Planta - Bashan Foundation

Planta (2007) 226:553–557 DOI 10.1007/s00425-007-0526-y

R A P I D CO M MU N I C A T I O N

A cryptic intracellular green alga in Ginkgo biloba: ribosomal DNA markers reveal worldwide distribution Jocelyne Trémouillaux-Guiller · Volker A. R. Huss

Received: 9 January 2007 / Accepted: 10 April 2007 / Published online: 15 May 2007 © Springer-Verlag 2007

Abstract Intracellular symbioses involving eukaryotic microalgae and a variety of heterotrophic protists and invertebrates are widespread, but are unknown in higher plants. Recently, we reported the isolation and molecular identiWcation of a Coccomyxa-like green alga from in vitro cell cultures of Ginkgo biloba L. This alga resides intracellularly in an immature “precursor” form with a nonfunctional chloroplast, implying that algal photosynthetic activity has no role in this endosymbiosis. In necrotizing Ginkgo cells, precursors evolved into mature algae, proliferated, and were liberated into the culture medium after host cell bursting. In the present paper we demonstrate by molecular methods a worldwide distribution of the alga in planta. EndosymbiontspeciWc sequences of ribosomal DNA could be traced in Ginkgo tissues of each specimen examined from diVerent geographic locations in Europe, North America, and Asia. The Ginkgo/Coccomyca association represents a new kind of intracellular, vertically inherited symbiosis. Storage bodies, probably of lipid nature, present in the cytoplasm of each partner suggest a possible involvement of the endosymbiont in metabolic pathways of its host. Keywords Coccomyxa · Endosymbiosis · Ginkgo · Ribosomal DNA

J. Trémouillaux-Guiller Département de Biologie Moléculaire et de Biochimie Végétale, UPRES-2106, Université F. Rabelais, Faculté des Sciences Pharmaceutiques, 31 Av. Monge, 37200 Tours, France e-mail: jocelyne.tremouillauxguiller@univ-tours.fr V. A. R. Huss (&) Lehrstuhl für Molekulare PXanzenphysiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany e-mail: varhuss@biologie.uni-erlangen.de

Abbreviations ITS Internal transcribed spacer MS Murashige and Skoog liquid medium PCR Polymerase chain reaction ssrRNA Small subunit ribosomal RNA

Introduction Ginkgo is regarded as the oldest living tree species on the Earth dating back to at least the early Jurassic period, more than 170 million years ago (Zhou and Zheng 2003). Ginkgo species were once common in Asia, North America, and Europe, but vanished from most regions during the Ice Age, leaving a single surviving species, G. biloba, in China. From there, Ginkgo spread to Japan and Korea. At the end of the seventeenth century Ginkgo seeds from Japanese trees were introduced to Europe and from there it spread to diVerent countries in the Western world (Strømgaard and Nakanishi 2004). Most, if not all, modern Ginkgo trees in Europe and North America are descendants of just a few Japanese trees. Owing to the presence of unique terpenoids, ginkgolides and bilobalide, and Xavonoids, Ginkgo extracts are used for treatment of, e.g. vascular disorders and age-related dementias (Braquet 1997; Strømgaard and Nakanishi 2004). In the course of research for accumulating such compounds in in vitro cultures (Laurain et al. 1993, 1997; Balz et al. 1999) we have identiWed the green eukaryotic algae present inside Ginkgo cells. These algae had been taken into culture and were identiWed by small subunit ribosomal RNA (ss rRNA) analysis to belong to the green algal genus Coccomyxa (Trémouillaux-Guiller et al. 2002). Coccomyxa is a terrestrial coccal alga known as photobiont in some lichens (Friedl and Büdel 1996; Lohtander et al. 2003), but has also been described as intracellular parasite of bivalves (Gray

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et al. 1999). Within Ginkgo host cells, the alga resides in an immature “precursor” state: nucleus and mitochondria are not observed, and diVuse electron-dense areas mark thylakoid-like membranous structures of a nonfunctional chloroplast (Fig. 1a). Mature algae with eukaryotic traits and a normal functional chloroplast (Fig. 1b) were observed almost exclusively in host cells undergoing necrosis. Such algae could be easily cultured in inorganic, ammoniumcontaining medium allowing identiWcation and characterization of the endosymbionts (Trémouillaux-Guiller et al. 2002). As existence of a green algal endosymbiont in G. biloba was never reported or observed in planta, we used endosymbiont-speciWc sequences of ribosomal RNA genes to address the following questions: does the endosymbiont naturally occur in Ginkgo trees, in which tissues can it be identiWed, and is the symbiont ubiquitously distributed in Ginkgo trees worldwide?

