Hidenobu Uchida

The phylogenetic relationship between the Chlamydomonadales and Chlorococcales inferred from 18SrDNA sequence data

Nuclear‐encoded small subunit ribosomal RNA gene (185rDNA) sequences were determined for Chlamydomonas moewusii Gerloff and five chlorococcalean algae (Chlorococcum hypnosporum Starr; Chlorococcum oleofaciens Trainor et Bold; Chlorococcum sp.; Tetracystis aeria Brown et Bold; Protosiphon botryoides (Kützingl Klebs). All these algae are characterized by a clockwise CCW) flagellar apparatus. Phylogenetic trees were constructed from sequences from these algae together with 20 green algae. All algae with a CW flagellar apparatus form a monophyletic clade (CW group). Three principal clades can be recognized in the CW group, although no morphological character supports monophyly of any of these three clades. The 185rDNA trees clearly demonstrate the non‐monophyly of the Chlamydomonadales and Chlorococcales, suggesting that vegetative morphology does not reflect phylogenetic relationships in the CW group. The paraphyly or polyphyly of the genus Chlamydomonas and Chlorococcum are also revealed. Present analysis suggests that the presence or absence of a zoospores cell wall and the multinucleate condition have limited taxonomic values at higher taxonomic ranks.

PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA) INFERRED FROM rbcL GENE SEQUENCE DATA

The chloroplast‐encoded large subunit of the ribulose‐1, 5‐bisphosphate carboxylase / oxygenase (rbcL) gene was sequenced from 20 species of the colonial Volvocales (the Volvacaceae, Goniaceae, and Tetrabaenaceae) in order to elucidate phylogenetic relationships within the colonial Volvocales. Eleven hundred twenty‐eight base pairs in the coding regions of the (rbcL) gene were analyzed by the neighbor‐joining (NJ) method using three kinds of distance estimations, as well as by the maximum parsimony (MP) method. A large group comprising all the anisogamous and oogamous volvocacean species was resolved in the MP tree as well as in the NJ trees based on overall and synonymous substitutions. In all the trees constructed, Basichlamys and Tetrabaena (Tetrabaenaceae) constituted a very robust phylogenetic group. Although not supported by high bootstrap values, the MP tree and the NJ tree based on nonsynonymous substitutions indicated that the Tetrabaenaceae is the sister group to the large group comprising the Volvocaceae and the Goniaceae. In addition, the present analysis strongly suggested that Pandorina and Astrephomene are monophyletic genera whereas Eudorina is nonmonophyletic. These results are essentially consistent with the results of the recent cladistic analyses of morphological data. However, the monophyly of the Volvocaceae previously supported by four morphological synapomorphies is found only in the NJ tree based on nonsynonymous substitutions (with very low bootstrap values). The genus Volvox was clearly resolved as a polyphyletic group with V. rousseletii Pocock separated from other species of Volvox in the rbcL gene comparisons, although this genus represents a monophyletic group in the previous morphological analyses. Furthermore, none of the rbcL gene trees supported the monophyly of the Goniaceae; Astrephomene was placed in various phylogenetic positions.

PHYLOGENETIC ANALYSIS OF YAMAGISHLELLA AND PLATYDORINA (VOLVOCACEAE, CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Yamagishiella, based on Pandorina unicocca Rayburn et Starr, is distinguished from Eudorina by its isogamous sexual reproduction, whereas Platydorina exhibits anisogamous sexual reproduction. In the present study, we sequenced the large subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (rbcL) genes from five Japanese and North American strains of Y. unicocca (Rayburn et Starr) Nozaki, two Platydorina caudata Kofoid strains, and two strains of Eudorina unicocca G. M. Smith, as well as eight related colonial and unicellular species. Phylogenetic trees were constructed based on these sequence data and on previously published rbcL gene sequences from 23 volvocalean species in order to deduce phylogenetic relationships within the colonial Volvocales, with particular regard to the phylogenetic positions and status of the genera Yamagishiella and Platydorina. Two robust monophyletic groups of the anisogamous/oogamous volvocacean species were resolved in the maximum‐parsimony tree as well as in the neighbor‐joining distance tree. One of the two groups comprises three species of Volvox section Volvox, whereas the other is composed of other sections of Volvox as well as of all the species of Eudorina and Pleodorina. Platydorina, however, was positioned outside these two monopliyletic groups. Therefore, derivation of the Platydorina lineage may be earlier than that of such anisogamous/oogamous groups, or orgin of “anisogamy with sperm packets” in Platydorina may be independent of sperm packet evolution in Eudorina, Pleodorina, and Volvox. It was also resolved with high bootstrap values that all of the Y. unicocca strains form a monophyletic group positioned outside the large monophyletic group including Eudorina and Pleodorina. These reject the possibility of the reverse evolution of isogamy from anisogamy to give rise to Yamagishiella within the lineage of Eudorina.

