Biosynthesis and differential spatial distribution of the 3-deoxyanthocyanidins apigenidin and luteolinidin at the interface of a plant-cyanobacteria symbiosis exposed to cold.

Aquatic ferns of the genus Azolla (Azolla) form highly productive symbioses with filamentous cyanobacteria fixing N2 in their leaf cavities, Nostoc azollae. Stressed symbioses characteristically turn red due to 3-deoxyanthocyanidin (DA) accumulation, rare in angiosperms and of unknown function. To understand DA accumulation upon cold acclimation and recovery, we integrated laser-desorption-ionization mass-spectrometry-imaging (LDI-MSI), a new Azolla filiculoides genome-assembly and annotation, and dual RNA-sequencing into phenotypic analyses of the symbioses. Azolla sp. Anzali recovered even when cold-induced DA-accumulation was inhibited by abscisic acid. Cyanobacterial filaments generally disappeared upon cold acclimation and Nostoc azollae transcript profiles were unlike those of resting stages formed in cold-resistant sporocarps, yet filaments re-appeared in leaf cavities of newly formed green fronds upon cold-recovery. The high transcript accumulation upon cold acclimation of AfDFR1 encoding a flavanone 4-reductase active in vitro suggested that the enzyme of the first step in the DA-pathway may regulate accumulation of DAs in different tissues. However, LDI-MSI highlighted the necessity to describe metabolite accumulation beyond class assignments as individual DA and caffeoylquinic acid metabolites accumulated differentially. For example, luteolinidin accumulated in epithelial cells, including those lining the leaf cavity, supporting a role for the former in the symbiotic interaction during cold acclimation.

The crane fly glycosylated triketide δ-lactone cornicinine elicits akinete differentiation of the cyanobiont in aquatic Azolla fern symbioses.

The restriction of plant-symbiont dinitrogen fixation by an insect semiochemical had not been previously described. Here we report on a glycosylated triketide δ-lactone from Nephrotoma cornicina crane flies, cornicinine, that causes chlorosis in the floating-fern symbioses from the genus Azolla. Only the glycosylated trans-A form of chemically synthesized cornicinine was active: 500 nM cornicinine in the growth medium turned all cyanobacterial filaments from Nostoc azollae inside the host leaf-cavities into akinetes typically secreting CTB-bacteriocins. Cornicinine further inhibited akinete germination in Azolla sporelings, precluding re-establishment of the symbiosis during sexual reproduction. It did not impact development of the plant Arabidopsis thaliana or several free-living cyanobacteria from the genera Anabaena or Nostoc but affected the fern host without cyanobiont. Fern-host mRNA sequencing from isolated leaf cavities confirmed high NH4-assimilation and proanthocyanidin biosynthesis in this trichome-rich tissue. After cornicinine treatment, it revealed activation of Cullin-RING ubiquitin-ligase-pathways, known to mediate metabolite signaling and plant elicitation consistent with the chlorosis phenotype, and 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.

Azolla ferns testify: seed plants and ferns share a common ancestor for leucoanthocyanidin reductase enzymes.

Questions about in vivo substrates for proanthocyanidin (PA) biosynthesis and condensation have not been resolved and wide gaps in the understanding of transport and biogenesis in 'tannosomes' persist. Here we examined the evolution of PA biosynthesis in ferns not previously reported, asking what PAs are synthesised and how. Chemical and gene-expression analyses were combined to characterise PA biosynthesis, leveraging genome annotation from the floating fern Azolla filiculoides. In vitro assay and phylogenomics of PIP-dehydrogenases served to infer the evolution of leucoanthocyanidin reductase (LAR). Sporophyte-synthesised (epi)catechin polymers, averaging only seven subunits, accumulated to 5.3% in A. filiculoides, and 8% in A. pinnata biomass dry weight. Consistently, a LAR active in vitro was highly expressed in A. filiculoides. LAR, and paralogous fern WLAR-enzymes with differing substrate binding sites, represent an evolutionary innovation of the common ancestor of fern and seed plants. The specific ecological niche of Azolla ferns, a floating plant-microbe mat massively fixing CO2 and N2 , shaped their metabolism in which PA biosynthesis predominates and employs novel fern LAR enzymes. Characterisation of in vivo substrates of these LAR, will help to shed light on the recently assigned and surprising dual catalysis of LAR from seed plants.

