La caracterització funcional de dos gens en la peridermis, la ω hidroxilasa d'àcids grassos CYP86A33 -candidata per la funcionalització del carboni ω-terminal dels monòmers alifàtics de la suberina- i la ketoacyl-CoA sintasa StKCS6 -candidata per elongar àcids grassos o derivats llargs de suberina i ceres- es realitza per silenciament per RNA d'interferència en patata. La deficiència de CYP86A33 comporta una gran reducció dels monòmers principals de la suberina, l'àcid gras ω-hidroxilat i l'α,ω-diàcid C18:1, juntament amb una reducció total de la quantitat de suberina del 60%. Aquesta deficiència altera l'estructura lamel·lar típica de la suberina, així com també la funció barrera de la peridermis. La deficiència en StKCS6 comporta que els monòmers de la suberina de 28 carbonis o més llargs es redueixin i que els de 26 carbonis o més curts s'incrementin. Aquesta deficiència suggereix que la llargada dels compostos alifàtics pot contribuir a les propietats impermeabilitzants de la peridermis. / The functional characterization of two genes in the periderm, the ω-hydroxylase CYP86A33 -candidate for the functionalization of the ω-terminal carbon of suberin aliphatic compounds- and the putative ketoacyl-CoA synthase StKCS6 -candidate for the elongation of VLCFA and derivatives of suberin and waxes of periderm- is performed by RNA interference-mediated silencing in potato The CYP86A33 deficiency leads to a great reduction of the main suberin monomers, the C18:1 ω-hydroxyacid and α,ω-diacid, together with an overall decrease of the suberin total amount by 60%. The deficiency in these ω-oxidized fatty acids alters the typical suberin lamellar structure as well as the periderm water barrier function.StKCS6 deficiency leads to a decrease of suberin and wax compounds with chain-length C28 and higher and an increase of those with chain-length C26 and lower. This deficiency suggests that the aliphatics chain-length can contribute to the sealing properties of periderm.

Universiẗat, Diss., 2002--Würzburg.

The root epidermis, being the outermost cell layer of the organ, is in contact with the soil environment. The position of the epidermis determines its important roles, such as taking up water and ions from the surrounding soil, and defending against harmful microorganisms. What is the chemical composition of the walls in this layer? The chemical nature of the soybean epidermal wall modifying substance was investigated in this study with the use of histochemical tests coupled with electron microscopy, and chemical depolymerizations in combination with chromatography. Soybean (<em>Glycine max</em>) was used as a test species in the present studay. Results of histochemical and electron microscopical studies indicated that the epidermal walls are modified with suberin. The suberized epidermal walls were permeable to apoplastic tracers, differing from those of cells with suberized Casparian bands, possibly due to the spatial distribution or chemical components of the suberin. Suberin may occur in a diffuse form linked with other wall components in the epidermis. What is the chemical nature of this modification, and does it play a role in pathogen resistance? The root epidermal wall compositions of two soybean cultivars were compared; one (cv. Conrad) is resistant to <em>Phytophthora sojae</em> and the other (cv. OX 760-6) is susceptible to this root-rot oomycete. Their epidermal walls were isolated enzymatically and subjected to two different degradation methods, i. e. BF₃-MeOH transesterification and nitrobenzene oxidation. The compositions of depolymerisates of the cell walls determined by GC-MS indicated four dominant suberin monomers varying in chain length from C16 to C24. In all epidermal cell walls, &omega;-hydroxycarboxylic acids were more abundant than diacids, carboxylic acids and alcohols. Two of the monomers detected (hydroxycarboxylic acid and a,&omega;-dicarboxylic acid) are known to be characteristic suberin markers. The quantitative chemical compositions significantly differed in the epidermal cell walls of the two soybean varieties. Walls of the resistant cultivar (Conrad) had a greater quantity of both the aliphatic and aromatic components of the polymer than the susceptible cultivar (OX760-6), providing evidence to support the hypothesis that preformed suberin plays a role in plant defense.

