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Phycocyanobilin Is the Natural Precursor of the Phytochrome Chromophore in the Green Alga Mesotaenium caldariorum

1997; Elsevier BV; Volume: 272; Issue: 41 Linguagem: Inglês

10.1074/jbc.272.41.25700

1083-351X

Shu‐Hsing Wu, Michael T. McDowell, J. Clark Lagarias,

Photosynthetic Processes and Mechanisms

Compared with phytochromes isolated from etiolated higher plant tissues and a number of lower plant species, the absorption spectrum of phytochrome isolated from the unicellular green alga Mesotaenium caldariorum is blue-shifted (Kidd, D. G., and Lagarias, J. C. (1990) J. Biol. Chem.265, 7029–7035). The present studies were undertaken to determine whether this blue shift is due to a chromophore other than phytochromobilin or reflects a different protein environment for the phytochromobilin prosthetic group. Using reversed phase high performance liquid chromatography, we show that soluble protein extracts prepared from algal chloroplasts contain the enzyme activities for ferredoxin-dependent conversions of biliverdin IXα to (3Z)-phytochromobilin and (3Z)-phytochromobilin to (3Z)-phycocyanobilin.In vitro assembly of recombinant algal apophytochrome was undertaken with (3E)-phytochromobilin and (3E)-phycocyanobilin. The difference spectrum of the (3E)-phycocyanobilin adduct was indistinguishable from that of phytochrome isolated from dark-adapted algal cells, while the (3E)-phytochromobilin adduct displayed red-shifted absorption maxima relative to purified algal phytochrome. These studies indicate that phycocyanobilin is the immediate precursor of the green algal phytochrome chromophore and that phytochromobilin is an intermediate in its biosynthesis in Mesotaenium. Compared with phytochromes isolated from etiolated higher plant tissues and a number of lower plant species, the absorption spectrum of phytochrome isolated from the unicellular green alga Mesotaenium caldariorum is blue-shifted (Kidd, D. G., and Lagarias, J. C. (1990) J. Biol. Chem.265, 7029–7035). The present studies were undertaken to determine whether this blue shift is due to a chromophore other than phytochromobilin or reflects a different protein environment for the phytochromobilin prosthetic group. Using reversed phase high performance liquid chromatography, we show that soluble protein extracts prepared from algal chloroplasts contain the enzyme activities for ferredoxin-dependent conversions of biliverdin IXα to (3Z)-phytochromobilin and (3Z)-phytochromobilin to (3Z)-phycocyanobilin.In vitro assembly of recombinant algal apophytochrome was undertaken with (3E)-phytochromobilin and (3E)-phycocyanobilin. The difference spectrum of the (3E)-phycocyanobilin adduct was indistinguishable from that of phytochrome isolated from dark-adapted algal cells, while the (3E)-phytochromobilin adduct displayed red-shifted absorption maxima relative to purified algal phytochrome. These studies indicate that phycocyanobilin is the immediate precursor of the green algal phytochrome chromophore and that phytochromobilin is an intermediate in its biosynthesis in Mesotaenium. Other than acting as an energy source for photosynthesis, light is an important signal for many growth and developmental processes in plants. Plants thus possess multiple photoreceptors, which enable them to perceive and adapt to light intensity, direction, and quality in their environment (1$$Google Scholar). Phytochrome, the most well characterized of these light receptors, mediates a diverse array of photomorphogenetic responses in various organisms from green algae to higher plants (2Quail P.H. Boylan M.T. Parks B.M. Short T.W. Xu Y. Wagner D. Science. 1995; 268: 675-680Crossref PubMed Scopus (662) Google Scholar, 3Furuya M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 617-645Crossref Scopus (293) Google Scholar, 4Wada M. Kadota A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 169-191Crossref Google Scholar). Molecular analyses have established that most plants contain multiple phytochrome genes for which expression appears to be responsible for this diversity of phytochrome-mediated phenomena (3Furuya M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 617-645Crossref Scopus (293) Google Scholar,5Sharrock R.A. Quail P.H. Genes Dev. 1989; 3: 1745-1757Crossref PubMed Scopus (700) Google Scholar, 6Clack T. Mathews S. Sharrock R.A. Plant Mol. Biol. 1994; 25: 413-427Crossref PubMed Scopus (566) Google Scholar, 7Pratt L.H. Photochem. Photobiol. 