| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online April 9, 2008; 10.1104/pp.108.116194 Plant Physiology 147:573-584 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Pentratricopeptide Repeat Protein DELAYED GREENING1 Is Involved in the Regulation of Early Chloroplast Development and Chloroplast Gene Expression in Arabidopsis1,[W],[OA]Photosynthesis Research Center, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China (W.C., J.M., J.G., C.L., L.Z.); School of Life Sciences, Lanzhou University, Lanzhou 73000, China (D.Z.); and Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100086, China (F.C.)
An Arabidopsis (Arabidopsis thaliana) mutant that exhibited a delayed greening phenotype (dg1) was isolated from a population of activation-tagged Arabidopsis lines. Young, inner leaves of dg1 mutants were initially very pale, but gradually greened and mature outer leaves, more than 3 weeks old, appeared similar to those of wild-type plants. Sequence and transcription analyses showed that DG1 encodes a chloroplast protein consisting of eight pentratricopeptide repeat domains and that its expression depends on both light and developmental status. In addition, analysis of the transcript profiles of chloroplast genes revealed that plastid-encoded polymerase-dependent transcript levels were markedly reduced, while nucleus-encoded polymerase-dependent transcript levels were increased, in dg1 mutants. Thus, DG1 is probably involved in the regulation of plastid-encoded polymerase-dependent chloroplast gene expression during early stages of chloroplast development.
The formation of normal chloroplasts is crucial for higher plant growth and development. The chloroplast genomes of higher plants typically encode only about 100 genes, and the vast majority of the greater than 2,000 proteins that have functions in the chloroplast are encoded by nuclear genes, translated in the cytosol, and subsequently imported into the chloroplast (Abdallah et al., 2000  ). Thus, the development of functional chloroplasts (which is essential for the normal autotrophic growth and development of higher plants) is dependent on the coordinated expression of nuclear and chloroplast genes. The transcriptional levels of plastid-encoded genes change dramatically during the course of chloroplast development and are dependent on the stage of plastid development (Mullet, 1993; Emanuel et al., 2004; Zoschke et al., 2007), suggesting that adjustments to the transcriptional machinery play important roles in the regulation of chloroplast development. Two types of RNA polymerases have been identified in higher plant chloroplasts: plastid-encoded polymerases (PEPs) and nucleus-encoded polymerase (NEPs; Maliga, 1988; Hajdukiewicz et al., 1997; Hedtke et al., 1997). PEPs contain core subunits encoded by rpoA, rpoB, rpoC1, and rpoC2 genes, while NEPs are each composed of a single submit (Hedtke et al., 1997; Liere and Maliga, 1999). These two types of polymerases are responsible for the transcription of distinct sets of chloroplast genes (Allison et al., 1996; Hajdukiewicz et al., 1997). The chloroplast-encoded photosynthetic genes (such as psbA, psbD, and rbcL) are exclusively transcribed by PEPs, a few genes (mostly encoding components of the transcription/translation apparatus, such as rpoB) are exclusively transcribed by NEPs, and nonphotosynthetic housekeeping genes are mostly transcribed by both PEPs and NEPs. Since NEPs mainly transcribe plastid genes encoding proteins involved in transcription/translation, while PEP accounts for the transcription of photosynthetic genes (Kapoor et al., 1997; Liere and Maliga, 1999), correct timing of photosynthetic gene expression relies on an increase in PEP transcription activity during the course of chloroplast development (Mullet, 1993; Demarsy et al., 2006). Thus, PEPs may have important functions during early stages of chloroplast development.
Although PEPs are plastid encoded, the transcription of PEP-transcribed genes is also under the control of nuclear genes. A large number of proteins associated with the PEP catalytic core (Pfannschmidt et al., 2000
Pentratricopeptide repeat (PPR) proteins, which are defined by the tandem array of a PPR motif consisting of 35 amino acids, are widely distributed in higher plants. There are 466 members in Arabidopsis and 480 in rice (Oryza sativa), but their functions are largely unknown (Small and Peeters, 2000 Here, we describe a T-DNA insertion Arabidopsis mutant named dg1 (for delayed greening1) in which early chloroplast development is affected. The DG1 gene encodes a novel PPR protein that is probably involved in the regulation of PEP-dependent transcript accumulation.
