Inheritance of mitochondrial and chloroplast genomes in the isogamous brown alga Scytosiphon lomentaria (Phaeophyceae)

Patterns of inheritance of chloroplasts and mitochondria were examined by fluorescence microscopy and haplotype genome markers in the isogamous brown alga Scytosiphon lomentaria (Lyngbye) Link. Germination of the zygote in this species was unilateral, the growing thallus developed entirely from the germ tube, and the original zygote cell did not develop except for the formation of a hair. Inheritance of chloroplasts was biparental, and partitioning of the two parental chloroplasts into the first sporophytic cells was accidental: either the maternal or the paternal chloroplast was migrated from the zygote into the germ tube cell, whereas the other chloroplast remained in the original cell. In contrast, the mitochondrial genome in all cells of the sporophyte came only from the female gamete (maternal inheritance). These inheritance patterns are similar to those of the isogamous brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye. Maternal inheritance of mitochondria might be universal in brown algae.     

CHLOROPLAST INHERITANCE IN COSMARIUM TURPINII BRÉB.

Chloroplast inheritance was studied in Cosmarium turpinii Bréb. with respect to both vegetative and zygotic transmission. Analyses were carried out on (1) reciprocal haploid x diploid crosses with respect to the number and size of the zygotic chloroplasts, (2) differential survival of chloroplasts in hypnospores of different mating type strains, and (3) the position of the chloroplasts in diploid zygotes. Morphological evidence indicates that the chloroplasts in the mature zygote are derived from the (+) mating type gamete with an infrequent contribution from the (–) mating type gamete. Additional evidence suggests that the chloroplast material of each of 2 Bones arising from the zygote is derived from the plastid material of a semicell of a gamete. Differential survival of the chloroplasts is interpreted as a result of physiological differences between cells of complementary mating types.     

Disintegration of chloroplasts during zygote formation inSpirogyra verruculosa

The chloroplast disintegration during zygote maturation inSpirogyra verruculosa was investigated by electron microscopy. In the seven-day-old zygote about half of the chloroplasts commenced to disintegrate and to turn yellow, losing starch grains, and, then, were torn into fragments of various sizes, which had mostly vesiculated thylakoids and plastoglobules increasing in both size and number. At about two weeks after conjugation, in the cytoplasm, electron-dense structures, linear in section, appeared and vacuoles of various sizes developed. Each of the dense linear structures lying around a fragment seemed to form a cavity of crescent shape in section, and these cavities fused mutually into a large one, leading to the separation of the fragment from the bulk of cytoplasm. The vacuoles seemed to be, involved in the sequestration of the fragments by their fusion with the cavities and by the invagination, of tonoplast. The fragments entrapped by the vacuoles were rapidly broken down into the aggregation of residual membrane pieces, plastoglobules, and undigested starch grains. The maintained chloroplasts changed little in structure compared with the chloroplast of the vegetative cell, and were transmitted to the germling. It is suggested that the eliminated chloroplasts are derived exclusively from the male gamete.     

Aplanogamous sexual reproduction in Carteria eugametos (Volvocales,Chlorophyta)

Morphological details of sexual reproduction in Carteria eugametos (Volvocales, Chlorophyta) were studied under controlled laboratory conditions. Protoplasts of the two pairing, flagellate cells were released from cell walls to become isogametes. Such gametes were nonmotile and soon fused to form a completely immobile zygote. The zygote then secreted a cell wall to enter the dormant period. After dark treatment, the zygote produced four, eight or 16 quadriflagellate germ cells, and a transparent vesicle enclosing all the germ cells was released from the zygote wall. This type of zygote germination and aplanogamy in C. eugametos is unique and may be related to its peculiar phylogenetic position within the Volvocales (Chlamydomonadales).     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

Chloroplast gene transmission in Chlamydomonas reinhardtii: A random choice model

Chloroplast genes in Chlamydomonas reinhardtii are inherited uniparentally. In a laboratory cross the majority (>95%) of the zygotes transmit to the meiotic progeny only those chloroplast genes donated by the maternal parent. This occurs even though the parents are isogamous and the chloroplasts from the two parents fuse shortly after mating. Uniparental inheritance of the chloroplast genes can be altered by several methods. If maternal gametes are irradiated with ultraviolet light prior to mating, the proportion of zygotes transmitting chloroplast genes from the paternal parent rises dramatically. In this paper I examine in detail the effects of uv irradiation on both maternal and paternal gametes and the effect of photore-activation following the uv irradiation. The effect of uv irradiation can be largely reversed by photoreactivation, and both the starting time and the intensity of the photoreactivating light used are found to be critical. Examination of the frequencies of the different zygote types obtained with respect to chloroplast gene transmission following uv treatment of the maternal gametes shows that they fit a hypergeometric distribution, in which choices are made from a population without replacement. By rearranging the basic hypergeometric equation I was able to estimate that the choice is made from a population of 27 maternal and 2 paternal units of gene transmission. These units probably contain more than one genome each, since their number is much lower than the estimated number of genomes per cell. My model explains both the observed distribution of zygote types and the bias in favor of maternal alleles found in the progeny of a biparental zygote, and may have a wider application to other organelle genetic systems. I also suggest that the extreme degree of uniparental inheritance of chloroplast genes in Chlamydomonas found in the laboratory may not be seen in nature.     

