Bioproduction of prostaglandins in a transgenic liverwort, Marchantia polymorpha

Prostaglandins are biologically active substances used in a wide range of medical treatments. Prostaglandins have been supplied mainly by chemical synthesis; nevertheless, the high cost of prostaglandin production remains a factor. To lower the cost of prostaglandin production, we attempted to produce prostaglandins using a liverwort, Marchantia polymorpha L., which accumulates arachidonic acid, which is known as a substrate of prostaglandins. Here we report the first bioproduction of prostaglandins in plant species by introducing a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla into the liverwort. The transgenic liverworts accumulated prostaglandin F_2α, prostaglandin E₂ and prostaglandin D₂ which were not detected in the wild-type liverwort. Moreover, we succeeded in drastically increasing the bioproduction of prostaglandins using an in vitro reaction system with the extracts of transgenic liverworts.     

Chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha

Journal of Plant Research - Under low-light conditions, chloroplasts localize along periclinal cell walls at temperatures near 20 °C, but they localize along anticlinal cell walls near...     

Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris

Summary Characteristics of the native and reconstituted H⁺-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H⁺-ATPase retained characteristics similar to those of the native plasma membrane H⁺-ATPase following reconstitution into proteoliposomes. ATPase activity and H⁺ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO ₃ ^– was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H⁺ transport showed a pH optimum of 6.5, required a divalent cation (Co²⁺>Mg²⁺>Mn²⁺>Zn²⁺>Ca²⁺), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK _m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H⁺-ATPases were stimulated by monovalent ions. Native plasma membrane H⁺-ATPase showed an order of cation preference of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Cs⁺>Li⁺>choline⁺. This basic order was unchanged following reconstitution, with K⁺, NH ₄ ⁺ , Rb⁺ and Cs⁺ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H⁺ transport, both enzymes required a cation such as K⁺ for maximal transport activity, but were stimulated preferentially by Cl^– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H⁺-ATPase.     

Positional cues regulate dorsal organ formation in the liverwort Marchantia polymorpha

Journal of Plant Research - Bryophytes and vascular plants represent the broadest evolutionary divergence in the land plant lineage, and comparative analyses of development spanning this divergence...     

Characterization of non-dominant lethal mutations in the yeast plasma membrane H+-ATPase gene

Site-directed mutants of yeast ATPase were previously studied after introduction of mutant alleles into a yeast strain where these alleles were constitutively expressed while the expression of the wild-type chromosomal ATPase gene was turned off. As a functional H+ pump is essential, strong selective pressure leads to the accumulation of revertants during growth of cells harboring variants with low activity. Thus, constitutive expression of the mutant gene can select phenotypes which reflect events such as gene conversion or reversion. We have therefore re-evaluated the phenotypes of non-dominant lethal alleles in an alternative set of conditional expression systems. We show that eight of 11 previously described site-directed mutations behave as recessive lethal alleles.     

Characterization of energy conversion based on metabolic flux analysis in mixotrophic liverwort cells, Marchantia polymorpha

In order to characterize the contributions of respiratory and photosynthetic actions to energy conversions, the mixotrophic cells of Marchantia polymorpha were cultivated in the medium containing 10kg/m(3) glucose as an organic carbon source. The cultures were conducted with the supply of ordinary air (0.03% CO(2)) at constant incident light intensities of 50 and 180W/m(2). From the results of metabolic analysis, it was found that the cell yield based on ATP synthesis was estimated to be 6.3x10(-3)kg-dry cells/mol-ATP in these cultures. Under the examined conditions, energy conversion efficiency through respiration was larger than that through photosynthesis, and efficiency of overall energy conversion to ATP was maximized when the sum of energies from glucose and light captured by the cells was approximately 7.2x10(5)J/(hkg-dry cells). Taking into account the efficiency of overall energy conversion, a batch culture of M. polymorpha in a bioreactor was carried out by regulating incident light intensity ranging from 9 to 58W/m(2). In the culture with light regulation, the cell yield of 6.2x10(-9)kg-dry cells/J was achieved on the basis of energy provided to the system throughout the culture, and this value was 2.3 and 9.3 times as large as those obtained in the cultures under constant incident light intensities of 50 and 180W/m(2), respectively.     

