Early nodulin gene expression and the action of nod factors in Vicia sativa

Bacteria of the genera Rhizobium, Bradyrhizobium or Azorhizobium secrete lipo-oligosaccharide signal molecules, which play a pivotal role in the induction of early steps of root nodule formation on legumes. In these nodules the bacteria are hosted and a proper environment is provided for the bacteria to fix atmospheric nitrogen into ammonia, making the plant for its growth independent of nitrogen compounds in the soil.The goal of the research described in this thesis is to provide insight in the mechanism by which the Nod factors secreted by Rhizobium bacteria initiate root nodule formation. Such kind of study requires a plant-bacteria system in which both the morphological and molecular changes induced by the Nod factor can be examined. Working on the Nod factors secreted by Rhizobium leguminosarum bv. viciae the choice of the host plant is limited to the genera Pisum, Lathyrus, Lens and Vicia. We decided that Viciasativa (vetch) would be the most useful plant for our studies, since the plant is small and various morphological changes, like root hair deformation and the formation of nodule primordia, that are induced by the Nod factors can easily be observed (Chapter 1).Nod factors can induce the expression of early nodulin genes. These genes are expressed during different developmental stages of root nodule formation and the expression of these genes can therefore be used as molecular markers of root nodule development and Nod factor induced processes. To study Nod factor induced plant responses in V.sativa on the molecular level, the homologues of the early nodulin genes of pea (Pisum sativum) were isolated from V. sativa and their expression pattern was studied by in situ hybridization during root nodule development (Chapter 2).To obtain information about the pathway from Nod factor to early nodulin gene expression, the mechanisms controlling ENOD12 expression were studied. With a promoter analysis in transgenic V.hirsuta root nodules we have identified that the 200 bp immediately upstream of the transcription start are sufficient to induce nodule specific and Nod factor induced expression (Chapter 3). For the isolation of transcription factors involved in controlling ENOD12 expression an expression library was screened and a preliminary characterization of cDNA clones encoding polypeptides that bind to the PsENOD12 promoter is described in Chapter 4.In Chapter 5 early nodulin gene expression during Nod factor induced morphological changes, like root hair deformation and nodule primordia induction, was studied. Furthermore we examined whether RNA and protein synthesis are required for root hair deformation and for the activation of the early nodulin genes. These studies provided new insights about the mode of action of Nod factors.In Chapter 6 is discussed to what extent V. sativa is a suitable host plant to study the mode of action of Nod factors and in which way the studies reported in this thesis have contributed to elucidate the mechanism by which Nod factors induce a diversity of plant responses

Marktkansen voor etnisch voedsel en etnische diensten

De bevolkingssamenstelling van de Nederlandse steden is afgelopen jaren sterk veranderd. De stedelijke vraag naar producten en diensten van het platteland is ook sterk veranderd. Uit de quick scan is gebleken dat de 'ethnic food & services' een belangrijk onderdeel vormen van de nieuwe vraag. Dit dient niet alleen ten behoeve van de nichemarkt van ethnic food & services maar kan tegelijkertijd als een strategisch middel ingezet worden om de algemene stadplattelandsrelaties aan te halen, uit te breiden en te verbetere

Stadsvarkens : schakel in een circulaire economie : ruimte in wet- en regelgeving voor stadsvarkens : een advies aan Stichting Stadsvarkens Ede

De Stichting Stadsvarkens te Ede wil weten aan welke regelgeving moet worden voldaan om vier varkens in een bosperceel te houden en te voeden met reststromen. Het doel is het sluiten van kringlopen binnen een circulaire economie. Dit rapport gaat in op de wet- en regelgeving rond diervoeding en op de afzet van het vlees in de setting van Stadsvarkens. Daarnaast wordt het voeren van reststromen in relatie tot diergezondheid belicht. Door studenten is binnen een ACT-traject gekeken naar relevante wetgeving en is een viertal scenario’s geschetst voor het houden van stadsvarkens die gevoerd worden met lokale reststromen. De scenario’s zijn door de onderzoekers tegen het licht gehouden en van kanttekeningen en een advies voorzien. Dit heeft geresulteerd in een ander advies, omdat verschillende knelpunten de eerdere scenario’s niet mogelijk maken

An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases

Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.)