Amphidiniopsis uroensis sp. nov. and Amphidiniopsis pectinaria sp. nov. (Dinophyceae): Two new benthic dinoflage llates from Japan

Two new armoured, heterotrophic sand‐dwelling marine dinoflagellates, Amphidiniopsis uroensis Toriumi, Yoshimatsu et Dodge sp. nov. and Amphidiniopsis pectinaria Toriumi, Yoshimatsu et Dodge sp. nov. were collected from Japanese sandy beaches, and their morphological features observed by light microscopy and scanning electron microscopy (SEM). The cell size of A. uroensis is 28–31 μm in length and 23–28 μm in width. The plate formula is Po 3′, 3a, 6″, 3c, 4s (+1 acc.), 5″′, 2″″. The thecal surface is ornamented with small processes, pores and spines, however, the surface of plate 2a is smooth. The epitheca possesses a narrow ridge that is extended along on the suture between 1′ and 3′. Plate 1″ connects with the right sulcal (Sd) and right sulcal accessory (Sda) plates, so the cingulum is incomplete. A nucleus is situated in the central part of the cell. There are a few small spines at the antapex. There are no stigma or chloroplasts. Amphidiniopsis pectinaria cells are 33–40 urn in length and 29–35 μm in width. The plate formula is Po 4′, 3a, 7″, 3c, 4s (+1 acc.), 5″′, 2″″. Plate 1″ connects directly with Sd and Sda plates, so the cingulum is incomplete. The thecal surface is ornamented with small processes, spines and pores. The epitheca is provided with a narrow ridge that is extended along on the suture between plates 1′, 4′ and 7″. The ornamentation on the antapical plates is unique. It is arranged in 10 straight rows on the hypotheca; each row has a strong spine at its posterior end. In addition, there is a long spine at the antapex. There are no stigma or chloroplasts. A nucleus is located in the central part of the cell.     

Frequency distributions for chloroplast genes in Chlamydomonas zygote clones: Evidence for random drift

Mitochondria and chloroplasts of eucaryotic cells contain populations of DNA molecules. In certain cases, e.g., the chloroplasts of Chlamydomonas reinhardtii and the mitochondria of Saccharomyces cerevisiae, organelles contributed by the two parents are known to fuse in the zygote, creating a single population of DNA molecules. In a cross, this population will include molecules of both parental genotypes. There is reason to suspect that organelle DNA molecules in this population are selected randomly for replication and recombination. This would result in random changes in the frequency of a particular allele or genotype within the organelle gene pool of a single zygote and also within its clone of progeny cells. A given gene frequency would increase in some zygote clones and decrease in others, analogous to random drift of gene frequencies in small Mendelian populations. To test this, we have examined the distribution of chloroplast gene frequencies among the zygote clones produced in each of a number of crosses of Chlamydomonas. These distributions are typically U or L shaped as predicted by the random drift hypothesis. They include uniparental zygote clones, in which a chloroplast allele from one parent has been fixed (frequency 100%) and the alternative allele from the other parent has been lost (frequency 0%). Among the remaining (biparental) zygote clones, there is a linear distribution of allele frequencies, showing a great increase in variance over the input frequencies. In these experiments both biparental and uniparental zygotes show a bias favoring chloroplast alleles from the mt⁺ (maternal) parent, and there is no statistically significant mode at the allele frequency of 0.5 corresponding to the equal input of alleles from the maternal and paternal (mt^?) parents. The observed distributions support the hypothesis that both uniparental inheritance and the high variance of allele frequencies among zygote clones are due to random drift of allele frequencies, coupled with a directional force which favors fixation of the maternal allele. In addition, statistical analysis of the data shows a strong but incomplete tendency for linked chloroplast markers to be fixed or lost together in uniparental zygotes. Possible cellular and molecular mechanisms for these observations are discussed.     

