The Effect of Oxygen Radicals on Proteolysis in Isolated Oat Chloroplasts

Chloroplast DNAs (ctDNAs) from several pteridophytes were analysedby cross-hybridization with the liverwort frxC gene as probe.Hybridization signals were observed without exception, suggestingthe presence of the frxC gene in ctDNAs of non-flowering vascularplants as in liverwort. A frxC homolog was also identified intwo gymnosperms. (Received July 5, 1991; Accepted January 28, 1992)     

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

Identification of a nifDK-Like Gene (ORF467) Involved in the Biosynthesis of Chlorophyll in the Cyanobacterium Plectonema boryanum

The frxC gene, which is found in chloroplast DNA (ctDNA) andin cyanobacteria, encodes a protein that is required for thelight-independent reduction of protochlorophyllide (Pchlide)to chlorophyllide a (Chlide). A DNA fragment downstream of frxCin the filamentous cyanobacterium Plectonema boryanum was clonedand analyzed. Sequencing of the DNA fragment revealed an openreading frame (ORF) that encoded a protein of 467 amino acidresidues (designated ORF467), which showed extensive homologyto the proteins encoded by genes on ctDNAs (ORF465 in liverwort,gidA in pine and chlN in Chlamydomonas reinhardtii) and to ORF469protein of the cyanobacterium Synechocystis sp. strain PCC 6803.We isolated a targeted mutant YFM6D-3 in which ORF467 was inactivatedby the insertion of a kanamycin-resistance gene into the codingregion. YFM6D-3 exhibited a phenotype similar to that of YFC1004,an frxC-disrupted mutant, which did not synthesize chlorophyll(Chl) and accumulated Pchlide, a precursor to Chl, in the dark.These phenotypic characteristics of YFM6D-3 indicate that thelight-independent reduction of Pchlide requires not only theFrxC protein but also the ORF467 protein. The amino acid sequencesof the homologues of ORF467 exhibit low but significant similarityto those of the and ß subunits of nitrogenase MoFe-protein,suggesting a phylogenetic relationship between the light-independentPchlide reductase and nitrogenase, as is observed between theFrxC protein and the Fe-protein of nitrogenase. ¹Institute for Protein Research, Osaka University, Suita, Osaka,565 Japan     

The nifH-Like (frxC) Gene Is Involved in the Biosynthesis of Chlorophyll in the Filamentous Cyanobacterium Plectonema boryanum

The frxC gene found in the DNA of the liverwort chloroplastencodes a protein of unknown function. The deduced amino acidsequence shows significant homology to that of the nitrogenaseFe-protein encoded by the nifH gene. We previously identifiedthe frxC and nifH genes in the filamentous cyanobacterium Plectonemaboryanum. We describe here the isolation of targeted mutantsof frxC (YFC1004) and nifH (YFH201) which were generated byinsertion of a kanamycin-resistance gene into the structuralportion of the respective genes. As expected, YFH201 cannotgrow under nitrogen-fixing conditions. However, YFC1004 growsas well as the wild type does under nitrogen-fixing, photoautotrophicand chemoheterotrophic conditions, indicating that the FrxCprotein is essential neither for nitrogen fixation nor for majorenergytransduction systems, such as photosynthesis and respiration.YFC1004 synthesizes chlorophyll (Chi) normally in the lightbut not in the dark, and it accumulates a precursor to Chi,protochlorophyllide (Pchlide) in the dark. These phenotypiccharacteristics of YFC1004 suggest that the cyanobacterium hastwo pathways for the reduction of Pchlide: a light-dependentand a lightindependent system. The FrxC protein appears to beinvolved in the light-independent reduction of Pchlide. (Received September 24, 1991; Accepted November 11, 1991)     

Stabilizing Effect of Lipopolysaccharide on the l-Glycerol 3-Phosphate Acyltransferase of Escherichia coli Membrane

A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase. Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known. Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469–0RF248) was isolated from the cyanobacterium Synechocystis PCC6803. In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively. Synechocystis frxC showed 31% amino acid sequence identity with nifHl from Clostridium pasteurianum. Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C. pasteurianum nifK product, which is the β subunit of a molybdenum-iron protein of a nitrogenase complex. Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Homologues of the green algal gidA gene and the liverwort frxC gene are present on the chloroplast genomes of conifers

