15th International Conference on Arabidopsis Research - TAIR

T10-015
A method to isolate chloroplasts from specific cell
types of Arabidopsis thaliana
Elisabeth B. Truernit(1), Julian M. Hibberd(1)
1-Department of Plant Sciences, University of Cambridge, Downing Site, Cambridge CB2 3EA
In multicellular organisms, cells and their organelles differentiate to perform
specific functions. In plants this is exemplified within a leaf where different
cell types contain chloroplasts of different sizes and structures, enabling photosynthesis
to be efficient (1). Photosynthetic properties also vary between
cell types, for example, recently it has been shown that cells located around
the vascular bundles in petioles and stems of C3 plants show characteristics
of C4 photosynthesis (2). However, to date one of the limitations to studying
photosynthetic properties of individual cell-types is our inability to isolate
chloroplasts from specific cell types.
In order to address this problem we are developing a method to tag
chloroplasts from specific cell-types and subsequently isolate them. As a
tag the gene coding for the yellow fluorescent protein (YFP) was fused to a
gene that codes for an outer envelope protein from pea. The fusion protein
inserts into the outer chloroplast envelope membrane with YFP located on the
cytosolic side of the membrane. We are using tissue specific promoters and
enhancer-trap lines to express the fusion construct in chloroplasts of different
cell-types of Arabidopsis thaliana leaves and stems. Magnetic beads coated
with anti-GFP antibody are used to isolate the YFP-tagged chloroplasts from
a mixed population. Our initial results show that we are able to specifically
isolate YFP-labelled chloroplasts.
(1) Terashima et al., (1986) Plant Cell Physiol., 27: 1023-1031
(2) Hibberd & Quick, (2001) Nature, 415: 451-454
T10 Novel Tools, Techniques and Resources
T10-016
Plant Bimolecular Fluorescence Complementation
(PBFC) ¯ a system for detecting protein-protein
interactions in plants
Keren Shichrur(1), Keren Bracha-Drori(1), Moran Oliva(1), Aviva Katz(1), Ruthie
Angelovici(1), Nir Ohad(1), Shaul Yalovsky(1)
1-Department of Plant Sciences, Tel Aviv University Israel
Protein function is often mediated via formation of stable or transient complexes.
Currently, however, there are no simple and reliable methodologies for
detecting protein-protein interactions in vivo in plants. To detect protein-protein
interactions in vivo, we have adapted the recently published bimolecular
fluorescence complementation (BiFC) assay (Hu et al., 2002) to plants. The
Yellow Fluorescent Protein (YFP) was separated into two non-overlapping
N- and C-terminal domains designated YN and YC, respectively. To reconstitute
YFP fluorescence, YN and YC were fused with putative interacting
protein pairs and co-expressed in plants. The feasibility of the PBFC system
was demonstrated with two known interacting proteins pairs: Arabidopsis
protein farnesyltransferase (PFT) A and B subunits and the polycomb proteins
FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Reconstitution
of YFP fluorescence occurred following transient expression of either
protein pair in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis.
The fluorescence emission spectra of native and reconstituted YFPs were
identical, confirming their specificity. Furthermore, no YFP fluorescence was
detected following co-expression of non-fused YN and YC or non-interacting
protein pairs. Monoclonal antibody tags fused to YN and YC enabled cross
verification of protein expression and interactions by immunoblots and coimmunoprecipitations.
Reconstitution of YFP fluorescence was detected in the
respective subcellular compartment (cytoplasm and nuclei) for each protein
pair. The simplicity PBFC and the ability to detect protein-protein interactions
at different subcellular compartments make it an attractive method for analysis
of protein interactions and networks.
Note added: The usefulness of BiFC in plant protein-protein interaction
research is also demonstrated on a poster presented by Christina Chaban
, Michael Walter, Katia Schütze, Oliver Batistic, Claudia Oecking , Wolfgang
Werr, Jörg Kudla and Klaus Harter, “Bimolecular fluorescence complementation
- a novel tool for in planta protein interaction studies”.
Hu C.-D., Chinenov Y., Kerppola T.K. (2002) Molecular Cell, 9: 789-798.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin