The Pentratricopeptide Repeat Protein DELAYED GREENING1Is Involved in the Regulation of Early Chloroplast Development and Chloroplast Gene Expression in Arabidopsis1[W][OA]
Wei Chi,Jinfang Ma,Dongyuan Zhang,Jinkui Guo,Fan Chen,Congming Lu,and Lixin Zhang* Photosynthesis Rearch Center,Key Laboratory of Photosynthesis and Environmental Molecular Physiology,Institute of Botany,Chine Academy of Sciences,Beijing100093,China(W.C.,J.M.,J.G.,
C.L.,L.Z.);School of Life Sciences,Lanzhou University,Lanzhou73000,China(
D.Z.);and Institute of Genetics and Developmental Biology,Chine Academy of Sciences,Beijing100086,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 dg1mutants were initially very pale,but gradually greened and mature outer leaves,more than3weeks old,appeared similar to tho of wild-type plants.Sequence and transcription analys showed that DG1encodes 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 polymera-dependent transcript levels were markedl
nothing in the worldy reduced,while nucleus-encoded polymera-dependent transcript levels were incread,in dg1mutants.Thus,DG1is probably involved in the regulation of plastid-encoded polymera-dependent chloroplast gene expression during early stages of chloroplast development.
The formation of normal chloroplasts is crucial for higher plant growth and development.The chloro-plast genomes of higher plants typically encode only about100genes,and the vast majority of the greater than2,000proteins that have functions in the chloro-plast are encoded by nuclear genes,translated in the cytosol,and subquently imported into the chloro-plast(Abdallah et al.,2000).Thus,the development of functional chloroplasts(which is esntial for the nor-mal 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 cour of chloroplast development and are dependent 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-meras have been identified in higher plant chlo-roplasts:plastid-encoded polymeras(PEPs)and nucleus-encoded polymera(NEPs;Maliga,1988; Hajdukiewicz et al.,1997;Hedtke et al.,1997).PEPs contain core subunits encoded by rpoA,rpoB,rpoC1, and rpoC2ge
chinemalenes,while NEPs are each compod of a single submit(Hedtke et al.,1997;Liere and Maliga, 1999).The two types of polymeras are responsible for the transcription of distinct ts 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 nonphotosyn-thetic houkeeping genes are mostly transcribed by both PEPs and NEPs.Since NEPs mainly transcribe plastid genes encoding proteins involved in transcrip-tion/translation,while PEP accounts for the transcrip-tion of photosynthetic genes(Kapoor et al.,1997;Liere and Maliga,1999),correct timing of photosynthetic gene expression relies on an increa in PEP transcrip-tion activity during the cour of chloroplast develop-ment(Mullet,1993;Demarsy et al.,2006).Thus,PEPs may have important functions during early stages of chloroplast development.
Although PEPs are plastid encoded,the transcrip-tion of PEP-transcribed genes is also under the control of 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
1This work was supported by the National Natural Science Foundation of China(grant nos.30725003
and30670164)and the Frontier Project of Knowledge Innovation Engineering of the Chi-ne Academy of Sciences(grant no.KJCX2–SW–w29).
*Corresponding author;e-mail zhanglixin@ibcas.ac.
The author responsible for distribution of materials integral to the findings prented in this article in accordance with the policy described in the Instructions for Authors(www.plantphysiol)is: Lixin Zhang(zhanglixin@ibcas.ac).
[W]The online version of this article contains Web-only data.
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www.plantphysiol/cgi/doi/10.1104/pp.108.116194
et al.,2006)and others that transiently interact with PEPs,such as sigma factors,are encoded by nuclear genes and are likely to mediate the direct control of plastid gene expression.The replacement of sigma factors associated with PEPs has been propod to account for the switching of transcription patterns during chloroplast development.In null mutants of Arabidopsis(Arabidopsis thaliana)sigma factor6(AtSig6), light-dependent chloroplast development has been found to be signifi
cantly delayed concomitant with reductions in the accumulation of PEP-dependent transcripts,indicating that AtSig6plays an esntial role in the regulation of PEP-dependent gene ex-pression(Ishizaki et al.,2005).In addition,knockout mutations of pTAC2,pTAC6,and pTAC12(three com-ponents of the transcriptionally active plastid chro-mosome,pTAC,in Arabidopsis)have been found to be edling lethal,and the affected genes all appear to be required for proper functioning of the PEP tran-scription machinery(Pfalz et al.,2006).Thefindings indicate that the regulation of PEP-dependent gene expression is much more complex than previously thought,and the nuclear genes involved in this pro-cess remain to be identified.