1995; 61: 10-21Crossref Scopus (72) Google Scholar). The ability of phytochrome to sense the light environment is due to a covalently attached linear tetrapyrrole chromophore, which allows the photoreceptor to photointerconvert between red light-absorbing Pr 1The abbreviations used are: Pr, red light-absorbing form; Pfr, far red light-absorbing form; PΦB, phytochromobilin; PCB, phycocyanobilin; BV, biliverdin IXα; HPLC, high performance liquid chromatography; TES,N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. form and far red light-absorbing Pfr form. Light-grown plants deficient in phytochrome chromophore biosynthesis exhibit aberrant growth and development (reviewed in Refs. 8Koorneef M. Kendrick R.E. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 601-628Crossref Google Scholar and 9Terry M.J. Plant Cell Environ. 1997; 20: 740-745Crossref Scopus (81) Google Scholar). It is well established that (3E)-phytochromobilin (PΦB) is the immediate precursor of the chromophore of phytochrome A from higher plants, and it has implicitly been assumed that other phytochromes also contain a PΦB chromophore (reviewed in Ref. 10Terry M.J. Maines M.D. Lagarias J.C. J. Biol. Chem. 1993; 268: 26099-26106Abstract Full Text PDF PubMed Google Scholar). Two types of observations suggest that some phytochromes could contain an alternative bilin chromophore. First, the ability to reconstitute photoactive phytochromes with phycobilin pigments supports this hypothesis. One of these is the phycobiliprotein chromophore precursor, (3E)-phycocyanobilin (PCB), assembly of which with apophytochrome A yields a photoactive species with blue-shifted absorption maxima for both Pr and Pfr forms (11Elich T.D. Lagarias J.C. J. Biol. Chem. 1989; 264: 12902-12908Abstract Full Text PDF PubMed Google Scholar). Second, phytochromes with blue-shifted absorption spectra have been reported for the fern Adiantum capillus-veneris (12Oyama H. Yamamoto K.T. Wada M. Plant Cell Physiol. 1990; 31: 1229-1238Google Scholar), the mosses Ceratodon purpureus (13Lamparter T. Podlowski S. Mittmann F. Schneiderpoetsch H. Hartmann E. Hughes J. J. Plant Physiol. 1995; 147: 426-434Crossref Scopus (36) Google Scholar) and Atrichum undulatum (14Lindemann P. Braslavsky S.E. Hartmann E. Schaffner K. Planta. 1989; 178: 436-442Crossref PubMed Scopus (14) Google Scholar), light-grown higher plants (15Cordonnier M.M. Greppin H. Pratt L.H. Biochemistry. 1986; 25: 7657-7666Crossref Scopus (40) Google Scholar, 16Tohuhisa J.G. Quail P.H. Planta. 1987; 172: 371-377Crossref PubMed Scopus (30) Google Scholar), and the green alga Mesotaenium caldariorum (17Taylor A.O. Bonner B.A. Plant Physiol. 1967; 42: 762-766Crossref PubMed Google Scholar, 18Kidd D.G. Lagarias J.C. J. Biol. Chem. 1990; 265: 7029-7035Abstract Full Text PDF PubMed Google Scholar). With the notable exception of green algal phytochrome, these blue shifts were restricted to the Pfr forms, a result that can readily be ascribed to limited proteolysis or denaturation of phytochrome during its purification (19Vierstra R.D. Quail P.H. Planta. 1982; 156: 158-165Crossref PubMed Scopus (59) Google Scholar). The observations that full-length algal phytochrome displays blue-shifted Pr and Pfr absorption maxima (18Kidd D.G. Lagarias J.C. J. Biol. Chem. 1990; 265: 7029-7035Abstract Full Text PDF PubMed Google Scholar), and that the action spectra for chloroplast reorientation in Mesotaenium is similarly blue-shifted (20.Machemer, C. (1964) Reagents Affecting the Rotation of the Chloroplasts in Mesotaenium. M.Sc. thesis, Harvard University, Cambridge, MA.Google Scholar), suggest that this atypical phytochrome spectrum is an inherent property of the native algal photoreceptor. The present study was undertaken to establish the molecular basis for the blue-shifted absorption maxima of the green algal phytochrome. In one avenue of investigation, we examined algal plastid protein extracts for the presence of enzymatic activities capable of converting biliverdin IXα (BV), a known intermediate in the biosynthesis of the phytochrome chromophore in higher plants, to PΦB and other bilin products. These studies take advantage of an improved HPLC assay system for PΦB synthase (21Wu S.