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A new triterpenoid from the fern Adiantum lunulatum and evaluation of antibacterial activity.

A new triterpenoid, 22,29xi-epoxy-30-norhopane-13beta-ol (1) was isolated together with six known compounds viz., fern-9(11)-en-6alpha-ol. fern-9(11)-ene, fern-9(11)-en-25-oic acid, fern-9(11)-en-28-ol, filicenol-B, adiantone and oxidation product of fern-9(11)-en-6alpha-ol obtained as 6-oxofern-9(11)-ene from the whole plant of Adiantum lunulatum, and their structures were elucidated by means of spectroscopic analysis and antibacterial evaluation of these compounds were conducted.

Anti-Bacterial Agents↗

Triterpenoids from Adiantum caudatum.

Three triterpenoids, 8alpha-hydroxyfernan-25,7beta-olide, 3alpha-hydroxy-4alpha-methoxyfilicane and 19alpha-hydroxyferna-7,9(11)-diene were isolated from the fresh fronds of Adiantum caudatum. Their structures were elucidated by spectroscopic techniques. Thirteen known triterpenoids were also identified from this fern.

Ferns↗

Triterpenes from Adiantum lunulactum.

The petrol extract of the whole plant of Adiantum lunuactum yielded a new hopane triterpenoid characterized as 6 alpha-acetoxy-16 beta,22-dihydroxy-3-ketoisohopane, along with the known 3beta,6 alpha,16 beta,22-tetrahydroxyisohopane (mollugogenol A). The structures were elucidated by spectral as well as chemical studies.

Ferns↗

Molecular dynamics simulation of the LOV2 domain from Adiantum capillus-veneris.

The mechanism for signal transduction from the LOV-domains toward the kinase region of phototropin is still not well understood. We have performed molecular dynamics (MD) simulations and CONCOORD calculations on the LOV2 domain of Adiantum capillus-veneris, with the goal to detect possible differences between the two forms of the LOV domain which may not show up in the static crystal structures. Since no such clear differences are found in the MD simulations also, we suggest that the real, biologically active conformation of the LOV domain within the whole phototropin is different from the crystal structure of the isolated LOV domains. The MD simulations do offer, however, insight into details of the dynamics of the dark and illuminated LOV domains, which are discussed in the light of recent experiments.

Journal Article↗

Diversity, genetic structure and evidence of outcrossing in British populations of the rock fern Adiantum capillus-veneris using microsatellites.

Microsatellites were isolated and a marker system was developed in the fern Adiantum capillus-veneris. Polymorphic markers were then used to study the genetic diversity and structure of populations within the UK and Ireland where this species grows at the northern edge of its range, requiring a specific rock habitat and limited to a few scattered populations. Three dinucleotide loci detected a high level of diversity (23 alleles and 28 multilocus genotypes) across the UK and Ireland, with nearly all variation partitioned among rather than within populations. Of 17 populations represented by multiple samples, all except four were monomorphic. Heterozygosity was detected in three populations, all within Glamorgan, Wales (UK), showing evidence of outcrossing. We make inferences on the factors determining the observed levels and patterns of genetic variation and the possible evolutionary history of the populations.

Alleles↗

Control of expression of a gene encoding an extensin by phytochrome and a blue light receptor in spores of Adiantum capillus-veneris L.

In the present study, using a newly developed fluorescent differential display technique, we have carried out large-scale screening for genes whose expression was regulated by phytochrome and antagonistically by a blue light receptor in the spores of the fern Adiantum capillus-veneris L. Spores after imbibition were briefly irradiated with red, red/blue or blue light and collected 8 h after the irradiation. Total RNA was isolated from each sample and used to make cDNA with an oligo-dT primer. The cDNA was then used as a template for PCR with the oligo-dT primer and 80 arbitrary primers. The resulting PCR products were analyzed by an automated fluorescent DNA sequencer. Among 8000 displayed bands, we identified 15 up-regulated and four down-regulated bands by red light, and this red light effect was irreversibly reversed by blue light. We cloned one of the up-regulated cDNA fragments and used it to screen a cDNA library prepared from the spores. The isolated insert is predicted to encode Ser-(Pro)n repeats and showed homology with cell wall-associated extensins. The expression of this cDNA was induced 8 h after a red light treatment and the red light induction was photoreversibly prevented by far-red light and photoirreversibly by blue light. The mRNA of this gene was detectable 4 h after red light irradiation and gradually increased in germinating spores.

Amino Acid Sequence↗

Complete nucleotide sequence of the chloroplast genome from a leptosporangiate fern, Adiantum capillus-veneris L.

