Exploring Synergistic Effects of Levan and Levan-Metabolizing Bacillaceae in Promoting Growth and Enhancing Immunity of Tomato and Wheat.

Boosting plant immunity by priming agents can lower agrochemical dependency in plant production. Levan and levan-derived oligosaccharides (LOS) act as priming agents against biotic stress in several crops. Additionally, beneficial microbes can promote plant growth and protect against fungal diseases. This study assessed possible synergistic effects caused by levan, LOS and five levan- and LOS-metabolizing Bacillaceae (Bacillus and Priestia) strains in tomato and wheat. Leaf and seed defense priming assays were conducted in non-soil (semi-sterile substrate) and soil-based systems, focusing on tomato-Botrytis cinerea and wheat-Magnaporthe oryzae Triticum (MoT) pathosystems. In the non-soil system, seed defense priming with levan, the strains (especially Bacillus velezensis GA1), or their combination significantly promoted tomato growth and protection against B. cinerea. While no growth stimulatory effects were observed for wheat, disease protective effects were also observed in the wheat-MoT pathosystem. When grown in soil and subjected to leaf defense priming, tomato plants co-applied with levan and the bacterial strains showed increased resistance to B. cinerea compared with plants treated with levan or single strains, and these effects were synergistic in some cases. For seed defense priming in soil, more synergistic effects on disease tolerance were observed in a non-fertilized soil as compared to a fertilized soil, suggesting that potential prebiotic effects of levan are more prominent in poor soils. The potential of using combinations of Bacilliaceae and levan in sustainable agriculture is discussed.

Insights in inulin binding and inulin oligosaccharide formation by novel multi domain endo-inulinases from Botrytis cinerea.

World-wide, pathogenic fungi such as Botrytis cinerea cause tremendous yield losses in terms of food production and post-harvest food decay. Many fungi produce inulin-type oligosaccharides (IOSs) from inulin through endo-inulinases which typically show a two domain structure. B.cinerea lacks a two domain endo-inulinase but contains a three domain structure instead. Genome mining revealed three and four domain (d4) enzymes in the fungal kingdom. Here, three and two domain enzymes were compared in their capacity to produce IOSs from inulin. Hill kinetics were observed in three domain enzymes as compared to Michaelis-Menten kinetics in two domain enzymes, suggesting that the N-terminal extension functions as a carbohydrate binding module. Analysis of the IOS product profiles generated from purified GF6, GF12, GF16 and GF18 inulins and extensive sugar docking approaches led to enhanced insights in the active site functioning, revealing subtle differences between the endo-inulinases from Aspergillus niger and B. cinerea. Improved insights in structure-function relationships in fungal endo-inulinases offer opportunities to develop superior enzymes for the production of specific IOS formulations to improve plant and animal health (priming agents, prebiotics).

The Complex GH32 Enzyme Orchestra from Priestia megaterium Holds the Key to Better Discriminate Sucrose-6-phosphate Hydrolases from Other ß-Fructofuranosidases in Bacteria.

Inulin is widely used as a prebiotic and emerging as a priming compound to counteract plant diseases. We isolated inulin-degrading strains from the lettuce phyllosphere, identified as Bacillus subtilis and Priestia megaterium, species hosting well-known biocontrol organisms. To better understand their varying inulin degradation strategies, three intracellular ß-fructofuranosidases from P. megaterium NBRC15308 were characterized after expression in Escherichia coli: a predicted sucrose-6-phosphate (Suc6P) hydrolase (SacAP1, supported by molecular docking), an exofructanase (SacAP2), and an invertase (SacAP3). Based on protein multiple sequence and structure alignments of bacterial glycoside hydrolase family 32 enzymes, we identified conserved residues predicted to be involved in binding phosphorylated (Suc6P hydrolases) or nonphosphorylated substrates (invertases and fructanases). Suc6P hydrolases feature positively charged residues near the structural catalytic pocket (histidine, arginine, or lysine), whereas other ß-fructofuranosidases contain tryptophans. This correlates with our phylogenetic tree, grouping all predicted Suc6P hydrolases in a clan associated with genomic regions coding for transporters involved in substrate phosphorylation. These results will help to discriminate between Suc6P hydrolases and other ß-fructofuranosidases in future studies and to better understand the interaction of B. subtilis and P. megaterium endophytes with sucrose and/or fructans, sugars naturally present in plants or exogenously applied in the context of defense priming.

Ethylene inhibits photosynthesis via temporally distinct responses in tomato plants.

