a putative mg chelatase subunit from arabidopsis thaliana cvplant physiol. (1996) 11 1 : 61-71 a...

Plant Physiol. (1996) 11 1 : 61-71

A Putative Mg Chelatase Subunit from Arabidopsis thaliana cv C24

Sequence and Transcript Analysis of the Gene, lmport of the Protein into Chloroplasts, and in Situ Localization of the Transcript and Protein

Lucien C. D. Cibson', Joanne 1. Marrison', Rache1 M. Leechl, Poul E. JensenZt3, Diane C. Bassham4, Marie Cibson', and C. Neil Hunter'*

Robert Hill lnstitute for Photosynthesis and Krebs lnstitute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S1 O 2UH, United Kingdom (L.C.D.G., M.G., C.N.H.); Department of Biology, University of York, Heslington, York YO1 5DD, United Kingdom (J.L.M.,

R.M.L.); Department of Ecology and Molecular Biology, Royal Veterinary and Agricultural University, Bülowsvej 13, DK-1870 Frederiksberg C, Denmark (P.E.J.); and Department of Biological Sciences, University of Warwick,

Coventry CV4 7AL, United Kingdom (D.C.B.)

We have isolated and sequenced a cDNA from Arabidopsis tbali- ana cv C24 that encodes a putative Mg chelatase subunit. The deduced amino acid sequence shows a very high level of identity to a gene previously characterized from Antirrbinum majus (olive) and also high similarity to bcbH, a bacterial gene involved in the Mg chelatase reaction of bacteriochlorophyll biosynthesis. We suggest that this gene be called CHL H. Northern blot analyses were used to investigate the expression of CHL H, another putative Mg chelatase gene, cb-42, and ferrochelatase. The CHL H transcript was observed to undergo a dramatic diurna1 variation, rising almost to i t s maximum level by the end of the dark period, then increasing slightly at the onset of the light and declining steadily t o a minimum by the end of the light period; in contrast, transcripts for cb-42 and ferrochelatase remained constant. A model i s proposed in which the CHL H protein plays a role in regulating the levels of chlorophyll during this cycle. In situ hybridization revealed that the transcripts are located over the surface of the chloroplasts, a feature in common with transcripts for the cb-42 gene. The CHL H protein was imported into the stromal compartment of the chloroplast and processed in an in vitro assay. lmmunoblotting showed that the distribution of CHL H protein between the stroma and chloroplast membranes varies depending on the concentration of Mg2+. In situ immunofluorescence was used to establish that the CHL H and CH-42 proteins are localized within the chloroplast in vivo.

' Supported by the Biotechnology and Biological Sciences Re- search Council of the United Kingdom.

Supported by the Danish Agricultura1 and Veterinary Re- search Council (grant no. 13-5005-1).

I'resent address: Robert Hill Institute for I'hotosynthesis and Krebs Institute for Biomolecular Research, Department of Molec- ular Biology and Biotechnology, University of Sheffield, Sheffield S10 2UH, UK.

Supported by a Biotechnology and Biological Sciences Re- search Council of the United Kingdom studentship.

* Corresponding author; e-mail c.n.hunter8sheffield.ac.uk; fax 44 -114 -272- 8697.

Chlorophyll pigments harvest light energy, which is used to drive the reductive fixation of carbon dioxide. These pigments are the most abundant on earth, yet rela- tively little is known about the enzymology of the committed steps of chlorophyll biosynthesis. Still less is known about the genetic control of this pathway. One important enzyme in this pathway is Mg-protoporphyrin chelatase, the enzyme that catalyzes the insertion of Mg into protoporphyrin IX. This lies at the branch point of the chlorophyll and heme biosynthetic pathways and is considered to be the first committed step of chlorophyll synthesis (see Fig. 1). Accordingly, this enzyme has been the subject of much study in plants (Fuesler et al., 1981; Walker and Weinstein 1991a, 1991b; Walker and Weinstein, 1994), although purification of the enzyme has proved difficult. At present, it has been possible to establish a continuous assay for the enzyme and to show that two components probably participate in the chelation reaction (Walker and Wein- stein, 1991b; Walker and Weinstein, 1995). It is also known that there is an ATP requirement for the activation of these components (Walker and Weinstein, 1994). However, the identities of these components and the genes that encode them have not been established.

The purple photosynthetic bacteria Rkodobacter capsulatus and Rkodobacter sphaeroides have been used to study the molecular genetics of bacteriochlorophyll biosynthesis, which has many intermediates in common with the chlorophyll biosynthetic pathway (Bauer et al., 1993). These bacteria provide a convenient model system for the study of these pathways partly because the bacteriochlorophyll biosynthesis genes are clustered on a small region of the chromosome, 45 kb in length (Marrs, 1981; Coomber and Hunter, 1989). Production of bacteriochlorophyll-minus mutants has allowed the correlation of mutant phenotypes with lesions in specific genes (Marrs, 1981; Zsebo and

Abbreviation: FITC, fluorescein isothiocyanate. 61

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62 Gibson et al. Plant Physiol. Vol. 11 1, 1996

O*’\OH O*‘bH

Protoporphyrin IX

Mg protoporphyrin IX Figure 1. The branch point of chlorophyll and heme biosynthesis. The steps leading to protoporphyrin are common to both pathways, whereupon Mg is inserted to form chlorophyll or iron is chelated to form heme.

Hearst, 1984; Coomber et al., 1990; Bollivar et al., 1994). These genes have been assigned to steps in the pathway on the basis of bacteriochlorophyll intermediates accumulated by the mutants. Recently, there has been a step forward in the analysis of this pathway, as a result of overexpression of some bch genes in Escherichia coli, followed by studies of enzyme activity. The bchM genes of R. capsulatus and R. sphaeroides have been overexpressed in E. coli, and extracts from the resulting strains are able to catalyze the methyl- ation of added Mg protoporphyrin, resulting in the formation of Mg protoporphyrin monomethyl ester. bchM has therefore been assigned to the methyltransferase step in bacteriochlorophyll biosynthesis (Bollivar et al., 1994; Gib- son and Hunter, 1994). A similar approach has been used to demonstrate that the genes bchI, bchD, and bchH encode components of the Mg chelatase. bchl and bchD were expressed in one E. coli strain and bchH in another, extracts of these strains were combined, and the mixture was able to catalyze the insertion of Mg into protoporphyrin IX (Gib- son et al., 1995).

