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• W2001808493 abstract "Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process. The corresponding product, magnesium protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. To get more information on the chlorophyll biosynthesis pathway and on magnesium protoporphyrin IX derivative functions, we have identified an magnesium protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from magnesium protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cytochrome b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that magnesium protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of magnesium protoporphyrin IX methyl ester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the magnesium chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step. Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process. The corresponding product, magnesium protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. To get more information on the chlorophyll biosynthesis pathway and on magnesium protoporphyrin IX derivative functions, we have identified an magnesium protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from magnesium protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cytochrome b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that magnesium protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of magnesium protoporphyrin IX methyl ester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the magnesium chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step. Chloroplast development depends on the coordinated synthesis of chlorophylls and cognate proteins and on their specific integration into photosynthetic complexes (for review, see Ref. 1Paulsen H. Aro E.M. Anderson B. in Advances in Photosynthesis and Respiration: Regulation of Photosynthesis. Kluwer Academic Publishers, Dordrecht, The Netherlands2001: 219-233Google Scholar). Chlorophylls are magnesium tetrapyrrole molecules resulting from condensation of ALA. 2The abbreviations used are: ALA, δ-aminolevulinic acid; MS, Murashige and Skoog; EYFP, enhanced yellow fluorescent protein; LHC, light-harvesting complex; HPLC, high performance liquid chromatography; RT, reverse transcription. Insertion of magnesium into protoporphyrin IX, the last common intermediate of hemes and chlorophylls, is catalyzed by magnesium chelatase, a membrane-interacting multisubunit enzyme containing 3 different soluble proteins: CHLH, CHLI, and CHLD (2Gibson L.C. Willows R.D. Kannangara C.G. Von Wettstein D. Hunter C.N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1941-1944Crossref PubMed Scopus (159) Google Scholar, 3Willows R.D. Gibson L.C. Kanangara C.G. Hunter C.N. Von Wettstein D. Eur. J. Biochem. 1996; 235: 438-443Crossref PubMed Scopus (101) Google Scholar, 4Papenbrock J. Grafe S. Kruse E. Hanel F. Grimm B. Plant J. 1997; 12: 981-990Crossref PubMed Scopus (77) Google Scholar, 5Nakayama M. Masuda T. Bando T. Yamagata H. Ohta H. Takamiya K. Plant Cell Physiol. 1998; 39: 275-284Crossref PubMed Scopus (68) Google Scholar). In the next step, magnesium protoporphyrin IX is methylated by a membrane Ado-Met-dependent methyltransferase. In Arabidopsis the gene At4g25080 or CHLM has been identified as encoding the methyltransferase enzyme, and the product of this single gene has been reported to be present in the two chloroplast membrane systems; that is, the envelope and the thylakoids (6Block M.A. Tewari A.K. Albrieux C. Marechal E. Joyard J. Eur. J. Biochem. 2002; 269: 240-248Crossref PubMed Scopus (69) Google Scholar). Whereas a sequence homologous to CHLM has been identified in tobacco (7Alawady A. Reski R. Yaronskaya E. Grimm B. Plant Mol. Biol. 2005; 57: 679-691Crossref PubMed Scopus (38) Google Scholar), the existence in plants of an additional and quite dissimilar gene for magnesium protoporphyrin IX methylation has not been totally excluded. Expression analysis of tobacco CHLM revealed posttranslational activation of methyltransferase activity during greening and direct interaction of CHLM with the CHLH subunit of magnesium chelatase (7Alawady A. Reski R. Yaronskaya E. Grimm B. Plant Mol. Biol. 2005; 57: 679-691Crossref PubMed Scopus (38) Google Scholar). Furthermore, analysis of tobacco plants expressing CHLM antisense or sense RNA led to the conclusion that methyltransferase activity is involved in the direct regulation of expression of some genes such as CHLH and LHCB (8Alawady A.E. Grimm B. Plant J. 2005; 41: 282-290Crossref PubMed Scopus (93) Google Scholar). The meaning of the dual localization of the methyltransferase in the chloroplast envelope and the thylakoids is puzzling for different reasons. On one hand, chlorophylls are expected to be synthesized in the thylakoids where chlorophyll-binding proteins are inserted into photosystems. On the other hand, the envelope is devoid of chlorophyll but contains several chlorophyll precursors from protoporphyrin IX to protochlorophyllide (9Pineau B. Gerard-Hirne C. Douce R. Joyard J. Plant Physiol. 