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(American Journal of Botany. 2002;89:727-733.)
© 2002 Botanical Society of America, Inc.

Anatomy and Morphology

Discovery of an endophytic alga in Ginkgo biloba1

Jocelyne Trémouillaux-Guiller2,5, Thomas Rohr3, René Rohr4 and Volker A. R. Huss3

2Département de Biologie Moléculaire et de Biochimie Végétale, UPRES-2106, Université F. Rabelais, Faculté des Sciences Pharmaceutiques, 31, Av. Monge F-37200 Tours, France; 3Institut für Botanik und Pharmazeutische Biologie der Universität, Staudtstrasse 5, D-91058 Erlangen, Germany; 4Laboratoire d'Interactions et Micropropagation, UMR-5557, Ecologie Microbienne, Faculté des Sciences, Bat. 405, Université Claude Bernard Lyon I, F-69622 Villeurbanne, France

Received for publication September 4, 2001. Accepted for publication December 13, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Although intracellular associations with mycorrhizal fungi are known for Ginkgo biloba, no other endosymbiotic relationships have ever been reported for this "living fossil." A protoplast culture derived from haploid explants has now revealed the existence of a green alga in vitro, whose eukaryotic status was confirmed by transmission electron microscopic studies. Phylogenetic 18S rDNA sequence analyses showed this alga to be closely related to the lichen photobiont Coccomyxa. Algae, which in host cells exist as more or less undifferentiated "precursor" forms, proliferated within necrosing G. biloba cells of a subculture derived from a zygotic embryo and were finally released into the medium. Light and electron microscopic observations showed that G. biloba cells rapidly filled up with countless green particles whose number increased up to the bursting of the hypertrophic host cells. At the beginning of reproduction no algae were visible in the nutritive medium, demonstrating that the proliferation started inside the G. biloba cells and excluding the possibility of an exogenous contamination. Occasionally, mature algae together with their precursor forms were detected by transmission electron microscopy in intact host cells of a green callus. The algae were easily identified by their similarity to the cultured algae. Eukaryotic algae have never been reported to date to reside inside higher plant cells, whereas several algal associations are well known from the animal kingdom.

Key Words: alga • cell culture • Coccomycxa • endophyte • eukaryote • Ginkgoaceae • Ginkgo biloba • SSU rRNA • TEM


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Ginkgo biloba of the family Ginkgoaceae is the single survivor of the order Ginkgoales that reached its peak from Upper Jurassic to lower Cretaceous. Called a "living fossil," this tree, native to Eastern China, is characterized by conspicuous features such as a zoidogamous fertilization process discovered by Hirase (1896) , leaves with a dichotomous nervation, and unique molecules (ginkgolides and bilobalide) effective in treating certain aging disorders (Braquet, 1997 ). Moreover, this dioecious tree possesses an aquatic fertilization mode, a photosynthetic female prothallus, and motile spermatozoids similar to ferns. Numerous pharmaceutical studies (reviewed by Juretzek, 1997 ) and some work on haploid tissue cultures (Tulecke, 1953, 1957 ), ultrastructure (Rohr, 1978 ), and protoplast biotechnology (Laurain, Trémouillaux-Guiller, and Chénieux, 1993 ; Trémouillaux-Guiller, 1997 ) have been published on Ginkgo biloba. Some fungal species have been found in Ginkgo biloba roots as parasites or as symbionts in arbuscular-vesicular mycorrhizae (Bonfante-Fasolo and Fontana, 1985 ; Fontana, 1985 ), as reviewed by Aoki (1997) . In contrast, intracellular endophytic algae are neither known from Ginkgo nor from any other land plants except for some occasional reports of, e.g., the xanthophyte Myxochloris living in the hyaline cells of Sphagnum (reviewed by Round, 1992 ) and the chlorophyte Chlorochytrium growing intracellularly in some bryophytes and, most conspicuous, invading sub-epidermal tissue of Lemna (reviewed by Chapman and Waters, 1992 ). However, the original reports of these cases are old, and none of them has been reconfirmed and investigated using modern methods such as transmission electron microscopy or molecular systematics.