Phenotype of dg1
The dg1 mutant was selected by its high chlorophyll fluorescence phenotype from a population of pSKI015 T-DNA-mutagenized Arabidopsis lines from the Arabidopsis Biological Resource Center (Peng et al., 2006
As shown in Figure 1B, the wild-type cotyledons were green when grown on Suc-supplemented Murashige and Skoog (MS) medium. However, the cotyledons of 3-d-old dg1 seedlings grown on this medium were white, then turned yellow at the 5th d. After 7 d, the cotyledons of dg1 became pale-green, and at 12 d, they had acquired an almost normal green coloration, albeit slightly paler than that of wild-type plants (Fig. 1B). The delayed greening phenotype of dg1 cotyledons suggested that DG1 may play a critical role in chloroplast development in early stages of seedling growth. To test this hypothesis, dg1 seeds were germinated on medium both with and without supplementary Suc, since exogenously supplied Suc is required to establish autotrophic growth when the early development of seedlings is arrested (Li et al., 1995 ). The development of dg1 seedlings on medium without supplementary Suc was severely inhibited compared with their development in the presence of exogenous Suc. After growth for 12 d without Suc, the cotyledons of dg1 mutants still remained yellow and no true leaves had appeared (Fig. 1C).
To investigate the effects of the dg1 mutation on chloroplast development, we measured the chloroplasts and examined their ultrastructure in both wild-type and mutant plants by transmission electron microscopy. The observations showed that the chloroplasts were smaller in dg1 mutants than in wild-type plants in both the cotyledons of 5-d-old seedlings (wild type, 5.0 ± 0.3 µm; dg1, 3.0 ± 0.5 µm) and young, 3-week-old leaves of 4-week-old plants (Fig. 2 ). In addition, transmission electron microscopy observations of ultrathin sections of chloroplasts in 5-d-old cotyledons showed that those in wild-type plants had well-structured thylakoid membranes, composed of grana connected by stroma lamellae. However, the thylakoid membrane organization in the chloroplasts of 5-d-old dg1 cotyledons was disturbed, and their thylakoid membranes were much less abundant. In contrast, the thylakoid membrane organization and abundance in mature 4-week-old leaves of dg1 mutants were similar to those in wild-type plants (Fig. 2).
Cloning of the DG1 Gene The genetic analysis showed that dg1 was a single recessive mutant, and cosegregation of the phosphinotricin resistance marker of the T-DNA with the mutant phenotype indicated that the mutation was due to the T-DNA insertion. The insertion site in dg1 was identified by thermal asymmetric interlaced (TAIL)-PCR isolation of the genomic sequences flanking the T-DNA borders and subsequent sequence analysis, which showed that the T-DNA was inserted in the 5' untranslated region of At5g67570, –3 bp relative to the ATG start codon (Fig. 3A ). No expression of the At5g67570 gene was detected in dg1 mutants by northern-blot analysis, although the levels of transcripts of its neighboring genes (At5g67560 and At5g67580) appeared to be similar to those of wild-type plants (Fig. 3B).
Database analysis of the Arabidopsis genome revealed that two partially overlapping cDNA clones (AK22212 and NM-126157) with different lengths occur at this locus. The 3' region of the NM-126157 clone overlaps with the 5' region of the AK22212 clone. Reverse transcription (RT)-PCR and sequence analysis of PCR products revealed that the full-length cDNA of At5g67570 does indeed consist of these two combined clones (Supplemental Fig. S1) and that two types of transcripts are present due to alternative splicing (Fig. 3C). One (designated transcript 1) appears to be functional, with an open reading frame that putatively encodes a polypeptide of 798 amino acids, while the other (transcript 2) encodes a truncated polypeptide with 205 amino acids. Transcript 2 has three additional exons, which act as introns (introns 1, 5, and 8) in transcript 1, and a nine-nucleotide insertion (5'-TCACTTTAG-3') in the 5' region of exon 4 (Fig. 3C). Sequence alignment revealed that the nine-nucleotide short sequence corresponds to the 3' region of intron 3, which indicated that two different splicing sites exist at the 3' end of intron 3 and that the nine-nucleotide sequence may be due to its alternative splicing. Such a phenomenon of alternative splicing was also observed in rice SDHB and peach (Prunus persica) ETR1, which are associated with differences in tissue localization and responses to environmental stresses, respectively (Kubo et al., 1999 ; Bassett et al., 2002). Besides the alternative splicing above, DG1 had unusual GC/AG borders at the 5' and 3' splicing sites in intron 7 (see www.arabidopsis.org/servlets/tairobject), rather than the GT/AG borders in the vast majority of eukaryotic introns. In Arabidopsis, nonconventional splicing sites amount to 0.7% of all splice sites (Alexandrov et al., 2006). Unusual borders of CA/CC and AT/AC in splicing sites have also been detected in rice AGPP and Arabidopsis AtLIM15, respectively (Anderson et al., 1991; Sato et al., 1995). The sequences of transcripts 1 and 2 are provided in Supplemental Figure S1. Only one signal, corresponding to transcript 1, was detected in northern-blot analyses of wild-type plants, and no signal was detected at the expected position of transcript 2 (Fig. 3B), which may be due to the very low-level expression of transcript 2. To further examine the expression of these two transcripts, RT-PCR using primers spanning intron 1 was performed (Fig. 3C). RT-PCR analysis revealed that transcript 2 accumulated to very low levels compared with transcript 1 in wild-type plants and that neither transcript was expressed in dg1 mutants (Fig. 3D), in accordance with the results of northern-blot analysis (Fig. 3B). To quantify the relation between transcript 1 and transcript 2, real-time RT-PCR with specific primers was performed. Our results showed that the abundance of transcript 2 was about 7% of that of transcript 1. To confirm that the inactivity of At5g67570 was responsible for the mutant phenotype of dg1, we genetically complemented the line with the full-length At5g67570 cDNA under the control of the cauliflower mosaic virus 35S promoter and obtained seven independent transgenic plants. Subsequent phenotypic observations and chlorophyll fluorescence analyses confirmed that wild-type traits had been restored in the complemented mutant (Fig. 1, A and C). Thus, it can be concluded that the disruption of At5g67570 is indeed responsible for the dg1 mutant phenotype.