1993; 102: 821-828Crossref PubMed Scopus (30) Google Scholar). In addition most of the enzymes of this part of the chlorophyll synthesis pathway, i.e. protoporphyrinogen oxidoreductase, magnesium chelatase, magnesium protoporphyrin IX methyl ester cyclase, and protochlorophyllide oxidoreductase, are present in or interact with the envelope (10Matringe M. Camadro J.M. Block M.A. Joyard J. Scalla R. Labbe P. Douce R. J. Biol. Chem. 1992; 267: 4646-4651Abstract Full Text PDF PubMed Google Scholar, 11Tottey S. Block M.A. Allen M. Westergren T. Albrieux C. Scheller H.V. Merchant S. Jensen P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 16119-16124Crossref PubMed Scopus (156) Google Scholar, 12Joyard J. Block M. Pineau B. Albrieux C. Douce R. J. Biol. Chem. 1990; 265: 21820-21827Abstract Full Text PDF PubMed Google Scholar, 13Barthelemy X. Bouvier G. Radunz A. Docquier S. Schmid G.H. Franck F. Photosynth. Res. 2000; 64: 63-76Crossref PubMed Scopus (25) Google Scholar). One reason for the occurrence of the chlorophyll intermediates in the envelope could be that some chlorophyll-binding proteins that are synthesized in the cytosol need to associate with chlorophyll or chlorophyll precursors during import through the envelope. Supporting this hypothesis, manipulation of Chlamydomonas in vivo systems, and mutagenesis of specific residues in the LHCB protein have shown that accumulation of physiological amounts of LHCB by plastids requires interaction of the protein with chlorophyll within the inner membrane of the envelope (14White R.A. Wolfe G.R. Komine Y. Hoober J.K. Photosynth. Res. 1996; 47: 267-280Crossref PubMed Scopus (23) Google Scholar, 15Hoober J.K. Eggink L.L. Photosynth. Res. 1999; 61: 197-215Crossref Scopus (57) Google Scholar). More recently, it was demonstrated that chlorophyllide a oxygenase is involved in the regulated import and stabilization of the chlorophyllide b binding light-harvesting proteins LHCB1 (LHCII) and LHCB4 (CP29) in chloroplasts (16Reinbothe C. Bartsch S. Eggink L.L. Hoober J.K. Brusslan J. Andrade Paz R. Monnet J. Reinbothe S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4777-4782Crossref PubMed Scopus (64) Google Scholar). Beside their direct role in pigment synthesis, some chlorophyll intermediates have been proposed to play an additional role in intracellular signaling. In Chlamydomonas, magnesium protoporphyrin IX and magnesium protoporphyrin IX methyl ester were shown to substitute for light in the induction of the nuclear gene HSP70 (17Kropat J. Oster U. Rudiger W. Beck C.F. Plant J. 2000; 24: 523-531Crossref PubMed Google Scholar). In Arabidopsis, an increased accumulation of magnesium protoporphyrin IX has been reported in Norfluorazon-treated plants in which photooxidation of the plastid compartment leads to the repression of nuclear photosynthesis-related genes (18Mayfield S.P. Taylor W.C. Eur. J. Biochem. 1984; 144: 79-84Crossref PubMed Scopus (165) Google Scholar, 19Strand A. Asami T. Alonso J. Ecker J.R. Chory J. Nature. 2003; 421: 79-83Crossref PubMed Scopus (446) Google Scholar). Furthermore, a genetic screen based on the use of Norfluorazon has allowed the identification of a series of Arabidopsis gun (for genome-uncoupled) mutants that are deficient in chloroplast-to-nucleus signaling. Several of the corresponding mutations have been shown to affect genes coding for the protoporphyrin IX manipulating proteins CHLH, CHLD, and GUN4 (19Strand A. Asami T. Alonso J. Ecker J.R. Chory J. Nature. 2003; 421: 79-83Crossref PubMed Scopus (446) Google Scholar, 20Mochizuki N. Brusslan J.A. Larkin R. Nagatani A. Chory J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2053-2058Crossref PubMed Scopus (520) Google Scholar, 21Larkin R.M. Alonso J.M. Ecker J.R. Chory J. Science. 2003; 299: 902-906Crossref PubMed Scopus (396) Google Scholar). In these mutants, Norfluorazon treatment induced only a moderate increase in magnesium protoporphyrin IX levels, correlated with partial derepression of transcription of the nuclear photosynthesis-related genes. This indicates a role of magnesium protoporphyrin IX accumulation in the repression of these genes (19Strand A. Asami T. Alonso J. Ecker J.R. Chory J. Nature. 