We determined the complete nucleotide sequence of the chloroplast genome of the leptosporangiate fern, Adiantum capillus-veneris L. (Pteridaceae). The circular genome is 150,568 bp, with a large single-copy region (LSC) of 82,282 bp, a small-single copy region (SSC) of 21,392 bp and inverted repeats (IR) of 23,447 bp each. We compared the sequence to other published chloroplast genomes to infer the location of putative genes. When the IR is considered only once, we assigned 118 genes, of which 85 encode proteins, 29 encode tRNAs and 4 encode rRNAs. Four protein-coding genes, all four rRNA genes and six tRNA genes occur in the IR. Most (57) putative protein-coding genes appear to start with an ATG codon, but we also detected five other possible start codons, some of which suggest tRNA editing. We also found 26 apparent stop codons in 18 putative genes, also suggestive of RNA editing. We found all but one of the tRNA genes necessary to encode the complete repertoire required for translation. The missing trnK gene appears to have been disrupted by a large inversion, relative to other published chloroplast genomes. We detected several structural rearrangements that may provide useful information for phylogenetic studies.

Base Sequence↗

Isolation and characterization of the cDNA for an A-like cyclin in Adiantum capillus-veneris L.

We have isolated and characterized the cDNA for a homolog of a cyclin from the fern Adiantum capillus-veneris L. Three fragments of cDNAs for cyclin homologs were amplified by the PCR from the cDNA of germinating spores with degenerate primers that encoded the highly conserved region in the so-called cyclin box. We isolated a full-length cDNA for cyclin from a lambda ZAPII-cDNA library that has been derived from spores using an amplified fragment as a probe. The cDNA insert (Cyc-Ac1) in one positive clone was 2.0 kb in length, having an open reading frame of 1,599 bp that encoded 532 amino acids. The putative CycAc1 protein had a cyclin box and a destruction motif, and it was homologous to A-type cyclins. Northern blot analysis was performed to study the expression of CycAc1 during the first cell cycle in the haploid generation of the fern. CycAc1 mRNA was not detectable in dormant spores, during the first G1 phase, and at the onset of the S phase in imbibed spores after breaking of dormancy. This mRNA became detectable after the termination of the S phase, and it accumulated during the second G1 phase. Although CycAc1 was classified as an A-like cyclin, the failure to detect CycAc1 mRNA at the onset of the S phase suggests that CycAc1 might not play a role in the replication of DNA during the S phase.

Amino Acid Sequence↗

Photoresponses of transgenic Arabidopsis overexpressing the fern Adiantum capillus-veneris PHY1.

The phytochrome gene (PHY1) cDNA from the fern Adiantum capillus-veneris encodes an amino acid sequence that shows equal similarity (50-60%) to all five Arabidopsis phytochromes (PHYA-E). The A. capillus-veneris PHY1 cDNA was transformed into Arabidopsis ecotype Landsberg erecta to investigate its activity in angiosperms. Three of the resulting lines contained at least 8 times more spectrally active phytochrome than the wild type, indicating that A. capillus-veneris phytochrome can incorporate the chromophore of the host plants. Hypocotyl growth inhibition of these transgenic lines was investigated under red and far-red light. The results indicated dominant negative activity of A. capillus-veneris phy1 on the phytochrome A response in the host plants under continuous far-red light. However, the fern phytochrome did not interfere with the red-light repression of hypocotyl growth mediated by endogenous phytochrome B, and it failed to complement a phyB mutant phenotype. These observations suggest that the phy1 phytochrome molecule is too diverged from those of Arabidopsis to be fully functional.

Arabidopsis↗

Chloroplast-avoidance response induced by high-fluence blue light in prothallial cells of the fern adiantum capillus-veneris as analyzed by microbeam irradiation

Chloroplast movement was induced by partial cell illumination using a high-fluence blue microbeam in light-grown and dark-adapted prothallial cells of the fern Adiantum capillus-veneris. Chloroplasts inside the illuminated area moved out (high-fluence response [HFR]), whereas those outside moved toward the irradiated area (low-fluence response [LFR]), although they stopped moving when they reached the border. These results indicate that both HFR and LFR signals are generated by high-fluence blue light of the same area, and that an LFR signal can be transferred long-distance from the beam spot, although an HFR signal cannot. The lifetime of the HFR signal was calculated from the traces of chloroplast movement induced by a brief pulse from a high-fluence blue microbeam to be about 6 min. This is very short compared with that of the LFR (30-40 min; T. Kagawa, M. Wada [1994] J Plant Res 107: 389-398). These data indicate that the signal transduction pathways of the HFR and the LFR must be distinct.

