Domestication of the floating fern symbiosis Azolla

Ferns from the Azolla genus are highly productive without nitrogen fertilizer because filamentous cyanobacteria, Nostoc azollae, associated with the shoot stem cells, invade leaf cavities for N2-fixation, and reproductive structures for generational transfer. Previously used as nitrogen biofertilizer, their domestication is now considered for circular economy including the sustainable production of plant protein. The symbiosis recently transgressed into molecular research. Sequences from metagenomes of several species are available to study the contribution of the microbiome components to the symbiosis traits. A first assembly and annotation of the reference genome A. filiculoides was released; it allowed reconstruction of tannin biosynthesis, which determines Azolla biomass quality as a feed. Here, we begin with describing novel research areas required to integrate agrosystem development with domestication. We next describe first achievements to control the life cycle of the symbiosis in relation to dissemination, storage and pre-breeding. We then identify key traits of the symbiosis that will need to be considered to achieve yield stability, and discuss these traits with the little mechanistic insight available thus far. We conclude that for rapid breeding, the next vital development will be genome editing of fern host and cyanobacterial symbiont and describe our first steps towards this end

Crane fly semiochemical overrules plant control over cyanobiont in Azolla symbioses

Semiochemicals from insects that restrict plant symbiont dinitrogen fixation had not been known. Here we report on a the glycosylated triketide δ-lactone only found in Nephrotoma cornicina crane flies, cornicinine, that causes chlorosis in the floating-fern symbioses from the genus Azolla.Cornicinine was chemically synthesized, as well as its aglycone and diastereoisomer. Only the glycosylated trans-A form was active: 500 nM cornicinine in the growth medium turned the dinitrogen-fixing cyanobacterial filaments from Nostoc azollae inside the host leaf cavities into akinete-like cells. Cornicinine further inhibited akinete germination in Azolla sporelings, precluding re-establishment of the symbiosis during sexual reproduction. It did not affect the plant Arabidopsis thaliana or several free-living cyanobacteria from the genera Anabaena or Nostoc. Chlorosis occurred in hosts on nitrogen with and devoid of cyanobiont. Cornicinine, therefore, targeted host mechanisms resulting in coordinate cyanobiont differentiation.Sequence profiling of messenger RNA from isolated leaf cavities confirmed high NH4-assimilation and proanthocyanidin biosynthesis in this trichome-rich tissue. Leaf-cavity transcripts in ferns grown on cornicinine reflected activation of Cullin-RING ubiquitin-ligase pathways, known to mediate metabolite signaling and plant elicitation consistent with the chlorosis phenotype. Transcripts accumulating when akinetes are induced, in leaf cavities of ferns on cornicinine and in megasporocarps, were consistent with increased JA-oxidase, sulfate transport and exosome formation.The work begins to uncover molecular mechanisms of cyanobiont differentiation in a seed-free plant symbiosis important for wetland ecology or circular crop-production today, that once caused massive CO2 draw-down during the Eocene geological past.Significance Coordinated differentiation of host and filamentous cyanobacteria underlies the development of ecologically important symbioses; this includes the floating ferns Azolla which share their wetland habitat with Nephrotoma cornicina craneflies containing the glycosylated triketide δ-lactone semiochemical, cornicinine. Cornicinine overrules cyanobiont differentiation thus inhibiting symbiosis N2-fixation and sexual reproduction; its mode of action resembles plant elicitation as suggested by transcriptional profiling of cells lining the cyanobiont cavities using a new release of the fern host genome.Competing Interest StatementThe authors have declared no competing interest

Secondary metabolites that shape the permanent symbioses between Azolla ferns and nitrogen-fixing cyanobacteria

Azolla has various applications such as green fertilizer, substrate for potting soil, animal feed or even human consumption. The high protein content and growth speed combined with the independence on agricultural land and nitrogen fertilizer, give it strong potential as sustainable protein crop to replace imported soy. Being the only obligate plant-cyanobacteria symbiosis it is furthermore an interesting study subject for fundamental research. Here we first focus on the chemistry, biosynthesis and spatial distribution of Azolla specialized metabolites that determine the usability of Azolla biomass and protein. We then research how secondary metabolism changes by environmental cues typical for the Netherlands such as a cold winter. We further focus on the biotic interaction between the crane fly Nephrotoma cornicina and Azolla, which share the same wetland habitat. A specialized metabolite from this insect overrules the coordinate differentiation of Nostoc azollae and therefore is of particular interest to reveal how the fern host might control the life cycle of the cyanobacteria. This essential mechanism has not been researched before. Finally, we explore Azolla transformation because of its importance to reveal gene function and the associated molecular mechanisms

Laser Post-Ionization Combined with Matrix-Free Laser Desorption/Ionization Enhances the Mass Spectrometry Imaging of Plant Metabolites

While the benefits of mass spectrometry imaging (MSI) coupled with recently described laser post-ionization (LPI) techniques (e.g., MALDI-2) have been well explored for the study of mammalian systems, their benefits for spatial metabolomics of plants have not. Here, we demonstrate that matrix-free laser desorption/ionization (LDI) coupled with LPI can significantly increase the number of plant metabolites detectable in an MSI experiment, compared to LDI alone, including for many flavonoids. Moreover, while many aromatic compounds are detected as their radical cations, a result of the photoionization processes accessible using LPI, many compounds (e.g., non-UV active compounds) also experience a significant increase in the abundance of their protonated ions. This suggests that endogenous UV active compounds, such as flavonoids can act as a MALDI-like matrix in promoting charge transfer upon excitation by the laser pulse used for LPI. MSI datasets using LDI-LPI acquired from Azolla filiculoides reveal rich spectra, containing several thousand peaks, with very few background-related signals, including many poly glycosylated flavonoids. This work provides an avenue to significantly enhance the capabilities for studying region-specific flavonoid metabolism within plants

The effects of cervical mucous glycodelin-A, granulocyte colony-stimulating factor and L-selectin on pregnancy outcomes in in-vitro fertilization-embryo transfer cycles

Objective: To investigate the benefits of cervical mucous glycodelin-A, granulocyte colony-stimulating factor (G-CSF) and L-selectin levels as a marker of endometrial receptivity in in-vitro fertilization/embryo transfer cycles (IVF-ET). Material and Methods: In this prospective cohort study cervical mucus samples were collected during oocyte pick up and embryo transfer from 56 IVF-ET patients. Blood samples were collected during embryo transfer only. The glycodelin-A, G-CSF and L-selectin levels in cervical mucus and blood samples were measured and the results were compared with the pregnancy rates. Results: Cervical mucus G-CSF and L-Selectin levels measured at embryo transfer were significantly higher than those measured during oocyte pick-up (p=0.04 and p=0.002, respectively). Mean cervical mucous G-CSF levels were significantly higher in pregnant patients compared to non pregnant patients during embryo transfer (p=0.024). When 1365 pg/ml was set as cut-off point for G-CSF levels in predicting pregnancy, sensitivity and specificity were 76% and 61.3%, respectively. However, there were no significant relationship between the glycodelin-A and L-selectin levels in the cervical mucus and pregnancy outcomes. Conclusion: Investigating markers of endometrial receptivity in the cervical mucus during embryo transfer is a novel and non-invasive method in IVF cycles. G-CSF may be an important marker in predicting the implantation. Copyright © 2012 by Türkiye Klinikleri