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THE PLANT CELL, Vol 4, Issue 7 851-864, Copyright © 1992 by American Society of Plant Biologists
The Use of Antisense mRNA to Inhibit the Tonoplast H+ ATPase in Carrot
J. P. Gogarten, J. Fichmann, Y. Braun, L. Morgan, P. Styles, S. L. Taiz, K. DeLapp and L. Taiz
Sinsheimer Laboratories, Biology Department, University of California, Santa Cruz, California 95064
Carrot root cells were transformed with the coding or 5[prime] noncoding
regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the
antisense orientation behind the cauliflower mosaic virus 35S promoter.
Bafilomycin-sensitive ATPase, H+-pumping, and 14C-O-methyl-glucose uptake
activities were specifically inhibited in the tonoplast fractions of mutant
cell lines. Protein gel blotting confirmed that the expression of the A
subunit was inhibited in the tonoplast fraction, but not in the Golgi
fraction. Two-dimensional protein gel blots of total microsomes of
wild-type and control transformant cell lines revealed two major
immunoreactive polypeptides in the acidic pl range. In contrast, highly
purified tonoplast membranes contained only the less acidic polypeptide.
Because the less acidic polypeptide was preferentially diminished in the
two antisense cell lines, we infer that the antisense constructs
specifically blocked expression of a tonoplast-specific isoform of the
V-ATPase A subunit in carrot. Regenerated plants containing the antisense
constructs exhibited altered leaf morphologies and reduced cell expansion.
The altered phenotype was correlated with the presence of the antisense
construct.
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