the pentratricopeptide repeat protein delayed greening1 is

The Pentratricopeptide Repeat Protein DELAYEDGREENING1 Is Involved in the Regulation of EarlyChloroplast Development and Chloroplast GeneExpression in Arabidopsis1[W][OA]

Wei Chi, Jinfang Ma, Dongyuan Zhang, Jinkui Guo, Fan Chen, Congming Lu, and Lixin Zhang*

Photosynthesis Research Center, Key Laboratory of Photosynthesis and Environmental MolecularPhysiology, 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 ofGenetics 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 populationof activation-tagged Arabidopsis lines. Young, inner leaves of dg1 mutants were initially very pale, but gradually greened andmature outer leaves, more than 3 weeks old, appeared similar to those of wild-type plants. Sequence and transcription analysesshowed that DG1 encodes a chloroplast protein consisting of eight pentratricopeptide repeat domains and that its expressiondepends on both light and developmental status. In addition, analysis of the transcript profiles of chloroplast genes revealedthat 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 forhigher plant growth and development. The chloro-plast genomes of higher plants typically encode onlyabout 100 genes, and the vast majority of the greaterthan 2,000 proteins that have functions in the chloro-plast are encoded by nuclear genes, translated in thecytosol, and subsequently imported into the chloro-plast (Abdallah et al., 2000). Thus, the development offunctional chloroplasts (which is essential for the nor-mal autotrophic growth and development of higherplants) is dependent on the coordinated expression ofnuclear and chloroplast genes. The transcriptionallevels of plastid-encoded genes change dramaticallyduring the course of chloroplast development and aredependent on the stage of plastid development (Mullet,1993; Emanuel et al., 2004; Zoschke et al., 2007), sug-gesting that adjustments to the transcriptional ma-chinery play important roles in the regulation of

chloroplast development. Two types of RNA poly-merases have been identified in higher plant chlo-roplasts: plastid-encoded polymerases (PEPs) andnucleus-encoded polymerase (NEPs; Maliga, 1988;Hajdukiewicz et al., 1997; Hedtke et al., 1997). PEPscontain core subunits encoded by rpoA, rpoB, rpoC1,and rpoC2 genes, while NEPs are each composed of asingle submit (Hedtke et al., 1997; Liere and Maliga,1999). These two types of polymerases are responsiblefor the transcription of distinct sets of chloroplastgenes (Allison et al., 1996; Hajdukiewicz et al., 1997).The chloroplast-encoded photosynthetic genes (suchas psbA, psbD, and rbcL) are exclusively transcribed byPEPs, a few genes (mostly encoding components of thetranscription/translation apparatus, such as rpoB) areexclusively transcribed by NEPs, and nonphotosyn-thetic housekeeping genes are mostly transcribed byboth PEPs and NEPs. Since NEPs mainly transcribeplastid genes encoding proteins involved in transcrip-tion/translation, while PEP accounts for the transcrip-tion of photosynthetic genes (Kapoor et al., 1997; Liereand Maliga, 1999), correct timing of photosyntheticgene expression relies on an increase in PEP transcrip-tion activity during the course of chloroplast develop-ment (Mullet, 1993; Demarsy et al., 2006). Thus, PEPsmay have important functions during early stages ofchloroplast development.

Although PEPs are plastid encoded, the transcrip-tion of PEP-transcribed genes is also under the controlof nuclear genes. A large number of proteins associ-ated with the PEP catalytic core (Pfannschmidt et al.,2000; Loschelder et al., 2004; Suzuki et al., 2004; Pfalz

1 This work was supported by the National Natural ScienceFoundation of China (grant nos. 30725003 and 30670164) and theFrontier Project of Knowledge Innovation Engineering of the Chi-nese Academy of Sciences (grant no. KJCX2–SW–w29).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Lixin Zhang ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.116194

Plant Physiology, June 2008, Vol. 147, pp. 573–584, www.plantphysiol.org � 2008 American Society of Plant Biologists 573

et al., 2006) and others that transiently interact withPEPs, such as sigma factors, are encoded by nucleargenes and are likely to mediate the direct control ofplastid gene expression. The replacement of sigmafactors associated with PEPs has been proposed toaccount for the switching of transcription patternsduring chloroplast development. In null mutants ofArabidopsis (Arabidopsis thaliana) sigma factor 6 (AtSig6),light-dependent chloroplast development has beenfound to be significantly delayed concomitant withreductions in the accumulation of PEP-dependenttranscripts, indicating that AtSig6 plays an essentialrole in the regulation of PEP-dependent gene ex-pression (Ishizaki et al., 2005). In addition, knockoutmutations of pTAC2, pTAC6, and pTAC12 (three com-ponents of the transcriptionally active plastid chro-mosome, pTAC, in Arabidopsis) have been found tobe seedling lethal, and the affected genes all appear tobe required for proper functioning of the PEP tran-scription machinery (Pfalz et al., 2006). These findingsindicate that the regulation of PEP-dependent geneexpression is much more complex than previouslythought, and the nuclear genes involved in this pro-cess remain to be identified.