A pseudomolecule‐scale genome assembly of the liverwort Marchantia polymorpha

Marchantia polymorpha has recently become a prime model for cellular, evo‐devo, synthetic biological, and evolutionary investigations. We present a pseudomolecule‐scale assembly of the M. polymorpha genome, making comparative genome structure analysis and classical genetic mapping approaches feasible. We anchored 88% of the M. polymorpha draft genome to a high‐density linkage map resulting in eight pseudomolecules. We found that the overall genome structure of M. polymorpha is in some respects different from that of the model moss Physcomitrella patens. Specifically, genome collinearity between the two bryophyte genomes and vascular plants is limited, suggesting extensive rearrangements since divergence. Furthermore, recombination rates are greatest in the middle of the chromosome arms in M. polymorpha like in most vascular plant genomes, which is in contrast with P. patens where recombination rates are evenly distributed along the chromosomes. Nevertheless, some other properties of the genome are shared with P. patens. As in P. patens, DNA methylation in M. polymorpha is spread evenly along the chromosomes, which is in stark contrast with the angiosperm model Arabidopsis thaliana, where DNA methylation is strongly enriched at the centromeres. Nevertheless, DNA methylation and recombination rate are anticorrelated in all three species. Finally, M. polymorpha and P. patens centromeres are of similar structure and marked by high abundance of retroelements unlike in vascular plants. Taken together, the highly contiguous genome assembly we present opens unexplored avenues for M. polymorpha research by linking the physical and genetic maps, making novel genomic and genetic analyses, including map‐based cloning, feasible.     

Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila

Summary Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol P_i/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H⁺ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H⁺-ATPase. Unusual properties of this enzyme are: (i) theK _m for ATP is around 60 M, considerably lower than in other plasma membrane H⁺-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K⁺ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H⁺-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.     

Antifungal bis[bibenzyls] from the Chinese liverwort Marchantia polymorpha L

Bioassay-guided separation of the antifungal constituents of the Chinese liverwort Marchantia polymorpha L. (Marchantiaceae) led to the isolation of seven bis[bibenzyl]-type macrocycles. On the basis of NMR and MS analyses, the three new compounds plagiochin E (1), 13,13'-O-isoproylidenericcardin D (4), and riccardin H (7) were identified, together with four known compounds: marchantin E (2), neomarchantin A (3), marchantin A (5), and marchantin B (6). Their antifungal activities against Candida albicans were determined by TLC bioautography.     

Coordination between growth and stress responses by DELLA in the liverwort Marchantia polymorpha

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Characterization of the cAMP phosphodiesterase domain in plant adenylyl cyclase/cAMP phosphodiesterase CAPE from the liverwort Marchantia polymorpha

Cyclic AMP (cAMP) acts as a second messenger and is involved in the regulation of various physiological responses. Recently, we identified the cAMP-synthesis/hydrolysis enzyme CAPE, which contains the two catalytic domains adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) from the liverwort Marchantia polymorpha. Here we characterize the PDE domain of M. polymorpha CAPE (MpCAPE-PDE) using the purified protein expressed in E. coli. The K_m and V_max of MpCAPE-PDE were 30 µM and 5.8 nmol min^?1 mg^?1, respectively. Further, we investigated the effect of divalent cations on PDE activity and found that Ca²⁺ enhanced PDE activity, suggesting that Ca²⁺ may be involved in cAMP signaling through the regulation of PDE activity of CAPE. Among the PDE inhibitors tested, only dipyridamole moderately inhibited PDE activity by approximately 40% at high concentrations. Conversely, 3-isobutyl-1-methylxanthine (IBMX) did not inhibit PDE activity.