Strong hybridization signals were obtained from total DNA of two conifers, lodgepole pine (Pinus contorta) and Norway spruce (Picea abies), in a Southern blot analysis using a probe derived from the chloroplast gidA gene of the green alga Chlamydomonas reinhardtii. The pine fragments detected by the probe were found to originate from the chloroplast genome and, as judged by the signal intensity, this was also true for the spruce fragments. Sequence analysis of the hybridizing pine chloroplast DNA region revealed an open reading frame potentially encoding a 459 amino acid polypeptide, highly homologous to that deduced from the algal gene and to ORF465 of liverwort chloroplast DNA. Upstream of the gidA sequence, we found a trnN(GUU) gene and an open reading frame of 291 codons which was 78% identical to the frxC gene of liverwort. Since ORF465 is located immediately downstream of trnN and frxC in liverwort, the genetic organization of this region is very similar in the two plants. In contrast, neither the gidA nor the frxC gene is present in the chloroplast DNA of tobacco or rice. It was recently reported that deletions in the gidA region of the chloroplast genome of Chlamydomonas reinhardtii abolish the light-independent pathway of chlorophyll synthesis which exists in many algae and lower plants. The presence of the gidA gene on the chloroplast genomes of conifers may therefore be of significance with respect to the ability of these plants to synthesize chlorophyll in the dark.     

Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in the Liverwort, Marchantia paleacea var. diptera

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase(EC 1.6.99.1 [EC] ) was isolated from suspension-cultured cells ofthe liverwort, Marchantia paleacea var. diptera. In contrastto the situation in most higher plants, the liverwort gene wasexpressed in a light-dependent manner. ²Present address: Department of Biological Science, Facultyof Science, Kumamoto University, Kurokami, Kumamoto, 860-8555Japan.     

Identification of the chlB Gene and the Gene Product Essential for the Light-Independent Chlorophyll Biosynthesis in the Cyanobacterium Plectonema boryanum

We cloned a 6.0-kb HindIII fragment from the cyanobacteriumPlectonema boryanum using the chloroplast chlB (ORF513) geneof the liverwort (Marchantia polymorpha) as a probe. An openreading frame (ORF508) encoding a polypeptide of 508 amino acidresidues was found within the nucleotide sequence of the 4,437-bpHindIII-EcoRV subfragment. The deduced amino acid sequence ofORF508 shows very high similarity to that encoded by the liverwortchlB gene (72.7%). A mutant, YFB14, in which ORF508 was inactivatedby the insertion of a kanamycin-resistance cartridge, was unableto synthesize chlorophyll, accumulating protochlorophyllidein darkness while synthesizing chlorophyll normally in the light.Thus, the chlB gene is the third gene that is essential forthe light-independent reduction of protochlorophyllide. Theother two genes are chlL and chlN, and the results suggest thatthe light-independent protochlorophyllide reductase consistsof at least three subunits, which are encoded by chlL, chlNand chlB. Using an antiserum prepared against a ChlB-6xHis fusionprotein expressed in Escherichia coli, we detected a proteinwith an apparent molecular weight of 58,000 in the membranefraction of the cyanobacterium. These results indicate thateither the cytoplasmic or thylakoid membranes could be the siteof the light-independent reduction of protochlorophyllide. (Received November 16, 1995; Accepted February 7, 1996)     

Anatomy of Ricciocarpus natans (L.) Corda, Studied by Scanning Electron Microscopy

The anatomy of the vegetative thallus in the life-forms of theamphibious liverwort Ricciocarpus natans was studied by scanningelectron microscopy. Particular attention was given to the structureof the epidermal stomatoid pores and the minute internal poresin the aerenchyma. The aquatic and terrestrial life-forms werefound to differ in (1) number and size of ventral scales, (2)presence of rhizoids and (3) width of the thallus margin. scanning electron microscopy, Ricciocarpus natans (L.) Corda, Bryophyta, liverwort, stomatoid pores, aerenchyma, structural adaptations     

