Search

Homologous genes for the peptidoglycan precursor flippase MurJ, and peptidoglycan hydrolases: lytic transglycosylase MltB, and DD-carboxypeptidase VanY are required for chloroplast division in the moss Physcomitrella patens. The moss Physcomitrella patens is used as a model plant to study plastid peptidoglycan biosynthesis. In bacteria, MurJ flippase transports peptidoglycan precursors from the cytoplasm to the periplasm. In this study, we identified a MurJ homolog (PpMurJ) in the P. patens genome. Bacteria employ peptidoglycan degradation and recycling pathways for cell division. We also searched the P. patens genome for genes homologous to bacterial peptidoglycan hydrolases and identified genes homologous for the lytic transglycosylase mltB, N-acetylglucosaminidase nagZ, and LD-carboxypeptidase ldcA in addition to a putative DD-carboxypeptidase vanY reported previously. Moreover, we found a ß-lactamase-like gene (Pplactamase). GFP fusion proteins with either PpMltB or PpVanY were detected in the chloroplasts, whereas fusion proteins with PpNagZ, PpLdcA, or Pplactamase localized in the cytoplasm. Experiments seeking PpMurJ-GFP fusion proteins failed. PpMurJ gene disruption in P. patens resulted in the appearance of macrochloroplasts in protonemal cells. Compared with the numbers of chloroplasts in wild-type plants (38.9 ± 4.9), PpMltB knockout and PpVanY knockout had lower numbers of chloroplasts (14.3 ± 6.7 and 28.1 ± 5.9, respectively). No differences in chloroplast numbers were observed after PpNagZ, PpLdcA, or Pplactamase single-knockout. Chloroplast numbers in PpMltB/PpVanY double-knockout cells were similar to those in PpMltB single-knockout cells. Zymogram analysis of the recombinant PpMltB protein revealed its peptidoglycan hydrolase activity. Our results imply that PpMurJ, PpMltB and PpVanY play a critical role in chloroplast division in the moss P. patens.

ANGUSTIFOLIA (AN) is a plant-specific subfamily of the CtBP/BARS/AN family, characterized by a plant-specific C-terminal domain of approximately 200 amino acids. Previously, we revealed that double knockout (DKO) lines of Physcomitrium (Physcomitrella) patens ANGUSTIFOLIA genes (PpAN1-1 and PpAN1-2) show defects in gametophore height and the lengths of the seta and foot region of sporophytes, by reduced cell elongation. In addition to two canonical ANs, the genome of P. patens has two atypical ANs without a coding region for a plant-specific C-terminus (PpAN2-1 and PpAN2-2); these were investigated in this study. Similar to PpAN1s, both promoters of the PpAN2 genes were highly active in the stems of haploid gametophores and in the middle-to-basal region of young diploid sporophytes that develop into the seta and foot. Analyses of PpAN2-1/2-2 DKO and PpAN quadruple knockout (QKO) lines implied that these four AN genes have partially redundant functions to regulate cell elongation in their expression regions. Transgenic strains harboring P. patens α-tubulin fused to green fluorescent protein, which were generated from a QKO line, showed that the orientation of the microtubules in the gametophore tips in the PpAN QKO lines was unchanged from the wild-type and PpAN1-1/1-2 DKO plants. In addition to both PpAN2-1 and PpAN2-2, short Arabidopsis AN without the C-terminus of 200 amino acids could rescue the Arabidopsis thaliana an-1 phenotypes, implying AN activity is dependent on the N-terminal regions.

Neptunomonas sp. BPy-1 is an epiphytic bacterium isolated from in vitro culture of the red alga Pyropia yezoensis. It uses ethanol as a sole carbon source and promotes the growth of host alga. A related bacterium, Neptunomonas sp. BZm-1, was isolated from leaves of Zostera marina found in the Yatsushiro Sea (Japan). BZm-1 showed 99% 16S rRNA sequence identity with Neptunomonas sp. BPy-1. Similar to BPy-1, BZm-1 grew in artificial seawater (ASW) medium containing ethanol or butanol. When thalli were treated with a multi-enzyme cleaner, the growth of treated thalli was retarded, but the addition of BZm-1 to the medium promoted growth. To explore the benefits of epiphytic bacteria, indoleacetic acid (IAA) production by isolated bacteria was examined under conditions of limited nutrients. Salkowski assays and GC-MS analysis revealed that both BZm-1 and BPy-1 excreted IAA during growth in ASW medium containing glucose or ethanol in the presence of tryptophan. In ASW medium containing tryptophan but lacking a carbon source, neither isolate grow, but produced IAA. ASW medium includes nitrate as the sole nitrogen source. In the absence of carbon source, different nitrogen forms in the presence of tryptophan did not affect IAA production by the two isolates. These findings indicate that IAA production by the two isolates is strictly dependent on tryptophan but less affected by carbon and nitrogen sources. Based on the different origins of BPy-1 and BZm-1, this mode of IAA production seems to be conserved among relatives of BPy-1.

Chloroplasts are believed to be descendants of ancestral cyanobacteria that have a peptidoglycan layer between the outer and the inner membranes. In particular, cyanelles having peptidoglycan in Cyanophora paradoxa are considered as evidence for the endosymbiotic origin of chloroplasts. The moss Physcomitrella patens has a complete set of genes involved in the synthesis of peptidoglycan, but a peptidoglycan layer has not been observed by conventional electron microscopy to date. Recently, a new metabolic labeling technique using a fluorescent probe was applied to visualize putative peptidoglycan surrounding the chloroplasts. The exact localization of the peptidoglycan, however, has not been clearly identified. Here we examined conventional electron micrographs of two types of moss materials (mutants or ampicillin-treated plants), one presumably having peptidoglycan and the other presumably lacking peptidoglycan, and analyzed in detail, by single-pixel densitometry, the electron density of the chloroplast envelope membranes and the intermembrane space. Statistical analysis showed that the relative electron density within the intermembrane space with respect to that of the envelope membranes was significantly higher in the materials presumably having peptidoglycan than in the materials presumably devoid of peptidoglycan. We consider this difference as bona fide evidence for the presence of peptidoglycan between the outer and the inner envelope membranes in the wild-type chloroplasts of the moss, although its density is lower than that in bacteria and cyanelles. We will also discuss this low-density peptidoglycan in the light of the phylogenetic origin of peptidoglycan biosynthesis enzymes.