Variation in the structures of plant fructans and their degree of polymerization (DP) can be explained as the result of diverse combinations of fructosyltransferases (FTs) with different properties. Although FT genes have been isolated in a range of plant species, sucrose:fructan 6-fructosyltransferase (6-SFT) cDNAs have only been functionally characterized in a few species such as wheat. A novel FT cDNA possessing 6-SFT activity has been identified and characterized from the temperate forage grass, timothy (Phleum pratense L.). The cDNA of an FT homolog, PpFT1, was isolated from cold-acclimated timothy. A recombinant PpFT1 protein expressed in Pichia pastoris showed 6-SFT/sucrose:sucrose 1-fructosyltransferase (1-SST) activity and produced linear β(2,6)-linked levans from sucrose with higher DPs than present in graminans formed in vitro by wheat 6-SFT (Wft1). PpFT1 and Wft1 showed remarkably different acceptor substrate specificities: PpFT1 had high affinity for 6-kestotriose to produce levans and low affinity for 1-kestotriose, whereas Wft1 preferentially used 1-kestotriose as an acceptor. The affinity of the PpFT1 recombinant enzyme for sucrose as a substrate was lower than that of the Wft1 recombinant enzyme. It is also confirmed that timothy seedlings had elevated levels of PpFT1 transcripts during the accumulation of fructans under high sucrose and cold conditions. Our results suggest that PpFT1 is a novel cDNA with unique enzymatic properties that differ from those of previously cloned plant 6-SFTs, and is involved in the synthesis of highly polymerized levans in timothy

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.)

<p>Abstract</p> <p>Background</p> <p>We have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.</p> <p>Results</p> <p>A cDNA, named <it>aleh1</it>, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The <it>aleh1 </it>encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (<it>pI</it>) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of <it>aleh1 </it>was produced in <it>Pichia pastoris</it>, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates.</p> <p>The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that <it>aleh1 </it>encoded 1-FEH.</p

Fructan and its relationship to abiotic stress tolerance in plants

Numerous studies have been published that attempted to correlate fructan concentrations with freezing and drought tolerance. Studies investigating the effect of fructan on liposomes indicated that a direct interaction between membranes and fructan was possible. This new area of research began to move fructan and its association with stress beyond mere correlation by confirming that fructan has the capacity to stabilize membranes during drying by inserting at least part of the polysaccharide into the lipid headgroup region of the membrane. This helps prevent leakage when water is removed from the system either during freezing or drought. When plants were transformed with the ability to synthesize fructan, a concomitant increase in drought and/or freezing tolerance was confirmed. These experiments indicate that besides an indirect effect of supplying tissues with hexose sugars, fructan has a direct protective effect that can be demonstrated by both model systems and genetic transformation

Are Small GTPases Signal Hubs in Sugar-Mediated Induction of Fructan Biosynthesis?

External sugar initiates biosynthesis of the reserve carbohydrate fructan, but the molecular processes mediating this response remain obscure. Previously it was shown that a phosphatase and a general kinase inhibitor hamper fructan accumulation. We use various phosphorylation inhibitors both in barley and in Arabidopsis and show that the expression of fructan biosynthetic genes is dependent on PP2A and different kinases such as Tyr-kinases and PI3-kinases. To further characterize the phosphorylation events involved, comprehensive analysis of kinase activities in the cell was performed using a PepChip, an array of >1000 kinase consensus substrate peptide substrates spotted on a chip. Comparison of kinase activities in sugar-stimulated and mock(sorbitol)-treated Arabidopsis demonstrates the altered phosphorylation of many consensus substrates and documents the differences in plant kinase activity upon sucrose feeding. The different phosphorylation profiles obtained are consistent with sugar-mediated alterations in Tyr phosphorylation, cell cycling, and phosphoinositide signaling, and indicate cytoskeletal rearrangements. The results lead us to infer a central role for small GTPases in sugar signaling