oxidative phosphorylation and thus increase energy production to compensate for the loss of photosynthesis in the inhibitor-treated albino seedlings. It is possible that the physiological role of a putative plastid signal may be involved in coordinating the expression of respiratory genes according to photosynthetic activity. AOX is a cyanide-insensitive terminal oxidase that oxidizes its substrate without the generation of a proton motive force (Vanlerberghe and McIntosh 1997; Siedow and Umbach 2000). The expression of Arabidopsis AOX1a

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is regulated by a variety of biotic and abiotic stresses (Millar et al. 2011; Ng et al. 2014). It is also a marker gene for the study of mitochondrial retrograde regulation in plants (Rhoads and Subbaiah 2007). NF and Lin are commonly used inhibitors to trigger the chloroplast-tonucleus retrograde signaling pathways. As the expression of AOX1a is up-regulated by dysfunctional mitochondria (Ng et al. 2014), the strong up-regulation of AOX1a by dysfunctional chloroplasts (e.g., in NF- and Lin-treated seedlings) raises a possibility that both chloroplasts and mitochondria may use separate pathways or they may share the same pathway and signal to each other to induce the expression of AOX1a. It has been shown that the transcription factor ABA INSENSITIVE 4 (ABI4) plays a central role in integrating chloroplast retrograde signaling pathways (Koussevitzky et al. 2007). Interestingly, independent studies have revealed that ABI4 is also involved in mediating mitochondrial retrograde signaling to induce the expression of AOX1a (Giraud et al. 2009). Thus, the transcription factor ABI4 may play an important role in the convergence of chloroplast and mitochondrial retrograde signaling pathways. Still, the induction of AOX1a may represent a nonspecific response to a variety of stress conditions as the activation of alternative respiration may alleviate the effects of oxidative stress generated by dysfunctional chloroplasts or mitochondria. Nonetheless, the functions of chloroplasts and mitochondria are closely connected in energy, metabolism, and redox status (Raghavendra and Padmasree 2003; van Lis and Atteia 2004; Noguchi and Yoshida 2008). The AOX pathway has been implied to play a key role in optimizing photosynthesis and chloroplast protection under extreme environments (Xu et al. 2011). It is tempting to speculate that the AOX pathway may be also involved in coordinating the retrograde signaling pathways between chloroplasts and mitochondria. Unlike the effects on the accumulation of mitochondrial transcripts, treatments of NF and Lin did not significantly affect mitochondrial RNA splicing. Although the editing of some mitochondrial transcripts was slightly affected, no complete loss of editing was observed in NF- or Lin-treated Arabidopsis seedlings. Still, we observed a more severe effect on the editing of nad6-103, and ccmFc-1172 in Lintreated seedlings. This is consistent with the notion that some distinct signals may exist in NF- and Lin-treated seedlings (Tseng et al. 2013). The mechanism by which NF and Lin affect mitochondrial RNA editing is not clear. One possibility is that signals derived from dysfunctional chloroplasts may directly affect RNA editing in the mitochondrion. Alternatively, NF and Lin may affect the expression and/or activity of trans-factors (e.g., editing enzymes or editing factors) encoded by the nuclear genes

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(Takenaka et al. 2013; Barkan and Small 2014), which in turn regulate the editing of mitochondrial transcripts. Nevertheless, the slight differences in nad4-107, nad6-103, and ccmFc-1172 editing extents may not have any physiological consequences. The major effect of NF and Lin on mitochondrial gene expression was the increase in transcript abundance. The transcription of mitochondrial genes relies exclusively on the nucleus-encoded RNA polymerase (NEP). The effects of NF and Lin on the expression of mitochondrial genes were reminiscent of their effects on NEP-dependent gene expression in chloroplasts (Tseng et al. 2013). The expression of NEP-dependent genes in both chloroplasts and mitochondria was increased or unaltered in NF- and Lintreated seedlings. These results suggest that the functional state of chloroplasts not only affects the expression of nuclear genes via the chloroplast-to-nucleus retrograde signaling pathway but also affect the expression of mitochondrial genes through the chloroplast-to-mitochondrion signaling pathway. The transcriptional and post-transcriptional regulation (e.g., maturation, editing, splicing, and stability of RNA) of mitochondrial genes is almost exclusively controlled by factors encoded by the nuclear genes. Still, we cannot exclude the possibility that the effects of NF and Lin on mitochondrial gene expression and RNA editing are indirectly derived from the chloroplast-to-nucleus and then nucleus-to-mitochondrion signaling pathways. We know very little about the signaling events between chloroplasts and mitochondria. It will be interesting to further investigate the interactions between chloroplasts and mitochondria at the molecular level. Acknowledgments This work was supported by Grants from National Science Council and Academia Sinica (98-CDA-L04). We thank the Cell Biology Core at the Institute of Plant and Microbial Biology for assistance in transmission electron microscopy.