A ZYGOTE-SPECIFIC PROTEIN WITH HYDROXYPROLINE-RICH GLYCOPROTEIN DOMAINS AND LECTIN-LIKE DOMAINS INVOLVED IN THE ASSEMBLY OF THE CELL WALL OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

The cell wall of Chlamydomonas reinhardtii zygotes, which forms rapidly after the fusion of wall‐free gametes, provides a tractable system for studying the properties and assembly of hydroxyproline‐rich glycoproteins, the major proteinaceous components of green algal and plant cell walls. We report the cloning of the zsp2 gene and the analysis of its ZSP‐2 product, a 58.9 kDa polypeptide that is synthesized exclusively by zygotes. The protein contains two (SP)x repeats, establishing it as a member of the cell wall hydroxyproline‐rich glycoproteins family. It also contains a 4‐fold iteration of an amino acid sequence centered around cysteine residues, a configuration found in both plant and animal lectins. Furthermore, we report four observations on pellicle composition and production. First, cell‐free preparations of the pellicle matrix are rich in hydroxyproline, arabinose, and galactose and contain bundles of very long fibrils. Second, glutathione blocks pellicle formation and results in the accumulation of long fibrils in the growth medium. Third, antibody to ZSP‐2 also blocks pellicle formation. Fourth, ZSP‐2 immunolocalizes to the boundary between the outer layers of the wall proper and the pellicle matrix. These observations are consistent with the possibility that the Cys‐rich (glutathione‐sensitive) lectin‐like domains of ZSP‐2 may bind to sugar residues on the long fibrils and anchor them to the cell wall, thereby initiating and maintaining pellicle formation.

A pair of invertedly repeated genes in Chlamydomonas reinhardtii encodes a zygote-specific protein whose expression is UV-sensitive.

Abstract Uniparental inheritance of the chloroplast genome has been observed in a wide variety of green plants. In Chlamydomonas this phenomenon, which can be selectively inhibited by UV irradiation of mt+ gametes, has been shown cytologically to be due to the preferential degradation of mt−-derived chloroplast nucleoids in young zygotes. The zygote-specific pair of zys1 genes, zys1A and zys1B, is expressed earliest among five genes isolated from a “10-min” zygote library. We report here that the ZYS1 protein, which is encoded by the invertedly duplicated zys1 gene, accumulates in zygotes and is localized in nuclei. In addition, when mt+ gametes (but not mt− gametes) are UV-irradiated before mating, only very limited accumulation of ZYS1 protein can be detected in the resulting zygotes.

PHYLOGENETIC ANALYSIS OF EUDORINA SPECIES (VOLVOCACEAE, CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Species and varieties in the genus Eudorina Ehrenberg (Volvocaceae, Chlorophyta) were evaluated on the basis of phylogenetic analyses of the large subunit ofribulose‐1,5‐bis‐phosphate carboxylase/oxygenase (rbcL) gene sequences from 14 strains of four Eudorina species, as well as from nine species of Pleodorina and Volvox. The sequence data suggested that 10 of the 14 Eudorina strains form three separate and robust monophyletic groups within the nonmonophyletic genus Eudorina. The first group comprises all three strains of E. unicocca G. M. Smith; the second group consists of one of the E. elegans Ehrenberg var. elegans strains, the E. cylindrica Korshikov strain, and both E. illinoisensis (Kofoid) Pascher strains; and the third group consists of two monoecious varieties of E. elegans [two strains of E. elegans var. synoica Goldstein and one strain of E. elegans var. carteri (G. M. Smith) Goldstein]. In addition, E. illinoisensis represents a poly‐ or paraphyletic species within the second group. The remaining four strains, all of which are assigned to E. elegans var. elegans, are nonmonophyletic. Although their position in the phylogenetic trees is more or less ambiguous, they are ancestral to other taxa in the large anisogamous/oogamous monophyletic group including Eudorina, Pleodorina, and Volvox (except for sect. Volvox). Thus, the four Eudorina groups resolved in the present molecular phylogeny do not correspond with the species concepts of Eudorina based on vegetative morphology, but they do reflect the results of the previous intercrossing experiments and modes of monoecious and dioecious sexual reproduction.

