Presence of Inulin-Type Fructo-Oligosaccharides and Shift from Raffinose Family Oligosaccharide to Fructan Metabolism in Leaves of Boxtree (Buxus sempervirens)

from raffinose family oligosaccharide to fructan metabolism in leaves of boxtree (Buxus sempervirens) Wim Van den Ende1,* Marlies Coopman1, Rudy Vergauwen1, André Van Laere11 KU Leuven, Laboratory of Molecular Plant Biology, Institute of Botany and Microbiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium* Correspondence: Wim Van den Ende, Laboratory of Molecular Plant Biology,Institute of Botany and Microbiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium tel +32 16321952; fax +32 16321967;[email protected]: inulin, oligosaccharides, stress, RFO, fructanAbstractFructans are known to occur in 15% of flowering plants and their accumulation is often associated with stress responses. Typically, particular fructan types occur within particular plant families. The family of the Buxaceae, harbouring Pachysandra terminalis, an accumulator of graminan- and levan-type fructans, also harbours boxtree (Buxus sempervirens), a cold and drought tolerant species. Surprisingly, boxtree leaves do not accumulate the expected graminan- and levan-type fructans but small inulin fructo-oligosaccharides (FOS: 1-kestotriose and nystose) and raffinose family oligosaccharides (RFO: raffinose and stachyose) instead. The seasonal variation in concentrations of glucose, fructose, sucrose, FOS and RFO were followed. Raffinose and stachyose peaked during the winter months, while FOS peaked at a very narrow time-interval in spring, immediately preceded by a prominent sucrose accumulation. Sucrose may function as a reserve carbohydrate in winter and early spring leaves. The switch from RFO to fructan metabolism in spring strongly suggests that fructan and RFO fulfil distinct roles in boxtree leaves. RFO may play a key role in the cold acclimation of winter leaves while temporal fructan biosynthesis in spring might increase sink strength to sustain the formation of new shoots

Sweet immunity : inulin boosts resistance of lettuce (Lactuca sativa) against grey mold (Botrytis cinerea) in an ethylene-dependent manner

The concept of Sweet Immunity postulates that sugar metabolism and signaling influence plant immune networks. In this study, we tested the potential of commercially available inulin-type fructans to limit disease symptoms caused by Botrytis cinerea in lettuce. Spraying mature lettuce leaves, with inulin-type fructans derived from burdock or chicory was as effective in reducing grey mold disease symptoms caused by Botrytis cinerea as spraying with oligogalacturonides (OGs). OGs are well-known defense elicitors in several plant species. Spraying with inulin and OGs induced accumulation of hydrogen peroxide and levels further increased upon pathogen infection. Inulin and OGs were no longer able to limit Botrytis infection when plants were treated with the ethylene signaling inhibitor 1-methylcyclopropene (1-MCP), indicating that a functional ethylene signaling pathway is needed for the enhanced defense response. Soluble sugars accumulated in leaves primed with OGs, while 1-MCP treatment had an overall negative effect on the sucrose pool. Accumulation of -aminobutyric acid (GABA), a stress-associated non-proteinogenic amino acid and possible signaling compound, was observed in inulin-treated samples after infection and negatively affected by the 1-MCP treatment. We have demonstrated for the first time that commercially available inulin-type fructans and OGs can improve the defensive capacity of lettuce, an economically important species. We discuss our results in the context of a possible recognition of fructans as Damage or Microbe Associated Molecular Patterns

Purification, cloning and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.)

Fructans, β2-1 and/or β2-6 linked polymers of fructose, are produced by fructosyltransferases (FTs) from sucrose. They are important storage carbohydrates in many plants. Fructan reserves, widely distributed in plants, are believed to be mobilized via fructan exohydrolases (FEHs). The purification, cloning, and functional characterization of a 6-FEH from wheat (Triticum aestivum L.) are reported here. It is the first FEH shown to hydrolyse exclusively β2-6 bonds found in a fructan-producing plant. The enzyme was purified to homogeneity using ammonium sulphate precipitation, ConA affinity-, ion exchange-, and size exclusion chromatography and yielded a single band of 70 kDa following SDS-PAGE. Sequence information obtained by mass spectrometry of in-gel trypsin digests demonstrated the presence of a single protein. Moreover, these unique peptide sequences, together with some ESTs coding for them, could be used in a RT-PCR based strategy to clone a 1.7 kb cDNA. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed—as did the native enzyme from wheat—a very high activity of the produced protein against bacterial levan, 6-kestose, and phlein whilst sucrose and inulin were not used as substrates. Therefore the enzyme is a genuine 6-FEH. In contrast to most FEHs from fructan-accumulating plants, this FEH is not inhibited by sucrose. The relative abundance of 6-FEH transcripts in various tissues of wheat was investigated using quantitative RT-PC

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

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

Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne)

The main storage compounds in Lolium perenne are fructans with prevailing β(2-6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgar

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

Linking Autophagy to Abiotic and Biotic Stress Responses

Autophagy is a process in which cellular components are delivered to lytic vacuoles to be recycled and has been demonstrated to promote abiotic/biotic stress tolerance. Here, we review how the responses triggered by stress conditions can affect autophagy and its signaling pathways. Besides the role of SNF-related kinase 1 (SnRK1) and TOR kinases in the regulation of autophagy, abscisic acid (ABA) and its signaling kinase SnRK2 have emerged as key players in the induction of autophagy under stress conditions. Furthermore, an interplay between reactive oxygen species (ROS) and autophagy is observed, ROS being able to induce autophagy and autophagy able to reduce ROS production. We also highlight the importance of osmotic adjustment for the successful performance of autophagy and discuss the potential role of GABA in plant survival and ethylene (ET)-induced autophagy

Physiological basis of chilling tolerance and early-season growth in miscanthus

Background and Aims: The high productivity of Miscanthus x giganteus has been at least partly ascribed to its high chilling tolerance compared with related C-4 crops, allowing for a longer productive growing season in temperate climates. However, the chilling tolerance of M. x giganteus has been predominantly studied under controlled environmental conditions. The understanding of the underlying mechanisms contributing to chilling tolerance in the field and their variation in different miscanthus genotypes is largely unexplored. Methods: Five miscanthus genotypes with different sensitivities to chilling were grown in the field and scored for a comprehensive set of physiological traits throughout the spring season. Chlorophyll fluorescence was measured as an indication of photosynthesis, and leaf samples were analysed for biochemical traits related to photosynthetic activity (chlorophyll content and pyruvate, Pi dikinase activity), redox homeostasis (malondialdehyde, glutathione and ascorbate contents, and catalase activity) and water-soluble carbohydrate content. Key Results: Chilling-tolerant genotypes were characterized by higher levels of malondialdehyde, raffinose and sucrose, and higher catalase activity, while the chilling-sensitive genotypes were characterized by higher concentrations of glucose and fructose, and higher pyruvate, Pi dikinase activity later in the growing season. On the early sampling dates, the biochemical responses of M. x giganteus were similar to those of the chilling-tolerant genotypes, but later in the season they became more similar to those of the chilling-sensitive genotypes. Conclusions: The overall physiological response of chilling-tolerant genotypes was distinguishable from that of chilling-sensitive genotypes, while M. x giganteus was intermediate between the two. There appears to be a trade-off between high and efficient photosynthesis and chilling stress tolerance. Miscanthus x giganteus is able to overcome this trade-off and, while it is more similar to the chilling-sensitive genotypes in early spring, its photosynthetic capacity is similar to that of the chilling-tolerant genotypes later on