The root epidermis, being the outermost cell layer of the organ, is in contact with the soil environment. The position of the epidermis determines its important roles, such as taking up water and ions from the surrounding soil, and defending against harmful microorganisms. What is the chemical composition of the walls in this layer? The chemical nature of the soybean epidermal wall modifying substance was investigated in this study with the use of histochemical tests coupled with electron microscopy, and chemical depolymerizations in combination with chromatography. Soybean (<em>Glycine max</em>) was used as a test species in the present studay. Results of histochemical and electron microscopical studies indicated that the epidermal walls are modified with suberin. The suberized epidermal walls were permeable to apoplastic tracers, differing from those of cells with suberized Casparian bands, possibly due to the spatial distribution or chemical components of the suberin. Suberin may occur in a diffuse form linked with other wall components in the epidermis. What is the chemical nature of this modification, and does it play a role in pathogen resistance? The root epidermal wall compositions of two soybean cultivars were compared; one (cv. Conrad) is resistant to <em>Phytophthora sojae</em> and the other (cv. OX 760-6) is susceptible to this root-rot oomycete. Their epidermal walls were isolated enzymatically and subjected to two different degradation methods, i. e. BF₃-MeOH transesterification and nitrobenzene oxidation. The compositions of depolymerisates of the cell walls determined by GC-MS indicated four dominant suberin monomers varying in chain length from C16 to C24. In all epidermal cell walls, &omega;-hydroxycarboxylic acids were more abundant than diacids, carboxylic acids and alcohols. Two of the monomers detected (hydroxycarboxylic acid and a,&omega;-dicarboxylic acid) are known to be characteristic suberin markers. The quantitative chemical compositions significantly differed in the epidermal cell walls of the two soybean varieties. Walls of the resistant cultivar (Conrad) had a greater quantity of both the aliphatic and aromatic components of the polymer than the susceptible cultivar (OX760-6), providing evidence to support the hypothesis that preformed suberin plays a role in plant defense.