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales. Two distinct nearly full-length Rhizobiales genomes were identified in leaf-pocket-enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2 -fixation. 15 N2 incorporation was active in ferns with N. azollae but not in ferns without. N2 O was not detectably released from surface-sterilized ferns with the Rhizobiales. N2 -fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2 O.

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

BACKGROUND: Since available arable land is limited and nitrogen fertilizers pollute the environment, cropping systems ought to be developed that do not rely on them. Here we investigate the rapidly growing, N2 -fixing Azolla/Nostoc symbiosis for its potential productivity and chemical composition to determine its potential as protein feed. RESULTS: In a small production system, cultures of Azolla pinnata and Azolla filiculoides were continuously harvested for over 100 days, yielding an average productivity of 90.0-97.2 kg dry weight (DW) ha-1 d-1 . Under ambient CO2 levels, N2 fixation by the fern's cyanobacterial symbionts accounted for all nitrogen in the biomass. Proteins made up 176-208 g kg-1 DW (4.9 × total nitrogen), depending on species and CO2 treatment, and contained more essential amino acids than protein from soybean. Elevated atmospheric CO2 concentrations (800 ppm) significantly boosted biomass production by 36-47%, without decreasing protein content. Choice of species and CO2 concentrations further affected the biomass content of lipids (79-100 g kg-1 DW) and (poly)phenols (21-69 g kg-1 DW). CONCLUSIONS: By continuous harvesting, high protein yields can be obtained from Azolla cultures, without the need for nitrogen fertilization. High levels of (poly)phenols likely contribute to limitations in the inclusion rate of Azolla in animal diets and need further investigation. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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

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.

Azolla domestication towards a biobased economy?

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 37 649 unigenes from RNAseq of microsporocarps, megasporocarps and sporophytes was assembled, then validated. Spores obtained year-round germinated in vitro within 26 d. In vitro fertilization rates reached 25%. Cryopreservation permitted storage for at least 7 months. 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.

The trehalose 6-phosphate/SnRK1 signaling pathway primes growth recovery following relief of sink limitation.

Trehalose 6-P (T6P) is a sugar signal in plants that inhibits SNF1-related protein kinase, SnRK1, thereby altering gene expression and promoting growth processes. This provides a model for the regulation of growth by sugar. However, it is not known how this model operates under sink-limited conditions when tissue sugar content is uncoupled from growth. To test the physiological importance of this model, T6P, SnRK1 activities, sugars, gene expression, and growth were measured in Arabidopsis (Arabidopsis thaliana) seedlings after transfer to cold or zero nitrogen compared with sugar feeding under optimal conditions. Maximum in vitro activities of SnRK1 changed little, but T6P accumulated up to 55-fold, correlating with tissue Suc content in all treatments. SnRK1-induced and -repressed marker gene expression strongly related to T6P above and below a threshold of 0.3 to 0.5 nmol T6P g(-1) fresh weight close to the dissociation constant (4 µm) of the T6P/ SnRK1 complex. This occurred irrespective of the growth response to Suc. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth in response to Suc accumulation under sink-limited conditions. To test this hypothesis, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm to analyze the role of T6P/SnRK1 in relief of growth restriction. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that the T6P/SnRK1 signaling pathway responds to Suc induced by sink restriction that enables growth recovery following relief of limitations such as low temperature.

Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120.

In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.

Trehalose 6-phosphate is required for the onset of leaf senescence associated with high carbon availability.

