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

231 Overexpression of Arabidopsis SOB5 Suggests the Involvement of a Novel Family of Plant
Proteins in Cytokinin-mediated Development.
Jingyu Zhang 2 , Elizabeth Wrage 2 , Radomira Vankova 1 , Jiri Malbeck 1 , Michael Neff 2
1
Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany AS CR, Rozvojova 135, 165
02 Prague 6, Czech Republic, 2 Department of Biology, Washington University, St. Louis, MO 63130
Activation tagging, a gene-overexpression mutagenesis tool, has been used to identify extragenic suppressors of the
long-hypocotyl phenotype conferred by the photoreceptor mutant phyB-4. This mutant screen allows us to cast a broad
net in search of potentially redundant genes involved in seedling development. The sob5-D mutation confers phenotypes
typical of transgenic plants with elevated levels of the plant hormones, cytokinins. The sob5-D mutation is caused by
the overexpression of a novel gene, SOB5, which is part of previously uncharacterized family of plant-specific proteins.
A translational fusion between SOB5 and the green fluorescent protein reporter was localized in the cytoplasm as well
as associated with the plasma membrane when transiently expressed in onion epidermal cells. Analysis of transgenic
plants harboring a SOB5:SOB5-B-glucuronidase (GUS) translational fusion under the control of the SOB5 promoter
region showed GUS activity in vegetative tissues (hydathodes and trichomes of leaves, shoot meristems and roots) as
well as in floral tissues (pistil tips, developing anthers and sepal vasculature). Cytokinin quantification analysis revealed
that adult sob5-D plants accumulated higher levels of trans-zeatin riboside, trans-zeatin riboside monophosphate and
isopentenyladenine 9-glucoside when compared to the wild type. Consistent with this result, AtIPT3 and AtIPT7 were
found to be up-regulated in a tissue-specific manner in sob5-D. Physiological analysis of sob5-D demonstrated reduced
responsiveness to exogenous cytokinin in both root-elongation and callus-formation assays. Though our data suggest a
role for a novel gene, SOB5, in cytokinin-mediated plant development, knock-down and knock-out mutants in SOB5
are phenotypically similar to the wild type, suggesting that other SOB-five-like (SOFL) proteins in Arabidopsis may be
functionally redundant with SOB5. In support of this hypothesis, overexpression of AtSOFL1 and AtSOFL2 confers
phenotypes similar to sob5-D. We are further testing this hypothesis by generating double and triple knock-down and
knock-out mutants between SOB5, AtSOFL1 and AtSOFL2.
232 Antisense-expression of the OSCP(δ) Subunit of Mitochondrial ATP Synthase Promotes
Mitochondrial Division in Arabidopsis
Mary Robison, Matthew Smid, Xingyuan Ling, David Wolyn
Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada, N1G 2W1
The mitochondrial and chloroplastic ATP synthases of plants are homologous multi-subunit complexes consisting of
intrinsic F 0 and extrinsic F 1 segments connected by a stator stalk. The oligomycin sensitivity-conferring protein (OSCP)
subunit which forms part of the stator stalk in mitochondria is homologous to the δ subunit in chloroplasts. To specifically
reduce mitochondrial ATP synthesis, we transformed Arabidopsis with an antisense copy of the mitochondrial OSCP
(AF380647) under the control of a dexamethasone-inducible promoter. Seed homozygous for the transgene, imbibed
on dexamethasone-containing media in the light, died shortly after germination, but imbibition in the dark resulted in
etiolated seedlings of short stature. Treatment of established soil- or hydroponically-grown plants with dexamethasone
resulted in slow growth and development, strap-like or cupped leaves that often had wavy margins, slight chlorosis, and
cup-like sepals that restricted normal pollination. The magnitude of response was dose-dependent. Crossing the anti-OSCP
plants with a line expressing GFP targeted to the mitochondrion (Logan and Leaver, 2000) allowed us to examine effects
on mitochondrial morphology. Dexamethasone-treated plants had increased numbers of small mitochondria, usually
arranged in chains. Northern analysis of genes related to mitochondrial fission or fusion demonstrated up-regulation of
several fission-related DRP genes after dexamethasone treatment whereas the fusion-related genes were unaffected.
X. Ling, current address:Department of Botany, PO Box 3165, University of Wyoming, Laramie WY 82071-3165