Chlorophyll d in an Epiphytic Cyanobacterium of Red Algae

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Abstract
Chlorophyll (Chl) a is found in all oxygenic photosynthetic organisms. Manning and Strain in 1943 ([ 1 ][1]) and others ([ 2 ][2]–[ 4 ][3]) reported Chl d (3-desvinyl-3-formyl Chl a) as the second Chl pigment in several species of marine macrophytic red algae, and it is now considered a genuine pigment of red algae ([ 5 ][4]–[ 7 ][5]). However, Chl d is not always detected in red algae ([ 2 ][2]) and is identical to one of the Chl a derivatives by the permanganate oxidation ([ 8 ][6]), raising controversy whether Chl d might be an artefact in the extraction process ([ 5 ][4]). In 1996, the Chl d–dominated cyanobacterium Acaryochloris marina was isolated from a colonial ascidian ([ 9 ][7]). A. marina and macrophytic red algae are phylogenetically distant from each other, so a question arose about the distribution of Chl d in red algae. Here, we have identified the source of Chl d in red algae as an epiphytic cyanobacterium living on the thalli of the red algae. Microscopic observation revealed several types of pigmented colonies living on the thallus surface of the red alga Ahnfeltiopsis flabelliformis comprising cyanobacteria, centric diatoms, and filamentous green algae. A typical example of cyanobacterial colonies ([Fig. 1A][8]) has a defined, rounded shape approximately 30 μm in diameter ([Fig. 1B][8]) with a specific fluorescence peak at 729 nm at room temperature ([Fig. 1C][8], red line), characteristic of Chl d. However, on areas of bare thallus ([Fig. 1B][8], circle with broken line), fluorescence peaked at 687 nm ([Fig. 1C][8], blue line) with a shoulder at approximately 740 nm, a spectrum common to Chl a of oxygenic photosynthetic organisms. This clearly demonstrates a lack of Chl d in red algal thalli. Chl d was obtained by methanol extraction of the cyanobacterial colony ([Fig. 1D][8], red line), but the bare thallus portion gave no signal for Chl d ([Fig. 1D][8], blue line). A second sample of cyanobacterial colonies in the amorphous shape gave identical results (fig. S1). ![ Fig. 1. ][9] Fig. 1. Microscopic observation and fluorescence spectra of epiphytes attached to the surface of Ahnfeltiopsis flabelliformis (see fig. S1). ( A ) Thallus under a stereomicroscope. Bar, 1 mm. ( B ) Magnified photograph under a bright-field microscope. Bar, 50 μm. ( C ) Microscopic fluorescence spectra of epiphytes (red line) and red algal thallus (blue line). ( D ) Absorption spectra of the methanol extracts from thalli with (red line) and without (blue line) epiphytes. A Chl d–containing cyanobacterium was isolated from colonies of fresh samples, and clonal cultures revealed a fluorescence maximum at 729 nm and an absorption maximum at 711 nm (fig. S2), very similar to authentic A. marina . Pigment analysis by high-performance liquid chromatography confirmed the organism contained Chl d as a predominant pigment (data not shown). The SSU rDNA sequence was determined (DDBJ, accession number AB112435, 1258 bases, approximately 90% of the total), and its phylogenetic position was analyzed (fig. S2). The new isolate was homologous with A. marina MBIC11017 (DDBJ, accession number AB058298), showing a difference of only 12 bases (99% identity). The isolate was designated Acaryochloris sp. strain Awaji, and formed a clade with A. marina MBIC11017, as supported by high bootstrap and quartet puzzling values; the clade was independent of other cyanobacterial subclades. Acaryochloris sp. was found not only on Ahnfeltiopsis flabelliformis but also on other marine red algae, Callophyllis japonica and Carpopeltis prolifera , as verified by typical Chl d fluorescence from their epiphytic colonies and methanol extraction (data not shown). Our findings show that Chl d is not a constituent of red algae, leading us to propose the correction of the general consensus regarding the distribution of Chl pigments in oxygenic photosynthetic organisms. Chl d biosynthesis is monophyletic in the clade of Acaryochloris and does not occur in eukaryotes. Acaryochloris , which is unique in its use of far-red light, probably lives in a variety of unexplored niches in coastal waters. Supporting Online Material [www.sciencemag.org/cgi/content/full/303/5664/1633/DC1][10] Materials and Methods Figs. S1 and S2 References 1. [↵][11] W. M. Manning, H. H. Strain, J. Biol. Chem. 151, 1 (1943). [OpenUrl][12][FREE Full Text][13] 2. [↵][14] H. H. Strain, Chloroplast Pigments and Chromatographic Analysis, 32nd Annual Priestley Lectures (Pennsylvania State Univ. Press, University Park, PA, 1958). 3. A. S. Holt, Can. J. Bot. 39, 327 (1961). [OpenUrl][15][CrossRef][16] 4. [↵][17] V. B. Estigneev, N. A. Cherkashina, Biochemistry (in Russian) 35, 48 (1970). [OpenUrl][18] 5. [↵][19] R. E. Ley, Phycology (Cambridge University Press, Cambridge, ed. 3, 1999). 6. B. B. Buchanan et al ., Biochemistry & Molecular Biology of Plants (American Society of Plant Physiologists, Rockville, MD, 2000). 7. [↵][20] R. E. Blankenship, Molecular Mechanisms of Photosynthesis (Blackwell Science, Oxford, 2002). 8. [↵][21] A. S. Holt, H. V. Morley, Can. J. Chem. 37, 507 (1959). [OpenUrl][22] 9. [↵][23] H. Miyashita et al ., Nature 383, 402 (1996). [OpenUrl][24][CrossRef][25][Web of Science][26] 10. We thank A. Tanaka for critical reading of the manuscript; T. Sakawa, S. Fujita, S. Okubo, and T. Murakami for technical assistance; M. Watanabe, M. Kamiya, and H. Kawai for technical advice; and Y. Ushihara and T. Nakano for collecting algae. This workwas in part financially supported by the Salt Science Research Foundation (A.M.), the NEDO project (H.M.), and MEXT (M.M.). [1]: #ref-1 [2]: #ref-2 [3]: #ref-4 [4]: #ref-5 [5]: #ref-7 [6]: #ref-8 [7]: #ref-9 [8]: #F1 [9]: pending:yes [10]: http://www.sciencemag.org/cgi/content/full/303/5664/1633/DC1 [11]: #xref-ref-1-1 "View reference 1 in text" [12]:...

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