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio-lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1-1 and 1-2). Both PpAN1 genes complemented the A. thaliana an-1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1-promoter- uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1-1 and PpAN1-2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip-growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α-tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1-1/1-2 double-knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.

The endosymbiotic theory states that plastids are derived from a single cyanobacterial ancestor that possessed a cell wall. Peptidoglycan (PG), the main component of the bacteria cell wall, gradually degraded during plastid evolution. PG-synthesizing Mur genes have been found to be retained in the genomes of basal streptophyte plants, although many of them have been lost from the genomes of angiosperms. The enzyme encoded by bacterial MurE genes catalyzes the formation of the UDP-N-acetylmuramic acid (UDP-MurNAc) tripeptide in bacterial PG biosynthesis. Knockout of the MurE gene in the moss Physcomitrella patens resulted in defects of chloroplast division, whereas T-DNA-tagged mutants of Arabidopsis thaliana for MurE revealed inhibition of chloroplast development but not of plastid division, suggesting that AtMurE is functionally divergent from the bacterial and moss MurE proteins. Here, we could identify 10 homologs of bacterial Mur genes, including MurE, in the recently sequenced genomes of Picea abies and Pinus taeda, suggesting the retention of the plastid PG system in gymnosperms. To investigate the function of gymnosperm MurE, we isolated an ortholog of MurE from the larch, Larix gmelinii (LgMurE) and confirmed its presence as a single copy per genome, as well as its abundant expression in the leaves of larch seedlings. Analysis with a fusion protein combining green fluorescent protein and LgMurE suggested that it localizes in chloroplasts. Cross-species complementation assay with MurE mutants of A. thaliana and P. patens showed that the expression of LgMurE cDNA completely rescued the albefaction defects in A. thaliana but did not rescue the macrochloroplast phenotype in P. patens. The evolution of plastid PG and the mechanism behind the functional divergence of MurE genes are discussed in the context of information about plant genomes at different evolutionary stages.

Plant growth regulators (PGRs) play a pivotal role in vascular plants, regulating growth, development, and stress responses; however, the role of PGRs in algae remains largely unexplored. Here, the role of ethylene, a simple plant growth regulator, was demonstrated in sexual reproduction of the marine red alga Pyropia yezoensis. Application of the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) promoted the formation of spermatia and zygotospores in the gametophytes as well as ethylene production, whereas the growth rate was repressed in comparison to gametophytes not treated with ACC. In addition, gametophytes treated with ACC and mature gametophytes showed enhanced tolerance to oxidative stress. Gene expression profiles revealed upregulation of genes involved in cell division and stress response in gametophytes treated with ACC and in mature gametophytes. These results indicate that ethylene plays an important role in the regulation of gamete formation and protection against stress-induced damage during the sexual reproductive stage. Considered together, these findings demonstrate that ethylene is involved in regulating the switching from a vegetative to a sexual reproductive phase in P. yezoensis.

It is believed that the plastids in green plants lost peptidoglycan (i.e., a bacterial cell wall-containing d-amino acids) during their evolution from an endosymbiotic cyanobacterium. Although wall-like structures could not be detected in the plastids of green plants, the moss Physcomitrella patens has the genes required to generate peptidoglycan (Mur genes), and knocking out these genes causes defects in chloroplast division. Here, we generated P. patens knockout lines (∆Pp-ddl) for a homolog of the bacterial peptidoglycan-synthetic gene encoding d-Ala:d-Ala ligase. ∆Pp-ddl had a macrochloroplast phenotype, similar to other Mur knockout lines. The addition of d-Ala-d-Ala (DA-DA) to the medium suppressed the appearance of giant chloroplasts in ∆Pp-ddl, but the addition of l-Ala-l-Ala (LA-LA), DA-LA, LA-DA, or d-Ala did not. Recently, a metabolic method for labeling bacterial peptidoglycan was established using ethynyl-DA-DA (EDA-DA) and click chemistry to attach an azide-modified fluorophore to the ethynyl group. The ∆Pp-ddl line complemented with EDA-DA showed that moss chloroplasts are completely surrounded by peptidoglycan. Our findings strongly suggest that the moss plastids have a peptidoglycan wall containing d-amino acids. By contrast, no plastid phenotypes were observed in the T-DNA tagged ddl mutant lines of Arabidopsis thaliana.

Class A penicillin-binding proteins (PBPs) are active in the final step of bacterial peptidoglycan biosynthesis. They possess a transglycosylase (TG) domain to polymerize the glycan chains and a transpeptidase (TP) domain to catalyze peptide cross-linking. We reported that knockout of the Pbp gene in the moss Physcomitrella patens (ΔPpPbp) results in a macrochloroplast phenotype by affecting plastid division. Here, expression of PpPBP-GFP in ΔPpPbp restored the wild-type phenotype and GFP fluorescence was observed mainly in the periphery of each chloroplast. Stable transformants expressing Anabaena PBP with the plastid-targeting sequence, or PpPBP replacing the Anabaena TP domain exhibited partial recovery, while chloroplast number was recovered to that of wild-type plants in the transformant expressing PpPBP replacing the Anabaena TG domain. Transient expression experiments with site-directed mutagenized PpPBP showed that mutations in the conserved amino acids in both domains interfered with phenotype recovery. These results suggest that both TG and TP functions are essential for function of PpPBP in moss chloroplast division.

Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes. The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S. maritima is related to European and India S. maritima, while Japanese S. maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S. maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S. maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding choline monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S. maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

Superoxide dismutases (SODs) catalyze the dismutation of superoxide and play an important role in reducing oxidative stress in plants. Based on in-gel SOD activity staining, chloroplasts of the moss Physcomitrella patens have two CuZn-SODs as the major SOD isozymes and minor SODs, including a Fe-SOD and two Mn-SODs. To investigate the contribution of chloroplastic SODs to stress tolerance in P. patens, we generated a double mutant lacking chloroplastic CuZn-SOD genes. The mutant did not show any differences in comparison to the wild type based on the growth of protonemata on normal and high-salt media, extractable activities of the other SODs after culture on normal and high-salt media, and inhibition of Fv/Fm under stress conditions (high-salt, high-light, and high-temperature). These results indicate that chloroplastic CuZn-SODs do not play a principal role in oxidative stress tolerance in chloroplasts under the investigated conditions. These findings explain the previously reported unusual response of P. patens to copper deficiency, in which chloroplastic CuZn-SODs are preferentially inactivated but cytosolic CuZn-SODs are unaffected.

The established culture of gametophytes of the red alga Pyropia yezoensis (TU-1) is superficially colonized by epiphytic bacteria. By 16S rRNA sequencing, 6 bacterial species were identified, and a culturable bacterium, Neptunomonas sp. BPy-1, was isolated. The 16S rRNA sequences of BPy-1 showed 100% identity with that of Neptunomonas sp. 0536, a probiotic bacterium found in greenshell mussels in New Zealand. Physiological tests revealed that 22 characters were identical between BPy-1 and 0536, but that 4 characters differed. BPy-1 cannot grow in the artificial seawater used for the culture of gametophytes. BPy-1 can grow in the artificial seawater with ethanol or butanol but not in methanol or propanol. To determine the effect of BPy-1 on gametophyte growth, BPy-1 colonization was reduced by 80% using a multi-enzyme cleaner. Changing the cleaner concentration yielded two types of gametophytes, a compressed or callus-like form and a nearly normal form. BPy-1 promoted the growth of the treated gametophytes with relatively normal form, while it showed less effect on compressed gametophytes. These findings suggested that BPy-1 promotes the growth of damaged gametophytes but does not affect the development of normal gametophytes.

Superoxide dismutases (SODs) are metallo-enzymes that catalyze the dismutation of superoxide radicals. In Arabidopsis thaliana, the expression of CuZn-SOD in both the chloroplast and cytosol was reported to be down-regulated by microRNA398 (miR398) during growth on low copper. The moss Physcomitrella patens contains chloroplastic and cytosolic CuZn-SOD genes, but lacks miR398. From analysis of P. patens microRNA, miR1073 was predicted to target CuZn-SOD mRNAs. We noticed that two chloroplastic CuZn-SOD genes contain the miR1073 target sequence in the 3' untranslated region; however, the cytosolic isozyme genes lack this sequence. In this study, we investigated the involvement of miR1073 in the expression of CuZn-SOD genes in P. patens. When protonemata of P. patens were cultured on a copper-depleted medium, SOD activity and mRNA levels of chloroplastic CuZn-SODs were decreased markedly. In contrast, cytosolic CuZn-SODs showed little or no change in mRNA levels or SOD activity. The precursor transcript and the mature form of miR1073 were induced by copper deficiency. The chloroplastic CuZn-SOD (PpCSD1) mRNA was cleaved at the miR1073 target site under copper deficiency. These results suggest that miR1073 is involved in the down-regulation of PpCSD1 expression. In addition to PpCSD1 mRNA, antisense RNAs of PpCSD1 were also detected under normal conditions; however, under copper deficiency, they were cleaved within the open reading frame (ORF) region. The cleavage of sense PpCSD1 mRNA was also detected within the ORF region. Although only miR1073 exists in the database, it is presumed that RNA cleavage, other than that mediated by miR1073, is involved in the regulation of PpCSD1 expression.

Chloroplasts are believed to be descendants of ancestral cyanobacteria that had peptidoglycan layer between the outer and the inner membranes. Historically, the glaucophyte Cyanophora paradoxa and the rhizopod Paulinella chromatophora were believed to harbor symbiotic cyanobacteria having peptidoglycan, which were conventionally named “cyanelles”. In addition, the complete set of genes involved in the synthesis of peptidoglycan has been found in the moss Physcomitrella patens and some plants and algae. The presence of peptidoglycan-like structures was demonstrated by a new metabolic labeling technique in P. patens. However, many green algae and all known red algae lack peptidoglycan-related genes. That is the reason why we questioned the origin of peptidoglycan-synthesizing enzymes in the chloroplasts of the green algae and plants. We performed phylogenetic analysis of ten enzymes involved in the synthesis of peptidoglycan exploiting the Gclust homolog clusters and additional genomic data. As expected, all the identified genes encoded in the chromatophore genome of P. chromatophora were closely related to cyanobacterial homologs. In the green algae and plants, only two genes, murA and mraY, were found to be closely related to cyanobacterial homologs. The origins of all other genes were diverse. Unfortunately, the origins of C. paradoxa genes were not clearly determined because of incompleteness of published genomic data. We discuss on the probable evolutionary scenarios to explain the mostly non-cyanobacterial origins of the biosynthetic enzymes of chloroplast peptidoglycan: A plausible one includes extensive multiple horizontal gene transfers during the early evolution of Viridiplantae.