2003; 421: 79-83Crossref PubMed Scopus (446) Google Scholar, 22Susek R.E. Ausubel F.M. Chory J. Cell. 1993; 74: 787-799Abstract Full Text PDF PubMed Scopus (471) Google Scholar). However, this demonstration was based on manipulation of plants with Norflurazon that has obvious pleiotropic effects. Moreover, because of the possibility of substrate channeling occurring between magnesium chelatase and magnesium protoporphyrin IX methyltransferase, it was not possible to clearly distinguish the specific contributions of magnesium protoporphyrin IX and its methyl ester. Supporting the intricacy of the regulation, it has recently been shown that the barley xantha-l mutant, defective in the magnesium protoporphyrin IX methyl ester cyclization step, has a non-gun phenotype in the presence of Norflurazon (23Rzeznicka K. Walker C.J. Westergren T. Kannangara C.G. Von Wettstein D. Merchant S. Gough S.P. Hansson M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5886-5891Crossref PubMed Scopus (90) Google Scholar, 24Gadjieva R. Axelsson E. Olsson U. Hansson M. Plant Physiol. Biochem. 2005; 43: 901-908Crossref PubMed Scopus (40) Google Scholar). In the absence of Norflurazon, the mutant has a high level of LHCB gene expression despite the accumulation of magnesium protoporphyrin IX methyl ester. In the present work, we identify a chlm null mutant and analyze the role of the methyltransferase enzyme in chlorophyll synthesis and chloroplast development. We also assess the ability of the mutant to carry out plastid-to-nucleus signaling. The results reveal a high level of repression of nuclear photosynthesis-related genes and a specific accumulation of the CHLH subunit of the magnesium chelatase. Functional implications of these results are discussed. Plant Material and Growth Conditions—The Arabidopsis thaliana chlm mutant (ABN42 line) of the ecotype Wassilewskija (WS) was obtained from the Institut National de la Recherche Agronomique, Versailles, France. Plants were grown on Murashige and Skoog (MS, Duchefa) agar containing 0.5% sucrose for 5 days under medium light (70 μmol of photons m–2 s–1) before transfer to dim light (5 μmol of photons m–2 s–1). Kanamycin selection of T-DNA containing seedlings was performed on medium supplemented with kanamycin (Sigma) at 50 mg liter–1. For Norflurazon treatment, seeds from CHLM/chlm heterozygous plants were germinated on standard MS-agar, and after 5 days of culture seedlings were transferred onto new plates including 5 μm Norflurazon and cultivated for 6 more days under strong light (100 μmol of photons m–2s–1). For complementation experiments, a pCambia derivative was obtained by cloning a EcoRI-HindIII fragment harboring the cauliflower mosaic virus 35S-labeled promoter-GUS-NOST fusion from pBI221 (Clontech) in an EcoRI-HindIII-digested pCambia 1200 vector. The XbaI-EcoRI CHLM cDNA from the SK+ derivative obtained by Block et al. (6Block M.A. Tewari A.K. Albrieux C. Marechal E. Joyard J. Eur. J. Biochem. 2002; 269: 240-248Crossref PubMed Scopus (69) Google Scholar) was then inserted into an SstI-XbaI-digested pCambia derivative using an EcoRI-SstI adaptor (5′-AATTAGCT-3′). The obtained binary vector was introduced into the GV3121 strain of Agrobacterium tumefaciens to transform CHLM/chlm heterozygous plants. The transformed plants were PCR-screened to identify CHLM/chlm heterozygous plants and self-pollinated. The LHCII-LUC construct was introduced into the chlm mutant background by crossing a heterozygous CHLM/chlm plant with a homozygous CAB-LUC transgenic plant (25Millar A.J. Kay S.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15491-15496Crossref PubMed Scopus (225) Google Scholar). The progeny was PCR-screened for the presence of chlm T-DNA, and seeds were collected from positive plants. The next generation segregates for both chlm T-DNA and the CAB-LUC transgene and was used for the experiment. The sequence encoding the putative CHLH transit peptide was amplified using TL350 (5′-GGATCCATGGCTTCGCTTGTGTATTCTCCATTC-3′) and TL352 (TCTAGAGACGATTTTCACCGTTCGGAAC-3′). The corresponding PCR fragment was subcloned into pGEM-T easy vector (Promega) and sequenced. The BamHI-XbaI fragment was then ligated in-frame with the XbaI-SacI EYFP gene (Clontech) into the BamHI-SacI sites of a binary vector derived from pCambia 1300 by introduction of a HindIII-EcoRI cauliflower mosaic virus 35S-labeled promoter and Nos-T terminator cassette from EL103 (26Mittler R. Lam E. Plant Cell. 1995; 7: 1951-1962Crossref PubMed Scopus (172) Google Scholar). The obtained binary vector was introduced into the GV3121 strain of A. tumefaciens to transform CHLM/chlm heterozygous plants. The transformed plants were PCR-screened to identify CHLM/chlm heterozygous plants and self-pollinated. PCR Analyses—Genomic DNA was amplified using the Tag6 primer (5′-CACTCAGTCTTTCATCTACGGCA-3′) and a specific primer (TL251 5′-GGAACAGCTTCGATGAGCCTAGAG-3′) to screen for the presence of chlm T-DNA insertion. The endogenous copy of the gene as well as the cDNA used for complementation were followed by PCR reactions using TL251 and TL252 (5′-GTGAGACTGATGAAGTGAATCG-3′). Standard PCR conditions were used. Pigment Analysis—Extraction was done according to Moran and Porath (27Moran R. Porath D. Plant Physiol. 