This assignment has direct relevance to two studies carried out on higher plants. Two genes have been isolated from plants that show significant similarity to subunits of the Rhodobacter Mg chelatase; the nuclear gene cs/ ch-42 from Arabidopsis thaliana (Koncz et al., 1990) is similar to bchI and olive from Antirrhinum majus is similar to bchH (Hudson et al., 1993). These genes have also been corre- lated with lesions in chlorophyll synthesis as a result of the chlorophyll-deficient phenotypes that arise from insertion of T-DNA or the Tam3 transposon into Arabidopsis and Antirrhinum, respectively. In view of these similarities and the demonstrated involvement of the bacterial homologs of these genes in bacteriochlorophyll synthesis, it seems likely that both ch-42 and olive encode subunits of the Mg chelatase.

In this paper, we have extended our previous bacterial studies to the model plant A. thaliana (cv C24); the olive homolog, which we propose to cal1 CHL H, has been cloned and sequenced. Expression of CHL H was investigated at the leve1 of transcription. The subcellular location of the CHL H and ch-42 transcripts were investigated using in

situ hybridization. We show that the protein is imported into chloroplasts, and that both the CHL H and CH-42 proteins were colocalized within the chloroplasts by immunofluorescence.

MATERIALS AND METHODS

Cloning of the CHL H cDNA

A AZAPII library of cDNA prepared from green leaf tissue of Arabidopsis thaliana cv C24 was a gift from Dr. Christine Raines (University of Essex, UK). Approximately 107 plaque- forming units were used as the template in a PCR, using the oligonucleotides 5’-CGGAATTC(GAT)GA(GCAT)C(GT)(G- CAT)C(GT)(CT)TT(GCAT)GC(GAT)AT(GCAT)GT-3’ and 5‘- CGGAAGCTT(CT)GG(GCAT)TA(CT)GA(GA)GG(GCAT)G- A(CT)CC(GCAT)AT-3’. One-fiftieth of h s PCR was used as the template in a nested PCR, using the oligonucleotides

AA(GA)CA-3’ and 5’-ACTGCTGCAGTT(GA)TT(GCAT)- GC(GCAT)GC(GA)TA(GA)TA(GA)TA-3’. A 120-bp product was obtained from the nested PCR, which was used to screen 2.5 X 105 clones of the AZAPII library. Two clones were isolated, one of which was determined to contain the entire open reading frame. The pBluescript plasmid containing this cDNA was excised using the Ex-Assist helper phage supplied by Stratagene. A nested set of deletions of this plasmid was generated using exonuclease 111 (Heni- koff, 1984) as described by Sambrook et al. (1989). These were used to determine the nucleotide sequence of the cDNA on both strands by the method of Sanger et al. (1977), using the Sequenase kit supplied by United States Biochemical. A11 DNA manipulations were carried out by standard methods (Sambrook et al., 1989).

5’ -AITCGAAITCGA (GA)TT(CT) ATGCC (GCAT)GG(GCAT)-

RNA Preparation and Northern Analysis

Seeds of A. thaliana cv C24 were planted in moist peat. For diurna1 rhythm experiments, seedlings were grown under a 16-h day/8-h night cycle at 24 +- 2°C and samples (3 g) from 25-d-old plants were harvested at 2-h intervals during a 24-h time period. The plants were watered with Hoagland mineral solution on alternate days. During har- vesting of plants in darkness, a dim, green safelight was used. Total RNA was prepared from leaves as described by Schuler and Zielinski (1989). Samples (25 pg/lane) were electrophoresed on a 1% formaldehyde gel and blotted onto Hybond N (Amersham) by standard methods (Sam- brook et al., 1989). Prehybridization and hybridization of the filters were carried out at 66°C in 5X SSPE (1X SSPE = 0.18 M NaC1, 10 mM sodium phosphate, pH 7.7, 1 mM EDTA), 5X Denhardt’s solution (1X Denhardt’s solution = 0.2 g /L BSA, 0.2 g /L Ficoll, 0.2 g / L PVP), 0.1% SDS, and 50 p g carrier DNA/mL. Radiolabeling of probes with 32P was performed using the Megaprime kit (Amersham). Washes were performed at 66°C with 0.1X SSPE and 0.1% SDS in the final wash. Equal loading of RNA samples was con- firmed by probing an identical blot with a soybean tubulin cDNA (Guiltinan et al., 1897). Sizes of transcripts were estimated by comparison with the 0.24- to 9.5-kb RNA ladder from GIBCO-BRL.



Mg Chelatase Subunit from Arabidopsis 63

lmport Studies Using Pea Chloroplasts

The precursor of the CHL H protein was synthesised in vitro by transcription of the cDNA clone followed by translation of capped transcripts in a reticulocyte lysate (Amer- sham) in the presence of [35S]Met. Import of the labeled precursor protein into intact pea chloroplasts was carried out as described by Robinson and Barnett (1988). After the import reaction was completed, the chloroplasts were washed and fractionated as previously described (Robin- son and Barnett, 1988). Equivalent amounts of each sample were separated on 7% SDS polyacrylamide gels (Laemmli, 1970), after which the gels were fixed, treated with the fluorofor Amplify (Amersham), dried, and exposed to x- ray film. Assays for cleavage of the precursor by partially purified pea stromal processing peptidase were as described by Musgrove et al. (1989).

Expression of CHL H in Escherichia coli

The primers 5’-GATCTAGACATATGCACGGTGCTG- TATCTGGAAACGGCCT-3’ and 5’-ACCAGAATTCAAGT- GGATGCCAAATTTCG-3’ were used to amplify the 5’ end of the C H L H cDNA. The product, of approximately 600 bp, was digested with NdeI and BamHI and cloned into pET3a (Rosenberg et al., 1987). The resulting plasmid was digested with BamHI and the remainder of CHL H was inserted, giving rise to a plasmid encoding a version of CHL H that we predicted would approximate the mature protein. This protein was produced in Escherichia coli BL21 (DE3) (Studier and Moffat, 1986) and used as a control in immunoblotting.

Preparation of Antibodies

Antibodies against the OLI protein were a gift from Dr. Andrew Hudson (University of Edinburgh, UK). Antibod- ies against the CH-42 protein were obtained by amplifying part of the ch-42 gene (Koncz et al., 1990) by PCR using the oligonucleotides 5‘-AGTTCTGCAGGGAACAAGTAGT- AGGGAAGTTTG-3’ and 5‘-TTAGGAATTCTCAGCTGA- AAATCTCGGCGAA-3’. f i e PCR fragment obtained was inserted into pET14b (Novagen, Madison, WI) and the CH-42 protein was overexpressed in E. coli. The protein was purified using the His tag system of Novagen. Immu- nization of rabbits and preparation of CH-42 antiserum were carried out at the Serum Institute (Copenhagen, Denmark).