Journal Article↗

Phytochrome action on the timing of cell division in adiantum gametophytes.

When filamentous protonemata of Adiantum capillus-veneris L. precultured under continuous red light were transferred to the dark, the apical cell divided about 24 to 36 hours thereafter. The time of the cell division was delayed for several hours by a brief exposure to far red light given before the dark incubation. The effect of far red light was reversed by a small dose of red light given immediately after the preceding far red light. The effects of red and far red light were repeatedly reversible, indicating that the timing of cell division was regulated by a phytochrome system. When a brief irradiation with blue light was given before the dark incubation, the cell division occurred after 17 to 26 hours in darkness. A similar red far red reversible effect was also observed in the timing of the blue light-induced cell division. Thus, the timing of cell division appeared to be controlled by phytochrome and a blue light-absorbing pigment.

Journal Article↗

The occurrence of a ca-dependent period in the red light-induced late g1 phase of germinating adiantum spores.

The first cell cycle of Adiantum spores, which is induced by red light (R), was studied with regard to its temporal dependence on Pfr (the active form of phytochrome) and Ca(2+). Responses were quantified with increases of the spore content of DNA, thus limiting the investigation to the reactions taking place before the S phase of the cell cycle (i.e. during G0/G1/S transitions). Spores hydrated for more than 2 days in the standard medium (includes 1 millimolar free Ca(2+)) showed, after stimulation with a saturating R pulse, an increase of DNA beginning at about 25 hours and saturating at about 55 hours. Reversal by far-red light of the inductive effect of R was used to examine the temporal requirement for Pfr. Spores became dependent on the supply of external Ca(2+) when incubated in a low Ca medium (32 nanomolar free Ca(2+) with 10 millimolar EGTA); this culture condition was used, after observing that the DNA increase occurs similarly if Ca(2+) is supplied after the R pulse, to examine the temporal requirement for external Ca(2+). It was concluded that the G1 phase of the spore is separated into three subphases: (a) the Pfr-dependent period which immediately follows the R pulse and varies among individual spores from a few to about 25 hours, (b) the Ca(2+)-dependent period (about 10 hours) which occurs immediately before the S phase, and (c) a gap (15-20 hours) between the two periods. In the Ca(2+)-dependent period, spores require the presence of extracellular Ca(2+). This period occurs only during a determined time after the R pulse, and the competence of spores to enter the S phase is lost sharply if external Ca(2+) is not available continuously during this period.

Journal Article↗

Cryptochrome nucleocytoplasmic distribution and gene expression are regulated by light quality in the fern Adiantum capillus-veneris.

Numerous cellular responses are reportedly regulated by blue light in gametophytes of lower plants; however, the molecular mechanisms of these responses are not known. Here, we report the isolation of two blue light photoreceptor genes, designated cryptochrome genes 4 and 5 (CRY4 and CRY5), from the fern Adiantum capillus-veneris. Because previously we identified three cryptochrome genes, this fern cryptochrome gene family of five members is the largest identified to date in plants. The deduced amino acid sequences of the five genes show remarkable similarities with previously identified cryptochromes as well as class I photolyases. Like the other plant cryptochromes, none of the cryptochromes of this fern possesses photolyase activity. RNA gel blot analysis and competitive polymerase chain reaction analysis indicate that the expression of the newly identified CRY4 and CRY5 genes is regulated by light and is under phytochrome control. The intracellular distribution of reporter beta-glucuronidase (GUS)-CRY fusion proteins indicates that GUS-CRY3 and GUS-CRY4 localize in fern gametophyte nuclei. The nuclear localization of GUS-CRY3 is regulated in a light-dependent manner. Together with our physiological knowledge, these results suggest that CRY3, CRY4, or both might be the photoreceptor that mediates inhibition of spore germination by blue light.

Amino Acid Sequence↗

Photoreaction of the cysteine S-H group in the LOV2 domain of Adiantum phytochrome3.

Phy3-LOV2 is the chromophore domain of a blue-light photoreceptor for tropic responses of plants. FMN is noncovalently bound to phy3-LOV2, and the protein structure is characteristic of the LOV (light-oxygen-voltage) domain. Primary photoreaction is considered to be adduct formation between FMN and a cysteine, while deprotonation of the cysteine S-H group was suggested. On the basis of the infrared spectral analysis, however, we have shown that the cysteine of phy3-LOV2 is in the protonated S-H form, and not in the thiolate form in the ground state. Upon formation of S390, the S-H group disappears, presumably forming the adduct between FMN and Cys966. S390 can be trapped at 150 K, and the protein structure of S390 may not be changed drastically at 295 K.

Adiantum↗