In Tumoren an Arabidopsis thaliana, induziert über Agrobacterium tumefaciens (Stamm C58), ist von den 49 bekannten Lipidtransferproteinen (LTPs) nur die Expression von LTP2 stark erhöht (Deeken et al., 2006). Mutanten ohne LTP2-Transkripte (ltp2KO) entwickeln deutlich kleinere Tumore als der Wildtyp. Durch die permanenten Zellstreckungs- und Dehnungsprozesse besitzen Tumore keine intakte Epidermis (Efetova et al., 2007). Dies wiederum führt zum Verlust einer vollständigen Cuticula-Schicht, welche von der Epidermis produziert wird und dieser als Barriere zur Umwelt aufgelagert ist. Um den transpirationsbedingten Wasserverlust zu minimieren, werden in Tumoren langkettige Aliphaten in die äußeren Zellschichten eingelagert (Efetova et al., 2006). Ein ähnliches Szenario findet um Verwundungsareale statt (Kolattukudy et al., 2001). Die Gen-Expression von LTP2 wird nicht durch tumorinduzierende Agrobakterien ausgelöst. Faktoren wie Verwundung, sowie die Applikation des Trockenstress-Phytohormons Abscisinsäure (ABA) begünstigen die LTP2-Gen-Expression positiv. Außerdem ist der LTP2-Promotor in Gewebe aktiv, in welchem sekundäre Zellwandmodifikationen auftreten, sowie insbesondere in Abscissionsschichten von welkenden Organen. Ungerichtete Lipidanalysen der ltp2KO-Mutante im Vergleich zum Wildtyp zeigten nur signifikante Veränderungen in der Menge definierter Sphingolipide – obwohl bislang eine Beteiligung von LTP2 am Transfer von Phospholipiden postuliert wurde. Allerdings kann das LTP2-Protein, wie Protein-Lipid-Overlay-Analysen demonstrierten, weder komplexen Sphingolipide noch Sphingobasen binden. Neben Sphingobasen sind auch langkettige Fettsäuren Bestandteile von Sphingolipiden und diese sind wiederum Bindepartner von LTP2. Um eine eventuelle Beteiligung von LTP2 an der Bildung von Suberin von Tumoren zu zeigen, wurde dieses analysiert. Die GC-MS-Analysen des Tumor-Suberins haben jedoch veranschaulicht, dass durch das Fehlen von LTP2-Transkripten das Lipidmuster nicht beeinträchtigt wird. Eine Überexpression von LTP2 im gesamten Kormophyten war trotz drei unabhängiger experimenteller Ansätze nicht möglich. Daher wurde das Protein ektopisch in epidermalen Zellen exprimiert (CER5Prom::LTP2). Die Transgenen CER5Prom::LTP2 wiesen einige morphologische Besonderheiten auf, wie verminderte Oberflächenhydrophobizität, aberrante Blüten- und Blattmorphologien etc., die typisch für Wachsmutanten sind. GC-MS-Analysen der cuticulären Wachse dieser transgenen Pflanzen zeigten, einen erhöhten Gehalt an C24- und C26-Fettsäuren, wohingegen die korrespondierenden Aliphaten wie Aldehyde und Alkane dezimiert waren. Unterstützend zeigten Lokalisationsanalysen, dass das LTP2-Protein an/in der Plasmamembran assoziiert ist. Somit kann die These aufgestellt werden, dass LTP2 langkettigen, unverzweigten Aliphaten (Fettsäuren) an der Grenzfläche Plasmamembran/Zellwand transferiert, die zur Versieglung und Festigung von Zellwänden benötigt werden. / Out of 49 known lipid transfer protein (LTP) only the expression of LTP2 is highly increased in tumors induced on Arabidopsis thaliana via Agrobacterium tumefaciens (strain C58; Deeken et al., 2006). Mutants with no LTP2 transcripts (ltp2KO) develop significantly smaller tumors than the wild-type. Due to the permanent cell stretch and elongation processes tumors do not possess an intact epidermal layer (Efetova et al., 2007). This leads to the loss of a complete cuticle layer, which is produced by the epidermis and builds up a barrier to the environment. To minimize the transpirational water loss, long-chain aliphatic compounds are incorperated into the outer cell layers of tumors (Deeken et al., 2006). The gene expression of LTP2 is not triggered by tumor-inducing agrobacteria. Instead, factors such as wounding and the application of the phytohormone abscisic acid (ABA) induce the LTP2 gene expression. In addition, the LTP2 promoter is highly active in tissue, in which secondary cell wall modifications occur, and in the abscission zone of wilting organs. Untargeted lipid analyzes of ltp2KO mutant in comparison to the wild type showed significant changes in the amount of defined sphingolipids only - although the involvement of LTP2 has been postulated for the transfer of phospholipids. However, the LTP2 protein, as protein-lipid overlay analysis demonstrated, binds neither complex sphingolipids nor sphingobases. Instead LCFAs, which are part of sphingolipids are binding partners of LTP2. In order to show a possible involvement of LTP2 in the formation of tumor-suberin GC-MS analyzes were performed. These demonstrated that the composition of the lipid-pool is not altered in ltp2KO plants. Overexpression of LTP2 was not possible in spite of three independent experimental approaches. The protein was instead expressed ectopically in epidermal cells (CER5Prom::LTP2). The transgenes CER5Prom::LTP2 showed some morphological abnormities, such as reduced surface hydrophobicity, aberrant flowers and leaf morphologies, which are typical for wax mutants. GC-MS analyzes of the cuticular wax of those transgenic lines revealed an increased amount of C24- and C26- fatty acids. Furthermore LTP2 was localized at the plasma membrane. Thus, this thesis proposes a role of LTP2 in the transfer of long chain, unbranched aliphatics (fatty acids), which are needed to seal up and strengthen cell walls at the interface plasma membrane and cell wall.

No description available.

