Coralloid Roots Research Articles

Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals.

Cycads are known to host symbiotic cyanobacteria, including Nostocales species, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition to Nostoc spp., symbiotic cyanobacteria of the genus Aulosira were identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includes Aulosira spp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

Exploring the Influence of Ecological Niches and Hologenome Dynamics on the Growth of Encephalartos villosus in Scarp Forests

Information on how bacteria in plants and soil, along with extracellular enzymes, affect nutrient cycling in Encephalartos villosus growing in phosphorus deficient and acidic scarp forests is lacking. Bacteria in coralloid roots, rhizosphere, and non-rhizosphere soils were isolated to determine the potential role of soil bacterial communities and their associated enzyme activities in nutrient contributions in rhizosphere and non-rhizosphere soils. The role of soil characteristics and associated bacteria on E. villosus nutrition and nitrogen source reliance was investigated. Encephalartos villosus leaves, coralloid roots, rhizosphere, and non-rhizosphere soils were collected at two scarp forests. Leaf nutrition, nitrogen source reliance, soil nutrition, and extracellular enzyme activities were assayed. A phylogenetic approach was used to determine the evolutionary relationship between identified bacterial nucleotide sequences. The clustering pattern of isolated bacterial strains was primarily dictated by the ecological niches from which they originated (rhizosphere soil, non-rhizosphere soil, and coralloid roots), thus indicating that host-microbe interactions may be a key driver of this pattern, in line with the hologenome theory. There were insignificant differences in the phosphorus and nitrogen cycling enzyme activities in E. villosus rhizosphere and non-rhizosphere soils in both localities. Significantly positive correlations were recorded between nitrogen and phosphorus cycling enzymes and phosphorus and nitrogen concentrations in rhizosphere and non-rhizosphere soils. Additionally, more than 70% of the leaf nitrogen was derived from the atmosphere. This study challenged the conventional expectation that environmental filters alone dictate microbial community composition in similar habitats and revealed that host-microbe interactions, as proposed by the hologenome theory, are significant drivers of microbial community structuring. The isolated bacteria and their plant growth promoting traits play a role in E. villosus nutrition and nitrogen source reliance and secrete nutrient cycling enzymes that promote nutrient availability in rhizosphere and non-rhizosphere soils.

Discovery of Cyanobacteria Associated with Cycads and Description of Three Novel Species in Desmonostoc (Nostocaceae)

Heterocyte-forming cyanobacteria form symbiotic relationships with several lineages of plants. Here, twenty (20) strains of endosymbiotic cyanobacteria (cyanobionts) with Nostoc-like morphologies were isolated from the highly specialized coralloid roots of five host species in Cycadales—Cycas debaoensis, C. fairylakea, C. elongata, Ceratozamia robusta, and Macrozamia moorei. Molecular phylogeny based on the 16S rRNA gene placed these strains into seven different taxa within the Nostocaceae, specifically under the genera Desmonostoc and Dendronalium. The percent dissimilarity and unique patterns in the secondary structures of the D1-D1′, Box-B, V2, and V3 helices, which were based on the 16S–23S rRNA internal transcribed spacer (ITS) regions, supported three distinct species in Desmonostoc. These three morphologically distinct novel species are described in this report: Desmonostoc debaoense sp. nov., Desmonostoc meilinense sp. nov., and Desmonostoc xianhuense sp. nov. Other investigated strains were phylogenetically identified as members of the recently discovered genus Dendronalium and represent the first report of association of that genus with cycads. Our findings suggest that the order Cycadales hosts diverse species of cyanobionts in their coralloid roots and that many potential unreported or novel taxa are present in cycads occurring in their natural habitat and await discovery.

