Two atypical ANGUSTIFOLIA without a plant-specific C-terminus regulate gametophore and sporophyte shapes in the moss Physcomitrium (Physcomitrella) 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.

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens.

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.

Genes encoding lipid II flippase MurJ and peptidoglycan hydrolases are required for chloroplast division in the moss Physcomitrella patens.

KEY MESSAGE: 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.

Moss Chloroplasts Are Surrounded by a Peptidoglycan Wall Containing D-Amino Acids.

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.

Production of indoleacetic acid by strains of the epiphytic bacteria Neptunomonas spp. isolated from the red alga Pyropia yezoensis and the seagrass Zostera marina.

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.

Single-Pixel Densitometry Revealed the Presence of Peptidoglycan in the Intermembrane Space of the Moss Chloroplast Envelope in Conventional Electron Micrographs.

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.

Genes Sufficient for Synthesizing Peptidoglycan are Retained in Gymnosperm Genomes, and MurE from Larix gmelinii can Rescue the Albino Phenotype of Arabidopsis MurE Mutation.

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.

Both the transglycosylase and transpeptidase functions in plastid penicillin-binding protein are essential for plastid division in Physcomitrella patens.

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.

Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt.

KEY MESSAGE: 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.

Overexpression of UDP-glucose pyrophosphorylase from Larix gmelinii enhances vegetative growth in transgenic Arabidopsis thaliana.

KEY MESSAGE: A UDP-glucose pyrophosphorylase gene ( LgUGPase ) was identified from Larix gmelinii, and its function in enhancing vegetative growth and cellulose biosynthesis was confirmed by analyzing transgenic Arabidopsis thaliana overexpressed LgUGPase . UDP-glucose pyrophosphorylase (UGPase), an important regulatory enzyme in carbohydrate metabolism, catalyzes the reversible production of glucose 1-phosphate and the conversion of uridine triphosphate to uridine diphosphate glucose and pyrophosphate. In this study, a larch UGPase (LgUGPase) gene was isolated from Larix gmelinii. The 1,443-bp open reading frame encodes a protein of 480 amino acids with a predicted molecular weight of 53.7 kDa and shows striking sequence similarity to UGPase proteins from Pinus taeda and Picea sitchensis. Semiquantitative reverse transcription-polymerase chain reaction showed that the LgUGPase gene was expressed primarily in the larch stem in addition to its root and leaf. Southern blot analysis indicated that LgUGPase is encoded by two genes in the L. gmelinii genome. Overexpression of LgUGPase enhanced vegetative growth in transgenic Arabidopsis and increased the contents of soluble sugars and cellulose, and thickened parenchyma cell walls. These results revealed that L. gmelinii UGPase participates in sucrose/polysaccharide metabolism and cell wall biosynthesis, suggesting that LgUGPase may be a good candidate gene for improvement of fiber cell development in plants.

Involvement of microRNA in copper deficiency-induced repression of chloroplastic CuZn-superoxide dismutase genes in the moss Physcomitrella patens.

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.

ANGUSTIFOLIA, a plant homolog of CtBP/BARS, functions outside the nucleus.

CtBP/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 would function in the nucleus as a transcriptional co-repressor, as CtBPs function in animals; however, precise verification has been lacking. In this paper, we isolated a homologous gene (MAN) of AN from liverwort, Marchantia polymorpha. Transformation of the Arabidopsis an-1 mutant with 35S-driven MAN completely complemented the an-1 phenotype, although it lacks the putative nuclear localization signal (NLS) that exists in AN proteins isolated from other plant species. We constructed several plasmids for expressing modified ANs with amino acid substitutions in known motifs. The results clearly indicated that modified AN with mutations in the putative NLS-like domain could complement the an-1 phenotype. Therefore, we re-examined localization of AN using several techniques. Our results demonstrated that AN localizes on punctuate structures around the Golgi, partially overlapping with a trans-Golgi network resident, which highlighted an unexpected link between leaf development and membrane trafficking. We should reconsider the roles and evolutionary traits of AN based on these findings.

Loss of the plastid envelope protein AtLrgB causes spontaneous chlorotic cell death in Arabidopsis thaliana.

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 (ß-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.

Plastid peptidoglycan.

It is now widely accepted that an endosymbiotic cyanobacterium evolved into the plastid of the primary photosynthetic eukaryotes: glaucocystophytes, red algae, and green plants. It has been thought that during the evolution of plants, the peptidoglycan wall (or murein) was lost from the endosymbiont immediately after the branching off of the glaucocystophytes, which have peptidoglycan-armed plastids termed cyanelles. However, we found that the moss Physcomitrella patens has all of the genes for peptidoglycan biosynthesis with the exception of one racemase. The aim of the present review is to summarize recent findings on plastid peptidoglycan and to present a hypothesis for the evolution of plastids containing peptidoglycan. Gene knockout experiments for the Mur(ein) genes, including MurE in P. patens, showed that the peptidoglycan synthesis pathway is related to plastid division, although no structure can be detected between the inner and outer envelopes of the chloroplasts by electron microscopy. On the other hand, MurE in Arabidopsis thaliana has a function in plastid gene expression and not in division. Based on data regarding plant genomes and antibiotic treatment experiments of plastid division, we propose that the loss of peptidoglycan occurred independently at least three times during plant evolution: from the lineage of red algae, from the chlorophytes, and during land plant evolution.