Here we describe a eukaryotic and endophytic unicellular alga whose intense proliferation and release is closely linked to the death of Ginkgo biloba host cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Primary explant cultures
Protoplast isolation from microspore mother cells
Microsporophylls or catkins (i.e., reproductive male organs) were taken, in 1993, from a Ginkgo biloba tree in the Botanical Garden of Tours (France) and surface sterilized with 7.5% (mass/volume) calcium hypochlorite solution by vigorously shaking for 5–10 min and rinsing three times with sterilized distilled water. Each microsporophyll was gently burst with a forceps, liberating large quantities of microspore mother cells, from which protoplasts were isolated after incubating in various enzyme mixtures containing 2% (mass/volume) cellulase «Onozuka» R10 (Yakult Honsha, Tokyo, Japan), 1% (mass/volume) pectolyase (Sigma, St. Louis, Missouri, USA), 1% (mass/volume) cytohelicase or 2% (mass/volume) Macerozyme R10 (Yakult Honsha), 1% (mass/volume) driselase (Sigma) or 2.5% (mass/volume) cellulase, 1.25% (mass/volume) Macerozyme R10, 0.2% (mass/volume) cellulase or 0.25% (mass/volume) pectolyase, each in a solution of 0.6 mol/L mannitol and 0.034 mol/L CaCl2*2H2O for 4–16 h. Then, the protoplasts were inoculated in the liquid Bourgin and Nitsch (1967) medium with or without growth regulators (Trémouillaux-Guiller, Laurain, and Chénieux, 1996 ).

Green microsporophyll tissue-derived calli
After sterilization as described above, young microsporophylls were vigorously burst (in 2000). Large quantities of microspore mother cells were released in liquid Westvaco (WV5) medium (Duchefa-Kalys Biotechnologies, Roubaix, France), including vitamins and supplemented with 26.85 µmol/L naphtalene acetic acid (NAA) and 0.045 µmol/L thidiazuron. Four months later, only one of six microspore mother cell cultures developed green calli, whereas two cultures released algae in the extracellular medium.

Hypocotyl and cotyledon cultures
In 1999, zygotic embryos were extracted at the cotyledonary stage from ovules harvested in China and plated on solid Ball (1959) medium. Then, the hypocotyls or cotyledons were independently transferred into liquid MS culture medium (Murashige and Skoog, 1962 ) supplemented with 9.05 µmol/L 2,4-D dichlorophenoxyacetic acid, 0.46 µmol/L kinetin, and 0.44 µmol/L benzyladenine and agitated.

Establishment of cellular suspension cultures
Zygotic embryo-derived "CGS" suspension
Zygotic embryos were carefully extracted from sterilized ovules (i.e., megagametophytes) harvested in November 1995 from a G. biloba female tree of the Botanical Garden of Tours (France), then cultured on solid MS medium supplemented with 0.48 µmol/L kinetin and 5.37 µmol/L NAA to allow callus development. Subsequently, the embryo-derived calli led to "compact globular structures" (termed CGS) after transfer to agitated liquid MS medium supplemented with growth regulators.

Protoplast isolation from a prothallus-derived cell suspension
Ovules were harvested in July 1994 from a female tree in the Botanical Garden of Tours and sterilized before extraction of prothalli. After cutting the prothalli in two, the halves were placed on solid MS medium supplemented with 10.74 µmol/L NAA and 0.93 µmol/L kinetin. After 2 mo, a green callus was derived from a prothallus half-section. A fast proliferating embryogenic cell suspension was initiated by introducing 0.65 g of green callus into 25 mL MS liquid medium supplemented with growth regulators. It grows in darkness. Protoplasts could be easily isolated from a 6-d-old female subculture and were incubated for 8 h in an enzyme mixture containing 0.5 g/L (mass/volume) cellulase «Onozuka» R10 (Yakult Honsha), 0.25 g/L (mass/volume) Macerozyme R10 (Yakult Honsha), 0.5 mol/L sorbitol, and 0.034 mol/L CaCl2*2H2O (pH 5.5). Purified protoplasts were cultured in the liquid Nitsch and Nitsch (1969) medium supplemented with 20 mg/L of 7-aza-indole, a substance that is able to inhibit auxin biosynthesis according to Kochba and Spiegel-Roy (1977) . The protoplast-derived culture was maintained in the agitated and hormone-free MS medium.