Pentratricopeptide repeat(PPR)proteins,which are defined by the tandem array of a PPR motif consisting of35amino acids,are widely distributed in higher plants.There are466members in Arabidopsis and480 in rice(Oryza sativa),but their functions are largely unknown(Small and Peeters,2000;Lurin et al.,2004). Most of the PPR proteins,predicted to be located in the plastid or mitochondria,play esntial roles in the posttranscriptional regulation of organelle gene expres-sion.In the chloroplast,PPR proteins have been found to 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),a
nd ribosome accumulation (Williams and Barkan,2003).In the mitochondria,EMP4 is required for the correct expression of a small subt of mitochondrial transcripts in the maize(Zea mays)en-dosperm and OPT43is required for the trans-splicing of the mitochondrial nad1intron1in Arabidopsis(de Longevialle et al.,2007;Gutie´rrez-Marcos et al.,2007). Here,we describe a T-DNA inrtion 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 tran-script accumulation.
RESULTS
Phenotype of dg1
The dg1mutant was lected by its high chlorophyll fluorescence phenotype from a population of pSKI015 T-DNA-mutagenized Arabidopsis lines from the Arab-idopsis Biological Resource Center(Peng et al.,2006).However,dg1mutants exhibit high chlorophyllfluo-rescence,and associated phenotypic traits,in a devel-opmentally regulated manner.As shown in Figure1A, there were clear phenotypic differences between the young and old mature leaves in both4-and6-week-old plants.Growth of the dg1mutants was retarded, and their young leaves exhibited a clearly chlorotic p
henotype under normal growth conditions(Fig.1A). In addition,F v/F m ratios(indicating the maximum potential capacity of the photochemical reactions of PSII)of the young,chlorotic,central,1-to3-week-old leaves of the dg1mutants were substantially lower than tho of their wild-type counterparts.However, the F v/F m ratios gradually incread and approached wild-type levels(0.85)in the more normal,mature, outer,older than3-week-old green leaves(Fig.1A). Thus,the mutant showed a delayed greening pheno-type and(hence)was named dg1.
As shown in Figure1B,the wild-type cotyledons were green when grown on Suc-supplemented Murashige and Skoog(MS)medium.However,the cotyledons of 3-d-old dg1edlings grown on this medium were white,then turned yellow at the5th d.After7d,the cotyledons of dg1became pale-green,and at12d,they had acquired an almost normal green coloration,albeit slightly paler than that of wild-type plants(Fig.1B). The delayed greening phenotype of dg1cotyledons suggested that DG1may play a critical role in chloro-plast development in early stages of edling growth. To test this hypothesis,dg1eds 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 edlings is arrested(Li et al.,1995).The development of dg1edlings on medium without supplementary Suc was verely inhibited compared with their de-velopment in the prence
of exogenous Suc.After growth for12d without Suc,the cotyledons of dg1 mutants still remained yellow and no true leaves had appeared(Fig.1C).
Impaired Chloroplast Development in dg1
To investigate the effects of the dg1mutation on chlo-roplast development,we measured the chloroplasts and examined their ultrastructure in both wild-type and mutant plants by transmission electron micros-copy.The obrvations showed that the chloroplasts were smaller in dg1mutants than in wild-type plants in both the cotyledons of5-d-old edlings(wild type, 5.060.3m m;dg1,3.060.5m m)and young,3-week-old leaves of4-week-old plants(Fig.2).In addition, transmission electron microscopy obrvations of ul-trathin ctions of chloroplasts in5-d-old cotyledons showed that tho in wild-type plants had well-struc-tured thylakoid membranes,compod of grana con-nected by stroma lamellae.However,the thylakoid membrane organization in the chloroplasts of5-d-old dg1cotyledons was disturbed,and their thylakoid mem-branes were much less abundant.In contrast,the thylakoid
Chi et al.
membrane organization and abundance in mature 4-week-old leaves of dg1mutants were similar to t
ho in wild-type plants (Fig.2).
Cloning of the DG1Gene
The genetic analysis showed that dg1was a single recessive mutant,and cogregation of the phosphino-tricin resistance marker of the T-DNA with the mutant phenotype indicated that the mutation was due to the T-DNA inrtion.The inrtion site in dg1was identi-
fied by thermal asymmetric interlaced (TAIL)-PCR iso-lation of the genomic quences flanking the T-DNA borders and subquent quence analysis,which showed that the T-DNA was inrted in the 5#untrans-lated region of At5g67570,23bp relative to the ATG start codon (Fig.3A).No expression of the At5g67570gene was detected in dg1mutants by northern-blot analysis,although the levels of transcripts of its neigh-boring genes (At5g67560and At5g67580)appeared to be similar to tho of wild-type plants (Fig.3B).