ORGANELLE DIVISION AND CYTOPLASMIC INHERITANCE : THE ORIGIN AND BASIS OF THE TRANSMISSION OF ORGANELLE GENOMES

ur current understanding of cytoplasmic inheritance is that most instances result from the transmission of mitochondrial and plastid genomes from parent(s) to progeny. The prevalent view regarding the origin of the mitochondria and plastids is that they were modified from prokaryotic endosymbionts during eukaryotic evolution (Gray 1992, Margulis 1970). According to this hypothesis, mitochondria and plastids originated as ac purple bacteria and cyanobacteria, respectively (Figure 1-I). This hypothesis suggests that originally, during asexual cell division in unicellular eukaryotes, organelles reproduced in the parent cell and then separated into daughter cells (Figure 1-II). This process is called cytoplasmic inheritance in asexual reproduction. During the evolution from haploid to diploid organisms, sexual reproduction, which allows variation within a population, must have arisen in unicellular eukaryotes that display unicellular isogamy (i.e., the gametes are similar in size or activity; Figure 1-III), multicellular anisogamy (i.e., the gametes are slightly different in size or activity; Cytoplasmic inheritance

Comparison of abp1 primary sequences from monocotyledonous and dicotyledonous species

Summary The cDNA fragments of auxin-binding protein (ABP1) were isolated by degenerated-primer mediated reverse transcription-polymerase chain reaction (RT-PCR) from dicotyledonous plant species, i.e. cress, mung bean, pea, radish and soybean, and monocotyledonous species, i.e. oat and rice. Cloned abp 1 cDNA fragments were sequenced and compared with known abp 1 clones from A. thaliana , maize, strawberry and tobacco at the amino acid level. In all plant species studied, the amino acid sequences of newly isolated abp 1 clones were highly conserved at two regions (region A: Thr-Pro-Ile-His-Arg-His-Ser-Cys-Glu-Glu-Ile/Val-Phe-Ile/Thr/Val-Val-Leu/Pro/Val-Lys-Gly-Xaa-Gly-Thr-LeuAy; region B: His-Glu-Asp-Leu-Gln-Phe/Val-Leu-Asp/Val-Ile/Val-Ile-Ser-Arg-Pro-Pro), which were previously reported to be important for auxin-binding. On the other hand, some putative residues of amino acids in the region between regions A and B could be found to be specific in dicot and monocot species, respectively. Southern-blot analysis indicated a small abp 1 gene family in all species studied. Northern-blot analysis indicated that the size of abp 1 mRNA transcripts in all species studied was conserved at approximately 850bp. The phyloge-netic tree of ABP1 was analyzed by the UPGMA method.

The Mitochondrial Endonuclease M20 Participates in the Down-regulation of Mitochondrial DNA in Pollen Cells

The mitochondrial endonuclease M20 is an H-N-H/N nuclease that degrades linear and circular DNA and participates in mitochondrial DNA regulation during pollen development. Maintaining the appropriate number of mitochondrial DNA (mtDNA) molecules is crucial for supporting mitochondrial metabolism and function in both plant and animal cells. For example, a substantial decrease in mtDNA levels occurs as a key part of pollen development. The molecular mechanisms regulating mtDNA copy number are largely unclear, particularly with regard to those that reduce mtDNA levels. Here, we identified and purified a 20-kD endonuclease, M20, from maize (Zea mays) pollen mitochondria. We found M20 to be an His-Asn-His/Asn (H-N-H/N) nuclease that degrades linear and circular DNA in the presence of Mg2+ or Mn2+. Arabidopsis (Arabidopsis thaliana) AtM20, which shared high sequence similarity with maize M20, localized to the mitochondria, had a similar H-N-H/N structure, and degraded both linear and circular DNA. AtM20 transcript levels increased during pollen development, in parallel with a rapid reduction in mtDNA. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome-editing techniques were used to generate knockout lines of AtM20 (atm20), which exhibited a significant delay in the reduction in mtDNA levels in pollen vegetative cells but normal mtDNA levels in somatic cells. The delayed reduction in pollen mtDNA levels was rescued by the transgenic expression of AtM20 in atm20 plants. This study thus uncovers an endonucleolytic DNase in plant mitochondria and its crucial role in reducing mtDNA levels, pointing to the complex mechanism regulating mtDNA levels in plants.