Trehalose metabolites in Arabidopsis - elusive, active and central: July 14 2009

Trehalose is an alpha, alpha-1, 1-linked glucose disaccharide. In plants, trehalose is synthesized in two steps. Firstly, trehalose-6-phosphate synthase (TPS) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate (T6P); secondly, T6P-phosphatase (TPP) converts T6P into trehalose and Pi. Trehalose is further cleaved into glucose by trehalase. In extracts of most plants, including Arabidopsis, levels of both trehalose and T6P are low, nearing detection limits, and this has delayed research into their function. Trehalose is transported widely in plants, but transport of T6P is not thought to occur except possibly at the subcellular level. Feeding trehalose to Arabidopsis seedlings alters carbon allocation with massive starch accumulation in cotyledons and leaves and absence of starch and growth in shoot and root apices. The Arabidopsis genome has experienced extensive radiation of genes likely encoding enzymes of T6P metabolism: 4 and 10 genes are found with homology to TPS and TPP respectively and 7 genes are found with homology to both TPS and TPP. Complementation of Saccharomyces cerevisiae mutants has shown that AtTPS1, AtTPPA and AtTPPB are functional enzymes. In contrast just a single gene encoding a protein with trehalase activity has been found. Whilst most TPS proteins appear cytosolic, strikingly, some TPPs appear targeted to chloroplasts; trehalase on the other hand is extracellular. Transporters of trehalose and T6P have yet to be described. Arabidopsis tps1 mutants are embryo lethal and results suggest that T6P is essential for several other steps in development including root growth and floral transition. Accordingly, altering T6P content has a profound effect on plant habitus and impacts metabolite profiles, sugar utilization and photosynthesis. These large effects have hindered dissection of cause and effect. In contrast, plants with large alterations in sucrose-6-phosphate concentrations are indistinguishable from wild type, suggesting very different functions for these compounds. Recently, T6P at low micromolar concentrations has been shown in vitro and in vivo to inhibit SnRK1 of the SNF1/AMPK group of protein kinases. This supports a function for T6P as a sugar signaling molecule integrating metabolism and development in plants in relation to carbon supply. Genetic engineering of Arabidopsis as well as tobacco, potato and rice with TPS or TPS/TPP protein fusions reveals that trehalose metabolism also mediates multiple abiotic stress tolerances. Trehalose applications also mediate biotic stress resistances. Both Escherichia coli and Saccharomyces cerevisiae TPS/TPP protein fusions can be used to engineer stress tolerance suggesting that metabolites rather than proteins of the trehalose pathway are key stress tolerance elicitors. Results underscore the central role of trehalose metabolites in integrating carbon metabolism and stress responses with plant development

Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase

Trehalose is the most widespread disaccharide in nature, occurring in bacteria, fungi, insects, and plants. Its precursor, trehalose 6-phosphate (T6P), is also indispensable for the regulation of sugar utilization and growth, but the sites of action are largely unresolved. Here we use genetic and biochemical approaches to investigate whether T6P acts to regulate starch synthesis in plastids of higher plants. Feeding of trehalose to Arabidopsis leaves led to stimulation of starch synthesis within 30 min, accompanied by activation of ADP-glucose pyrophosphorylase (AGPase) via posttranslational redox modification. The response resembled sucrose but not glucose feeding and depended on the expression of SNF1-related kinase. We also analyzed transgenic Arabidopsis plants with T6P levels increased by expression of T6P synthase or decreased by expression of T6P phosphatase (TPP) in the cytosol. Compared with wild type, leaves of T6P synthase-expressing plants had increased redox activation of AGPase and increased starch, whereas TPP-expressing plants showed the opposite. Moreover, TPP expression prevented the increase in AGPase activation in response to sucrose or trehalose feeding. Incubation of intact isolated chloroplasts with 100 μM T6P significantly and specifically increased reductive activation of AGPase within 15 min. Results provide evidence that T6P is synthesized in the cytosol and acts on plastidial metabolism by promoting thioredoxin-mediated redox transfer to AGPase in response to cytosolic sugar levels, thereby allowing starch synthesis to be regulated independently of light. The discovery informs about the evolution of plant metabolism and how chloroplasts of prokaryotic origin use an intermediate of the ancient trehalose pathway to report the metabolic status of the cytosol

