Coralloid Roots Research Articles

Unlocking a high bacterial diversity in the coralloid root microbiome from the cycad genus Dioon.

Cycads are among the few plants that have developed specialized roots to host nitrogen-fixing bacteria. We describe the bacterial diversity of the coralloid roots from seven Dioon species and their surrounding rhizosphere and soil. Using 16S rRNA gene amplicon sequencing, we found that all coralloid roots are inhabited by a broad diversity of bacterial groups, including cyanobacteria and Rhizobiales among the most abundant groups. The diversity and composition of the endophytes are similar in the six Mexican species of Dioon that we evaluated, suggesting a recent divergence of Dioon populations and/or similar plant-driven restrictions in maintaining the coralloid root microbiome. Botanical garden samples and natural populations have a similar taxonomic composition, although the beta diversity differed between these populations. The rhizosphere surrounding the coralloid root serves as a reservoir and source of mostly diazotroph and plant growth-promoting groups that colonize the coralloid endosphere. In the case of cyanobacteria, the endosphere is enriched with Nostoc spp and Calothrix spp that are closely related to previously reported symbiont genera in cycads and other early divergent plants. The data reported here provide an in-depth taxonomic characterization of the bacterial community associated with coralloid root microbiome. The functional aspects of the endophytes, their biological interactions, and their evolutionary history are the next research step in this recently discovered diversity within the cycad coralloid root microbiome.

Cycad Coralloid Roots Contain Bacterial Communities Including Cyanobacteria and Caulobacter spp. That Encode Niche-Specific Biosynthetic Gene Clusters.

Cycads are the only early seed plants that have evolved a specialized root to host endophytic bacteria that fix nitrogen. To provide evolutionary and functional insights into this million-year old symbiosis, we investigate endophytic bacterial sub-communities isolated from coralloid roots of species from Dioon (Zamiaceae) sampled from their natural habitats. We employed a sub-community co-culture experimental strategy to reveal both predominant and rare bacteria, which were characterized using phylogenomics and detailed metabolic annotation. Diazotrophic plant endophytes, including Bradyrhizobium, Burkholderia, Mesorhizobium, Rhizobium, and Nostoc species, dominated the epiphyte-free sub-communities. Draft genomes of six cyanobacteria species were obtained after shotgun metagenomics of selected sub-communities. These data were used for whole-genome inferences that suggest two Dioon-specific monophyletic groups, and a level of specialization characteristic of co-evolved symbiotic relationships. Furthermore, the genomes of these cyanobacteria were found to encode unique biosynthetic gene clusters, predicted to direct the synthesis of specialized metabolites, mainly involving peptides. After combining genome mining with detection of pigment emissions using multiphoton excitation fluorescence microscopy, we also show that Caulobacter species co-exist with cyanobacteria, and may interact with them by means of a novel indigoidine-like specialized metabolite. We provide an unprecedented view of the composition of the cycad coralloid root, including phylogenetic and functional patterns mediated by specialized metabolites that may be important for the evolution of ancient symbiotic adaptations.

Generation and characterization of expressed sequence tags (ESTs) from coralloid root cDNA library of Cycas debaoensis

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN (duplex-specific nuclease) normalization method combined with the SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique. The titer of the original cDNA library was about 1.5 × 106 cfu·mL−1 and the average insertion size was about 1 kb with a high recombination rate (97%). The 5011 high-quality expressed sequence tags (ESTs) were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally annotated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 (50.1%) unigenes were associated with 4082 Gene Ontology (GO) terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups (COG) functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase (RLK) genes were screened. Furthermore, a total of 94 simple sequence repeats (SSRs) were discovered, of which 20 loci were successfully amplified in C. debaoensis. This study is the first EST analysis for the coralloid roots of C. debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C. debaoensis and related cycad species.

Highly diverse endophytes in roots of Cycas bifida (Cycadaceae), an ancient but endangered gymnosperm.

As an ancient seed plant, cycads are one of the few gymnosperms that develop a root symbiosis with cyanobacteria, which has allowed cycads to cope with harsh geologic and climatic conditions during the evolutionary process. However, the endophytic microbes in cycad roots remain poorly identified. In this study, using next-generation sequencing techniques, we investigated the microbial diversity and composition of both the coralloid and regular roots of Cycas bifida (Dyer) K.D. Hill. Highly diverse endophytic communities were observed in both the coralloid and regular roots. Of the associated bacteria, the top five families were the Nostocaceae, Sinobacteraceae, Bradyrhizobiaceae, Bacillaceae, and Hyphomicrobiaceae. The Nectriaceae, Trichocomaceae, and Incertae sedis were the predominant fungal families in all root samples. A significant difference in the endophytic bacterial community was detected between coralloid roots and regular roots, but no difference was observed between the fungal communities in the two root types. Cyanobacteria were more dominant in coralloid roots than in regular roots. The divergence of cycad root structures and the modified physiological processes may have contributed to the abundance of cyanobionts in coralloid roots. Consequently, the colonization of cyanobacteria inhibits the assemblage of other endophytes. Our results contribute to an understanding of the species diversity and composition of the cycad-endophyte microbiome and provide an abbreviated list of potential ecological roles of the core microbes present.

Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta.

ABSTRACTWe report here the whole-genome sequence of Nostoc cycadae strain WK-1, which was isolated from cyanobacterial colonies growing in the coralloid roots of the gymnosperm Cycas revoluta. It can provide valuable resources to study the mutualistic relationships and the syntrophic metabolisms between the cyanobacterial symbiont and the host plant, C. revoluta.

A new species of Gastrodia (Orchidaceae: Gastrodieae, Epidendroideae) from Java

Gastrodia Brown (1810: 330) is a mycoheterotrophic genus of Orchidaceae that consists of ca. 50 species (Tan et al. 2012, Suetsugu 2014, Huang et al. 2015). It is characterized by a fleshy tuber or coralloid root system, fused sepals and petals, and two mealy pollinia without caudicles. Since most Gastrodia species have completely fused sepals and petals, observation of the perianth is difficult, and identification of this genus is often imprecise. As a result of re-examination of floral characters, several new taxa in this genus have been reported in China, Taiwan, Japan, Madagascar and other countries during the last decade (Meng et al. 2007, Hsu et al. 2010, Hsu & Kuo 2011, Yeh et al. 2011, Hsu et al. 2012, Tan et al. 2012, Suetsugu 2013, 2014, Huang et al. 2015, Martos et al. 2015). Most of these new taxa belong to the section Codonanthus (Schlechter 1911), which has a short inflorescence (<40 cm).

Plastic Responses Mediated by Identity Recognition in Below-Ground Competition in Cycas micronesica K.D. Hill

We studied the ability of Cycas micronesica roots to recognize the identity of competitor roots using pot culture. Pairs of seedlings were grown in single pots for 10.5 months, and morphological traits of the focal plant were determined when the non-focal plant was a half-sibling, a close relative from a mother 170 m away, a distant relative from a habitat 40 km away, or a different species ( Cycas edentata) from Mindanao, Philippines. Most of the measured response traits exhibited similar trends and confirmed that C. micronesica plant roots were able to recognize the identity of competitors. For example, focal plants grown in pots with C. edentata competitors exhibited 66% increase in fine root dry weight, 54% increase in coralloid root dry weight, and 49% increase in total plant dry weight, compared to focal plants grown in competition with half-siblings. Our methods have shown that plot design in ex situ conservation and botanic garden settings would improve by positioning C. micronesica plants adjacent to non-relatives. Recruitment may improve within in situ conservation plots if managers distribute seeds away from the maternal parent so that emerging seedlings are not forced into immediate competition with half-siblings. These findings may be useful for designing more complex experiments to quantify the influence of these plastic root responses on plant nutrient status and physiology.

Cyanobionts from the coralloid roots of Cycadales: is there any molecular evidence of cyanotoxins?

Cyanobionts from the coralloid roots of Cycadales: is there any molecular evidence of cyanotoxins?

Cycads in vitro

The Cycadales are a group of botanical and evolutionary importance; however, many species face the threat of extinction due to poaching and habitat destruction. The current investigation reviews previous work on in vitro production of cycads, which holds great potential for mass production and germplasm conservation of these unique plants. Megagametophytes and zygotic embryos have been used as explants in most studies, while seedling tissue and new leaf tissue of mature trees have also been used. Callus, coralloid roots and somatic embryos have been formed in vitro but direct organogenesis appears to be the most promising method for mass production and germplasm preservation of the cycads, as recent studies have reported the acclimatization of numerous plantlets less than 200 days after initial culture of zygotic embryos.Plant Tissue Cult. & Biotech. 24(2): 287-301, 2014 (December)

Free sugar profile in cycads.