Materials and methods Sampling Juvenile or fertilized G. biloba L. ovules were harvested from female trees in Botanical or private gardens, as well as from Weld or alley trees from diVerent geographic locations (i.e., China, France, Italy, Japan, and the United States) and surface-sterilized before excising zygotic embryos of 6– 22 mm in length from the gametophyte tissues. Microsporophylls were taken from male trees and sterilized, as were all plant samples, by successive treatment with 70% (v/v) ethanol for 1 min, 10 or 20% (v/v) Domestos commercial bleach solution [0.5–2% (v/v) NaOCl; Lever Bros., Warrington, UK] for 5 min under vigorous shaking, and with sterilized distilled water, before being quickly frozen in

Fig. 1 Electron micrographs of two endosymbiotic algae freshly escaped from in vitro-cultured Ginkgo biloba cells. a Immature precursor alga showing within its cytoplasm large lipid droplets (black arrows) and electron-dense material (white arrows), which subsequently

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liquid nitrogen. Details on the geographic location, time of harvest and type of tissue of all G. biloba specimens studied are shown in Table 1. DNA isolation Approximately 300–500 mg of either sterilized frozen G. biloba tissues or of sedimented cell pellet of the endosymbiont (isolate CMS-93) cultured in MS medium (Murashige and Skoog 1962) were ground to Wne powder. DNA was extracted with the DNeasy Plant minikit (Qiagen). In total, 18, 17, and 11 independent DNA extractions were performed from embryos, and from reproductive and vegetative tissues, respectively. Gene ampliWcation and sequencing Two sets of primers were designed to speciWcally amplify endosymbiont DNA sequences from Ginkgo tissues by PCR. The Wrst set (5⬘-TCACGCCTGGGCTCCCCAG-3⬘ and 5⬘-GAACCGCCGGAAGCCGCCA-3⬘) with an expected ampliWcation product size of 352 bp was targeted against regions of the ss rRNA gene with suYcient sequence heterogeneity among symbiont (GenBank accession number AJ302939) and host (D16448). The second primer set with an expected ampliWcation product size of 387 bp was targeted against a region close to the end of the ss rRNA gene (5⬘-GTTAAACCCTCCCACCTAG-3⬘) and to the terminal region of ITS1 (5⬘-TGGTTTGAAGGCAGCGCTT-3⬘) of the symbiont (AJ880282). Amounts of DNA template from Ginkgo tissues were up to 1.5 g compared to 1–10 ng from the cultured alga used as control. Among the tested DNA polymerases, either the Invitrogen Taq polymerase (Invitrogen Life Technology) or the TaKaRa Ex Taq polymerase (TaKaRa Biomedicals, SaintGermain-en-Laye, France) worked best for the PCR reactions.

evolved into thylakoids. b Mature alga with a nucleus (black arrowhead), mitochondria (white arrows), a cup-shaped chloroplast (white arrowhead), and lipid droplets (black arrows). Scale bar = 1.0 m

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Table 1 Details on the geographic location, time of harvest, and type of tissue isolated from G. biloba specimens Continent

Country

Asia

China

North America

Europe

Location

Tissue

Time of harvest

PCRresult

Sequencing

ss rDNA; ITS1

Commercial seeds (Vilmorin)

Embryo

Autumn

+

Wuhan-Hubei; in Welds

Cotyledons

Autumn

+

Japan

Hiroshima; Botanical Garden

Embryo

Autumn

+

USA

Yellow Springs (Ohio); alley tree

Embryo

Autumn

+


Hypocotyl

June

+


Roots

June

¡

Tours; Botanical GardenSt. Cyr; Private Garden

Embryo

November

+

ss rDNA; ITS1

Tours; Botanical Garden

Prothallus

July

+

ss rDNA; ITS1


Ovule

August

+

ss rDNA


Microsporophyll

March-April

+

ss rDNA


Pollen

April-May

+

ss rDNA; ITS1


Hypocotyl

June

+


Older leaves

July

¡

Commercial seeds (Vilmorin)Bologna-Ferrara; Private Garden

Embryo

Autumn

+


Cotyledons

Autumn

+


Hypocotyl

Autumn

¡ ¡

France

Italy

Germany

ITS1

ss rDNA

Erlangen; Botanical Garden

Older leaves

August


Young leaves

April

+

ss rDNA


Pollen

April

+

ss rDNA

Commercial plant; Garden Centre

Roots

October

¡

Detection of the Coccomyxa endosymbiont is indicated by a positive PCR reaction with endosymbiont-speciWc primers for either 18S rDNA, ITS1, or both. Sequences of several PCR products were exemplarily determined and were all completely identical with the respective sequence of the cultured endosymbiont

