Liverwort Blasia Research Articles

Comparative genomic insights into culturable symbiotic cyanobacteria from the water fern Azolla.

Species of the floating, freshwater fern Azolla form a well-characterized symbiotic association with the non-culturable cyanobacterium Nostoc azollae, which fixes nitrogen for the plant. However, several cyanobacterial strains have over the years been isolated and cultured from Azolla from all over the world. The genomes of 10 of these strains were sequenced and compared with each other, with other symbiotic cyanobacterial strains, and with similar strains that were not isolated from a symbiotic association. The 10 strains fell into three distinct groups: six strains were nearly identical to the non-symbiotic strain, Nostoc (Anabaena) variabilis ATCC 29413; three were similar to the symbiotic strain, Nostoc punctiforme, and one, Nostoc sp. 2RC, was most similar to non-symbiotic strains of Nostoc linckia. However, Nostoc sp. 2RC was unusual because it has three sets of nitrogenase genes; it has complete gene clusters for two distinct Mo-nitrogenases and an alternative V-nitrogenase. Genes for Mo-nitrogenase, sugar transport, chemotaxis and pili characterized all the symbiotic strains. Several of the strains infected the liverwort Blasia, including N. variabilis ATCC 29413, which did not originate from Azolla but rather from a sewage pond. However, only Nostoc sp. 2RC, which produced highly motile hormogonia, was capable of high-frequency infection of Blasia. Thus, some of these strains, which grow readily in the laboratory, may be useful in establishing novel symbiotic associations with other plants.

A Genetic and Chemical Perspective on Symbiotic Recruitment of Cyanobacteria of the Genus Nostoc into the Host Plant Blasia pusilla L.

Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin.

Symbiosis between the cyanobacterium Nostoc and the liverwort Blasia requires a CheR-type MCP methyltransferase

In response to environmental change, the cyanobacterium Nostoc punctiforme ATCC 29133 produces highly adapted filaments known as hormogonia that have gliding motility and serve as the agents of infection in symbioses with plants. Hormogonia sense and respond to unidentified plant-derived chemical signals that attract and guide them towards the symbiotic tissues of the host. There is increasing evidence to suggest that their interaction with host plants is regulated by chemotaxis-related signal transduction systems. The genome of N. punctiforme contains multiple sets of chemotaxis (che)-like genes. In this study we characterize the large che5 locus of N. punctiforme. Disruption of NpR0248, which encodes a putative CheR methyltransferase, results in loss of motility and significantly impairs symbiotic competency with the liverwort Blasia pusilla when compared with the parent strain. Our results suggest that chemotaxis-like elements regulate hormogonia function and hence symbiotic competency in this system.

Arabinogalactan Proteins Occur in the Free-Living Cyanobacterium Genus Nostoc and in Plant–Nostoc Symbioses

Arabinogalactan proteins (AGP) are a diverse family of proteoglycans associated with the cell surfaces of plants. AGP have been implicated in a wide variety of plant cell processes, including signaling in symbioses. This study investigates the existence of putative AGP in free-living cyanobacterial cultures of the nitrogen-fixing, filamentous cyanobacteria Nostoc punctiforme and Nostoc sp. strain LBG1 and at the symbiotic interface in the symbioses between Nostoc spp. and two host plants, the angiosperm Gunnera manicata (in which the cyanobacterium is intracellular) and the liverwort Blasia pusilla (in which the cyanobacterium is extracellular). Enzyme-linked immunosorbent assay, immunoblotting, and immunofluorescence analyses demonstrated that three AGP glycan epitopes (recognized by monoclonal antibodies LM14, MAC207, and LM2) are present in free-living Nostoc cyanobacterial species. The same three AGP glycan epitopes are present at the Gunnera-Nostoc symbiotic interface and the LM2 epitope is detected during the establishment of the Blasia-Nostoc symbiosis. Bioinformatic analysis of the N. punctiforme genome identified five putative AGP core proteins that are representative of AGP classes found in plants. These results suggest a possible involvement of AGP in cyanobacterial-plant symbioses and are also suggestive of a cyanobacterial origin of AGP.