-H. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8989-8994Crossref PubMed Scopus (27) Google Scholar), the plastid-localized enzyme responsible for the reductive conversion of BV to PΦB in higher plants (22Terry M.J. Lagarias J.C. J. Biol. Chem. 1991; 266: 22215-22221Abstract Full Text PDF PubMed Google Scholar, 23Terry M.J. McDowell M.T. Lagarias J.C. J. Biol. Chem. 1995; 270: 11111-11118Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). With this assay, which can readily distinguish between (3Z)- and (3E)-isomers of PΦB and PCB, the production of PCB by a non-phycobiliprotein containing green algal species has been documented for the first time. M. caldariorum strain 41 was obtained from the University of Texas at Austin Culture Collection of Algae. Liquid suspension cultures were grown at 16–18 °C in Sager and Granick medium II (24Sager R. Granick S. Ann. N. Y. Acad. Sci. 1953; 56: 831-838Crossref PubMed Scopus (331) Google Scholar) supplemented with 2% yeast extract as recommended (17Taylor A.O. Bonner B.A. Plant Physiol. 1967; 42: 762-766Crossref PubMed Google Scholar) under cool white fluorescent continuous light (50–100 μmol m−2 s−1). Cyanidium caldarium strain CPD was a generous gift from Dr. S. I. Beale (Brown University, Providence, RI) and was grown at 37 °C in pH 2.0 glucose-based heterotrophic medium (25Beale S.I. Chen N.C. Plant Physiol. 1983; 71: 263-268Crossref PubMed Google Scholar). Mesotaenium suspension cultures (300 ml/flask) were allowed to grow to late log phase in continuous light. For isolation of intact chloroplasts, algal cultures were placed in darkness for 7 days to consume the starch storage granules. Dark-adapted cells were harvested by centrifugation at 160 ×g for 1 min and washed with deionized H2O. Cell pellets were resuspended in 50 ml of protoplast buffer (0.3m potassium succinate, pH 5.6, 0.2 m sorbitol, 2 mm CaCl2) containing 0.5% Onozuka RS cellulase (Karlan) and incubated with shaking (60 rpm) under green safe light for 4–6 h at room temperature until >90% of the cells had become protoplasts. Protoplasts were harvested by centrifugation in a swinging-bucket rotor at 425 × g for 3 min, washed with 50 ml of protoplast buffer, and re-centrifuged. Protoplast pellets were resuspended in 11 ml of cold protoplast lysis buffer (25 mm TES-KOH, pH 7.3, 2.4 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1% polyvinylpyrrolidone 40, 2 mg/ml bovine serum albumin, 1 μg/ml leupeptin) by pipetting and inverting for 2 min, whereupon 1 ml of 4 m sorbitol was immediately added for osmotic stabilization of chloroplasts. Intact chloroplasts were collected by centrifugation in a swinging-bucket rotor at 760 × gfor 3 min. Chloroplasts were then osmotically lysed by adding 25 ml of chloroplast lysis buffer (100 mm potassium phosphate, pH 7.3, 1 mm EDTA, 1 mm Na2EDTA, 1 mm MgCl2, 1.5 mg/ml sodium ascorbate, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride) and incubated on ice for 10–20 min. The supernatant obtained, after centrifugation at 100,000 × g for 1 h, represented the chloroplast soluble protein fraction. This solution was further concentrated by using an Amicon ultrafiltration cell with a YM 10 membrane. Phosphate buffer was chosen for chloroplast lysis because of its necessity for solubilizing bilin reductase activity in higher plant. 