BLAST searches of the complete Arabidopsis sequence revealed that only one copy of the DG1 gene is present in the nuclear genome, which encodes a putative polypeptide of 798 amino acids with a calculated molecular mass of 92 kD, containing eight PPR motifs (Fig. 4 ). Protein alignments showed that it shares significant identity with the Medicago truncatula protein MtrDRAFT_AC147000g14V1 (73% similarity) and the rice protein Os05g0315100 (64% similarity). Another (hypothetical) Arabidopsis protein, At1g30610, was also found to have high similarity (59%) to DG1.
To examine the cellular localization of the DG1 protein, we constructed a chimeric gene expressing a fusion protein consisting of the 300 N-terminal amino acids of DG1 and GFP under the control of the 35S promoter. The plasmid containing the chimeric gene was transformed into wild-type Arabidopsis plants, and leaves of stably transformed plants were examined by confocal laser-scanning microscopy. The fusion protein was colocalized with the chloroplastic chlorophyll in the mesophyll cells, in accordance with results obtained when the GFP was fused to the transit peptide of the small subunit of Arabidopsis ribulose bisphosphate carboxylase (Fig. 5E ; Lee et al., 2002 ). In contrast, GFP signals accumulated specifically in the nucleus when GFP was fused to the nuclear localization signal of the fibrillarin protein from Arabidopsis (Pih et al., 2000). In addition, GFP signals were found to accumulate in both the cytoplasm and the nucleus when Arabidopsis was transformed with the control vector without a specific targeting sequence, and no GFP signal was detected in wild-type, untransformed plants. Thus, these findings suggest that DG1 is targeted to the chloroplast.
Embryogenesis Was Affected in dg1
BLAST searches of the Arabidopsis database revealed that At5g67570 was cataloged as the candidate gene EMB1408 (for EMBRYO-DEFECTIVE1408) involved in embryo development. To confirm its involvement in the development of embryos, siliques from homozygous dg1 plants were dissected approximately 7 d after pollination, and cleared ovules were examined by differential interference contrast microscopy using a Nomarski optics microscope. The results showed that about 40% of the ovules were arrested at various stages, from the early globular stage to the heart stage, in dg1 plants, while the remaining 60% apparently developed normally, similar to wild-type ovules (Table I
). The arrested embryos subsequently shriveled and finally degenerated. Impaired embryo development has also been observed in other mutants in which plastid development is affected (Uwer et al., 1998
Expression Profiles of DG1
Light, which triggers the differentiation of nonphotosynthetic proplastids into fully functional photosynthetic chloroplasts, is one of the most important signals influencing chloroplast development (López-Juez and Pyke, 2005
To study the expression of DG1 at different developmental stages, levels of DG1 transcripts in leaves ranging from 1 to 4 weeks old were evaluated by northern-blot analysis. The expression level of DG1 decreased as the age and developmental state of both the plants and leaves increased. The DG1 transcript content of 4-week-old plants was only about 10% of that detected in 1-week-old plants (Fig. 6B), and levels of DG1 transcripts in the mature leaves were only approximately 20% of those in the young leaves of 6-week-old plants (Fig. 6C).