Microscopy observations
Light microscopy
In vitro cultures of Ginkgo biloba were observed with an inverted microscope Olympus IM (Olympus Optical, Tokyo, Japan) or Leica DM IRB (Leica Mikroscopie, Wetzlar, Germany) and micrographs were taken with an Olympus C-35 (Olympus Optical) and a Nixon F-601 M camera (Nixon-France S.A., Champigny-sur-Marne, France).

Transmission electron microscopy (TEM)
Free endophytic algae, cell aggregates, or suspensions of Ginkgo biloba were fixed by immersion in 1% formaldehyde and 4% paraformaldehyde in 0.1 mol/L phosphate buffer at room temperature for 10 min and then at 4°C overnight. After washing in 0.1 mol/L phosphate buffer and 0.2% NaCl, the cells were post-fixed with 2% osmium tetroxide in 0.1 mol/L phosphate buffer for 1 h. Cells were dehydrated with increasing alcohol concentrations of an alcohol/propylene-oxide mixture (50, 70, 90, and 100% alcohol) and embedded in epoxy resin (epon) at 60°C for 24 h. Ultrathin sections (75 nm) obtained with an ultramicrotome Ultracut Reichert and Reichert-Jung (Reichert-Leica, Vienna, Austria) were stained each 10 min with uranyl acetate and lead citrate. Direct observations and electron micrographs were made with a JEM 1010 (JEOL, Tokyo, Japan) or a Philips CM 120 transmission electron microscope (Philips, Eidhoven, The Netherlands).

Phylogenetic analyses
DNA isolation, amplification, and sequencing
DNA of cultured algae was isolated and purified as described (Huss et al., 1986 ). Nuclear 18S rRNA genes were amplified from total genomic DNA by the polymerase chain reaction (PCR) with eukaryote specific amplification primers (Huss et al., 1999 ). The amplified DNA fragments were sequenced in an ABI Prism 310 Genetic Analyzer (Perkin-Elmer, Foster City, California, USA) according to the manufacturer's recommendations. Sequences of both strands were determined using oligonucleotide primers complementary to conserved regions of the 18S rRNA (Huss et al., 1999 ). The sequences were manually aligned using the sequence editor program distributed by G. Olsen (Olsen et al., 1992 ). Reference sequences were taken from GenBank except Coccomyxa spec., which was generously provided by T. Friedl prior to publication. GenBank accessions for the endophytic algal strains CMS-93 and BC-98 are GBAN-AJ302939 and GBAN-AJ302940, respectively.

Reconstruction of phylogenetic trees
Phylogenetic trees were inferred by the neighbor-joining (NJ), the maximum parsimony (MP), and the maximum likelihood (ML) method using PAUP, version 4.0 b3a (Swofford, 2000 ). For the ML topology shown in Fig. 7 the following settings were used: the transition/transversion rate was set to 2, nucleotide frequencies and the proportion of invariant sites were estimated by maximum likelihood; the stepwise addition of sequences was random. For bootstrap analyses, each 1000 replicates were calculated with MP (261 parsimony-informative characters) and NJ (Kimura two-parameter method with a gamma shape parameter of 0.5 for among-site variation and stepwise addition of input sequences replicated ten times), and 100 replicates with ML.



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  		Fig. 7. Phylogenetic position of the endophytic algae. A phylogenetic tree was derived from small subunit rRNA gene sequences of Ginkgo biloba endophytes CMS-93 and BC-98 and several representative green algae (Chlorophyta). Algae found as endosymbionts of invertebrates (Hydra) or as lichen photobionts are typed in bold. Branches drawn with thick lines are supported by bootstrap values of more than 80% obtained with three independent methods of reconstructing the evolutionary history (maximum parsimony, neighbor joining, and maximum likelihood). One hundred percent bootstrap support with all three methods is indicated above branches. The ulvophycean algae Gloeotilopsis planctonica and Ulothrix zonata were used as outgroups