Naturen är en källa till en mängd komplexa molekyler som har potential att användas inom industrin. En del av dessa molekyler kan utvinnas från suberin som bland annat finns i träd och i särskilt hög grad i björknäver. Genom att lösa upp näver i natriumhydroxid kan suberin sönderdelas i mindre beståndsdelar vilka sedan kan extraheras.  En av dessa beståndsdelar är 9,10-epoxi-18-hydroxioktadekansyra (EFA), vilken har tre olika funktionella grupper: en epoxid-, en hydroxid- och en karboxylgrupp.  De tre omnämnda funktionella grupperna påvisar det breda potentiella användningsområdet för denna molekyl.  EFA skulle därmed kunna vara intressant att utnyttja i utvecklingen av nya, gröna material. Målet med detta projekt är att extrahera EFA från näver för att sedan via enzymkatalys syntetisera oligomerer med dimetyladipat (DA). Under projektet utfördes ett flertal extraktioner med varierande resultat vilket visar på metodens känslighet. Troligen har pH samt den använda näverns individuella egenskaper stor inverkan på extraktionen och dess utbyte. Den fjärde extraktionen gav 0,44 g EFA vilket innebar ett utbyte på 12%. EFA polymeriserades via enzymatisk katalys med CalB (Lipas B från Candida Antarctica) vilket gav en polymer som efter 1H-NMR-analys kunde konstateras ha en bibehållen epoxidgrupp. Genom att använda DA som ändgrupp och samtidigt reglera det stökiometriska förhållandet mellan reaktanterna, kunde polymerisationsgraden kontrolleras.  Genom MALDI-ToF-analys kunde det fastslås att det fanns en trend typisk för enzymkatalyserade polymerer i det erhållna spektrumet. Denna trend indikerade att monomeren EFA fanns kvar men även att en polymerisation ägt rum och oligomerer med polymerisationsgrader ett och två hade bildats.

Verticillium longisporum ist ein bodenbürtiger, phytopathogener Pilz, der Pflanzen der Familie der Brassicaceen befällt. Er dringt durch die Wurzel ein und verbreitet sich in der Pflanze über das Xylem. In dieser Arbeit wurden die metabolischen Veränderungen in der Modellpflanze Arabidopsis thaliana während der Pflanzen-Pilz-Interaktion analysiert. Hierfür wurde die

La conductivité hydraulique racinaire (Lpr) traduit la capacité de transport d’eau de la racine. Lors de son trajet du sol vers le xylème, l’eau diffuse au sein de l’apoplasme ou au travers des cellules (voie de cellule-à-cellule). Au niveau de l’endoderme, la diffusion apoplasmique de l’eau est bloquée par le cadre de Caspari et des lamelles de subérine. La voie de cellule-à-cellule dépend principalement de l’activité des aquaporines régulées en partie par des interactions protéiques. Ce travail caractérise de nouveaux interactants de l’aquaporine racinaire PIP2;1 : le récepteur kinase RKL1 et 4 protéines de fonction inconnue appartenant à la sous-famille 1 des Casparian Strip membrane domain Protein Like (CASPL1) (CASPL1-B1/B2/D1/D2). RKL1 est exprimée dans l’endoderme, est capable d’interagir physiquement avec PIP2;1 et stimule in vitro le transport d’eau par l’aquaporine. Cependant, l’inactivation de RKL1 n’affecte pas la Lpr sans que cela ne puisse être expliqué par une redondance fonctionnelle avec son plus proche homologue, RLK902. Une étude bibliographique suggère que l’interaction entre RKL1 et PIP2;1 interviendrait dans une voie de signalisation en réponse à une attaque pathogène. Concernant les CASPL, D1 est exprimé dans tous les tissus, alors que B1, B2 et D2 semblent uniquement exprimés dans des territoires subérisés. Ce profil suggère une implication de B1, B2 et D2 dans une régulation des aquaporines et de la subérisation. Au niveau moléculaire, D2, malgré son interaction physique avec PIP2;1, ne module pas le transport d’eau par l’aquaporine. En revanche, B1 interagit préférentiellement avec PIP2;1 sous une forme phosphorylée et stimule le transport d’eau par l’aquaporine. Au niveau de la plante entière, l’inactivation d’un ou deux gènes CASPL n’affecte ni la Lpr., ni la subérisation. Par contre, l’inactivation de PIP2;1 et PIP2;2 révèle un effet inhibiteur de ces aquaporines sur la subérisation. Cette étude a permis de décrire de nouveaux mécanismes originaux de régulation des aquaporines. Elle pose également, la question de l’existence d’une relation entre les transports d’eau par la voie apoplasmique et par les aquaporines. / The root hydraulic conductivity (Lpr) reflects the water transport capacity of the root. During its transfer from the soil to the xylem, water can diffuse in the apoplasm or through the cells (cell-to-cell pathway). At the endodermis, the apoplastic diffusion of water is blocked by the Casparian Strip and suberin lamellae. The cell-to-cell pathway mainly relies on aquaporin activity which can be regulated by protein interactions. This study aims at characterizing new interactants of the root aquaporin PIP2;1: the receptor kinase RKL1 and 4 proteins of unknown function belonging to the Casparian Strip membrane domain Protein Like 1 sub-family (CASPL1-B1/B2/D1/D2). RKL1 is expressed in the endodermis, can physically interact with PIP2;1 and stimulates its water transport function in vitro. However a loss-of-function of RKL1 does not affect the Lpr., independently of a putative functional redundancy with its closest homolog RLK902. Concerning CASPL, D1 is expressed in every tissue of the root whereas B1, B2 and D2 appear to be specifically expressed in suberized tissues. This suggests a putative role of these isoforms in aquaporin regulation and suberisation. At the molecular level, D2 does not modulate PIP2;1 water transport activity despite a physical interaction between the two partners. By contrast, B1 interacts with PIP2;1 preferentially in its phosphorylated form and enhances the water transport activity of the aquaporin. At the plant level, disrupting one or two CASPL genes neither impact the Lpr nor affect the suberisation. However, the loss of function of both PIP2;1 and PIP2;2 reveals a negative effect of these aquaporins on suberisation. In conclusion, this study, uncovered novel regulation mechanisms of aquaporins. It also raises the question of the existence of a putative relationship between water transport by the apoplastic pathway and by aquaporins.