Ethylene is a volatile plant hormone that regulates many developmental processes and responses toward (a)biotic stress. Studies have shown that high levels of ethylene repress vegetative growth in many important crops, including tomato (Solanum lycopersicum), possibly by inhibiting photosynthesis. We investigated the temporal effects of ethylene on young tomato plants using an automated ethylene gassing system to monitor the physiological, biochemical, and molecular responses through time course RNA-seq of a photosynthetically active source leaf. We found that ethylene evokes a dose-dependent inhibition of photosynthesis, which can be characterized by 3 temporally distinct phases. The earliest ethylene responses that marked the first phase and occurred a few hours after the start of the treatment were leaf epinasty and a decline in stomatal conductance, which led to lower light perception and CO2 uptake, respectively, resulting in a rapid decline of soluble sugar levels (glucose, fructose). The second phase of the ethylene effect was marked by low carbohydrate availability, which modulated plant energy metabolism to adapt by using alternative substrates (lipids and proteins) to fuel the TCA cycle. Long-term continuous exposure to ethylene led to the third phase, characterized by starch and chlorophyll breakdown, which further inhibited photosynthesis, leading to premature leaf senescence. To reveal early (3 h) ethylene-dependent regulators of photosynthesis, we performed a ChIP-seq experiment using anti-ETHYLENE INSENSITIVE 3-like 1 (EIL1) antibodies and found several candidate transcriptional regulators. Collectively, our study revealed a temporal sequence of events that led to the inhibition of photosynthesis by ethylene and identified potential transcriptional regulators responsible for this regulation.

Far-Red Light Mediated Carbohydrate Concentration Changes in Leaves of Sweet Basil, a Stachyose Translocating Plant.

Photosynthetic active radiation (PAR) refers to photons between 400 and 700 nm. These photons drive photosynthesis, providing carbohydrates for plant metabolism and development. Far-red radiation (FR, 701-750 nm) is excluded in this definition because no FR is absorbed by the plant photosynthetic pigments. However, including FR in the light spectrum provides substantial benefits for biomass production and resource-use efficiency. We investigated the effects of continuous FR addition and end-of-day additional FR to a broad white light spectrum (BW) on carbohydrate concentrations in the top and bottom leaves of sweet basil (Ocimum basilicum L.), a species that produces the raffinose family oligosaccharides raffinose and stachyose and preferentially uses the latter as transport sugar. Glucose, fructose, sucrose, raffinose, and starch concentrations increased significantly in top and bottom leaves with the addition of FR light. The increased carbohydrate pools under FR light treatments are associated with more efficient stachyose production and potentially improved phloem loading through increased sucrose homeostasis in intermediary cells. The combination of a high biomass yield, increased resource-use efficiency, and increased carbohydrate concentration in leaves in response to the addition of FR light offers opportunities for commercial plant production in controlled growth environments.

Constitutive Defense Mechanisms Have a Major Role in the Resistance of Woodland Strawberry Leaves Against Botrytis cinerea.

The necrotrophic fungus Botrytis cinerea is a major threat to strawberry cultivation worldwide. By screening different Fragaria vesca genotypes for susceptibility to B. cinerea, we identified two genotypes with different resistance levels, a susceptible genotype F. vesca ssp. vesca Tenno 3 (T3) and a moderately resistant genotype F. vesca ssp. vesca Kreuzkogel 1 (K1). These two genotypes were used to identify the molecular basis for the increased resistance of K1 compared to T3. Fungal DNA quantification and microscopic observation of fungal growth in woodland strawberry leaves confirmed that the growth of B. cinerea was restricted during early stages of infection in K1 compared to T3. Gene expression analysis in both genotypes upon B. cinerea inoculation suggested that the restricted growth of B. cinerea was rather due to the constitutive resistance mechanisms of K1 instead of the induction of defense responses. Furthermore, we observed that the amount of total phenolics, total flavonoids, glucose, galactose, citric acid and ascorbic acid correlated positively with higher resistance, while H2O2 and sucrose correlated negatively. Therefore, we propose that K1 leaves are more resistant against B. cinerea compared to T3 leaves, prior to B. cinerea inoculation, due to a lower amount of innate H2O2, which is attributed to a higher level of antioxidants and antioxidant enzymes in K1. To conclude, this study provides important insights into the resistance mechanisms against B. cinerea, which highly depend on the innate antioxidative profile and specialized metabolites of woodland strawberry leaves.

Characterization of inulolytic enzymes from the Jerusalem artichoke-derived Glutamicibacter mishrai NJAU-1.