To identify nuclear genes involved in plastid function, we analyzed Arabidopsis thaliana mutants with albino, pale green or variegated leaves using the Activator/Dissociation (Ac/Ds) transposon tagging system. In this study, we focused on mutants with a Ds insertion in the gene At1g32080 (AtLrgB), which encodes a homolog of the bacterial membrane protein LrgB. Although the detailed function of bacterial LrgB remains unclear, it is speculated that LrgB functions against cell death and lysis in cooperation with LrgA. Reverse transcription-PCR (RT-PCR) and promoter-GUS (β-glucuronidase) analyses showed that AtLrgB is expressed in leaves, stems and flowers, but not in roots. Moreover, its expression in leaves continued until senescence. We used three Ac/Ds-tagged mutants (atlrgB) that showed the same phenotypes. During the continuous observation of seedlings under short-day conditions, we found that the cotyledons and true leaves of the mutant plants during early development showed immediate greening, similar to wild-type plants, after which some parts showed a chlorotic phenotype. In contrast, true leaves at the late stage of plant development did not show degreening. When the atlrgB mutant was grown under continuous light, its chlorotic phenotype was suppressed. Transformation with normal AtLrgB restored these phenotypes. Trypan blue staining and electron microscopic observations indicated that chlorotic cell death occurred in the white sectors. The phenotypes of atlrgB resembled those in lesion mimic mutants, suggesting that AtLrgB functions against cell death, similar to the bacterial Lrg system.

SUMMARYCtBP/BARS is a unique protein family in having quite diversified cellular functions, intercellular localizations,and developmental roles. ANGUSTIFOLIA (AN) is the sole homolog of CtBP/BARS from Arabidopsis thaliana,although it has plant AN-specific motifs and a long C-terminus. Previous studies suggested that AN wouldfunction in the nucleus as a transcriptional co-repressor, as CtBPs function in animals; however, preciseverification has been lacking. In this paper, we isolated a homologous gene (MAN)ofAN from liverwort,Marchantia polymorpha. Transformation of the Arabidopsis an-1 mutant with 35S-driven MAN completelycomplemented the an-1 phenotype, although it lacks the putative nuclear localization signal (NLS) that existsinANproteinsisolatedfromother plantspecies.WeconstructedseveralplasmidsforexpressingmodifiedANswith amino acid substitutions in known motifs. The results clearly indicated that modified AN with mutationsin the putative NLS-like domain could complement the an-1 phenotype. Therefore, we re-examinedlocalization of AN using several techniques. Our results demonstrated that AN localizes on punctuatestructures around the Golgi, partially overlapping with a trans-Golgi network resident, which highlighted anunexpected link between leaf development and membrane trafficking. We should reconsider the roles andevolutionary traits of AN based on these findings.Keywords: ANGUSTIFOLIA, CtBP/BARS, Golgi, Marchantia polymorpha, TGN.INTRODUCTIONLeaf development is divided into two processes, cell prolif-eration and cell differentiation. In the cell differentiationprocess, genetic pathways controlling leaf cell shape havebeen revealed by the characterization of two mutants ofArabidopsis thaliana (hereafter Arabidopsis): angustifolia(an) and rotundifolia3 (rot3). The an mutant was originallyisolated by Re´dei (1962) as a mutant having a narrow leafand twisted fruit. Later studies reported that an has tric-homes with two branches (Hu¨lskampet al., 1994) and its leafcells are narrower and thicker than wild-type cells (Tsukayaet al., 1994; Tsuge et al., 1996). In contrast, rot3 has short,rounded leaves resulting from reduced cell expansion alongthe leaf length direction (Tsuge et al., 1996). These axis-specific defects in cell expansion demonstrate two inde-pendent polarity-dependent cell expansion systems (Tsugeet al., 1996; Tsukaya, 2002).

Dynamin family proteins in eukaryotic cells assemble into rings or spirals on the surface of membranes and pinch the membranes. We found 21 dynamin-related protein (DRP) genes in the Physcomitrella patens genome. Phylogenetic analysis indicated that three of them (PpDRP5B-1, PpDRP5B-2, and PpDRP5B-3) showed robust monophyly with Arabidopsis thaliana DRP5B and Cyanidioschyzon merolae CmDnm2, both of which are related to plastid division. Quantitative RT-PCR analysis showed that the amounts of DRP5B-3 transcripts were 14-fold and 8-fold higher than those of DRP5B-1 and DRP5B-2, respectively. We generated PpDRP5B knockout transformants for each of these genes. Subapical protonemata cells in wild-type plants had an average of 47 chloroplasts. The cells in the PpDRP5B-3 knockout transformant had slightly enlarged chloroplasts, with an average chloroplast number of 28, whereas the PpDRP5B-1 and 5B-2 knockout lines had no effect on chloroplast number in P. patens. To analyze function of each PpDRP5B gene, we generated double- and triple-knockout lines. Whereas there were 32 chloroplasts in a cell of the PpDRP5B-1/5B-2 double-knockout lines, the triple-knockout line had only a few macrochloroplasts. A transient expression assay with the triple-knockout line demonstrated that the PpDRP5B-3 gene could recover the normal chloroplast phenotype.

Although maternal or uniparental inheritance of mitochondrial genomes is a general rule, biparental inheritance is sometimes observed in protists and fungi, including yeasts. In yeast, recombination occurs between the mitochondrial genomes inherited from both parents. Mitochondrial fusion observed in yeast zygotes is thought to set up a space for DNA recombination. In the last decade, a universal mitochondrial fusion mechanism has been uncovered, using yeast as a model. On the other hand, an alternative mitochondrial fusion mechanism has been identified in the true slime mold Physarum polycephalum. A specific mitochondrial plasmid, mF, has been detected as the genetic material that causes mitochondrial fusion in P. polycephalum. Without mF, fusion of the mitochondria is not observed throughout the life cycle, suggesting that Physarum has no constitutive mitochondrial fusion mechanism. Conversely, mitochondria fuse in zygotes and during sporulation with mF. The complete mF sequence suggests that one gene, ORF640, encodes a fusogen for Physarum mitochondria. Although in general, mitochondria are inherited uniparentally, biparental inheritance occurs with specific sexual crossing in P. polycephalum. An analysis of the transmission of mitochondrial genomes has shown that recombinations between two parental mitochondrial genomes require mitochondrial fusion, mediated by mF. Physarum is a unique organism for studying mitochondrial fusion.

