Cloning of the Gene Encoding a Protochlorophyllide

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In photosynthetic bacteria containing Bchl, the chlorin B-ring of chlorophyllide is further reduced to form bacteriochlorin. Thus, the reduction of Pchlide is the last.

Plant Cell Physiol. 39(2): 177-185 (1998) JSPP © 1998

Cloning of the Gene Encoding a Protochlorophyllide Reductase: the Physiological Significance of the Co-Existence of Light-Dependent and -Independent Protochlorophyllide Reduction Systems in the Cyanobacterium Plectonema boryanum Yuichi Fujita1, Hidenori Takagi and Toshiharu Hase Division of Enzymology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565 Japan the biosynthetic pathway of Chi and bacteriochlorophyll (Bchl). In this reaction, the porphyrin D-ring of Pchlide is converted to chlorin by stereo-specific reduction. Chlorophyllide (Chlide), the product of this reduction, is subsequently converted to Chi by phytylation in oxygenic phototrophs. In photosynthetic bacteria containing Bchl, the chlorin B-ring of chlorophyllide is further reduced to form bacteriochlorin. Thus, the reduction of Pchlide is the last step in the Mg-branch common to all phototrophic organisms. There are two different enzymes for Pchlide reduction; one is a light-dependent Pchlide reductase (LPOR; EC 1.3.1.33) and the other is a light-independent one (DPOR). LPOR is one of a unique group of enzymes requiring light for catalysis. In etioplasts of angiosperms, LPOR exists as a ternary complex with its substrates, Pchlide and NADPH. Upon exposure to light, a stereo-specific reduction occurs to form Chlide and NADP + (for reviews, see Griffiths 1991). LPOR consists of a single polypeptide with a molecular mass of about 35 kDa; the amino acid sequences deduced from cDNA sequences are now available for some plants and algae (Schulz et al. 1989, Darrah et al. 1990, Benli et al. 1991, Spano et al. 1992a, b, Forreiter and Apel 1993, Teakle and Griffiths 1993, Armstrong et al. 1995, Kuroda et al. 1995, Holtorf et al. 1995, Li and Timko 1996). In contrast, there is little information about DPOR, which is a determinant enzyme for greening ability in the dark. Recent molecular genetic analyses have revealed that three genes, chlL, chlN and chlB, encode putative subunits for DPOR (for reviews, see Fujita 1996, Reinbothe and Reinbothe 1996). These genes are present in plastid genomes in eukaryotic species.

Key words: Chlorophyll biosynthesis — Cyanobacterium — Plectonema boryanum — por — Protochlorophyllide reductase.

Chi synthesis in angiosperms stops at the step of Pchlide reduction in the dark, resulting in etiolation, because they have only LPOR as their sole Pchlide reductase. In contrast, most other oxygenic photosynthetic organisms carry both reductases and are able to synthesize Chi in both the light and the dark. Anoxygenic phototrophs such as purple non-sulfur bacteria have only DPOR. From the distribution of the genes for these two enzymes in photosynthetic organisms, the following evolutionary scenario has been written (Fujita 1996, Reinbothe et al. 1996a); in the early evolution of photosynthetic organisms, only DPOR was used for the biosynthesis of Bchl (or Chi) at first in

Protochlorophyllide (Pchlide) reduction is a step in Abbreviations: Bchl, bacteriochlorophyll; Chlide, chlorophyllide; DPOR, light-independent protochlorophyllide reductase; LPOR, light-dependent protochlorophyllide reductase; ORF, open reading frame; Pchlide, protochlorophyllide; 6xHis, six consecutive histidine residues. 1 Corresponding author. 177

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Cyanobacteria have two protochlorophyllide (Pchlide) reductases catalyzing the conversion of Pchlide to chlorophyllide, a key step in the biosynthetic pathway of chlorophylls (Chls); a light-dependent (LPOR) and a light-independent (DPOR) reductase. We found an open reading frame (ORF322) in a 2,131-bp EcoRl fragment from the genomic DNA of the cyanobacterium Plectonema boryanum. Because the deduced amino acid sequence showed a high similarity to those of various plant LPORs and the LPOR activity was detected in the soluble fraction of Escherichia coli cells over-expressing the ORF322 protein, ORF322 was defined as the por gene encoding LPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolated by targeted mutagenesiss to investigate the physiological importance of LPOR. YFP12 grew as well as wild type under low light conditions (10-25 fiE m~2 s"1). However, its growth was significantly retarded as a result of a significant decrease in its Chi content under higher light conditions (85-130 ^Em" 2 s"'). Furthermore, YFP12 stopped growing and suffered from photobleaching under the highest light intensity (170 ^lE m~2 s"1). In contrast, a chlL-disrupted (DPOR-less) mutant YFC2 grew as well as wild type irrespective of light intensity. From these phenotypic characteristics, we concluded that, although both LPOR and DPOR contribute to Chi synthesis in the cells growing in the light, the extent of the contribution by LPOR increases with increasing light intensity; without it, the cells are unable to grow under light intensities of more than 130