2M. T. McDowell and J. C. Lagarias, unpublished data. For a standard 1-ml assay, chloroplast soluble protein extracts (730 μl) containing roughly 1 mg of total protein as determined by Bradford assay (26Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) were used as enzyme source. Reaction mixtures consisted of a NADPH-regenerating system (6.5 mm glucose 6-phosphate, 0.82 mmNADP+, and 1.1 units/ml glucose-6-phosphate dehydrogenase), 10 μm bovine serum albumin, 4.6 μm spinach ferredoxin, and 0.025 units/ml ferredoxin-NADP+ reductase (all final concentrations). Assays were initiated by addition of BV (or other bilin) in Me2SO to give a final bilin concentration of 5–10 μm and Me2SO concentration of 1% (v/v). Reaction mixtures were incubated at 28 °C under green safe light for the appropriate time indicated and stopped by placing on ice. Crude bilins were isolated with a C18 Sep-Pak Light cartridge (Waters-Millipore Corp., Milford, MA) as described (23Terry M.J. McDowell M.T. Lagarias J.C. J. Biol. Chem. 1995; 270: 11111-11118Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Crude bilins were analyzed by C18 reversed phase HPLC using a Varian 5000 liquid chromatograph equipped with a 4.6 mm × 250-mm Phenomenex Ultracarb 5μ ODS20 and a 4.6 mm × 30-mm guard column of the same material. The mobile phase consisted of a 50:50 mixture of acetone and 20 mm formic acid in water. Column eluates were monitored at 380 nm with Varian UV100 absorbance detector. Two different recombinant phytochrome apoproteins were used for in vitroassembly experiments. Oat apophytochrome A was prepared from Saccharomyces cerevisiae strain 29A (MATα leu2-3 leu2-112 his3-1 ade1-101 trp1-289) expressing pMphyA3 (27Wahleithner J.A. Li L. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10387-10391Crossref PubMed Scopus (72) Google Scholar). For Mesotaenium apophytochrome, an XhoI-NotI fragment containing MCphy1b cDNA was subcloned into an engineered pMAC105 vector 3S. H. Wu and J. C. Lagarias, unpublished data. digested with SalI and NotI to generate pMMCphy1b. This plasmid was then used to transform S. cerevisiae strain 29A, and Mesotaenium apophytochrome was expressed as described (27Wahleithner J.A. Li L. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10387-10391Crossref PubMed Scopus (72) Google Scholar). For in vitro spectrophotometric assays, ammonium sulfate was added to the yeast soluble protein fraction to produce a concentration of 0.23 g/ml. The mixture was incubated on ice for at least 1 h, and precipitates were collected by centrifugation for 30 min at 17,000 × g at 4 °C. Protein pellets were dissolved in TEGE buffer (25 mm Tris-HCl, pH 8.0, 25% ethylene glycol, 1 mm EDTA) containing 1 mm dithiothreitol and 1 mm phenylmethylsulfonyl fluoride. Holophytochrome concentrations were determined with a HP8450A UV/visible spectrophotometer using the absorbance difference assay described previously (28Li L. Lagarias J.C. J. Biol. Chem. 1992; 267: 19204-19210Abstract Full Text PDF PubMed Google Scholar). For in vitro assembly, (3E)-PCB, (3E)-PΦB, or bilin pigment as indicated was added to reaction mixture to produce a final concentration of 4 μm. Assay mixtures were incubated at room temperature under green safe light for 30 min prior to spectrophotometric measurements. BV was prepared from bilirubin and purified as described (29McDonagh A.F. Palma L.A. Biochem. J. 1980; 189: 193-208Crossref PubMed Scopus (90) Google Scholar). Crude (3E)-PCB and (3E)-PΦB were obtained by methanolysis of Spirulina platensis (10Terry M.J. Maines M.D. Lagarias J.C. J. Biol. Chem. 1993; 268: 26099-26106Abstract Full Text PDF PubMed Google Scholar) and Porphyridium cruentum (30Cornejo J. Beale S.I. Terry M.J. Lagarias J.C. J. Biol. Chem. 1992; 267: 14790-14798Abstract Full Text PDF PubMed Google Scholar), respectively. Crude pigments were further purified by C-18 reversed phase HPLC as described above, except that a 10 mm × 250-mm semipreparative column with a 10 mm × 50-mm guard column was used. (3Z)-PCB was obtained by co-incubation of BV with Cyanidium extracts (31Rhie G. Beale S.I. J. Biol. Chem. 1992; 267: 16088-16093Abstract Full Text PDF PubMed Google Scholar) and purified by HPLC as described above. (3Z)-PΦB was similarly biosynthesized from BV using a partially purified oat etioplast fraction containing PΦB synthase. 