Retrotransposons are mobile genetic elements which transpose via an RNA intermediate. Because the transposition of retrotransposons causes insertional mutagenesis in a genome, the activation of retrotransposons has been used for basic and applied genome studies in plant biology. Some retrotransposons were shown to be transcriptionally activated by biotic and abiotic stress factors, such as UV light, ozone, pathogens, wounding, and drought. A pulsed streamerlike discharge in water is known to generate physical phenomena, such as high electric fields, UV, ozone, other chemically active species, and shock waves. In this paper, an underwater pulsed discharge treatment was used as a new tool for the activation of retrotransposons in a Porphyra yezoensis genome. The results of reverse-transcription polymerase chain reaction for the discharge treatment of P. yezoensis showed that a copia-like retrotransposon, called PyRE1G1, was transcriptionally activated by the underwater pulsed discharge without cell damage. The effects of pulse parameters on the mRNA accumulation of PyRE1G1 showed that a certain charging voltage and larger applied pulse numbers gave sufficient results for the transcriptional activation of PyRE1G1. The maximum activation of the PyRE1G1 expression was induced 1 h after the discharge treatment.

In the moss Physcomitrella patens, 10 Mur genes involved in peptidoglycan biosynthesis were found, and the MurE and Pbp genes are related to plastid division. Although the MraY and MurG genes were missing in our previous expressed sequence tag screening, they were discovered in the P. patens genome in this study, indicating that P. patens has a full set of genes capable of synthesizing peptidoglycan. In addition, a second MurA gene (PpMurA2) was found. Whereas Northern analyses indicated that PpMurA1, PpMurG and PpMraY were expressed, transcripts of PpMurA2 were detected only when RT-PCR was employed. Whereas GFP fusion proteins with either PpMurA1 or PpMraY were detected in chloroplasts, the PpMurA2 fusion proteins were located in the cytoplasm. Protonema cells in the wild-type plants had an average of 46 chloroplasts. PpMurA1 gene-disrupted lines had

The molecular organization of mitochondrial-nucleoids (mt-nucleoids) isolated from BY-2 cultured tobacco (Nicotiana tabacum L.) cells was analyzed. Fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) revealed that isolated mt-nucleoids retained the compact structural organization as that observed in vivo. Treatments of mt-nucleoids with DNase I, proteinase K and 2 M NaCl resulted in the disappearance, partial swelling, or complete dispersion of their DAPI-fluorescence, respectively, whereas treatment with RNase A did not affect their compact appearance. These results suggest that the compact structure of mt-nucleoids is maintained by electrostatic interaction between DNA and proteins. When mt-nucleoids that were treated with micrococcal nuclease (MNase) were analyzed by agarose gel electrophoresis, a ladder-like electrophoretic pattern of DNA, whose minimum fragment size was about 75 bp, was observed. This result suggests the presence of nucleosome-like, repetitive structures in the mt-nucleoids. Treatment of mt-nucleoids with increasing concentrations of NaCl gradually solubilized various proteins, including those with DNA-binding activities, which was accompanied by gradual dispersion of the mt-nucleoid structure. This result suggests that a variety of DNA-binding proteins with differing degrees of binding strength are present in the mt-nucleoids, and are involved in the compact organization of DNA-protein complexes at various levels. Detailed analyses of mt-nucleoids by Southwestern blotting, SDS-DNA PAGE, and DNA-cellulose affinity chromatography revealed the presence of many different DNA-binding proteins in the 2 M NaCl-soluble fraction of mt-nucleoids, which are expected to be involved in the compact organization of mt-nucleoids at various levels.

We investigated the effect of antibiotics that specifically inhibit peptidoglycan synthesis on plastid division in pteridophytes by comparing the numbers of plastids in normal and antibiotic-treated pteridophyte cells. Plastid numbers were unaffected in Moniliformopses cells but decreased in Lycophytina cells after treatment with the β-lactam antibiotic ampicillin. However, cells of all four pteridophyte species exhibited decreased plastid numbers when treated with the antibiotic fosfomycin. Together, these results suggest that the Mur genes may be related to plastid division in pteridophytes, as demonstrated for certain bryophytes.

A genomic PCR fragment of 4581 bp, referred to as PyRE10G, was isolated from the red alga Porphyra yezoensis. PyRE10G contained a putative open reading frame encoding gag, protease, integrase, reverse transcriptase (RT), and RNase H, but one stop codon was present in the integrase region. Southern blot analysis revealed that PyRE10G exists as a single copy in the genome. From the order of gene arrangement of polyproteins, PyRE10G appears to be a copia-like retrotransposon. Amino acid sequences of PyRE10G RT and RNase H were closely related to those of copia-like retrotransposons. In contrast, the phylogenetic tree suggested that PyRE10G integrase stands within the gypsy elements and outside the copia group. PyRE10G is the first example of a chimeric composition of copia- and gypsy-like polyprotein genes in a single element, supporting the hypothesis that long terminal repeat-containing retrotransposons have evolved by fusion of ancestral RT/RNase H and other polyprotein genes.

Enzymes encoded by bacterial MurE genes catalyze the ATP-dependent formation of uridine diphosphate-N-acetylmuramic acid-tripeptide in bacterial peptidoglycan biosynthesis. The Arabidopsis thaliana genome contains one gene with homology to the bacterial MurE:AtMurE. Under normal conditions AtMurE is expressed in leaves and flowers, but not in roots or stems. Sequence-based predictions and analyses of GFP fusions of the N terminus of AtMurE, as well as the full-length protein, suggest that AtMurE localizes to plastids. We identified three T-DNA-tagged and one Ds-tagged mutant alleles of AtMurE in A. thaliana. All four alleles show a white phenotype, and A. thaliana antisense AtMurE lines showed a pale-green phenotype. These results suggest that AtMurE is involved in chloroplast biogenesis. Cells of the mutants were inhibited in thylakoid membrane development. RT-PCR analysis of the mutant lines suggested that the expression of genes that depend on a multisubunit plastid-encoded RNA polymerase was decreased. To analyze the functional relationships between the MurE genes of cyanobacteria, the moss Physcomitrella patens and higher plants, a complementation assay was carried out with a P. patens (Pp) MurE knock-out line, which exhibits a small number of macrochloroplasts per cell. Although the Anabaena MurE, fused with the N-terminal region of PpMurE, complemented the macrochloroplast phenotype in P. patens, transformation with AtMurE did not complement this phenotype. These results suggest that AtMurE is functionally divergent from the bacterial and moss MurE proteins.