Mind-altering Cycads? Preliminary Evidence of Psychoactive Effects in Cycadales

This article examines historical and contemporary evidence for the importance of cycads as sources of psychoactive experiences for ritual and recreational purposes. Methods used include critical analysis and comparison of published and gray literature as well as ethnographic accounts gathered by the author in Mexico and South Africa. The consumption of cycad parts (pollen, coralloid root, and caudex) to derive an intentional psychoactive effect as a drug may implicate the neurotoxic amino acid BMAA, while fermentation and distillation of cycad starch result in intoxication from alcohol. Cycad ingestion is related to medicinal uses, magico-religious practice, and sexual stimulation as an aphrodisiac. Interconnections between these functions, the ceremonial importance of cycads, and conservation implications are discussed. Conclusions include the need to better understand effects on users and on cycad populations, and the potential pitfalls and opportunities psychoactive uses present for community-based conservation.

Core Endophytic Bacteria and Their Roles in the Coralloid Roots of Cultivated Cycas revoluta (Cycadaceae).

As a gymnosperm group, cycads are known for their ancient origin and specialized coralloid root, which can be used as an ideal system to explore the interaction between host and associated microorganisms. Previous studies have revealed that some nitrogen-fixing cyanobacteria contribute greatly to the composition of the endophytic microorganisms in cycad coralloid roots. However, the roles of host and environment in shaping the composition of endophytic bacteria during the recruitment process remain unclear. Here, we determined the diversity, composition, and function prediction of endophytic bacteria from the coralloid roots of a widely cultivated cycad, Cycas revoluta Thunb. Using next-generation sequencing techniques, we comprehensively investigated the diversity and community structure of the bacteria in coralloid roots and bulk soils sampled from 11 sites in China, aiming to explore the variations in core endophytic bacteria and to predict their potential functions. We found a higher microbe diversity in bulk soils than in coralloid roots. Meanwhile, there was no significant difference in the diversity and composition of endophytic bacteria across different localities, and the same result was found after removing cyanobacteria. Desmonostoc was the most dominant in coralloid roots, followed by Nostoc, yet these two cyanobacteria were not shared by all samples. Rhodococcus, Edaphobacter, Niastella, Nordella, SH-PL14, and Virgisporangium were defined as the core microorganisms in coralloid roots. A function prediction analysis revealed that endophytic bacteria majorly participated in the plant uptake of phosphorus and metal ions and in disease resistance. These results indicate that the community composition of the bacteria in coralloid roots is affected by both the host and environment, in which the host is more decisive. Despite the very small proportion of core microbes, their interactions are significant and likely contribute to functions related to host survival. Our study contributes to an understanding of microbial diversity and composition in cycads, and it expands the knowledge on the association between hosts and symbiotic microbes.

Core Microbiome and Microbial Community Structure in Coralloid Roots of Cycas in Ex Situ Collection of Kunming Botanical Garden in China.

Endophytes are essential in plant succession and evolution, and essential for stress resistance. Coralloid root is a unique root structure found in cycads that has played a role in resisting adverse environments, yet the core taxa and microbial community of different Cycas species have not been thoroughly investigated. Using amplicon sequencing, we successfully elucidated the microbiomes present in coralloid roots of 10 Cycas species, representing all four sections of Cycas in China. We found that the endophytic bacteria in coralloid roots, i.e., Cyanobacteria, were mainly composed of Desmonostoc_PCC-7422, Nostoc_PCC-73102 and unclassified_f__Nostocaceae. Additionally, the Ascomycota fungi of Exophiala, Paraboeremia, Leptobacillium, Fusarium, Alternaria, and Diaporthe were identified as the core fungi taxa. The Ascomycota fungi of Nectriaceae, Herpotrichiellaceae, Cordycipitaceae, Helotiaceae, Diaporthaceae, Didymellaceae, Clavicipitaceae and Pleosporaceae were identified as the core family taxa in coralloid roots of four sections. High abundance but low diversity of bacterial community was detected in the coralloid roots, but no significant difference among species. The fungal community exhibited much higher complexity compared to bacteria, and diversity was noted among different species or sections. These core taxa, which were a subset of the microbiome that frequently occurred in all, or most, individuals of Cycas species, represent targets for the development of Cycas conservation.