Trehalose 6-phosphate (T6P) is an important regulator of plant metabolism and development. T6P content increases when carbon availability is high, and in young growing tissue, T6P inhibits the activity of Snf1-related protein kinase (SnRK1). Here, strong accumulation of T6P was found in senescing leaves of Arabidopsis (Arabidopsis thaliana), in parallel with a rise in sugar contents. To determine the role of T6P in senescence, T6P content was altered by expressing the bacterial T6P synthase gene, otsA (to increase T6P), or the T6P phosphatase gene, otsB (to decrease T6P). In otsB-expressing plants, T6P accumulated less strongly during senescence than in wild-type plants, while otsA-expressing plants contained more T6P throughout. Mature otsB-expressing plants showed a similar phenotype as described for plants overexpressing the SnRK1 gene, KIN10, including reduced anthocyanin accumulation and delayed senescence. This was confirmed by quantitative reverse transcription-polymerase chain reaction analysis of senescence-associated genes and genes involved in anthocyanin synthesis. To analyze if the senescence phenotype was due to decreased sugar sensitivity, the response to sugars was determined. In combination with low nitrogen supply, metabolizable sugars (glucose, fructose, or sucrose) induced senescence in wild-type and otsA-expressing plants but to a smaller extent in otsB-expressing plants. The sugar analog 3-O-methyl glucose, on the other hand, did not induce senescence in any of the lines. Transfer of plants to and from glucose-containing medium suggested that glucose determines senescence during late development but that the effects of T6P on senescence are established by the sugar response of young plants.

Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice.

Oshox22 belongs to the homeodomain-leucine zipper (HD-Zip) family I of transcription factors, most of which have unknown functions. Here we show that the expression of Oshox22 is strongly induced by salt stress, abscisic acid (ABA), and polyethylene glycol treatment (PEG), and weakly by cold stress. Trans-activation assays in yeast and transient expression analyses in rice protoplasts demonstrated that Oshox22 is able to bind the CAAT(G/C)ATTG element and acts as a transcriptional activator that requires both the HD and Zip domains. Rice plants homozygous for a T-DNA insertion in the promoter region of Oshox22 showed reduced Oshox22 expression and ABA content, decreased sensitivity to ABA, and enhanced tolerance to drought and salt stresses at the seedling stage. In contrast, transgenic rice over-expressing Oshox22 showed increased sensitivity to ABA, increased ABA content, and decreased drought and salt tolerances. Based on these results, we conclude that Oshox22 affects ABA biosynthesis and regulates drought and salt responses through ABA-mediated signal transduction pathways.

Growth arrest by trehalose-6-phosphate: an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway.

The strong regulation of plant carbon allocation and growth by trehalose metabolism is important for our understanding of the mechanisms that determine growth and yield, with obvious applications in crop improvement. To gain further insight on the growth arrest by trehalose feeding, we first established that starch-deficient seedlings of the plastidic phosphoglucomutase1 mutant were similarly affected as the wild type on trehalose. Starch accumulation in the source cotyledons, therefore, did not cause starvation and consequent growth arrest in the growing zones. We then screened the FOX collection of Arabidopsis (Arabidopsis thaliana) expressing full-length cDNAs for seedling resistance to 100 mm trehalose. Three independent transgenic lines were identified with dominant segregation of the trehalose resistance trait that overexpress the bZIP11 (for basic region/leucine zipper motif) transcription factor. The resistance of these lines to trehalose could not be explained simply through enhanced trehalase activity or through inhibition of bZIP11 translation. Instead, trehalose-6-phosphate (T6P) accumulation was much increased in bZIP11-overexpressing lines, suggesting that these lines may be insensitive to the effects of T6P. T6P is known to inhibit the central stress-integrating kinase SnRK1 (KIN10) activity. We confirmed that this holds true in extracts from seedlings grown on trehalose, then showed that two independent transgenic lines overexpressing KIN10 were insensitive to trehalose. Moreover, the expression of marker genes known to be jointly controlled by SnRK1 activity and bZIP11 was consistent with low SnRK1 or bZIP11 activity in seedlings on trehalose. These results reveal an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway involving T6P, SnRK1, and bZIP11.

Wheat grain development is characterized by remarkable trehalose 6-phosphate accumulation pregrain filling: tissue distribution and relationship to SNF1-related protein kinase1 activity.

