Plant Cell Physiol. 38(3): 274-281 (1997) JSPP © 1997

Pigment Composition of a Novel Oxygenic Photosynthetic Prokaryote Containing Chlorophyll d as the Major Chlorophyll Hideaki Miyashita', Kyoko Adachi 2 , Norihide Kurano 1 , Hisato Ikemoto 1 , Mitsuo Chihara 13 and Shigetoh Miyachi 4 1 2 3

4

Marine Biotechnology Institute, Kamabhi Laboratories, Heita, Kamaishi, Iwate, 026 Japan Marine Biotechnology Institute, Shimizu Laboratories, Sodeshi, Shimizu, Shizuoka, 424-19 Japan Japanese Red Cross College of Nursing, Shibuya-ku, Tokyo, 150 Japan

Marine Biotechnology Institute, Bunkyo-ku, Tokyo, 113 Japan

The principal pigment found in the majority of oxygenic photosynthetic organisms is known to be chlorophyll a. However, we isolated a new oxygenic photosynthetic prokaryote that contained chlorophyll (/ as a predominant pigment with chlorophyll a being a minor pigment. Chlorophyll d had previously been noted but its natural occurrence and function remained unclear. Cells of the new prokaryote had an absorption maximum at red region of 714-718 nm due to chlorophyll d absorption, but no characteristic absorption peak of chlorophyll a around 680 nm was observed. Chlorophyll d of the new organism was identified spectrophotometrically in several solvents and its chemical structure was confirmed by NMR and FABMS analysis. The cell also contained a chlorophyll c-like pigment, zeaxanthin and a-carotene but not chlorophyll b and /^-carotene. The content of chlorophyll d accounted for more than 2% of the cell dry weight, while the content of chlorophyll a was less than 0.1%. The chlorophyll a/d ratio remained between 0.03 and 0.09 under different culture conditions. The light absorption characteristics and the high content of chlorophyll d along with the small content of chlorophyll a indicated the existence of a new light utilization mechanism involving chlorophyll d. Key word: Chlorophyll d — In vivo absorption — NMR — Oxygenic photosynthesis — Pigment composition — Prokaryote.

Chlorophyll a is an essential pigment for oxygenic photosynthesis. Therefore it is contained as a predominant pigment in all oxygenic photosynthetic organisms (Lee 1989). Oxygenic phototrophs, consequently, show a characteristic absorption maximum of red light around 680 nm due to a bathochromic shift of chlorophyll a bound to the photosynthetic apparatus (French et al. 1972). Chlorophyll d was first reported as a minor accessory pigment in various species of red macroalgae (Manning and Strain 1943). However, the evidence for its existence was inconsistent. And it was suggested that chlorophyll d could be an artifact produced by the extraction process of the pigment, since one of the oxidation derivatives of chlorophyll a gave an ab274

sorption spectrum identical with that of chlorophyll d (Holt and Morley 1959, Holt 1961). The in vivo occurrence of chlorophyll d as well as its photosynthetic function has been unclear for longer than half a century. Recently, we isolated a new oxygenic photosynthetic prokaryote containing chlorophyll d as a major green pigment (Miyashita et al. 1996). In this report, pigment composition of the organism was characterized and the in vivo occurrence and the chemical structure of chlorophyll d were confirmed. Also the possibility of a new light-utilization system using chlorophyll d was discussed. Materials and Methods Isolation and cultivation of cells—The new oxygenic photosynthetic prokaryote was isolated from a suspension of algae squeezed out of Lissoclinum patella, a colonial ascidian, collected in 1993 from the coast of Palau in the western Pacific Ocean. Cells of the algal suspension were enriched in K medium (Keller et al. 1987). Clonal culture of the new organism was established by serial single-colony transfer on agar plates (0.7%). The cells were grown in K+ESM medium (Miyashita et al. 1995) with a gentle bubbling of air. The culture was kept under white fluorescent light (~80/imol photons nT 2 s~') with a 12/12 h light/dark cycle at 28°C. Spectrophotometry—Absorption spectrum of a cell suspension was made spectrophotometrically (DU-640, Beckman, U.S.A.). Prior to the measurement, cell aggregation was dispersed by ultrasonication of the cells for a few seconds. This absorption spectrum was compared with those of a cyanophycean alga, Synechocystis sp. ATCC27266 and a green alga, Nannochloris maculata CCAP251/3. Electron microscopy—Cells were prefixed with glutaraldehyde [1% in 50 mM phosphate buffer (pH 7.2)] for 2 h and postfixed with osmic acid (2% in the same phosphate buffer) for 2 h. The fixed cells were harvested by centrifugation, washed with water and then dehydrated by a series of alcohol washes. For the observation of thylakoids and plasmalemma, cells were initially fixed with glutaraldehyde (as above), and then with potassium permanganate (1% in the phosphate buffer) for 2 h. The fixed cells were washed with water and dehydrated by a series of acetone extractions. Fixed cell samples were mounted in Spurr's resin (Spurr 1969). Ultrathin sections were double-stained with uranyl acetate and lead citrate (Reynolds 1963). Pigment analysis—Cells were harvested by centrifugation and washed with filtered seawater. Pigments were extracted with cold methanol (4°C) for a few minutes. After centrifugation, the supernatant was immediately injected into the HPLC system with