Pentratricopeptide repeat (PPR) proteins, which aredefined by the tandem array of a PPR motif consistingof 35 amino acids, are widely distributed in higherplants. There are 466 members in Arabidopsis and 480in rice (Oryza sativa), but their functions are largelyunknown (Small and Peeters, 2000; Lurin et al., 2004).Most of these PPR proteins, predicted to be located inthe plastid or mitochondria, play essential roles in theposttranscriptional regulation of organelle gene expres-sion. In the chloroplast, PPR proteins have been foundto be involved in RNA splicing (Hashimoto et al., 2003;Meierhoff et al., 2003; Schmitz-Linneweber et al.,2006), RNA editing (Kotera et al., 2005; Okuda et al.,2007), RNA processing (Fisk et al., 1999), RNA stability(Yamazaki et al., 2004), and ribosome accumulation(Williams and Barkan, 2003). In the mitochondria, EMP4is required for the correct expression of a small subsetof mitochondrial transcripts in the maize (Zea mays) en-dosperm and OPT43 is required for the trans-splicingof the mitochondrial nad1 intron 1 in Arabidopsis (deLongevialle et al., 2007; Gutierrez-Marcos et al., 2007).

Here, we describe a T-DNA insertion Arabidopsismutant named dg1 (for delayed greening1) in whichearly chloroplast development is affected. The DG1gene encodes a novel PPR protein that is probablyinvolved in the regulation of PEP-dependent tran-script accumulation.

RESULTS

Phenotype of dg1

The dg1 mutant was selected by its high chlorophyllfluorescence phenotype from a population of pSKI015T-DNA-mutagenized Arabidopsis lines from the Arab-idopsis Biological Resource Center (Peng et al., 2006).

However, dg1 mutants exhibit high chlorophyll fluo-rescence, and associated phenotypic traits, in a devel-opmentally regulated manner. As shown in Figure 1A,there were clear phenotypic differences between theyoung and old mature leaves in both 4- and 6-week-old plants. Growth of the dg1 mutants was retarded,and their young leaves exhibited a clearly chloroticphenotype under normal growth conditions (Fig. 1A).In addition, Fv/Fm ratios (indicating the maximumpotential capacity of the photochemical reactions ofPSII) of the young, chlorotic, central, 1- to 3-week-oldleaves of the dg1 mutants were substantially lowerthan those of their wild-type counterparts. However,the Fv/Fm ratios gradually increased and approachedwild-type levels (0.85) in the more normal, mature,outer, older than 3-week-old green leaves (Fig. 1A).Thus, the mutant showed a delayed greening pheno-type and (hence) was named dg1.

As shown in Figure 1B, the wild-type cotyledons weregreen when grown on Suc-supplemented Murashigeand Skoog (MS) medium. However, the cotyledons of3-d-old dg1 seedlings grown on this medium werewhite, then turned yellow at the 5th d. After 7 d, thecotyledons of dg1 became pale-green, and at 12 d, theyhad acquired an almost normal green coloration, albeitslightly paler than that of wild-type plants (Fig. 1B).The delayed greening phenotype of dg1 cotyledonssuggested that DG1 may play a critical role in chloro-plast development in early stages of seedling growth.To test this hypothesis, dg1 seeds were germinated onmedium both with and without supplementary Suc,since exogenously supplied Suc is required to establishautotrophic growth when the early development ofseedlings is arrested (Li et al., 1995). The developmentof dg1 seedlings on medium without supplementarySuc was severely inhibited compared with their de-velopment in the presence of exogenous Suc. Aftergrowth for 12 d without Suc, the cotyledons of dg1mutants still remained yellow and no true leaves hadappeared (Fig. 1C).