References Barkan A (2011) Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold. Plant Physiol 155:1520–1532 Barkan A, Small I (2014) Pentatricopeptide repeat proteins in plants. Annu Rev Plant Biol 65:415–442 Beisel KG, Schurr U, Matsubara S (2011) Altered turnover of bcarotene and Chl a in Arabidopsis leaves treated with lincomycin or norflurazon. Plant Cell Physiol 52:1193–1203 Binder S, Brennicke A (2003) Gene expression in plant mitochondria: transcriptional and post-transcriptional control. Philos Trans R Soc B 358:181–189 ˚ (2014) Interaction between Blanco NE, Dı´az MG, Whelan J, Strand A plastid and mitochondrial retrograde signaling pathways during changes to plastid redox status. Philos Trans R Soc B 369:20130231

Photosynth Res Bonen L (2008) Cis- and trans-splicing of group II introns in plant mitochondria. Mitochondrion 8:26–34 Bradbeer JW, Atkinson YE, Bo¨rner T, Hagemann R (1979) Cytoplasmic synthesis of plastid polypeptides may be controlled by plastid-synthesized RNA. Nature 279:816–817 Chi W, Sun X, Zhang L (2013) Intracellular signaling from plastid to nucleus. Annu Rev Plant Biol 64:559–582 Dietz KJ (2015) Efficient high light acclimation involves rapid processes at multiple mechanistic levels. J Exp Bot 66:2401–2414 Falcon de Longevialle A, Meyer EH, Andre´s C, Taylor NL, Lurin C, Millar AH, Small ID (2007) The pentatricopeptide repeat gene OTP43 is required for trans-splicing of the mitochondrial nad1 intron 1 in Arabidopsis thaliana. Plant Cell 19:3256–3266 Falcon de Longevialle A, Small ID, Lurin C (2010) Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles. Mol Plant 3:691–705 Giraud E, Van Aken O, Ho LH, Whelan J (2009) The transcription factor ABI4 is a regulator of mitochondrial retrograde expression of ALTERNATIVE OXIDASE1a. Plant Physiol 150:1286–1296 Gonzalez DH, Welchen E, Attallah CV, Comelli RN, Mufarrege EF (2007) Transcriptional coordination of the biogenesis of the oxidative phosphorylation machinery in plants. Plant J 51:105–116 Gould SB, Waller RF, McFadden GI (2008) Plastid evolution. Annu Rev Plant Biol 59:491–517 Guseinova IM, Suleimanov SY, Aliyev JA (2005) The effect of norflurazon on protein composition and chlorophyll organization in pigment–protein complex of photosystem II. Photosynth Res 84:71–76 Hedtke B, Wagner I, Bo¨rner T, Hess WR (1999) Inter-organellar crosstalk in higher plants: impaired chloroplast development affects mitochondrial gene and transcript levels. Plant J 19:635–643 Hess WR, Bo¨rner T (1999) Organellar RNA polymerases of higher plants. Int Rev Cytol 190:1–59 Hsieh MH, Goodman HM (2005) The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis. Plant Physiol 138:641–653 Hsieh MH, Chang CY, Hsu SJ, Chen JJ (2008) Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial genes in ispD and ispE albino mutants in Arabidopsis. Plant Mol Biol 66:663–673 Hsieh WY, Liao JC, Chang CY, Harrison T, Boucher C, Hsieh MH (2015) The SLOW GROWTH 3 pentatricopeptide repeat protein is required for the splicing of mitochondrial nad7 intron 2 in Arabidopsis. Plant Physiol doi:10.1104/pp.15.00354 (Epub ahead of print) Jung S (2004) Effect of chlorophyll reduction in Arabidopsis thaliana by methyl jasmonate or norflurazon on antioxidant systems. Plant Physiol Biochem 42:225–231 Koprivova A, des Francs-Small C, Calder G, Mugford ST, Tanz S, Lee BR, Zechmann B, Small I, Kopriva S (2010) Identification of a pentatricopeptide repeat protein implicated in splicing of intron 1 of mitochondrial nad7 transcripts. J Biol Chem 285:32192–32199 Koussevitzky S, Nott A, Mockler TC, Hong F, Sachetto-Martins G, Surpin M, Lim J, Mittler R, Chory J (2007) Signals from chloroplasts converge to regulate nuclear gene expression. Science 316:715–719 Kuhn K, Carrie C, Giraud E, Wang Y, Meyer EH, Narsai R, des Francs-Small C, Zhang B, Murcha MW, Whelan J (2011) The RCC1 family protein RUG3 is required for splicing of nad2 and complex I biogenesis in mitochondria of Arabidopsis thaliana. Plant J 67:1067–1080 Leister D (2003) Chloroplast research in the genomic age. Trends Genet 19:47–56

Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans 11:591–592 Millar AH, Whelan J, Soole KL, Day DA (2011) Organization and regulation of mitochondrial respiration in plants. Annu Rev Plant Biol 62:79–104 Ng S, De Clercq I, Van Aken O, Law SR, Ivanova A, Willems P, Giraud E, Van Breusegem F, Whelan J (2014) Anterograde and retrograde regulation of nuclear genes encoding mitochondrial proteins during growth, development, and stress. Mol Plant 7:1075–1093 Noctor G, De Paepe R, Foyer CH (2007) Mitochondrial redox biology and homeostasis in plants. Trends Plant Sci 12:125–134 Noguchi K, Yoshida K (2008) Interaction between photosynthesis and respiration in illuminated leaves. Mitochondrion 8:87–99 Nott A, Jung HS, Koussevitzky S, Chory J (2006) Plastid-to-nucleus retrograde signaling. Annu Rev Plant Biol 57:739–759 Raghavendra AS, Padmasree K (2003) Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation. Trends Plant Sci 8:546–553 Rhoads DM, Subbaiah CC (2007) Mitochondrial retrograde regulation in plants. Mitochondrion 7:177–194 Rossel JB, Wilson PB, Hussain D, Woo NS, Gordon MJ, Mewett OP, Howell KA, Whelan J, Kazan K, Pogson BJ (2007) Systemic and intracellular responses to photooxidative stress in Arabidopsis. Plant Cell 19:4091–4110 Sato S, Nakamura Y, Kaneko T, Asamizu E, Tabata S (1999) Complete structure of the chloroplast genome of Arabidopsis thaliana. DNA Res 6:283–290 Siedow JN, Umbach AL (2000) The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity. Biochim Biophys Acta 1459:432–439 Stern DB, Goldschmidt-Clermont M, Hanson MR (2010) Chloroplast RNA metabolism. Annu Rev Plant Biol 61:125–155 Sugita M, Sugiura M (1996) Regulation of gene expression in chloroplasts of higher plants. Plant Mol Biol 32:315–326 Sullivan JA, Gray JC (1999) Plastid translation is required for the expression of nuclear photosynthesis genes in the dark and in roots of the pea lip1 mutant. Plant Cell 11:901–910 Sung TY, Tseng CC, Hsieh MH (2010) The SLO1 PPR protein is required for RNA editing at multiple sites with similar upstream sequences in Arabidopsis mitochondria. Plant J 63:499–511 Szechyn´ska-Hebda M, Karpin´ski S (2013) Light intensity-dependent retrograde signalling in higher plants. J Plant Physiol 170:1501–1516 Takenaka M, Zehrmann A, Verbitskiy D, Ha¨rtel B, Brennicke A (2013) RNA editing in plants and its evolution. Annu Rev Genet 47:335–352 Timmis JN, Ayliffe MA, Huang CY, Martin W (2004) Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet 5:123–135 Tseng CC, Lee CJ, Chung YT, Sung TY, Hsieh MH (2013) Differential regulation of Arabidopsis plastid gene expression and RNA editing in non-photosynthetic tissues. Plant Mol Biol 82:375–392 van Lis R, Atteia A (2004) Control of mitochondrial function via photosynthetic redox signals. Photosynth Res 79:133–148 Vanlerberghe GC, McIntosh L (1997) Alternative oxidase: from gene to function. Annu Rev Plant Physiol Plant Mol Biol 48:703–734 Woodson JD, Chory J (2008) Coordination of gene expression between organellar and nuclear genomes. Nat Rev Genet 9:383–395 Xu F, Yuan S, Lin HH (2011) Response of mitochondrial alternative oxidase (AOX) to light signals. Plant Signal Behav 6:55–58

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