CHLOROPLAST SPECIALIZATION IN DICOTYLEDONS POSSESSING THE C4-DICARBOXYLIC ACID PATHWAY OF PHOTOSYNTHETIC CO2 FIXATION

The C₄-dicarboxylic-acid pathway of photosynthetic CO₂ fixation found in tropical grasses has recently been demonstrated in members of the Amaranthaceae and Chenopodiaceae. In the tropical grasses this CO₂-fixation pathway is correlated with specialized leaf anatomy and chloroplast structure. This investigation was undertaken to determine if leaf cells of some representatives of these other families had structural features similar to those of tropical grasses. The leaf anatomy of Amaranthus edulis and a variety of Atriplex species is very similar and it resembles that of grasses such as sugar cane. Prominent bundle sheaths are surrounded by a layer of palisade cells. Bundle-sheath cells of Am. edulis have large chloroplasts containing much starch, but the chloroplasts have grana. The palisade cells have much smaller chloroplasts containing very little starch. The bundle-sheath cell chloroplasts of At. lentiformis have grana, their profiles tend to be ovoid, and they contain abundant starch grains. The palisade cell chloroplasts contain little starch and their profiles are discoid. The bundle-sheath cells of both species contain mitochondria which are much larger than those in the palisade cells. The chloroplasts in both types of cells in both species have a highly developed peripheral reticulum. This reticulum is composed of anastomosing tubules which are contiguous with the inner plastid membrane. The leaf anatomy and cell ultrastructure of these dicots are similar to those of the tropical grasses possessing this new photosynthetic carbon-fixation pathway. These morphological features are interpreted as adaptations for the rapid transport of precursors and end products of photosynthesis. A hypothesis is presented stating that the unique morphological and biochemical characters of these plants represent adaptations for efficient and rapid carbon fixation in environments where water stress frequently limits photosynthesis.     

PHYLOGENETIC ANALYSIS OF THE GENUS EUGLENA (EUGLENOPHYCEAE) WITH PARTICULAR REFERENCE TO THE TYPE SPECIES EUGLENA VIRIDIS1

Euglena viridis (subgenus Euglena) serves as the type species for the genus Euglena. In this study, molecular phylogenetic analyses using a small subunit (SSU) and a combined SSU–partial large subunit rDNA data set for members of the genus Euglena showed that strains identified as E. viridis on the basis of morphology are distributed between two separate nonsister clades. Although all the E. viridis strains examined were morphologically indistinguishable and possessed spherical mucocysts and stellate chloroplasts with one paramylon center, there was a high degree of sequence divergence between the E. viridis strains in different clades, making this a cryptic species. Like E. viridis, all taxa from the subgenus Euglena are characterized by having one or more stellate chloroplasts with paramylon grains clustered around the center of the chloroplast. These additional taxa were divided into four clades in all the molecular analyses. Strains of Euglena stellata formed two nonsister clades whose members had a single aggregate chloroplast with paramylon center and spindle‐shaped mucocysts. A geniculata clade included species with one or two stellate chloroplasts with paramylon centers and spherical mucocysts, and the cantabrica clade had members with one stellate chloroplast with paramylon center and spherical mucocysts often arranged in spiral rows. Interspersed among these were three additional clades bearing taxa from the subgenus Calliglena that contains members with discoid plastids and pyrenoids that may or may not be capped with paramylon. These taxa formed a laciniata clade, mutabilis clade, and gracilis clade. This study demonstrates that E. viridis and E. stellata are cryptic species that can only be distinguished at the molecular level. Because E. viridis is the designated type species for the genus Euglena, we designated an epitype for E. viridis.     

PHYLOGENETIC TRANSITIONS IN THE CHLOROPLASTS OF THE ANTHOCEROTALES I. THE NUMBER AND ULTRASTRUCTURE OF THE MATURE PLASTIDS

Representatives of three genera of anthooerotes were examined: Phaeoceros, Notothylas, and Megaceros. Species of the first two genera were found to exemplify the typical anthocerote plastid condition. This condition is characterized by the presence in each cell of the gametophyte of only a single large chloroplast containing a “multiple” pyrenoid. The genus Megaceros, however, proved to be quite different. In two species of Megaceros the pyrenoid was observed to be composed of a highly subdivided thylakoid system of even greater complexity than the “multiple” pyrenoids of Phaeoceros. In another species only an indistinct “pyrenoid-like” area was noted while in a fourth species no evidence was found for any internal differentiation. Associated with these changes in plastid structure there are corresponding alterations in the number and the size of the chloroplasts. Together they indicate an evolutionary trend away from a primitive, algal-like condition to a more advanced land plant form.     