Impaired Chloroplast Development in dg1

To investigate the effects of the dg1 mutation on chlo-roplast development, we measured the chloroplastsand examined their ultrastructure in both wild-typeand mutant plants by transmission electron micros-copy. The observations showed that the chloroplastswere smaller in dg1 mutants than in wild-type plantsin both the cotyledons of 5-d-old seedlings (wild type,5.0 6 0.3 mm; dg1, 3.0 6 0.5 mm) and young, 3-week-old leaves of 4-week-old plants (Fig. 2). In addition,transmission electron microscopy observations of ul-trathin sections of chloroplasts in 5-d-old cotyledonsshowed that those in wild-type plants had well-struc-tured thylakoid membranes, composed of grana con-nected by stroma lamellae. However, the thylakoidmembrane organization in the chloroplasts of 5-d-olddg1 cotyledons was disturbed, and their thylakoid mem-branes were much less abundant. In contrast, the thylakoid

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574 Plant Physiol. Vol. 147, 2008

membrane organization and abundance in mature4-week-old leaves of dg1 mutants were similar to thosein wild-type plants (Fig. 2).

Cloning of the DG1 Gene

The genetic analysis showed that dg1 was a singlerecessive mutant, and cosegregation of the phosphino-tricin resistance marker of the T-DNA with the mutantphenotype indicated that the mutation was due to theT-DNA insertion. The insertion site in dg1 was identi-

fied by thermal asymmetric interlaced (TAIL)-PCR iso-lation of the genomic sequences flanking the T-DNAborders and subsequent sequence analysis, whichshowed that the T-DNA was inserted in the 5# untrans-lated region of At5g67570, 23 bp relative to the ATGstart codon (Fig. 3A). No expression of the At5g67570gene was detected in dg1 mutants by northern-blotanalysis, although the levels of transcripts of its neigh-boring genes (At5g67560 and At5g67580) appeared to besimilar to those of wild-type plants (Fig. 3B).

Database analysis of the Arabidopsis genome re-vealed that two partially overlapping cDNA clones

Figure 1. Phenotypes of the dg1mutant and wild-type (WT) plants.A, Photographs and chlorophyll fluo-rescence images of dg1 mutant andwild-type plants grown for 2, 4, and6 weeks in the growth chamber.Fluorescence was measured by theFluorCam700MF and visualized us-ing a pseudocolor index as indi-cated at the bottom. Bars 5 1 cm.B, Photographs of seedlings in earlystages of growth on MS mediumwith 2% Suc. Bars 5 0.2 cm. C,Seedlings grown for 12 d on MSmedium with and without 2% Suc.Bars 5 1 cm.

Figure 2. Transmission electron microscopic imagesof chloroplasts in 5-d-old cotyledons and young andmature green leaves of 4-week-old dg1 mutant andwild-type (WT) plants.

Involvement of DG1 in Chloroplast Development

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(AK22212 and NM-126157) with different lengths oc-cur at this locus. The 3# region of the NM-126157 cloneoverlaps with the 5# region of the AK22212 clone.Reverse transcription (RT)-PCR and sequence analysisof PCR products revealed that the full-length cDNA ofAt5g67570 does indeed consist of these two combinedclones (Supplemental Fig. S1) and that two types oftranscripts are present due to alternative splicing (Fig.3C). One (designated transcript 1) appears to be func-

tional, with an open reading frame that putativelyencodes a polypeptide of 798 amino acids, while theother (transcript 2) encodes a truncated polypeptidewith 205 amino acids. Transcript 2 has three addi-tional exons, which act as introns (introns 1, 5, and8) 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-nucleotideshort sequence corresponds to the 3# region of intron 3,

Figure 3. Molecular cloning ofDG1. A, Schematic diagram (not toscale) showing the T-DNA insertedinto the 5# untranslated region ofAt5g67570, genes (in boxes), and theT-DNA insertion locus (bar toppedby a triangle). LB, Left border; RB,right border. B, Northern-blot anal-ysis of At5g67570 and its neighboringgenes in dg1 mutant and wild-type(WT) plants. 25S rRNA stained withethidium bromide is shown as a load-ing control. C, Schematic diagramsof DG1 genomic organization withexons (black boxes) and introns(lines between exons) and its alter-natively spliced transcripts. Thethree additional exons of transcript2 are shown as gray boxes. Theblack triangle represents the nine-nucleotide insertion in transcript 2.Arrows indicate the locations ofprimers used for PCR analysis. D,RT-PCR analysis of DG1 transcripts.The expression of two forms of tran-scripts with different lengths wasexamined using primers spanningintron 1 (PU and PD, shown in C).1 and 2 indicate the positions oftranscripts 1 and 2, respectively. E,Relative quantities of two transcriptsof DG1 by real-time RT-PCR analy-sis. Transcript 1 is given a value as100, and values are means 6 SE ofthree replicates.