To assess the possibility that the delayed chloroplast development in dg1 mutants may be reflected at the level of photosynthetic gene expression, we also examined the expression of photosynthetic genes in dg1 by northern-blot analysis. Since there was a clear difference in Fv/Fm ratios between young leaves (1–3 weeks old) and mature green leaves (>3 weeks old) in dg1 plants, differences in plastid transcript profiles were compared between young and old mature leaves as well as between mutant and wild-type plants. As shown in Figure 7A , there were no significant differences in the contents of PEP-dependent transcripts in mature green leaves of dg1 mutants, apart from slight increases in psbA and psaA transcripts. However, the levels of psbA, psbB, psbC, psbEFJL, psaA, and petA transcripts were significantly lower in young leaves of dg1 mutants than in those of wild-type plants (Fig. 7A). Furthermore, psbA transcripts were detected in the cotyledons of wild-type plants after growth for 3 d, and their levels increased with further development, while in dg1 cotyledons psbA was expressed much more weakly until the plants were 7 d old (Fig. 7B). The delayed accumulation of PEP-dependent transcripts was consistent with the delayed greening phenotype of dg1.
In addition, our results showed that the contents of rpoB transcripts were almost identical in mature green leaves of dg1 and wild-type plants, but they were considerably higher in the young leaves of dg1 plants. In contrast, levels of clpP and accD transcripts were higher in both young and mature leaves of dg1 plants (Fig. 7C). These findings are interesting, since clpP is transcribed by both PEP and NEP, while rpoB and accD are transcribed exclusively by NEP. Unlike the plastid genes mentioned above, transcript levels of the nuclear genes were largely unaltered in both young and mature leaves of dg1 plants (Fig. 7D).
Nucleus-encoded factors play essential roles in the regulation of chloroplast development, which requires the coordinated expression of both nucleus-encoded and chloroplast-encoded genes. In the study reported here, we identified a novel Arabidopsis mutant with delayed greening, dg1, and isolated the affected gene, which encodes a PPR protein targeted to the chloroplast. Subsequent genetic and molecular analyses showed that DG1 is probably involved in the regulation of early stages of chloroplast development and plastid gene expression.
The most distinct characteristic of dg1 was retarded greening of both its cotyledons and rosette leaves. The young leaves were initially chlorotic, then gradually greened during development in dg1 plants, and the mature, green leaves appeared similar to those of wild-type plants. Analyses of chlorophyll fluorescence in dg1 mutants revealed that photosynthetic activity was reduced in their young leaves (Fig. 1). In addition, ultrastructural observations showed that thylakoid membranes were less abundant in the young leaves of the mutants than in wild-type counterparts, but their abundance gradually increased as the leaves developed and eventually reached levels similar to wild-type levels (Fig. 2). These results suggest that DG1 plays important roles in early stages of chloroplast development and that functional chloroplast development was dependent on leaves reaching a mature or close to mature developmental state in the dg1 mutants.
Several mutants with impaired chloroplast development have been described. Sig6-1 mutants, which have no functional copies of the plastid sigma factor gene AtSIG6, have chlorotic young cotyledons, but a wild-type phenotype is restored in 8-d-old seedlings (Ishizaki et al., 2005
The expression of both nucleus- and chloroplast-encoded photosynthetic genes was tightly linked with the developmental status of the chloroplasts. In dg1 mutants, the levels of PEP-dependent transcripts were reduced, levels of NEP-dependent transcripts were increased, and levels of nuclear gene transcripts were not clearly altered (Fig. 7). It is generally accepted that NEP activities are largely responsible for the transcription of housekeeping genes during early stages of chloroplast development and that PEP activities increase while NEP activities decrease during subsequent chloroplast development (Hanaoka et al., 2005
Three interpretations can be made to the observation of decreased accumulation of PEP-dependent transcripts. PEP is a multisubunit (bacterial-type) polymerase. Recently, in addition to the chloroplast-encoded catalytic core subunits, a number of polypeptides have been shown to be associated with transcriptionally active chloroplast chromosomes (Pfannschmidt et al., 2000
The developmental status of the chloroplast has been shown to control a set of nuclear genes that encode chloroplast-localized proteins via a process known as retrograde signaling (Surpin et al., 2002
Plant Materials and Growth Conditions Mutant and wild-type Arabidopsis (Arabidopsis thaliana ecotype Columbia) plants were grown in soil under short-day conditions (10-h-light/14-h-dark cycles) with a photon flux density of 120 µmol m–2 s–1 at a constant temperature of 22°C. To ensure synchronized germination, the seeds were incubated in darkness for 48 h at 4°C after sowing. For growth on agar plates, seeds were surface sterilized and sown on MS medium containing 2% Suc (except in the Suc-free treatment) and 0.8% agar.