Databa analysis of the Arabidopsis genome re-vealed that two partially overlapping cDNA
clones汉英转换器
Figure 1.Phenotypes of the dg1mutant and wild-type (WT)plants.A,Photographs and chlorophyll fluo-rescence images of dg1mutant and wild-type plants grown for 2,4,and 6weeks in the growth chamber.Fluorescence was measured by the FluorCam700MF and visualized us-ing a pudocolor index as indi-cated at the bottom.Bars 51cm.B,Photographs of edlings in early stages of growth on MS medium with 2%Suc.Bars 50.2cm.C,Seedlings grown for 12d on MS medium with and without 2%Suc.Bars 51
cm.
Figure 2.Transmission electron microscopic images of chloroplasts in 5-d-old cotyledons and young and mature green leaves of 4-week-old dg1mutant and wild-type (WT)plants.
strandedqualitycontrolInvolvement of DG1in Chloroplast Development
(AK22212and NM-126157)with different lengths oc-cur at this locus.The 3#region of the NM-126157clone overlaps with the 5#region of the AK22212clone.Rever transcription (RT)-PCR and quence analysis of PCR products revealed that the full-length cDNA of At5g67570does indeed consist of the two combined clones (Supplemental Fig.S1)and that two types of transcripts are prent due to alternative splicing (Fig.3C).One (designated transcript 1)appears to be func-tional,with an open reading frame that putatively encodes a polypeptide of 798amino acids,while the other (transcript 2)encodes a truncated polypeptide with 205amino acids.Transcript 2has three addi-tional exons,which act as introns (introns 1,5,and 8)in transcript 1,and a nine-nucleotide inrtion (5#-TCACTTTAG-3#)in the 5#region of exon 4(Fig.3C).Sequence alignment revealed that the nine-nucleotide short quence corresponds to the 3#region of intron
3,
Figure 3.Molecular cloning of DG1.A,Schematic diagram (not to scale)showing the T-DNA inrted into the 5#untranslated region of At5g67570,genes (in boxes),and the T-DNA inrtion locus (bar topped by a triangle).LB,Left border;RB,right border.B,Northern-blot anal-ysis of At5g67570and its neighboring genes in dg1mutant and wild-type (WT)plants.25S rRNA stained with ethidium bromide is shown as a load-ing control.C,Schematic diagrams of DG1genomic organization with exons (black boxes)and introns (lines between exons)and its alter-natively spliced transcripts.The three additional exons of transcript 2are shown as gray boxes.The black triangle reprents the nine-nucleotide inrtion in transcript 2.Arrows indicate the locations of primers ud for PCR analysis.D,RT-PCR analysis of DG1transcripts.The expression of two forms of tran-scripts with different lengths was examined using primers spanning intron 1(PU and PD,shown in C).1and 2indicate the positions of transcripts 1and 2,respectively.E,Relative quantities of two transcripts of DG1by real-time RT-PCR analy-sis.Transcript 1is given a value as 100,and values are means 6SE of three replicates.
Chi et al.
which indicated that two different splicing sites exist at the3#end of intron3and that the nine-nucleoti
de quence may be due to its alternative splicing.Such a phenomenon of alternative splicing was also obrved in rice SDHB and peach(Prunus persica)ETR1,which are associated with differences in tissue localization and respons to environmental stress,respectively (Kubo et al.,1999;Bastt et al.,2002).Besides the alternative splicing above,DG1had unusual GC/AG borders at the5#and3#splicing sites in intron7(e www.arabidopsis/rvlets/tairobject),rather than the GT/AG borders in the vast majority of eukaryotic introns.In Arabidopsis,nonconventional splicing sites amount to0.7%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 quences of transcripts 1and2are provided in Supplemental Figure S1. Only one signal,corresponding to transcript1,was detected in northern-blot analys of wild-type plants, and no signal was detected at the expected position of transcript2(Fig.3B),which may be due to the very low-level expression of transcript2.To further exam-ine the expression of the two transcripts,RT-PCR using primers spanning intron1was performed(Fig. 3C).RT-PCR analysis revealed that transcript2accu-mulated to very low levels compared with transcript 1in wild-type plants and that neither transcript was expresd in dg1mutants(Fig.3D),in accordance with the results of northern-blot analysis(Fig.3B).To quan-tify the relation between transcript1and transcript2, real-time RT-PCR with specific primers was per-formed.Our results showed that the abundance of transcript2was about7%of that of transcript1.
