(3Z)- and (3E)-Phytochromobilin Are Intermediates in the Biosynthesis of the Phytochrome Chromophore (1995)

Using a high performance liquid chromatography (HPLC)-based assay, we have demonstrated that isolated oat etioplasts convert the linear tetrapyrrole biliverdin IXα to (3E)-phytochromobilin, the proposed precursor of the chromophore of the plant photoreceptor phytochrome. In addition to (3E)-phytochromobilin, the synthesis of a second phytochromobilin was detected by its ability to functionally assemble with recombinant oat apophytochrome A. The structure of this new pigment has been determined to be the 3Z isomer of phytochromobilin by absorption and 1H NMR spectroscopy. Like (3E)-phytochromobilin, assembly of HPLC-purified (3Z)-phytochromobilin with apophytochrome yielded a holoprotein that is spectrally indistinguishable from native oat phytochrome A. However, the postchromatographic conversion of (3Z)- to (3E)-phytochromobilin appears to be responsible for this result. Kinetic HPLC analyses have demonstrated that (3Z)-phytochromobiIin is synthesized prior to the 3E isomer by oat etioplasts. We therefore propose that (3Z)-phytochromobilin is the immediate product of biliverdin IXα reduction by the enzyme phytochromobilin synthase. This implicates the presence of an isomerase that catalyzes the conversion of (3Z)- to (3E)-phytochromobilin, the immediate precursor of the phytochrome A chromophore. Using a high performance liquid chromatography (HPLC)-based assay, we have demonstrated that isolated oat etioplasts convert the linear tetrapyrrole biliverdin IXα to (3E)-phytochromobilin, the proposed precursor of the chromophore of the plant photoreceptor phytochrome. In addition to (3E)-phytochromobilin, the synthesis of a second phytochromobilin was detected by its ability to functionally assemble with recombinant oat apophytochrome A. The structure of this new pigment has been determined to be the 3Z isomer of phytochromobilin by absorption and 1H NMR spectroscopy. Like (3E)-phytochromobilin, assembly of HPLC-purified (3Z)-phytochromobilin with apophytochrome yielded a holoprotein that is spectrally indistinguishable from native oat phytochrome A. However, the postchromatographic conversion of (3Z)- to (3E)-phytochromobilin appears to be responsible for this result. Kinetic HPLC analyses have demonstrated that (3Z)-phytochromobiIin is synthesized prior to the 3E isomer by oat etioplasts. We therefore propose that (3Z)-phytochromobilin is the immediate product of biliverdin IXα reduction by the enzyme phytochromobilin synthase. This implicates the presence of an isomerase that catalyzes the conversion of (3Z)- to (3E)-phytochromobilin, the immediate precursor of the phytochrome A chromophore. The phytochrome photoreceptor performs a vital role in a plant’s adaptive response to its light environment (1Sage L.C. Pigment of the Imagination: A History of Phytochrome Research. Academic Pres, San Diego1992Google Scholar, 2Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Martinus Nijhoff Publishers, Dordrecht, The Netherlands1994Crossref Google Scholar). Comprised of linear tetrapyrrole chromophore that is covalently attached to a polypeptide of about 124 kDa, phytochrome functions as a detector of red and far-red wavelengths of light (3Furuya M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 617-645Crossref Scopus (294) Google Scholar). The molecular basis of phytochrome action depends upon the ability to photointerconvert between two stable isomers, the red light absorbing form of phytochrome, Pr, 1The abbreviations used are: Prthe red light absorbing form of phytochromePfrthe far-red light absorbing form of phytochromeBVbiliverdin IXαHPLChigh performance liquid chromatographyMOPS3-(N-morpholino)propanesulfonic acidPCBphycocyanobilinPEBphycoerythrobilinPΦβphytochromobilinTESN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and the far-red light absorbing form, Pfr. In higher plants, phytochrome is encoded by a multigene family, consisting of at least five individual members (4Quail P.H. Annu. Rev. Genet. 