1980; 65: 478-479Crossref PubMed Google Scholar). 3 mg of cotyledons of 8-day plants were ground in 200 μl of dimethylformamide and incubated for 16 h in the dark and on ice. The supernatant was collected by centrifugation for 15 min at 13,000 × g, and its absorbance in visible light was recorded. Tetrapyrole Intermediate Analysis—Approximately 20 mg of frozen leaf material was homogenized in 500 μl of acetone, 0.125 m NH4OH (9:1, v/v) in the dark and centrifuged at 4 °C. The supernatant was diluted with 1 ml of grinding medium and extracted 3 times with 1 ml of hexane. After the complete elimination of the hexane phase, the acetone phase was either directly used for fluorescence measurement or dried under argon before solubilization in methanol, 5 mm tetrabutylammonium phosphate (70:30 v/v) for HPLC analysis. Florescence emission spectra were recorded with a spectrofluorometer MOS-450 from Biologic at room temperature from 570 to 690 nm upon excitation at either 402 nm for detection of protoporphyrin IX or 416 nm for detection of magnesium protoporphyrin IX and magnesium protoporphyrin IX methyl ester or 440 nm for detection of chlorophyllide. HPLC analysis was as described in Block et al. (6Block M.A. Tewari A.K. Albrieux C. Marechal E. Joyard J. Eur. J. Biochem. 2002; 269: 240-248Crossref PubMed Scopus (69) Google Scholar) except that elution was monitored by absorbance detection at 420 nm and by fluorescence detection (λexcitation 420 nm/λemission 595 nm or λexcitation 420 nm/λemission 625 nm). Standards of protorporphyrin IX (Sigma), of magnesium protoporphyrin IX (Porphyrin Product Inc.), of magnesium protoporphyrin IX methyl ester prepared and identified as described in Block et al. (6Block M.A. Tewari A.K. Albrieux C. Marechal E. Joyard J. Eur. J. Biochem. 2002; 269: 240-248Crossref PubMed Scopus (69) Google Scholar) were used. When required, plants were incubated overnight with 10 mm ALA and 5 mm magnesiumCl2 in 10 mm Hepes, pH 7.0, before extraction. Protein Extraction and Immunoblot—Proteins were extracted from leaf material according to the method described by Hurkman and Tanaka (28Hurkman W.J. Tanaka C.K. Plant Physiol. 1986; 81: 802-806Crossref PubMed Google Scholar). Protein analysis and immunoblotting were performed as described previously (6Block M.A. Tewari A.K. Albrieux C. Marechal E. Joyard J. Eur. J. Biochem. 2002; 269: 240-248Crossref PubMed Scopus (69) Google Scholar). Primary antibodies were used at the following dilution: anti-CHLM 1/1000, anti-KARI 1/1000, anti-HSP70b 1/1000, anti-CF1 1/10000, anti-CRD1 1/1000, anti-IE37 1/3000, anti-OEP21 1/500, anti-PORA-PORB 1/1000, anti-PORC 1/1000, anti-LHCB2 (Agrisera) 1/5000, anti-LHCA2 (Agrisera) 1/2000, anti-cytochrome f 1/5000, anti-CP43 1/500, anti-D1 (Agrisera) 1/1000, anti-CHLH 1/200, anti-CHLD 1/500, anti-CHLI 1/200, anti-green fluorescent protein (Euromedex) 1/3000. Primary antibodies were detected using peroxidase-conjugated goat anti-rabbit IgG (Jackson Immuno Research) or goat anti-mouse IgG (Bio-Rad) and the chemiluminescent detection system (ECL, Amersham Biosciences). RT-PCR and Northern Blot Analysis—For RT-PCR analysis, total RNA was isolated using the Invisorb Spin Plant RNA Mini kit following the manufacturer's instructions (Invitek) followed by a DNase treatment using the RQ1 RNase-free DNase (Promega). A PCR reaction was performed on RNA with primers for the RPL21 gene (5′-TCCACTGCGTCGACGTCTCG-3′ and 5′-CTCCAACTGCAACATTGGGCG-3′) to check for the absence of genomic DNA. Reverse transcription was performed on 500 ng of total RNA using the ProSTAR First-Strand RT-PCR kit following the manufacturer's instructions (Stratagene). Semiquantitative RT-PCR was calibrated by non-saturating PCR reaction (22 cycles) using primers designed to amplify the EF1–4α cDNAs (5′-CTGCTAACTTCACCTCCCAG-3′ and 5′-TGGTGGGTACTCAGAGAAGG-3′). A parallel set of reactions without the addition of reverse transcriptase was run as a quality control. To analyze CHLM expression, semiquantitative RT-PCR was performed with TL251 and TL252 primers (see above). The luciferase reporter gene expression was analyzed using two specific primers (5′-CCCGGCGCCATTCTATCC-3′ and 5′-TGAAATCCCTGGTAATCCG-3′). For Northern blot analysis, total RNA was extracted as described by Nagy et al. (29Nagy F. Boutry M. Hsu M.Y. Wong M. Chua N.H. EMBO J. 1987; 6: 2537-2542Crossref PubMed Scopus (77) Google Scholar). 