Preparation of Arabidopsis Chloroplasts and lmmunoblotting

Chloroplasts were prepared from 21-d-old Arabidopsis leaves by the method of Kunst et al. (1988), resuspended in 10 mM Tricine, pH 7.6, 2 mM EDTA containing either 1 or 5 mM MgCI, and subjected to two freeze-thaw cycles. Membranes were pelleted by centrifugation in a microfuge for 1 min, and the supernatants, referred to as the stromal fractions, were retained. The pelleted membranes were washed and resuspended in the appropriate buffer. Equiv- alent amounts of whole chloroplasts and stromal and mem-

brane fractions were electrophoresed in a 10% SDS-polyacrylamide gel (Laemmli, 1970). Transfer of proteins to Hybond C (Amersham) and incubation of the blot with antibodies were done by standard methods (Sambrook et al., 1989). CHL H was detected using horseradish peroxi- dase-conjugated goat anti-mouse antisera and the en- hanced chemiluminescence detection reagents supplied by Amersham.

In Situ Labeling of Transcripts and Proteins

A. thaliana (L.) cv Landsberg erecta seedlings were grown as described by Pyke et al. (1991). Whole first leaves were harvested 16 d after sowing and fixed in 3% (w/v) para- formaldehyde, 50% (v/v) ethanol, and 5% (v/v) acetic acid and embedded in PEG 1500 as previously described (Mar- rison and Leech, 1994). Transverse tissue sections (7 pm) were placed onto dampened polysine slides (Merck, Poole, UK) and left to dry on a hot plate at 40°C. In situ hybridization was performed with antisense and sense digoxi- genen-labeled RNA probes synthesized by an in vitro transcription reaction using 1 pg of linearized template DNA, digoxigenin-11-UTP, and T3 or T7 RNA polymerase, ac- cording to the manufacturer’s protocol (Boehringer Mann- heim). 25s rRNA transcripts were localized using the 0.9-kb BamHI-EcoRI insert from pTA71 subcloned into pBS- (KS+) (Gerlach and Bedbrook, 1979; Rode et al., 1987). Hybridized probe was detected using anti-digoxigenin- alkaline phosphatase conjugate and overnight color devel- opment with nitroblue tetrazolium chloride and 5-bromo- 4-chloro-3-indolyl-phosphate as described by Marrison and Leech (1994). Starch was stained in rehydrated tissue sections using Lugol’s iodine (Sigma) (6% [w/v] KI and 4% [w/v] iodine, diluted 4-fold before use).

Immunolocalization was carried out as described by Marrison and Leech (1992). Antiserum raised against the OLI protein was used to detect the CHL H protein. Sections were incubated overnight at 4°C with a 1:200 dilution of OLI or CH-42 antiserum in 0.5% (w / v) BSA/ PBS or in 0.5% (w/v) BSA/PBS alone (control). CHL H was visualized using FITC-conjugated goat anti-mouse antiserum and CH-42 was visualized using FITC-conjugated anti-rabbit antiserum (Sigma) diluted in BSA / PBS as recommended by the supplier. Sections were viewed using a Nikon FXA microscope with epifluorescence attachment, high-pres- sure mercury lamp and filter combination dichroic mirror 510,450- to 490-nm excitation filter, and barrier filter 515IF. Photomicrographs were taken using Kodak Ektachrome 400 color slide film with automatic exposure setting.

RESULTS

Sequence Analysis

The sequence of the C H L H cDNA was determined, and the data are shown in Figure 2A. The gene contains 1382 codons, which corresponds to a predicted molecular weight of 153,507 for the CHL H preprotein. The overall leve1 of identity with the Antirrhinum OLI protein is 85%, and CHL H also has 41 and 40% identity with the BchH proteins from R. sphaeroides and R. capsulatus, re-



Gibson et al. Plant Physiol. Vol. 11 1 , 1996

A GCGGCCGCCGATGAGAGAATCATAAACTCCCACTTGGAGCTCAAAAAGTGTAAGAGACAACCAAC~TAAACGATTCATCTCTTCTCCTATCCTCTCCTCTTCGAATTCAACGTTTGGAG 120 AATCCAGCAGCCGCAAAATGGCTTCGCTTGTGTATTCTCCATTCACTCTATCCACTTCTAAAGCAGAGCATCTCTCTTCGCTCACTAACAGTACCARACATTCTTTCCTCCGGAAGAFJ\C 240

M A S L V Y S P F T L S T S K A E H L S S L T N S T K H S F L R K K H

ACAGATCAACCAAACCAGCCAAATCTTTCTTCAAGGTGAAATCTGCTGTATCTGGARACGGCCTCTTCACACAGACGAACCCGGAGGT~GTCGTATAGTTCCGATCAAGAGAGACAACG 360 R S T K P A K S F F K V K S A V S G N G L F T Q T N P E V R R I V P I K R D N V

TTCCGACGGTGAAAATCGTCTACGTCGTCCTCGAGGCTCAGTACCAGTCTTCTCTCAGTGAAGCCGTGCRATCTCTCAACAAGACTTCGAGATTCGCATCCTACGAAGTGGTTGGATACT 480 P T V K I V Y V V L E A Q Y Q S S L S E A V Q S L N K T S R F A S Y E V V G Y L

TGGTCGAGGAGCTTAGAGACAAGRACACTTACAACAACTTCTGCGAAGACCTTARAGACGCCRACATCTTCATTGGTTCTCTGATCTTCGTCGAGGAATTGGCGATTARAGTTAAGGATG 600 V E E L R D K N T Y N N F C E D L K D A N I F I G S L I F V E E L A I K V K D A

CGGTGGAGAAGGAGAGAGACAGGATGGACGCAGTTCTTGTCTTCCCTTCAATGCCTGAGGTAATGAGACTGAACAAGCTTGGATCTTTTAGTATGTCTCAATTGGGTCAGTC~GTCTC 720 V E K E R D R M D A V L V C P S M P E V M R L N K L G S F S M S Q L G Q S K S P

CGTTTTTCCAACTCTTCAAGAGGAAGIL9ACRRGGCTCTGCTGGTTTTGCCGATAGTATGTTGAAGCTTGTTAtiGACTTTGCCTAAGGTTTTGAAGTACTTACCTAGTGACAAGGCTCAAti 840 F F Q L F K R K K Q G S A G F A D S M L K L V R T L P K V L K Y L P S D K A Q D