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

Metabolite transport and associated sugar signalling systems underpinning source/ sink interactions

Metabolite transport between organelles, cells and source and sink tissues not only enables pathway co-ordination but it also facilitates whole plant communication, particularly in the transmission of information concerning resource availability. Carbon assimilation is co-ordinated with nitrogen assimilation to ensure that the building blocks of biomass production, amino acids and carbon skeletons, are available at the required amounts and stoichiometry, with associated transport processes making certain that these essential resources are transported from their sites of synthesis to those of utilization. Of the many possible posttranslational mechanisms that might participate in efficient co-ordination of metabolism and transport only reversible thiol-disulphide exchange mechanisms have been described in detail. Sucrose and trehalose metabolism are intertwined in the signalling hub that ensures appropriate resource allocation to drive growth and development under optimal and stress conditions, with trehalose-6-phosphate acting as an important signal for sucrose availability. The formidable suite of plant metabolite transporters provides enormous flexibility and adaptability in inter-pathway coordination and source-sink interactions. Focussing on the carbon metabolism network, we highlight the functions of different transporter families, and the important of thioredoxins in the metabolic dialogue between source and sink tissues. In addition, we address how these systems can be tailored for crop improvement

Diversity and activity of sugar transporters in nematode-induced root syncytia

The plant-parasitic nematode Heterodera schachtii stimulates plant root cells to form syncytial feeding structures which synthesize all nutrients required for successful nematode development. Cellular re-arrangements and modified metabolism of the syncytia are accompanied by massive intra- and intercellular solute allocations. In this study the expression of all genes annotated as sugar transporters in the Arabidopsis Membrane Protein Library was investigated by Affymetrix gene chip analysis in young and fully developed syncytia compared with non-infected Arabidopsis thaliana roots. The expression of three highly up-regulated (STP12, MEX1, and GTP2) and three highly down-regulated genes (SFP1, STP7, and STP4) was analysed by quantitative RT-PCR (qRT-PCR). The most up-regulated gene (STP12) was chosen for further in-depth studies using in situ RT-PCR and a nematode development assay with a T-DNA insertion line revealing a significant reduction of male nematode development. The specific role of STP12 expression in syncytia of male juveniles compared with those of female juveniles was further shown by qRT-PCR. In order to provide evidence for sugar transporter activity across the plasma membrane of syncytia, fluorescence-labelled glucose was used and membrane potential recordings following the application of several sugars were performed. Analyses of soluble sugar pools revealed a highly specific composition in syncytia. The presented work demonstrates that sugar transporters are specifically expressed and active in syncytia, indicating a profound role in inter- and intracelluar transport processes

Foliar application of microdoses of sucrose to reduce codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) damage to apple trees

Abstract BACKGROUND The effects of foliar applications of microdoses of sucrose to reduce the damage by the codling moth have been reported from nine trials carried in France and Algeria from 2009 to 2014. The activity of sucrose alone was assessed by comparison with an untreated control and some treatments with the Cydia pomonella granulovirus or a chemical insecticide. The addition of sucrose to these different treatments was also investigated. RESULTS The application of sucrose at 0.01% reduced the means of infested fruits with a value of Abbott's efficacy of 41.0 ± 10.0%. This involved the induction of resistance by antixenosis to insect egg laying. Indeed, it seems that acceptance of egg laying on leaves treated with sucrose was reduced. The addition of sucrose to thiacloprid improved its efficacy (59.5% ± 12.8) by 18.4%. However, the sucrose had no added value when associated with C. pomonella granulovirus treatments. CONCLUSION Foliar applications of microdoses of sucrose every 20 days in commercial orchards can partially protect against the codling moth. Its addition to thiacloprid increases the efficacy in integrated control strategies, contrary to C. pomonella granulovirus treatments. This work opens a route for the development of new biocontrol strategies