20 μg of total RNA were separated on an agarose gel. RNA blot hybridization and membrane washing were performed on Hybond-N membrane (Amersham Biosciences). The IE37 probe was obtained by PCR amplification on a RT reaction by using specific primers (5′-CACCTAGACTCTCGGTGGC-3′ and 5′-TTTGGGAACGATCTGATCC-3′). The 25 S probe was used as a loading control (30Logemann J. Mayer J.E. Schell J. Willmitzer L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1136-1140Crossref PubMed Google Scholar). Microscopy—Leaves were observed with an immersion 40× objective at room temperature by confocal laser scanning microscopy using a Leica TCS-SP2 operating system. EYFP and chlorophyll fluorescence were excited, respectively, at 488 and 633 nm. Fluorescence emission was collected from 500 to 535 nm for EYFP and from 643 to 720 nm for chlorophyll. Characterization of a Magnesium Protoporphyrin IX Methyltransferase Null Mutant—An Arabidopsis chlm mutant line was identified by screening the flanking sequence tag data base derived from the T-DNA insertion lines of the Versailles collection (31Bechtold N. Ellis J. Pelletier G. C. R. Acad. Sci. (Paris). 1993; 316: 1194-1199Google Scholar, 32Samson F. Brunaud V. Balzergue S. Dubreucq B. Lepiniec L. Pelletier G. Caboche M. Lecharny A. Nucleic Acids Res. 2002; 30: 94-97Crossref PubMed Scopus (205) Google Scholar) using the CHLM cDNA as query sequence (6Block M.A. Tewari A.K. Albrieux C. Marechal E. Joyard J. Eur. J. Biochem. 2002; 269: 240-248Crossref PubMed Scopus (69) Google Scholar) (Fig. 1A). PCR genotyping and kanamycin selection resulted in the selection of plants that were heterozygous for insertion of a T-DNA in the CHLM gene. Sequencing of the PCR-genotyping product and further PCR analysis showed that the insertion of the T-DNA in the single intron present in the coding sequence resulted in the deletion of the 5′ part of the gene including the entire first exon (Fig. 1A). A mutant line with 3:1 kanamycin segregation as expected for a single genomic insertion was chosen for further analyses. Its progeny shows one-third of the kanamycin-resistant plants being non-green (Fig. 1B), and the yellow/white plants always exhibited a homozygous mutant genotype (Fig. 1C). Thus, the mutation in the CHLM gene is recessive and linked to the albino phenotype. In the homozygous mutant plants, RT-PCR indicated an absence of full-length CHLM transcript (Fig. 1A, middle) and immunoblotting confirmed the absence of CHLM protein (Fig. 1, A, right, and C). Because the yellow/white plants could not produce seeds, homozygous mutants were only obtained from heterozygous plants. During silique maturation, some white seeds were visible, suggesting the absence of chlorophyll in the cotyledons of the homozygous seeds (Fig. 1B). However, after desiccation, the seed color heterogeneity was no longer visible, making it impossible to visualize the mutant at this stage. When germinated on MS-agar containing 0.5% sucrose and under medium light (70 μmol of photons m–2 s–1), the homozygous seedlings turned white and stopped growth after cotyledon development. However, when the plants were transferred after 5 days in medium light to dim light (5 μmol of photons m–2 s–1), they could develop, although very slowly and with a stunted appearance; they then formed small yellow leaves (Fig. 1B). The absence of chlorophyll in the yellow homozygous plants was verified by spectrophotometry (Fig. 2A). Complementation of the mutant with the CHLM cDNA sequence resulted in expression of the CHLM protein and restoration of the green phenotype (Fig. 1C). Altogether, these results indicate that this chlm mutant is an magnesium protoporphyrin IX methyltransferase null mutant and that the CHLM gene plays a unique role in the chlorophyll biosynthetic pathway in higher plants. Accumulation of Magnesium Protoporphyrin IX—Fluorescence analysis and HPLC separation of acetone extracts showed that magnesium protoporphyrin IX was the main chlorophyll intermediate present in the mutant (Fig. 2, B and C). When the mutant was fed with ALA to increase the synthesis of intermediates, we observed an increase of magnesium protoporphyrin IX, whereas magnesium protoporphyrin IX methyl ester and other downstream chlorophyll intermediates were absent (Fig. 2C). Conversely, in the wild type, fluorescent spectra indicated the presence of downstream chlorophyll intermediates, and magnesium protoporphyrin IX methyl ester was detected by HPLC when the wild type was fed with ALA. Altogether the data indicated that in the chlm mutant, the chlorophyll pathway is indeed blocked in the methyltransferase step, leading to accumulation of magnesium protoporphyrin IX. Absence of Chlorophyll-containing Protein Complexes in the chlm Mutant—To evaluate chloroplast development in the mutant, we analyzed the expression of a series of proteins in leaves of