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Cyanobacterial por gene for chlorophyll synthesis

Cyanobacteria are the most primitive organisms among oxygenic phototrophs to carry both reductases (Fujita et al. 1992, Suzuki and Bauer 1995), and amenable to molecular genetic techniques (Thiel 1994). They provide a good system for addressing the above questions. In the previous work, we have cloned the three genes for DPOR from the cyanobacterium Plectonema boryanum and isolated a series of mutants lacking each gene (Fujita et al. 1992, 1993, 1996). In this study, we cloned the cyanobacterial por gene encoding LPOR and compared the phenotype of a por-disrupted mutant (LPOR-less) in comparison with a c/i/L-disrupted (DPOR-less) mutant. We demonstrate here that contribution of LPOR to Chi synthesis increases in accord with an increase in the light intensity and that LPOR is indispensable for growth under high light intensities over

Materials and Methods Cultivation of the cyanobacterium—Plectonema boryanum IAM-M101 strain dg5 was cultivated as described previously (Fujita et al. 1991, 1996). To determine the growth curve, cultures in flat bottles were bubbled with 2% (vol/vol) CO2 in air under continuous illumination provided by fluorescent lamps (ca. 10170^Em~ 2 s~', Homolux FL40S-PG; National, Osaka). PCR and cloning of the 2.1-kb EcoRI fragment—The nucleotide sequences of a pair of degenerate primers (22mer) with BamHl sites (underlined) at their 5'-ends were as follows: 5'GTGGATCCAA(T/C)CA(T/C)(T/C)TIGGICA-3', TTGGATCCAT(A/G)CAICCIGG-(A/G)TA-3'. These sequences were derived from the two conserved regions of known plant LPORs (NHLGH and YPGCI). PCR was carried out with the following thermal cycle (Compton 1990); first three cycles (95°C; 2 min, 37°C; 1 min, and 72°C; 2 min) including a ramp time (5.0°C s~') followed by the standard 27 cycles (95°C; 1 min, 55°C; 1 min, and 72°C; 2 min). The amplified DNA fragment (354 bp), which had been digested with BamHl, was subcloned to the BamHl site of pBlue-