Four DNA fragments encoding a reverse transcriptase (RT)-like gene related to that of long terminal repeat (LTR) retrotransposons were isolated from the red alga Porphyra yezoensis by genomic PCR. Southern blot analysis suggested that one clone exists as a single copy per genome. Its full-length cDNA (PyRE2A) contained RT/RNase H-like sequences, which are most closely related to those of the Volvox LTR retrotransposon, although two stop codons were present within the RT region. We did not find any sequence related to LTR retrotransposons other than RT/RNase H in RyRE2A. These results indicate that PyRE2A is a single RT/RNase H-like gene and a defective progenitor of LTR retrotransposons.

The general consensus is that a cyanobacterium phagocytosed by a host cell evolved into the plastids of red and green algae, land plants, and glaucophytes. In contrast to the plastids of glaucophytes, which retain a cyanobacterial-type peptidoglycan layer, no wall-like structures have been detected in plastids from other sources. Although the genome of Arabidopsis thaliana contains five genes that are essential for peptidoglycan synthesis, MurE, MurG, two genes for d -Ala- d -Ala ligase (Ddl), and the gene for translocase I (MraY), their functions have not been determined. We report that the moss Physcomitrella patens has nine homologous genes related to peptidoglycan biosynthesis: MurA, B, C, D, E, and F, Ddl, genes for the penicillin-binding protein Pbp, and dd -carboxypeptidase (Dac). Corroborating a computer prediction, analysis of the GFP fusion proteins with the N terminus of PpMurE or of PpPbp suggests that these proteins are located in the chloroplasts. Gene disruption of the PpMurE gene in P. patens resulted in the appearance of macrochloroplasts both in protonema and in leaf cells. Moreover, gene knockout of the P. patens Pbp gene showed inhibition of chloroplast division in this moss; however, no Pbp gene was found in A. thaliana .

We report a new protocol for the stable transformation of Larix gmelinii. Thirty mature zygotic embryos precultured for 3 days on solid medium supplemented with benzyladenine were bombarded with plasmids pUC-GHG (GUS, HPT, and GFP genes) or pBI221-HPT (HPT and GUS genes). After a 2-month culture on selection medium, hygromycin-resistant calli appeared on the surfaces of the necrotic embryos. The frequencies of embryos with resistant calli were 18.4% and 17.4% in the transformations with pUC-GHG and pBI221-HPT DNA, respectively. More than 20 adventitious shoots formed from each of the transgenic calli. Of 17 elongated shoots selected for culturing on a rooting medium, five shoots rooted after 2 months. Expression of the GFP and GUS genes was detected in the resistant tissues by microscopic observations and by a histological GUS activity assay, respectively. PCR and Southern analysis confirmed the stable insertion of the introduced DNA into the genome.

We investigated two genes that encode a homolog of the bacterial cell division site determinant MinD, PpMinD1 and PpMinD2, which were previously identified in the moss Physcomitrella patens. Southern analysis suggested that the P. patens genome does not contain minD genes other than PpMinD1 and PpMinD2. Molecular phylogenetic analysis and the results of a transient expression assay using MinD : green fluorescent protein (GFP) fusions implied that PpMinD1 and PpMinD2 are cyanobacterium-derived, closely related genes encoding chloroplast-targeted MinD proteins. Disruption of the genomic PpMinD1 or PpMinD2 locus had no detectable effects on plastid division. This implies that there is functional redundancy in the two minD genes in P. patens.

Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O.Pki and Shojovaka) was investigated. Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP). The regeneration frequency of calli of cvs. Senshuki and O.Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium. By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars. These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months. Most (97%) of the regenerated plantlets had a normal diploid karyotype (2 n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv. Shojovaka had a tetraploid karyotype.

Liverwort, Marchantia paleacea var. diptera, contains Mn-superoxide dismutase (Mn-SOD) in mitochondria, Fe-SOD in chloroplast and CuZn-SOD in cytosol. An Mn-SOD gene (MpMnSOD) was isolated from the liverwort. Using this clone together with the liverwort Fe-SOD and CuZn-SOD genes as probes, the expression of three SOD genes was investigated. Under heterotrophic conditions, the transcript of three SOD genes was accumulated light independently. On the other hand, under photoautotrophic conditions, the transcript levels of Fe-SOD and Mn-SOD increased in the light while that of CuZn-SOD decreased. The reverse occurred in the dark. In contrast to the transcript level, the activity of the three SODs was barely affected by light. The transcription inhibitor, cordycepin, inhibited either the light-promoted accumulation of Fe-SOD and Mn-SOD transcript or the light-induced reduction of the CuZn-SOD transcript. Photosynthetic electron transport inhibitors, DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, inhibited the photo-response in three SOD genes. These results suggest that the transcript abundance of three SOD genes in liverwort is regulated by photosynthetic electron transport but the mechanism regulating the transcript abundance of the CuZn-SOD gene is different from that of the Fe-SOD and Mn-SOD genes.

Three DNA fragments encoding part of a reverse transcriptase similar to that of Ty1- copia retrotransposons were isolated from suspension-cultured cells of the liverwort Marchantia paleacea var. diptera. Digestion of total DNA with methylation-sensitive and -insensitive isoschizomers revealed that, like those of the subtelomeric region, CpG sequences in retrotransposon genes were hypermethylated. The methylation status and expression of these retrotransposons was less affected by 5-azadeoxycytidine treatment compared to the subtelomeric region, which was considerably demethylated.

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions.