Juvenile Gametophyte Development in the Blasiales (Marchantiophyta; Metzgeriidae). 1. Receptacular Gemma/Gemmaling Ontogeny of Blasia pusilla

Abstract The pattern of gemma/gemmaling ontogeny in the liverwort Blasia pusilla L. is described from its origin as a primordium within the gemma receptacle to the formation of a juvenile gametophyte. Data were obtained from herbarium specimens, field-grown and axenically cultured plants, using a combination of light and scanning electron microscopy techniques. A consistent, uniform, fundamental developmental pattern was elucidated that is characterized by the production, within the receptacle, of a discoid to ellipsoid, stalked gemma composed of distinct tiers of thick-walled cells and margined by lateral columns of thin-walled, potentially active cells. After dispersal from the receptacle, lateral growth ensues from a gemmaling initial that is produced via quadrant formation from one lateral margin cell. Ultimately, an adult cuneate apical cell is honed that generates segments the same as in the adult plant to produce the juvenile gametophyte typical of Blasia. Both the gemmaling and sporeling ontogenie...

Diversity and Specificity in Cyanobacterial Symbioses

DIVERSITY AND SPECIFICITY IN CYANOBACTERIAL SYMBIOSES U. Rasmussen and C. Johansson U.Rasmussen (corresponding author; e-mailullarasmussen@ botan su se) and C Johansson, Department ofBotany,Stockholm University, 106 91 Stockholm, Sweden INTRODUCTION Cyanobacteria constitute one of the largest sub groups of Gram-negative photosynthetic prokary otes and are of great evolutionary antiquity, dating back to the Precambrian (2.8-3.5 billion years ago). They are found worldwide in highly diverse ecosystems, from aquatic (limnic and marine) habi tats to terrestrial systems and from polar to tropical regions of the globe. In terrestrial systems, nitro gen-fixing Nostoc isby far themost common genus and includes cyanobacteria capable of forming symbioses with a broad range of plants and other organisms. Hosts include fungi (Geosiphon species and lichenised fungi), bryophytes (liverworts and hornworts), aquatic ferns (genus Azolla), gym nosperms (cycads) and angiosperms (genus Gunnera) (Bergman et al. 1996). CELL DIFFERENTIATION IN NOSTOC The key to the success of the genus Nostoc, whether in the free-living form or in symbiosis, is most likely its extreme structural and functional plasticity. Influenced by environmental changes, Nostoc undergoes differentiation into several differ ent cell and filament types (P1. I).When grown under nitrogen-limited conditions or in symbiosis, vegetative cells differentiate to form specialised nitrogen-fixing heterocysts at a frequency of ap proximately 5% in the free-living form and up to 80% in symbiosis with hosts such as Gunnera spe cies. In addition, the entire vegetative filament can differentiate into amotile stage, the hormogonium, which serves as a means of dispersal under unfa vourable conditions and constitutes the 'infective unit' during establishment of the symbiosis. Finally, akinetes are differentiated under adverse conditions and function as spores. Akinete differentiation is an unusual stage in symbiotic associations and is known to play a role only in Azolla, in which akinetes function as cyanobacterial inocula for the next sporophyte generation. In addition, the cyanobacteria must change their metabolism from that of a free-living, phototrophic organism to that of a symbiotic, heterotrophic organism in order to be symbiotically competent. The mechanisms in volved in this shift are largely unknown. SPECIFICITY The specificity of the cyanobacterium-host inter action has been investigated by isolating and grow ing the two symbiotic partners separately and then reconstituting the symbiotic association under lab oratory conditions. Nostoc strains isolated from a cycad, the bryophyte Anthoceros and a lichen all infect Gunnera. Similarly, strains isolated from cycads, Gunnera and a lichen infect Anthoceros species. Hence, Nostoc in general exhibits a very low host specificity, with no correlation to the taxonomic status of the host. Furthermore, the host has a low specificity for its cyanobiont. Even certain strains of Calothrix and Chlorogloeopsis can infect both the homwort Phaeoceros and the liverwort Blasia (West and Adams 1997). Moreover, the cyanobiont shows great adap tive flexibility because the same strain has the capacity to infect different host organs. The cyanobacteria are found extracellularly among the fungal hyphae in lichens, in the cavities of bryophyte thalli and Azolla leaves and in the spe cialised roots of cycads, whereas they are found intracellularly in the fungus Geosiphon and in the stem glands of Gunnera. Taken together, these facts indicate that the host is the (more) selective part ner. Cyanobacteria are unique among symbiotic bacteria in their ability to infect a broad range of hosts and organs. GENETIC DIVERSITY The extreme morphological flexibility of Nostoc makes identification and taxonomy based onmorph ology problematic and unreliable. In the recent literature, many symbiotic isolates (from Gunnera, cycads, bryophytes and the fungus Geosiphon) have been referred to as N. punctiforme, but a pheno type-based characterisation has proved inadequate. No genetic analysis has yet been used to identify the taxonomic status of different symbiotic isolates. At present, the taxonomic interrelatedness of the symbiotic Nostoc strains is largely unknown and does not extend below the genus level. More precise genetic information must therefore be ob tained to establish a more reliable classification system. In recent years, molecular methods have contrbuted to amore detailed classification of the genus Nostoc (Table 1). BIOLOGY AND ENVIRONMENT PPOCEENDINGS OF THE ROYAL IRISHACADEMY, VOL 102B, No 1, 53-56 (2002) 0) ROYAL IRISHACAsEMY 53 BIOLOGY AND ENVIRONMENT P1...