script II SK+ (Stratagene, La Jolla, CA, U.S.A.) to construct pNPR58. The inserted fragment excised from pNPR58 was labeled with digoxigenin-11-dUTP (DNA Labeling and Detection Kit Non-radioactive; Boehringer Mannheim Yamanouchi, Tokyo) to use as a probe for the screening of a genomic library of P. boryanum constructed with lambda DASH II (Stratagene). Genomic DNA prepared from cells of P. boryanum was partially digested with Sau3Al and ligated with the arms of lambda DASH II, followed by in vitro packaging with GIGA-PACK GOLD (Stratagene). Plaque hybridization was carried out using the digoxigenin-labeled DNA fragment as the probe. Five positive plaques were isolated. The 2.1-kb EcoKl fragment, which was commonly contained in all clones, was subcloned into theiTcoRI site of pBluescript II SK+ to form pNPR510. The nucleotide sequence of the 2.1-kb fragment was determined by a Dye-terminator method with a DNA sequencer model 373 A (Perkin Elmer Applied Biosystems, Foster City, CA, U.S.A.). The sequences of each reaction were assembled and analyzed by GENETYX ver 8.0 (Software DC, Tokyo). Expression of LPOR in E. coli and preparation of the antiserum—The POR-6xHis fusion protein was expressed in E. coli JM105 by the use of an expression vector pQE-60 (QIAGEN Inc., Chatsworth, CA, U.S.A.). A pair of primers (5'-GTTGTTCCATGGCACAGGATCAAAAACCC-3', 5-TTAGGATCCAGCGAGTCCAACGAGCTT-3') was used to amplify a DNA fragment (972 bp) that includes the entire coding region of por (322 aa) with Ncol and BamHl sites (underlined). The amplified 972-bp fragment was subcloned into the Ncol and BamHl sites of pQE-60 to generate pPOR I, which encodes a POR fusion protein with the 6xHis tag via a tetrapeptide linker (GSRS) at the C-terminus of the LPOR protein. E. coli JM105 carrying pPOR I was cultivated as described in Fujita et al. (1996). The E. coli cells were harvested by centrifugation and disrupted by sonication (Sonifier 350; Branson Sonic Power Co., Danbury, CT, U.S.A.) in 50 mM sodium phosphate (pH 6.8) and 300 mM NaCl. The supernatant was recovered by centrifugation at 20,000 xg for 10 min. The POR-6xHis protein was purified by affinity chromatography and gel filtration to give a preparation that yielded a single band in SDS-PAGE. Polyclonal antibodies against the POR-6xHis protein were raised as described previously (Fujita et al. 1996). Enzymatic assay of LPOR—The substrate Pchlide for the LPOR assay was prepared from the culture medium of darkgrown YFC2. This mutant, whose chlL gene had been inactivated by gene targeting, does not synthesize Chi and anomalously accumulates Pchlide in the dark (Fujita et al. 1992, 1996). This accumulated Pchlide is excreted into the culture medium in large amounts. YFC2 was cultivated as described previously (Fujita et al. 1992) for two weeks. The culture medium was recovered as the supernatant by centrifugation at 10,000 x g for 20 min. The yellow pigment was extracted in the equal volume of diethylether. Water contaminating in the ether fraction was removed as ice by short storage at -20°C. Ether was evaporated to dryness under an argon stream, and the dried pigment was dissolved in acetone. The concentration of Pchlide was determined by absorbance at 625 nm (Brouers and Michel-Wolwertz 1983). The assay mixture contained 3 fiM of Pchlide, 20 /iM NADPH (or NADH), 0.05% Triton X-100, and an appropriate volume of E. coli lysate in a buffer of 50 mM HEPES-NaOH (pH 7.5) and 2 mM MgC^. The reaction was carried out at 30°C illuminated with a tungsten lamp (ca. 3,200 lux). After incubation, the reaction was terminated by the addition of a four-fold volume of acetone. Fluorescence emission spectra of the supernatant were re-

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anoxygenic photosynthetic bacteria. LPOR arose in an ancestor of cyanobacteria that began to perform oxygenic photosynthesis. From that point, both enzymes have co-existed during the evolution from cyanobacteria to gymnosperms. In the early stage of evolution of eukaryotic phototrophs, the por gene encoding LPOR was transferred to the nuclear genome, while the three genes for DPOR were retained in the plastid DNA. On the way from gymnosperms to angiosperms, DPOR was lost for unknown reasons, resulting in the loss of greening ability in the dark. This scenario suggests that each enzyme has a distinct physiological role in phototrophic organisms under various growth conditions. Investigating the physiological significance of the co-existence of these two reductases in extant phototrophs might give us important clues to understanding the reasons why LPOR appeared and DPOR has been lost. However, there are only some fragmentary observations suggestive of physiological significance (Ford et al. 1981).

Cyanobacterial por gene for chlorophyll synthesis

Results PCR and cloning of the por gene—Using a pair of degenerate oligonucleotides corresponding to the highly conserved amino acid sequences of plant LPORs, a 354-bp DNA fragment was amplified from the genomic DNA of P. boryanum. This fragment was used as a probe to screen a genomic library of P. boryanum. Five lambda clones were isolated. A 2.1-kb EcoRl fragment, which was commonly contained in all clones, was subcloned into pBluescript II SK+. The complete nucleotide sequence of the fragment (2,131 bp) was determined (Fig. 1). There was an open reading frame (ORF322) encoding a polypeptide of 322 amino acid residues. The amino acid sequence of ORF322 showed the highest similarity (73.6%) to that of YFP12

I

I

URF6 0.5 kb

por (ORF322) -i

Fig. 1 A physical map of the 2.1-kb £coRI fragment of pNPR510 including the por (ORF322) gene. ORFs found in this fragment are shown by a thick arrow (shaded) and a box. A solid triangle indicates the site at which the neo gene cartridge was introduced in YFP12. The sequence data will appear in the DDBJ/ EMBL/GenBank Nucleotide Sequence Database under the accession number AB005556.