Chlorophyll d as a predominant pigment a reversed-phase column, TSKgel ODS-80Ts (15 cm x 4.6 mm, 5 fttn particle size; Tosoh, Japan). The pigments were eluted with methanol: water (9 : 1) for 0.5 min, with a linear gradient to 100% methanol for 4.5 min, and then with 100% methanol. The initial flow rate, 1 ml min~', was changed to 2 ml min" 1 at 9 min after the sample injection. Eluted pigments were detected with a UV-visible detector (UV8010, Tosoh, Japan) at 440 nm and a photodiode array detector (SPD-M6A, Shimadzu, Japan). Absorption spectra of the methanol extract and of the pigments fractionated from HPLC stream were made with a spectrophotometer (DU-640, Beckman, U.S.A.). Pigments were identified by comparing the retention times and absorption spectra with those of known pigments extracted from Prochloron didemni (collected at Palau) containing chlorophylls a and b, zeaxanthin, Mg 2,4-divinyl pheoporphyrin a5 monomethyl ester (MgDVP), or from Prochlorococcus sp. SB strain (Shimada et al. 1995) containing divinyl-chlorophylls (DVChl.) a and b, zeaxanthin and a-carotene. Comparison was also made to existing data (Rowan 1989). Identification of a major green pigment—A major green pigment of the new organism was fractionated from the stream of the HPLC with a reversed phase column (TSKgel, ODS-80TM, 4.7 mm x 30 cm, Tosoh, Japan) and purified by fractionation with the HPLC several times. Absorption peaks of the pigment in several solvents were compared with those of the published data (Rowan 1989). Acidification product of the pigment (phaeophytin) was prepared by addition of a few drops of 0.5 M HC1. Since there was no available authentic chlorophyll d nor a reference algal strain which contained chlorophyll d, chemical structure of the major pigment was confirmed by nuclear magnetic resonance (NMR) spectroscopy and fast atom bombardment mass spectroscopy (FABMS), in comparison with the data obtained from authentic chlorophyll a. The NMR spectra of the pigment in acetone-rf6 were recorded on a Varian Unity 500 NMR Spectrometer. The FABMS was measured with a JEOL JMS-SX102 mass spectrometer. Quantification of pigments—Each pigment was quantified based on the peak area of HPLC analysis using the method of Mantoura and Llewellyn (1983). A tentative extinction coefficient for chlorophyll d, E 1 ^ (440 nm)=400, obtained from the measurement of the relationship between the dry weight of purified chlorophyll d injected into HPLC and the peak area with our system was applied for the quantification of the pigment. For calculation of the a-carotene content, the extinction coefficient for /?-carotene, E1% (440nm) = 2,200 (Mantoura and Llewellyn 1983), was used, since there was no appropriate data for the a-carotene quantification. Extraction of water-soluble pigment—Water-soluble pigments were extracted by hard grinding of the cells with quartz sand in 20 mM acetate buffer, pH 5.2. After precipitating at 2,000xg for 5 min at 4°C, the supernatant was centrifuged again at 20,000xg for 2 h at 4°C. The absorption spectrum of the resulting supernatant was compared with the water-soluble fraction obtained from Anacystis nidulans R2 IAM200 (University of Tokyo) by the same procedures. Photosynthetic activity measurement—Photosynthetic activity was measured using the oxygen electrode system obtained from Rank Brothers Inc. (Cambridge, U.K.) under a halogen lamp (JCD100V, Iwasaki-denki, Japan) at 28°C. Irradiation strength was measured with a LI-1000 multichannel dataloger (RI-COR, U.S.A.) with a LI-192SA sensor (RI-COR, U.S.A.). Before the measurement, aggregates of the cells were dissociated by ultrasonication for a few seconds, then washed arid resuspended in fresh culture medium.