4M. T. McDowell and J. C. Lagarias, manuscript in preparation. HPLC-purified bilins were diluted with 2–3 volumes of 0.1% trifluoroacetic acid, applied to C-18 Sep-Pak cartridge, and washed with 0.1% trifluoroacetic acid, and bilins were eluted with 60:40 acetonitrile:0.1% trifluoroacetic acid. Eluted pigments were concentrated in vacuo with a Speed Vac concentrator (Savant), wrapped with foil, and stored at −20 °C. For biosynthetic studies, bilins were dissolved in Me2SO to give a final concentration of 1 mm. Bilins were quantitated in 2% (v/v) HCl in methanol using molar absorption coefficients of 46,773m−1 cm−1 at 368 nm for (3Z)-PCB (32Weller J.P. Gossauer A. Chem. Ber. 1980; 113: 1603-1611Crossref Scopus (69) Google Scholar), 47,900 m−1cm−1 at 374 nm for (3E)-PCB (33Cole W.J. Chapman D.J. Siegelman H.W. J. Am. Chem. Soc. 1967; 89: 3643-3645Crossref Scopus (70) Google Scholar), 66,200m−1 cm−1 at 377 nm for BV (29McDonagh A.F. Palma L.A. Biochem. J. 1980; 189: 193-208Crossref PubMed Scopus (90) Google Scholar), 38,019 m−1 cm−1 at 382 nm for (3Z)-PΦB (32Weller J.P. Gossauer A. Chem. Ber. 1980; 113: 1603-1611Crossref Scopus (69) Google Scholar), and 64,565 m−1cm−1 at 386 nm for (3E)-PΦB (32Weller J.P. Gossauer A. Chem. Ber. 1980; 113: 1603-1611Crossref Scopus (69) Google Scholar). Previous studies have shown that the biosynthetic pathway for the chromophore of phytochrome resides in the plastid compartment of higher plants (22Terry M.J. Lagarias J.C. J. Biol. Chem. 1991; 266: 22215-22221Abstract Full Text PDF PubMed Google Scholar). To test the hypothesis that the green alga Mesotaenium is capable of synthesizing phytochrome chromophore precursor(s) other than PΦB, we prepared chloroplast soluble protein extracts to study the metabolism of BV. An NADPH-regenerating system, ferredoxin, and ferredoxin-NADP+reductase were included in our assay mixture, since reduced ferredoxin has been shown to be required for the synthesis of PCB in red algae (34Beale S.I. Cornejo J. J. Biol. Chem. 1991; 266: 22328-22332Abstract Full Text PDF PubMed Google Scholar) and (3Z)-PΦB in higher plants.4 After prolonged incubation of chloroplast extracts with BV, the total pigment mixture was partially purified and analyzed for its ability to assemble with recombinant oat apophytochrome A. Fig.1 A shows the difference spectrum of the oat phytochrome A-bilin adduct(s) produced. Comparisons with the spectra of PCB and PΦB adducts of recombinant oat apophytochrome A show that this spectrum is indistinguishable with that of the PCB adduct (Fig. 1 B). This result strongly suggests that the Mesotaenium chloroplast soluble fraction contains enzymatic activities that can metabolize BV either to PCB or a novel bilin, for which assembly with apophytochrome A yields a phytochrome species with a difference spectrum very similar to the PCB adduct. To identify the pigment(s) produced by incubation of the Mesotaenium chloroplast soluble protein extracts with BV, a more detailed time-course study was conducted (Fig.2). At time zero, the predominant pigment detected by HPLC was the reaction substrate BV. A new pigment, labeled A, emerged after 5 min of incubation and increased during the initial 15-min period. Pigment A was accompanied by another minor pigment, labeled C, with an earlier retention time. A third pigment, labeled B, appeared after most of the BV had been metabolized. Pigment B became the predominant species along with the production of a fourth pigment, labeled D. At later time points, pigments B and D appeared to be the end products of the BV conversion. The kinetics of BV turnover and the production of pigments A and B are illustrated in Fig.3. These data strongly suggest that the metabolism of BV proceeds via pigment A, which is subsequently converted to pigment B. That these conversions were enzyme-mediated was established by the inability of heat-denatured chloroplast soluble protein extracts to convert BV to pigments A–D (data not shown). Spectrophotometric analysis of the pigments eluting prior to 10 min (Fig. 2) revealed these to be rubinoid pigments, possibly thiol adducts, which were not further characterized.Figure 3Kinetic plot of BV metabolism by Mesotaenium plastid extracts. This plot was generated by plotting the percent