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which indicated that two different splicing sites existat the 3# end of intron 3 and that the nine-nucleotidesequence may be due to its alternative splicing. Such aphenomenon of alternative splicing was also observedin rice SDHB and peach (Prunus persica) ETR1, whichare associated with differences in tissue localizationand responses to environmental stresses, respectively(Kubo et al., 1999; Bassett et al., 2002). Besides thealternative splicing above, DG1 had unusual GC/AGborders at the 5# and 3# splicing sites in intron 7 (seewww.arabidopsis.org/servlets/tairobject), rather thanthe GT/AG borders in the vast majority of eukaryoticintrons. In Arabidopsis, nonconventional splicing sitesamount to 0.7% of all splice sites (Alexandrov et al.,2006). Unusual borders of CA/CC and AT/AC insplicing sites have also been detected in rice AGPP andArabidopsis AtLIM15, respectively (Anderson et al.,1991; Sato et al., 1995). The sequences of transcripts1 and 2 are provided in Supplemental Figure S1.

Only one signal, corresponding to transcript 1, wasdetected in northern-blot analyses of wild-type plants,and no signal was detected at the expected position oftranscript 2 (Fig. 3B), which may be due to the verylow-level expression of transcript 2. To further exam-ine the expression of these two transcripts, RT-PCRusing primers spanning intron 1 was performed (Fig.3C). RT-PCR analysis revealed that transcript 2 accu-mulated to very low levels compared with transcript1 in wild-type plants and that neither transcript wasexpressed in dg1 mutants (Fig. 3D), in accordance withthe results of northern-blot analysis (Fig. 3B). To quan-tify the relation between transcript 1 and transcript 2,real-time RT-PCR with specific primers was per-formed. Our results showed that the abundance oftranscript 2 was about 7% of that of transcript 1.

To confirm that the inactivity of At5g67570 wasresponsible for the mutant phenotype of dg1, wegenetically complemented the line with the full-lengthAt5g67570 cDNA under the control of the cauliflowermosaic virus 35S promoter and obtained seven inde-pendent transgenic plants. Subsequent phenotypicobservations and chlorophyll fluorescence analysesconfirmed that wild-type traits had been restored inthe complemented mutant (Fig. 1, A and C). Thus, itcan be concluded that the disruption of At5g67570 isindeed responsible for the dg1 mutant phenotype.

DG1 Encodes a PPR Protein Targeted to the Chloroplast

BLAST searches of the complete Arabidopsis se-quence revealed that only one copy of the DG1 gene ispresent in the nuclear genome, which encodes a pu-tative polypeptide of 798 amino acids with a calculatedmolecular mass of 92 kD, containing eight PPR motifs(Fig. 4). Protein alignments showed that it sharessignificant identity with the Medicago truncatula pro-tein MtrDRAFT_AC147000g14V1 (73% similarity) andthe rice protein Os05g0315100 (64% similarity). An-other (hypothetical) Arabidopsis protein, At1g30610,was also found to have high similarity (59%) to DG1.

To examine the cellular localization of the DG1protein, we constructed a chimeric gene expressing afusion protein consisting of the 300 N-terminal aminoacids of DG1 and GFP under the control of the 35Spromoter. The plasmid containing the chimeric genewas transformed into wild-type Arabidopsis plants,and leaves of stably transformed plants were exam-ined by confocal laser-scanning microscopy. The fu-sion protein was colocalized with the chloroplasticchlorophyll in the mesophyll cells, in accordance withresults obtained when the GFP was fused to the transitpeptide of the small subunit of Arabidopsis ribulosebisphosphate carboxylase (Fig. 5E; Lee et al., 2002). Incontrast, GFP signals accumulated specifically in thenucleus when GFP was fused to the nuclear localiza-tion signal of the fibrillarin protein from Arabidopsis(Pih et al., 2000). In addition, GFP signals were foundto accumulate in both the cytoplasm and the nucleuswhen Arabidopsis was transformed with the controlvector without a specific targeting sequence, and noGFP signal was detected in wild-type, untransformedplants. Thus, these findings suggest that DG1 istargeted to the chloroplast.

