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First published online March 3, 2006; 10.1105/tpc.105.039073 The Plant Cell 18:970-991 (2006) © 2006 American Society of Plant Biologists Nuclear Photosynthetic Gene Expression Is Synergistically Modulated by Rates of Protein Synthesis in Chloroplasts and Mitochondria[W]
a Abteilung für Pflanzenzüchtung und Genetik, Max-Planck-Institut für Züchtungsforschung, D-50829 Cologne, Germany 1 To whom correspondence should be addressed. E-mail leister{at}lrz.uni-muenchen.de; fax 49-89-17861-362.
Arabidopsis thaliana mutants prors1-1 and -2 were identified on the basis of a decrease in effective photosystem II quantum yield. Mutations were localized to the 5'-untranslated region of the nuclear gene PROLYL-tRNA SYNTHETASE1 (PRORS1), which acts in both plastids and mitochondria. In prors1-1 and -2, PRORS1 expression is reduced, along with protein synthesis in both organelles. PRORS1 null alleles (prors1-3 and -4) result in embryo sac and embryo development arrest. In mutants with the leaky prors1-1 and -2 alleles, transcription of nuclear genes for proteins involved in photosynthetic light reactions is downregulated, whereas genes for other chloroplast proteins are upregulated. Downregulation of nuclear photosynthetic genes is not associated with a marked increase in the level of reactive oxygen species in leaves and persists in the dark, suggesting that the transcriptional response is light and photooxidative stress independent. The mrpl11 and prpl11 mutants are impaired in the mitochondrial and plastid ribosomal L11 proteins, respectively. The prpl11 mrpl11 double mutant, but neither of the single mutants, resulted in strong downregulation of nuclear photosynthetic genes, like that seen in leaky mutants for PRORS1, implying that, when organellar translation is perturbed, signals derived from both types of organelles cooperate in the regulation of nuclear photosynthetic gene expression.
Photosynthesis is regulated at multiple levels. In the chloroplast itself, rates of photosynthesis are adjusted by posttranslational modification of proteins (Buchanan et al., 1994  ; Wollman, 2001; Aro and Ohad, 2003), by structural reorganization of the photosynthetic protein complexes (Niyogi, 1999), and by metabolic regulation within and downstream of the Calvin cycle (Paul and Pellny, 2003). Moreover, the expression of plastome-encoded photosynthetic genes is regulated by transcriptional (Pfannschmidt et al., 1999; Pfannschmidt and Liere, 2005), posttranscriptional (Deng and Gruissem, 1987; Rochaix, 2001), and translational (Danon, 1997; Choquet and Wollman, 2002) mechanisms. In addition, plastid-to-nucleus (retrograde) signaling modulates the expression of nuclear genes coding for photosynthetic proteins (Leister, 2005). The concentrations of carbon metabolites (Rolland et al., 2002), components, and/or products of the light phase of photosynthesis, such as tetrapyrroles (Strand et al., 2003), reactive oxygen species (ROS) (Apel and Hirt, 2004), and the redox state of the thylakoid membranes (Pfannschmidt, 2003), all contribute to retrograde signaling. However, not all signals require light, since inhibition of translation in plastids activates a light-independent retrograde signaling pathway (Gray et al., 2002). For instance, in the albostrians mutant of barley (Hordeum vulgare), which is deficient in plastid ribosomes, the expression of nuclear photosynthetic genes is drastically downregulated (Bradbeer et al., 1979; Hess et al., 1991, 1994). Similar effects are observed when light- or dark-adapted wild-type plants are treated with inhibitors of plastid translation (Oelmüller et al., 1986; Adamska, 1995; Gray et al., 1995; Yoshida et al., 1998; Sullivan and Gray, 1999).
