Research Projects in the Geldner lab
Wave lines: Mapping the complexity of the plant membrane system
Eukaryotic cells maintain a dynamic and complex network of membrane compartments, such as the endoplasmic reticulum, Golgi apparatus, endosomes, and vacuole/lysosomes. These compartments separate and organize proteins within the cell and impose temporal as well as spatial restrictions on protein activities. Compartment identities are maintained in spite of a constant exchange of material. This process of continuous generation and consumption of compartments defies any simple analogies with static macroscopic structures and membrane compartments should rather be compared to waves, whose very essence is the dynamic flow of material through them.
The complexity of the membrane compartments of plant cells are only insufficiently appreciated, especially for cells in the context of the whole plant. One of the problems is that many membrane compartments have an inconspicuous morphology, especially at the current resolution of fluoresent live-imaging. Therefore, they can only be distinguished by co-localisation with spectrally distinct fluorescent markers. At the Salk Institute, we developed a vector series for plant transformation for a non-Gateway-based recombination . This vector set allows the use of a recently developed Arabidopsis ORF library for large scale generation of fluorescent fusion constructs in plants. These vectors are an alternative to Gateway. Their CRE/lox system is non-patented, cheap and compatible with the Arabidopsis ORF collection, currently encompassing about 13.000 clones. We used this system (Liu et al., 1998) to generate a large set of membrane compartment markers. This set allows the definition of compartments and trafficking pathways in a developmental context and does not rely on transient expression in protoplasts or cultured cells. Our set of transgenic plants with yellow, red and blue variants of compartment markers is now established and has been published (Geldner et al., 2009).
Wave constructs and seeds are available at the Nottingham Arabidopsis Stockcenter (NASC)
American researchers can order from there while the stock are being transferred to the ABRC.
When using our Wave construct and lines or our pNIGEL vector series, please cite our paper in The Plant Journal:
Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.
Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J.
Plant J. 2009 Jul;59(1):169-78. Epub 2009 Feb 26. PMID: 19309456
|Wave number||gene name||AGI||EYFP (Yellow)||mCherry (Red)||mCerulean (blue)||mTFP1 (bluegreen)|
|1||tag only (free FP)||n.a.||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|2||RabF2b (ARA7)||At4g19640||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|3||Rab C1||At1g43890||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|7||RabF2a (Rha1)||AT5g45130||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|11||Rab G3c||At3g16100||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|18||Got1p homolog||At3g03180||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|24||Rab A5d||At2g31680||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|25||Rab D1||At3g11730||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|27||Rab E1d||At5g03520||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|29||Rab D2a||At1g02130||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|33||Rab D2b||At5g47200||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|34||Rab A1e||At4g18430||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
|129||Rab A1g||At3g15060||DNA/seeds||DNA/seeds||DNA/seeds||DNA only|
FOR THE LATEST, UPDATED INFORMATION ON THE WAVE LINES, DOWNLOAD THIS PDF:
Wave_line_info_sheet_May_2010_1.pdf (826 Kb)
The pNIGEL recombination vector series
The pNIGEL vectors were developed to allow optimal co-localisation between lines carrying different fluorescent protein fusion constructs. The three fluorescent proteins chosen can be easily separated spectrally. The Cerulean and mTFP1 vectors contain an additional 3xHA tag, the EYFP vector contains an additional 3xMyc epitope. Vectors of different colours carry different resistances, such as to allow selection of crossing products and/or sequential transformations with different constructs. Marker expression is driven by the intron-bearing, endogenous UBQ10 promoter, in order to promote stable expression levels and minimise overexpression artifacts while obtaining sufficiently strong signals. pNIGEL vectors are based on the pGREEN II vector series, in which the bacterial Kan Resistance was exchanged against Amp Resistance. For additional information, please refer to the publication (Geldner et al., 2009).
For a complete list of pNIGEL vectors, please refer to this pdf file:
pNIGEL_vector_series.pdf (1200 Kb)
Wave constructs are produced by recombination of a given pNIGEL with the respective ORF, cloned into the pUNI51 vector by the SSP consortium. Below is shown part of the resulting fusion construct using the Wave131Y construct as example (NPSN12 ORF fused to pNIGEL07):
Example sequences of recombined Wave constructs can be downloaded below as genbank files. In order to obtain the exact sequence of your construct of interest, just exchange the example NPSN12 ORF with your ORF of interest (including start and stop codon).