Chlorophyll a fluorescence images were obtained at room temperature using a modulated imaging fluorometer (FluorCam; Photon System Instruments) according to the instructions provided by the manufacturer. Values of Fo (for minimum fluorescence yield) and Fm (for maximum fluorescence yield) were averaged to improve the signal-to-noise ratio. Image data acquired in each experiment were normalized to a false color scale, with arbitrarily assigned extreme values of 0.3 (lowest) and 0.85 (highest). This resulted in the highest and lowest Fv/Fm values being represented by the red and blue extremes of the color scale, respectively.
Chlorophyll fluorescence was measured using a PAM 2000 portable chlorophyll fluorometer (Walz) connected by a leaf-clip holder (2030-B; Walz) with a trifurcated fiber optic cable (2010-F; Walz; Peng et al., 2006
DNA fragments flanking the T-DNA were obtained by a series of three nested TAIL-PCRs (Liu et al., 1995 Total RNA isolation and RT-PCR were performed according to the manufacturer's instructions. The specific primers used to amplify the cDNA of DG1 were as follows: sense primer, 5'-CTTGCACTATCCTCCTCTTCGCAAC-3'; antisense primer, 5'-TCATTCTTTCTTACTACTCTCTTCC-3'. The primers spanning intron 1 were as follows: PU, 5'-AGAGTGCCACCGGAATTTGAACCAG-3'; and PD, 5'-TTCCTGTGGCCTCCTCGCAAAC-3'.
Two different primer pairs were used to quantify the expression of transcript 1 and transcript 2. One primer pair was designed to specifically amplify transcript 2 (forward, 5'-CCAACATAAACGAACTGCTG-3'; reverse, 5'-ACATCTTAAGACCACCCTACAT-3'), whereas the other primer pair amplifies both transcripts (forward, 5'-GCAGGCCGCTCGAGATTAC-3'; reverse, 5'-CATCAACATGACCACCGTTCA-3'). The amplification of ELONGATION FACTOR1-
Samples of wild-type and mutant leaves were prepared for transmission electron microscopy by cutting them into small pieces, fixing in 2.5% glutaraldehyde in phosphate buffer for 4 h at 4°C, rinsing and incubating them in 1% OsO4 overnight at 4°C, rinsing again in phosphate buffer, dehydrating in an ethanol series, infiltrating with a graded series of epoxy resin in epoxy propane, and then embedding them in Epon 812 resin. Thin sections were obtained using a diamond knife and a Reichert OM2 ultramicrotome, stained in 2% uranyl acetate, pH 5.0, followed by 10 mM lead citrate, pH 12, then viewed with a transmission electron microscope (JEM-1230; JEOL).
Siliques from wild-type and mutant plants were dissected with hypodermic needles and cleared in Hoyer's solution (7.5 g of gum arabic, 100 g of chloral hydrate, and 5 mL of glycerol in 30 mL of water) as described by Meinke (1994)
The full-length DG1 cDNA amplified using PCR was cloned into the pBI121 vector under the control of the cauliflower mosaic virus 35S promoter. The resultant construct was transferred into Agrobacterium tumefaciens strain C58, then introduced into dg1 homozygous plants using the floral dip method (Clough and Bent, 1998
DNA encoding the N-terminal region of the DG1 protein (amino acids 1–300) was amplified by PCR and ligated into the GFP fusion vector p221-GFP, which encodes a GFP under the constitutive control of the cauliflower mosaic virus 35S promoter. Two variants were then constructed: one in which the nuclear localization signal of the fibrillarin protein (Pih et al., 2000
Total RNAs were extracted, fractionated, and transferred onto nylon membranes, which were probed with 32P-labeled cDNA probes. Following high-stringency hybridization and washing, all of the blots were exposed to x-ray film for 1 to 3 d. The hybridization probes were prepared using a random priming labeling kit (Promega) with the PCR fragments of encoding regions of target genes. The sequences of the PCR primers used to amplify the genes are presented in Supplemental Table S1. Sequence data for the alignments performed in this study can be found in the GenBank/EMBL data libraries under the following accession numbers: NP_201558 for Arabidopsis At5g67570; NP_001043842 for rice Os01g0674700; NP_001055170 for rice Os05g035100; ABE79582 for M. truncatula MtrDRAFT_AC147000g14V1; and NP_174349 for Arabidopsis At1g30610. Mutant lines used in this study can be found in The Arabidopsis Information Resource as Arabidopsis seed stock CS31100.
The following materials are available in the online version of this article.
We thank Prof. Weicai Yang for helpful discussions. We thank the Arabidopsis Biological Resource Center for the Arabidopsis seeds. We also thank Zeal Quest Scientific Technology Co. Ltd. for technical assistance. Received January 11, 2008; accepted April 1, 2008; published April 9, 2008.