Trehalose 6-phosphate (T6P) is a sugar signal that regulates metabolism, growth, and development and inhibits the central regulatory SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11). To better understand the mechanism in wheat (Triticum aestivum) grain, we analyze T6P content and SnRK1 activities. T6P levels changed 178-fold 1 to 45 d after anthesis (DAA), correlating with sucrose content. T6P ranged from 78 nmol g(-1) fresh weight (FW) pregrain filling, around 100-fold higher than previously reported in plants, to 0.4 nmol g(-1) FW during the desiccation stage. In contrast, maximum SnRK1 activity changed only 3-fold but was inhibited strongly by T6P in vitro. To assess SnRK1 activity in vivo, homologs of SnRK1 marker genes in the wheat transcriptome were identified using Wheat Estimated Transcript Server. SnRK1-induced and -repressed marker genes were expressed differently pregrain filling compared to grain filling consistent with changes in T6P. To investigate this further maternal and filial tissues were compared pre- (7 DAA) and during grain filling (17 DAA). Strikingly, in vitro SnRK1 activity was similar in all tissues in contrast to large changes in tissue distribution of T6P. At 7 DAA T6P was 49 to 119 nmol g(-1) FW in filial and maternal tissues sufficient to inhibit SnRK1; at 17 DAA T6P accumulation was almost exclusively endospermal (43 nmol g(-1) FW) with 0.6 to 0.8 nmol T6P g(-1) FW in embryo and pericarp. The data show a correlation between T6P and sucrose overall that belies a marked effect of tissue type and developmental stage on T6P content, consistent with tissue-specific regulation of SnRK1 by T6P in wheat grain.

Metabolism control over growth: a case for trehalose-6-phosphate in plants.

How plants relate their requirements for energy with the reducing power necessary to fuel growth is not understood. The activated glucose forms and NADPH are key precursors in pathways yielding, respectively, energy and reducing power for anabolic metabolism. Moreover, they are substrates or allosteric regulators of trehalose-phosphate synthase (TPS1) in fungi and probably also in plants. TPS1 synthesizes the signalling metabolite trehalose-6-phosphate (T6P) and, therefore, has the potential to relate reducing power with energy metabolism to fuel growth. A working model is discussed where trehalose-6-phosphate (T6P) inhibition of SnRK1 is part of a growth-regulating loop in young and metabolically active heterotrophic plant tissues. SnRK1 is the Snf1 Related Kinase 1 and the plant homologue of the AMP-dependent protein kinase of animals, a central energy gauge. T6P accumulation in response to high sucrose levels in a cell inhibits SnRK1 activity, thus promoting anabolic processes and growth. When T6P levels drop due to low glucose-6-phosphate, uridine-diphosphoglucose, and altered NADPH or due to restricted TPS1 activity, active SnRK1 promotes catabolic processes required to respond to energy and carbon deprivation. The model explains why too little or too much T6P has been found to be growth inhibitory: Arabidopsis thaliana embryos and seedlings without TPS1 are growth arrested and Arabidopsis seedlings accumulating T6P on a trehalose medium are growth arrested. Finally, the insight gained with respect to the possible role of T6P metabolism, where it is known to alter developmental and environmental responses of plants, is discussed.

Determination of trehalose-6-phosphate in Arabidopsis thaliana seedlings by hydrophilic-interaction liquid chromatography-mass spectrometry.

A hydrophilic-interaction chromatography (HILIC) method coupled to electrospray ionization mass spectrometry (ESI-MS) was developed for the determination of trehalose-6-phophate (Tre6P) in Arabidopsis thaliana seedlings. The method was optimized for MS detection and separation of Tre6P from its isomers, such as sucrose-6-phosphate, by testing eluent pH, type of organic solvent and alkalinizer, and gradient conditions. Tre6P could be resolved from matrix components within 28 min by using a water-acetonitrile gradient (0.2 ml/min) at pH 12 with piperidine as alkalinizer. The method was validated for concentrations between 25 and 4,000 nM Tre6P in A. thaliana seedling extracts. Seedlings were extracted with consecutive liquid-liquid and solid-phase extractions, and analyzed with HILIC-MS. Obtained accuracy (80-120 %) and precision (<24 %) demonstrated the suitability of HILIC-MS for determining Tre6P level variations in plants. The limit of detection (LOD) was 3.5 nM Tre6P in extracts corresponding to 4.1 pmol.g(-1) fresh plant weight (FW). This is a considerable improvement with respect to anion-exchange chromatography (AEC)-MS (40 nM) and capillary electrophoresis-MS (80 nM). Furthermore, HILIC-MS analysis times were shorter than with AEC-MS (30 and 60 min, respectively). The applicability of the HILIC-MS method was demonstrated by the analysis of extracts from seedlings grown on medium containing 100 mM sorbitol or trehalose, resulting in mean Tre6P concentrations of 0.2 and 1.9 nmol.g(-1) FW, respectively. Similar concentrations were found with AEC-MS. HILIC-MS was also evaluated at a high flow rate (2.0 ml/min). This high-speed method resolved the Suc6P and Tre6P peaks within 3 min yielding a detection limit of 1.3 nM Tre6P.