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
First detection of algae
Green microorganisms were directly isolated from primary explants of different ploidy levels and genotypes or from well-established Ginkgo biloba cell suspensions. At first, these organisms were observed in a protoplast culture derived from microspore mother cells. These protoplasts, which were unable to divide in such a nutritive medium, disappeared after 10 wk, and instead green spherical structures with a clear center and up to eight peripheral particles became apparent. These particles, which probably corresponded to autospores whose proliferation and release was limited by the hypertonic medium, could be identified after deplasmolysis as individual algae. They grew under continuous light in agitated liquid medium with or without carbohydrates (glucose or sucrose), whereby a fast-growing alga strain, referred to as CMS-93, was established. In vitro, the algal growth was dependent on an ammonium source but was notably slowed down or stopped by nitrate.

Eukaryotic status
By TEM, the immobile cells, 4.10 ± 0.8 µm long and 3.6 ± 0.8 µm wide, were shown to be eukaryotic. They possessed a true nucleus, several mitochondria with tubular cristae, and a voluminous chloroplast with stacked thylakoids (Fig. 1). No grana could be identified. In some algae, starch granules were visible between the thylakoids. Some pictures showed a nucleus with one or two nucleoli and endoplasmic reticulum (not shown). The mitochondrial tubular cristae indicated that an intensive lipid synthesis occurred. All organelles as well as lipid droplets were included in a more or less dense cytosol that was surrounded by a plasma membrane and a thin cell wall of 33 nm up to 40–50 nm depending on the maturation degree of the alga.



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  		Fig. 1. A mature green alga, belonging to the BC-98 strain, shows most of the characteristic organelles of eukaryotic plant cells: a nucleus (N), mitochondria (M) with tubular cristae, and a voluminous chloroplast (Ch) with thylakoids. Note the presence of lipid droplets (LD) in a cytoplasm surrounded by a homogeneous cell wall (CW). Scale bar = 500 nm

 
Proliferation and expulsion phase
Later, algae appeared in a zygotic embryo-derived CGS suspension of G. biloba that seemed to be undergoing necrosis. Light microscopic observations of this subculture grown in agitated liquid MS medium showed that approximately 10% of the G. biloba cells rapidly filled up with countless green particles (Fig. 2A–D) similar to chloroplasts, whose number increased up to the bursting of the hypertrophic host cell (Fig. 2E). No algae were visible in the nutritive medium prior to the burst of host cells (Fig. 2A–D), demonstrating that the algal proliferation started inside G. biloba cells and excluded a possible exogenous contamination. Figure 2A–E chronologically ranks different stages of the developmental process that led to the massive intracellular division and expulsion of the algae from dead G. biloba cells. First, a degenerative host cell (Fig. 2A), prior to its complete necrosis, exhibited a yellowish cytoplasm that was pushed aside by the intensive algal growth. As development proceeded, some algae escaped from a hypertrophic cell whose cell wall had opened (Fig. 2D), while Fig. 2E shows cellular remnants and many algae in the culture medium after breakdown of the cell wall. The TEM analyses confirmed that the algae proliferated inside G. biloba cells (Fig. 2F). From this embryo-derived CGS suspension culture, a second strain of algae, BC-98, was isolated and maintained.



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  		Fig. 2. Algal proliferation phase, observed with an inverted microscope from the zygotic embryo-derived subculture cells. (A) Necrotizing host cell, exhibiting a yellowish cytoplasm and massive growth of the algae. (B) G. biloba cell completely filled with algae contrasting another necrotic but alga-free cell that is entirely empty. (C) Incomplete internal proliferation of algae permitting a partial view of the host cell wall. Note the total absence of algae in the nutritive medium at the beginning of algal proliferation (A–C). (D) Hypertrophic cell with an opening allowing the escape of algae into the nutritive medium. (E) Remnants of a burst cell and numerous free algae in the medium. (F) TEM picture confirming the growth of algae and the presence of voluminous precursors inside host cells. Remnants of cell wall are surrounding some algae. The cell wall of G. biloba delimits the internal from the alga-free external medium. Figure Abbreviations: A, alga(e); BC, burst cell; C, cell; CW, cell wall; Cy, cytoplasm; EM, external medium; IM, internal medium; LC, hypertonic cell; NC, necrotic cell; Op, opening; Rmb, remnants of cell walls; VP, voluminous precursor. Respective scale bars are 12.5 µm (A)–(E) and 4 µm (F)