Über Plastidenstapel beiBotrydiopsis alpina sowie Anlage und Vermehrung der Stigmen bei dieser undHeterococcus (Xanthophyceae)

Under certain conditions inBotrydiopsis alpina stacks of chloroplasts are formed. They consist of up to 8 elements. In contrast to what is known from other algae in zoosporangia of this species and ofHeterococcus caespitosus, stigmata are formed in early developmental stages. They are reproduced together with the chloroplasts, in which they occupy a position at the edge and near the existing or future incision. At the side of the old stigma a new one is formed, and partitioning of the chloroplast between these two leads to their distribution to the daughter chloroplasts. Young daughter cells in the zoosporangia ofBotrydiopsis alpina contain one chloroplast which undergoes a last unequal division giving rise to one astigmate and usually somewhat smaller and to one stigmate chloroplast. In both species the capacity for locomotion may be suppressed, the presumptive zoospores thereby becoming aplanospores. Autospores in the proper sense were not observed. Their development quite generally is different from that of aplanospores (and zoospores), and both types of spores should be distinguished.

Herrn Professor Dr.Lothar Geitler zum 80. Geburtstag in Verehrung gewidmet.     

SEXUAL REPRODUCTION AND MEIOSIS IN PERIDINIUM INCONSPICUUM LEMMERMANN (DINOPHYCEAE)

In Peridinium inconspicuum Lemmermann, sexual reproduction occurs in both nitrogen-enriched and nitrogen-deficient media. In this homothallic strain, protoplasmic fusion begins between two thecate gametes; but zygote formation is completed in a space outside the fusing pair. This diploid cell can form a plated theca which is shed as the cell enlarges. This spherical zygote then forms a new non-plated theca. The process of ecdysis and the formation of a new non-plated theca is repeated several times. During this process the zygote gradually elongates and by cytoplasmic infurrowing becomes peanut-shaped. Eventually two cells are formed. The first and second meiotic divisions are greatly separated in time. The first meiotic division occurs in the spherical non-thecate zygote. The second meiotic division can occur in the peanut-shaped zygote before it completes cytokinesis. This meiotic division may not be synchronous, occasionally resulting in a trinucleate stage. Eventually four flagellated, haploid products are produced.     

Observations on the morphology and vegetative cell division of the diatomDonkinia recta

Wild and cultured material ofDonkinia recta (Donk.) Grun. has been examined using light microscopy, and both the structure of the living cell and its vegetative cell division are described. Unlike most naviculoid diatoms,D. recta has four chloroplasts per cell, each with four oblong pyrenoids, and division of the chloroplasts follows rather than precedes mitosis and cell division. These two unusual features are probably linked. The species is briefly discussed in relation to other naviculoid taxa in the light of these findings.     

On the function of the ‘eye-spots’ in agonistic behaviour in the fire-mouth cichlid (Cichlasoma Meeki)

During frontal threatening, individuals of the fire-mouth cichlid (Cichlasoma meeki) dilate their opercula, thereby demonstrating two conspicous ‘eye-spots’. In a hostile situation, a fish confronted with an opponent whose eye-spots have been experimentally removed is more likely to react with overt aggression towards the other. Fights also follow a more violent course than does a ‘normal’ fight between two intact males. A tentative explanation to these results is advanced which supposes that the eye-spots may exert an ‘intimidating’ effect upon the opponent during threat and fighting. This hypothesis is also supported by an experiment where males were confronted with a mirror. In this situation the distance between the mirror and the fish during frontal threat was significantly shorter in fishes lacking eye-spots, indicating that the intimidating effect emerging from the mirror image was diminished. In a third set of experiments when two males (one intact and one without eye-spots) competed for the only suitable territory site in an aquarium, the intact male eventually dominated the other in five of six aquariums.     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。     

EVIDENCE THAT THE NUCLEOMORPHS OF CHLORARACHNION REPTANS (CHLORARACHNIOPHYCEAE) ARE VESTIGIAL NUCLEI: MORPHOLOGY,DIVISION AND DNA-DAPI FLUORESCENCE1

Chlorarachnion reptans Geitler shows affinities to both the Chlorophyceae and the chloroplast endoplasmic reticulum-containing chromophyte algae in possessing chlorophyll b and chloroplasts which are limited by four membranes, respectively. In the periplastidal compartment surrounding each of the four to eight chloroplasts of a C. reptans cell are putative eukaryotic-sized ribosomes, scattered tubules and vesicles, and a small double-membrane-limited nucleus-like organelle named the nucleomorph. The nucleomorphs display 4′-6-diamidino-2-phenylindole (DAPI)fluorescence which is sensitive to DNase digestion, but not to treatment with RNase. The nucleomorphs also contain a fibrillogranular body which resembles a nucleolus. Nucleomorph division occurs by the sequential infolding of the inner and outer envelope membranes and subsequent constriction in two, with no involvement of microtubules. In all these characteristics, the nucleomorphs of C. reptans are similar to the cryptomonad nucleomorph which has been hypothesized to be the vestigial nucleus of an ancestral red alga which gave rise to the chloroplasts of the Cryptophyceae. The presence of chlorophyll b and the contents and morphology of C. reptans chloroplast compartments suggest a green algal origin for the chloroplasts of these cells. The discovery of a second organism with a DNA-containing, nucleus-like organelle in its chloroplast compartment lends strong support to the hypothesis that the chloroplasts of many algae have evolved from eukaryotic endosymbionts.     

