Summary Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized.A metagenomic approach was taken complemented by detection of N2O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales.Two distinct nearly full‐length Rhizobiales genomes were identified in leaf‐pocket‐enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2‐fixation. 15N2 incorporation was active in ferns with N. azollae but not in ferns without. N2O was not detectably released from surface‐sterilized ferns with the Rhizobiales.N2‐fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2O.

Questions about in vivo substrates for proanthocyanidin (PA) biosynthesis and condensation have not been resolved and wide gaps in the understanding of transport and biogenesis in 'tannosomes' persist. Here we examined the evolution of PA biosynthesis in ferns not previously reported, asking what PAs are synthesised and how. Chemical and gene-expression analyses were combined to characterise PA biosynthesis, leveraging genome annotation from the floating fern Azolla filiculoides. In vitro assay and phylogenomics of PIP-dehydrogenases served to infer the evolution of leucoanthocyanidin reductase (LAR). Sporophyte-synthesised (epi)catechin polymers, averaging only seven subunits, accumulated to 5.3% in A. filiculoides, and 8% in A. pinnata biomass dry weight. Consistently, a LAR active in vitro was highly expressed in A. filiculoides. LAR, and paralogous fern WLARenzymes with differing substrate binding sites, represent an evolutionary innovation of the common ancestor of fern and seed plants. The specific ecological niche of Azolla ferns, a floating plant-microbe mat massively fixing CO 2 and N 2 , shaped their metabolism in which PA biosynthesis predominates and employs novel fern LAR enzymes. Characterisation of in vivo substrates of these LAR, will help to shed light on the recently assigned and surprising dual catalysis of LAR from seed plants.

Water ferns of the genus Azolla and the filamentous cyanobacteria Nostoc azollae constitute a model symbiosis that enabled the colonization of the water surface with traits highly desirable for the development of more sustainable crops: their floating mats capture CO2 and fix N2 at high rates using light energy. Their mode of sexual reproduction is heterosporous. The regulation of the transition from the vegetative phase to the spore forming phase in ferns is largely unknown, yet a prerequisite for Azolla domestication, and of particular interest as ferns represent the sister lineage of seed plants. Sporocarps induced with far red light could be crossed so as to verify species attribution of strains from the Netherlands but not of the strain from the Anzali lagoon in Iran; the latter strain was assigned to a novel species cluster from South America. Red-dominated light suppresses the formation of dissemination stages in both gametophyte- and sporophyte-dominated lineages of plants, the response likely is a convergent ecological strategy to open fields. FR-responsive transcripts included those from MIKCC homologues of CMADS1 and miR319-controlled GAMYB transcription factors in the fern, transporters in N. azollae, and ycf2 in chloroplasts. Loci of conserved microRNA (miRNA) in the fern lineage included miR172, yet FR only induced miR529 and miR535, and reduced miR319 and miR159. Phylogenomic analyses of MIKCC TFs suggested that the control of flowering and flower organ specification may have originated from the diploid to haploid phase transition in the homosporous common ancestor of ferns and seed plants.

Semiochemicals from insects that restrict plant symbiont dinitrogen fixation had not been known. Here we report on a the glycosylated triketide delta-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.

SummaryRegulation of the transition from vegetative to spore-forming phases in ferns is largely unknown yet a pre-requisite for their domestication. External and endogenous cues regulating the formation of sporocarps were researched in Azolla ferns, that are heterosporous and constitute a symbiosis with the cyanobacteria Nostoc azollae.Once external cues were identified, small RNA and mRNA from eukaryotic and prokaryotic partners in the symbiosis were analyzed for differential accumulation upon transition to sexual reproduction.filiculoides fern sporocarp formation required far-red light (FR) and was suppressed by medium nitrogen; FR-induced sporocarps were viable in crosses that verified species attribution of Dutch strains but not those from Iran’s Anzali lagoon. FR-responsive transcripts encoded the Azolla MIKCC homologue of CMADS1, miRNA319-controlled GAMYB in the fern, ycf2 in chloroplasts, and transporters in N. azollae. Loci of conserved microRNA in the fern lineage included miR172, but FR only induced mirR529 and miR535, and reduced miR159 and miR319.The clade of the FR-responsive MIKCC radiated separately in ferns from the seed plant clade containing AtSOC1 and floral homeotic genes A, C, D and E, yet the data supports that regulons controlling seed plant flowering originate from the diploid to haploid phase transition in the common ancestor of seed plants and ferns.