Domestication of the floating fern symbiosis Azolla

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 N2-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 towards this end

Azolla domestication towards a biobased economy?

Brouwer P, Bräutigam A, Külahoglu C, et al. Azolla domestication towards a biobased economy? New Phytologist. 2014;202(3):1069-1082.Due to its phenomenal growth requiring neither nitrogen fertilizer nor arable land and its biomass composition, the mosquito fern Azolla is a candidate crop to yield food, fuels and chemicals sustainably. To advance Azolla domestication, we research its dissemination, storage and transcriptome. Methods for dissemination, cross-fertilization and cryopreservation of the symbiosis Azolla filiculoides-Nostoc azollae are tested based on the fern spores. To study molecular processes in Azolla including spore induction, a database of 37649 unigenes from RNAseq of microsporocarps, megasporocarps and sporophytes was assembled, then validated. Spores obtained year-round germinated in vitro within 26d. In vitro fertilization rates reached 25%. Cryopreservation permitted storage for at least 7months. The unigene database entirely covered central metabolism and to a large degree covered cellular processes and regulatory networks. Analysis of genes engaged in transition to sexual reproduction revealed a FLOWERING LOCUS T-like protein in ferns with special features induced in sporulating Azolla fronds. Although domestication of a fern-cyanobacteria symbiosis may seem a daunting task, we conclude that the time is ripe and that results generated will serve to more widely access biochemicals in fern biomass for a biobased economy

Metabolic Adaptation, a Specialized Leaf Organ Structure and Vascular Responses to Diurnal N-2 Fixation by Nostoc azollae Sustain the Astonishing Productivity of Azolla Ferns without Nitrogen Fertilizer

Brouwer P, Bräutigam A, Buijs VA, et al. Metabolic Adaptation, a Specialized Leaf Organ Structure and Vascular Responses to Diurnal N-2 Fixation by Nostoc azollae Sustain the Astonishing Productivity of Azolla Ferns without Nitrogen Fertilizer. Frontiers in Plant Science. 2017;8: 442.Sustainable agriculture demands reduced input of man-made nitrogen (N) fertilizer, yet N-2 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. N-15(2) 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 N-2-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

Fern genomes elucidate land plant evolution and cyanobacterial symbioses

Li F-W, Brouwer P, Carretero-Paulet L, et al. Fern genomes elucidate land plant evolution and cyanobacterial symbioses. NATURE PLANTS. 2018;4(7):460-472

Cytokinin-induced promotion of root meristem size in the fern Azolla supports a shoot-like origin of euphyllophyte roots

de Vries J, Fischer AM, Roettger M, et al. Cytokinin-induced promotion of root meristem size in the fern Azolla supports a shoot-like origin of euphyllophyte roots. New Phytologist. 2016;209(2):705-720.The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises the questions of how auxin and cytokinin govern fern root system architecture and whether this can tell us something about the origin of that root. Using Azolla filiculoides, we characterized the influence of IAA and zeatin on adventitious fern root meristems and vasculature by Nomarski microscopy. Simultaneously, RNAseq analyses, yielding 36 091 contigs, were used to uncover how the phytohormones affect root tip gene expression. We show that auxin restricts Azolla root meristem development, while cytokinin promotes it; it is the opposite effect of what is observed in Arabidopsis. Global gene expression profiling uncovered 145 genes significantly regulated by cytokinin or auxin, including cell wall modulators, cell division regulators and lateral root formation coordinators. Our data illuminate both evolution and development of fern roots. Promotion of meristem size through cytokinin supports the idea that root meristems of euphyllophytes evolved from shoot meristems. The foundation of these roots was laid in a postembryonically branching shoot system

Arabidopsis Trehalose-6-Phosphate Synthase 1 Is Essential for Normal Vegetative Growth and Transition to Flowering

In resurrection plants and yeast, trehalose has a function in stress protection, but the absence of measurable amounts of trehalose in other plants precludes such a function. The identification of a trehalose biosynthetic pathway in angiosperms raises questions on the function of trehalose metabolism in nonresurrection plants. We previously identified a mutant in the Arabidopsis trehalose biosynthesis gene AtTPS1. Plants homozygous for the tps1 mutation do not develop mature seeds (Eastmond et al., 2002). AtTPS1 expression analysis and the spatial and temporal activity of its promoter suggest that this gene is active outside the seed-filling stage of development as well. A generally low expression is observed in all organs analyzed, peaking in metabolic sinks such as flower buds, ripening siliques, and young rosette leaves. The arrested tps1/tps1 embryonic state could be rescued using a dexamethasone-inducible AtTPS1 expression system enabling generation of homozygous mutant plants. When depleted in AtTPS1 expression, such mutant plants show reduced root growth, which is correlated with a reduced root meristematic region. Moreover, tps1/tps1 plants are retarded in growth and remain generative during their lifetime. Absence of Trehalose-6-Phosphate Synthase 1 in Arabidopsis plants precludes transition to flowering

Aquatic weeds as novel protein sources: Alkaline extraction of tannin-rich Azolla