How the right evolved partners in Cycads and Legumes drive enhanced growth in a harsh environment

Cycads are ancient plants that establish symbiotic associations with plant growth-promoting (PGP) microbes. These ancient associations are rarely contrasted with more recent associations involving PGP microbes and legumes. This study investigated if Vigna unguiculata growing in Encephalartos villosus rhizosphere and non-rhizosphere soils shares similar symbionts with E. villosus and if there is any sanction by plants towards certain soil bacteria. Also, the biomass accumulation and plant nutrition of V. unguiculata growing in these soils was investigated. Vigna unguiculata seeds were grown in E. villosus rhizosphere and non-rhizosphere soils. Thereafter, growth characteristics and plant nutrition were analyzed. Vigna unguiculata plants grown in E. villosus rhizosphere and non-rhizosphere soils were nodulated by Paenibacillus, Bacillus, Peribacillus, Brevibacillus, Alkalihalobacillus, and Lysinibacillus species identified in E. villosus coralloid roots. Bacteria isolated from nodules and coralloid roots were phylogenetically close, regardless of the soil from which these bacteria came. That supports the filter theory by which specific environmental conditions select certain microbial groups to establish symbiotic interactions with plants. No significant differences were observed in the total plant biomass, however, V. unguiculata plants grown in rhizosphere and non-rhizosphere soils invested significantly more resources in belowground biomass that could be related to the extra nitrogen coming from the biological nitrogen fixation that is devoted to roots. This study shows that V. unguiculata and E. villosus growing in similar soil conditions may share the same symbionts promoting plant nutrient assimilation and growth, this opens an idea of a common evolution of the two species and their symbionts.

Encephalartos natalensis, Their Nutrient-Cycling Microbes and Enzymes: A Story of Successful Trade-Offs.

Encephalartos spp. establish symbioses with nitrogen (N)-fixing bacteria that contribute to soil nutrition and improve plant growth. Despite the Encephalartos mutualistic symbioses with N-fixing bacteria, the identity of other bacteria and their contribution to soil fertility and ecosystem functioning is not well understood. Due to Encephalartos spp. being threatened in the wild, this limited information presents a challenge in developing comprehensive conservation and management strategies for these cycad species. Therefore, this study identified the nutrient-cycling bacteria in Encephalartos natalensis coralloid roots, rhizosphere, and non-rhizosphere soils. Additionally, the soil characteristics and soil enzyme activities of the rhizosphere and non-rhizosphere soils were assayed. The coralloid roots, rhizosphere, and non-rhizosphere soils of E. natalensis were collected from a population of >500 E. natalensis in a disturbed savanna woodland at Edendale in KwaZulu-Natal (South Africa) for nutrient analysis, bacterial identification, and enzyme activity assays. Nutrient-cycling bacteria such as Lysinibacillus xylanilyticus; Paraburkholderia sabiae, and Novosphingobium barchaimii were identified in the coralloid roots, rhizosphere, and non-rhizosphere soils of E. natalensis. Phosphorus (P) cycling (alkaline and acid phosphatase) and N cycling (β-(D)-Glucosaminidase and nitrate reductase) enzyme activities showed a positive correlation with soil extractable P and total N concentrations in the rhizosphere and non-rhizosphere soils of E. natalensis. The positive correlation between soil enzymes and soil nutrients demonstrates that the identified nutrient-cycling bacteria in E. natalensis coralloid roots, rhizosphere, and non-rhizosphere soils and associated enzymes assayed may contribute to soil nutrient bioavailability of E. natalensis plants growing in acidic and nutrient-poor savanna woodland ecosystems.

Structure and Biosynthesis of Desmamides A-C, Lipoglycopeptides from the Endophytic Cyanobacterium Desmonostoc muscorum LEGE 12446.