Embryogenesis Was Affected in dg1

BLAST searches of the Arabidopsis database re-vealed that At5g67570 was cataloged as the candidategene EMB1408 (for EMBRYO-DEFECTIVE1408) in-volved in embryo development. To confirm its in-volvement in the development of embryos, siliquesfrom homozygous dg1 plants were dissected approx-imately 7 d after pollination, and cleared ovules wereexamined by differential interference contrast micros-copy using a Nomarski optics microscope. The resultsshowed that about 40% of the ovules were arrested atvarious stages, from the early globular stage to theheart stage, in dg1 plants, while the remaining 60%apparently developed normally, similar to wild-typeovules (Table I). The arrested embryos subsequentlyshriveled and finally degenerated. Impaired embryodevelopment has also been observed in other mutantsin which plastid development is affected (Uwer et al.,1998; Apuya et al., 2001; Hormann et al., 2004; Baldwinet al., 2005; Kovacheva et al., 2005; Kobayashi et al.,2007). Thus, genes that affect the function and/ordevelopment of plastids may also be required forembryo development (Uwer et al., 1998; Apuya et al.,2001; Hormann et al., 2004; Baldwin et al., 2005;Kovacheva et al., 2005; Kobayashi et al., 2007).

Expression Profiles of DG1

Light, which triggers the differentiation of nonphoto-synthetic proplastids into fully functional photosyn-thetic chloroplasts, is one of the most important signalsinfluencing chloroplast development (Lopez-Juez andPyke, 2005). To examine the effects of light on theexpression of DG1 in wild-type plants, the accumulationof DG1 transcripts during the light-induced greening of



etiolated seedlings was investigated. DG1 transcriptsaccumulated at very low levels in the etiolated seed-lings, increased after illumination for 4 h, and reachedmaximal levels after 16 h of illumination (Fig. 6A).

To study the expression of DG1 at different devel-opmental stages, levels of DG1 transcripts in leavesranging from 1 to 4 weeks old were evaluated bynorthern-blot analysis. The expression level of DG1decreased as the age and developmental state of boththe plants and leaves increased. The DG1 transcriptcontent of 4-week-old plants was only about 10% ofthat detected in 1-week-old plants (Fig. 6B), and levelsof DG1 transcripts in the mature leaves were onlyapproximately 20% of those in the young leaves of6-week-old plants (Fig. 6C).

Expression of Chloroplast Genes in dg1

To assess the possibility that the delayed chloroplastdevelopment in dg1 mutants may be reflected at the

level of photosynthetic gene expression, we also ex-amined the expression of photosynthetic genes in dg1by northern-blot analysis. Since there was a cleardifference in Fv/Fm ratios between young leaves (1–3weeks old) and mature green leaves (.3 weeks old) indg1 plants, differences in plastid transcript profileswere compared between young and old mature leavesas well as between mutant and wild-type plants. Asshown in Figure 7A, there were no significant differ-ences in the contents of PEP-dependent transcripts inmature green leaves of dg1 mutants, apart from slightincreases in psbA and psaA transcripts. However, thelevels of psbA, psbB, psbC, psbEFJL, psaA, and petAtranscripts were significantly lower in young leaves ofdg1 mutants than in those of wild-type plants (Fig. 7A).Furthermore, psbA transcripts were detected in thecotyledons of wild-type plants after growth for 3 d,and their levels increased with further development,while in dg1 cotyledons psbA was expressed muchmore weakly until the plants were 7 d old (Fig. 7B).

Figure 4. Amino acid sequence alignment of DG1 PPR domains. Comparison of the amino acid sequence of the DG1 PPRdomains with homologous sequences from O. sativa, M. truncatula, and Arabidopsis. Black boxes indicate strictly conservedamino acids among these proteins. Bars above the sequences labeled A to H indicate the eight PPR domains of DG1.

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The delayed accumulation of PEP-dependent tran-scripts was consistent with the delayed greening phe-notype of dg1.

In addition, our results showed that the contents ofrpoB transcripts were almost identical in mature greenleaves of dg1 and wild-type plants, but they wereconsiderably higher in the young leaves of dg1 plants.In contrast, levels of clpP and accD transcripts werehigher in both young and mature leaves of dg1 plants(Fig. 7C). These findings are interesting, since clpP istranscribed by both PEP and NEP, while rpoB and accDare transcribed exclusively by NEP. Unlike the plastidgenes mentioned above, transcript levels of the nu-

clear genes were largely unaltered in both young andmature leaves of dg1 plants (Fig. 7D).