275 Results

Morphological characteristics of the new isolate—The cell of the new phototrophic organism was unicellular and spheroidal or ellipsoidal, 1.5-2.0 pm in diameter and 2.03.0nm in length (Fig. 1A). Electron microscopy revealed the prokaryotic nature of the new organism (Fig. IB, C). Its genomic DNA was dispersed cytoplasmically and not enclosed in a nuclear envelope (Fig. IB). No organized chromosomes, mitochondria or other membrane-limited organ elles were visible. Layers of thylakoid-like structures were appressed peripherally (Fig. 1C). No protein entities similar to phycobilisomes could be observed on the thylakoid-like membranes. In vivo absorption—The cell suspension was green and had an absorption maximum in the red region at 716 nm (Fig. 2). Cell suspensions of Synechocystis and Nannochloris have absorption maxima at 675 nm and 685 nm, respectively. The absorption maximum of the new prokaryote was located at least 30 nm longer wavelength than those of Synechocystis and Nannochloris. The new prokaryote has an absorption minimum at 678 nm where absorption peaks are normally found in known oxygenic photoautotrophs. Pigment analysis—Methanol extract of the cells had

Fig. 1A-C Micrographs of the new oxygenic photosynthetic prokaryote cells. (A) Photomicrograph. Scale bar=5 fim. (B) Electron micrograph of a longitudinal section showing the cytoplasmic space (glutaraldehyde/osmic acid fixation), c: carboxysomelike structure, n: nucleoide. t: thylakoid-like membranes. Scale bar=0.3//m. (C) Electron micrograph of the transverse section showing thylakoid-like membranes and plasmalemma (glutaraldehyde/potassium permanganate fixation). Arrowheads: thylakoidlike membranes. Arrows: plasmalemma. Scale bar=0.5/im.

276

Chlorophyll d as a predominant pigment

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Fig. 4 HPLC profile of pigments extracted with methanol from the cells of the new oxygenic photosynthetic prokaryote (A), Prochloron sp. (B) and Prochlorococcus sp. (C).

from the two reference strains, the pigment was identified as zeaxanthin. The third pigment was the major green pigment of the cell. It had similar retention time to that of chlorophyll b or divinyl-chlorophyll b, but the absorption spectrum (Fig. 5C) was obviously different from those of the chlorophyll pigments. Chlorophyll b was not detected. The fourth pigment was identified as chlorophyll a (Fig. 4, Fig. 5D). The fifth pigment was identical to a-carotene from Prochlorococcus in both retention time and absorption spectrum (Fig. 4, Fig. 5E). /J-Carotene was not detected. Identification of chlorophyll d—The absorption spectra of the major green pigment measured in several solvents were the same as those reported for chlorophyll d (Table 1). Absorption peaks of its acidification product in dimethyl ether was identical to phaeophytin d (Table 1). The 'H-NMR spectrum of the pigment (Table 2) was very similar to that of chlorophyll a, the differences being in the absence of a vinyl proton signal and the presence of a formyl signal. The assignments of the macrocycle and short side-chains of chlorophyll d were straightforward using correlation spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC) and heteronuclear multiple-bond multiple quantum coherence (HMBC), on the basis of known 'H and 13C chemical shifts of chlorophyll a (Lotjonen et al. 1987). The complete assignments of

350 400 450 500 550 600 650 700 750 Wavelength (nm) Fig. 5 Absorption spectra of pigments fractionated at each peak from the stream of HPLC. The number in each parenthesis corresponds to the peak number of pigment in Fig. 4A. For identification of the peaks, see Table 3. protons of the phytyl side-chain were made by the agreement of the chemical shifts between chlorophyll d and chlorophyll a, which was confirmed by HSQC and HMBC. The position of the formyl group was confirmed by nuclear Overhauser effect (NOE) spectroscopy, as shown in Fig. 6A. The results showed that the chemical structure of the pigment is 2-desvinyl-2-formyl chlorophyll a (3-desvinyl-3-formyl chlorophyll a by current nomenclature under International Union of Pure and Applied Chemistry; IUPAC) (Fig. 6B) which was identical with the chemical structure for chlorophyll d proposed in 1959 (Holt and Morley 1959). The molecular formula of the pigment was

278

Chlorophyll d as a predominant pigment

Table 2 karyote

'H and " C NMR assignments for the predominant green pigment in the new oxygenic photosynthetic pro-

Position

Chemical shift (ppm) 'H S (multiplicity, J in Hz)

10

147.33

3.68 (s) 11.40 (s) 10.20 (s) 3.33 (s) 3.86 (q, 7.6) 1.73 (t,7.6)