Maxime Versluys

Distinguishing between endo- and exo-type enzymes within the glycoside hydrolase (GH) classification presents significant challenges. Traditional methods, often based on endpoint activity measurements, do not capture the full range of products generated, leading to inconsistencies in classification. Not all exo-acting fructanases and glucanases produce monosaccharides (like fructose or glucose), while endo-acting enzymes do not solely produce higher-degree polymerization oligosaccharides. In practice, both enzyme types can yield a variety of products throughout the reaction, complicating classification efforts. To address these challenges, we propose a refined nomenclature system for GH enzymes, including fructanases and glucanases, based on good practices and initial product formation. This system classifies enzymes into four categories for each type: Fr, Fn, Fn,n+1 and F1 for fructanases, and Gr, Gn, Gn,n+1 and G1 for glucanases, based on their mode of action (endo- or exo-based) and initial product profiles. Our refined nomenclature system will advance enzyme structure–function research and support the production and application of fructan and glucan oligosaccharides as prebiotics, priming agents, and potential signaling molecules in microbe–microbe and plant–microbe interactions. Ultimately, this system could benefit agronomy and the food industry, contributing to health improvements.

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.

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.

Fructan metabolism in bacteria and plants relies on fructosyltransferases and fructanases. Plant fructanases (fructan exohydrolase, FEH) only hydrolyse terminal fructose residues. Levan (β-2,6 linkages) is the most abundant fructan type in bacteria. Dicot fructan accumulators, such as chicory (Cichorium intybus), accumulate inulin (β-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.

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6^T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.

Fructans are multifunctional fructose‐based water soluble carbohydrates found in all biological kingdoms but not in animals. Most research has focused on plant and microbial fructans and has received a growing interest because of their practical applications. Nevertheless, the origin of fructan production, the so‐called “fructan syndrome,” is still unknown. Why fructans only occur in a limited number of plant and microbial species remains unclear. In this review, we provide an overview of plant and microbial fructan research with a focus on fructans as an adaptation to the environment and their role in (a)biotic stress tolerance. The taxonomical and biogeographical distribution of fructans in both kingdoms is discussed and linked (where possible) to environmental factors. Overall, the fructan syndrome may be related to water scarcity and differences in physicochemical properties, for instance, water retaining characteristics, at least partially explain why different fructan types with different branching levels are found in different species. Although a close correlation between environmental stresses and fructan production is quite clear in plants, this link seems to be missing in microbes. We hypothesize that this can be at least partially explained by differential evolutionary timeframes for plants and microbes, combined with potential redundancy effects.

This perspective paper proposes that endogenous apoplastic fructans in fructan accumulating plants, released after stress-mediated cellular leakage, or increased by exogenous application, can act as damage-associated molecular patterns (DAMPs), priming plant innate immunity through ancient receptors and defense pathways that most probably evolved to react on microbial fructans acting as microbe-associated molecular patterns (MAMPs). The proposed model is placed in an evolutionary perspective. How this type of DAMP signaling may contribute to cross-tolerance and multistress resistance effects in plants is discussed. Besides apoplastic ATP, NAD and fructans, apoplastic polyamines, secondary metabolites and melatonin may be considered potential players in DAMP-mediated stress signaling. It is proposed that mixtures of DAMP priming formulations hold great promise as natural and sustainable alternatives for toxic agrochemicals.