1 This work was supported by the National Natural Science Foundation of China (grant nos. 30725003 and 30670164) and the Frontier Project of Knowledge Innovation Engineering of the Chinese Academy of Sciences (grant no. KJCX2–SW–w29).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Lixin Zhang (zhanglixin{at}ibcas.ac.cn).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.116194 * Corresponding author; e-mail zhanglixin{at}ibcas.ac.cn.
Abdallah F, Salamini F, Leister D (2000) A prediction of the size and evolutionary origin of the proteome of chloroplasts of Arabidopsis. Trends Plant Sci 5: 141–142[CrossRef][ISI][Medline] Alexandrov NN, Troukhan ME, Brover VV, Tatarinova T, Flavell RB, Feldmann KA (2006) Features of Arabidopsis genes and genome discovered using full-length cDNAs. Plant Mol Biol 60: 69–85[CrossRef][ISI][Medline] Allison LA, Simon LD, Maliga P (1996) Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants. EMBO J 15: 2802–2809[ISI][Medline] Anderson JM, Larsen R, Laudencia D, Kim WT, Morrow D, Okita TW, Preiss J (1991) Molecular characterization of the gene encoding a rice endosperm-specific ADP glucose pyrophosphorylase subunit and its developmental pattern of transcription. Gene 97: 199–205[CrossRef][ISI][Medline] Apuya NR, Yadegari R, Fischer RL, Harada JJ, Zimmerman JL, Goldberg RB (2001) The Arabidopsis embryo mutant schlepperless has a defect in the chaperonin-60a gene. Plant Physiol 126: 717–730 Baba K, Schmidt J, Espinosa-Ruiz A, Villarejo A, Shiina T, Gardestrom P, Sane AP, Bhalerao RP (2004) Organellar gene transcription and early seedling development are affected in the rpoT; 2 mutant of Arabidopsis. Plant J 38: 38–48[CrossRef][ISI][Medline] Baldwin A, Wardle A, Patel R, Dudley P, Park SK, Twell D, Inoue K, Jarvis P (2005) A molecular-genetic study of the Arabidopsis Toc75 gene family. Plant Physiol 138: 715–733 Bassett CL, Artlip TS, Callahan AM (2002) Characterization of the peach homologue of the ethylene receptor, PpETR1, reveals some unusual features regarding transcript processing. Planta 215: 679–688[CrossRef][ISI][Medline] Bellaoui M, Keddie JS, Gruissem W (2003) DCL is a plant-specific protein required for plastid ribosomal RNA processing and embryo development. Plant Mol Biol 53: 531–543[CrossRef][ISI][Medline] Boudreau E, Nickelsen J, Lemaire SD, Ossenbuhl F, Rochaix JD (2000) The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability. EMBO J 19: 3366–3376[CrossRef][ISI][Medline] Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][ISI][Medline] Courtois F, Merendino L, Demarsy E, Mache R, Lerbs-Mache S (2007) Phage-type RNA polymerase RPOTmp transcribes the rrn operon from the PC promoter at early developmental stages in Arabidopsis. Plant Physiol 145: 712–721 de Longevialle AF, Meyer EH, Andres 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: 3256–3265 Demarsy E, Courtois F, Azevedo J, Buhot L, Lerbs-Mache S (2006) Building up of the plastid transcriptional machinery during germination and early plant development. Plant Physiol 142: 993–1003 Emanuel C, Weihe A, Graner A, Hess WR, Börner T (2004) Chloroplast development affects expression of phage-type RNA polymerases in barley leaves. Plant J 38: 460–472[CrossRef][ISI][Medline] Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300: 1005–1016[CrossRef][ISI][Medline] Fisk DG, Walker MB, Barkan A (1999) Molecular cloning of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression. EMBO J 18: 2621–2630[CrossRef][ISI][Medline] Gutiérrez-Marcos JT, Dal Prà M, Giulini A, Costa LM, Gavazzi G, Cordelier S, Sellam O, Tatout C, Paul W, Perez P, et al (2007) empty pericarp4 encodes a mitochondrion-targeted pentatricopeptide repeat protein necessary for seed development and plant growth in maize. Plant Cell 19: 196–210 Hajdukiewicz PTJ, Allison LA, Maliga P (1997) The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids. EMBO J 16: 4041–4048[CrossRef][ISI][Medline] Hanaoka M, Kanamaru K, Fujiwara M, Takahashi H, Tanaka K (2005) Glutamyl-tRNA mediates a switch in RNA polymerase use during chloroplast biogenesis. EMBO Rep 6: 545–550[CrossRef][ISI][Medline] Hashimoto