Control of theAzollasymbiosis sexual reproduction: ferns to shed light on the origin of floral regulation?

Abstract

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.

Crane fly semiochemical overrules plant control over cyanobiont inAzollasymbioses

“…At the shoot tips, the cabbage-like crop of leaves tightly conceals the important shoot-apical Nostoc colony (SANC) and large ST. The small and likely motile SANC filaments inoculate newly forming leaf initials and sporocarps, for vertical transfer of Nostoc to the next generation (Dijkhuizen et al, 2021). Inside the cavities of the upper leaf lobes, mature N 2 -fixing Nostoc filaments are typically found along with a variety of ST.…”

“…The four Azolla species used in this study were A. filiculoides (Li et al, 2018), A. pinnata originating from the Botanical Gardens of Antwerp (Belgium), an Azolla species from the Anzali lagoon (Iran) which was phylogenetically analyzed but could not be assigned to any of the described Azolla species (Dijkhuizen et al, 2021) and an unknown Azolla species collected from the Botanical Gardens of Bordeaux (France). Adult Azolla sporophytes were grown in modified IRRI-medium as previously described (Brouwer et al, 2017) Distinguishably yellow-colored clumps of megasporocarps, held together by the glochidia of the massulae, could then be hand-picked from the shallow water and used.…”

“…Dual RNA-Sequencing of far-red light grown sporophytes, micro and megasporocarps A. filiculoides ferns were grown on light with a far-red component to induce sporulation as described in Dijkhuizen et al, 2021. Micro and megasporocarps were manually picked from the sporulating ferns during a period of two h, two h into the light period, snap frozen along with the sporophytes collected two h into the light period.…”

“…Material was sampled from independent cultures so as to obtain three independent biological replicates for each of the megasporocarp, microsporocarp and sporophyte samples. RNA was extracted, then libraries synthesized and dual RNA sequenced as described in Dijkhuizen et al, 2021. Data from this experiment is deposited under accession nr. (provided upon acceptance of the manuscript).…”

“…No reuse allowed without permission.The copyright holder for this preprint this version posted February 21, 2023. ; https://doi.org/10.1101/2023.02.19using poly-A enriched RNA; read-count normalization was based on read counts of 1100 genes most expressed in the assay (see Materials and Methods). Profiles from the sporocarps versus sporophyte were obtained from was based on total read counts aligning to the fern genome as inDijkhuizen et al, 2021, with for each feature the read counts per million reads aligning to the genome. (Supplied as an excel file along with its source data).…”