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

Context dependency of host-parasite interactions: Impact of Microcystis aeruginosa on parasitism in Daphnia magna

In the last decade, it has become clear that changing environmental conditions may affect host-parasite interactions. Nevertheless, it is often not clear in what way they affect these interactions. In freshwater ecosystems, changing circumstances, such as nutrient enrichment, can induce toxic cyanobacterial blooms. We here investigate whether the widespread and frequently studiednbsp;species Microcystis aeruginosa has an effect on an infectious disease in Daphnia magna, a filter-feeding zooplankton species living in freshwater. The parasite that we study causes White Bacterial Disease (WBD), a virulent infectious disease in Daphnia that induces peculiar phenotypic effects in the adipose tissue. In the first part of the thesis, we evaluated the impact of direct and indirect effects of different M. aeruginosa strains onD. magna exposed to WBD. Microcystins are one of the most common toxins produced by cyanobacteria. However, not all cyanobacterial negative effects are attributed to these toxins. Cyanobacteria can also produce other less harmful bio-active metabolites and they are low quality food for zooplankton due to the absence of polyunsaturated fatty acids and sterols. To evaluate these non-toxic impacts, a non-microcystin-producing M. aeruginosa strain was tested. Results demonstratednbsp;M. aeruginosa protected D. magna against parasitism. Increasing M. aeruginosanbsp;reduced the percentage of infected individuals and antagonistic effects between M. aeruginosa and thenbsp;were found on different life-history characteristics of D. magna. Plating experiments showed a directnbsp;effect of M. aeruginosa on bacterial growth, which may explain the direct, antagonistic effect. In the next chapter, we investigated the effect of a microcystin-producing M. aeruginosa strain and its microcystin-lacking mutant on the susceptibility of D. magna to WBD. As direct effects of these strains against bacteria were absent, focus wasnbsp;to indirect effects. We focused on differences in clearance rate. Clearance rates cannbsp;disease as most Daphnia parasites are taken up by grazing. We comparednbsp;populations, each originating from a different pond. The results demonstrated that the population with a higher clearance rate in the presence of the microcystin producing M. aeruginosa strain, was more susceptible to disease than the population with a lower clearance rate, as they were earlier infected and produced less offspring upon simultaneous exposure to the parasite and the microcystin producing M. aeruginosa strain. These results show that the presence of cyanobacteria can indirectlynbsp;an organism more susceptible to disease due to thenbsp;of trait changes in this organism. Then, attempts were made to characterize WBD in Daphnia. Literature indicates that WBD may be caused by a coccoid bacterium. Thus, the bacterial community of WBD infected and control D. magna was analyzed by Denaturing Gradient Gel Electrophoresis and compared. Thenbsp;suggested the involvement of the genera Flavobacterium and Emticicia, both members of the Bacteroidetes. Asnbsp;is characterized by changes in the adipose tissue, the degree of lipid oxidation in WBD infected and control D. magna wasnbsp;Results showed that WBD infected D. magna contained morenbsp;fatty acids than control D. magna. Moreover, Bacteroidetes were found in the adipose tissue of WBD infected D. magna. Nevertheless, so far we cannot exclude that Flavobacterium and/orEmticicia are only opportunists associated with WBD andnbsp;other agents induce the disease. Therefore, alternative analyses (e.g. 16S RNA gene pyrosequencing of infected and control individuals) are suggested. Innbsp;last chapter, the establishment of an additional host-parasite study system Microcystis and its cyanophages (viruses of cyanobacteria) is described. We aimed to develop a Microcystis-cyanophage model system to study environmental influences on host-parasite interactions. We succeeded to isolate Microcystis strains and cyanophage strains from 22 ponds in Belgiumnbsp;started to optimize the model system. The Microcystis strains werenbsp;and identified via the 16S-23S rDNAnbsp;transcribed spacer (ITS). These data showed geographical diversity, as there was a difference between West-Middle Belgian strains and East Belgian strains, but many Microcystis strains from the same pond/region, shared the same ITS sequence. We attempted to distinguishnbsp;strains further by a higher resolution DNA fingerprinting technique (HIP1), but as not all cultures were axenic, we could not relynbsp;this technique.nbsp;also succeeded tonbsp;cyanophagesnbsp;double layer plaques assay andnbsp;quantify them via epifluorescence microscopy, but we were not able to characterize them molecularly. In conclusionnbsp;can state that cyanobacteria influence disease in Daphnia either in a positive or a negative way depending on the cyanobacterial strainnbsp;and the fact if they cause direct or indirect effects on the D. magna-WBD interaction. Further, our results suggest that the genera Flavobacterium and Emticicia may be involved innbsp;disease of WBD, but further researchnbsp;needed to confirm if thesenbsp;are the causative agents. Finally, we can state that Microcystisnbsp;cyanophages, but we could not develop a successful technique to perform experiments to study environmental effects on Microcystis-cyanophage interactions or on multi-trophic interactions between cyanophages-Microcystis-Daphnia-parasites.nrpages: 199status: publishe

Presence of inulin-type fructo-oligosaccharides and shift from raffinose family oligosaccharide to fructan metabolism in leaves of boxtree (Buxus sempervirens)

Fructans 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, harboring Pachysandra terminalis, an accumulator of graminan- and levan-type fructans, also harbors 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 (RFOs: raffinose and stachyose) instead. The seasonal variation in concentrations of glucose, fructose, sucrose, FOS and RFOs 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 fructans and RFOs fulfill distinct roles in boxtree leaves. RFOs 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.status: publishe