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.

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.

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 N 2 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 toward this end.