plants grown 20 days in medium/dim light conditions. After SDS-PAGE and Coomassie staining, we observed that the profiles of total protein extracts from wild type and mutant were similar (Fig. 3A). The large subunit of Rubisco was apparently present at a similar level in both types of plants. However, a major difference was visible at the molecular size expected for LHCII proteins (Fig. 3A, arrow). Immunoblot detection confirmed considerable reduction in the accumulation of a number of major thylakoid-associated proteins including components of photosystem I (LHCI), photosystem II (LHCII, D1, CP43), and the cytochrome b6f complex (Cyt f) (Fig. 3B). In contrast, no significant change was visible in the levels of stromal proteins KARI and HSP70β, chloroplast envelope proteins IE37 and OEP21, or thylakoid protein CF1 from CF1-CF0 ATPase (Fig. 3B). The chlm null mutant was therefore devoid of the major chlorophyll-containing protein complexes. The chlm Mutant Behaves as a “Super Repressor” of Nuclear Photosynthesis Genes—Several processes can induce depletion in chlorophyll-containing proteins, from gene expression to protein turn over. Northern blot analysis indicated that very little change was observed for the expression of chloroplast genes PSBA, PSBD, and PETA, whereas the corresponding proteins (D1, CP43, and cytochrome f (Cyt f)) were absent (compare Figs. 3B and 4A, top). This result confirms that the accumulation of plastid-encoded chloroplast proteins is mostly regulated at a translational or posttranslational level. In contrast, the levels of LHCB transcripts and, to a lesser extent, RBCS transcripts, were reduced in the chlm mutant, whereas the levels of mRNA for the chloroplast inner envelope protein IE37 (33Block M.A. Dorne A.J. Joyard J. Douce R. J. Biol. Chem. 1983; 258: 13281-13286Abstract Full Text PDF PubMed Google Scholar) and of 25 S cytosolic ribosomal RNA were not affected (Fig. 4A, bottom). To verify that the low amount of LHCB mRNA in the chlm mutant was related to transcriptional repression, we crossed a heterozygous CHLM/chlm plant with a line harboring an LHCB promoter-luciferase construct and compared the LHCB promoter-dependent expression of the luciferase reporter gene in the wild-type and chlm mutant backgrounds. We observed no luciferase mRNA in the chlm mutant (Fig. 4B), indicating that the decrease of LHCB mRNA accumulation in the mutant is due to transcriptional repression. Moreover, in an assay for the gun (genome uncoupled) phenotype as defined by Susek et al. (22Susek R.E. Ausubel F.M. Chory J. Cell. 1993; 74: 787-799Abstract Full Text PDF PubMed Scopus (471) Google Scholar), we compared LHCB mRNA accumulation in wild-type and chlm mutant plants grown either in the absence or in the presence of Norfluorazon (Fig. 4C). We observed that photobleaching by Norflurazon induced even stronger repression of the LHCB gene in the chlm mutant compared with the wild type, indicating that this mutant does not show a gun phenotype (compare Fig. 4C, second and fourth lanes). This is not surprising because this mutant is expected to accumulate greater amounts of magnesium protoporphyrin IX through a direct effect on the biosynthetic pathway. Interestingly, the extent of repression observed in the chlm mutant in the absence of Norflurazon is even stronger than that observed in wild-type plants treated with Norfluorazon. This indicates that the chlm mutant behaves more like a super repressor of LHCB gene expression. In conclusion, in the chlm null mutant, the chloroplast-to-nucleus retrograde signaling seems to be turned on without any additional treatment. Up-regulation of CHLH—Besides the role of magnesium protoporphyrin IX in the plastid-to-nucleus signaling pathway, published data suggest an additional role for certain enzymes in the chlorophyll synthesis pathway, such as the magnesium chelatase subunits (20Mochizuki N. Brusslan J.A. Larkin R. Nagatani A. Chory J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2053-2058Crossref PubMed Scopus (520) Google Scholar). We analyzed how these proteins are affected in the chlm mutant. Immunoblot analysis showed that the CHLD and CHLI subunits of the magnesium chelatase, the CRD1 subunit of the cyclase, and the POR proteins are present in similar amounts, whereas the CHLH magnesium chelatase subunit is five to eight times more abundant in the mutant than in the wild type (Fig. 5A). In the chlm-complemented mutant, the level of CHLH protein is reduced, although not quite to the wild-type level (Fig. 5B), indicating that CHLM depletion leads to a specific increase in the level of the CHLH subunit of the magnesium chelatase. Northern blot analyses indicated that, in the chlm mutant compared with the wild type, there were similar