ATGCTCGTCTCTACATCTTGAGTTTACAGTTTTGGCTTGGAGGCTCTCCTGATAATCTTCAGAATTTTGTT~GATGATTTCTGGATCTTATGTTCCGAGCTTTGARAGGTGTC~TCG 960 A R L Y I L S L Q F W L G G S P D N L Q N F V K M I S G S Y V P A L K G V K I E

AGTATTCGGATCCGGTTTTGTTCTTGGATACTGGAATTTGGCATCCACTTGCTCCAACCATGTACGATGATGTGAAGGAGTACTGGAGTACTGGAACTGGTATGACACTAGAAGGGACACCAATGACT 1 0 8 0 Y S D P V L F L D T G I W H P L A P T M Y D D V K E Y W N W Y D T R R D T N D S

CACTCAAGAGGkWlGATGCAACGGTTGTCGGTTTAGTCTTC~AGAGGAGTCACATTGTCACTGGTGATGATAGTCACTATGTG%TGTTATCATtiGAGCTTGAGGCTAGAGGTf;CT~GG 1200 L K K K D A T V V G L V L Q R S H I V T G D D S H Y V A V I M E L E A R G A K V

TCGTTCCTATATTCGCAGGAGGGTTGGATTTCTCTGGTCCAGTAGAGARATATTTCGTAGACCCGGTGTCGAAACAGCCCATCGTAAACTCTGCTGTCTCCTTGACTGGTTTTGCTCTTG 1320 V P I F A G G L D F S G P V E K Y F V D P V S K Q P I V N S A V S L T G F A L V

TTGGTGGACCTGCAAGGCAGGATCATCCCAGGGCTATCGAAGCCCTGAAAAAGCTCGATGTTCCTTACCTTGTGGCAGTACCACTGGTGTTCCAGACGACAGAGGAATGGCTARAGAGCA 1440 G G P A R Q D H P R A I E A L K K L D V P Y L V A V P L V F Q T T E E W L K S T

CACTTGGTCTCCATCCCATCCAGGTGGCTCTGCAGGTTGCCCTCCCTGAGCTTGATGGAGCGATGGAC~CAATCGTTTTCGCTGGTCGTGACCCTAGAACAGGGAAGTCACATGCTCTCC 1560 L G L H P I Q V A L Q V A L P E L D G A M E P I V F A G R D P R T G K S H A L H

ACAAGAGAGTGGAAGCAACTCTGCATCAGAGCGATTCGATGGGGTGAGCTCAAAAG~CT~G~AGAGAAGAAGCTGGCAATCACTGTTTTCAGTTTCCCACCTGAT~GGTAATG 1 6 8 0 K R V E Q L C I R A I K W G E L K R K T K A E K K L A I T V F S F P P D K G N V

TAGGGACTGCAGCTTACCTCAATGTGTTTGCTTCCATCTTCTCGGTGTTAAGAGACCTCAAGAGAGATGGCTACAATGTTGAAG~CTTCCTGAGAATGCAGAGACTCTTATTGAAGAAA 1 8 0 0 G T A A Y L N V F A S I F S V L R D L K R ~ G Y N V E G L P E N A E T L I E E I

TCATTCATGACAAGGAGGCTCAGTTCAGCAGCCCTAACCTCAATGTAGCTTACAAAATGGGAGTCCGTGAGTACCAAGACCTCACTCCTTATGC~TCCCCTGGAAG~CTGGGGGA 1 9 2 0 I H D K E A Q F S S P N L N V A Y K M G V R E Y Q D L T P Y A N P L E E N W [ ; K

AACCTCCGGGGAACCTTAACTCAGATGGAGAGAACCTTCTTGTCTATGGRAAAGCGTACGGTAATGTTTTCATCGGAGTGCAACCAACATTTGGGTATGAAGGTGATCC~~AT~iAGl~CTl~CCAT 2040 P P G N L N S D G E N L L V Y G K A Y G N V F I G V Q ? T F G Y E G D ? M R L L

TTTTCTCCAAGTCAGCAAGTCCTCATCACGGTTTTGCTGCTTACTACTCTTATGTAG~GATCTTCELAAGCTGATGCTGTTCTTCATTTTGGAACACATtiGTTCTCTCGAGTTTATGC 2160 F S K S A S P H H G F A A Y Y S Y V E K I F K A D A V L H F G T H G S L E F M P

CCGGGAAGCAAGTGGGAATGAGTGATGCTTGTTTTCCCGACAGTCTTATCGGGAACATTCCC~TGTCTACTATTATGCAGCTAACAATCCCTCTGAAGCTACCATTGC~GAGGAGAA 2280 G K Q V G M S D A C F P D S L I G N I P N V Y Y Y A A N N P S E A T I A K R R S

GTTATGCCMCACCATCAGTTATTTGACTCCTCCAGCTGAGAATr~TGGTCTATACAAAGGGCTGAGCAGGTGAGTGAGCTGATATCGT~CTATCAGTCTCTGAAGGACACGGGGAGAC 24Olj Y A N T I S Y L T P P A E N A G L Y K G L K Q V S E L I S S Y @ S L K D T G R G

GTCCACAGATCGTCAGTTCCATCATCAAGCACAGCTAAGCAATGTAATCTTGATAAGGATGTGGATCTTCCAGATGAAGGCTTGGAGTTGTCACCT~GACAGAGATTCTGTGGTTGGGA 2520 P Q I V S S I I S T A K Q C N L D K D V D L P D E G L E L S P K D R D S V V G K

AAGTTTATTCCAAGATTATGGAGATTGAATCAAGAGCTTTTGCCGTGCGGGCTTCACG~ATTGGAGÂGCCTCCATCCGCCATGG~CCTGTGGCCACACTGG~RACATTGCT~TCTAG 2640 V Y S K I M E I E S R L L P C G L H V I G E P P S A M E A V A T L V N I A A L D

ATCGTCCGGAGGATGAGATTTCAGCTCTTCCTTCTATATTAGCTGAGTGTGTTGGAAGGGAGATAGAGGATGTTTTACAGAGGAAGCGACAAGGGTATCTTGAGCGATGTAGAGCTTCTCA 2760 R P E D E I S A L P S I L A E C V G R E I E D V Y R G S D K G I L S D V E L L K

AAGAGATCACTGATGCCTCACGTGGC~TGTTTCCGCCTTTGTGG~CAAC~TAGC~GGACAGGTGGTGGATGTGTCTGACAAGCTTACCTCtiCTTCTTGGGTTTf;GAA~A 2 8 8 0 E I T D A S R ~ ~ A V S A F V E K T T N S K G Q V V D V S ~ K L T S L L ~ ~ F G I N