DISCUSSION

Nucleus-encoded factors play essential roles in theregulation of chloroplast development, which requiresthe coordinated expression of both nucleus-encodedand chloroplast-encoded genes. In the study reportedhere, we identified a novel Arabidopsis mutant withdelayed greening, dg1, and isolated the affected gene,which encodes a PPR protein targeted to the chloro-plast. Subsequent genetic and molecular analysesshowed that DG1 is probably involved in the regula-tion of early stages of chloroplast development andplastid gene expression.

Normal Chloroplast Development Was Delayed in dg1

The most distinct characteristic of dg1 was retardedgreening of both its cotyledons and rosette leaves. Theyoung leaves were initially chlorotic, then graduallygreened during development in dg1 plants, and themature, green leaves appeared similar to those of wild-type plants. Analyses of chlorophyll fluorescence indg1 mutants revealed that photosynthetic activity wasreduced in their young leaves (Fig. 1). In addition,ultrastructural observations showed that thylakoidmembranes were less abundant in the young leavesof the mutants than in wild-type counterparts, but

Figure 5. Cellular localization of DG1 by GFP assays. Fluorescencesignals were visualized using confocal laser-scanning microscopy.Green fluorescence indicates GFP, red fluorescence shows chloroplastautofluorescence, and orange/yellow fluorescence shows images withthe two types of fluorescence merged. A, GFP signals from the DG1-GFP fusion protein. B, Control with the transit peptide of ribulosebisphosphate carboxylase small subunit. C, Control with the nuclearlocalization signal of fibrillarin. D, Control lacking the transit peptide.E, Wild type with no transformation. Bars 5 5 mm.

Figure 6. Expression profiles of DG1. A, Transcript levels of DG1during light-induced greening of etiolated Arabidopsis seedlings. Aftergrowth in darkness for 5 d, the etiolated Arabidopsis seedlings wereilluminated for 4, 8, 16, and 24 h. B, Transcript levels of DG1 in leavesof different developmental stages. 25S rRNA stained with ethidiumbromide is shown as a loading control. C, Transcript levels of DG1 inyoung and mature green leaves of a 6-week-old wild-type plant.



their abundance gradually increased as the leavesdeveloped and eventually reached levels similar towild-type levels (Fig. 2). These results suggest thatDG1 plays important roles in early stages of chloro-plast development and that functional chloroplastdevelopment was dependent on leaves reaching amature or close to mature developmental state in thedg1 mutants.

Several mutants with impaired chloroplast devel-opment have been described. Sig6-1 mutants, whichhave no functional copies of the plastid sigma factorgene AtSIG6, have chlorotic young cotyledons, but awild-type phenotype is restored in 8-d-old seedlings(Ishizaki et al., 2005). Repression of the ATP-dependentClp proteases, Clp4 and Clp6, of Arabidopsis report-edly results in a chlorotic phenotype in young leaves,which moderates upon maturity (Sjogren et al., 2006;Zheng et al., 2006), but no such chlorosis has beenobserved in the cotyledons of Clp6 antisense plants(Sjogren et al., 2006). The severity of the chloroticphenotype in young leaves in the cited mutants sug-gests that the affected chloroplast proteins may playimportant roles in early stages of chloroplast develop-ment. A possible explanation for the delayed chloro-plast development is that other proteins may partlycompensate for their absence during late stages ofdevelopment. For instance, another sigma factor islikely to have overlapping functions with AtSIG6 thatcompensate for its deficiency in AtSIG6 mutants whenthey are 8 or more days old (Ishizaki et al., 2005). Giventhe complexity of Clp protease complements, it is also

Table I. The frequency of aborted seeds in dg1 siliques

Embryos about 7 d after pollination were examined by whole-mountseed clearing. Siliques from five different plants were scored for thepresence of normal and aborted seeds at different stages.

Phenotypic

Class

Normal Seeds Aborted Seeds

Wild Type dg1 Wild Type dg1

Preglobular 51 (19%) 81 (11%) 3 (1%) 96 (13%)Globular 64 (24%) 103 (14%) 81 (11%)Heart 75 (28%) 125 (17%) 73 (10%)Torpedo 40 (15%) 74 (10%) 37 (5%)Cotyledon 33 (13%) 66 (9%)Total 263 (99%) 449 (61%) 3 (1%) 287 (39%)

Figure 7. Expression of chloroplast genes in dg1mutant and wild-type (WT) plants. Total RNA samplesfrom wild-type and dg1 mutant plants were sizefractionated by agarose gel electrophoresis, trans-ferred to a nylon membrane, and probed with 32P-labeled cDNA probes. A, PEP-dependent chloroplastgenes. B, psbA transcription accumulation duringearly seedling development. C, NEP-dependent chlo-roplast genes. D, Nuclear genes.