 
Precursor and mature forms
During the proliferation and expulsion phase, transition forms from undifferentiated "precursor" forms to mature algae were revealed by electron microscopy. These forms obviously corresponded to different maturation degrees (Fig. 3). The size of free precursor forms inside necrotic host cells was about 3.8 ± 0.6 µm in length. Lipid droplets were apparent at all stages, largely filling the cytoplasm of some precursors (Fig. 3D). Most of the immature algae escaped from the host cell, sometimes with several layers of mother cell wall remnants attached to their surface (Fig. 3C). Precursor algal cells were found at various stages of cell division (Fig. 3A–C), and their eukaryotic status was confirmed through the presence of a central nucleus and mitochondria (Fig. 3D). The dividing precursor cells contained electron-dense membrane systems corresponding to future thylakoids and were associated with lipid droplets in a fine-granular cytoplasm whose appearance was similar to the stroma of G. biloba plastids (Fig. 3B–C). Figure 3A shows an immature and voluminous (10.5 µm long and 5.3 µm wide) algal precursor inside an intact G. biloba cell that contained several electron dense areas corresponding at least partly to future thylakoids of daughter algae. Cytokinesis was characterized by a wide invagination in the equatorial region. In addition, some short cytoplasmic projections are visible at the periphery of this precursor cell that might be involved in the division process and result from the adhesion of the precursor's plasma membrane to the cell wall (Fig. 3A). Nevertheless, such cytoplasmic projections as well as lipid droplets were found in several more or less mature precursors and could be considered algal markers. Moreover, the existence of precursor algal cells inside the host cells indicates that this alga was a natural intracellular endophyte of G. biloba. At this stage, no mature algae could be detected inside intact host cytoplasm by TEM.



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  		Fig. 3. Alga precursor forms. (A) Voluminous precursor form inside a living cell of G. biloba with a wide invagination, short cytoplasmic projections, and electron dense areas corresponding, at least partly, to future thylakoids of several daughter cells. (B) Dividing precursor inside a necrotic G. biloba cell (note the host cell wall) exhibiting a short invagination in the equatorial region (between two future daughter cells) and lipid droplets near future thylakoids in a clear cytoplasm. (C) After complete cytokinesis, several cell wall layers delimit the new precursors possessing future thylakoids and lipid droplets. (D) Transition form of an alga (between precursor and mature alga) showing a large nucleus, several mitochondria with tubular cristae, numerous lipid droplets, and cytoplasmic projections. Figure Abbreviations: CW, cell wall; CWR, cell wall remnants; CP, cytoplasmic projection; FT, future thylakoids; INV, invagination; LD, lipid droplets; M, mitochondrion; N, nucleus. Scale bars = 1 µm for (A) and 500 nm for (B)–(D)

 
Mature alga inside a living host cell
Subsequently, thin sections from a green callus derived from microsporophyll tissues (probably from green tapetal cells) and cultured in liquid WV5 medium without agitation revealed the presence of a mature alga inside a living cell of G. biloba (Fig. 4B–C) easily identified by its similarity to the cultured algae, in particular to those that were only recently released into the extracellular medium (Fig. 4A).The alga, about 5 µm in length and 4 µm in width, was enclosed in the intact host cell cytoplasm close the large amyloplastids (Fig. 4B). The cytoplasm of the host cells stored large amounts of lipid droplets resembling those of the algae (Fig. 5).