Photosynthesis provides substrates for mitochondrial respiration but also depends on a range of compounds synthesized by mitochondria. Thus, the two organelles are metabolically interdependent (Hoefnagel et al., 1998
The relationship between mitochondrial function and photosynthesis has been studied using inhibitors of mitochondrial oxidative electron transport (Padmasree and Raghavendra, 1999 Despite the pronounced metabolic interdependence of plastids and mitochondria and the ability of the latter to influence photosynthesis, little is known about the signals that mediate communications between mitochondria and plastids or about the coordination of the activities of the two organelles by signals other than carbon metabolites. We have identified and characterized mutant alleles of PRORS1, an Arabidopsis thaliana gene coding for a prolyl-tRNA synthetase that is imported into chloroplasts and mitochondria, and used these mutants to elucidate novel aspects of interorganellar crosstalk. Complete absence of PRORS1 disrupts early plant development and causes lethality. Leaky prors1 mutants show defects in photosynthesis due to the simultaneous impairment of translation in plastids and mitochondria. Concomitantly, a specific and marked drop was detected in the levels of transcripts of nuclear genes for proteins involved in the light reactions of photosynthesis. The mutants mrpl11-1 and prpl11-1, which are defective in the ribosomal L11 proteins of mitochondria and plastids, respectively, were employed to separate the contributions of the two organelles to the downregulation of nuclear photosynthetic transcripts. Comparison of the transcript profiles of single prors1, prpl11-1, and mrpl11-1 mutants and the prpl11-1 mrpl11-1 double mutant indicates that plastids and mitochondria are able to generate signals that act synergistically to modulate nuclear photosynthetic gene expression.
Identification and Phenotypic Analysis of Mutants for the PAM15/18 Locus Screening of a collection of T-DNA–mutagenized Arabidopsis lines (ecotype Columbia-0 [Col-0]) for plants with alterations in the effective quantum yield of PSII (  II) (Varotto et al., 2000) led to the identification of the mutants photosynthesis altered mutant15 (pam15) and pam18 (Figure 1A
). Both photosynthetic phenotypes segregated as monogenic, recessive traits. Crosses between homozygous pam15 and pam18 plants resulted in F1 plants with a pam phenotype, indicating that the same genetic locus was affected in both cases. The phenotype of pam18 was the more severe. Thus, pam18 plants showed a stronger reduction in growth rate than pam15 plants (Figure 1A) and more marked changes in photosynthetic parameters. This is illustrated by the relative decrease in II: wild type, 0.75 ± 0.02; pam15/pam15, 0.58 ± 0.01; pam15/pam18, 0.51 ± 0.02; pam18/pam18, 0.46 ± 0.01. Analysis of chlorophyll fluorescence in light-adapted plants revealed, in addition to the altered II, a significant reduction in the maximum quantum yield of PSII (Fv/Fm) in both mutants (pam15/pam15, 0.65 ± 0.02; pam18/pam18, 0.52 ± 0.01; wild type, 0.79 ± 0.01), implying a defect in energy transfer within PSII. In addition, an increase in the reduction of QA and of the plastoquinone pool, measured as 1-qP, was observed in pam18 leaves (pam15, 0.06 ± 0.02; pam18, 0.09 ± 0.02; wild type, 0.05 ± 0.01). HPLC analysis of light-adapted pam15 and pam18 leaves revealed a decrease in chlorophyll concentration (chlorophyll a+b), indicating a reduction in the abundance of thylakoid antenna proteins (Table 1
). Moreover, in leaves of both mutant lines, the reduction in the chlorophyll a/b ratio and the ß-carotene content suggested a decrease in levels of PSII core proteins. The relative increase in the level of xanthophyll cycle pigments (violaxanthin + antheraxanthin + zeaxanthin) with respect to neoxanthin and lutein is likely to be the result of increased photooxidative stress in the mutants (see below). This, together with the restoration of photosynthetic efficiency (measured as Fv/Fm) during the night (Figure 1B) and the wild type–like behavior of pam15 plants when grown under low light (data not shown), indicated that illumination exacerbates the molecular defect(s) caused by mutations at the PAM15/18 locus. One direct consequence of the impaired light reactions of photosynthesis in pam15 and pam18 plants on carbohydrate metabolism was a persistent reduction in leaf starch content measured over the course of the day (Figure 1C).