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possible that other members of the Clp protease familyhave functional redundancy with either Clp4 or Clp6in their respective mutants. Furthermore, our proteinalignments showed that another protein with homol-ogy to DG1, At1g30610, is present in the Arabidopsisgenome (Fig. 4). Sequence analysis revealed thatAt1g30610 is a hypothetical protein with five PPRdomains targeted to the chloroplast according to pre-dictions by Target P (Emanuelsson et al., 2000). Thus, itis likely that At1g30610 has functional redundancywith DG1. However, the possibility that DG1 is notrequired for later developmental stages of chloroplastscannot be excluded, since the expression level of DG1decreased as their development proceeded (Fig. 6B).

PEP-Dependent Transcripts Were Dramatically Reducedin dg1

The expression of both nucleus- and chloroplast-encoded photosynthetic genes was tightly linked withthe developmental status of the chloroplasts. In dg1mutants, the levels of PEP-dependent transcripts werereduced, levels of NEP-dependent transcripts were in-creased, and levels of nuclear gene transcripts werenot clearly altered (Fig. 7). It is generally accepted thatNEP activities are largely responsible for the tran-scription of housekeeping genes during early stages ofchloroplast development and that PEP activities in-crease while NEP activities decrease during subse-quent chloroplast development (Hanaoka et al., 2005).Thus, NEP activities play important roles in the earlystages of chloroplast development. Accordingly, themutations of RpoT2, a NEP polymerase that is duallytargeted to chloroplasts and mitochondria, result inonly moderate defects in chloroplast gene expressionand delayed greening of the leaves (Baba et al., 2004;Courtois et al., 2007), while the knockout mutants andtransgenic lines with low levels of solely chloroplast-targeted NEP polymerase reportedly exhibit moresevere defects in chloroplast development and geneexpression (Hricova et al., 2006; Courtois et al., 2007;Swiatecka-Hagenbruch et al., 2008). In dg1 mutants,however, levels of PEP-dependent transcriptions werereduced (Fig. 7A), but not levels of NEP-dependenttranscripts, suggesting that NEP functions efficientlyin dg1. Thus, PEP is likely to have an important role inearly stages of chloroplast development as well asNEP. In accordance with this hypothesis, the PEPtranscription system is present in very early stages ofplant development, even in dry seeds and duringgermination (Demarsy et al., 2006).

Three interpretations can be made to the observationof decreased accumulation of PEP-dependent tran-scripts. PEP is a multisubunit (bacterial-type) polymer-ase. Recently, in addition to the chloroplast-encodedcatalytic core subunits, a number of polypeptides havebeen shown to be associated with transcriptionallyactive chloroplast chromosomes (Pfannschmidt et al.,2000; Pfalz et al., 2006). Inactivation of pTAC2, a PPRprotein, resulted in a seedling-lethal phenotype, re-

sembling those of rpo mutants (Pfalz et al., 2006). Thissuggests that pTAC2 is an intrinsic component of PEP.DG1 is not detected in the transcriptionally activechromosome (Pfalz et al., 2006). Thus, DG1 may beinvolved in the regulation of PEP transcription ma-chinery during early stages of chloroplast develop-ment, and the decrease of PEP-dependent transcriptsmight be due to impairment of the PEP transcriptionmachinery in dg1. Another possible explanation forthe reduced levels of PEP-dependent transcripts in thedg1 mutants is that the stability of the transcripts maybe lower, or the rate of mRNA turnover enhanced,in them. In support of this hypothesis, a number ofnucleus-encoded factors that affect the stability ofchloroplast gene transcripts have been identified. Forexample, in Chlamydomonas, a 140-kD tetratricopeptiderepeat protein, Nac2, is strictly required for the stabi-lization of psbD transcripts via a large protein complexthat is associated with RNA (Boudreau et al., 2000),and a peptide chain release factor 2 has been shown toaffect the stability of UGA-containing transcripts inArabidopsis chloroplasts (Meurer et al., 2002). Thus,DG1 may be involved in regulation of the stability ofPEP-dependent transcripts via its PPR motif associat-ing with its mRNA targets. Finally, it is also possiblethat the reduced levels of PEP-dependent transcriptsare due to inefficient assembly of the plastid ribosome,since (for instance) translation of PEP subunits islimited, and may account for reduced PEP activities,in ribosome-deficient mutants (Hess et al., 1993; Zubkoand Day, 2002).