area of three major pigments in the whole time-course studies shown in Fig. 2 versus the reaction time. Percent area was obtained by dividing integrated area of individual pigments peaks by the total integrated area of the whole HPLC profile. The three curves represent different bilin pigments with symbols as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Several lines of research were undertaken to address the identities of the pigments derived from BV in this green alga. First, pigments A and B were HPLC-purified and tested for their ability to assemble with recombinant oat apophytochrome A. Fig. 4 A shows that oat apophytochrome A adducts of pigments A and B exhibited difference spectra typical for PΦB and PCB adducts, respectively (see Fig.1 A for comparison). Second, the absorption spectra of pigments A and B were determined and proved indistinguishable from those of (3Z)-PΦB and (3Z)-PCB (Fig.4 B and data not shown). Third, HPLC co-injection experiments with the products of BV metabolism by soluble chloroplast extracts were performed using authentic (3Z)- and (3E)-isomers of PCB and PΦB (Fig. 5). The retention times of pigments A and B were shown to be identical with those for (3Z)-PΦB and (3Z)-PCB (Fig. 5, c and d). These experiments also demonstrate that pigments C and D represent the (3E)-isomers PΦB and PCB, respectively (Fig.5, e and f). That (3Z)-PΦB is a bona fide precursor of (3Z)-PCB in Mesotaenium was established by the time-dependent conversion of purified (3Z)-PΦB to (3Z)-PCB (Fig.6). Taken together, it is clear that Mesotaenium chloroplasts possess the enzyme activities for conversion of BV to (3Z)-PΦB and subsequently to (3Z)-PCB.Figure 5HPLC co-injection of BV metabolites with authentic bilin standards. Mesotaenium chloroplast soluble protein extracts were incubated with BV for 30 min under standard bilin reductase conditions as described under “Experimental Procedures,” and the products were separated by HPLC (a). Co-injection experiments were performed by mixing the crude pigment products with 1 μmol of BV IXα (b), (3Z)-PΦB (c), (3Z)-PCB (d), (3E)-PΦB (e) or (3E)-PCB (f) prior to HPLC analysis. Peaks that increase are indicated with an asterisk. The increase in peak height at 14.7 min in c was due to chemical isomerization of (3Z)-PΦB to (3E)-PΦB during sample preparation for HPLC.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Kinetic plot of (3Z)-PΦB metabolism by plastid extracts. Standard bilin reductase assays were performed as described under “Experimental Procedures” except that 3 μm (3Z)-PΦB was used as reaction substrate. A, HPLC traces for initial time point (T0) and end time point (T20). B, relative integrated area was plotted against reaction time in minutes (min).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although Mesotaenium chloroplasts synthesize PCB from BV, it was still unclear whether PCB is the chromophore precursor for algal phytochrome. To address this issue, recombinant algal apophytochrome was therefore expressed in yeast and used for in vitro assembly with either PΦB or PCB. Fig.7 A indicates that, when assembled with PΦB, the spectrum was quite similar to the native spectrum for the higher plant phytochrome A (compare with Fig.1 B). When assembled with PCB, the difference spectrum of recombinant Mesotaenium phytochrome was indistinguishable from that of native algal phytochrome (Fig. 7 B). Together with the metabolism of BV to PCB by algal plastid extracts, these results demonstrate that PCB is the immediate chromophore precursor for the algal photoreceptor phytochrome. This work was undertaken to determine the nature of the observed blue-shifted difference spectrum of phytochrome from the green alga Mesotaenium. We have demonstrated that this blue shift arises from the use of PCB as the precursor of the algal phytochrome chromophore rather than an altered chromophore environment. To our knowledge, this is the first report of the existence of PCB in an organism lacking phycobiliproteins and the first phytochrome identified to use a chromophore other than PΦB. Our studies also establish that Mesotaenium