Funktionelle Charakterisierung des AtPIN1-Proteins aus Arabidopsis thaliana

Die AtPIN1-cDNA wurde unter der Kontrolle des CaMV35S-Promotors in A. thaliana exprimiert. In transgenen Pflanzen wurde in Western-Blot Analysen eine erhöhte AtPIN1- Menge in Wurzeln nachgewiesen. Immuncytochemisch wurde ectopisch exprimiertes AtPIN1 in Wurzelspitzen und Sproßachsen, genauso wie endogenes AtPIN1 polar, an basalen Zellenden nachgewiesen. Keimlinge von stark AtPIN1-überexprimiernde Linien zeigten gravitrop gestörtes Wurzelwachstum und eine erhöhte Anzahl von Seitenwurzeln. Weiterhin wurde bei diesen Linien in Wurzelspitzen qualitativ eine verstärkte- und ectopische Aktivität des Auxin-sensitiven DR5-Promotorelements festgestellt. In Sproßachsen von AtPIN1- überexprimierenden Pflanzen wurde eine konzentrationsabhängig verringerte Sensitivität des polaren Auxintransports gegenüber dem spezifischen Inhibitor NPA gemessen. Weiterhin wurde AtPIN1 unter der Kontrolle eines Glucocorticoid-induzierierbaren Promotors in A. thaliana exprimiert in Western-Blot-Analysen auf Blättern wurde eine induzierbare AtPIN1- Expression nachgewiesen. Mit Glucorticoid behandelte Pflanzen zeigten hyponastisches Blattwachstum. Von diesen Pflanzen wurde eine Suspensionskultur angelegt und mittels Tritium-markiertem Auxin in Kompetitionsanalysen mit überschüßigem Auxin der saturierbare Auxinefflux gemessen, wobei die AtPIN1-überexprimierende Suspensionkultur einen erhöhten Efflux aufwies. Die Inhibierbarkeit durch NPA war durch AtPIN1- Überexpression in konzentrationsabhängiger Weise verringert. In Wurzelspitzen von A. thaliana, welche die Serin/Threonin-Kinase PINOID überexprimieren, wurde immuncytochemisch die Lokalisierung von AtPIN1, 2 und 4 untersucht. In polarisierten Zellen waren im Wildtyp apikal lokalisierte AtPIN-Proteine in diesen Pflanzen am basalen Zellende lokalisiert. In Sproßachsen wurde eine revertierte AtPIN1-Lokalisierung festgestellt, wobei der polare Auxntransport nicht verändert war

Protocol: An improved and universal procedure for whole-mount immunolocalization in plants

Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment

MOESM2 of Protocol: an improved and universal procedure for whole-mount immunolocalization in plants

Additional file 2. Supplementary protocols

The TORNADO1 and TORNADO2 Genes Function in Several Patterning Processes during Early Leaf Development in Arabidopsis thaliana

In multicellular organisms, patterning is a process that generates axes in the primary body plan, creates domains upon organ formation, and finally leads to differentiation into tissues and cell types. We identified the Arabidopsis thaliana TORNADO1 (TRN1) and TRN2 genes and their role in leaf patterning processes such as lamina venation, symmetry, and lateral growth. In trn mutants, the leaf venation network had a severely reduced complexity: incomplete loops, no tertiary or quaternary veins, and vascular islands. The leaf laminas were asymmetric and narrow because of a severely reduced cell number. We postulate that the imbalance between cell proliferation and cell differentiation and the altered auxin distribution in both trn mutants cause asymmetric leaf growth and aberrant venation patterning. TRN1 and TRN2 were epistatic to ASYMMETRIC LEAVES1 with respect to leaf asymmetry, consistent with their expression in the shoot apical meristem and leaf primordia. TRN1 codes for a large plant-specific protein with conserved domains also found in a variety of signaling proteins, whereas TRN2 encodes a transmembrane protein of the tetraspanin family whose phylogenetic tree is presented. Double mutant analysis showed that TRN1 and TRN2 act in the same pathway

An Integrated View of Gene Expression and Solute Profiles of Arabidopsis Tumors: A Genome-Wide Approach

Transformation of plant cells with T-DNA of virulent agrobacteria is one of the most extreme triggers of developmental changes in higher plants. For rapid growth and development of resulting tumors, specific changes in the gene expression profile and metabolic adaptations are required. Increased transport and metabolic fluxes are critical preconditions for growth and tumor development. A functional genomics approach, using the Affymetrix whole genome microarray (∼22,800 genes), was applied to measure changes in gene expression. The solute pattern of Arabidopsis thaliana tumors and uninfected plant tissues was compared with the respective gene expression profile. Increased levels of anions, sugars, and amino acids were correlated with changes in the gene expression of specific enzymes and solute transporters. The expression profile of genes pivotal for energy metabolism, such as those involved in photosynthesis, mitochondrial electron transport, and fermentation, suggested that tumors produce C and N compounds heterotrophically and gain energy mainly anaerobically. Thus, understanding of gene-to-metabolite networks in plant tumors promotes the identification of mechanisms that control tumor development