Expression of Photosynthetic Functions Is Downregulated at Both Protein and RNA Levels in pam15 and pam18 Thylakoid proteins isolated from light-adapted wild-type and mutant plants at the eight-leaf rosette stage were subjected to immunoblot analysis with antibodies specific for proteins of photosystem I (PSI) and PSII, cytochrome b6f, and  - and ß-subunits of ATPase (ATPase +ß) (Figure 2
). A marked decrease in the amounts of all tested photosynthetic proteins was observed in pam18, while the effects in pam15 were less severe (Table 2
). These changes in thylakoid protein composition, which affected both plastid- and nucleus-encoded products (Table 2), can account for the altered kinetics of chlorophyll fluorescence observed in the two mutants.
To check whether the decrease in the level of nuclear-encoded thylakoid proteins was associated with downregulation of their corresponding genes, DNA array analysis (Kurth et al., 2002 ; Richly et al., 2003) was performed on a set of 3292 nuclear genes, most of which code for chloroplast-targeted proteins. Comparison of the data obtained from the protein and microarray analyses revealed covariation of photosynthetic proteins and their transcripts in both pam mutants (Table 2) (i.e., the decrease in protein abundance was reflected in reductions in mRNA levels, and the more prominent decrease in photosynthetic protein amounts in pam18 was associated with a more pronounced reduction in transcript levels). Together, these findings suggest that the reduced amounts of nuclear-encoded photosynthetic proteins seen in the chloroplasts of pam15 and pam18 plants are associated with lower levels of the transcripts of the corresponding nuclear genes.
The Leaky Mutations pam15 and pam18 Are Due to T-DNA Insertions in the 5'-Untranslated Region of PRORS1
Database analysis of the Arabidopsis genome revealed that At5g52520 is a single-copy gene. Its coding sequence is entirely covered by overlapping ESTs, and transcripts of the gene were detected in green tissues and roots (Figure 3C). The At5g52520 protein contains 543 amino acids and shares significant sequence identity with known prolyl-tRNA synthetases (ProRSs) from eukaryotes and prokaryotes, including Thermus thermophilus (identity/similarity of 55.8/64.2%) (Yaremchuk et al., 2001 ), Giardia intestinalis (36.7/46.9%) (Bunjun et al., 2000), Aeropyrum pernix (38.5/50.7%) (Yokozawa et al., 2003), and putative ProRS proteins from Oryza sativa (77.8/82.9%), Chlorobium tepidum (53.8/62.3%), and Clostridium sticklandii (52.4/62.1%) (Figure 4
). All of these proteins have the catalytic domain (motifs 1 to 3; Eriani et al., 1990) and anticodon binding motif (Cusack et al., 1998) characteristic of class IIa aminoacyl-tRNA synthetases (aaRSs) (reviewed in Ibba and Soll, 2000). Because protein synthesis occurs in three different compartments (cytosol, mitochondria, and chloroplasts) in plants, we searched for additional ProRS sequences in the genome of Arabidopsis. The genes At3g62120 and At5g10880 code for proteins that show moderate homology to At5g52520 (identity/similarity of 43.7/54.8% and 38.2/47.3%, respectively) and are more closely related to eukaryotic bifunctional glutamyl-prolyl-tRNA synthetases.
The findings described above indicate that the pam15 and pam18 phenotypes are due to the altered transcription of the At5g52520 gene, prompting us to designate At5g52520 as PRORS1, the pam15 mutant as prors1-1, and the pam18 allele as prors1-2.
Complete Loss of PRORS1 Function in the Knockout Mutants prors1-3 and -4 Affects Female Gametogenesis At least 500 progeny plants obtained by self-fertilizing prors1-3/PRORS1 and prors1-4/PRORS1 plants, either grown on soil or in sterile culture, were genotyped by PCR. No homozygous prors1-3 or -4 plants were identified, implying that disruption of the PRORS1 gene causes lethality. Adult prors1-3/PRORS1 and prors1-4/PRORS1 plants behaved like the wild type with respect to plant size, leaf pigmentation, and photosynthetic performance. Crosses between prors1-3/PRORS1 or prors1-4/PRORS1 plants and prors1-1 or prors1-2 plants resulted in plants that were more severely affected in growth and photosynthesis than homozygous prors1-1 or -2 plants, indicating that a direct correlation between transcript, protein abundance, and phenotype exists in these diploids.