possesses a novel biosynthetic pathway for the synthesis of PCB. Instead of the major route employed by the red algae which synthesizes PCB via the intermediates of 15,16-dihydrobiliverdin and phycoerythrobilin (34Beale S.I. Cornejo J. J. Biol. Chem. 1991; 266: 22328-22332Abstract Full Text PDF PubMed Google Scholar, 35Beale S.I. Cornejo J. J. Biol. Chem. 1991; 266: 22333-22340Abstract Full Text PDF PubMed Google Scholar, 36Beale S.I. Cornejo J. J. Biol. Chem. 1991; 266: 22341-22345Abstract Full Text PDF PubMed Google Scholar), Mesotaenium converts BV to (3Z)-PΦB, which is subsequently converted to (3Z)-PCB (Fig. 8). While similar to that proposed for phytochrome chromophore biosynthesis in higher plants, an additional step for the reductive conversion of (3Z)-PΦB to (3Z)-PCB catalyzed by a hypothetical PΦB reductase has been included in the green algae phytochrome chromophore biosynthetic pathway. In this scheme, we have also included 3Z to 3E bilin isomerization steps based on the observed production of 3E isomers of PΦB and PCB. Whether one or both isomerization steps are enzyme-mediated and/or required for assembly with Mesotaenium apophytochrome is unknown at present. The ability of Mesotaenium to synthesize both PΦB and PCB raises the possibility that PΦB could also be used as a precursor of the algal phytochrome chromophore. This might occur should PΦB escape from the chloroplast compartment or should further reduction of PΦB to PCB be inhibited and/or regulated under various physiological conditions. Since the observed blue-shifted difference spectrum was observed for Mesotaenium holophytochrome isolated from the dark-adapted cells, and the enzyme used for BV metabolism was also obtained from dark-adapted cells, it is conceivable that PΦB can be used as a natural phytochrome chromophore precursor for cells grown under different conditions. These possibilities remain to be addressed in future study. The identification of an enzyme activity responsible for the reduction of PΦB to PCB raises a number of interesting questions. Does this represent a second bilin reduction enzyme or reflect a dual substrate specificity for PΦB synthase in green algae? What is the ecological advantage for this green alga to possess an enzyme that yields a blue-shifted phytochrome species? Could introduction of the green algal PΦB synthase gene into higher plants lead to the synthesis of phytochrome molecules with PCB as chromophore? A 10–16-nm blue shift in both red- and far red-absorbing forms by using PCB as chromophore could provide a powerful tool for studying the impact of light quality in plant physiology and development. Whether higher plants possess phytochrome molecules with different chromophores other than PΦB remains to be addressed. The presence of PCB in the non-phycobiliprotein containing green alga Mesotaenium raises the interesting question of whether green algae can synthesize PCB via same pathway as that reported for the red alga Cyanidium. Preliminary experiments in our laboratory failed to detect either 15,16-dihydrobiliverdin or phycoerythrobilin as BV metabolites by plastid extracts from Mesotaenium (data not shown). These results suggest that phycobilin-containing organisms that do not produce phycoerythrobilin might synthesize PCB via the intermediacy of PΦB. Furthermore, a closer examination of the PCB biosynthetic pathway in Cyanidium has revealed the evidence for the production of (3Z)-PΦB as the biosynthetic intermediate.2 Although it is not known at present, cyanobacteria may be capable of synthesizing PΦB based on the hypothesis that the cyanobacterial family of eubacteria are the progenitors of the rhodophyte plastids. Together with the presence of PΦB synthase activity in yeast, Pichia pastoris (21Wu S.-H. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8989-8994Crossref PubMed Scopus (27) Google Scholar), it is tempting to speculate that PΦB synthase, the enzyme catalyzing BV reduction to PΦB, was present in the primitive ancestor for all extant organisms. We thank Dr. Matthew Terry for initial studies on the chromophore biosynthetic pathway in Mesotaenium.