To confirm that the inactivity of At5g67570was responsible for the mutant phenotype of dg1,we genetically complemented the line with the full-length At5g67570cDNA under the control of the cauliflower mosaic virus35S promoter and obtained ven inde-pendent transgenic plants.Subquent phenotypic obrvations and chlorophyllfluorescence analys 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 At5g67570is indeed responsible for the dg1mutant phenotype.
DG1Encodes a PPR Protein Targeted to the Chloroplast BLAST arches of the complete Arabidopsis -quence revealed that only one copy of the DG1gene is prent in the nuclear genome,which encodes a pu-tative polypeptide of798amino acids with a calculated molecular mass of92kD,containing eight PPR motifs (Fig.4).Protein alignments showed that it shares significant identity with the Medicago truncatula pro-tein MtrDRAFT_AC147000g14V1(73%similarity)and the 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 DG1 protein,we constructed a chimeric gene expressing a fusion protein consisting of the300N-terminal amino acids of DG1and GFP under the control of the35S promoter.The plasmid containing the chimeric gene was transformed into wild-type Arabidop
sis plants, and leaves of stably transformed plants were exam-ined by confocal lar-scanning microscopy.The fu-sion protein was colocalized with the chloroplastic chlorophyll in the mesophyll cells,in accordance with results obtained when the GFP was fud to the transit peptide of the small subunit of Arabidopsis ribulo bisphosphate carboxyla(Fig.5E;Lee et al.,2002).In contrast,GFP signals accumulated specifically in the nucleus when GFP was fud to the nuclear localiza-tion signal of thefibrillarin 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 quence,and no GFP signal was detected in wild-type,untransformed plants.Thus,thefindings suggest that DG1is targeted to the chloroplast.take me home country road
Embryogenesis Was Affected in dg1
BLAST arches of the Arabidopsis databa re-vealed that At5g67570was cataloged as the candidate gene EMB1408(for EMBRYO-DEFECTIVE1408)in-volved in embryo development.To confirm its in-volvement in the development of embryos,siliques from homozygous dg1plants were discted approx-imately7d after pollination,and cleared ovules were examined by differential interference contrast micros-copy using a Nomarski optics microscope.The results showed that about40%of the ovules were arrested at various stages,from the early globular stage to the heart sta
ge,in dg1plants,while the remaining60% apparently developed normally,similar to wild-type ovules(Table I).The arrested embryos subquently shriveled andfinally degenerated.Impaired embryo development has also been obrved in other mutants in which plastid development is affected(Uwer et al., 1998;Apuya et al.,2001;Ho¨rmann et al.,2004;Baldwin et al.,2005;Kovacheva et al.,2005;Kobayashi et al., 2007).Thus,genes that affect the function and/or development of plastids may also be required for embryo development(Uwer et al.,1998;Apuya et al., 2001;Ho¨rmann 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 signals influencing chloroplast development(Lo´pez-Juez and Pyke,2005).To examine the effects of light on the expression of DG1in wild-type plants,the accumulation of DG1transcripts during the light-induced greening of Involvement of DG1in Chloroplast Development
person怎么读etiolated edlings was investigated.DG1transcripts accumulated at very low levels in the etiolated ed-lings,incread after illumination for4h,and reached maximal levels after16h of illumination(Fig.6A).即期信用证
To study the expression of DG1at different devel-opmental stages,levels of DG1transcripts in leaves ranging from1to4weeks old were evaluated by northern-blot analysis.The expression level of DG1 decread as the age and developmental state of both the plants and leaves incread.The DG1transcript content of4-week-old plants was only about10%of that detected in1-week-old plants(Fig.6B),and levels of DG1transcripts in the mature leaves were only approximately20%of tho in the young leaves of 6-week-old plants(Fig.6C).
ceilingExpression of Chloroplast Genes in dg1
To asss the possibility that the delayed chloroplast development in dg1mutants may be reflected at the level of photosynthetic gene expression,we also ex-amined the expression of photosynthetic genes in dg1 by northern-blot analysis.Since there was a clear difference in F v/F m ratios between young leaves(1–3 weeks old)and mature green leaves(.3weeks old)in dg1plants,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 Figure7A,there were no significant differ-ences in the contents of PEP-dependent transcripts in mature green leaves of dg1mutants,apart from slight increas in psbA and psaA transcripts.However,the levels of psbA,psbB,psbC,psbEFJL,psaA,and petA transcripts were significantly lower in young leaves of dg1mutants than in tho of wild-type pla英文歌曲流行
nts(Fig.7A). Furthermore,psbA transcripts were detected in the cotyledons of wild-type plants after growth for3d, and their levels incread with further development, while in dg1cotyledons psbA was expresd much more weakly until the plants were7d old(Fig.
7B).