The aquatic weed Azolla is a potential protein crop due to its prolific growth and high protein content, supported entirely by nitrogen-fixing symbionts. Alkaline protein extraction at pH 8 followed by acid precipitation allowed recovery of 16–26% of the biomass nitrogen, while at pH 10.5 nitrogen recovery improved to 35–54%. This pH effect was typical of ferns of the family Salviniaceae, and may be explained by high concentrations of condensed tannins (CTs) in the biomass that precipitate protein at mild pH. Two approaches were tested to increase protein yield and reduce protein binding by CTs. Pre-extraction with aqueous acetone (70 v/v%) removed 76–85% of the CTs and subsequent alkaline extraction at pH 12.5 and 95 °C recovered 38% of the biomass nitrogen. Extraction with 1.5% of PEG as a CT-binding agent, also permitted to recover 38% of the nitrogen, under milder conditions of pH 8 and 45 °C

ω20-Hydroxy and ω9,ω10-dihydroxy biomarker lipids in ferns from the Salviniaceae family

All seven species of floating ferns from the genus Azolla (family Salviniaceae) produce a unique series of long chain mid-chain ω20-hydroxy compounds (ω20-alkanols, 1,ω20-diols, ω20-hydroxy fatty acids) and structurally related ω9,ω10-dihydroxy compounds (ω9,ω10-diols, 1,ω9,ω10-triols and ω9,ω10-dihydroxy fatty acids). These very long chain fatty acid (VLCFA) derivatives occur in the ferns’ waxes in free and esterified form. The specific distribution of these lipids differed between species belonging to each of the two sections in the Azolla genus: in species of the section Azolla and Rhizosperma, the ratio of C31 over C35 ω20-alkanols averaged 7.0 and 0.40, and the ratio of C26 over C28 ω20-hydroxy fatty acids averaged 2.7 and 1.0, respectively. Similar compounds were identified in species of another genus in the Salviniaceae family, Salvinia, suggesting that their biosynthetic pathway evolved early during Salviniaceae evolution (>89 Ma). Salvinia species contain ω20-hydroxy and ω9,ω10-dihydroxy compounds in smaller concentrations and in a much different distribution compared to Azolla; the C31 1,ω20-diol is unique to Salvinia species. Closely related fern species from the genera Marsilea, Pilularia and Regnellidium did not contain these compounds, nor did unrelated aquatic plants from the genera Lemna and Pistia. All mid-chain hydroxy compounds detected in extant Azolla have been traced previously in Arctic Eocene sediments from the so-called ‘Azolla Event’ (48.5 Ma), implying that they are well preserved in the geological record and may therefore serve as Azolla biomarkers. Our findings indicate that ω20-hydroxy and ω9,ω10-dihydroxy compounds in sediments could be used as biomarkers of the whole Salviniaceae family. Subsequently, the clear differences in compound distribution between the Azolla and Salvinia genera and the more subtle ones between the two Azolla sections, may allow assigning the compound's origin at the genus (and possibly section) level, depending on the preservation of compound classes in the sediment and the timing of the Azolla or Salvinia deposition. This is exemplified by a sediment interval of the so-called ‘Salvinia bed’ (Eemian), which contained trace amounts of the C31 1,ω20-diol, but none of the ω20-hydroxy and ω9,ω10-dihydroxy compounds common to Azolla, indicating the value of C31 1,ω20-diol as a biomarker for distinguishing Salvinia from Azolla

ω20-Hydroxy and ω9,ω10-dihydroxy biomarker lipids in ferns from the Salviniaceae family

All seven species of floating ferns from the genus Azolla (family Salviniaceae) produce a unique series of long chain mid-chain ω20-hydroxy compounds (ω20-alkanols, 1,ω20-diols, ω20-hydroxy fatty acids) and structurally related ω9,ω10-dihydroxy compounds (ω9,ω10-diols, 1,ω9,ω10-triols and ω9,ω10-dihydroxy fatty acids). These very long chain fatty acid (VLCFA) derivatives occur in the ferns’ waxes in free and esterified form. The specific distribution of these lipids differed between species belonging to each of the two sections in the Azolla genus: in species of the section Azolla and Rhizosperma, the ratio of C31 over C35 ω20-alkanols averaged 7.0 and 0.40, and the ratio of C26 over C28 ω20-hydroxy fatty acids averaged 2.7 and 1.0, respectively. Similar compounds were identified in species of another genus in the Salviniaceae family, Salvinia, suggesting that their biosynthetic pathway evolved early during Salviniaceae evolution (>89 Ma). Salvinia species contain ω20-hydroxy and ω9,ω10-dihydroxy compounds in smaller concentrations and in a much different distribution compared to Azolla; the C31 1,ω20-diol is unique to Salvinia species. Closely related fern species from the genera Marsilea, Pilularia and Regnellidium did not contain these compounds, nor did unrelated aquatic plants from the genera Lemna and Pistia. All mid-chain hydroxy compounds detected in extant Azolla have been traced previously in Arctic Eocene sediments from the so-called ‘Azolla Event’ (48.5 Ma), implying that they are well preserved in the geological record and may therefore serve as Azolla biomarkers. Our findings indicate that ω20-hydroxy and ω9,ω10-dihydroxy compounds in sediments could be used as biomarkers of the whole Salviniaceae family. Subsequently, the clear differences in compound distribution between the Azolla and Salvinia genera and the more subtle ones between the two Azolla sections, may allow assigning the compound's origin at the genus (and possibly section) level, depending on the preservation of compound classes in the sediment and the timing of the Azolla or Salvinia deposition. This is exemplified by a sediment interval of the so-called ‘Salvinia bed’ (Eemian), which contained trace amounts of the C31 1,ω20-diol, but none of the ω20-hydroxy and ω9,ω10-dihydroxy compounds common to Azolla, indicating the value of C31 1,ω20-diol as a biomarker for distinguishing Salvinia from Azolla