or barely lower levels of CHLH and CHLD transcripts (Fig. 5C). Therefore, the up-regulation of the CHLH subunit occurs at a post-transcriptional level in the chlm mutant. CHLH Is Matured and Accumulates Inside Plastids—As an initial step to elucidate the regulatory mechanism leading to this accumulation, we checked if CHLH is located inside and/or outside plastids in the chlm mutant. Because isolation of plastids from these plants was not possible, we indirectly addressed this question by testing for the presence of a unprocessed CHLH precursor in the chlm mutant. The precursor protein was expected at 154 kDa and the mature protein at 144 kDa. When running on a high molecular weight-resolving gel, CHLH was visualized around 144 kDa, clearly below the 150-kDa marker, and the size of the protein was similar in both the wild type and the mutant, suggesting that most of the CHLH protein, if not all, is matured and most probably addressed to plastids. The accumulation of processed CHLH protein in the chlm mutant could be related either to an increased stability of the protein in the chlm plastids or a more efficient import of the precursor form into chlm plastids. To verify the capability of the CHLH transit peptide for sorting the protein to chloroplasts, we expressed this peptide fused to the N-terminal end of EYFP in wild-type and chlm plants and analyzed the localization of the fusion protein. In the wild-type, EYFP and chlorophyll fluorescence colocalized, indicating a chloroplast targeting of the EYFP protein. Aggregation of part of the EYFP in the chloroplasts is likely because additional highly fluorescent spots of EYFP were visible in the chlorophyll fluorescence area (Fig. 6B). In the chlm mutant where chlorophyll is absent, the same image of EYFP fluorescence was observed, suggesting the same plastid localization (data not shown). To discriminate between an increased stability of CHLH or a more efficient import into chlm plastids, we compared the accumulation of the EYFP fusion protein in CHLH transit peptide-EYFP transgenic plants cosegregating for the chlm T-DNA (Fig. 6C). The size of the EYFP fusion protein in each plant was similar and corresponded to EYFP without the transit peptide, and its accumulation was not different. Our conclusion is that the CHLH transit peptide is still capable of addressing the precursor protein into chloroplasts even in the chlm mutant context and that its import capacity is not modified in the chlm mutant. Therefore, the accumulation of the CHLH protein in the chlm mutant probably occurs at a posttranslocational level. CHLM Is a Key Gene for Chlorophyll Biosynthesis and Chloroplast Development—Our results show that chlorophyll formation is totally dependent on the CHLM gene product in Arabidopsis. The inactivation of this gene prevents setting up of chlorophyll-binding proteins in the thylakoids, whereas most other proteins in the chloroplast remain relatively stable. Not only photosystem I and II with their associated light harvesting complex are affected but also the cytochrome b6f complex that contains very low amounts of chlorophyll (34Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2076) Google Scholar, 35Zouni A. Witt H.T. Kern J. Fromme P. Krauss N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1771) Google Scholar, 36Pierre Y. Chabaud E. Herve P. Zito F. Popot J.L. Biochemistry. 2003; 42: 1031-1041Crossref PubMed Scopus (10) Google Scholar). Chlorophyll is required for maturation of chlorophyll-binding proteins, for correct folding of the complexes, and for their insertion in the thylakoids (for review, see Ref. 1Paulsen H. Aro E.M. Anderson B. in Advances in Photosynthesis and Respiration: Regulation of Photosynthesis. Kluwer Academic Publishers, Dordrecht, The Netherlands2001: 219-233Google Scholar). It also has a stabilizing effect on complexes; complexes lacking chlorophyll proteins becoming substrates for proteases. This was reported for chloroplast-encoded proteins such as D1, CP43, and cytochrome f (37Eichacker L.A. Helfrich M. Rudiger W. Muller B. J. Biol. Chem. 1996; 271: 32174-32179Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and it may explain the absence of these proteins in the mutant since we effectively observed that the corresponding mRNAs were expressed at relatively normal levels. Modification of Expression of Nuclear-encoded Photosynthetic Genes—On the opposite, the level of LHCB mRNAs is greatly decreased in the mutant due to transcriptional regulation. Because the mutant specifically accumulates magnesium protoporphyrin IX without any treatment, our result provides an independent indication that magnesium protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously suggested in Norfluorazon-treated plants (19Strand A. Asami T. Alonso J. Ecker J.R. Chory J. Nature. 