The sucrose-regulated Arabidopsis transcription factor bZIP11 reprograms metabolism and regulates trehalose metabolism.

• The Arabidopsis basic region-leucine zipper transcription factor 11 (bZIP11) is known to be repressed by sucrose through a translational inhibition mechanism that requires the conserved sucrose control peptide encoded by the mRNA leader. The function of bZIP11 has been investigated in over-expression studies, and bZIP11 has been found to inhibit plant growth. The addition of sugar does not rescue the growth inhibition phenotype. Here, the function of the bZIP11 transcription factor was investigated. • The mechanism by which bZIP11 regulates growth was studied using large-scale and dedicated metabolic analysis, biochemical assays and molecular studies. • bZIP11 induction results in a reprogramming of metabolism and activation of genes involved in the metabolism of trehalose and other minor carbohydrates such as myo-inositol and raffinose. bZIP11 induction leads to reduced contents of the prominent growth regulatory molecule trehalose 6-phosphate (T6P). • The metabolic changes detected mimic in part those observed in carbon-starved plants. It is proposed that bZIP11 is a powerful regulator of carbohydrate metabolism that functions in a growth regulatory network that includes T6P and the sucrose non-fermenting-1 related protein kinase 1 (SnRK1).

Far-Red Light-Induced Azolla filiculoides Symbiosis Sexual Reproduction: Responsive Transcripts of Symbiont Nostoc azollae Encode Transporters Whilst Those of the Fern Relate to the Angiosperm Floral Transition.

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.

Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry.

A method for the detection of trehalose-6-phosphate (T6P) in tissue of the model plant Arabidopsis thaliana is presented. Liquid-liquid extraction (LLE) and mixed mode solid-phase extraction (SPE) were used for sample pretreatment followed by anion exchange chromatography (AEC) coupled with electrospray ionization mass spectrometry (MS) for highly selective quantitative analysis. LLE of plant material was performed with chloroform/acetonitrile/water (3:7:16, v/v/v) followed by SPE with Oasis MAX material, which significantly reduced the complexity of the extracts. On-line coupling of MS with gradient AEC using a sodium hydroxide eluent was accomplished with a postcolumn ion suppressor. The method allows specific quantification of T6P with good linearity for spiked plant extracts, from 80 nM to 1.3 microM (r(2)>0.98). The limit of detection in plant extracts was 40 nM. The recovery of the method was above 80% for relevant T6P levels. The method was applied to the determination of T6P in seedlings from four mutant A. thaliana lines (TRR1-4) resisting growth arrest caused by external supply of trehalose. Results reveal that T6P accumulation differed substantially in the four mutant lines and wild type (WT). It is concluded that the mutants circumvent the growth arrest observed in WT seedlings on 100mM trehalose by different mechanisms.

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.

Fern genomes elucidate land plant evolution and cyanobacterial symbioses.

Ferns are the closest sister group to all seed plants, yet little is known about their genomes other than that they are generally colossal. Here, we report on the genomes of Azolla filiculoides and Salvinia cucullata (Salviniales) and present evidence for episodic whole-genome duplication in ferns-one at the base of 'core leptosporangiates' and one specific to Azolla. One fern-specific gene that we identified, recently shown to confer high insect resistance, seems to have been derived from bacteria through horizontal gene transfer. Azolla coexists in a unique symbiosis with N2-fixing cyanobacteria, and we demonstrate a clear pattern of cospeciation between the two partners. Furthermore, the Azolla genome lacks genes that are common to arbuscular mycorrhizal and root nodule symbioses, and we identify several putative transporter genes specific to Azolla-cyanobacterial symbiosis. These genomic resources will help in exploring the biotechnological potential of Azolla and address fundamental questions in the evolution of plant life.