Segregation analyses based on PCR genotyping were performed on the progeny of self-fertilized prors1-3/PRORS1 and prors1-4/PRORS1 plants and revealed that the numbers of heterozygous plants were lower than expected for a 2:1 (heterozygote:wild type) segregation ratio (Table 3
). The progenies derived from PRORS1/PRORS1 (
Development of the Embryo Sac and the Embryo Is Arrested in Null Mutants for PRORS1 We used scanning electron microscopy to analyze prors1-3/PRORS1 siliques at different developmental stages. Siliques containing seeds at the globular stage of normal embryo development were found to include small abnormal seeds and aborted ovule-like structures (Figures 5A and 5B). Visual inspection of  300 siliques at this stage showed that, in all cases, two to four abnormal seeds (corresponding to 4 to 8% of the total structures present) were observed per silique, whereas the frequency of the aborted ovule-like structures ranged from 20 to 30%. In younger siliques, abnormal seeds were difficult to distinguish from the wild type, whereas in older siliques, a fraction of seeds had disintegrated or were in the process of disintegrating. To further examine the phenotypic consequences of absence of the PRORS1 protein during seed development, 94 abnormal seeds were investigated as cleared whole mounts. Microscopy comparison revealed that all of the abnormal embryos scored were arrested at preglobular stages (Figures 5C and 5D; data not shown). In addition, the endosperm of abnormal seeds was drastically reduced (Figures 5C and 5D). Identical results were obtained for siliques of self-fertilized heterozygous prors1-4 plants. These observations indicate that the PRORS1 protein is necessary for normal seed development. In combination with the results of the PCR genotyping of the progenies of self-fertilized heterozygous prors1 knockout plants (see previous section), the findings allowed us to conclude that absence of the PRORS1 protein results in lethality prior to the seedling stage.
To further characterize the aborted ovule-like structures, optical sections of cleared, whole-mount ovules at stages 3-I and 3-VI (Schneitz et al., 1995 ) were stained with Mayer's hemalaun. At stage 3-I, only wild type–like ovules carrying the degenerating tetrad and the mononuclear embryo sac were observed in siliques of self-fertilized heterozygous prors1-3 plants, indicating that early megaspore development and meiosis proceed normally (data not shown). By contrast, of the 879 ovules scored at stage 3-VI, only 70% had a normal-looking seven-nuclear embryo sac (Figure 5E); the others had a patch of rather nondescript tissue in its place (Figure 5F). Because formation of a mononuclear embryo sac was always observed, this patch is likely to represent an embryo sac that failed to develop any further and degenerated. While these data indicate that the PRORS1 protein is essential for efficient embryo sac development, a fraction of the mutant embryo sacs look like the wild type, indicating incomplete penetrance of the mutation. Since 50% of the ovules in siliques of self-fertilized heterozygous prors1-3 and -4 plants should carry the wild-type allele, but 70% of the ovules scored at stage 3-VI had a normal-looking seven-nuclear embryo sac, we conclude that 40% of mutant ovules behave like the wild type. Indeed, these mutant ovules can be still fertilized by wild-type pollen and develop into embryos that are heterozygous for the mutation, which in turn give rise to the unexpected ratio of homozygous wild-type to heterozygous plants when fertilized with pollen from homozygous wild-type plants (as reported in the previous section). Conversely, complete absence of the PRORS1 protein (prors1-3 or -4 ovules fertilized by mutant pollen grains) leads to development arrest at preglobular stages.
PRORS1 Is Targeted to Both Mitochondria and Chloroplasts
To experimentally investigate the subcellular localization of PRORS1, the precursor protein was synthesized and radiolabeled in vitro and mixed with import-competent mitochondria and chloroplasts according to the procedure described by Rudhe et al. (2002)
A Decrease in PRORS1 Level Is Associated with Partial Loss of Proteins of the Mitochondrial Respiratory Chain and Perturbs Protein Synthesis in Plastids The role of PRORS1 in mitochondrial function was investigated by analyzing the polypeptide composition of the respiratory chain in prors1-1 and -2 mitochondria. Digitonin-solubilized mitochondria were fractionated by Blue-native PAGE in the first dimension and by SDS-PAGE in the second dimension (Figure 7A ). A decrease in the abundance of inner membrane complexes was noted in both prors1-1 (15% reduction) and prors1-2 (50% reduction) mitochondria. The levels of all of the visualized complexes were decreased to the same extent, implying a general impairment of oxidative phosphorylation. Similar results were obtained by probing total protein extracts with antibodies raised against the mitochondrial encoded cytochrome oxidase II subunit (COXII) and the ribosomal protein S12 (RPS12) (Figure 7B).