The developmental status of the chloroplast hasbeen shown to control a set of nuclear genes thatencode chloroplast-localized proteins via a processknown as retrograde signaling (Surpin et al., 2002).However, as shown in Figure 7D, the transcript levelsof the nuclear genes were not affected, although thechloroplast development was delayed, in dg1 mutants,suggesting that the retrograde signaling mechanismscontrolling nuclear gene expression are intact in dg1. Asimilar absence of effects on the expression of nucleargenes encoding chloroplast proteins has also beenobserved in pac and dcl mutants, which have defectivechloroplasts (Reiter et al., 1994; Meurer et al., 1998;Bellaoui et al., 2003), and the retrograde signalingmechanisms controlling palisade cell developmentare affected, but not those regulating nuclear geneexpression.

MATERIALS AND METHODS

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 mmol m22 s21 at a constant temper-

ature 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 Fluorescence Analysis

Chlorophyll a fluorescence images were obtained at room temperature

using a modulated imaging fluorometer (FluorCam; Photon System Instru-

ments) 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).

Before each measurement, leaves were dark adapted for 10 min. The Fo was

measured under measuring light (650 nm) with very low intensity (0.8 mmol

m22 s21). To estimate the Fm, a saturating pulse of white light (3,000 mmol m22

s21 for 1 s) was applied. The maximal photochemical efficiency of PSII was

calculated from the ratio of Fv to Fm [Fv/Fm 5 (Fm 2 Fo)/Fm].

TAIL-PCR and RT-PCR

DNA fragments flanking the T-DNA were obtained by a series of three

nested TAIL-PCRs (Liu et al., 1995). The products of the final PCR were

subcloned into the pGEM-T Easy vector (Promega), sequenced, and subjected

to BLAST analysis.

Total RNA isolation and RT-PCR were performed according to the manu-

facturer’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 span-

ning intron 1 were as follows: PU, 5#-AGAGTGCCACCGGAATTTGAAC-

CAG-3#; and PD, 5#-TTCCTGTGGCCTCCTCGCAAAC-3#.

Real-Time RT-PCR

Two different primer pairs were used to quantify the expression of tran-

script 1 and transcript 2. One primer pair was designed to specifically amplify

transcript 2 (forward, 5#-CCAACATAAACGAACTGCTG-3#; reverse, 5#-ACA-

TCTTAAGACCACCCTACAT-3#), whereas the other primer pair amplifies both

transcripts (forward, 5#-GCAGGCCGCTCGAGATTAC-3#; reverse, 5#-CATCA-

ACATGACCACCGTTCA-3#). The amplification of ELONGATION FACTOR1-a

was used as an internal control for normalization. Experimental process and

data analysis were performed according to Livak and Schmittgen (2001). The

primers were designed using Primer Express software. PCRs were performed

with an ABI 7900 sequence detection system (Applied Biosystems) according to

the manufacturer’s protocol.

Transmission Electron Microscopy

Samples of wild-type and mutant leaves were prepared for transmission

electron microscopy by cutting them into small pieces, fixing in 2.5% glutar-

aldehyde 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).

Analysis of Embryo Development

Siliques from wild-type and mutant plants were dissected with hypoder-

mic 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). Embryo development was studied microscopically using an

Olympus BH-2 microscope equipped with Nomarski optics.

Complementation of the dg1 Mutant

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). Transformed plants were selected on MS medium

containing 50 mg/mL kanamycin monosulfate. Seven resistant plants were

transferred to soil and grown in a greenhouse to produce seeds. The success

of the complementation was confirmed by measurements of chlorophyll

fluorescence.

Subcellular Localization of GFP Proteins

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 mo-

saic virus 35S promoter. Two variants were then constructed: one in which the

nuclear localization signal of the fibrillarin protein (Pih et al., 2000) and the

other in which the transit peptide of the small subunit of ribulose bisphos-

phate carboxylase of Arabidopsis (Lee et al., 2002) were also fused to the

vector. The resulting fusion constructions and the control vector (GFP with no

additional coding sequence) were introduced into wild-type Arabidopsis. The

GFP in the examined samples was excited with the 488-nm laser line, and GFP

emission was detected using a 505- to 530-nm bandpass filter.

Northern-Blot Analysis

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.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Sequences of two transcripts of DG1.

Supplemental Table S1. Primers used to prepare the hybridization

probes.

ACKNOWLEDGMENTS

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.

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the pentratricopeptide repeat protein delayed greening1 is

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