Certain cyanobacteria of the secondary metabolite-rich order Nostocales can establish permanent symbioses with a large number of cycads, by accumulating in their coralloid roots and shifting their metabolism to dinitrogen fixation. Here, we report the discovery of two new lipoglycopeptides, desmamides A (1) and B (2), together with their aglycone desmamide C (3), from the nostocalean cyanobacterium Desmonostoc muscorum LEGE 12446 isolated from a cycad (Cycas revoluta) coralloid root. The chemical structures of the compounds were elucidated using a combination of 1D and 2D NMR spectroscopy and mass spectrometry. The desmamides are decapeptides featuring O-glycosylation of tyrosine (in 1 and 2) and an unusual 3,5-dihydroxy-2-methyldecanoic acid residue. The biosynthesis of the desmamides was studied by substrate incubation experiments and bioinformatics. We describe herein the dsm biosynthetic gene cluster and propose it to be associated with desmamide production. The discovery of this class of very abundant (>1.5% d.w.) bacterial lipoglycopeptides paves the way for exploration of their potential role in root endosymbiosis.

Macro-propagation and Conservation of Cycad Species at Botanical Garden, Yogi Vemana University, Kadapa, Andhra Pradesh, India

Cycads are the most threatened plants of all living organisms and they are on the brink of extinction in the wild. Cycads need urgent conservation and this can only be accomplished by in situ as well as ex situ conservation techniques. As a consequence of human activity, most of the Indian cycads are threatened with extinction. To oppose the current issues the authors collected three cycad germ plasms from different localities of Andhra Pradesh and carried out a similar kind of germination process. The present study’s intention was to propagate more number of seedlings through seed germination in less time to augment the species population in their natural population. Seed germination response was best by using fleshly outer coat removed cycad seeds (without sarcotesta) while directly collected from trees. The most preferable growing medium is red soil, sand and cocopeat (1:1:1). The seedlings were raised through this method and developed coralloid roots faster which suggests their survivability. The present study provideed a low-cost standard methodology followed for speedy and effective germination and obtained positive results. These methods would help in restoring species in their natural habitats as well as avoiding species extinction in wild.

Seed Germination in CITES Listed <i>Cycas beddomei</i> and its Bearing in its Conservation

Cycas beddomei Dyer, is an endemic and endangered, CITES (Appendix I) listed species from the Eastern Ghats of India. For large scale production of seedlings, the authors proposed a new seed germination technique with the objectives of disabling seed dormancy, quickening germination and improving germination percentage. The seedlings raised through this method develop coralloid roots faster that guarantee their survivability. The method is cost effective and suggestable for large scale species restoration programmes of this otherwise threatened species.

Polyphasic approach identifies two new species of Desmonostoc (Nostocales, Cyanobacteria) in the coralloid roots of Cycas fairylakea (Cycadales)

ABSTRACT Endogenously formed coralloid roots in living cycads are known to be infected and maintained by a mutual partnership with photosynthetic cyanobacteria. In this study, two strains of filamentous, nitrogen-fixing endosymbiotic cyanobacteria from the green zone of the coralloid roots of Cycas fairylakea were isolated and grown in culture. The phylogenetic placement based on the 16S rRNA gene region and the secondary structures of the ITS between the 16S and 23S rRNA gene, in addition to traditional morphological characterization, were used to analyse the two strains. Results showed that the 16S rRNA gene sequences of the two strains, CF01 and CF06, displayed ≥97% similarity to the genus Desmonostoc. The phylogeny inferred by maximum likelihood, bayesian inference and neighbour-joining placed the two strains on a separate node away from other species of Desmonostoc. Hormogonial filaments were evident in the two symbiotic species. In addition, a few unique morphological structures in the CF01 and CF06 strains were observed. The D1-D1ʹ, Box-B and V3 helices generated from the ITS of 16S–23S sequences did not fit those of any described species of Desmonostoc, and this led to proposing the two strains as novel species. In compliance with the International Code of Nomenclature for algae, fungi, and plants, the two species are named Desmonostoc lechangense sp. nov. and Desmonostoc aggregatum sp. nov.