To measure the rates of protein synthesis in mutant and wild-type plastids, in vivo translation assays were performed (Figures 8A to 8C). Leaves were collected from plants at the eight-leaf rosette stage, incubated with [35S]Met and illuminated for different times (0, 15, 30, 45, and 60 min). Subsequently, thylakoid membranes were isolated and polypeptides fractionated by SDS-PAGE. Accumulation of labeled D1 protein was reduced by 20% in prors1-1 and by 70% in prors1-2 plants.
mRNA Expression Profiles in the Leaky prors1 Mutant Reveal Specific Downregulation of Photosynthetic Genes To study the effects of the altered functional state of mitochondria and chloroplasts on nuclear gene expression, the data obtained with the 3292-gene DNA array, used for the analysis of transcript abundance of nuclear genes encoding photosynthetic proteins (see above), were evaluated in more detail. Because in both leaky mutants the severity of the photosynthetic lesions seen in the light and dark differed (see Figure 1B), expression profiles were analyzed in light- and dark-adapted plants (see Methods). In light-adapted prors1-1 plants, 2170 genes displayed significant differential expression relative to wild-type plants. A similar number of genes (2124) showed differential expression in light-adapted prors1-2 plants. Direct comparison of the prors1-1 and -2 expression profiles showed that 1805 genes were differentially expressed in both genotypes, and 1763 of these showed the same trend in expression (1631 upregulated and 132 downregulated), as expected for mutations that reduce the expression of the same gene. In dark-adapted mutant plants, slightly fewer genes displayed significant differential expression (prors1-1, 2090; prors1-2, 1862). Both the number of genes differentially expressed in both genotypes (1286) and the fraction of genes exhibiting the same trend in expression (1219) were smaller than in light-grown plants (the entire set of data is available online at the Gene Expression Omnibus repository, http://www.ncbi.nlm.nih.gov/geo/; see Methods for accession numbers).
When the differentially expressed genes were grouped into 15 functional categories (Figure 9
), 80 to 90% of the genes in each category were upregulated in both mutants, irrespective of whether the plants were light- or dark-adapted. By contrast, in light-adapted mutant plants
Taken together, the mRNA profiling data indicate that the mitochondrial and chloroplast defects caused by partial loss of PRORS1 result in downregulation of genes for the light reactions of photosynthesis. This type of regulation is seen in both light- and dark-adapted prors1 plants, indicating that the transcriptional response is independent of light and of photooxidative stress.
Mutational Dissection of the Specific Contributions of Mitochondria and Chloroplasts to the Modulation of Nuclear Photosynthetic Gene Expression
Screening of the insertion flanking database SIGnAL (http://signal.salk.edu/cgi-bin/tdnaexpress) led to the isolation of the Salk_090016 line (ecotype Col-0), which carries a T-DNA element inserted at position +484 relative to the start codon of the single-copy gene At4g35490 (see Supplemental Figure 1A online). This line, designated as mrpl11-1, shows a 90% reduction in MRPL11 transcripts (see Supplemental Figure 1B online) coding for the protein MRPL11, a component of the 50S subunit of the mitochondrial ribosome. The MRPL11 protein contains 155 amino acid residues, shares significant sequence homology with other plant and prokaryotic L11 proteins (see Supplemental Figure 2 online; Porse et al., 1999
As a consequence of the marked reduction of MRPL11 gene expression, mutant plants are reduced in size and show a darker leaf coloration than the wild type (Figure 10D
). Agrobacterium tumefaciens–mediated transformation of mrpl11-1 plants with MRPL11 cDNA fused to the 35S promoter of Cauliflower mosaic virus fully restored wild-type leaf coloration and growth. Analysis of the mitochondrial electron transport chain revealed a marked and general decrease in the abundance of the protein complexes in mutant plants with respect to the wild type (Figure 7A), implying that mitochondrial activity is altered. The reduction in mitochondrial protein abundance was confirmed by protein gel blot analysis of total protein extracts (Figure 7B). However, mrpl11-1 plants showed essentially normal photosynthetic performance, as indicated by the parameters Fv/Fm (mrpl11-1, 0.80 ± 0.02; wild type, 0.79 ± 0.01),