The rhizosphere context of inulin-accumulating plants, such as Jerusalem artichoke (Helianthus tuberosus), is an ideal starting basis for the discovery of inulolytic enzymes with potential for bio fructose production. We isolated a Glutamicibacter mishrai NJAU-1 strain from this context, showing exo-inulinase activity, releasing fructose from fructans. The growth conditions (pH 9.0; 15 °C) were adjusted, and the production of inulinase by Glutamicibacter mishrai NJAU-1 increased by 90% (0.32 U/mL). Intriguingly, both levan and inulin, but not fructose and sucrose, induced the production of exo-inulinase activity. Two exo-inulinase genes (inu1 and inu2) were cloned and heterologously expressed in Pichia pastoris. While INU2 preferentially hydrolyzed longer inulins, the smallest fructan 1-kestose appeared as the preferred substrate for INU1, also efficiently degrading nystose and sucrose. Active site docking studies with GFn- and Fn-type small inulins (G is glucose, F is fructose, and n is the number of ß (2-1) bound fructose moieties) revealed subtle substrate differences between INU1 and INU2. A possible explanation about substrate specificity and INU's protein structure is then suggested. KEY POINTS: • A Glutamicibacter mishrai strain harbored exo-inulinase activity. • Fructans induced the inulolytic activity in G. mishrai while the inulolytic activity was optimized at pH 9.0 and 15 °C. • Two exo-inulinases with differential substrate specificity were characterized.

Production of a high molecular weight levan by Bacillus paralicheniformis, an industrially and agriculturally important isolate from the buffalo grass rhizosphere.

A new exopolysaccharide (EPS) producing Gram-positive bacterium was isolated from the rhizosphere of Bouteloua dactyloides (buffalo grass) and its EPS product was structurally characterized. The isolate, designated as LB1-1A, was identified as Bacillus paralicheniformis based on 16S rRNA gene sequence and phylogenetic tree analysis. The EPS produced by LB1-1A was identified as a levan, having ß(2 → 6) linked backbone with ß(2 → 1) linkages at the branch points (4.66%). The isolate LB1-1A yielded large amount (~ 42 g/l) of levan having high weight average molecular weight (Mw) of 5.517 × 107 Da. The relatively low degree of branching and high molecular weight of this levan makes B. paralicheniformis LB1-1A a promising candidate for industrial applications.

Fructan oligosaccharide priming alters apoplastic sugar dynamics and improves resistance against Botrytis cinerea in chicory.

Carbohydrates such as fructans can be involved in priming or defence stimulation, and hence potentially provide new strategies for crop protection against biotic stress. Chicory (Cichorium intybus) is a model plant for fructan research and is a crop with many known health benefits. Using the chicory-Botrytis cinerea pathosystem, we tested the effectiveness of fructan-induced immunity, focussing on different plant and microbial fructans. Sugar dynamics were followed after priming and subsequent pathogen infection. Our results indicated that many higher plants might detect extracellular levan oligosaccharides (LOS) of microbial origin, while chicory also detects extracellular small inulin-type fructooligosaccharides (FOS) of endogenous origin, thus differing from the findings of previous fructan priming studies. No clear positive effects were observed for inulin or mixed-type fructans. An elicitor-specific burst of reactive oxygen species was observed for sulfated LOS, while FOS and LOS both behaved as genuine priming agents. In addition, a direct antifungal effect was observed for sulfated LOS. Intriguingly, LOS priming led to a temporary increase in apoplastic sugar concentrations, mainly glucose, which could trigger downstream responses. Total sugar and starch contents in total extracts of LOS-primed leaves were higher after leaf detachment, indicating they could maintain their metabolic activity. Our results indicate the importance of balancing intra- and extracellular sugar levels (osmotic balance) in the context of 'sweet immunity' pathways.

Sweet Immunity Aspects during Levan Oligosaccharide-Mediated Priming in Rocket against Botrytis cinerea.

New strategies are required for crop protection against biotic stress. Naturally derived molecules, including carbohydrates such as fructans, can be used in priming or defense stimulation. Rocket (Eruca sativa) is an important leafy vegetable and a good source of antioxidants. Here, we tested the efficacy of fructan-induced immunity in the Botrytis cinerea pathosystem. Different fructan types of plant and microbial origin were considered and changes in sugar dynamics were analyzed. Immune resistance increased significantly after priming with natural and sulfated levan oligosaccharides (LOS). No clear positive effects were observed for fructo-oligosaccharides (FOS), inulin or branched-type fructans. Only sulfated LOS induced a direct ROS burst, typical for elicitors, while LOS behaved as a genuine priming compound. Total leaf sugar levels increased significantly both after LOS priming and subsequent infection. Intriguingly, apoplastic sugar levels temporarily increased after LOS priming but not after infection. We followed LOS and small soluble sugar dynamics in the apoplast as a function of time and found a temporal peak in small soluble sugar levels. Although similar dynamics were also found with inulin-type FOS, increased Glc and FOS levels may benefit B. cinerea. During LOS priming, LOS- and/or Glc-dependent signaling may induce downstream sweet immunity responses.