Algae or Protozoa: Phylogenetic Position of Euglenophytes and Dinoflagellates as Inferred from Mitochondrial Sequences

The chloroplasts of euglenophytes and dinoflagellates have been suggested to be the vestiges of endosymbiotic algae acquired during the process of evolution. However, the evolutionary positions of these organisms are still inconclusive, and they have been tentatively classified as both algae and protozoa. A representative gene of the mitochondrial genome, cytochrome oxidase subunit I (coxI), was chosen and sequenced to clarify the phylogenetic positions of four dinoflagellates, two euglenophytes and one apicomplexan protist. This is the first report of mitochondrial DNA sequences for dinoflagellates and euglenophytes. Our COXI tree shows clearly that dinoflagellates are closely linked to apicomplexan parasites but not with algae. Euglenophytes and algae appear to be only remotely related, with euglenophytes sharing a possible evolutionary link with kinetoplastids. The COXI tree is in general agreement with the tree based on the nuclear encoded small subunit of ribosomal RNA (SSU rRNA) genes, but conflicts with that based on plastid genes. These results support the interpretation that chloroplasts present in euglenophytes and dinoflagellates were captured from algae through endosymbioses, while their mitochondria were inherited from the host cell. We suggest that dinoflagellates and euglenophytes were originally heterotrophic protists and that their chloroplasts are remnants of endosymbiotic algae. Received: 24 March 1997 / Accepted: 21 April 1997     

Unusual chloroplast structures in endosymbiotic dinoflagellates: A clue to evolutionary differentiation within the genusSymbiodinium (Dinophyceae)

Symbiodinium microadriaticum Freudenthal is widely regarded to represent one pandemic species of endosymbiotic dinoflagellates. Thin-sectioned and freeze-fractured chloroplasts of symbionts derived from different hosts reveal the envelope to be composed of three membraneous layers, the middle one featuring an uncommon cleavage pattern. Unusual thylakoid arrangement and inclusions indicate intrinsic differences in the chloroplasts among these algae. The results are discussed in the light of evolutionary differentiation withinSymbiodinium.     

TAXONOMIC REVISION OF THE CONJUGATOPHYCEAN FAMILY PENIACEAE ON THE BASIS OF CELL WALL ULTRASTRUCTURE1

An ultrastructural investigation of the cell wall of Penium silvae-nigrae Raban. and P. spinospermum Josh. showed that these species possess true pores with a pore apparatus and overlapping semi-cell walls. It follows that these two taxa belong not to the Peniaceae, but to the Desmidiaceae sensu stricto; they are referred to the genus Actinotaenium Teil. on account of the shape of their cells and chloroplasts. Two other species previously included in Penium Bréb. are referred to Actinotaenium. Although their cell wall structure could not be studied, they are distinguished from “typical” representatives of Penium by the following photomicroscopically observable complex of features: (pseudo-) girdle bands none, cell wall pores in longitudinal rows, zygospores not globose but of irregular shape. The following new combinations ensued: Actinotaenium borgeanum (Skuja), A. phymatosporum (Nordst.), A. silvae-nigrae (Raban.), A. silvaenigrae var. parallelum (Krieger) and A. spinospermum (Josh.). In addition the diagnosis of the genus Penium was emended and P. margaritaceum (Ehr.) ex Bréb. was selected as the lectotype species. The family Gonatozy-gaceae is merged into the Peniaceae on the basis of cell wall structure.     