ATGAGCCATGGGTTGAGTATTTGTCCAACACCAAGTTCTACAGGGCGAACAGAGATAAGCTCAGAACAGTGTTTGGTTTCCTT~~GAGAGTGCCTGAAGTTGGTGGTCATGGACAAC~~AAC 3000 E P W V E Y L S N T K F Y R A N R D K L R T V F G F L G E C L K L V V M D N E L

T A G G G A G T C T A A T G C A A G C T T T G G A A G G C A A G T A C G T C G A G C C T G G C C C C G G A G G T G A T C C C A T C A T C A A A G G C T C A G G 3120 G S L M Q A L E G K Y V E P G P G G D P I R N P K V L P T G K T S I A L D P Q A

CTATTCCCACAACAGCAGCAATGGCAAGTGCCAAGATTGTGGTTGAGAGGTTGGTAGAGAGACAGAAGCTCG~CGAAGGGRRATATCCCGAGACRATCGCGCTTGTTCTTTGGGGAA 3240 I P T T A A M A S A K I V V E R L V E R Q K L E N E G K Y P E T I A L V L W G T

CTGACAACATCAAAACATATGGGGAGTCTCTCTTGGCCAGGTTCTTTGGATGATTGGTGTGAGACCAATTGCTGATACTTTTGG~GAGTG~CCGTGTCGAGCCTGTGAGCTTAGIL4GAAC 3360 D N I K T Y G E S L G Q V L W M I G V R P I A D T F G R V N R V E P V S L E E I J

Figure 2. (Continued on facing page.)




TAGGIL4GGCCGAGGATCGATGTAGTTGTTAACTGCTCAGGGGTCTTCCGTGATCTCTTTATCAACCAGATGAACCTTCTTGACCGAGCTATC~GATGGTGGCGGACGTAGATGAGCCCG G R P R I D V V V N C S G V F R D L F I N Q M N L L D R A I K M V A D V D E P V

TAGAGCk4AATTTTGTAA~~GAAACACGCGTTGGAACAAGCAGAGGCGCTTGf;CATTGATATTAGAGAti~~AGCGACAAGAGTTTTCTCAAACGCTTCAGGGTCATACTCAGCCAACATCA E Q N F V R K H A L E Q A E A L G I D I R E A A T R V F S N A S G S Y S A N I S

GTCTTGCTGTTGAAAACTCGTCATGGAACGATGAGAAACAGCTTCAGGACATGTACTTGAGCCGCAAATCtiTTTGCGTTTGATAGTGATGCTCCTGGAGCAGGAATGGCTGAGAAGAAGC L A V E N S S W N D E K Q L a D M Y L S R K S F A F D S D A P G A G M A E K K Q

AGGTCTTTGAGATGGCTCTTAGCACTGCAGAAGTCACCTTCCAGAACCTGGATTCTTCAGAGATTTCTTTGACTGATGTGAGCCACTACTTCGATTCTGACCCTACAAATCTAGTTCAGA V F E M A L S T A E V T F Q , N L D S S E I S L T D V S H Y F D S D P T N L V Q S

GTTTGAGGAAGGATAAGAAGAAACCAAGCTCTTACATTGCTGACACTACAACTC~AAACr~GCAGGTGAGGACACTATCTGAGACACAGTGAGr~TGGACGCAAGAACAAAGCTGCTGAATC L R K D K K K P S S Y I A D T T T A N A Q V R T L S E T V R L D A R T K L L N P

CARRGTGGTACGAAGGRATGATGTCAAGTGGATATGAAGGAGTTCGTTCGTGAGATAGAG~GAGACTGTCC~CACT~T~G~~TGGAGT[;C~CGTCAGGTCAAGTAGACAATTGGGTCTACG K W Y E G M M S S G Y E G V R E I E K R L S N T V G W S A T S G Q V D N W V Y E

AGGAGGCCAACTCAACTTTCATCCAAGACGACGAGGAGATGCTG~CCGTCTCATGAACACCAATCCCAACTCCTTCAGG~Tr~TTCAGACTTTCTTGGAGGCC~TGGTCGTG~TACT E A N S T F I Q D E E M L N R L M N T N P N S F R K M L Q T F L E A N G R G Y W

GGGACACTTCCGCTGAAAACATAGAGAAGCTCAAGGAATTGTACTCGCAGGTGGAAGACAAGATCGAAGGGATCGATCGATAAACAATGGGATATAAGCCTTTCTTCTTGTAAAT~~AACT D T S A E N I E K L K E L Y S Q V E D K I E G I D R I

TAAAACTTTATTTTTGTGTATTACAGTATTCTACCAAACGACATCATCTCTCTGTGAATAAGAGAATGTTTTGGCTGACATTTTCTGACTTTAAAAAAAAAAAAAAAA 4 4 2 8

B At ,MASLVYSPFT LSTSKAEHLS SLTNSTKHSF LRKKHRSTKP AKSFFKVKSA 50 Syn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . At VSGNGLFTQT NPEVRRIVPI KRDNVPTVKI VYWLEAQYQ SSLSEAVQSL 100 Syn ..... MFTNV KSTIPRVDPE ALNGRQLLKV VYWLESQYQ SALSAAVRNI 45 Rs . . . . . . . . MH GE.VSGPAGT P . . . . . GYSI AIVTLDAHAA GPVAFUAPRL 36 Rc . . . . . . . . MH DESMSGTMPL PPHRPGGYNV VIITLDQHAA GPAARALPRL 42

Figure 2. A, DNA sequence of the CHL H cDNA (upper line) and deduced amino acid sequence (lower line). The arrow shows the putative processing site of the transit peptide. B, N-terminal portion of an alignment between Arabidopsis CHL H (At), Synechocystsk ChlH (Syn), R. sphaeroides BchH (Rs), and R. capsulatus BchH (Rc). Conserved amino acid residues are in boldface.

3490

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spectively. CHL H also exhibits 65% identity with ChlH, a putative Mg chelatase subunit from Synechocystis PCC 6803 (Jensen et al., 1996a). Hudson et al. (1993) noted the strong similarity between the OLI protein and CobN, which is a subunit of the cobaltochelatase enzyme from Pseudomonas dentrificans (Crouzet et al., 1991). Similarly, the Arabidopsis CHL H protein shows 27% identity with CobN. The significance of these homologies to the role played by the CHL H protein will be commented on in ”Discussion.”