Phenotypic and genotypic comparison of symbiotic and free-living cyanobacteria from a single field site

PCR amplification techniques were used to compare cyanobacterial symbionts from a cyanobacterium-bryophyte symbiosis and free-living cyanobacteria from the same field site. Thirty-one symbiotic cyanobacteria were isolated from the hornwort Phaeoceros sp. at several closely spaced locations, and 40 free-living cyanobacteria were isolated from the immediate vicinity of the same plants. One of the symbiotic isolates was a species of Calothrix, a genus not previously known to form bryophyte symbioses, and the remainder were Nostoc spp. Of the free-living strains, two were Calothrix spp., three were Chlorogloeopsis spp. and the rest were Nostoc spp. All of the symbiotic and all but one of the free-living strains were able to reconstitute the symbiosis with axenic cultures of both Phaeoceros and the liverwort Blasia sp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the regions flanking the 16S-23S rRNA internal transcribed spacer. With one exception, the two techniques produced complementary results and confirmed for the first time that a diversity of symbiotic cyanobacteria infect Phaeoceros in the field. Symbionts from adjacent colonies were different as often as they were the same, showing that the same thallus could be infected with many different cyanobacterial strains. Strains found to be identical by the techniques employed here were often found as symbionts in different thalli at the same locale but were never found free-living. Only one of the free-living strains, and none of the symbiotic strains, was found at more than one sample site, implying a highly localized distribution of strains.

Phenolic constituents of the liverwort: Four novel cyclic bisbibenzyl dimers from Blasia pusilla L

Four novel, cyclic, bisbibenzyl dimers (pusilatins A-D), six bibenzyl derivatives, apigenin 7-O-β- d-glucoside , shikimic acid and five orsellinic acid derivatives have been isolated from the methanolic extract of the liverwort Blasia pusilla L. and their structures characterized by a combination of spectral data, chemical modification and X-ray crystallographic analysis. The previously assigned structure of pusilatin D was revised. Pusilatins B and C showed DNA polymerase β inhibitory activity.

A method for studying chemotaxis in nitrogen fixing cyanobacterium-plant symbioses

A modified version of the Pfeffer technique has been developed to demonstrate a strong chemotactic response of hormogonia from the filamentous nitrogen fixing cyanobacterium Nostocsp. towards exudates of one of its symbiotic partners, the liverwort Blasia pusilla(L.). This technique offers the potential to study this mutualistic symbiosis further, in particular at those primary stages of the interaction which have not been investigated previously. It will also enable other plants, for example wheat, to be screened for the production of chemotactic signals and thereby raise the possibility of extending the host-range of this nitrogen fixing cyanobacterium.

Structures of four novel macrocyclic bis(bibenzyl) dimers, pusilatins A–D from the liverwort Blasia pusilla

Four novel macrocyclic bis(bibenzyl) dimers, named pusilatins A–D have been isolated from the liverwort, Blasia pusilla, and their structures established by a combination of high resolution NMR spectra, X-ray crystallographic analysis and chemical degradation.