The Predominance of Proteobacteria and Cyanobacteria in the Cycas dolichophylla Coralloid Roots Revealed by 16S rRNA Metabarcoding

The Predominance of Proteobacteria and Cyanobacteria in the Cycas dolichophylla Coralloid Roots Revealed by 16S rRNA Metabarcoding

Diversity, Phylogeny and Antagonistic Activity of Fungal Endophytes Associated with Endemic Species of Cycas (Cycadales) in China.

The culture-based approach was used to characterize the fungal endophytes associated with the coralloid roots of the endemic Cycas debaoensis and Cycas fairylakea from various population sites in China. We aim to determine if the assemblages of fungal endophytes inside these endemic plant hosts are distinct and could be explored for bioprospecting. The isolation method yielded a total of 284 culturable fungal strains. Identification based on the analysis of the internal transcribed spacer (ITS) rDNA showed that they belonged to two phyla, five classes, eight orders and 22 families. At least 33 known genera and 62 different species were confirmed based on >97% ITS sequence similarity. The most frequent and observed core taxa in the two host species regardless of their population origin were Talaromyces, Penicillium, Fusarium, Pochonia and Gliocladiopsis. Seventy percent was a rare component of the fungal communities with only one or two recorded isolates. Contrary to common notions, diversity and fungal richness were significantly higher in C. debaoensis and C. fairylakea collected from a botanical garden, while the lowest was observed in C. debaoensis from a natural habitat; this provides evidence that garden management, and to a minor extent, ex-situ conservation practice, could influence fungal endophyte communities. We further selected nineteen fungal isolates and screened for their antagonistic activities via a co-cultivation approach against the phytopathogens, Diaporthe sp. and Colletotrichum sp. Among these, five isolates with high ITS similarity matches with Hypoxylon vinosupulvinatum (GD019, 99.61%), Penicillium sp. (BD022, 100%), Penicillifer diparietisporus (GD008, 99.46%), Clonostachys rogersoniana (BF024, 99.46%) and C. rosea (BF011, 99.1%), which showed exceptional antagonistic activities against the phytopathogenic fungi with a significant inhibition rate of 70–80%. Taken together, our data presented the first and most comprehensive molecular work on culturable fungal endophytes associated with the coralloid roots of cycads. Our study also demonstrated that about 5% of fungal endophytes were not detected by the high-throughput sequencing approach, implying the equal importance of a culture-dependent approach to study fungal communities of cycads. We further highlighted the potential role of endemic and rare plants to discover and isolate unique plant-associated fungal taxa with excellent biocontrol properties.

Full-Length 16S rRNA and ITS Gene Sequencing Revealed Rich Microbial Flora in Roots of Cycas spp. in China.

Cycads have developed a complex root system categorized either as normal or coralloid roots. Past literatures revealed that a great diversity of key microbes is associated with these roots. This recent study aims to comprehensively determine the diversity and community structure of bacteria and fungi associated with the roots of two Cycas spp. endemic to China, Cycas debaoensis Zhong & Chen and Cycas fairylakea D.Y. Wang using high-throughput amplicon sequencing of the full-length 16S rRNA (V1-V9 hypervariable) and short fragment ITS region. The total DNA from 12 root samples were extracted, amplified, sequenced, and analyzed. Resulting sequences were clustered into 61 bacteria and 2128 fungal OTUs. Analysis of community structure revealed that the coralloid roots were dominated mostly by the nitrogen-fixer Nostocaceae but also contain other non-diazotrophic bacteria. The sequencing of entire 16S rRNA gene identified four different strains of cyanobacteria under the heterocystous genera Nostoc and Desmonostoc. Meanwhile, the top bacterial families in normal roots were Xanthobacteraceae, Burkholderiaceae, and Bacillaceae. Moreover, a diverse fungal community was also found in the roots of cycads and the predominating families were Ophiocordycipitaceae, Nectriaceae, Bionectriaceae, and Trichocomaceae. Our results demonstrated that bacterial diversity in normal roots of C. fairylakea is higher in richness and abundance than C. debaoensis. On the other hand, a slight difference, albeit insignificant, was noted for the diversity of fungi among root types and host species as the number of shared taxa is relatively high (67%). Our results suggested that diverse microbes are present in roots of cycads which potentially interact together to support cycads survival. Our study provided additional knowledge on the microbial diversity and composition in cycads and thus expanding our current knowledge on cycad-microbe association. Our study also considered the possible impact of ex situ conservation on cyanobiont community of cycads.