Ions in the soil solution traverse many layers (epidermis, exodermis, central cortex, and endodermis) within the root to reach the stele. The endodermis is present in almost all vascular plants while the exodermis is found only in majority of angiosperm roots tested. The maturation of the exodermis and the death of epidermis alter the plasma membrane surface areas (PMSA) potentially available for ion uptake. Do these changes reduce the ion uptake in proportion to the loss of absorptive surface areas? To answer this question onion (Allium cepa L cv. Wolf) adventitious root segments representing above features: Immature Exodermis Live Epidermis (IEXLEP), Mature Exodermis Live Epidermis (MEXLEP), Mature Exodermis Dead Epidermis (MEXDEP) were excised. Using a compartmental elution technique, radioactive sulphate and phosphate present in various internal compartments were quantified. Quantities of ions moved across the plasma membrane, a summation of quantities in the cytoplasm, ‘vacuole’, and ‘bound’ compartments, indicated that the maturation of the exodermis reduces the uptake of sulphate but not phosphate. In contrast, epidermal death reduced the movement of both ions across the plasma membranes. Although there is a reduction in the available PMSA with the maturation of the exodermis and death of the epidermis, these events do not necessarily reduce the ion movement into the plasma symplast. The endodermal cells of onion roots deposit suberin lamellae as secondary walls. As seen in cross-sections some cells remain without these lamellae and are known as ‘passage cells’. What is the pattern of suberin lamella deposition along the root? Is the suberin lamella a continuous layer? To answer these questions, endodermal layers isolated from onion adventitious roots were used in the present study. These layers were observed using four stains (Sudan Red 7B, Fluorol yellow 088 [Fy], berberine, and Nile red) and three microscopes (compound-white light, compound-epifluorescence and confocal scanning). In differentiating cells with and without suberin lamellae in endodermal layers Sudan Red 7B served the best results for compound-white light microscope, Fy for compound-epifluorescence microscope and Nile for confocal laser scanning microscope (CLSM). Suberin lamellae deposition initiated almost in a random manner; they continued to be deposited resulting in the production of longitudinal files alternating with files with passage cells, and were ultimately deposited in almost all cells at a distance of 255 mm from the tip. The suberin lamellae are perforated with pores, a consistent feature even as far as 285 mm from the tip. These pores may serve as portals for water, ions, and pathogen movement.