1991; 25: 389-409Crossref PubMed Scopus (284) Google Scholar). These molecular properties enable this unique photoreceptor family to modulate many aspects of plant growth and development in response to both light quality and quantity in the natural environment. the red light absorbing form of phytochrome the far-red light absorbing form of phytochrome biliverdin IXα high performance liquid chromatography 3-(N-morpholino)propanesulfonic acid phycocyanobilin phycoerythrobilin phytochromobilin N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid The biosynthesis of phytochrome requires two pathways, one for the apoprotein and another for phytochromobilin (PΦβ), the immediate precursor of the protein-bound chromophore (5Terry M.J. Wahleithner J.A. Lagarias J.C. Arch. Biochem. Biophys. 1993; 306: 1-15Crossref PubMed Scopus (95) Google Scholar). These two components are brought together in the plant cytosol where the mature photoreceptor is assembled. While the chemical structure of the protein-bound phytochrome chromophore has been known for some time (6Lagarias J.C. Rapoport H. J. Am. Chem. Soc. 1980; 102: 4821-4828Crossref Scopus (261) Google Scholar, 7Rudiger W. Thummler F. Cmiel E. Schneider S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6244-6248Crossref PubMed Google Scholar), the structure of PΦβ was only recently verified (8Cornejo J. Beale S.I. Terry M.J. Lagarias J.C. J. Biol. Chem. 1992; 267: 14790-14798Abstract Full Text PDF PubMed Google Scholar). In that study, a pigment with the 3E-PΦβ structure (see Fig. 1) was isolated from the red alga Porphyridium cruentum and shown to properly assemble with recombinant oat apophytochrome A. The resulting holophytochrome exhibited a spectrophotometric signature that was indistinguishable from that of oat phytochrome isolated from dark grown seedlings. Based on these investigations, we proposed that 3.E-PΦβ is the immediate precursor of the phytochrome chromophore. Until now, the intermediacy of 3E-PΦβ in the pathway of phytochrome chromophore biosynthesis has been primarily based upon an indirect coupled assay in which its production was detected after assembly with apophytochrome (9Terry M.J. Lagarias J.C. J. Biol. Chem. 1991; 266: 22215-22221Abstract Full Text PDF PubMed Google Scholar). That study demonstrated that PΦβ is derived from biliverdin IXα (BV; see Fig. 1 for structure), a conversion that is accomplished by the plastid-localized enzyme, PΦβ synthase. In this report, we present direct experimental evidence for the conversion of BV to 3.E-PΦβ by oat etioplast preparations using a newly developed HPLC assay. These studies reveal that 3Z-PΦβ pigment is also produced during the incubation of BV with etioplasts (see Fig. 1 for structure). Based upon these investigations and the kinetics of the synthesis of the two PΦβ isomers, a revised pathway for the biosynthesis of the phytochrome chromophore is proposed. Bilin Preparations—For all biochemical assays, reagent grade biliverdin IXα was prepared from bilirubin (Vega Biochemicals, Tucson, AZ) as described previously (10Elich T.D. McDonagh A.F. Palma L.A. Lagarias J.C. J. Biol. Chem. 1989; 264: 183-189Abstract Full Text PDF PubMed Google Scholar). For 1H NMR spectroscopy and data shown in Fig. 4, BV was further purified by HPLC as described under “HPLC Analyses.” 3E-PΦβ was purified from P. cruentum as described previously (8Cornejo J. Beale S.I. Terry M.J. Lagarias J.C. J. Biol. Chem. 1992; 267: 14790-14798Abstract Full Text PDF PubMed Google Scholar, 11Terry M.J. Maines M.D. Lagarias J.C. J. Biol. Chem. 1993; 268: 26099-26106Abstract Full Text PDF PubMed Google Scholar). The molar absorption coefficient of 64,600 m–1 cm–1 at 386 nm in 2% HCl in methanol was used to estimate the concentration of the 3E-PΦβ standard used for HPLC analyses (12Weller J.