Cycads: Ancient Plants With Bacteria Living in Their Roots

Have you ever seen a picture of a dinosaur and realized that there is a palm-like plant by its side? Maybe you have walked around your neighborhood and seen the same kind of plant there? Incredibly, those ancient plants, called cycads, have been around for millions of years. A single cycad plant can live up to 2,000 years! We think that the secret to cycads’ survival and long life lies inside a very special structure called the coralloid root, which has microbes living inside it. We studied these coralloid roots and found a high diversity of bacterial species living inside of them, more than anyone had ever imagined. When we took a closer look at these bacteria, we found that they can produce many compounds that can help them communicate among themselves and with the plant, transport nutrients, and perform other functions that are still a mystery.

Specialized bacteriome uncovered in the coralloid roots of the epiphytic gymnosperm, Zamia pseudoparasitica

AbstractEpiphytes face several constraints regarding nutrient acquisition: They are disconnected from soil minerals and they have to mainly rely on nutrients leached by precipitation and microbes. The cycad, Zamia pseudoparasitica Yates, is the only known strictly epiphytic gymnosperm, and it is endemic to Panamanian rainforests. Cycads have evolved specialized coralloid roots that host endophytic cyanobacteria specialized in nitrogen fixation. We collected coralloid roots from plants in the Omar Torrijos National Park, Provincia de Coclé. DNA was extracted from fresh inner coralloid roots, and the bacteriome was described using two molecular markers: rbcL‐rbcX (targeting cyanobacteria) and 16S (all bacteria). Sixteen samples were sequenced for rbcL‐rbcX yielding sequences belonging to a monophyletic group within the order Nostocales. One hundred and sixty‐five amplicon sequence variants (ASVs) were found in at least two of our 27 samples amplified for 16S. Nostocales, Rhizobiales, and Acetobacterales were the three most diverse and abundant orders of bacteria found within the coralloid roots, and the candidate phylum WPS‐2 was also found in many samples. We performed a de novo assembly from a single culture of the endophytic cyanobacteria. A phylogenomic analysis of the isolate places the cyanobacterium in a sister clade to mostly symbiotic taxa from mosses, liverworts, and lichens. Additionally, the isolate has genes putatively involved in symbiotic signaling, hormogonium differentiation, ammonium transport, nitrogen fixation, heterocyst differentiation, sulfate transport, and secondary metabolites. Although dominated by organisms with the capacity to fix nitrogen, coralloid roots are also inhabited by a diverse community of other taxa which may also play biologically important roles.

Exploring cycad foliage as an archive of the isotopic composition of atmospheric nitrogen.