M, Endo T, Peltier G, Tasaka M, Shikanai T (2003) A nucleus-encoded factor, CRR2, is essential for the expression of chloroplast ndhB in Arabidopsis. Plant J 36: 541–549[CrossRef][ISI][Medline] Hedtke B, Börner T, Weihe A (1997) Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis. Science 277: 809–811 Hess WR, Prombona A, Fieder B, Subramanian AR, Börner T (1993) Chloroplast RPS15 and the RPOB/C1/C2 gene cluster are strongly transcribed in ribosome deficient plastids: evidence for a functioning non-chloroplast encoded RNA polymerase. EMBO J 12: 563–571[ISI][Medline] Hörmann F, Küchler M, Sveshnikov D, Oppermann U, Li Y, Soll J (2004) Tic32, an essential component in chloroplast biogenesis. J Biol Chem 279: 34756–34762 Hricová A, Quesada V, Micol JL (2006) The SCABRA3 nuclear gene encodes the plastid Rpo Tp RNA polymerase, which is required for chloroplast biogenesis and mesophyll cell proliferation in Arabidopsis. Plant Physiol 141: 942–958 Ishizaki Y, Tsunoyama Y, Hatano K, Ando K, Shinmyo A, Kobori M, Takeba G, Nakahira Y, Shiina T (2005) A nuclear-encoded sigma factor, Arabidopsis SIG6, recognizes sigma-70 type chloroplast promoters and regulates early chloroplast development in cotyledons. Plant J 42: 133–144[CrossRef][ISI][Medline] Kapoor S, Suzuki JY, Sugiura M (1997) Identification and functional significance of a new class of non-consensus-type plastid promoters. Plant J 11: 327–337[CrossRef][ISI][Medline] Kobayashi H, Kondo M, Fukuda H, Nishimura M, Ohta H (2007) Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis. Proc Natl Acad Sci USA 104: 17216–17221 Kotera E, Tasaka M, Shikana T (2005) A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature 433: 326–330[CrossRef][Medline] Kovacheva S, Bédard J, Patel R, Dudley P, Twell D, Ríos G, Koncz C, Jarvis P (2005) In vivo studies on the roles of Tic110, Tic40 and Hsp93 during chloroplast protein import. Plant J 41: 412–428[ISI][Medline] Kubo N, Harada K, Hirai A, Kadowaki K (1999) A single nuclear transcript encoding mitochondrial RPS14 and SDHB of rice is processed by alternative splicing: common use of the same mitochondrial targeting signal for different proteins. Proc Natl Acad Sci USA 96: 9207–9211 Lee KH, Kim DH, Lee SW, Kim ZH, Hwang I (2002) In vivo import experiments in protoplasts reveal the importance of the overall context but not specific amino acid residues of the transit peptide during import into chloroplasts. Mol Cells 14: 388–397[ISI][Medline] Li H, Culligan K, Dixon RA, Chory J (1995) CUE1: a mesophyll cell-specific positive regulator of light-controlled gene expression in Arabidopsis. Plant Cell 7: 1599–1610[Abstract] Liere K, Maliga P (1999) In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters. EMBO J 18: 249–257[CrossRef][ISI][Medline] Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8: 457–463[CrossRef][ISI][Medline] Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2– López-Juez E, Pyke KA (2005) Plastids unleashed: their development and their integration in plant development. Int J Dev Biol 49: 557–577[CrossRef][ISI][Medline] Loschelder H, Homann A, Ogrzewalla K, Link G (2004) Proteomics-based sequence analysis of plant gene expression: the chloroplast transcription apparatus. Phytochemistry 65: 1785–1793[CrossRef][ISI][Medline] Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, Bruyere C, Caboche M, Debast C, Gualberto J, Hoffmann B, et al (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16: 2089–2103 Maliga P (1988) Two plastid RNA polymerases of higher plants: an evolving story. Trends Plant Sci 3: 4–6[Medline] Meierhoff K, Felder S, Nakamura T, Bechtold N, Schuster G (2003) HCF152, an Arabidopsis RNA binding pentatricopeptide repeat protein involved in the processing of chloroplast psbB-psbT-psbH-petB-petD RNAs. Plant Cell 15: 1480–1495 Meinke DW (1994) Seed development in Arabidopsis thaliana. In EM Meyerowitz, CR Somerville, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 253–259 Meurer J, Grevelding C, Westhoff P, Reiss B (1998) The PAC protein affects the maturation of specific chloroplast mRNAs in Arabidopsis thaliana. Mol