Anhydrous β-guanine crystals in a marine dinoflagellate: Structure and suggested function

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Abstract

Guanine crystals are used by certain animals, including vertebrates, to produce structural colors or to enhance vision, because of their distinctive reflective properties. Here we use cryo-SEM, cryo- FIB SEM and Raman spectroscopic imaging to characterize crystalline inclusions in a single celled photosynthesizing marine dinoflagellate species. We demonstrate spectroscopically that these inclusions are blocky crystals of anhydrous guanine in the β-polymorph. Two-dimensional cryo-SEM and three-dimensional cryo-FIB-SEM serial block face imaging show that the deposits of anhydrous guanine crystals are closely associated with the chloroplasts. We suggest that the crystalline deposits scatter light either to enhance light exploitation by the chloroplasts, or possibly for protection from UV radiation. This is consistent with the crystal locations within the cell, their shapes and their sizes. As the dinoflagellates are extremely abundant in the oceans and are a major group of photosynthesizing marine organisms, the presence of guanine crystals in this marine organism may have broad significance.

Introduction

Dinoflagellates are extremely abundant in the oceans and as they photosynthesize, they are among the most important marine primary producers (Taylor, 1987). Some dinoflagellate species are also known to produce spectacular luminescence (Haddock et al., 2010, Valiadi and Iglesias-Rodriguez, 2013). Dinoflagellates have two main life stages, the thecate/motile stage and the cyst/resting stage. In the thecate stage, they have two flagella and a cell covering made of cellulose; the theca.

Since the first studies of dinoflagellate ultrastructure using transmission electron microscopy (TEM), “organic” crystalline inclusions (‘crystal-like bodies’ or ‘crystal-like particles’) were described (Clode et al., 2009, DeSa et al., 1968, DeSa et al., 1963, Inouye and Pienaar, 1983, Kopp et al., 2013, Pokorny and Gold, 1973, Schmitter, 1971, Taylor, 1968, Yamashita et al., 2009, Zinssmeister et al., 2013). Other studies identified calcium oxalate crystals in the dinoflagellate family Suessiales based on their solubility in different acids (Doyle and Doyle, 1940) and histochemical silver staining (Taylor, 1968). Subsequent investigations of the same crystalline inclusions using Nano-SIMS, EELS and GC–MS revealed that the crystalline deposits are actually composed of uric acid (Clode et al., 2009, Kopp et al., 2013). The function of these uric acid crystals is unknown. One possibility is that uric acid deposits are part of the nitrogen cycling between cnidarians hosts and their dinoflagellate symbionts (Clode et al., 2009, Kopp et al., 2013). In the motile stage of the genus Symbiodinium an eyespot formed by uric acid crystals in an arrangement of 5–7 regularly spaced rows has been found (Yamashita et al., 2009). This assembly effectively absorbs and reflects blue-green wavelengths and is structurally connected to the flagellar apparatus, which indicates a possible role in the cell phototactic behavior. Perhaps the best known crystalline materials produced by dinoflagellates are the calcitic outer shells of the cysts that are formed by one family of dinoflagellates, the Thoracosphaeraceae (Inouye and Pienaar, 1983, Wall et al., 1970, Zinssmeister et al., 2013). Resting cysts are able to survive from several months up to a 100 years in the sediments until germination (Lundholm et al., 2011). The calcitic and organic-walled cysts are important micropaleontological components of marine sediments (Dias-Brito, 2000, Keupp, 1991, Keupp, 1981, Vink et al., 2004a, Zonneveld et al., 2005). Here we focus on some organic crystalline materials produced by dinoflagellates.

The presence of guanine crystals in the dinoflagellate species Gonyaulax polyedra was first noted by DeSa et al., 1968, DeSa et al., 1963. The guanine crystals were assumed to be an essential part of the bioluminescence system, which is referred to as a scintillon. Guanine crystals were subsequently found in several other, non-luminescent dinoflagellate species, raising questions about their functional role (Pokorny and Gold, 1973, Schmitter, 1971). Guanine deposits in dinoflagellates have, to date, been considered as a waste or storage product of the cell’s nitrogen metabolism (Schmitter, 1971).