-P. Gossauer A. Chem. Ber. 1980; 113: 1603-1611Crossref Scopus (69) Google Scholar). Etioplast Isolation—Oat seeds (Avena sativa L. cv Garry) were germinated and grown in the dark on vermiculite for 6 days at 25 °C. Shoot tissue was homogenized at 4 °C in a precooled Waring blender with an equal volume to fresh weight tissue of cold homogenization medium (50 mm MOPS/KOH buffer, pH 8.0, containing 500 mm sucrose, 1 mm MgCl2, and 142 mm 2-mercaptoethanol). The homogenate was filtered through four layers of cheesecloth and two layers of Miracloth and centrifuged at 8000 × g for 1 min. The pellet was resuspended in homogenization medium lacking 2-mercaptoethanol but including 0.2% (w/v) bovine serum albumin. This suspension was centrifuged at 100 × g for 1 min to remove cell debris. The supernatant was removed and centrifuged at 1500 × g for 2 min to give a pellet containing mostly intact plastids. Plastids were kept as a pellet on ice until required. Phytochromobilin Synthase Assay—Plastids were resuspended in PΦβ synthase assay buffer (20 mm TES, 10 mm HEPES/NaOH buffer, pH 7.7, containing 500 mm sorbitol, 1 mm phenylmethanesulfonyl fluoride, 0.5 mm dithiothreitol, and 2 μμ leupeptin) containing 3000 units/ml catalase and an NADPH regenerating system (1.2 mm NADP+, 9.3 mm glucose 6-phosphate, and 1.5 units/ml glucose-6-phosphate dehydrogenase). The reaction was initiated by addition of 1 mm BV in Me2SO to give a final concentration of 10 μμ BV. Reaction mixtures were incubated at 28 °C in the dark for various lengths of time. Reaction volumes were routinely 2.5 or 5 ml for analytical assays, 40 ml for preparative assays, and 1 ml for kinetic measurements. Argon was typically used to remove oxygen from the reaction tubes both prior to and at hourly intervals throughout the assay. Following completion of the incubation period, samples were centrifuged at 39,000 × g for 15 min to remove the plastids. Bilins were partially purified on a C18 Sep-Pak cartridge (Waters-Millipore Corp., Milford, MA) using the following procedure. Prior to application of the assay supernatant, the Sep-Pak cartridge was washed with acetonitrile, water, and 50 mm N-methylmorpholine/acetate buffer, pH 7.7. After application of the extract, the Sep-Pak cartridge was then sequentially washed with 50 mm N-methylmorpholine/acetate buffer, pH 7.7 (2 × 3 ml), 0.1% (v/v) trifluoroacetic acid (2 × 3 ml), and acetonitrile/0.1% trifluoroacetic acid (20:80; 2 × 2 ml). The crude bilin mixture was typically eluted with 3 ml acetonitrile/0.1% trifluoroacetic acid (60:40) and dried in vacuo. Holophytochrome Assembly Assay—Recombinant apophytochrome was prepared from the yeast Saccharomyces cerevisiae strain 29A containing the full-length oat phytochrome (phyA3) cDNA plasmid pM-phyA3 as described previously (13Li L. Lagarias J.C. J. Biol. Chem. 1992; 267: 19204-19210Abstract Full Text PDF PubMed Google Scholar). All phytochrome reconstitution experiments were performed under green safelight in a final volume of 500 μl of assembly buffer (25 mm Tris/HCl buffer, pH 7.8, containing 25% (v/v) ethylene glycol, 1 mm dithiothreitol, 1 mm EDTA, and 1 mm phenylmethanesulfonyl fluoride). Crude and HPLC-purified bilin preparations were added by diluting stock solutions in Me2SO by 100-fold to give an final bilin concentration of approximately 5 μμ. Apophytochrome was added from a 17.5 μg/ml stock in assembly buffer (described above). The final concentrations of apophytochrome and bilins in specific experiments are given in the appropriate figure legend. Following incubation for 15 min on ice, samples were centrifuged at 5 °C for 15 min at 200,000 × g before measurement of phytochrome difference spectra. HPLC Analyses—Crude bilin samples were analyzed by C18 reverse phase HPLC using a Varían 5000 liquid Chromatograph and a Beckman Ultrasphere