The prpl11-1 mutation affects the plastid counterpart of MRPL11 and has been described previously (Pesaresi et al., 2001 ). PRPL11 is a component of the 50S subunit of the chloroplast ribosome. Its amino acid sequence contains a typical chloroplast transit peptide (see Supplemental Figure 2 online), and the protein is specifically imported into chloroplasts (see Supplemental Figure 3B online; Aronsson and Jarvis, 2002). The prpl11-1 plants have pale-green leaves and are drastically reduced in size in comparison with the wild type (Figure 10E). Analysis of leaf chlorophyll fluorescence revealed a marked reduction of Fv/Fm in mutant plants (prpl11-1, 0.69 ± 0.01; wild type, 0.79 ± 0.01), implying a defect in energy transfer within PSII. In addition, the II value is markedly decreased in prpl11-1 (0.55 ± 0.01 versus 0.75 ± 0.02 in the wild type), whereas the reduction state of the plastoquinone pool (1-qP) is only slightly increased (prpl11-1, 0.06 ± 0.01; wild type, 0.05 ± 0.01). Two-dimensional PAGE analysis revealed a drastic reduction (between 40 and 50%) in the level of plastome-encoded thylakoid proteins (Pesaresi et al., 2001) as a consequence of the impairment of translation in the plastids (Figure 8E). Analysis of the polypeptide composition of the mitochondrial electron transport chain, however, did not show any major difference between prpl11-1 and wild-type plants (Figures 7A and 7B), indicating that the mitochondrial activity remains unaffected.
The prpl11-1 mrpl11-1 double mutant, generated by crosses between prpl11-1 and mrpl11-1 plants, exhibited pale-green leaves and a drastic reduction in size, like prpl11-1 (Figure 10F). Furthermore, the photosynthetic performance of the double mutant is identical to that of prpl11-1 plants (Fv/Fm, 0.69 ± 0.03 versus 0.79 ± 0.01 in the wild type; Thus, while mrpl11-1 specifically impairs mitochondrial gene expression without affecting photosynthesis, the converse is true of the prpl11-1 mutation. The prpl11-1 mrpl11-1 double mutants (like prors1 plants) are defective in both mitochondrial (Figure 7B) and plastid gene expression.
Chloroplasts and Mitochondria Act Synergistically in Regulating the Expression of Photosynthetic Genes
In the prpl11-1 mutant, transcripts of only 18 of the 27 genes analyzed were downregulated, most of them accumulating to 60 to 80% of wild-type levels; the exceptions were PSAF, PSAK, LHCB2.2, LHCB6, and PSBT2, which were more strongly affected. Levels of transcripts for the antenna proteins Lhca3, Lhcb3, and Lhcb5, the G and O subunits of PSI (PsaG and PsaO), the O subunit of the oxygen-evolving complex (PsbO2), and the low molecular weight PSII subunits PsbR, PsbW, and PsbX were not affected by the prpl11-1 mutation. In mrpl11-1 leaves, fewer genes were downregulated: five encoding subunits of PSI (Lhca1, Lhca2, PsaD1, PsaE1, and PsaN) and four coding for subunits of PSII (Lhcb1.2, PsbQ2, PsbR, and PsbT2). Moreover, the abundance of transcripts was reduced by only 20 to 40%, implying that alterations in mitochondrial activity alone do not have a major influence on the transcription of nuclear photosynthetic genes. However, when mitochondrial and chloroplast functions are simultaneously impaired, as in the case of the prpl11-1 mrpl11-1 double mutant, all the nuclear photosynthetic genes analyzed were markedly downregulated to a similar extent to that seen in prors1 plants. Thus, in prpl11-1 mrpl11-1 leaves, transcript levels of most of the genes tested were reduced by up to 80%. Taken together, the data clearly demonstrate that only the concomitant impairment of mitochondrial and plastid protein synthesis, in prors1 mutants and the prpl11 mrpl11 double mutant, results in a drastic downregulation of most of the nuclear photosynthetic genes. This suggests the existence of a synergistic contribution of plastid and mitochondrial protein synthesis to the transcriptional regulation of nuclear photosynthetic genes.