     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

Multicopy genes uniquely amplified in the Y chromosome-specific repeats of the liverwort Marchantia polymorpha

Sex of the liverwort Marchantia polymorpha is determined by the sex chromosomes Y and X, in male and female plant, respectively. Approximately half of the Y chromosome is made up of unique repeat sequences. Here, we report that part of the Y chromosome, represented by a 90-kb insert of a genomic clone pMM2D3, contains five putative genes in addition to the ORF162 gene, which is present also within the Y chromosome-specific repeat region. One of the five putative genes shows similarity to a male gamete-specific protein of lily and is expressed predominantly in male sex organs, suggesting that this gene has a male reproductive function. Furthermore, Southern blot analysis revealed that these five putative genes are amplified on the Y chromosome, but they also probably have homologs on the X chromosome and/or autosomes. These observations suggest that the Y chromosome evolved by co-amplifying protein-coding genes with unique repeat sequences.     

Large-scale isolation of the Neurospora plasma membrane H+-ATPase

A method for the purification of relatively large quantities of the Neurospora crassa plasma membrane proton translocating ATPase is described. Cells of the cell wall-less sl strain of Neurospora grown under O2 to increase cell yields are treated with concanavalin A to stabilize the plasma membrane and homogenized in deoxycholate, and the resulting lysate is centrifuged at 13,500g. The pellet obtained consists almost solely of concanavalin A-stabilized plasma membrane sheets greatly enriched in the H+-ATPase. After removal of the bulk of the concanavalin A by treatment of the sheets with alpha-methylmannoside, the membranes are treated with lysolecithin, which preferentially extracts the H+-ATPase. Purification of the lysolecithin-solubilized ATPase by glycerol density gradient sedimentation yields approximately 50 mg of enzyme that is 91% free of other proteins as judged by quantitative densitometry of Coomassie blue-stained gels. The specific activity of the enzyme at this stage is about 33 mumol of P1 released/min/mg of protein at 30 degrees C. A second glycerol density gradient sedimentation step yields ATPase that is about 97% pure with a specific activity of about 35. For chemical studies or other investigations that do not require catalytically active ATPase, virtually pure enzyme can be prepared by exclusion chromatography of the sodium dodecyl sulfate-disaggregated, gradient-purified ATPase on Sephacryl S-300.     

Characterization of an essential arginine residue in the plasma membrane H+-ATPase of Neurospora crassa

Treatment of the plasma membrane H+-ATPase of Neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees C, pH 7.0, leads to a marked inhibition of ATPase activity. MgATP, the physiological substrate of the enzyme, protects against inactivation. MgADP, a competitive inhibitor of ATPase activity with a measured Ki of 0.11 mM, also protects, yielding calculated KD values of 0.125 and 0.115 mM in the presence of phenylglyoxal and 2,3-butanedione, respectively. The excellent agreement between Ki and KD values makes it likely that MgADP exerts its protective effect by binding to the catalytic site of the enzyme. Loss of activity follows pseudo-first order kinetics with respect to phenylglyoxal and 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration yield slopes of 0.999 (phenylglyoxal) and 0.885 (2,3-butanedione), suggesting that the modification of one reactive site/mol of H+-ATPase is sufficient for inactivation. This stoichiometry has been confirmed by direct measurements of the incorporation of [14C]phenylglyoxal. Taken together, the results support the notion that one arginine residue, either located at the catalytic site or shielded by a conformational change upon nucleotide binding, plays an essential role in Neurospora H+-ATPase activity.     

Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.     

Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L.

Thalli of the haploid liverwort Marchantia polymorpha were successfully used for direct particle bombardment with plasmid pMT, which carries a hygromycin phosphotransferase gene (hpt) controlled by the CaMV 35S promoter and the NOS polyadenylation region. Hygromycin-resistant cell masses arose from the thallus surface and developed directly into hygromycin-resistant thalli. Southern blot analyses indicated that these thalli carried at least 1–4 copies of the hpt gene, which were stably transmitted to their asexual thallus progenies via gemma propagation for three generations. This transformation and direct plant regeneration protocol is expected to be a valuable tool for the molecular analysis of this lower land plant.