WAVE markers lines for membrane compartments

by Niko Geldner

Department of Plant Molecular Biology, University of Lausanne, UNIL-Sorge, Biophore, 1015 Lausanne, Switzerland

The process of continuous generation and consumption of membrane compartments within a cell defies simple analogies with static macroscopic structures and membrane compartments are more appropriately compared to waves, whose very essence is the dynamic flow of material through them.

Membrane compartments of plant cells are highly complex, but their complexity is still insufficiently resolved by currently available marker sets, especially when analysing cells in the context of the whole plant. Good marker proteins are crucial, however, since many membrane compartments have an inconspicuous morphology at the current resolution of fluoresent live-imaging. Therefore, they can only be distinguished by co-localisation with spectrally distinct fluorescent markers. This is especially true for the endosomal system of plants, although this membrane system is highly relevant for intercellular communication and thus for plant development and responses to the environment.
For an overview on endosomes, see the section by Marisa Otegui.
Vectors
A new vector series has been developed for plant transformation for a non-Gateway-based recombination system. The pNIGEL vector set allows the use of a recently developed Arabidopsis ORF library for large scale generation of fluorescent fusion constructs in plants. Their CRE/lox system is cheap and the compatible Arabidopsis ORF collection currently encompassing about 13.000 clones. We used this system (Liu et al., 1998) to generate a large set of fusion construct for compartment marker candidates, with a focus on the endosomal system. This set allows the definition of compartments and trafficking pathways in a developmental context, although the constructs can also be used for transient expression in protoplasts or other cellular systems. The set of transgenic plants with yellow, red and blue variants of compartment markers is now established and has been published (Geldner et al., 2009). Expression from the pNIGEL vector is driven from the UBQ10 promoter. This endogenous, intron-bearing promoter shows moderate over-expression, compared to the viral 35S promoter. UBQ10 expression appears stable and homogenous and its expression domain is very broad. The fluorophores (FPs) used in the pNIGEL vectors are EYFP as the yellow fluorophore (pNIGEL 07). As a red FP mCherry was chosen (pNIGEL 17). Its red-shifted excitation and emission provides a very low background fluorescence in non-chlorophyllic tissues. This, together with its very good photo-stability makes mCherry a well-performing red fluorophore. mCerulean was chosen as an alternative to the weak and problematic ECFP (pNIGEL 18). However, it only performed slightly better that than ECFP and the mCerulean marker lines show only weak signals. As another option, we have generated constructs using the blue-green mTFP1 (pNIGEL 19). This FP should be clearly superior to mCerulean and ECFP. However, transgenic plants are in the process of being generated and it remains to be seen whether mTFP1 is showing a clearly superior performance in plants.
Probes for Vacuoles
Pictures of Arabidopsis root meristematic cells of F1 plants from crosses between EYFP (WaveY) and mCherry (WaveR) lines. Example show the non-overlapping distribution of a Wave129R (RabA1g) marked endosomal compartment with a new Golgi stack marker, Wave18R (Got1p) – as compared to the control cross of the Y and R variant of the Golgi marker with itself. Wave18 and Wave 129 are also clearly distinguishable by their different responses to the trafficking inhibitor Brefeldin A, with Wave 18Y marked compartment arranging at the periphery of a Wave129R marked core.

The Wave constructs are available at the Nottingham Arabidopsis Stockcenter (NASC).  Also American researchers can order from there while the stock are being transferred to the ABRC. Seeds of the Wave lines are currently bulked for submission to NASC. If you are interested in receiving seeds in the meanwhile, please send an e-mail to: (Niko.Geldner@unil.ch). More information about the Wave marker lines can be found at http://www.unil.ch/dbmv/page52928_en.html

Wave marker lines
Overview of WAVE marker lines

External Links : Endosome Membrane proteins