Up to 20 PCR reactions were performed for each DNA isolated from embryos and reproductive and vegetative tissues to check the presence of the respective 352 and 387 bp PCR bands. AmpliWcation products were sequenced either by Genome Express (Grenoble, France) with a 3730 XLTM Sequencer (Applied Biosystems) or by ourselves with an ABI Prism 310 Genetic Analyzer (Applied Biosystems).

Results PCR ampliWcation and sequence comparisons We designed two sets of primers speciWcally to amplify the endosymbiont DNA from Ginkgo tissues by PCR. The Wrst set of primers was targeted to regions of the endosymbiont ss rRNA genes with suYcient sequence heterogeneity compared to the corresponding Ginkgo sequence (Chaw et al. 1993). With these primers, diVerent tissues collected from several Ginkgo trees from Asia, Europe, and North America were assayed for presence of the endosymbiont. In order to obtain reasonable PCR

products, it was necessary to enhance DNA quantities from G. biloba tissue extracts up to 1,000-fold compared to the endosymbiont control DNA. Moreover, the choice of particular Taq polymerase was critical for obtaining a positive result. Using optimized PCR conditions, DNA samples taken from reproductive tissues such as microsporophylls, pollen grains, ovules, and prothallus, as well as from complete zygotic embryos yielded the expected 352 bp band for all Ginkgo specimens studied (Fig. 2a, b). Sequences obtained from the ampliWcation products were completely identical among each other and to those from the cultured symbionts (Table 1). For vegetative tissues, the presence of the endosymbiont could be conWrmed so far only for cotyledons and young leaves (data not shown). However, the negative PCR reactions for older leaves might be caused by a dilution eVect of the fast growing leaves and thus by very low endosymbiont amounts in these tissues. Failure to obtain PCR products from organs that are utmost exposed to their environment such as leaves, on the other hand, serves as a reliable negative control to exclude exogenous contamination as the source for the presence of the alga.

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Fig. 2 Molecular detection of endosymbiotic algae in various Ginkgo biloba tissues collected from diVerent geographic locations. a, b A 352 bp fragment of the ss rRNA gene sequence ampliWed with symbiont-speciWc PCR primers from total DNA extracts from Italian embryo (IE), French microsporophyll (FM), French prothallus (FP), and Chinese embryo (CE), and from French prothallus (Pr), ovule (Ov), micro-

sporophyll (Mi), and pollen grains (Po). c, d A 387 bp fragment of the symbiont ITS1 sequence was ampliWed from embryos collected in China (CE), Italy (IE), France (FE), and the United States (AE), and from prothallus (Pr), ovule (Ov), pollen grains (Po), and microsporophyll (Mi) from French trees. DNA of cultured symbionts (strain CMS-93) as control c S size marker (100 bp ladder)

A common ancestry of endosymbionts in modern Ginkgo trees

Discussion

As outlined above, most Ginkgo trees have a separate evolutionary history of only about 300 years. If the ancestor of these trees already contained the endosymbiont, we would not expect to Wnd diVerences within the conserved ss rRNA gene even in geographically separate specimens. The ITS region of the ribosomal cistron is much more variable and widely used for phylogenetic inference at low taxonomic levels, including biogeographic studies (Álvarez and Wendel 2003). A second primer set was therefore directed against the ITS1 region of the alga. This region is similar to only about 70% between the endosymbiont and the lichen-forming Coccomyxa glaronensis (Lohtander et al. 2003), while their ss rRNA genes share a sequence similarity of 99.5%. Using this primer set, we obtained ampliWcation products of the expected size 387 bp for several tissues of Ginkgo specimens from diVerent geographic locations (Fig. 2c, d). All ITS1 sequences ampliWed from Ginkgo tissues again completely matched the sequence from the cultured symbiont (Table 1). This could imply a recent origin of the event that led to the establishment of this intracellular symbiosis. Alternatively, and more plausibly, the observed genetic homogeneity of endosymbiont populations might be caused by the short evolutionary history most Ginkgo trees share. According to this idea, the Japanese ancestor(s) of modern Ginkgo trees, from which seeds were Wrst brought to Europe and their oVspring then distributed all over the world, did already contain the endosymbiont. Even the variable ITS1 DNA did not accumulate mutations within the following 300 years of separate evolution till today. Investigation of wild-growing Ginkgo that might still exist in China should resolve this question.

