75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

117 Interactions between the Microtubule plus end localising proteins EB1s and SPR1 in
Arabidopsis
Despoina Kaloriti 1 , David Rancour 2 , Sebastian Bednarek 2 , John Sedbrook 1
1
Department of Biological Sciences, Illinois State University, Normal, IL, 61790, USA, 2 Department of
Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706-1544, USA
Microtubules (MTs) are involved in many processes of plant growth and development including cell expansion, cell
division and organelle movement and secretion. The plant MTs form four basic arrays during the cell cycle: the cortical
array, preprophase band, mitotic spindle and phragmoplast. Recent studies revealed that MTs in the cortical array exhibit
dynamics at both ends. The plus end undergoes dynamic instability (periods of growth, pausing and shrinking), while the
minus end undergoes slow depolymerisation (Shaw et al., 2003). In animals and fungi, dynamic instability is regulated
by a collection of proteins called +TIPS (plus end tracking proteins) that are preferentially localized to MT plus ends.
Little is known about what +TIPS exist in plants and how they regulate MT related processes. We have recently identified
a novel, plant specific +TIP named SPIRAL (SPR1) in a forward genetics screen looking for Arabidopsis mutants
affected in root cell expansion (Sedbrook et al., 2004). In addition to SPR1, Arabidopsis contains three +TIP proteins,
which are homologues to the animal EB1 (End Binding) proteins. The function of EB1-like genes in plants has not yet
been clarified, although GFP imaging studies of two of the three AtEB1 isoforms showed that they localise to MT plus
ends with similar dynamics as seen with SPR1 (Chan et al., 2003; Mathur et al., 2003). We focused on identifying the
relationship between SPR1 and EB1-like proteins in Arabidopsis. We phenotypically characterised plants with T-DNA
insertional mutations in each of the three AtEB1 genes. Only one AtEB1 mutant exhibited a slight cell expansion defect
related to root growth, while AtEB1 triple mutant roots exhibited the single AtEB1 mutant phenotype. Double mutant
analysis was also performed between the AtEB1 mutants and spr1-6, with one AtEB1/spr1-6 double mutant exhibiting
a severe root cell expansion defect, suggestive of a genetic interaction. In addition, all three AtEB1 homologues and
SPR1-6 were tested in a Yeast Two Hybrid system to investigate possible protein-protein interactions. Our Yeast Two
Hybrid results will be presented and discussed.
118 Trasngenic Expression of a Calcium-Binding Peptide: A Link Between ER Calcium Stores and
Drought Tolerance
Sang-Yoon Lee, Heike Winter-Sederoff, Dominique Robertson
Department of Botany, North Carolina State University
The development of osmotic stress tolerant crop plants is an important agricultural goal because of the increasing
global demand for water, increased salinity in soil and declining aquifer reserves. Several calcium regulated protein
kinases have been identified in Arabidopsis that affect osmotic stress tolerance. These kinases are regulated by transient
cytosolic calcium oscillations that occur in response to a wide range of environmental stimuli. Cytosolic calcium
concentration is maintained at a low level by Ca2+ transport proteins and the sequestering of higher calcium levels
in intracellular compartments. We used a calcium binding peptide (CBP) derived from calreticulin, a calcium storage
protein, to increase calcium levels in the endoplasmic reticulum (ER). The CBP was fused to the green fluorescent protein
(GFP) and expressed in Arabidopsis thaliana. Compared to GFP vector control and wild type plants, the CBP plants
showed increased root growth and better survival under osmotic stress conditions in 150 mM sorbitol medium, 75 mM
and 150 mM NaCl media, and in soil. Analysis of gene expression demonstrated that CBP expression in the ER altered
transcription of selected osmotic stress-inducible genes, including CIPK6 (a serine/threonine protein kinase), DREB1A,
Dehydrin, rd29a, and a Myb transcription factor. Some of these genes were upregulated in CBP transgenic plants in the
absence of stress. These results identify a new link between ER calcium, root growth, and drought tolerance. Currently,
we are investigating how ER CBP affects cytosolic calcium and whether constitutive expression of CIPK6 can increase
drought tolerance.