Gen Genet 258: 342–351[CrossRef][ISI][Medline] Meurer J, Lezhneva L, Amann K, Godel M, Bezhani S, Sherameti I, Oelmuller R (2002) A peptide chain release factor 2 affects the stability of UGA-containing transcripts in Arabidopsis chloroplasts. Plant Cell 14: 3255–3269 Mullet JE (1993) Dynamic regulation of chloroplast transcription. Plant Physiol 103: 309–313[CrossRef][ISI][Medline] Okuda K, Myouga F, Motohashi R, Shinozaki K, Shikanai T (2007) Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proc Natl Acad Sci USA 104: 8178–8183 Peng L, Ma J, Chi W, Guo J, Zhu S, Lu Q, Lu C, Zhang L (2006) LOW PSII ACCUMULATION 1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana. Plant Cell 18: 955–969 Pfalz J, Liere K, Kandlbinder A, Dietz KJ, Oelmüller R (2006) pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression. Plant Cell 18: 176–197 Pfannschmidt T, Ogrzewalla K, Baginsky S, Sickmann A, Meyer HE, Link G (2000) The multisubunit chloroplast RNA polymerase A from mustard (Sinapis alba L.): integration of a prokaryotic core into a larger complex with organelle-specific functions. Eur J Biochem 267: 253–261[ISI][Medline] Pih KT, Yi MJ, Liang YS, Shin BJ, Cho MJ, Hwang I, Son D (2000) Molecular cloning and targeting of a fibrillarin homolog from Arabidopsis. Plant Physiol 123: 51–58 Reiter RS, Coomber SA, Bourett TM, Bartley GE, Scolnik PA (1994) Control of leaf and chloroplast development by the Arabidopsis gene pale cress. Plant Cell 6: 1253–1264[Abstract] Sato S, Hotta Y, Tabata S (1995) Structural analysis of a recA-like gene in the genome of Arabidopsis thaliana. DNA Res 2: 89–93[Abstract] Schmitz-Linneweber C, Williams-Carrier RE, Williams-Voelker PM, Kroeger TS, Vichas A, Barkan A (2006) A pentatricopeptide repeat protein facilitates the trans-splicing of the maize chloroplast rps12 pre-mRNA. Plant Cell 18: 2650–2663 Sjögren LL, Stanne TM, Zheng B, Sutinen S, Clarke AK (2006) Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis. Plant Cell 18: 2635–2649 Small ID, Peeters N (2000) The PPR motif: a TPR-related motif prevalent in plant organellar proteins. Trends Biochem Sci 25: 45–47[CrossRef] Surpin M, Larkin RM, Chory J (2002) Signal transduction between the chloroplast and the nucleus. Plant Cell (Suppl) 14: S327–S338 Suzuki JY, Ytterberg AJ, Beardslee TA, Allison LA, Wijk KJ, Maliga P (2004) Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme. Plant J 40: 164–172[CrossRef][ISI][Medline] Swiatecka-Hagenbruch M, Emanuel C, Hedtke B, Liere K, Börner T (2008) Impaired function of the phage-type RNA polymerase RpoTp in transcription of chloroplast genes is compensated by a second phage-type RNA polymerase. Nucleic Acids Res 36: 785–792 Uwer U, Willmitzer L, Altmann T (1998) Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development. Plant Cell 10: 1277–1294 Williams PM, Barkan A (2003) A chloroplast-localized PPR protein required for plastid ribosome accumulation. Plant J 36: 675–686[CrossRef][ISI][Medline] Yamazaki H, Tasaka M, Shikanai T (2004) PPR motifs of the nucleus-encoded factor, PGR3, function in the selective and distinct steps of chloroplast gene expression in Arabidopsis. Plant J 38: 152–163[CrossRef][ISI][Medline] Zheng B, MacDonald TM, Sutinen S, Hurry V, Clarke AK (2006) A nuclear-encoded ClpP subunit of the chloroplast ATP-dependent Clp protease is essential for early development in Arabidopsis thaliana. Planta 224: 1103–1115[CrossRef][ISI][Medline] Zoschke R, Liere K, Börner T (2007) From seedling to mature plant: Arabidopsis plastidial genome copy number, RNA accumulation and transcription are differentially regulated during leaf development. Plant J 50: 710–722[CrossRef][ISI][Medline] Zubko MK, Day A (2002) Differential regulation of genes transcribed by nucleus-encoded plastid RNA polymerase, and DNA amplification, within ribosome-deficient plastids in stable phenocopies of cereal albino mutants. Mol Gen Genet 267: 27–37
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
was used as an internal control for normalization. Experimental process and data analysis were performed according to Livak and Schmittgen (2001)