Protective effect of sodium selenite on ofloxacin-induced loss of chloroplast DNA inEuglena gracilis

Some xenobiotics induce permanent loss of chloroplasts inEuglena gracilis which results in the growth of white mutant colonies (bleaching effect). The effect of ofloxacin and sodium selenite on the organization of chloroplast DNA ofE. gracilis was studied by fluorescent microscopy. In the presence of ofloxacin DNA-specific staining with DAPI revealed a decrease in chloroplast DNA content and in the number of nucleoids per chloroplast. This was accompanied with a decrease of chloroplast number per cell and with the loss of stigma. Spectrophotometry of chlorophyll extract revealed changes in th ratio of chla: chlb. The presence of sodium selenite protected chloroplast DNA against the inhibitory effect of ofloxacin.     

THE INITIATION OF SPOROPHYTES BY OBLIGATE APOGAMY IN CHEILANTHES CASTANEA

The initiation of apogamous sporophytes in Cheilanthes castanea was recorded by daily photography of individual gametophytes. Whereas an ordinary embryo arises from a zygote, apogamous embryos of C. castanea originate from one to three initial cells which occur just behind the apical region of the prothallus. The initial (or initials) produce cells with small chloroplasts behind the sinus of the gametophyte. The appearance of cells with smaller chloroplasts than those normally found in gametophytes is the first indication that apogamy is occurring. The cells with small plastids produce a group of densely-cytoplasmic meristematic cells. The size of the meristematic mass increases until shoot and root apices of the apogamous embryo are organized.     

AUXOSPORE FORMATION BY THE SILICA‐SINKING,OCEANIC DIATOM FRAGILARIOPSIS KERGUELENSIS (BACILLARIOPHYCEAE)1

Size restoration by the auxospore that develops from the zygote is a crucial stage in diatom life cycles. However, information on sexual events in pelagic diatom species is very limited. We report for the first time auxospore formation by the pennate diatom Fragilariopsis kerguelensis (O'Hara) Hustedt during an iron‐induced bloom in the Southern Ocean (EIFEX, European Iron Fertilization EXperiment). Auxospores of F. kerguelensis resembled those described for Pseudo‐nitzschia species. The auxospore was characterized by an outer coating, the perizonium; two caps, one at each distal end; and four chloroplasts, one at each end and two in the central part. Different stages of auxospore elongation were recorded, with a length of 24–91 μm, but only the largest auxospores contained the initial cell, whose apical axis ranged between 76 and 90 μm. Gametangial cell walls were often attached to the auxospores and ranged from 10 to 31 μm in length. Auxospore abundances were consistently higher in the fertilized patch, where an increase in the F. kerguelensis population was observed, as compared with surrounding waters.     

Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.     

THE SEXUAL LIFE CYCLES OF PFIESTERIA PISCICIDA AND CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE)1

Sexual life cycle events in Pfiesteria piscicida and cryptoperidiniopsoid heterotrophic dinoflagellates were determined by following the development of isolated gamete pairs in single‐drop microcultures with cryptophyte prey. Under these conditions, the observed sequence of zygote formation, development, and postzygotic divisions was similar in these dinoflagellates. Fusion of motile gamete pairs each produced a rapidly swimming uninucleate planozygote with two longitudinal flagella. Planozygotes enlarged as they fed repeatedly on cryptophytes. In <12 h in most cases, each planozygote formed a transparent‐walled nonmotile cell (cyst) with a single nucleus. Zygotic cysts did not exhibit dormancy under these conditions. In each taxon, dramatic swirling chromosome movements (nuclear cyclosis) were found in zygote nuclei before division. In P. piscicida, nuclear cyclosis occurred in the zygotic cyst or apparently earlier in the planozygote. In the cryptoperidiniopsoids, nuclear cyclosis occurred inthe zygotic cyst. After nuclear cyclosis, a single cell division occurred in P. piscicida and cryptoperidiniopsoid zygotic cysts, producing two offspring that emerged as biflagellated cells. These two flagellated cells typically swam for hours and fed on cryptophytes before encysting. A single cell division in these cysts produced two biflagellated offspring that also fed before encysting for further reproduction. This sequence of zygote development and postzygotic divisions typically was completed within 24 h and was confirmed in examples from different isolates of each taxon. Some aspects of the P. piscicida sexual life cycle determined here differed from previous reports.     

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.