Different effects of eubacterial and eukaryotic DNA topoisomerase II inhibitors on chloroplasts ofEuglena gracilis

Inhibitors of eubacterial and eukaryotic DNA topoisomerases type II exhibited different effects on chloroplasts of the flagellateEuglena gracilis. Antibacterial agents (cinoxacin, nalidixic and oxolinic acids, ciprofloxacin, enoxacin, norfloxacin and ofloxacin) from the group of quinolones and coumarins (coumermycin A₁, clorobiocin and novobiocin) — all inhibitors of prokaryotic DNA topoisomerase II — were very potent eliminators of chloroplasts fromE. gracilis. In contrast, antitumor drugs (adriamycin, etoposide, teniposide and mitoxantrone) — antagonists of the eukaryotic counterpart — did not affect these semiautonomous photosynthetic organelles. These findings point out again the close evolutionary relationships between eubacteria and chloroplasts and are in agreement with the hypothesis of an endosymbiotic origin of chloroplasts.     

THE MESOZOIC RADIATION OF EUKARYOTIC ALGAE: THE PORTABLE PLASTID HYPOTHESIS1

Although all chloroplasts appear to have been derived from a common ancestor, a major schism occurred early in the evolution of eukaryotic algae that gave rise to red and green photoautotrophic lineages. In Paleozoic and earlier times, the fossil record suggests that oceanic eukaryotic phytoplankton were dominated by the green (chl b‐containing) algal line. However, following the end‐Permian extinction, a diverse group of eukaryotic phytoplankton evolved from secondary symbiotic associations in the red (chl c‐containing) line and subsequently rose to ecological prominence. In the contemporary oceans, red eukaryotic phytoplankton taxa continue to dominate marine pelagic food webs, whereas the green line is relegated to comparatively minor ecological and biogeochemical roles. To help elucidate why the oceans are not dominated by green taxa, we analyzed and compared whole plastid genomes in both the red and green lineages. Our results suggest that whereas all algal plastids retain a core set of genes, red plastids retain a complementary set of genes that potentially confer more capacity to autonomously express proteins regulating oxygenic photosynthetic and energy transduction pathways. We hypothesize that specific gene losses in the primary endosymbiotic green plastid reduced its portability for subsequent symbiotic associations. This corollary of the plastid “enslavement” hypothesis may have limited subsequent evolutionary advances in the green lineage while simultaneously providing a competitive advantage to the red lineage.     

THE STRUCTURAL RELATIONSHIPS OF THE INTERNAL MEMBRANE SYSTEMS OF IN SITU AND ISOLATED CHLOROPLASTS OF HORDEUM VULGARE

Healthy chloroplasts of Hordeum vulgare are compared with chloroplasts subjected to abnormal stresses such as in situ disruption, isolation, isolation plus washing in 0.5 m sucrose, and isolation plus washing in 0.5 m sucrose and distilled H₂O. Normal chloroplasts resemble those of Nicotiana rustica and Phaseolus vulgaris in being composed of compartmented grana connected by an anastomosing fretwork system. They differ in having a somewhat greater incidence of parallel frets and double partitions. Under conditions of stress both grana and fretwork undergo varying degrees of swelling, and the double partition maintains its structural integrity. Grana are more resistant to abnormal stresses than the fretwork. Fret connections with more than 3 grana do not generally occur, but in some micrographs a single pathway may be traced through several grana. Washing isolated chloroplasts in distilled water results in an enlargement involving compartments of 2 or more grana together with the associated fretwork membranes. These results indicate that the grana in mature chloroplasts of Hordeum vulgare, like those of Nicotiana rustica and Phaseolus vulgaris, are compartmented structural units and not a series of localized aligned thickenings in regular extensive discs. These enlargements are complex structures comprising the membranes and spaces of both grana and frets. The swelling indicates an increase of locular and fret channel substance and possibly an enlargement of membrane surfaces. Dried down on grids, the compartments and frets appear as flat discs with radial appendages.     

ULTRASTRUCTURE OF THE GREEN ALGA DICHOTOMOSIPHON TUBEROSUS WITH SPECIAL REFERENCE TO THE OCCURRENCE OF STRIATED TUBULES IN THE CHLOROPLAST1

The ultrastructure of the siphonous green alga Dichotomosiphon tuberosus (A. Br.) Ernst is compared with that of other siphonous plants. There is a characteristic association between the Golgi bodies and endoplasmic reticulum, but. the mitochondria are not involved in the association as they are in Vaucheria and the phycomycete Saprolegnia. An unusual structure and arrangement of the chloroplasts is described as well as a previously unreported type of “striated tubule” which occurs in most if not all chloroplasts, and amyloplasts. The structure of these tubules is compared with that of other tubules recently found in green algae and higher plants. In addition, cytoplasmic microtubules arranged in the longitudinal direction of the siphon suggest a function in cytoplasmic streaming.     

