Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase.

Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.

Spermine and Spermidine Priming against Botrytis cinerea Modulates ROS Dynamics and Metabolism in Arabidopsis.

Polyamines (PAs) are ubiquitous small aliphatic polycations important for growth, development, and environmental stress responses in plants. Here, we demonstrate that exogenous application of spermine (Spm) and spermidine (Spd) induced cell death at high concentrations, but primed resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis. At low concentrations, Spm was more effective than Spd. Treatments with higher exogenous Spd and Spm concentrations resulted in a biphasic endogenous PA accumulation. Exogenous Spm induced the accumulation of H2O2 after treatment but also after infection with B. cinerea. Both Spm and Spd induced the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase after treatment but also after infection with B. cinerea. The soluble sugars glucose, fructose, and sucrose accumulated after treatment with high concentrations of PAs, whereas only Spm induced sugar accumulation after infection. Total and active nitrate reductase (NR) activities were inhibited by Spm treatment, whereas Spd inhibited active NR at low concentrations but promoted active NR at high concentrations. Finally, γaminobutyric acid accumulated after treatment and infection in plants treated with high concentrations of Spm. Phenylalanine and asparagine also accumulated after infection in plants treated with a high concentration of Spm. Our data illustrate that Spm and Spd are effective in priming resistance against B. cinerea, opening the door for the development of sustainable alternatives for chemical pesticides.

Priming with γ-Aminobutyric Acid against Botrytis cinerea Reshuffles Metabolism and Reactive Oxygen Species: Dissecting Signalling and Metabolism.

The stress-inducible non-proteinogenic amino acid γ-aminobutyric acid (GABA) is known to alleviate several (a)biotic stresses in plants. GABA forms an important link between carbon and nitrogen metabolism and has been proposed as a signalling molecule in plants. Here, we set out to establish GABA as a priming compound against Botrytis cinerea in Arabidopsis thaliana and how metabolism and reactive oxygen species (ROS) are influenced after GABA treatment and infection. We show that GABA already primes disease resistance at low concentrations (100 µM), comparable to the well-characterized priming agent ß-Aminobutyric acid (BABA). Treatment with GABA reduced ROS burst in response to flg22 (bacterial peptide derived from flagellum) and oligogalacturonides (OGs). Plants treated with GABA showed reduced H2O2 accumulation after infection due to increased activity of catalase and guaiacol peroxidase. Contrary to 100 µM GABA treatments, 1 mM exogenous GABA induced endogenous GABA before and after infection. Strikingly, 1 mM GABA promoted total and active nitrate reductase activity whereas 100 µM inhibited active nitrate reductase. Sucrose accumulated after GABA treatment, whereas glucose and fructose only accumulated in treated plants after infection. We propose that extracellular GABA signalling and endogenous metabolism can be separated at low exogenous concentrations.

Fructans Prime ROS Dynamics and Botrytis cinerea Resistance in Arabidopsis.

Naturally derived molecules can be used as priming or defense stimulatory agents to protect against biotic stress. Fructans have gained strong interest due to their ability to induce resistance in a number of crop species. In this study, we set out to establish the role of fructan-induced immunity against the fungal pathogen Botrytis cinerea in Arabidopsis thaliana. We show that both inulin- and levan-type fructans from different sources can enhance Arabidopsis resistance against B. cinerea. We found that inulin from chicory roots and levan oligosaccharides from the exopolysaccharide-producing bacterium Halomonas smyrnensis primed the NADPH-oxidase-mediated reactive oxygen species (ROS) burst in response to the elicitors flg22, derived from the bacterial flagellum, and oligogalacturonides (OGs), derived from the host cell wall. Neither induced a direct ROS burst typical of elicitors. We also found a primed response after infection with B. cinerea for H2O2 accumulation and the activities of ascorbate peroxidase and catalase. Sucrose accumulated as a consequence of fructan priming, and glucose and sucrose levels increased in fructan-treated plants after infection with B. cinerea. This study shows that levan-type fructans, specifically from bacterial origin, can prime plant defenses and that both inulin and levan oligosaccharide-mediated priming is associated with changes in ROS dynamics and sugar metabolism. Establishing fructan-induced immunity in Arabidopsis is an important step to further study the underlying mechanisms since a broad range of biological resources are available for Arabidopsis.

