Sterols have been identified as major components of membrane lipids that are part of specialized membrane domains necessary for organizing events such as polar protein targeting and signal transduction in plants, fungi and animals. However a common modification of sterols is the addition of sugar moieties via glycosylation abundantly found in plants. An exact physiological role for such diversification of sterols in plants is still unknown. Using reverse genetics and transcriptomics we show that UDP-glucose: sterol glucosyltransferase encoded by UGT80B1 is necessary for correct epidermal patterning in Arabidopsis root. Patterning of hair cells (trichoblasts) and non-hair cells (atrichoblasts) in the epidermis of the Arabidopsis root involves signaling through SCRAMBLED (SCM), a plasma membrane localized LRR-RL kinase. Feedback regulation via the transcriptional regulatory complex containing R2R3-MYB transcription factor WEREWOLF (WER) represses SCM and activates the homeodomain-leucine-zipper protein GLABRA2 (GL2) in atrichoblasts. Evidence suggests symplastic connections between cells, known as plasmodesmata, establish passage ways for single-repeat R3-MYB transcription factors to activate SCM expression in trichoblasts. Mutations in UGT80B1 cause atypical localization patterns of GL2, WER, and SCM in the root epidermis. The ugt80B1 formed fewer trichoblasts in comparison to wild-type. A translational fusion of UGT80B1 to GFP localizes to the ER, plasma membrane and to sites that appear to be plasmodesmata-associated desmotubules. Ultrastructural analysis revealed abnormalities in plasmodesmata formation and morphology in ugt80B1 mutants. Steryl glucoside profiling indicated deficiencies in specific glycosylated sterol compounds in roots. This study identifies UGT80B1 as a novel membrane component that is critical for plasmodesmata morphogenesis and cell-fate determination in the root epidermis. A model is proposed in which UGT80B1 activity provides spatially discreet sterol and steryl glucoside architecture within the plasma membrane to anchor the SCM receptor and within plasmosdesmata to facilitate intercellular movement of R3-MYB regulatory proteins underlying proper differentiation of trichoblasts versus atrichoblasts. Moreover, evidence from reverse genetics, proteomics and live cell imaging point to a actin dependent localization of UGT80B1 at the vesicle rich zone of root hair tip. This localization actively supports root hair elongation via tip growth, possibly by membrane modifications required for vesicle trafficking.

This thesis aimed to investigate several fundamental and perplexing questions relating to the phloem loading and transport mechanisms of <i>Cucurbita maxima</i>, by combining metabolomic analysis with cell biological techniques. This putative symplastic loading species has long been used for experiments on phloem anatomy, phloem biochemistry, phloem transport physiology and phloem signalling. Symplastic loading species have been proposed to use a polymer trapping mechanism to accumulate RFO (raffinose family oligosaccharides) sugars to build up high osmotic pressure in minor veins which sustains a concentration gradient that drives mass flow. However, extensive evidence indicating a low sugar concentration in their phloem exudates is a long-known problem that conflicts with this hypothesis. Previous metabolomic analysis shows the concentration of many small molecules in phloem exudates is higher than that of leaf tissues, which indicates an active apoplastic loading step. Therefore, in the view of the phloem metabolome, a symplastic loading mechanism cannot explain how small molecules other than RFO sugars are loaded into phloem. <br><br> Most studies of phloem physiology using cucurbits have neglected the possible functions of vascular architecture in phloem transport. It is well known that there are two phloem systems in cucurbits with distinctly different anatomical features: central phloem and extrafascicular phloem. However, mistaken conclusions on sources of cucurbit phloem exudation from previous reports have hindered consideration of the idea that there may be important differences between these two phloem systems. <br><br> The major results are summarized as below:<br> 1) O-linked glycans in <i>C.maxima</i> were structurally identified as beta-1,3 linked glucose polymers, and the composition of glycans in cucurbits was found to be species-specific. Inter-species grafting experiments proved that these glycans are phloem mobile and transported uni-directionally from scion to stock.<br> 2) As indicated by stable isotopic labelling experiments, a considerable amount of carbon is incorporated into small metabolites in phloem exudates. However, the incorporation of carbon into RFO sugars is much faster than for other metabolites.<br> 3) Both CO2 labelling experiments and comparative metabolomic analysis of phloem exudates and leaf tissues indicated that metabolic processes other than RFO sugar metabolism play an important role in cucurbit phloem physiology.