Figure 2B shows an alignment between the N termini of CHL H from Arabidopsis, the Synechocystis ChlH protein, and the Rhodobacter BchH proteins. Given that the overall level of similarity between all of these proteins is quite high, the CHL H protein appears to have a 55-amino acid extension at the N terminus that may constitute the transit peptide. Indeed, this sequence does have typical features of a stroma-targeting signal because it has a high proportion of Ser/Thr and an overall positive charge (von Heijne et al., 1989) and the motif MASLV at the N terminus of the protein is similar to that found in severa1 other chloroplast transit peptides (Karlin-Neumann and Tobin, 1986). There does not appear to be a motif at the N terminus that fits with the cleavage-site motif suggested by Gavel and von

Heijne (1990). Therefore, we suggest that the processing site may be at the point where the N terminus of the Synechocystis protein aligns with CHL H; this is marked by an arrow in Figure 2A.

Northern Analysis of CHL H Expression

Figure 3 shows an analysis of CHL H expression by northern blotting that demonstrates that a large transcript of approximately 4.5 kb is present in leaves but not in roots. In addition, an investigation of fluctuations in transcript levels throughout a diurna1 cycle was carried out. RNA was prepared from plants harvested at 2-h intervals during an 8-h darkl16-h light cycle, blotted, and probed with the CHL H cDNA. An identical blot was probed with the tubulin cDNA to provide a constitutive control; the level of this transcript is constant, indicating that the amounts of different RNA samples on the blot are essentially the same. The level of the CHL H transcript increased strongly in the dark, almost reaching its maximum level by the end of the dark period. A further increase in the transcript level was observed slightly after the onset of the light, rising to a maximum after 1 h and then decreasing gradually during the light period. We also probed an identical blot with the



66 Gibson et al. Plant Physiol. Vol. 111, 1996

A B

Hours in dark Hours in light

light cycle, which is in marked contrast to the behavior ofthe CHL H transcript.

In Situ Detection of CHL Hand ch-42 Transcriptsand Proteins

The detection of transcripts and proteins using northernand western blotting techniques provides useful information that is effectively averaged for all of the cell populations in the leaves used for RNA or protein extraction. Asa result of recent developments in in situ detection methods, it is now possible to visualize both transcripts and theencoded proteins in each cell of the leaf. Variations intranscripts and proteins between cells within the leaf tissueand the exact subcellular locations within the cell of bothproteins and their transcripts (Marrison and Leech, 1994),can now be determined. Figure 4 shows the subcellularlocation of the CHL Hand ch-42 transcripts and their proteins. Sections of Arabidopsis leaves were prepared andhybridized with antisense and sense (control) digoxigeninlabeled RNA probes derived from the CHL Hand ch-42cDNAs and from the wheat 25S rRNA gene.

Low magnification clearly shows the leaf cells in sectionsstained by the antisense CHL Hand ch-42 probes (Fig. 4, Aand B), revealing that both transcripts are present in everyleaf mesophyll cell. At higher magnification (Fig. 4, C andD) it is clear that both CHL H transcripts and ch-42 transcripts are present in the cytoplasm. However, in the highresolution photomicrograph, it is also possible to see veryclearly that both CHL Hand ch-42 transcripts are clusteredaround the external surface of the chloroplast outer envelope membrane. In several sections, the transcripts are soconcentrated that they appear as a stained ring aroundsectioned chloroplasts (Fig. 4C, arrowheads) or over theentire surface of unsectioned chloroplasts (Fig. 4D, arrowheads). This pattern has already been described previouslyfor transcripts of the ch-42 gene (Marrison and Leech, 1994).The transcript patterns for CHL Hand ch-42 are easilydistinguishable from chloroplast-encoded transcripts, forexample for the Rubisco large subunit (Marrison andLeech, 1994), and also from the transcript pattern for thenuclear-encoded cytosolic 25S rRNA gene (Fig. 4D, inset),where the stain is uniformly distributed throughout thecytoplasm with no concentration at the chloroplast surface.The sections in Figure 4 have been probed under identicalexperimental conditions, allowing us to determine the degree of similarity of location for transcripts of the ch-42 and

(v)

7.5-L R 1 3 5 7 3 5 7 9 11 13 15

(i)

4.4-

(ii)

2.4-

(iii)

1.4-

(iv)

Figure 3. Northern analysis of CHL H expression. A, Lane L, leaftissuei lane R, root tissue. Sizes of RNA markers are indicated on theleft. B, Transcript levels during a diurnal cycle. RNA was preparedfrom plants harvested at the indicated times during an 8-h dark/16-hlight cycle and probed with CHL H (i), ch-42 (ii), caM (iii), ferrochelatase eDNA (iv), and tubulin eDNA (v).

ch-42 gene (Koncz et al., 1990); surprisingly, in view of theproposed role for the CH-42 protein in forming Mg chelatase along with the CHL H protein (see "Discussion"), thetranscript for this gene shows no diurnal variation, remaining almost constant throughout the cycle. As a control, an,identical blot was probed with the cab6 gene, which en-codes a chlorophyll alb binding protein associated with PSI(Jensen et al., 1992). In general, cab genes are induced bylight and their mRNA expression patterns are under thecontrol of a circadian rhythm (Kellmann et al., 1993); in thiscase (Fig. 3) the transcript level rose to a maximum after 5h, indicating that the cab6 transcript exhibited a diurnaloscillation. Finally, as a further control, the blot was probedwith the Arabidopsis cDNA encoding chloroplast ferrochelatase (Smith et al., 1994). This is the other enzyme at thebranch poiht of tetrapyrrole biosynthesis (Fig. 1) that catalyzes the insertion of Fe2 + into protoporphyrin IX to yieldprotoheme. Figure 3 shows that transcripts for ferrochelatase display almost no variation throughout the darkl

Figure 4. (Figure appears on facing page.) In situ hybridization (A-F) and immunolocalization (G and H) to 7-J.Lm PEGtransverse sections, of 16-d A. thaliana leaf tissue. In each picture, several leaf mesophyll cells containing chloroplasts arevisualized. A and C, Sections hybridized to the antisense strand of a CHL H digoxigenin-Iabeled RNA probe and detectedusing alkaline phosphatase. Band 0, Sections hybridized to the antisense strand of a ch-42 digoxigenin-labeled RNA probeand detected using alkaline phosphatase. Inset in 0, Section hybridized to the antisense strand of a 25S rRNA digoxigeninlabeled RNA probe and detected using alkaline phosphatase. E, Section hybridized to the sense strand of a CHL Hdigoxigenin-labeled RNA probe. F, Section hybridized to the sense strand of a ch-42 digoxigenin-Iabeled RNA probe. G,Immunolocalization of CHL H using FITC for visualization. H, lmmunolocalization of CH-42 using FITC for visualization.Insets in H, Nonimmune control (upper) and starch localization (lower) within the chloroplasts (brown stain). In C and 0,arrowheads indicate the location of transcripts on the chloroplast envelope surface. In G and H, the striations reflect thepresence of starch grains. m, Mesophyll celli cp, chloroplast. Scale bar = 20 J.Lm.