ODS column (5 μm; 10 mm × 250 mm). The mobile phase solvent consisted of ethanol/acetone/water/acetic acid (48/34717/1; v/v/v/v) with a flow rate of 4 ml/min. The column eluate was monitored at 380 nm with an Varian UV100 flow through absorbance detector. Peak areas were quantitated using a Hewlett Packard 3390A integrator. Before injection, all samples were dissolved in the HPLC solvent and centrifuged at 14,000 rpm for 15 s. HPLC-purified bilins were concentrated by diluting 1 to 4 with 0.1% (v/v) trifluoroacetic acid and applying to a C18 Sep-Pak cartridge. After washing with 0.1% trifluoroacetic acid, bilins were eluted with acetonitrile/0.1% trifluoroacetic acid (60: 40). The purified bilins were then dried in vacuo prior to spectroscopic analysis and/or reconstitution experiments. Spectroscopic Measurements—All absorbance measurements and phytochrome difference spectra were obtained using a Hewlett Packard 8450A UV-visible diode array spectrophotometer. Absorption spectra of 3Z- and 3E-PΦβ were taken in acetonitrile/0.1% trifluoroacetic acid (60:40; v/v) or 2% HCl in methanol at room temperature. Phytochrome difference spectra were measured at 5 °C as described previously (14Lagarias J.C. Kelly J.M. Cyr K.L. Smith Jr., W.O. Photochem. Photobiol. 1987; 46: 5-13Crossref Scopus (105) Google Scholar). 500 MHz 1H NMR spectra were obtained in pyridine-d5 (100.0 atom % D; Sigma catalog no. 23,699-3) at 28 °C using a GE Omega 500 spectrometer at the UCD NMR Facility. Protein Assay—Protein concentrations were determined using the BCA protein assay (Pierce) with bovine serum albumin as a standard (15Smith P.K. Krohn R.I. Hemanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olsen B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus Google Scholar). and 3E-PΦβ from conversion of biliverdin IXα to phytochromobilin by oat etioplast preparations was analyzed using the reverse phase HPLC described for the analysis of biosynthesis S.I. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). shown in Fig. 2 BV as a with a time of approximately min on this HPLC After incubation with for 1 at 28 °C in the presence of an NADPH regenerating BV is to a new pigment with a time of 28 min This was in samples where BV or were from the incubation These that the new pigment is by the conversion of BV by enzyme shown in Fig. 2 and the time of this new pigment is with that of 3E-PΦβ (see structure in Fig. In the absorption of the new pigment is indistinguishable from that of 3E-PΦβ Based on these and phytochrome reconstitution experiments (see is that the new pigment is with studies (9Terry M.J. Lagarias J.C. J. Biol. Chem. 1991; 266: 22215-22221Abstract Full Text PDF PubMed Google Scholar), etioplast preparations were analyzed with the HPLC These studies that BV is to 3E-PΦβ in this tissue as experimental etioplasts yielded a of 3.E-PΦβ by with oat etioplasts For this investigations have oat etioplasts to study the conversion of BV to A Phytochromobilin by further the of the 3E-PΦβ produced by incubation of BV with phytochrome reconstitution experiments were This incubation of the HPLC-purified pigment with recombinant apophytochrome and of a phytochrome difference studies have shown that BV assemble with apophytochrome (13Li L. Lagarias J.C. J. Biol. Chem. 1992; 267: 19204-19210Abstract Full Text PDF PubMed Google Scholar), the BV was analyzed as a These experiments that both yielded that a second PΦβ was with BV on HPLC shown in Fig. of of a preparative C18 column in partially these as and These two components and two pigment one that at 3 min and the at 25 min were and analyzed using the phytochrome assembly assay. The of these phytochrome reconstitution experiments are shown in the of Fig. in the described two pigment yielded the difference spectra were indistinguishable from that of phytochrome isolated from oat with at nm and at spectrophotometric analyses using