<br> 4) The underlying assumption that the central phloem of cucurbits continuously releases exudates after physical incision was proved wrong by rigorous experiments including direct observation by normal microscopy and combined multiple-microscopic methods. Errors in previous experimental confirmation of phloem exudation in cucurbits are critically discussed.<br> 5) Extrafascicular phloem was proved to be functional, as indicated by phloem-mobile carboxyfluorescein tracer studies. Commissural sieve tubes interconnect phloem bundles into a complete super-symplastic network.<br> 6) Extrafascicular phloem represents the main source of exudates following physical incision. The major transported metabolites by these extrafacicular phloem are non-sugar compounds including amino acids, O-glycans, amines.<br> 7) Central phloem contains almost exclusively RFO sugars, the estimated amount of which is up to 1 to 2 molar. The major RFO sugar present in central phloem is stachyose. <br> 8) Cucurbits utilize two structurally different phloem systems for transporting different group of metabolites (RFO sugars and non-RFO sugar compounds). This implies that cucurbits may use spatially separated loading mechanisms (apoplastic loading for extrafascicular phloem and symplastic loading for central phloem) for supply of nutrients to sinks. <br> 9) Along the transport systems, RFO sugars were mainly distributed within central phloem tissues. There were only small amounts of RFO sugars present in xylem tissues (millimolar range) and trace amounts of RFO sugars in cortex and pith. The composition of small molecules in external central phloem is very different from that in internal central phloem.<br> 10) Aggregated P-proteins were manually dissected from central phloem and analysed by both SDS-PAGE and mass spectrometry. Partial sequences of peptides were obtained by QTOF <i>de novo</i> sequencing from trypsin digests of three SDS-PAGE bands. None of these partial sequences shows significant homology to known cucurbit phloem proteins or other plant proteins. This proves that these central phloem proteins are a completely new group of proteins different from those in extrafascicular phloem. The extensively analysed P-proteins reported in literature to date are therefore now shown to arise from extrafascicular phloem and not central phloem, and therefore do not appear to be involved in the occlusion processes in central phloem. / Phloem transportiert ein ausgedehntes Spektrum an Molekülen zwischen Pflanzenorganen, um Wachstum und Entwicklung zu koordinieren. Folglich ist eine umfassende und unvoreingenommene Metabolom-Analyse notwendig, um unser Verständnis über den Transport von Stoffwechselprodukten sowie über Phloemtransport zu vertiefen. Phloemexsudate von Kürbispflanzen werden unter Verwendung der Metabolom-Analyse analysiert. Bei diesen Pflanzen wird angenommen, dass sie symplastische Beladungswege verwenden, um Photoassmilate als Ausgangsschritt des Phloemtransportes zu konzentrieren. Zwei neue Familien Callose-verwandter Substanzen, 1,3-Overknüpfte Glycane, sowie eine Reihe anderer kleinerer Metabolite werden in den Phloemexsudaten detektiert. Metabolom-Daten und physiologische Experimente widersprechen früher berichtetem Verständnis des Phloemexsudationsprozesses in Kürbispflanzen. Folglich bestätigt sich der Phloemexsudationsprozeß durch Kombination unterschiedlicher mikroskopischer Techniken. Kürbispflanzen besitzen zwei Phloemsysteme mit eindeutigen anatomischen Eigenschaften. Es zeigt sich, daß Phloemexsudate in Kürbissen hauptsächlich vom extrafaszikulären Phloem, nicht vom zentralen Phloem, stammen. In den letzten Jahrzehnten wurde gewöhnlich mißverstanden, daß Phloemexsudate vom zentralen Phloem stammen. Die eindeutigen metabolischen Profile der unterschiedlichen Phloemsysteme, die durch Metabolom-Analysen in der räumlichen Auflösung beobachtet werden, bestätigen die unterschiedlichen physiologischen Funktionen der zwei unterschiedlichen Phloemsysteme: das zentrale Phloem transportiert hauptsächlich Zucker, während das extrafaszikuläre Phloem ein ausgedehntes Spektrum von Metaboliten transportiert. Es kann auch ein unterschiedliches metabolisches Profil kleiner Moleküle zwischen internem und externem zentralem Phloem beobachtet werden. Von Strukturproteinen des zentralen Phloems wurden auch Proben genommen und mittels Massenspektrometrie analysiert. Diese Proteine erweisen sich als neuartige Proteine, die sich zu denen im extrafaszikulären Phloem unterscheiden. Dies bestätigt ferner den Funktionsunterschied der unterschiedlichen Phloemsysteme in Kürbispflanzen. Basierend auf diesen neuartigen Entdeckungen des Phloem-Metaboloms und dem vorhergehenden Wissen über den Phloemtransport in Kürbispflanzen, wird ein neues Modell vorgeschlagen, um den Mechanismus des Phloemtransports in der symplastischen Beladung zu verstehen.<br>

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