Three iron-sulfur proteins encoded by three ORFs in chloroplasts and cyanobacteria

A brief review is presented on the gene products of frxA, frxB and frxC found in chloroplasts. The product of frxA shows high sequence homologies to bacterial 2[4Fe-4S] ferredoxins, but it functions as iron-sulfur centers A and B in Photosystem I, transferring electrons to [2Fe-2S] ferredoxin. This protein is located on surface of the thylakoid membranes in a state being covered by two other proteins. Proteins homologous to frxB product are found in mitochondrial respiratory Complex I and the product of frxB may function in chlororespiration, but at present no clear function of this protein is known. The frxC gene product is found to function in light-independent chlorophyll synthesis as one of the subunits of protochlorophyllide reductase and is reviewed in comparison to nitrogenase. Several problems and future research direction in these areas are also presented.     

An Examination of some Symbiotic Systems for Fixation of Nitrogen

By the use of ¹⁵N the occurrence of fixation of elemental nitrogenhas been demonstrated in the lichens Collema granosum and Leptogiumlichenoides (both of which contain Nostoc as a symbiont), andalso in the liverwort Blasia pusilla (which has Nostoc-containingcavities in the thallus). The circumstances indicate that thefixation in these instances should be attributed to the Nostoc. No evidence of fixation was obtained in similar isotopic testson the mycorrhizal roots of intact plants of Calluna vulgarisand of Pinus sylvestris, although these, more especially inthe case of Calluna, have been held by some previous authorsto be nitrogen-fixing.     

Circulating Tumor DNA as Biomarkers for Cancer Detection

Detection of circulating tumor DNAs (ctDNAs) in cancer patients is an important component of cancer precision medicine ctDNAs. Compared to the traditional physical and biochemical methods, blood-based ctDNA detection offers a non-invasive and easily accessible way for cancer diagnosis, prognostic determination, and guidance for treatment. While studies on this topic are currently underway, clinical translation of ctDNA detection in various types of cancers has been attracting much attention, due to the great potential of ctDNA as blood-based biomarkers for early diagnosis and treatment of cancers. ctDNAs are detected and tracked primarily based on tumor-related genetic and epigenetic alterations. In this article, we reviewed the available studies on ctDNA detection and described the representative methods. We also discussed the current understanding of ctDNAs in cancer patients and their availability as potential biomarkers for clinical purposes. Considering the progress made and challenges involved in accurate detection of specific cell-free nucleic acids, ctDNAs hold promise to serve as biomarkers for cancer patients, and further validation is needed prior to their broad clinical use.     

Fractionation and Partial Characterization of Pectic Polysaccharides in Cell Walls from Liverwort (Marchantia polymorph) Cell Cultures

Konno, H., Yamasalu, Y. and Katoh, K. 1987. Fractionation andpartial characterization of pectic polysaccharides in cell wallsfrom liverwort (Marchantia polymorpha) cell cultures.—Jexp. Bot. 38: 711–722. Pectic polysaccharides were extracted from the starch-free cellwall preparation of cell suspension cultures of Marchantia polymorpha.The polysaccharides were fractionated by DEAE-Sephadex A-50ion-exchange chromatography yielding the five fractions, andthe degree of polymerization and glycosyl composition determinedfor each fraction. The neutral rich and acidic pectic polymerswere depolymerized by purified endoglucanase (l,4-ß-D-glucan4-glucanohydrolase, E.C. 3.2.1.4 [EC] .) and endopolygalacturonase(poly-l,4--Dgalacturonide glycanohydrolase, E.C. 3.2.1.15 [EC] ),respectively. The degraded pectic fractions were fractionatedby gel filtration chromatography on Bio-Gel A-5m and Bio-GelP-2, and glycosyl composition determined for each fraction.The results indicate that pectic polysaccharides contain glucose-richpolymer, rhamnogalacturonan and homogalacturonan in a ratioof 1:4:0–6. In addition, pectic polysaccharides were releasedas five pectic fragments from the cell walls by purified endopectatelyase (poly-l,4--D-galacturonide lyase, E.C. 4.2.2.2 [EC] ). Basedon the analysis of glycosyl composition of each fragment, thepectic polysaccharides of Marchantia cell walls are characterized Key words: Cell suspension culture, cell wall, liverwort, Marchantia polymorpha, pectic polysaccharides     