Moss chloroplasts should prove useful for studying the cyanobacteria-derived system in chloroplasts. To determine the effects of antibiotics that inhibit bacterial peptidoglycan synthesis, the numbers of chloroplasts in treated Physcomitrella patens cells were counted. Ampicillin and D-cycloserine caused a rapid decrease in the number of chloroplasts per cell. Fosfomycin affected half of the cells, while vancomycin affected a few cells. Conversely, bacitracin had no effect. With the decrease in chloroplast number, macrochloroplasts appeared in antibiotic-treated cells. Removal of the antibiotics resulted in the recovery of chloroplast number, suggesting that the decrease in number was directly dependent on the antibiotic treatment. Microscopic observations showed that the decrease in the number of chloroplasts resulted from cell division without chloroplast division. These results suggest that enzymes derived from the bacterial peptidoglycan synthesis pathway are related to moss chloroplast division.

Suspension-cultured cells of Polygonum hydropiper contain a high level of catechins (H cells), unlike tissues of the parental plants, which express a low level. A cell line (L cells) containing a tenfold lower level of catechins than H cells was isolated from H cells. To determine the mechanism regulating catechin biosynthesis, we have investigated the expression of genes involved in flavonoid biosynthesis in these two cell lines. Deduced amino acid sequences of the gene fragments encoding chalcone synthase, flavanone 3-hydroxylase and dihydroflavonol 4-reductase isolated from H cells showed a high similarity to those from other plants. Genomic Southern analysis indicated that there is no difference in the restriction patterns of these gene fragments between the two cell lines and seedlings. Throughout the culture period, all of the genes in the H cells were highly expressed, while those in the L cells were repressed. In the seedlings, all of the genes were expressed in a light-dependent fashion, while in H cells these genes were highly expressed regardless of the presence or absence of illumination. These findings indicate that the genes involved in catechin biosynthesis are coordinately regulated at the transcriptional level.

Mobile group I introns sometimes contain an open reading frame (ORF) possibly encoding a site-specific DNA endonuclease. However, previous phylogenetic studies have not clearly deduced the evolutionary roles of the group I intron ORFs. In this paper, we examined the phylogeny of group IA2 introns inserted in the position identical to that of the chloroplast-encoded rbcL coding region (rbcL-462 introns) and their ORFs from 13 strains of five genera (Volvox, Pleodorina, Volvulina, Astrephomene, and Gonium) of the colonial Volvocales (Chlorophyceae) and a related unicellular green alga, Vitreochlamys. The rbcL-462 introns contained an intact or degenerate ORF of various sizes except for the Gonium multicoccum rbcL-462 intron. Partial amino acid sequences of some rbcL-462 intron ORFs exhibited possible homology to the endo/excinuclease amino acid terminal domain. The distribution of the rbcL-462 introns is sporadic in the phylogenetic trees of the colonial Volvocales based on the five chloroplast exon sequences (6021 bp). Phylogenetic analyses of the conserved intron sequences resolved that the G. multicoccum rbcL-462 intron had a phylogenetic position separate from those of other colonial volvocalean rbcL-462 introns, indicating the recent horizontal transmission of the intron in the G. multicoccum lineage. However, the combined data set from conserved intron sequences and ORFs from most of the rbcL-462 introns resolved robust phylogenetic relationships of the introns that were consistent with those of the host organisms. Therefore, most of the extant rbcL-462 introns may have been vertically inherited from the common ancestor of their host organisms, whereas such introns may have been lost in other lineages during evolution of the colonial Volvocales. In addition, apparently higher synonymous substitutions than nonsynonymous substitutions in the rbcL-462 intron ORFs indicated that the ORFs might evolve under functional constraint, which could result in homing of the rbcL-462 intron in cases of spontaneous intron loss. On the other hand, the presence of intact to largely degenerate ORFs of the rbcL-462 introns within the three isolates of Gonium viridistellatum and the rare occurrence of the ORF-lacking rbcL-462 intron suggested that the ORFs might degenerate to result in the spontaneous intron loss during a very short evolutionary time following the loss of the ORF function. Thus, the sporadic distribution of the rbcL-462 introns within the colonial Volvocales can be largely explained by an equilibrium between maintenance of the introns by the intron ORF and spontaneous loss of introns when the introns do not have a functional ORF.

The transfer of Marchantia paleacea var. diptera cells to darkness caused a reversible repression in the accumulation of transcript for a gene, por, encoding the NADPH: protochlorophyllide oxidoreductase (EC 1.3.1.33). The photosynthetic inhibitor DCMU and DBMIB repressed the accumulation in light. In the presence of transcription inhibitor cordycepin, not only incubation in the dark but also addition of DCMU or DBMIB in light stimulated the degradation of the por transcript. These findings suggest that photosynthetic electron transport is involved in regulating the stability of the por transcript.

We studied the effects of the β-lactam antibiotic ampicillin on the morphology and number per cell of chloroplasts using cells of the liverwort Marchantia polymorpha in suspension culture. Cells in suspension culture without ampicillin contained a mean±SD of 20.6±9.5 chloroplasts. Those in culture with ampicillin showed a decrease in chloroplast number, reaching 7.5±6.5 after 14 d of treatment. Ampicillin-treated cells contained very large chloroplasts. Following removal of ampicillin on day 6, the chloroplast number recovered to that in untreated controls 8 d after removal. These results demonstrated inhibition of chloroplast division by this β-lactam antibiotic, suggesting that the bacterial peptidoglycan-related system has been conserved in the chloroplasts of liverwort.