A novel chicory fructanase can degrade common microbial fructan product profiles and displays positive cooperativity.

Fructan metabolism in bacteria and plants relies on fructosyltransferases and fructanases. Plant fructanases (fructan exohydrolase, FEH) only hydrolyse terminal fructose residues. Levan (ß-2,6 linkages) is the most abundant fructan type in bacteria. Dicot fructan accumulators, such as chicory (Cichorium intybus), accumulate inulin (ß-2,1 linkages), harbouring several 1-FEH isoforms for their degradation. Here, a novel chicory fructanase with high affinity for levan was characterized, providing evidence that such enzymes widely occur in higher plants. It is adapted to common microbial fructan profiles, but has low affinity towards chicory inulin, in line with a function in trimming of microbial fructans in the extracellular environment. Docking experiments indicate the importance of an N-glycosylation site close to the active site for substrate specificity. Optimal pH and temperature for levan hydrolysis are 5.0 and 43.7 °C, respectively. Docking experiments suggested multiple substrate binding sites and levan-mediated enzyme dimerization, explaining the observed positive cooperativity. Alignments show a single amino acid shift in the position of a conserved DXX(R/K) couple, typical for sucrose binding in cell wall invertases. A possible involvement of plant fructanases in levan trimming is discussed, in line with the emerging 'fructan detour' concepts, suggesting that levan oligosaccharides act as signalling entities during plant-microbial interactions.

Sweet Modifications Modulate Plant Development.

Plant development represents a continuous process in which the plant undergoes morphological, (epi)genetic and metabolic changes. Starting from pollination, seed maturation and germination, the plant continues to grow and develops specialized organs to survive, thrive and generate offspring. The development of plants and the interplay with its environment are highly linked to glycosylation of proteins and lipids as well as metabolism and signaling of sugars. Although the involvement of these protein modifications and sugars is well-studied, there is still a long road ahead to profoundly comprehend their nature, significance, importance for plant development and the interplay with stress responses. This review, approached from the plants' perspective, aims to focus on some key findings highlighting the importance of glycosylation and sugar signaling for plant development.

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.

Phosphorolytic degradation of leaf starch via plastidic α-glucan phosphorylase leads to optimized plant growth and water use efficiency over the diel phases of Crassulacean acid metabolism.

In plants with Crassulacean acid metabolism (CAM), it has been proposed that the requirement for nocturnal provision of phosphoenolpyruvate as a substrate for CO2 uptake has resulted in a re-routing of chloroplastic starch degradation from the amylolytic route to the phosphorolytic route. To test this hypothesis, we generated and characterized four independent RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with a >10-fold reduction in transcript abundance of plastidic α-glucan phosphorylase (PHS1). The rPHS1 lines showed diminished nocturnal starch degradation, reduced dark CO2 uptake, a reduction in diel water use efficiency (WUE), and an overall reduction in growth. A re-routing of starch degradation via the hydrolytic/amylolytic pathway was indicated by hyperaccumulation of maltose in all rPHS1 lines. Further examination indicated that whilst operation of the core circadian clock was not compromised, plasticity in modulating net dark CO2 uptake in response to changing photoperiods was curtailed. The data show that phosphorolytic starch degradation is critical for efficient operation of the CAM cycle and for optimizing WUE. This finding has clear relevance for ongoing efforts to engineer CAM into non-CAM species as a means of boosting crop WUE for a warmer, drier future.

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.

At the Crossroads of Survival and Death: The Reactive Oxygen Species-Ethylene-Sugar Triad and the Unfolded Protein Response.

Upon stress, a trade-off between plant growth and defense responses defines the capacity for survival. Stress can result in accumulation of misfolded proteins in the endoplasmic reticulum (ER) and other organelles. To cope with these proteotoxic effects, plants rely on the unfolded protein response (UPR). The involvement of reactive oxygen species (ROS), ethylene (ETH), and sugars, as well as their crosstalk, in general stress responses is well established, yet their role in UPR deserves further scrutiny. Here, a synopsis of current evidence for ROS-ETH-sugar crosstalk in UPR is discussed. We propose that this triad acts as a major signaling hub at the crossroads of survival and death, integrating information from ER, chloroplasts, and mitochondria, thereby facilitating a coordinated stress response.

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.