A Novel 3.5 kDa Protein Component of Cyanobacterial Photosystem I Complexes

A novel protein component of 3.5 kDa was detected in photosystemI complexes prepared from several cyanobacteria, viz. Synechococcusvulcanus, Synechococcus elongotus BP-1, Synechococcus sp. FCC7002 and Synechocystis sp. FCC 6803. The complete amino acidsequence of this component was determined by direct proteinsequencing. The sequences of the 3.5 kDa proteins from thesefour organisms were highly homologous to each other, featuringa hydrophobic domain in the middle. The cyanobacterial consensussequence exhibits significant homology to the presumed productof ORF32 in the chloroplast DNA of liverwort (Marchantia polymorpha),but no homologous ORF is present in the chloroplast DNA of tobaccoor rice. Since this protein appears to interact strongly withthe PS I reaction center complex, it may play some role in thefunction and maintenance of the structure of PS I. (Received May 25, 1992; Accepted August 18, 1992)     

Growth Behavior of a Liverwort, Jungermannia subulata Evans, in a Cell Suspension Culture. The Role of Organic Acids Required for Cell Growth

A green callus of a liverwort, Jungermannia subulata Evans,was induced from gametophytes, and a cell suspension culturewas obtained from the callus. Both callus and suspension-culturedcells grew in a modified Murashige and Skoog medium if organicacids of the TCA cycle were supplied. In the cell suspensionculture of J. subulata, ammonium was taken up preferentiallyby the cells particularly at the earliest stage of growth, whileonly a negligible amount of nitrate was utilized as long asammonium was present in the medium, and this unbalanced utilizationof the two nitrogen sources caused an abrupt drop in the pHof the medium. The organic acids of the TCA cycle supportedthe growth of this cell line by preventing the abrupt drop inthe pH of the medium. (Received June 13, 1981; Accepted October 8, 1981)     

Some Problems of Mitochondrial Growth

Observations on mitochondrial growth are recorded from the meristemof a liverwort(Anthoceros laevis) and from a Chrysophycean flagellate(Chrysochromulina brevifilum). No sign of de novo origin hasbeen obtained in either case. In both the commonest form ofmitochondrial reproduction is from growth leading to clustersof mutually attached mitochondria which are presumed to separatelater. Some stages in the formation of clusters are illustratedand some of the processes involved analysed in a preliminaryway. Mitochondrial growth is seen to be a somewhat more complicatedtotal process than has sometimes been thought.     

Purification of the Cytosolic CuZn-Superoxide Dismutase (CuZn-SOD) of Marachantia paleacea var. diptera and its Resemblance to CuZn-SOD from Chloroplasts

Suspension-cultured cells of Marchantia paleacea var. dipteracontain a single form of CuZn-superoxide dismutase (SOD; EC1.15.1.1 [EC] ) which is localized in the cytosol. SOD activity wasfound in cells cultured under heterotrophic, photoheterotrophicand photoautotrophic conditions. The CuZn-SOD was purified tohomogeneity from liverwort cells that had been cultued hetertrophically.Its molecular mass was 32.6 kDa, and it contained 17.5 kDa subunits,an indication that the enzyme is a homodimer. The enzyme hadpeaks of absorption at 252, 258 and 264 nm in the ultravioledregion, due to the presence of phenylalanine, and a peak at680 nm in the visible region, which is characteristic of CuZn-SODsfrom cholorplasts. The amino acid sequence of the amino-terminalregion of the enzyme exhibited a very high degree of homologyto those of cholorplast CuZn-SODs. An antiserum raised againstthe CuZn-SOD from liverwort cross-reacted more strongly withthe enzyme from spinach chloroplasts, than with the enzyme fromspinach cytosol. These results indicate that the CuZn-SOD ofliverwort resembles CuZn-SOD in chloroplasts even though theformer is located in the cytosol. (Received November 27, 1995; Accepted April 5, 1996)