LPOR from Synechocystis sp. strain PCC 6803 and considerable similarity (53-56%) to those of known LPORs from plants and green algae (Fig. 2). While eukaryotic LPORs encoded in nuclear genomes have transit peptides in their N-termini to target them to plastids, both ORF322 and LPOR from Synechocystis sp. strain PCC 6803 lack such a long N-terminal extension. Enzymatic assay of LPOR expressed in E. coli—To examine whether the protein encoded by ORF322 carries the enzymatic activity, the ORF322 protein was over-expressed in E. coli cells as a fusion protein with a 6xHis tag, and an extract of the cells was assayed for light-dependent Pchlide reduction activity. The assay mixture was continuously illuminated by a tungsten lamp. The conversion of Pchlide to Chlide was detected by spectrofluorometric measurement (Fig. 3, trace b). This conversion required light (trace c) and NADPH (trace d) but not NADH (trace e). This result indicated that ORF322 certainly encodes LPOR with the enzymatic activity. Thus, we designated ORF322 as the por gene according to the naming in Synechocystis sp. strain PCC 6803 (Suzuki and Bauer 1995). Immunochemical detection and subcellular localization of LPOR in cyanobacterial cells—The recombinant LPOR was purified from the extract of E. coli to raise an antibody against LPOR. Thus prepared antiserum was used to detect the LPOR protein in the cyanobacterial cells by Western blot analysis (Fig. 4). A major single band was detected in the total extract of wild type cells of P. boryanum. This band showed an apparent molecular mass of about 34 kDa, which was in good agreement with the theoretical value of LPOR (35,373 Da). This 34-kDa band was not detected in the total extract of the mutant YFP12, in which the por gene had been inactivated (Fig. 4, lane 3; see below). This results confirmed that the 34-kDa band is the protein encoded by the por gene. It is noteworthy that this 34-kDa protein was detected in the total extract of a DPOR-less mutant YFC2 as well as in wild type (Fig. 4, lane 2). To examine the subcellular localization of LPOR, fractionation of wild-type cells was carried out (Fig. 5). LPOR was detected mainly in the total membrane fraction and very slightly in the soluble fraction (Fig. 5B). In the two membrane fractions, LPOR was detected in both thylakoid and plasma membranes to a similar extent. A minor signal with a smaller size in the membranes might be a degradation product of the LPOR protein during cell fractionation, because no such signal was found in the total extract. The 33-kDa and CmpA (Omata et al. 1990) proteins were immunochemically detected as the marker proteins for thylakoid and plasma membranes, respectively (Fig. 5C). This control experiment showed that the membrane fractionation was almost completely successful with little cross-contamination. These combined results indicated that LPOR is localized in both membranes, suggesting that both mem-

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corded with a spectrofluorometer (model RF1500; Shimadzu, Kyoto). Preparation of membrane fractions from cyanobacterial cells and Western blot analysis—Soluble and insoluble membrane fractions of cyanobacterial cells were prepared as described in Fujita et al. (1996). Plasma and thylakoid membranes were prepared according to the protocol by Murata and Omata (1988). Western blotting was carried out as previously described (Fujita et al. 1996). Isolation and characterization of a por-disrupted mutants YFPJ2—To disrupt the por gene, a kanamycin cartridge excised from pMC19 (Fujita et al. 1992) by BamHl digestion was inserted into the unique flg/II site of pNPR510 to form pYFP12. The plasmid pYFP12 linearized by Kpnl was introduced into wild type cells by electroporation as describe previously (Fujita et al. 1992). Growth of wild type and mutants were estimated by a photoelectric colorimeter with a KS-66 filter (transmission spectral range: 640-700 nm; Klett meter, Manostat, New York, NY, U.S.A.). Chi contents were determined as described previously (Fujita et al. 1992). To detect Pchlide in the culture-media, four volumes of acetone was added to the culture media prepared by centrifugation, and fluorescence emission spectra were recorded as described above.

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PLECT SYNEC CHLAM PINUS PISUM CUCUM ARABI ARABI TRITI HORDE HORDE

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PLECT SYNEC CHLAM PINUS PISUM CUCUM ARABI ARABI TRITI HORDE HORDE

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1: MALTMSA1WSARAQVSSDAQAAPAVAVSGRTSSRVMPAPA1>ARSSVARTI>LVVCAATATAI>SI>SI^^ l:KTTLLQTHICaVAFAI0--KlDGHSASA-ia3SAFTj3VSLVOICaaeEFSF^ l:MALQTASMLPASFSIPKEGKIGASUOM-TLFGVSSI£DSlja3DFrS--SALRCK-E--LR0KVGAVRAErAAPATPAVH l:MAlflAASLVSPAl^IPKHa«SSVCljaK-SU^I-SFSDHIJ 130//E m~2 s~'), Pchlide reduction depends exclusively on LPOR, (2) under intermediate light conditions (25-130 /iEm~ 2 s~'), a major part of Pchlide reduction depends on LPOR, (3) in the dark, Pchlide is exclusively reduced by