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68 Cibson et al. Plant Physiol. Vol. 111, 1996

CHL H genes. After hybridization with the sense strand ofCHL H and ch-42 RNA probes (used as controls) and colordetection, no positive mRNA staining was observed andthe chloroplasts appeared pink in cross-section (Fig. 4, Eand F).

The subcellular location of chlorophyll biosynthetic en-zymes was also determined by probing thin leaf sections ofArabidopsis with antibodies raised against the OLI andCH-42 proteins (Fig. 4, G and H). The results show thatthese biosynthetic enzymes are confined to the chloroplastsin every mesophyll cell within the leaf; thus, every cell andevery chloroplast has the potential to synthesize chloro-phyll. The insets in Figure 4H show the tissue after incu-bation with 0.5% (w/v) BSA/PBS and FITC (immunolabel-ing control, upper inset) and after incubation with Lugol'siodine (lower inset), illustrating the starch grains withinthe chloroplasts (brown precipitate). The starch grains ac-count for the characteristic striated pattern seen in Arabi-dopsis chloroplasts of this age.

Import of the Nascent CHL H Preprotein into Chloroplasts

The use of an import assay followed by fractionation ofthe chloroplast provides more information on the organel-lar location of proteins. An in vitro study was carried outusing pea chloroplasts to demonstrate that the CHL Hprotein is imported into isolated chloroplasts. Using thefull-length cDNA clone as a template, transcripts wereproduced in vitro from the T7 promoter, and this in turnwas translated in a rabbit reticulocyte lysate in the presenceof [35S]Met to produce labeled CHL H precursor protein

1mMMg2' 5mM Mg2'

W S M S M H

B

-M

-M

Figure 5. A, Import into pea chloroplasts and in vitro processing ofthe CHL H protein. Lanes 1 and 4, In vitro-translated CHL H precur-sor; lane 2, washed chloroplasts analyzed after import of the CHL Hprotein; lane 3, protease-treated chloroplasts; lane 5, CHL H proteinafter incubation with partially purified stromal processing peptidase.B, Localization of the CHL H protein within chloroplasts. Lane 1,CHL H precursor; lane 2, washed chloroplasts; lane 3, protease-treated chloroplasts; lane 4, stromal fraction; lane 5, membranefraction. P, Precursor protein; M, mature protein.

~150kDa—

Figure 6. Immunoblot analysis of the location of the CHL H protein.W, Whole chloroplasts; S, stromal fractions; M, membrane fractions;H, E. co// extract containing expressed CHL H protein. The concen-tration of Mg2+ in the chloroplast lysis buffer is indicated.

(Fig. 5A, lanes 1 and 4). Figure 5A shows that upon incu-bation with chloroplasts the protein is processed to asmaller size (lane 2). This protein band can also be seenafter protease treatment of the chloroplasts (lane 3), indi-cating that the protein was protected from digestion and,therefore, was imported. Furthermore, the CHL H proteinwas cleaved in vitro by the partially purified stromal pro-cessing protease; lane 5 shows that approximately 50% ofthe labeled precursor was processed to a smaller protein. InFigure 5B the location of the protein within the chloroplastwas investigated; after the chloroplasts were incubatedwith labeled CHL H, the chloroplasts were washed, lysed,and separated into stroma and membrane fractions. A bandcorresponding to the mature-sized protein can be seen inthe stroma fraction (lane 4), whereas no such band could bedetected in the membrane fraction (lane 5).

Localization of CHL H by Immunoblotting

To confirm the location of CHL H within the chloroplast,Arabidopsis chloroplasts were prepared and fractionatedinto stroma and membrane fractions. Walker and Wein-stein (1991b) demonstrated that Mg chelatase activity re-quired soluble and membrane fractions of lysed pea chlo-roplasts. They later reported that Mg chelatase activity wasrendered entirely soluble if pea chloroplasts were lysed inbuffers containing low concentrations of Mg2+ (Walkerand Weinstein, 1995). Accordingly, the Arabidopsis chlo-roplasts were lysed in buffer containing either 1 or 5 mMMg2+. The immunoblot in Figure 6 shows that in 1 mMMg2+, CHL H is largely present in the stroma fraction,whereas in 5 mM Mg2+, a significant proportion of the CHLH protein is membrane associated.

DISCUSSION

We have cloned and sequenced a putative Mg chelatasesubunit, CHL H, from A. thaliana. Hudson et al. (1993) havecloned the equivalent gene from Antirrhinum majus andnoted its likely importance in chlorophyll synthesis, sincethey were able to correlate a mutation in the gene with achlorophyll-deficient phenotype. They speculated that itcould encode a Mg chelatase subunit due to homologieswith two bacterial genes involved in tetrapyrrole-metalchelation. One of these bacterial genes, bchH, has now beenpositively identified as a Mg chelatase subunit (Gibson etal., 1995). Furthermore, it seems likely that BchH binds theprotoporphyrin substrate during the Mg-chelation reaction(Gibson et al., 1995). The other bacterial protein, CobNfrom Pseudomonas denitrificans, is involved in the chelation




of cobalt into a porphyrin during coenzyme B,, biosynthesis and is also responsible for binding the porphyrin substrate (Debussche et al., 1992). The striking similarities between CHL H and BchH/CobN suggest that CHL H is a component of Mg chelatase in Arabidopsis and is presumably the protoporphyrin-binding component in this reaction. The extremely high level of identity between CHL H and OLI (85%), CHL H and Synechocystis ChlH (65%), and CHL H and BchH (40%) indicate that these Mg chelatase subunits are members of a highly conserved class of proteins. This is borne out by the fact that CH-42, another protein implicated in chlorophyll biosynthesis (Koncz et al., 1990; Armstrong et al., 1993), shows 50% identity to BchI, which is also a bacterial Mg chelatase subunit (Gibson et al., 1995). These proteins have evidently been highly conserved through evolution.

In Figure 3 it was shown that expression of CHL H is confined to photosynthetic tissues, since the transcript was detected in leaves but not in roots. This would be expected for a gene involved in the formation of the photosynthetic apparatus. In addition, the transcript undergoes a dramatic diurnal variation, rising almost to its maximum level by the end of the dark period, then increasing slightly at the onset of the light and declining steadily to a minimum by the end of the light period. This is somewhat similar to the behavior of the olive transcript from Antirrhinum, although the increase of olive mRNA in the dark was much more rapid. If the transcript levels reflect the amount of protein in the cell and CHL H is indeed the protoporphyrin-binding subunit, this may be a mechanism to regulate the amount of chlorophyll (see below). It is intriguing that ch-42 transcript levels remain almost constant. Koncz et al. (1990) demonstrated that ch-42 transcription was induced strongly in dark-adapted plants that were subsequently placed in the light. However, it seems that in plants that are established in a day/night cycle there is no response of this transcript to light, and we could reasonably assume that CH-42 levels remain constant.