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The kind of symbiosis described here between a cryptic green eukaryotic alga and a higher plant is unprecedented. Intracellular symbioses usually have co-evolved to the beneWt of at least one partner, resulting in host/symbiont relationships deWned as commensalism when neutral to the host, as parasitism when injurious to the host, and as mutualism when beneWcial for both partners (Parniske 2000). The type of symbiosis in the Ginkgo/Coccomyxa association is unknown but likely to be beneWcial for at least the host. Unicellular algae are widely found as intracellular symbionts in diverse groups of organisms such as invertebrates and protists (Huss 1999). Commonly in this kind of symbiosis, the algae complement their host’s diet by providing photosynthetically Wxed energy-rich carbon compounds, eventually becoming indispensable for some hosts, such as corals (Lewis and CoVroth 2004). Such a role for the Ginkgo endosymbiont can be ruled out for two reasons: Ginkgo itself is photoautotrophic, and the in situ precursor forms of the endosymbiont are likely to be photosynthetically inactive (Fig. 1a). During the development from precursors into mature algae, transition forms were observed by transmission electron microscopy (TEM) representing diVerent maturation degrees (Trémouillaux-Guiller et al. 2002). Although mature algae showed the typical eukaryotic organelles (Fig. 1b), neither nucleus nor mitochondria or plastids could be identiWed in the immature precursors (Fig. 1a). Instead, diVuse electron-dense areas were visible, which can be interpreted as thylakoid-like membranous structures of an obviously nonfunctional chloroplast. Upon maturation they evolved into thylakoid membranes, and chloroplasts became visible. An interesting phenomenon of

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membrane reorganization could be followed by TEM, although biogenesis of plastids is classically admitted to follow the division of pre-existing organelles. Moreover, TEM pictures showed numerous storage bodies, probably of lipid nature, in the cytoplasm of endosymbionts as well as in Ginkgo host cells (Trémouillaux-Guiller et al. 2002). Similar storage bodies were observed in Coccomyxa phycobionts of ascolichens, while they were absent in cultured isolates (Peveling and Galun 1976). This suggests a possible involvement of the endosymbiont in metabolic pathways of its host. Abundant lipid biosynthesis is indicated by mitochondria with tubular cristae present inside mature Ginkgo symbionts, implying a lipid nature of the storage bodies (Prince 1999). How could an alga become an inherent part of a tree like Ginkgo? Our hypothesis is linked to the unique fertilization process of Ginkgo. As a lichen alga, Coccomyxa without doubt sometimes lands upon a pollination droplet, which covers the micropyle of young Ginkgo ovules. The alga would be retracted together with the pollen inside the pollen chamber, where it may survive the 4–5 months until fertilization (Hori and Miyamura 1997; Soma 1997). Considering the size of Coccomyxa, which is about 20 times smaller than a Ginkgo spermatozoid, it seems possible that an algal cell fortuitously could have entered the egg cell along with sperm. Once in the egg cell, it had to escape digestion like any other intracellular symbiont, adjust reproduction rate, and become an inherent part of the host. Undoubtedly, such a process leading to a stable endosymbiosis is a rare event, but recurrently exempliWed by the diverse endosymbiotic associations found in nature. First detected in in vitro cell cultures of Ginkgo tissues, we now could demonstrate its existence in planta in geographically dispersed Ginkgo trees. Acknowledegments We thank Drs. Katsuhiko Kondo (University of Hiroshima, Japan), Terumitsu Hori (University of Tsukuba, Japan), Walter Tulecke (Antioch College, Yellow Springs, OH, USA), Philippe Damas (Pharmacie de Chinon, France), as well as Mr. Robert Nadreau (Université et Jardin Botanique de Tours, France) and Mrs. Marie Christine Maïquès (St. Cyr/Loire, France) for kindly providing G. biloba ovules from diVerent geographic locations, and Dr. Brigitte Arbeille (UFR de Médecine, Université F. Rabelais de Tours, France) for assistance in electron microscopy.

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