Metabolic Adaptation, a Specialized Leaf Organ Structure and Vascular Responses to Diurnal N2 Fixation by Nostoc azollae Sustain the Astonishing Productivity of Azolla Ferns without Nitrogen Fertilizer

Abstract

Sustainable agriculture demands reduced input of man-made nitrogen (N) fertilizer, yet N2 fixation limits the productivity of crops with heterotrophic diazotrophic bacterial symbionts. We investigated floating ferns from the genus Azolla that host phototrophic diazotrophic Nostoc azollae in leaf pockets and belong to the fastest growing plants. Experimental production reported here demonstrated N-fertilizer independent production of nitrogen-rich biomass with an annual yield potential per ha of 1200 kg−1 N fixed and 35 t dry biomass. 15N2 fixation peaked at noon, reaching 0.4 mg N g−1 dry weight h−1. Azolla ferns therefore merit consideration as protein crops in spite of the fact that little is known about the fern’s physiology to enable domestication. To gain an understanding of their nitrogen physiology, analyses of fern diel transcript profiles under differing nitrogen fertilizer regimes were combined with microscopic observations. Results established that the ferns adapted to the phototrophic N2-fixing symbionts N. azollae by (1) adjusting metabolically to nightly absence of N supply using responses ancestral to ferns and seed plants; (2) developing a specialized xylem-rich vasculature surrounding the leaf-pocket organ; (3) responding to N-supply by controlling transcripts of genes mediating nutrient transport, allocation and vasculature development. Unlike other non-seed plants, the Azolla fern clock is shown to contain both the morning and evening loops; the evening loop is known to control rhythmic gene expression in the vasculature of seed plants and therefore may have evolved along with the vasculature in the ancestor of ferns and seed plants.

Growing Azolla to produce sustainable protein feed: the effect of differing species and CO2 concentrations on biomass productivity and chemical composition

“…The N 2 ‐fixing symbiont N. azollae apparently provided all nitrogen incorporated in both species, and nitrate concentrations up to 3.4 mmol L −1 did not inhibit N 2 fixation. Previously, addition of 4 mmol L −1 nitrate resulted in increased nitrate reductase activity in A. filiculoides and a small increase in growth and biomass nitrogen content, but nitrate uptake was not quantified in this study . In A. pinnata 80–97% of the N incorporated into the biomass was shown to derive from N 2 fixation at 1 mmol L −1 NO 3 .…”

“…A unique feature of Azolla ferns is that they host nitrogen (N 2 )‐fixing cyanobacteria, Nostoc azollae , in the cavities of their leaves . These phototrophic symbionts fix N 2 during the day and likely release it as NH 4 + in the leaf cavities to be taken up by the ferns . Nitrogen fixation by N. azollae of 0.15–0.17 mg N h −1 per gram of dry biomass is remarkably high compared to 0.08 mg N h −1 per gram of dry biomass fixed by Rhizobia residing in the root nodules of soybean ( Glycine max ) .…”

“…[13][14][15] These phototrophic symbionts fix N 2 during the day and likely release it as NH 4 + in the leaf cavities to be taken up by the ferns. 16,17 Nitrogen fixation by N. azollae of 0.15-0.17 mg N h −1 per gram of dry biomass is remarkably high P Brouwer et al compared to 0.08 mg N h −1 per gram of dry biomass fixed by Rhizobia residing in the root nodules of soybean (Glycine max). 16,18 Effectively, the N. azollae symbiont is capable of supplying all the nitrogen (1100-1200 kg N ha −1 per year) required for the fern's rapid growth.…”

“…16,17 Nitrogen fixation by N. azollae of 0.15-0.17 mg N h −1 per gram of dry biomass is remarkably high P Brouwer et al compared to 0.08 mg N h −1 per gram of dry biomass fixed by Rhizobia residing in the root nodules of soybean (Glycine max). 16,18 Effectively, the N. azollae symbiont is capable of supplying all the nitrogen (1100-1200 kg N ha −1 per year) required for the fern's rapid growth. 16,19 As a result of their high growth and N 2 fixation rates, Azolla ferns have been used as a bio-fertilizer in rice paddies in south-east Asia for several centuries.…”

Jasmonic and salicylic acid response in the fern Azolla filiculoides and its cyanobiont

“…Azolla filiculoides is on the brink of becoming a model system for fern biology. It is fast growing (Wagner, ), has been the subject of multiple transcriptomic studies (Brouwer et al, ; Brouwer et al, ; de Vries, Fischer, et al, ), and is one of the few ferns whose genome is under investigation (F. ‐W. Li & Pryer, ; Sessa et al, ; Sessa & Der, ).…”