concentrated samples of these holophytochrome this to the that 3E-PΦβ (see and 3E-PΦβ is a phytochrome chromophore precursor (8Cornejo J. Beale S.I. Terry M.J. Lagarias J.C. J. Biol. Chem. 1992; 267: 14790-14798Abstract Full Text PDF PubMed Google Scholar), the assembly of was The of is in the experiments were also performed with the two in both of holophytochrome were produced a and was as BV the of holophytochrome produced is to a of from the PΦβ in The for the of holophytochrome obtained with a was further The Phytochromobilin difference of the holophytochrome and the time of this pigment with both the that is 3Z-PΦβ (see Fig. 1 for structure). The 3Z of the chromophore phycocyanobilin and phycoerythrobilin both 3E on a HPLC system S.I. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). The absorption of HPLC-purified is also with this shown in Fig. 4, both absorption of are to of 3.E-PΦβ in acetonitrile/0.1% trifluoroacetic acid v/v) The absorption of 3Z-PΦβ and with 3E (12Weller J.-P. Gossauer A. Chem. Ber. 1980; 113: 1603-1611Crossref Scopus (69) Google Scholar). the spectra of and 3E-PΦβ were in 2% HCl in the absorption spectra of and 3E-PΦβ are this with the the was by HPLC after in 2% HCl in This analysis that is in the solvent and in to 3.E-PΦβ Based on all of the taken appears that is the chemical of its 1H NMR in pyridine-d5 was determined and is in Fig. analysis of this that of bilin In addition to the new PΦβ two of these components to be known bilins BV and BV was to be a of this the BV partially in HPLC 3.E-PΦβ appears to have by a chemical of the new a that has been by HPLC (see The are present in in the NMR an that has the of specific to individual bilin The of a molar of 25% and new PΦβ in the The of the 1H NMR spectra of the components of are in All to BV and 3E-PΦβ were by with data (8Cornejo J. Beale S.I. Terry M.J. Lagarias J.C. J. Biol. Chem. 1992; 267: 14790-14798Abstract Full Text PDF PubMed Google Scholar, S.I. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google and NMR measurements of purified BV in pyridine-d5 the chemical of all for both bilins with of purified experiments and with the 1H NMR spectra of known bilins to the of the of the new PΦβ The new bilin the NMR signature of an two at and experiments shown in Fig. 6 and chemical of the coupled the of the two to the and 3 and J. These experiments that the is to the of in the at the is coupled with the in the at of the also the to a which the of this to the of 3E-PΦβ This that the environment of the of the new PΦβ is the as that of MHz 1H NMR data for HPLC-purified in of one or both PΦβ were with of for both PΦβ were for both PΦβ were of one or both PΦβ were with of for both PΦβ were for both PΦβ were for both PΦβ were for both PΦβ were of one or both PΦβ were with of of one or both PΦβ were with of for both PΦβ were of one or both PΦβ were with of for both PΦβ were for both PΦβ were for both PΦβ were of one or both PΦβ were with of for both PΦβ were for both PΦβ were for both PΦβ were for both PΦβ were for both PΦβ were for both PΦβ were for both PΦβ were for both PΦβ were of one or both PΦβ were with of for both PΦβ were in a new The is the only to the bilin that is the for both the of the new PΦβ from that of 3E-PΦβ the to which is coupled The chemical of these two is known to be a of the of the (8Cornejo J. Beale S.I. Terry M.J. Lagarias J.C. J. Biol. Chem. 1992; 267: 14790-14798Abstract Full Text PDF PubMed Google Scholar, S.I. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). in the chemical of the and for the new PΦβ are in with of linear The chemical of the of the new bilin to that of 3E-PΦβ also the of the 3Z the of the two in the of the 3Z at higher in the NMR solvent (see were also in the and and two of the four from these the NMR spectra of both are the 1H NMR data the that the bilin in the mixture is to for the and the for and containing bilins and in study, have been by to have been by to have been by to have been by to in a new 3Z-PΦβ That of synthesis of two PΦβ the which PΦβ isomer is synthesized this oat etioplasts were incubated with BV as described under with the following to PΦβ synthase 5 mm the 0.5 mm dithiothreitol, and the pH of the reaction was to The HPLC mobile phase used for this was to mm acid This of 3Z-PΦβ from BV and therefore of the two bilins Fig. the kinetics of 3Z- and 3E-PΦβ synthesis following the incubation of BV with oat etioplasts. For time the molar of the bilins and was by the individual areas by the molar absorption coefficient for After to the of BV present at the of the these were as a of incubation time This analysis that 3Z-PΦβ is produced prior to the time the of was the of 3Z-PΦβ was The to Fig. that 3E-PΦβ appears only after an incubation period, during which time the of the 3Z isomer These the that 3Z-PΦβ is the precursor of The reverse phase HPLC assay described the that 3E-PΦβ is a in the biosynthesis of the phytochrome chromophore as proposed (9Terry M.J. Lagarias J.C. J. Biol. Chem. 1991; 266: 22215-22221Abstract Full Text PDF PubMed Google Scholar, J. Beale S.I. Terry M.J. Lagarias J.C. J. Biol. Chem. 1992; 267: 14790-14798Abstract Full Text PDF PubMed Google Scholar). In these studies that a second PΦβ is produced during the incubation of BV with oat etioplast detected by its assembly with this new pigment has been to be 3Z-PΦβ by 1H NMR spectroscopy. Kinetic measurements are with the that the 3Z isomer of PΦβ is synthesized prior to the 3E This is by biochemical investigations to further PΦβ synthase from oat etioplasts have yielded enzyme that only convert BV to which that the 3Z isomer is the product of BV reduction by PΦβ a bilin that catalyzes the reduction of a has been in the red alga S.I. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). The product of this enzyme, which is the 3Z isomer of Based on this to the that 3Z-PΦβ is the direct product of BV reduction in is that 3E bilin are stable 3Z to the 3E isomer in in an mixture of the two is produced A. 1983; Scopus Google Scholar). the of 3Z-PΦβ was previously (12Weller J.-P. Gossauer A. Chem. Ber. 1980; 113: 1603-1611Crossref Scopus (69) Google Scholar), the present that 3Z-PΦβ to the 3E isomer in two solvent pyridine-d5 and 2% HCl in These the proposed shown in Fig. in which the conversion of BV to 3E-PΦβ in two In the we that BV reduction to 3Z-PΦβ is by the enzyme PΦβ synthase. the 3Z to 3E in the pathway of biosynthesis in the red alga are S.I. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, S.I. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, S.I. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar), we the that the second is accomplished by an enzyme that we propose to PΦβ of the properties of 3Z-PΦβ is that the absorption of the pigment from that of the phytochrome difference spectra after assembly to oat apophytochrome A is indistinguishable for the two This that both this which are the of this is that the isomer assemble with apophytochrome but that its assembly requires to the This be using a kinetic assay for holophytochrome which has recently been developed in J. and J. in A ability of 3Z-PΦβ to assemble with apophytochrome be with its role as an in the synthesis of the phytochrome chromophore precursor However, we to that only assembly to phytochrome A has been in this as P.H. 1989; PubMed Scopus Google Scholar, S. Plant Mol. Biol. 25: PubMed Scopus Google Scholar), the 3Z-PΦβ isomer as the immediate precursor of the protein-bound chromophore. studies have shown that the assembly of to recombinant phytochrome with an rate to that for phytochrome A from oat M. E. J. Biochem. 1993; PubMed Scopus (69) Google Scholar). This that 3E-PΦβ is the natural precursor of the of both A and We for the synthesis of BV and for in the development of for bilin in the of this