The sugars fructose, glucose, maltose, and sucrose were quantified in seven tissues of Zamia muricata Willd. to determine their distribution throughout various organs of a model cycad species, and in lateral structural roots of 18 cycad species to determine the variation in sugar concentration and composition among species representing every cycad genus. Taproot and lateral structural roots contained more sugars than leaf, stem, female strobilus, or coralloid roots. For example, taproot sugar concentration was 6.4-fold greater than stem sugar concentration. The dominant root sugars were glucose and fructose, and the only detected stem sugar was sucrose. Sucrose also dominated the sugar profile for leaflet and coralloid root tissue, and fructose was the dominant sugar in female strobilus tissue. Maltose was a minor constituent of taproot, leaflet, and female strobilus tissue, but absent in other tissues. The concentration of total free sugars and each of the four sugars did not differ among genera or families. Stoichiometric relationships among the sugars, such as the quotient hexoses/disaccharides, differed among organs and families. Although anecdotal reports on cycad starch have been abundant due to its historical use as human food and the voluminous medical research invested into cycad neurotoxins, this is the first report on the sugar component of the non-structural carbohydrate profile of cycads. Fructose, glucose, and sucrose are abundant in cycad tissues, with their relative abundance highly contrasting among organs. Their importance as forms of carbon storage, messengers of information, or regulators of cycad metabolism have not been determined to date.

A Long Shot? Could neurodegenerative disease be caused by a cyanobacterial toxin?

A Long Shot? Could neurodegenerative disease be caused by a cyanobacterial toxin?

Genetic diversity of symbiotic cyanobacteria in Cycas revoluta (Cycadaceae)

The diversity of cyanobacterial species within the coralloid roots of an individual and populations of Cycas revoluta was investigated based on 16S rRNA gene sequences. Sixty-six coralloid roots were collected from nine natural populations of cycads on Kyushu and the Ryukyu Islands, covering the entire distribution range of the species. Approximately 400 bp of the 5'-end of 16S rRNA genes was amplified, and each was identified by denaturing gradient gel electrophoresis. Most coralloid roots harbored only one cyanobiont, Nostoc, whereas some contained two or three, representing cyanobiont diversity within a single coralloid root isolated from a natural habitat. Genotypes of Nostoc within a natural population were occasionally highly diverged and lacked DNA sequence similarity, implying genetic divergence of Nostoc. On the other hand, Nostoc genotypes showed no phylogeographic structure across the distribution range, while host cycads exhibited distinct north-south differentiation. Cycads may exist in symbiosis with either single or multiple Nostoc strains in natural soil habitats.

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths >10cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.

The First Organismal Concept for an Extinct Species of Pinaceae:Pinus arnoldiiMiller

The Eocene Princeton Chert locality of southern British Columbia, Canada, provides data to develop organismal concepts for several species of fossil plants, including the first extinct species of Pinaceae, Pinus arnoldii Miller. This new species concept is based on a combination of interconnected organs, common histological features, and patterns of association among isolated plant parts. These data expand our knowledge of P. arnoldii from a morphospecies of ovulate cones to an extinct species of pine that produces woody stems with five-needled fascicles of leaves; simple pollen cones with two abaxial pollen sacs per microsporophyll, containing bisaccate pollen grains; seed cones with inflated scale apices, dorsal umbos that do not bear mucros, and a thick-walled sclerotic outer cortex; and coralloid roots with ectomycorrhizal associations. Organs preserved at various stages of development provide evidence for numerous aspects of growth and reproductive biology, including pollination biology and seed cone development. Cladistic analyses of morphological and molecular characters demonstrate that the novel combination of characters displayed by P. arnoldii causes this extinct species to fall near the base of the Pinus L. tree, either as the sister to all living species of the subgenus Pinus or outside the subgenera that have been defined by extant species of Pinus.

Host Selection of Symbiotic Cyanobacteria in 31 Species of the Australian Cycad Genus: Macrozamia (Zamiaceae)

The nitrogen-fixing cyanobacterium Nostoc is a commonly occurring terrestrial and aquatic cyanobacterium often found in symbiosis with a wide range of plant, algal, and fungal species. We investigated the diversity of cyanobacterial species occurring within the coralloid roots of different Macrozamia cycad species at diverse locations throughout Australia. In all, 74 coralloid root samples were processed and 56 endosymbiotic cyanobacteria were cultured. DNA was isolated from unialgal cultures and a segment of the 16S rRNA gene was amplified and sequenced. Microscopic analysis was performed on representative isolates. Twenty-two cyanobacterial species were identified, comprising mostly Nostoc spp. and a Calothrix sp. No correlation was observed between a cycad species and its resident cyanobiont species. The predominant cyanobacterium isolated from 18 root samples occurred over a diverse range of environmental conditions and within 14 different Macrozamia spp. Phylogenetic analysis indicated that endosymbionts were not restricted to previously described terrestrial species. An isolate clustering with Nostoc PCC7120, an aquatic strain, was identified. This is the first comprehensive study to identify the endosymbionts within a cycad genus using samples obtained from their natural habitats. These results indicate that there is negligible host specialization of cyanobacterial endosymbionts within the cycad genus Macrozamia in the wild.