Molecular nitrogen (N2 ) constitutes the majority of Earth's modern atmosphere, contributing ~0.79bar of partial pressure (pN2 ). However, fluctuations in pN2 may have occurred on 107 -109 year timescales in Earth's past, perhaps altering the isotopic composition of atmospheric nitrogen. Here, we explore an archive that may record the isotopic composition of atmospheric N2 in deep time: the foliage of cycads. Cycads are ancient gymnosperms that host symbiotic N2 -fixing cyanobacteria in modified root structures known as coralloid roots. All extant species of cycads are known to host symbionts, suggesting that this N2 -fixing capacity is perhaps ancestral, reaching back to the early history of cycads in the late Paleozoic. Therefore, if the process of microbial N2 fixation records the δ15 N value of atmospheric N2 in cycad foliage, the fossil record of cycads may provide an archive of atmospheric δ15 N values. To explore this potential proxy, we conducted a survey of wild cycads growing in a range of modern environments to determine whether cycad foliage reliably records the isotopic composition of atmospheric N2 . We find that neither biological nor environmental factors significantly influence the δ15 N values of cycad foliage, suggesting that they provide a reasonably robust record of the δ15 N of atmospheric N2 . Application of this proxy to the record of carbonaceous cycad fossils may not only help to constrain changes in atmospheric nitrogen isotope ratios since the late Paleozoic, but also could shed light on the antiquity of the N2 -fixing symbiosis between cycads and cyanobacteria.

Perspectives on Endosymbiosis in Coralloid Roots: Association of Cycads and Cyanobacteria.

Past endosymbiotic events allowed photosynthetic organisms to flourish and evolve in terrestrial areas. The precursor of chloroplasts was an ancient photosynthetic cyanobacterium. Presently, cyanobacteria are still capable of establishing successful symbioses in a wide range of hosts. One particular host plant among the gymnosperms is cycads (Order Cycadales) in which a special type of root system, referred to as coralloid roots, develops to house symbiotic cyanobacteria. A number of studies have explained coralloid root formation and cyanobiont invasion but the questions on mechanisms of this host-microbe association remains vague. Most researches focus on diversity of symbionts in coralloid roots but equally important is to explore the underlying mechanisms of cycads-Nostoc symbiosis as well. Besides providing an overview of relevant areas presently known about this association and citing putative genes involved in cycad-cyanobacteria symbioses, this paper aims to identify the limitations that hamper attempts to get to the root of the matter and suggests future research directions that may prove useful.

Isolation and characterization of 1-palmitoyl-2-linoleoyl-sn-glycerol as a hormogonium-inducing factor (HIF) from the coralloid roots of Cycas revoluta (Cycadaceae)

Coralloid roots are specialized tissues of cycads (Cycas revoluta) that are involved in symbioses with nitrogen-fixing Nostoc cyanobacteria. We found that a crude methanolic extract of coralloid roots induced differentiation of the filamentous cell aggregates of Nostoc species into motile hormogonia. Hence, the hormogonium-inducing factor (HIF) was chased using bioassay-based isolation, and the active principle was characterized as a mixture of diacylglycerols (DAGs), mainly composed of 1-palmitoyl-2-linoleoyl-sn-glycerol (1), 1-palmitoyl-2-oleoyl-sn-glycerol (2), 1-stearoyl-2-linolenoyl-sn-glycerol (3), and 1-stearoyl-2-linoleoyl-sn-glycerol (4). Enantioselectively synthesised compound 1 showed a clear HIF activity at 1 nmol (0.6 µg) disc−1 for the filamentous cells, whereas synthesised 2-linoleoyl-3-palmitoyl-sn-glycerol (1′) and 1-palmitoyl-2-linoleoyl-rac-glycerol (1/1′) were less active than 1. Conversely, synthesised 1-linoleoyl-2-palmitoyl-rac-glycerol (8/8′) which is an acyl positional isomer of compound 1 was inactive. In addition, neither 1-monoacylglycerols nor phospholipids structurally related to 1 showed HIF-like activities. As DAGs are protein kinase C (PKC) activators, 12-O-tetradecanoylphorbol-13-acetate (12), urushiol C15:3-Δ10,13,16 (13), and a skin irritant anacardic acid C15:1-Δ8 (14) were also examined for HIF-like activities toward the Nostoc cells. Neither 12 nor 13 showed HIF-like activities, whereas 14 showed an HIF-like activity at 1 nmol/disc. These findings appear to indicate that some DAGs act as hormogonium-inducing signal molecules for filamentous Nostoc cyanobacteria.