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  		Fig. 4. Mature alga inside a living G. biloba cell. (A) Freshly released alga with a voluminous chloroplast, numerous lipid droplets, and cell wall remnants. (B) Living host cell harboring a mature alga with lipid droplets and amyloplasts with voluminous starch granules. (C) Detail from B: mature alga with lipid droplets and a cytoplasmic projection. Figure Abbreviations: A, alga; AP, amyloplast; CWR, cell wall remnants; CH, chloroplast; CP, cytoplasmic projection; LD, lipid droplets; S, starch granule. Scale bars for (A)–(C) = 1, 2, and 1 µm, respectively

 


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  		Fig. 5. Ultrastructure of Ginkgo biloba cells. Inside the cytoplasm of cells derived from a green callus, several amyloplasts (AP) and lipid droplets (LD) similar to those found in the alga can be seen. Scale bar = 5 µm

 
Green algae could also be detected from other primary explants placed in agitated liquid media as hypocotyls and cotyledons or from a protoplast-derived cell suspension isolated from a prothallus-derived cell line. After 3 mo of in vitro culture in darkness, the dead cells derived from explants released a large number of precursor and mature algae, most of them displaying numerous lipid droplets in their cytoplasm (Fig. 6). Within a period of 10 yr, a total of six strains of algae were isolated from various G. biloba explants or cell suspensions and taken into culture.



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  		Fig. 6. Internal proliferation of algae. Several algae (A) with a large number of lipid droplets (LD) were observed in a necrotic host cell (note its cell wall [CW]) belonging to a primary 3-mo-old cotyledon culture of G. biloba. One alga displays a compact nucleolus (Nu). Scale bar = 2 µm

 
Phylogenetic position of isolated algae
The nuclear small subunit ribosomal RNA (SSU rRNA) gene sequences of both strains (CMS-93 and BC-98) of endophytic algae that were isolated from G. biloba cell cultures as described above were identical and had a length of 1798 base pairs (bp). The sequences were compared with all available green algal SSU rRNA gene sequences. Phylogenetic analyses based on a representative excerpt of these sequences showed (Fig. 7) that the endophytes belong to trebouxiophytes, a class of green algae with several members known to be involved in symbiotic associations with protists, invertebrates, and fungi (Gärtner, 1992 ; Friedl and Büdel, 1996 ; Huss, 1999 ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The continuous presence of the intracellular endophytes within G. biloba cells raises several questions. Firstly, why is it so difficult to detect mature algae inside living cells? We have demonstrated that proliferation and liberation of algae inevitably provoked the Ginkgo cell decay. Such events occurred only in the G. biloba cells cultured in liquid medium, agitated or not. On the other hand, no algae have been detected from root cell cultures up to now. It is conceivable that only some tissues of G. biloba harbor alga precursors, in particular the mother cells of the gametophytes. Thus, the observation of a mature alga in an intact host cell was probably a rare event, since the intense proliferation of algae would destroy the host cells. Without doubt, it is the more immature precursor form of the alga that can be tolerated in situ, and it could be mistaken for a plastid.

Secondly, it seems obvious that maturation and division of alga precursors probably requires environmental conditions rarely achieved. Indeed, necrosis after cell aging did not necessarily lead to algal proliferation. In the course of numerous cell culture experiments with G. biloba, no particular genotype, ploidy level, medium, primary explant, cell suspension, or growth regulator seemed to be a factor capable of triggering host cell death followed by proliferation of algae. Thirdly, a priori what symbiotic advantage may two photoautotrophic species have with respect to the carbon source in as much as the alga precursors apparently do not possess functional plastids? TEM pictures showed the simultaneous presence of lipid droplets in the cytosol of both partners (Figs. 5 and 6). This suggests a possible involvement of the endophyte in major metabolic pathways of G. biloba, as lipid (Ohlrogge and Browse, 1995 ) or terpenoid biosynthesis by the Rohmer's pathway (Rohmer et al., 1993 ) normally occur in plastids. It is known that among other organisms, e.g., some algae, mosses, and higher plants, including G. biloba, use both mevalonate and pyruvate (Rohmer's) pathways (Eisenreich, Rohdich, and Bacher, 2001 ).