2003; 421: 79-83Crossref PubMed Scopus (446) Google Scholar). Moreover, the chlm mutant behaves like a super repressor of the LHCB promoter and seems more efficient in repressing LHCB expression than plants treated with Norflurazon. The extent of repression may be due to a different level of accumulation of magnesium protoporphyrin IX in the plant. A second possibility may be linked to the complete absence of magnesium protoporphyrin IX methyl ester and its derivatives in the mutant. This suggests that one of these components acts as a positive effector of nuclear photosynthetic gene expression. Magnesium protoporphyrin methyl ester itself may be a positive effector. In support of this latter hypothesis (24Gadjieva R. Axelsson E. Olsson U. Hansson M. Plant Physiol. Biochem. 2005; 43: 901-908Crossref PubMed Scopus (40) Google Scholar), have shown that, in the absence of Norflurazon, the barley xantha l mutant that is defective in the magnesium protoporphyrin methyl ester cyclase, accumulates magnesium protoporphyrin IX methyl ester and has a high level of LHCB expression. In addition, Alawady and Grimm (8Alawady A.E. Grimm B. Plant J. 2005; 41: 282-290Crossref PubMed Scopus (93) Google Scholar) reported positive correlation between LHCB expression and methyltransferase activity in tobacco CHLM antisense and sense RNA mutants. The activity of magnesium protoporphyrin IX methyltransferase is obviously dependent on the availability of magnesium protoporphyrin IX but is also certainly adjusted to levels of Ado-Met and Ado-Hcy in the chloroplast (see, for instance, Ref. 38Ravanel S. Block M.A. Rippert P. Jabrin S. Curien G. Rebeille F. Douce R. J. Biol. Chem. 2004; 279: 22548-22557Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The differential effects of magnesium protoporphyrin IX and magnesium protoporphyrin IX methyl ester on LHCB expression would consequently finely attune the synthesis of light-harvesting complexes to the one-carbon metabolism. Increase of CHLH Level—In the chlm mutant we observed a strong increase in the level of mature CHLH, whereas that of CHLH mRNA was slightly reduced. Our data indicate that stabilization of the protein inside chloroplasts probably occurs in the chlm mutant. One possible mechanistic explanation for this accumulation could be traced to the increased level of magnesium protoporphyrin IX in this mutant. Indeed, a tight channeling of substrate between the magnesium chelatase and the methyltransferase has been suspected for a long time due to the absence of detectable magnesium protoporphyrin intermediates in standard conditions (39Gorchein A. Biochem. J. 1972; 127: 97-106Crossref PubMed Scopus (48) Google Scholar). More recently, the physical interaction between CHLM and CHLH has been shown (40Hinchigeri S.B. Hundle B. Richards W.R. FEBS Lett. 1997; 407: 337-342Crossref PubMed Scopus (48) Google Scholar, 7Alawady A. Reski R. Yaronskaya E. Grimm B. Plant Mol. Biol. 2005; 57: 679-691Crossref PubMed Scopus (38) Google Scholar). During diurnal growth, the magnesium chelatase activity peaks during the transfer from dark to light, whereas the methyltransferase activity maximum follows only a few hours later. During this period, the magnesium protoporphyrin IX level is transiently higher than that of magnesium protoporphyrin IX methyl ester (7Alawady A. Reski R. Yaronskaya E. Grimm B. Plant Mol. Biol. 2005; 57: 679-691Crossref PubMed Scopus (38) Google Scholar). It is possible that magnesium protophorphyrin IX binds to CHLH, preventing the transitory accumulation of free magnesium protoporphyrin IX when CHLM is not sufficiently present/active. This could both stabilize the rapidly turning over CHLH protein (5Nakayama M. Masuda T. Bando T. Yamagata H. Ohta H. Takamiya K. Plant Cell Physiol. 1998; 39: 275-284Crossref PubMed Scopus (68) Google Scholar) and protect it against major photooxidative damage generated by free magnesium protoporphyrin IX. Whatever the mechanism underlying CHLH accumulation, one can question whether its concomitant increase with magnesium protoporphyrin IX plays a role in mediating the plastid-to-nucleus regulatory pathway. Particular thanks are due to researchers who gave us aliquots of antibodies we used in this work: R. Dumas, M. Schroda, S. Merchant, T. Masuda, F.-A. Wollman, and M. Hansson. We acknowledge D. Vega for excellent technical assistance and F.-Y. Bouget for the LHCII-LUC line. We thank Dr. R. Cooke for copyediting the manuscript." @default.
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• W2001808493 title "Knock-out of the Magnesium Protoporphyrin IX Methyltransferase Gene in Arabidopsis" @default.
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