38. Search for signal substances that are associated with coralloid root formation as an event in cycad-cyanobacteria symbiosis

38. Search for signal substances that are associated with coralloid root formation as an event in cycad-cyanobacteria symbiosis

Rhizopalmoxylon singulare sp. nov. - coralloid palm roots from the Late Cretaceous Deccan Intertrappean beds of Nawargaon, India

Permineralized coralloid aerial palm roots forming a thick compact mantle have been described from the Deccan Intertrappean beds of Nawargaon, Maharashtra, India. A medium sized root exhibits rhizodermis, thick exodermis, thin outer and wide inner cortex; fibers in the inner cortex; air cavities in 3-7 concentric rings in the middle zone of inner cortex; polyarch stele with 12-15 xylem and phloem bundles and sclerenchymatous pith with 1-3 medullary bundles. Morpho-anatomical characters of the root suggest its affinity with coralloid roots probably of Hyphaene dichotoma (White) Furtado and Phoenix sylvestris (L.) Roxb. A combination of details of stelar and cortical region is found to be a better criterion to resolve the natural affinity of permineralized palm roots. Two new combinations of Rhizopalmoxylon, R. angiorhizon and R. macrorhizon are also suggested.

Three phases of research on β-N-methylamino-L-alanine (BMAA) - a neurotoxic amino acid

This paper discusses various aspects of the research that lead from the discovery of beta-N-methylamino-L-alanine (BMAA) to consider a variety of mechanisms that might explain the acute and chronic toxicities of this non-protein amino acid. Such is the fashion of science that current work represents the third phase of research on this compound over a period of more than 40 years. BMAA is now known to exist not only in the plant genus Cycas, where it is synthesized by symbiotic cyanobacteria in the coralloid roots of the plants, but to be widely distributed in the many sites at which free living cyanobacteria abound.

Morphology and ecology of the Antarcticycas plant

The Antarcticycas plant is reconstructed from the co-occurring vegetative organ taxa Antarcticycas schopfii and Yelchophyllum omegapetiolaris and the pollen cone taxon Delemaya spinulosa from permineralized peat of the Fremouw Formation, early Middle Triassic of Antarctica. The association of the organs in the same deposit, assignment of each taxon to Cycadales, and histological features shared among the taxa suggest they were all produced by the same plant. The Antarcticycas plant is reconstructed as a small plant, possibly with a subterranean stem; it was probably similar in habit to small-stemmed species of extant Zamia. Antarcticycas stems are inferred to exhibit apical rather than axillary branching based on reinterpretation of type material that shows a branching axis accompanied by anatomy indicative of the presence of cone domes when observed in extant cycads. The presence of a bulbil on one specimen indicates that branching also took place through adventitious budding. The Antarcticycas plant is similar in its anatomy to extant cycads, although contractile tissue and coralloid roots have yet to be identified and may not have been present in the fossil plant. The plant inhabited a warm temperate polar habitat with protracted periods of winter darkness for which there is no modern equivalent. Evidence for deciduousness in this taxon, previously suggested as a possible adaptation to its warm, light-limited environment, is equivocal. The possibilities that fire played a role in the Fremouw peat ecosystem and that the Antarcticycas plant may have been insect pollinated are explored.

Dichotomization of mycorrhizal and NPA-treated short roots in Pinus sylvestris

Conifers like Scots pine (Pinus sylvestris) have a complicated root system consisting of morphologically and anatomically different root types, of which the short roots have a very limited ability to elongate. Short roots have an important role in nature since they are able to establish ectomycorrhizal symbiosis, in which the growth of fungal mycelium between the epidermal cells and in the intercellular space between cortical cells leads to formation of dichotomous short roots, which may, through further splitting of the meristem, form coralloid root structures. Dichotomous short roots have been suggested to result from changes in either auxin or ethylene concentrations due to the fungal growth inside the root. NPA, the inhibitor of polar auxin transport, enhances the dichotomization of P. sylvestris short root tips similarly to the fungal growth in the root, thus confirming that auxin plays a role in short root morphogenesis. Ethylene is also known to have an important role in the regulation of root morphogenesis. In future the research dealing with the root system and ectomycorrhiza development in P. sylvestris must take into account that both auxin and ethylene are involved and that there is no contradiction in obtaining the same phenotype with both hormones. The expression analysis of PIN proteins, auxin efflux carries, could give valuable information about the role of auxin transport in regulating the root growth and morphogenesis of coniferous root system and mycorrhiza.