Crane fly semiochemical overrules plant control over cyanobiont inAzollasymbioses

Abstract

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.

High-resolution metabolite imaging: luteolinidin accumulates at the host-cyanobiont interface during cold-acclimation inAzollasymbioses

“…Relative amounts of transcripts the fern host invested per metabolic process, as defined by automatic Mercator annotation of Afi_v2 genes (Lohse et al, 2014; Güngör et al, 2023), after 14 days ( C14 ) and 21 days ( C21 ) cold-treatment were compared to the untreated control after 14 days ( U14 ). Photosynthesis (239 genes) was the most reduced process, while secondary metabolism (60 manually curated genes) was the most induced ( Figure S10; File S2 ), consistent with literature (Tran et al, 2020).…”

“…The methods was as in Güngör et al, 2023. Briefly, sporophytes were treated with cellulase, macerozyme and pectolyase for 19 h at 30°C after which leaf pockets were mechanically released and manually collected.…”

“…Acquired reads were demultiplexed, quality-filtered and trimmed to remove primers before alignment with STAR to the concatenated genomes of A. filiculoides v2, its chloroplast and N. azollae (Dobin et al, 2013; Güngör et al, 2023). For the spore samples only reads for N. azollae were extracted whilst for the cold-treatment reads were extracted for both N. azollae and A. filiculoides .…”

“…Bottom: the apigenidin peak appeared only after acid-heat treatment consistent with color-development. Güngör et al, 2023, showing enzymes related to the FBP, including 7 Af2OGDs and functionally characterized 2OGDs from angiosperms, lycophytes and bryophytes (Li et al, 2020). Branches with grey circles have more than 80% bootstrap support.…”

“…The second and final enzymatic step from flavan-4-ols to 3-deoxyanthocyanidins is generally attributed to ANS (Figure 1B). ANS belongs to the 2-oxoglutarate dependent dioxygenase (2OGD) superfamily, for which we calculated a phylogenetic tree including 29 identified A. filiculoides 2OGDs using the 1KP orthogroup database in Güngör et al, 2023. Clades with flavonoid-related 2OGDs were combined into a new pruned tree and included 7 A. filiculoides 2OGDs (Figure 7A). The tree showed that flavonol synthase (FLS) and ANS diverged in seed plants whilst A. filiculoides has an FLS/ANS-like gene (Afi_v2_s19G002650.1).…”