Downregulation of Nuclear Photosynthetic Genes Is Not Correlated with Increased Levels of ROS
Because in plants significant amounts of ROS are also generated in peroxisomes and mitochondria, expression of genes encoding the mitochondrial alternative oxidase (AOX1) and catalases (CAT1 and CAT2) was investigated. AOX1 transcripts were increased by approximately threefold in prors1-2, prpl11-1, and mrpl11-1 leaves, but no increase was detected in prors1-1 or prpl11-1 mrpl11-1 leaves. The abundance of CAT1 transcripts did not change markedly in prors1-1, prpl11-1, or prpl11-1 mrpl11-1 leaves, while a threefold to fourfold increase was observed in prors1-2 and mrpl11-1. Furthermore, CAT2 was strongly upregulated in mrpl11-1 leaves; in prpl11-1, a reduction in transcript accumulation was detected. The markedly enhanced expression of antioxidant genes in mrpl11-1, together with the essentially wild-type content of H2O2 in leaves, is consistent with a previous report showing that an increase in antioxidant gene expression is associated with a reduction in leaf H2O2 (Dutilleul et al., 2003b ). Furthermore, it is interesting to note that prors1-1 and prpl11-1 mrpl11-1 plants did not show any increase in antioxidant gene expression, suggesting that the concomitant impairment of both organelles might result in less oxidative stress than a defect in either organelle. The increased accumulation of antioxidant transcripts in prors1-2 might be attributable to the more severe phenotype.
Singlet oxygen ( Taken together, the data do not reveal any association between ROS production in leaves and downregulation of nuclear photosynthetic genes. Although prors1-2 and prpl11-1 mrpl11-1 mutant leaves display similar patterns of downregulation of nuclear photosynthetic genes, they contain different amounts of hydrogen peroxide and antioxidant transcripts. Furthermore, the highest expression of ROS-induced genes was observed in mrpl11-1 leaves, in which the expression of photosynthetic genes was virtually unaffected.
Photosynthesis is strictly dependent on the coordinated activity of the chloroplast, mitochondrial, and nuclear compartments (Raghavendra and Padmasree, 2003 ). This, in turn, implies the existence of signaling pathways that serve to integrate nuclear and organellar gene expression (Rodermel and Park, 2003). The preeminence of the nucleus in this interorganellar exchange is indisputable: nuclear proteins imported into plastids and mitochondria regulate the transcription and translation of organellar genes. Nevertheless, it is now well established that perturbation of a number of plastid processes, including tetrapyrrole biosynthesis, protein synthesis, and photosynthesis, influence the expression of nuclear genes encoding photosynthetic proteins (Leister, 2005). In addition, investigations of mitochondrial mutants, such as cms (Noctor et al., 2004) and ncs (Newton and Coe, 1986), have clearly indicated that mitochondrial performance influences the activities of plastids and nucleus. However, modeling of mitochondrion–plastid crosstalk has been hampered by the inability of available experimental strategies to unequivocally define individual signaling pathways (Gray et al., 2002). Norflurazon, which was used to initially define the plastid-to-nucleus signal, for instance, results in a broad range of secondary effects, including ROS production (Mullineaux and Karpinski, 2002), and impairment of transcription (Tonkyn et al., 1992) and translation (Frosch et al., 1979; Reiss et al., 1983) in plastids. Studies employing inhibitors of prokaryotic-like 70S ribosomes, such as lincomycin, chloramphenicol, erythromycin, and streptomycin, could not distinguish unambiguously between effects on plastid and mitochondrial protein synthesis. Furthermore, the genetic dissection of organelle signaling has often been hindered by the pleiotropic nature of many mutant phenotypes.