A 2,158 bp cDNA (PyBPO1) encoding a bromoperoxidase (BPO) of 625 amino acids was isolated from Pyropia yezoensis. Phylogenetic analysis using amino acid sequences of BPOs suggested that P. yezoensis and cyanobacteria were grouped in the same clade and separated from brown algae. Genomic Southern blot analysis suggested that PyBPO1 existed as a single copy per haploid genome. RT-PCR revealed that PyBPO1 was actively expressed in filamentous sporophytes but repressed in leafy gametophytes under normal growth conditions. High expression levels of PyBPO1 in sporophytes were observed when sporophytes were grown under gametophyte conditions, suggesting that preferential expression of PyBPO1 occurs during the sporophyte phase. BPO activity of cell-free extracts from sporophytes and gametophytes was examined by activity staining on native PAGE gel using o-dianisidine. One activity band was detected in sporophyte sample, but not in gametophyte sample. In addition, we found that bromide and iodide were effective substrate, but chloride was not. BPO activity was observed-likely in chloroplasts-when sporophyte cells were incubated with o-dianisidine and hydrogen peroxide. Cellular BPO staining showed the same halogen preference identified by in-gel BPO staining. Based on GS-MS analysis, bromoform was detected in medium containing sporophytes. Bromoform was not detected under dark culture conditions but was detected in the culture exposed to low light intensity (5 μmol m(-2) s(-1)) and increased under a moderate light intensity (30 μmol m(-2) s(-1)).

acetylserine (thiol) lyase (OASTL) genes were isolated by screening a genomic library of the primitive red alga Cyanidioschyzon merolae and named cmSAT2, and cmOASTL1 and cmOASTL2, respectively. cmSAT2 encoded a polypeptide of 406 amino acids. cmSAT2 was encoded on chromosome IX. cmOASTL1 and cmOASTL2 encoded for polypeptides of 389 and 390 amino acids, respectively. A molecular phylogenic tree of the amino acid sequences suggested that cmOASTL1 belongs to the group of eukaryotic plant OASTL while cmOASTL2 belongs to the bacterial type OASTL. cmOASTL1 and cmOASTL2 were encoded on chromosomes XVII and VIII, respectively. In Northern blot analyses, the probes for cmOASTL1 and cmOASTL2 hybridized with 1.4 kb and 1.3 kb bands, respectively. The identity of cmOASTL1 and cmOASTL2 was confirmed by genetic complementation in an OASTL- deficient mutant of Escherichia coli NK3 using sulfate or sulfide as a sole source of sulfur.

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.

Pulsed-field gel electrophoresis (PFGE) was used to examine the in vivo and in vitro conformations of Physarum polycephalum mitochondrial DNA (mtDNA). We used plugs containing isolated mitochondria, isolated mitochondrial nucleoids (mt-nuclei), and isolated mtDNA, in addition to whole cells. The mtDNA contained in the myxamoebae, plasmodia, isolated mitochondria, and isolated mt-nuclei was circular, but most of the isolated mtDNA had been site-specifically fragmented and linearized during DNA preparation and storage under low ionic strength conditions. Restriction mapping of Physarum mtDNA by the direct digestion of the isolated mt-nuclei from two different strains, DP89 x AI16 and KM88 x AI16, resulted in the circular form. A linear mitochondrial plasmid, mF, is known to promote mitochondrial fusion and integration of itself into the mtDNA in Physarum. Linearization of mtDNA by the integration of the mF plasmid was demonstrated when we used PFGE to analyze isolated mitochondria from the plasmodial strain DP89 x NG7 carrying the mF plasmid (mF+). The PFGE system can be used not only to determine whether the form of mtDNA is linear or circular but also to analyze the dynamic conformational changes of mtDNA.

In cultured Bright Yellow-2 (BY-2) tobacco (Nicotiana tabacum) cells, the depletion of auxin (2,4-dichlorophenoxyacetic acid) in the culture medium induces the accumulation of starch. This is accelerated by the addition of cytokinin (benzyladenine). Light and electron microscopic observations revealed that this amyloplast formation involves drastic changes in plastid morphology. The effects of auxin and cytokinin on amyloplast development were investigated by adding auxin or cytokinin to cells grown in a hormone-free culture. Auxin repressed amyloplast development, whereas cytokinin accelerated starch accumulation regardless of the timing of hormone addition. RNA gel-blot analysis revealed that the accumulation of the ADP-glucose pyrophosphorylase small subunit gene (AgpS), granule-bound starch synthase, and starch branching enzyme transcripts were also affected by hormonal conditions. High levels of AgpS, granule-bound starch synthase, and starch branching enzyme transcripts accumulated in amyloplast-developing cells grown in auxin-depleted conditions. Furthermore, the addition of auxin to the cells cultured in hormone-free medium reduced the level of AgpS transcripts, whereas the addition of cytokinin increased it, irrespective of the timing of hormone addition. These results suggest that auxin and cytokinin exert opposite effects on amyloplast development by regulating the expression of the genes required for starch biosynthesis.

Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles.

The chloroplast-encoded atpB gene was sequenced from 33 strains representing 28 species of the colonial Volvocales (the Volvocaceae and its relatives) to reexamine phylogenetic relationships as previously deduced by morphological data and rbcL gene sequence data.1128 base pairs in the coding regions of the atpB gene were analyzed by MP, NJ, and ML analyses. Although supported with relatively low bootstrap values (75% and 65% in the NJ and ML analyses, respectively), three anisogamous/oogamous volvocacean genera—Eudorina, Pleodorina, and Volvox, excluding the section Volvox (=Euvolvox, illegitimate name), constituted a large monophyletic group (Eudorina group). Outside the Eudorina group, a robust lineage composed of three species of Volvox sect. Volvox was resolved as in the rbcL gene trees, rejecting the hypothesis of the previous cladistic analysis based on morphological data that the genus Volvox is monophyletic. In addition, the NJ and ML trees suggested that Eudorina is a nonmonophyletic genus as inferred from the morphological data and rbcL gene sequences. Although phylogenetic status of the genus Gonium is ambiguous in the rbcL gene trees and the paraphyly of this genus is resolved in the cladistic analysis based on morphological data, the atpB gene sequence data suggest monophyly of Gonium with relatively low bootstrap values (56–61%) in the NJ and ML trees. On the basis of the combined sequence data (2256 base pairs) from atpB and rbcL genes, Gonium was resolved as a robust monophyletic genus in the NJ and ML trees (with 68–86% bootstrap values), and Eudorina elegans Ehrenberg represented a paraphyletic species positioned most basally within the Eudorina group. However, phylogenetic status and relationships of the families of the colonial Volvocales were still almost ambiguous even in the combined analysis.