PREDATION BY THE TOXIC DINOFLAGELLATE DINOPHYSIS FORTII ON THE CILIATE MYRIONECTA RUBRA AND OBSERVATION OF SEQUESTRATION OF CILIATE CHLOROPLASTS1

This is the first report of the propagation of the toxic dinoflagellate Dinophysis fortii Pavill. under laboratory conditions when fed on the marine ciliate Myrionecta rubra grown with the cryptophyte Teleaulax amphioxeia (W. Conrad) D. R. A. Hill. In contrast, reduced growth of D. fortii (max. of 3–4 divisions) and formation of small cells were observed in the absence of the ciliate or when provided with T. amphioxeia only as prey, showing that D. fortii cannot utilize T. amphioxeia as prey. In the TEM observation of D. fortii cells, which had fully fed on the ciliate prey, well‐developed chloroplasts (5–12 μm in length) were seen and three thylakoids were usually arranged in most of the chloroplasts observed, but chloroplasts having two thylakoids were sometimes confirmed. In cells starved for 4 weeks, decrease of chloroplast numbers and disappearance of large chloroplasts were observed, and only a few small chloroplasts (0.5–2 μm in length) remained in the marginal regions. In the observation of the sequestration process of the chloroplasts ingested from M. rubra by D. fortii, within 15 min after D. fortii captured M. rubra, incorporation of almost all of the chloroplasts was observed, while most of the other cell contents still remained in the M. rubra cell. After that, dispersion of the ingested chloroplasts toward the marginal regions was confirmed, suggesting that chloroplasts of M. rubra are ingested and dispersed in D. fortii cells in advance of the ingestion of the other cell contents to prevent them from being digested in food vacuoles. The ingested chloroplasts can also function as kleptoplastids.     

COMPARISON OF ENDOSYMBIOTIC AND FREE‐LIVING SYMBIODINIUM (DINOPHYCEAE) DIVERSITY IN A HAWAIIAN REEF ENVIRONMENT1

Many scleractinian corals must acquire their endosymbiotic dinoflagellates (genus Symbiodinium) anew each generation from environmental pools, and exchange between endosymbiotic and environmental pools of Symbiodinium (reef waters and sediments) has been proposed as a mechanism for optimizing coral physiology in the face of environmental change. Our understanding of the diversity of Symbiodinium spp. in environmental pools is poor by comparison to that engaged in endosymbiosis, which reflects the challenges of visualizing the genus against the backdrop of the complex and diverse micro‐eukaryotic communities found free‐living in the environment. Here, the molecular diversity of Symbiodinium living in the waters and sediments of a reef near Coconut Island, O‘ahu, Hawai‘i, sampled at four hourly intervals over a period of 5 d was characterized using a Symbiodinium‐specific hypervariable region of the chloroplast 23S. A comparison of Symbiodinium spp. diversity recovered from environmental samples with the endosymbiotic diversity in coral species that dominate the adjacent reef revealed limited overlap between these communities. These data suggest that the potential for infection, exchange, and/or repopulation of corals with Symbiodinium derived from the environment is limited at this location, a finding that is perhaps consistent with the high proportion of coral species in this geographic region that transmit endosymbionts from generation to generation.     