In this study, the role of chloroplast and mitochondrial protein synthesis in controlling nuclear photosynthetic gene expression has been investigated genetically by employing mutants primarily affected in either the mitochondrial (mrpl11) or chloroplast (prpl11) compartment or in both (prors1). The PRORS1 locus encodes a ProRS with an ambiguous targeting presequence that is recognized as an import signal by both mitochondria and chloroplasts (Figure 6). Many proteins have now been shown to be localized in both mitochondria and chloroplasts, and most of them belong to the aaRS family: MetRS (Menand et al., 1998
Since all proteins encoded by the chloroplast and mitochondrial genomes of Arabidopsis contain at least one Pro residue, a complete lack of ProRS activity in these compartments should result in lethality. Indeed, absence of PRORS1 impairs ovule development and arrests embryo development at preglobular stages, implying that neither of the other two putative ProRS enzymes, At3g62120 and At5g10880, can efficiently replace PRORS1. In addition to the prors1-3 and -4 mutations described in this article, knockout mutants for other mitochondrial aaRSs have been described; however, these were only impaired in embryo development. In the acd mutant, which shows reduced expression of the ALARS gene coding for an alanyl-tRNA synthetase targeted to both cytosol and mitochondria (Mireau et al., 1996
The two leaky prors1 alleles, prors1-1 and -2, were identified because of their photosynthetic phenotype. The drop in photosynthetic performance in prors1-1 and, to a greater extent, in prors1-2 mutants could be attributed to the direct impairment of chloroplast and mitochondrial translation. As an additional consequence of the mutation, however, a marked and specific downregulation of nuclear photosynthetic gene expression was detected in prors1-2 (and to a lesser extent also in prors1-1). The contribution of each organelle to the repression of nuclear photosynthetic gene expression was assessed by analyzing mutants that are defective specifically in translation in mitochondria or plastids. A defect in protein synthesis in either mitochondria (in the mrpl11 mutant) or chloroplasts (in the prpl11 mutant) failed to elicit a transcriptional response like that observed in prors1 leaves (Figure 12). Only when both organelles were affected (in the prpl11-1 mrpl11-1 double mutant) was a prors1-like transcriptional response observed, implying that the two organelles cooperate in regulating the expression of nuclear photosynthetic genes. Clearly, the prpl11-1 mrpl11-1 transcriptional response was not due to an additive effect of impaired mitochondria on plastid activity, since the prpl11-1 and prpl11-1 mrpl11-1 leaves were identical in terms of thylakoid protein composition and photosynthetic performance. Moreover, prors1-1 leaves showed a more general and marked downregulation of nuclear photosynthetic gene expression than prpl11-1, although the prpl11-1 chloroplast activities were more drastically impaired. The fact that a comparable decrease in the level of photosynthetic transcripts is observed in pea seedlings treated with lincomycin and erythromycin, which inhibit translation in both chloroplasts and mitochondria (Sullivan and Gray, 1999
In addition to organellar protein synthesis per se, the prors1 and prpl11 mrpl11 mutations also affect photosynthetic electron transfer, which itself is known to modulate the expression of nuclear genes coding for thylakoid proteins. Light intensity–dependent changes in LHCB1 transcription in the green alga Dunaliella tertiolecta are due to changes in the redox state of the plastoquinone pool and in the transthylakoid membrane potential (Escoubas et al., 1995 The use of mutants for the dissection of organelle-to-nucleus signaling has often raised the question whether the developmental stage of the organelles might influence nuclear gene expression. A specific and general downregulation of nuclear photosynthetic genes was observed even in prors1-1 leaves, in which the protein composition of mitochondria and plastids was only marginally altered and mutant plants grew like the wild type under low light levels. Therefore, it can be concluded that organelle function, rather than developmental stage, is involved in the observed transcriptional response. Taken together, our data indicate that alterations in rates of translation in plastids and mitochondria are able to modulate synergistically the transcription of nuclear photosynthetic genes independently of light. How these signals are transduced from the organelles to the nucleus and integrated at the molecular levels remains to be revealed. ROS have been suggested as signaling molecules in the crosstalk between organelles and the n |
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ABSTRACT
INTRODUCTION
assimilation (Kromer, 1995
) x prors1-3/PRORS1 (
) and PRORS1/PRORS1 (
) has been also reported to influence the expression of photosynthesis-related nuclear genes (op den Camp et al., 2003