Hudson et al. (1993) proposed that OLI could have a regulatory function. In view of the biochemical evidence that the bacterial homolog, BchH, binds protoporphyrin (Gibson et al., 1995), a mechanism is proposed below that involves such a binding reaction between CHL H and protoporphyrin in regulating flux down the tetrapyrrole biosynthetic pathways, primarily the chlorophyll branch. At the end of the dark period, the level of CHL H transcript has almost achieved its maximum, and the assumption made in this model is that the level of cognate protein also varies in the same manner as the transcript. The elevated level of CHL H is proposed to sequester protoporphyrin IX, thus depleting the pool of substrate for ferrochelatase, the level of which does not vary. The consequent drop in heme levels stimulates flux down the tetrapyrrole biosynthetic pathway, since heme has been shown to repress the activity of glutamyl-tRNA reductase (Pontoppidan and Kannan- gara, 1994). Therefore, at the end of the dark period the overall effect of increasing CHL H levels would be to stimulate the production of protoporphyrin, with a consequent effect on chlorophyll biosynthesis.

We propose that at the end of the light períod the oppo- site situation occurs, with lowered levels of CHL H and lowered 8-aminolevulinic acid and chlorophyll biosynthesis. Thus, CHL H could play an important role in matching the requirement for chlorophyll synthesis with fluctuations in light conditions. More work will have to be performed on the patterns of CHL H transcripts, but it is interesting to note that the activity of Mg chelatase does exhibit a diurnal variation in barley (Jensen et al., 1996b). Furthermore, there is some similarity between CHL H and cab6 in that both transcripts display significant oscillations of mRNA accumulation and both transcripts rise in the dark indepen- dently of light. This is often the case with transcripts that are under the influence of a circadian rhythm, such as the Arabidopsis PSII cab genes (Kay and Millar, 1992). PSI cab genes from Arabidopsis are less well characterized, but PSI cab genes are known to be under circadian control in tomato (Kellmann et al., 1993). This apparent synchroniza- tion of CHL H and cab transcript levels might reflect the fact that the LHC polypeptides are the major chlorophyll-binding proteins in the chloroplast.

In situ hybridization revealed that CHL H and ch-42 transcripts are localized over the surface of the chloroplasts. To our knowledge, this is the first visualization of transcripts encoding chloroplast proteins preferentially ac- cumulating at the chloroplast surface (Fig. 4, C and D). By analogy with the situation in mitochondria (Verner, 1993), this could indícate that the translation of CHL H and ch-42 transcripts is coupled to the import of the nascent polypeptides. It supports the view that within the mesophyll cell a co-translational mechanism may at least partly account for the import of polypeptides into the chloroplast. However, more work is needed to determine whether this is a general rule for other nuclear-encoded chloroplast proteins or whether this is a specific feature of the import of chlorophyll biosynthetic enzymes. The immunolocalization study (Fig. 4, G and H) clearly shows that both proteins are found inside the chloroplast. Previous immunofluorescence studies demonstrated that it is possible to identify Rubisco in chloroplasts and to correlate this with the distribution of transcripts encoding this protein (Marrison and Leech, 1994). The data in Figure 4 extend this technique to provide the first definitive identification of the localization of a chlorophyll biosynthetic enzyme within the chloroplast. This kind of approach opens the way to a description of transcript and protein levels for the chlorophyll biosynthetic pathway within developing plant tissue. The large number of starch grains within the chloroplasts (Fig. 4H, lower inset) give rise to the striated staining pattern that is seen in Figure 4, G and H. This pattern does not reflect the position of the thylakoid membranes within the chloroplasts.

The chloroplast import data in Figure 5 show that the CHL H preprotein is processed following import into the chloroplast. Since this protein is so large, it is not possible to make an accurate estimate of the size of the mature protein, although by comparison with Synechocystis and Rhodobacter sequences a possible cleavage site can be deduced (Fig. 2). Indeed, it is remarkable that the transcript



70 Gibson et al. Plant Physiol. Vol. 11 1, 1996

encoding a preprotein of this size w a s translated in vitro. This experiment, using a heterologous system, suggested that the CHL H protein is located in the stroma (Fig. 58). However, immunoblotting of fractionated Arabidopsis chloroplasts with OLI antibodies (Fig. 6) shows that, a t least in vitro, the distribution of CHL H between the stromal and membrane fractions depends on the concentration of Mg2+. It appears that the interaction between CHL H and the membrane is only peripheral, since CHL H is largely soluble at low Mg2+ concentration. It is possible that the Mg2+-dependent distribution of Mg chelatase activity observed by Walker and Weinstein (1995) is due to the CHL H protein becoming membrane associated i n high Mg2+ concentrations. In the light of our demonstration that association and dissociation of CHL H with the membrane appears to take place i n the 1 to 5 miv range of Mg2+ concentration, it is interesting t o note that the concentration of free Mg2' within the chloroplast varies between 1 and 6 mM (reviewed by Leegood et al., 1985). Koncz et al. (1990) reported that CH-42 is a stromal protein, and the equivalent protein from soybean has been shown t o reside in the stroma (Nakayama et al., 1995). Assuming that both CHL H and CH-42 are Mg chelatase subunits, they must presumably interact with one another. It will require further s tudy to determine the nature of the association between CHL H and CH-42.

ACKNOWLEDCMENTS

This work benefitted from the use of the SEQNET facility of the Biotechnology and Biological Sciences Research Council at Dares- bury. We would particularly like to thank Dr. Andrew Hudson for providing us with the olive cDNA and the OLI antibodies. We would also like to thank Dr. Alison Smith for providing us with the Arabidopsis ferrochelatase cDNA, Dr. Czaba Koncz for providing the ch-42 cDNA, Professor Chris Leaver for the wheat 25s rRNA gene, and Dr. Gamini Kannangara for help in production of the CH-42 antibodies.

Received October 2, 1995; accepted February 20, 1996. Copyright Clearance Center: 0032-08891961 111 10061 111. The accession number for the sequence reported in this article is

268495.

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a putative mg chelatase subunit from arabidopsis thaliana cvplant physiol. (1996) 11 1 : 61-71 a...

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