Wave131_in_pNIGEL_07__YFP_.gb (94 Kb)
The root endodermis as a polar epithelium
Epithelial polarity is a fundamental feature of multi-cellular life and model systems for animal epithelia have been intensely investigated for decades. Epithelial polarity in animals has presumably evolved from basic mechanisms of cell polarity that were present in unicellular organisms. However, epithelial polarity is more complex than individual cell polarity and a number of specialised modules and pathways have evolved in the animal lineage for its establishment and maintenance. The acquisition of complex multi-cellularity is a rare event during evolution, and higher plants are the other relevant group of organisms to have independently achieved this. However, nothing is known about the mechanisms whereby diverse plant tissue layers establish and maintain differences between outer and inner cellular surfaces.
One tissue that displays all the hallmarks of an epithelium is the root endodermis. The endodermis is an invariant feature of all vascular plants and fulfils a crucial barrier function, separating the extracellular (apoplastic) space of outer cell layers (connected to the soil), with the inner apoplastic space of the vascular bundles (the transport route to aerial tissues). This was recognised as early as 1910 by de Rufz de Lavison. In order to act as a barrier, endodermal cells secrete hydrophobic material in a highly localised and coordinated fashion, the "Casparian strip". A large body of descriptive and physiological data has been accumulated on endodermal cell layers in diverse plant species. The Casparian strip is composed of cork-like suberin, a compound polymer that is cross-linked into an extensive, supra-cellular network. The plasma membrane underlying the casparian strip appears more electron dense and ordered, suggesting the localised presence of protein scaffolds, tightly attached to the extracellular matrix. Our observations suggest that there is no lateral diffusion across the Membrane Adjacent the Casparian Strip (MACS) between outer and inner plasma membrane domains. The endodermis is therefore divided into separate outer (peripheral) and inner (central) subdomains. Accordingly, transporters should localise to one or the other membrane region, depending on their function in uptake or efflux of nutrients. This has indeed been shown for a few examples in recent years. However, there are no data regarding their targeting to specific subdomains. In addition, we ignore the molecular identities of the proteins associated with the Casparian strip. We will investigate the mechanisms that allow the endodermis to set-up the precisely aligned Casparian Strip sub-domains on the surface of its cells and how it localises proteins to either its central or peripheral surface. For this we are use advanced live-cell imaging that will becombined with forward and reverse genetic screens and proteomic approaches.
Receptor trafficking in plants
Endosomal trafficking of animal receptors is also intricately linked to the logic of their signal transduction pathways. It is used to regulate signalling length, amplitude, sensitivity as well as specificity of these pathways. Plants have independently evolved a large set of plant-specific receptors-kinases. More than 200 of these LRR-RLKs exist in Arabidopsis and this large number has developed in conjunction with multi-cellular development. A function and a molecular knowledge of their ligands and downstream partners has been only obtained for very few of them. The two best understood models are the brassinosteroid receptor BRI1 and the flagellin peptide receptor FLS2. Both have a very different physiological function and distinct downstream signalling components. BRI1 is a receptor for an important, growth promoting hormone, called brassinolide, which is more or less continuously present in growing tissues. BRI1's function is probably to report changes in the hormone concentration more than to signal it's acute presence or absence. Consistently, brassinosteroids seem to be produced in very much the same tissues as they are perceived and to act in an autocrine/paracrine fashion. FLS2 on the other hand has evolved to report presence of a microbial peptide in the event of a bacterial infection and its signalling behaviour appears to be thresholded rather than graded. Interestingly, this different functionality is reflected in a very distinct trafficking behaviour of these two receptors. Whereas our recent work established that BRI1 is trafficked in a ligand-independent fashion, but to always partition between an endosomal and plasma membrane localised pool (see picture, above), FLS2 was shown to localise exclusively to the plasma membrane, but to be rapidly internalised into endosomes upon ligand binding.
We have generated BRI1 constructs that allows its pulsed expression in root meristems, allowing to easily follow BRI1 endocytosis as its gradual disappearance after an expression pulse in roots. We will use this system in a forward genetic screen in order to identify factors required for endocytosis and degradation of this receptor kinase.