Our TEM analyses convincingly prove the eukaryotic and endophytic status of the observed algae as well as the existence of precursor forms, and at the same time exclude an exogenous contamination from intercellular spaces of G. biloba tissues or from the culture medium. The intracellular association of a green alga with G. biloba represents a new type of plant–plant interaction, as no eukaryotic algae have been reported to date to reside inside higher plant cells (with the possible exception of Lemna; see Introduction). Our molecular analyses indicate a close phylogenetic relationship of the G. biloba endophytes to the lichen photobiont Coccomyxa spec. (Fig. 7). Coccomyxa is a unicellular alga that often builds up gelatinous colonies and that propagates by autosporulation just as the G. biloba algae. Coccomyxa species live terrestrially or aerophytically and, in addition to lichens, can be isolated from soil, wood, and bark of trees. Thus, our isolates from G. biloba are phylogenetically linked to algae living in intimate or symbiotic associations with higher plants and fungi. Moreover, the phylogenetic relationship with several other algae known to be involved in (endo)symbiotic interactions with fungi and invertebrates (Fig. 7) may indicate a preadaptation of at least some of them to live intracellularly in certain host cells. However, as outlined above, the meaning of a symbiosis between the two partners studied here is unclear in contrast to the well-studied interactions of some green algae with lichens, protists, or invertebrates, reviewed by Huss (1999) and Reisser (1992) .

For the first time, we confirm the intracellular status of an endophytic green alga and its immature precursor form within living cells of G. biloba. Environmental factors that trigger algal proliferation and /or induce host-cell death remain to be explored.


   FOOTNOTES
 
1 The authors thank Dr. B. Arbeille, Dr. P. Y. Sizaret, S. Trassart, A. M. Carriot, and other members of the Electron Microscopy Department (Faculté de Médecine, Université F. Rabelais de Tours-France) for their technical assistance. This work is dedicated to Prof. Dr. Walter Tulecke, Antioch College (USA) for his sustained encouragement of our studies on the Ginkgo biloba model. Back

5 Author for reprint requests (phone: +33 (0) 247 367214, FAX: +33 (0) 247 276660; guiller{at}univ-tours.fr ) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Aoki T. 1997 Fungal association with Ginkgo biloba. In T. Hori, R. W. Ridge, W. Tulecke, P. Del Tredici, J. Trémouillaux-Guiller, and H. Tobe [eds.], Ginkgo biloba: a global treasure from biology to medicine, vol. 1, 251–257. Springer-Verlag, Tokyo, Japan

Ball E. 1959 Growth of the embryo of Ginkgo biloba under experimental conditions. III. Growth rate of the root and shoot upon media absorbed through the cotyledons. American Journal of Botany 46: 130-139[CrossRef][ISI]

Bonfante-Fasolo P. A. Fontana 1985 VAM fungi in Ginkgo biloba roots: their interactions at cellular level. Symbiosis 1: 53-67

Bourgin J. P. J. P. Nitsch 1967 Obtention de Nicotiana haploïdes à partir d'étamines cultivées in vitro. Annales de Physiologie Végétale 9: 377-382

Braquet P. G. 1997 Platelet activating factor and its antagonists: scientific background and clinical applications of ginkgolides. In R. W. Ridge, W. Tulecke, P. Del Tredici, J. Trémouillaux-Guiller, and H. Tobe [eds.], Ginkgo biloba: a global treasure from biology to medicine, vol. 1, 359–369. Springer-Verlag, Tokyo, Japan

Chapman R. L. D. A. Waters 1992 . Epi- and endobiotic chlorophytes. In W. Reisser [ed.], Algae and symbioses, 619–639. Biopress Limited, Bristol, UK

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Friedl T. B. Büdel 1996 Photobionts. In T. H. Nash III [ed.], Lichen biology, 8–23. Cambridge University Press, Cambridge, UK

Gärtner G. 1992 Taxonomy of symbiotic eukaryotic algae. In W. Reisser [ed.], Algae and symbioses, 325–338. Biopress Limited, Bristol, UK

Hirase S. 1896 Spermatozoid of Ginkgo biloba. Botanical Magazine Tokyo 10: 325 (in Japanese)

Huss V. A. R. 1999 Freshwater algal symbioses in protozoa and invertebrates. In J. Seckbach [ed.], Enigmatic microorganisms and life in extreme environments, 641–650. Kluwer Academic Publishers, Dordrecht, The Netherlands

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