HOST‐SPECIALIST LINEAGES DOMINATE THE ADAPTIVE RADIATION OF REEF CORAL ENDOSYMBIONTS

Bursts in species diversification are well documented among animals and plants, yet few studies have assessed recent adaptive radiations of eukaryotic microbes. Consequently, we examined the radiation of the most ecologically dominant group of endosymbiotic dinoflagellates found in reef‐building corals, Symbiodinium Clade C, using nuclear ribosomal (ITS2), chloroplast (psbA^ncr), and multilocus microsatellite genotyping. Through a hierarchical analysis of high‐resolution genetic data, we assessed whether ecologically distinct Symbiodinium, differentiated by seemingly equivocal rDNA sequence differences, are independent species lineages. We also considered the role of host specificity in Symbiodinium speciation and the correspondence between endosymbiont diversification and Caribbean paleo‐history. According to phylogenetic, biological, and ecological species concepts, Symbiodinium Clade C comprises many distinct species. Although regional factors contributed to population‐genetic structuring of these lineages, Symbiodinium diversification was mainly driven by host specialization. By combining patterns of the endosymbiont's host specificity, water depth distribution, and phylogeography with paleo‐historical signals of climate change, we inferred that present‐day species diversity on Atlantic coral reefs stemmed mostly from a post‐Miocene adaptive radiation. Host‐generalist progenitors spread, specialized, and diversified during the ensuing epochs of prolonged global cooling and change in reef‐faunal assemblages. Our evolutionary reconstruction thus suggests that Symbiodinium undergoes “boom and bust” phases in diversification and extinction during major climate shifts.     

ULTRASTRUCTURE OF THE SHOOT APEX OF TOMATO (LYCOPERSICON ESCULENTUM)

The vegetative shoot apical meristem of tomato (Lycopersicon esculentum Mill.) was examined at the ultrastructural level. The meristem consisted of a surface layer that was different from the rest of the meristem and was unique among the dicotyledonous species. The cells of the surface layer contained large distal vacuoles with relatively large electron-dense inclusions, proplastids with membrane-bound inclusions (MB), and differentiating chloroplasts. In addition, periclinal and oblique divisions were observed in the surface layer cells along with anticlinal divisions. The cells of the subsurface layers contained small vacuoles with fewer inclusions as well as proplastids of various shapes but without MB. Differentiating chloroplasts were not observed in these cells, but autophagic vacuoles at various stages of development were present. The normal complement of cell inclusions, e.g., the mitochondria, golgi bodies, endoplasmic reticulum (ER), ribosomes, and microtubules were observed in subsurface layers, and in many cells the ER was observed to be continuous with the outer membrane of the nuclear envelope and with the plasmalemma. Further below in the meristem, cells contained both the proplastids and differentiating chloroplasts with MB. In the latter, the outer membrane of the MB was found to be continuous with the developing lamellae, suggesting that MB probably serve as the storage centers for lamellae membranes. Near the base of the meristem, in the pith-rib meristem, enlarged cells containing large vacuoles and differentiated chloroplasts were present.     

The making of a chloroplast

Since its endosymbiotic beginning, the chloroplast has become fully integrated into the biology of the host eukaryotic cell. The exchange of genetic information from the chloroplast to the nucleus has resulted in considerable co‐ordination in the activities of these two organelles during all stages of plant development. Here, we give an overview of the mechanisms of light perception and the subsequent regulation of nuclear gene expression in the model plant Arabidopsis thaliana, and we cover the main events that take place when proplastids differentiate into chloroplasts. We also consider recent findings regarding signalling networks between the chloroplast and the nucleus during seedling development, and how these signals are modulated by light. In addition, we discuss the mechanisms through which chloroplasts develop in different cell types, namely cotyledons and the dimorphic chloroplasts of the C₄ plant maize. Finally, we discuss recent data that suggest the specific regulation of the light‐dependent phases of photosynthesis, providing a means to optimize photosynthesis to varying light regimes.     

Endosymbiosis,cell evolution,and speciation

In 1905, the Russian biologist C. Mereschkowsky postulated that plastids (e.g., chloroplasts) are the evolutionary descendants of endosymbiotic cyanobacteria-like organisms. In 1927, I. Wallin explicitly postulated that mitochondria likewise evolved from once free-living bacteria. Here, we summarize the history of these endosymbiotic concepts to their modern-day derivative, the “serial endosymbiosis theory”, which collectively expound on the origin of eukaryotic cell organelles (plastids, mitochondria) and subsequent endosymbiotic events. Additionally, we review recent hypotheses about the origin of the nucleus. Model systems for the study of “endosymbiosis in action” are also described, and the hypothesis that symbiogenesis may contribute to the generation of new species is critically assessed with special reference to the secondary and tertiary endosymbiosis (macroevolution) of unicellular eukaryotic algae.     