DPOR because LPOR requires light for catalysis, and (4) under low light conditions (10-25 /iE m~2 s~'), LPOR and DPOR operate cooperatively to compensate for their respective abilities. Since the growth rate of wild type cells increases with the light intensity and since the Chi content of wild-type cells is essentially constant (ca. 0.02 //g ml" 1 Klett"1) irrespective of light intensity (Fig.7B), cells growing under a higher light intensity must synthesize Chi more rapidly than those growing under a lower light intensity (see Figs. 6B, 9B). In addition, newly synthesized Chi might be used in part to compensate loss of Chi due to photooxidative degradation. Thus, the efficient reduction of Pchlide is necessary to supply this great demand for Chi; LPOR assures that cells can supply an appropriate amount of Chlide to synthesize Chi in accordance with light conditions. DPOR activity seems to be too low to fill the Chl-demand in high light (Fig. 7B). YFP12 cells grow under light intensity up to 13O^Em~ 2 s~' by excreting accumulated Pchlide into the culture medium in order to avoid photooxidative damage (Fig. 8). A marked increase in carotenoid content (about 3.5 fold on a Chi base) was observed in YFP12 grown at 85/iEm~ 2 s"' (data not shown), suggesting that these cells actually suffered from photooxidative stress (Hischberg and Chamovitz 1994). Under a light intensity of more than 130^Em~ 2 s~' YFP12 cells could not survive because of unavoidable photooxidative bleaching. It is suggested that cyanobacterial LPOR plays a role as a scavenger for Pchlide, as is the case for POR-A in higher plants. The three putative subunits of DPOR show significant similarities to three subunits of nitrogenase (Burke et al. 1993b, Fujita 1996), which is very sensitive to molecular oxygen. If this property is conserved, DPOR activity might be inhibited to some extent under light conditions in which oxygen is evolved from PSII. This might explain why an ancestor of cyanobacteria invented an oxygen-resistant Pchlide reductase, LPOR. Alternatively, one may assume that the expression level of genes for DPOR is suppressed or not enhanced in response to an increase in light intensity. To examine these possibilities further, investigation is needed in terms of gene expression, enzymatic activity, and turn-over rate of Chi under various light conditions. The authors thank Dr. Tatsuo Omata for the gift of antiserum against the CmpA protein of Synechococcus sp. strain PCC 7942 and Drs. Tomohiro Matsumura and Hiroaki Okuhara for constructing and sharing of the genomic library of P. boryanum. This work was supported by Grants-in-Aid for Scientific Research to Y.F. (No. 08836006) from the Ministry of Education, Science, and Culture of Japan.

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content (Fig. 5B), it is concluded that the majority of the total LPOR is distributed in thylakoid membranes in the cells. On the other hand, the activity of DPOR was detected in the plasma membrane of another cyanobacterium, Synechococcus sp. strain PCC 6301 (Peschek et al. 1989). It is tempting to speculate that the two reductases differ in subcellular localization in order to fulfill distinct roles. However, it is necessary to carry out a systematic analysis on localization of the activities and the polypeptides. Significance of co-existence of LPOR and DPOR in cyanobacteria—LPOR is the sole Pchlide reductase in angiosperms. It was recently found that some angiosperms, such as barley and A.thaliana, have LPOR isozymes, POR-A and POR-B (Holtorf et al. 1995, Armstrong et al. 1995). POR-A accumulates in large amounts in etioplasts of dark-grown seedlings and disappears rapidly in the early phase of the greening process upon illumination. On the other hand, POR-B persists throughout the greening process, even though its expression level is much lower than that of POR-A in etioplasts. This differential expression pattern suggested that the LPOR-isozymes play distinct physiological roles during chloroplast development. PORA might function as not only the enzyme for Pchlide reduction but also as a scavenger of hazardous pigment Pchlide, which causes photooxidation upon illumination. POR-B is likely to be the "true" Pchlide reductase driving Chi synthesis in chloroplasts (Reinbothe and Reinbothe 1996, Reinbothe et al. 1996b).

Cyanobacterial por gene for chlorophyll synthesis

References

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(Received September 26, 1997; Accepted November 21, 1997)

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