Sweet Immunity: The Effect of Exogenous Fructans on the Susceptibility of Apple (Malus × domestica Borkh.) to Venturia inaequalis.

There is an urgent need for novel, efficient and environmentally friendly strategies to control apple scab (Venturia inaequalis), for the purpose of reducing overall pesticide use. Fructans are recently emerging as promising "priming" compounds, standing out for their safety and low production costs. The objective of this work was to test a fructan-triggered defense in the leaves of apple seedlings. It was demonstrated that exogenous leaf spraying can reduce the development of apple scab disease symptoms. When evaluated macroscopically and by V. inaequalis-specific qPCR, levan-treated leaves showed a significant reduction of sporulation and V. inaequalis DNA in comparison to mock- and inulin-treated leaves, comparable to the levels in fosetyl-aluminum-treated leaves. Furthermore, we observed a significant reduction of in vitro mycelial growth of V. inaequalis on plates supplemented with levans when compared to controls, indicating a direct inhibition of fungal growth. Variations in endogenous sugar contents in the leaves were followed during priming and subsequent infection, revealing complex dynamics as a function of time and leaf ontogeny. Our data are discussed in view of the present theories on sugar signaling and fructan-based immunity, identifying areas for future research and highlighting the potential use of fructans in apple scab management in orchards.

Autophagy in Plants: Both a Puppet and a Puppet Master of Sugars.

Autophagy is a major pathway that recycles cellular components in eukaryotic cells both under stressed and non-stressed conditions. Sugars participate both metabolically and as signaling molecules in development and response to various environmental and nutritional conditions. It is therefore essential to maintain metabolic homeostasis of sugars during non-stressed conditions in cells, not only to provide energy, but also to ensure effective signaling when exposed to stress. In both plants and animals, autophagy is activated by the energy sensor SnRK1/AMPK and inhibited by TOR kinase. SnRK1/AMPK and TOR kinases are both important regulators of cellular metabolism and are controlled to a large extent by the availability of sugars and sugar-phosphates in plants whereas in animals AMP/ATP indirectly translate sugar status. In plants, during nutrient and sugar deficiency, SnRK1 is activated, and TOR is inhibited to allow activation of autophagy which in turn recycles cellular components in an attempt to provide stress relief. Autophagy is thus indirectly regulated by the nutrient/sugar status of cells, but also regulates the level of nutrients/sugars by recycling cellular components. In both plants and animals sugars such as trehalose induce autophagy and in animals this is independent of the TOR pathway. The glucose-activated G-protein signaling pathway has also been demonstrated to activate autophagy, although the exact mechanism is not completely clear. This mini-review will focus on the interplay between sugar signaling and autophagy.

UDP-Glucose: A Potential Signaling Molecule in Plants?

This perspective paper focuses on the most recent results suggesting a potential role for UDP-Glucose as a signaling molecule in plants. In animals, UDP-Glucose is well-established as an extracellular signaling molecule that is sensed by G-protein coupled receptors, activating several downstream defense mechanisms. Recent studies have shown that abnormal growth occurred in both vegetative and reproductive tissue of plants with reduced UDP-Glucose levels, and this could be rescued by exogenous UDP-Glucose. In plants with increased biomass accumulation, the genes involved in UDP-Glucose production were up-regulated. However, excessive endogenous accumulation of UDP-Glucose induced programmed cell death (PCD), and this could also be obtained by exogenous UDP-Glucose application. Plants with decreased UDP-glucose were insensitive to pathogen induced PCD. We speculate that UDP-Glucose acts as an extracellular signaling molecule in plants, and that it may be perceived as a damage-associated molecular pattern.