Poster: Membrane transport
Abs #
180: Molecular, biochemical and phenotypic definition of Arabidopsis type I V-PPase T-DNA insertion mutant, avp1-1: elucidation of a conditional phenotype
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Presenter: |
Green, Brian J., bjgreen@sas.upenn.edu |
Authors | Green, Brian J. (A) Drozdowicz, Yolanda M. (A) Rea, Philip A. (A) | | Affiliations: |
(A): Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
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We have succeeded in isolating T-DNA insertion mutant lines with markedly different properties for each of the three Arabidopsis vacuolar-type proton pyrophosphatase (V-PPase) genes, AVP1, AVP2 and AVP3. In studies focused on the first of these, the AVP1 knockout mutant, avp1-1, containing a T-DNA insertion 245 bp 5’ of the coding sequence of AVP1, we have demonstrated a severe attenuation of the steady state levels of AVP1 transcripts but not those of AVP2 or AVP3. Likewise, Western analyses of low-density membranes fractionated from avp1-1 mutant plants, using AVP1-specific antibody PAB-AVP1, establish a near-total abolition of the Mr 81,000 AVP1 polypeptide. Critically, from a functional standpoint, vacuolar membrane-associated potassium-activated PPi hydrolytic activity is severely impaired and potassium-activated proton pumping is abolished in the avp1-1 mutants versus the equivalent membrane preparations from wild-type plants. The results of phenotypic screens are consistent with what has been determined of the localization and functionality of AVP1 in that avp1-1 mutants are markedly more sensitive to nitrate in the growth medium than wild-type controls as indicated by chlorosis, growth retardation and eventually necrosis and death, implying that when vacuolar proton ATPase (V-ATPase) activity is diminished by the addition of this type-specific inhibitor to the medium, the requirement for AVP1 for sustained growth is greatest. The most straightforward interpretation of these findings is that AVP1 operates in parallel with the V-ATPase on the vacuolar membrane such that loss of one or other pump necessitates a greater reliance on the other.