Crystalline guanine is very interesting because of its combined hydrogen bonding and highly conjugated character. The pronounced tendency of guanine to form stacked planes of hydrogen bonded molecules is well exemplified in the crystal structure of anhydrous guanine (Guille et al., 2006, Hirsch et al., 2015). The multitude of hydrogen bonds endows the structure with great stability and with a pronounced anisotropy. The anisotropy of the crystal structures of anhydrous guanine is the reason why the crystals have very good reflector properties. When the crystals form as thin plates parallel to the hydrogen bonded planes, the refractive index in the direction perpendicular to the plate is extremely high (1.83) (Schmidt, 1949). Fish and other animals use guanine crystals mainly for light manipulation to produce structural colors and in vision to build mirrors that reflect light (Gur et al., 2017). White spiders use guanine crystals with prismatic morphology to scatter light, and this gives them their white color (Levy-Lior et al., 2010). As dinoflagellates are able to manipulate light both for photosynthesis and for luminescence, we decided to investigate whether or not dinoflagellates do indeed produce guanine crystals. With this in mind, we carried out a detailed structural and spectroscopic study of one species.

Here we investigate the nature and possible function of the crystal-like particles that were reported in the dinoflagellate species Calciodinellum operosum aff., a member of the family Thoracosphaeraceae (Inouye and Pienaar, 1983, Zinssmeister et al., 2013). C. operosum aff. is a photosynthetic autotroph and occurs typically in the surface waters (up to 75 m depth) of subtropical and tropical oceans (Dale, 1992). We show that the faceted particles are anhydrous β guanine prismatic crystals and that they are juxtaposed to the chloroplasts. We therefore raise the possibility that the crystals may function to scatter light into the chloroplast, and by so doing increase the light exploitation for the photosynthetic process.

Section snippets

Dinoflagellate cultivation

Calciodinellum operosum aff. was provided by the Culture Collection of Algae at the University of Cologne (CCAC, strain 4750B). Cultivation was performed in a 50/250 ml filter-cap cell culture flasks (Cellstar, greiner bio-one) with sterile filtrated (0.22 µm, Corning) artificial seawater (ASP-12) medium prepared according to the protocol of the CCAC (McFadden and Melkonian, 1986, McLachlan, 1973). An Innova 4230 incubator (23 °C) equipped with a Philips LED lamp (100 W, 4000 K, 1521 lm,

Results

Dinoflagellates have two main life stages: the thecate/motile stage and the cyst/resting stage. We studied cultures of C. operosum aff. that were almost exclusively composed of golden brown, photosynthetically active thecate cells undergoing vegetative division. Under the chosen growing conditions very few C. operosum aff. produce cysts.

Discussion

Here we show that the crystal-like particles in the vacuoles are crystals of the β-form of anhydrous guanine. To date, three crystal structures exist for guanine: the monohydrate form (Thewalt et al., 1971) and the α and β anhydrous structures (Guille et al., 2006, Hirsch et al., 2015). All biogenic guanine crystals examined so far adopt the β-form of anhydrous guanine (Gur et al., 2017). Both anhydrous polymorphs have a monoclinic symmetry. Extended hydrogen-bonded layers in the bc plane are

Conclusions

The crystalline inclusions observed in C. operosum aff. were identified in situ as anhydrous guanine in the biogenic β-form by Raman spectroscopy and electron diffraction. The cryo-SEM and cryo-FIB-SEM show a spatial correlation between the guanine-containing vesicles and the chloroplast network. The distribution and size range of the crystals are consistent with the guanine crystals functioning by scattering. Possible functions might be to scatter residual light back into the chloroplasts,

Acknowledgments

We thank Eyal Shimoni and Ifat Kaplan-Ashiri (Weizmann Institute of Science) for help with cryo-SEM and EDS measurements. The kind help of Barbara Melkonian (CCAC) with the provision and cultivation of the dinoflagellates is highly appreciated. Haim Weissman is gratefully acknowledged for technical assistance with the fluorescence spectrophotometer. Matthias Finger provided the SpectralImaging software and was a great help analyzing the Raman data. We thank Dr. Saskia Mimietz-Oeckler and

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