Intracellular Bacterial Symbionts of Aphids Possess Many Genomic Copies per Bacterium

Although Buchnera, the endosymbiotic bacteria of aphids, are close relatives of Escherichia coli, their genome size is only a seventh that of E. coli. In this study, we estimated the genomic copy number of Buchnera by dot-blot hybridization and fluorimetry using a video-intensified microscope photon-counting system and obtained convincing evidence that each cell of these bacteria contains an average of 120 genomic copies. Thus, the Buchnera symbiont, with many copies of a small-sized genome, is reminiscent of cell organelles such as mitochondria and chloroplasts. Received: 25 November 1998 / Accepted: 25 December 1998     

Glutamate synthase is plastid-encoded in a red alga: implications for the evolution of glutamate synthases

An actively transcribed gene (glsF) encoding for ferredoxin-dependent glutamate synthase (Fd-GOGAT) was found on the plastid genome of the multicellular red alga Antithamnion sp. Fd-GOGAT is not plastid-encoded in chlorophytic plants, demonstrating that red algal plastid genomes encode for additional functions when compared to those known from green chloroplasts. Moreover, our results suggest that the plant Fd-GOGAT has an endosymbiotic origin. The same may not be true for NADPH-dependent GOGAT. In Antithamnion glsF is flanked upstream by cpcBA and downstream by psaC and is transcribed monocistronically. Implications of these results for the evolution of GOGAT enzymes and the plastid genome are discussed.     

TAKAYAMA GEN. NOV. (GYMNODINIALES,DINOPHYCEAE), A NEW GENUS OF UNARMORED DINOFLAGELLATES WITH SIGMOID APICAL GROOVES,INCLUDING THE DESCRIPTION OF TWO NEW SPECIES1

A new potentially ichthyotoxic dinoflagellate genus, Takayama de Salas, Bolch, Botes et Hallegraeff gen. nov., is described with two new species isolated from Tasmanian (Australia) and South African coastal waters: T. tasmanica de Salas, Bolch et Hallegraeff, sp. nov. and T. helix, de Salas, Bolch, Botes et Hallegraeff, sp. nov. The genus and two species are characterized by LM and EM of field samples and laboratory cultures as well as large subunit rDNA sequences and HPLC pigment analyses of several cultured strains. The new Takayama species have sigmoid apical grooves and contain fucoxanthin and its derivatives as the main accessory pigments. Takayama tasmanica is similar to the previously described species Gymnodinium pulchellum Larsen, Gyrodinium acrotrochum Larsen, and G. cladochroma Larsen in its external morphology but differs from these in having two ventral pores, a large horseshoe‐shaped nucleus, and a central pyrenoid with radiating chloroplasts that pass through the nucleus. It contains gyroxanthin‐diester and a gyroxanthin‐like accessory pigment, both of which are missing in T. helix. Takayama helix has an apical groove that is nearly straight while still being clearly inflected. A ventral pore or slit is present. It has numerous peripheral, strap shaped, and spiraling chloroplasts with individual pyrenoids and a solid ellipsoidal nucleus. The genus Takayama has close affinities to the genera Karenia and Karlodinium.     

A MORPHOMETRIC STUDY OF THE ULTRASTRUCTURE OF ECHINOCEREUS ENGELMANNII (CACTACEAE). III. SUBAPICAL AND MATURE TISSUES

A stereological, morphometric study of the ultrastructure of subapical cells, xylem parenchyma, and cortical chlorenchyma of Echinocereus engelmannii shows that each of these cell types has a unique organellar composition. The cells of any of these tissues are unique not only in vacuolation (which is visible at the light microscope level), but also in the nucleus/cytoplasm ratio and the concentrations of mitochondria, chloroplasts, and dictyosomes. Furthermore, the differences between each of these cell types were large and statistically significant. It had previously been found that the cells of each zone of the shoot apical meristems of E. engelmannii are different from those of the other zones, but the present study suggests that, considering the large ranges of structure possible in the nonapical cells of this species, the apical meristem variation should be considered as only a minor difference and that the meristem zones are really quite similar to each other.