An Arabidopsis homolog of the bacterial peptidoglycan synthesis enzyme MurE has an essential role in chloroplast development.

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.

The peptidoglycan biosynthesis genes MurA and MraY are related to chloroplast division in the moss Physcomitrella patens.

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 <10 chloroplasts, whereas approximately 30 chloroplasts existed in the PpMurA2 knockout lines. The PpMurA1/A2 double-knockout lines had only a few macrochloroplasts, suggesting a redundant function for these two genes. Disruption of the PpMraY gene in P. patens resulted in the appearance of macrochloroplasts. Anabaena MraY fused to the N-terminal region of PpMraY and A. thaliana MraY could complement the macrochloroplast phenotype in the PpMraY knockout line. Electron microscopic observations showed no obvious differences in the shape or stacking of thylakoid membranes between all knockout transformants and wild-type plants, suggesting that these Mur genes are related only to plastid division in moss.

Ammonium induced expression of the red algal chloroplast gene Ycf18, a putative homolog of the cyanobacterial NblA gene involved in nitrogen deficiency-induced phycobilisome degradation.

In cyanobacteria, nutrient deficiency-induced phycobilisome degradation is controlled by the NblA gene. Red algae also have an NblA-related gene, Ycf18, in their chloroplast genomes. To elucidate the role of Ycf18, the expression pattern of Ycf18 in a red alga, Porphyra yezoensis, was investigated. Ycf18 expression was low in nitrate medium, but was greatly promoted in ammonium medium. Nitrogen starvation caused bleaching, but did not affect the expression of Ycf18. The responses of Ycf18 to nitrogen-starvation and the supply of ammonium were distinct from those of NblA, suggesting that Ycf18 has a role other than the regulation of phycobilisome degradation.

Two different clades of copia-like retrotransposons in the red alga, Porphyra yezoensis.

A copia-like retrotransposon referred to as PyRE1G1 was isolated from the genome of the red alga Porphyra yezoensis. PyRE1G1 is 4807 bp in length, with 204 bp long terminal repeats (LTRs) at both ends. PyRE1G1 has an open reading frame of 1401 residues encoding gag, protease, integrase, reverse transcriptase (RT), and RNase H. From the order of gene arrangement of proteins, PyRE1G1 appears to be a copia-like retrotransposon. Genomic Southern blot analysis suggests that PyRE1G1 consists of a small gene family. From the phylogenetic tree of RT sequences, PyRE1G1 is grouped in the clade of usual copia elements and distinct from the previously isolated red algal copia-like gene PyRE10G in that the latter is closely related to a new clade of aquatic animal-specific copia-like retrotransposons.

Isolation of Ef silicatein and Ef lectin as molecular markers for sclerocytes and cells involved in innate immunity in the freshwater sponge Ephydatia fluviatilis.

Sponges (phylum Porifera) have remarkable regenerative and reconstitutive abilities and represent evolutionarily the oldest metazoans. To investigate sponge stem cell differentiation, we have focused on the asexual reproductive system in the freshwater sponge Ephydatia fluviatilis. During germination, thousands of stem cells proliferate and differentiate to form a fully functional sponge. As an initial step of our investigation of stem cell (archeocyte) differentiation, we isolated molecular markers for two differentiated cell types: spicule-making sclerocyte cells, and cells involved in innate immunity. Sclerocyte lineage-specific Ef silicatein shares 45% to 62% identity with other sponge silicateins. As in situ hybridization of Ef silicatein specifically detects archeocytes possibly committed to sclerocytes, as well as sclerocytes with an immature or mature spicule, therefore covering all the developmental stages, we conclude that Ef silicatein is a suitable sclerocyte lineage marker. Ef lectin, a marker for the cell type involved in innate immunity, shares 59% to 65% identity with the marine sponge Suberites domuncula galactose-binding protein (Sd GBP) and horseshoe crab Tachypleus tridentatus tachylectin1/lectinL6. Since Sd GBP and tachylectin1 are known to bind to bacterial lipopolysaccharides and inhibit the growth of bacteria, Ef lectin may have a similar function and be expressed in a specialized type of cell involved in defense against invading bacteria. Ef lectin mRNA and protein are not expressed in early stages of development, but are detected in late stages. Therefore, Ef lectin may be specifically expressed in differentiating and/or differentiated cells. We suggest Ef lectin as a marker for cells that assume innate immunity in freshwater sponges.

Preferential expression of a bromoperoxidase in sporophytes of a red alga, Pyropia yezoensis.

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)).