Research Report Dr.
Institut fr Biologie III
D-79104 Freiburg i. Br.
against plant PM-H+-ATPase
1. Distribution of Plasma Membrane H+-ATPase
in Elodea Leafs and Stems
antibodies directed to plasma membrane H+-ATPase have been
generated, using purified protein from Zea mays coleoptiles as
antigen (Ltzelschwab, Ph.D. thesis, Freiburg 1990). These antibodies
preferentially recognize cells in the vascular bundles and stomatal guard
cells of Zea mays shoots and Cucurbita pepo hypocotyls. The
antibodies also preferentially recognize one of the two cell layers in
leaves of Elodea canadensis (Ltzelschwab, Ph.D. thesis, Freiburg
1990, Baur et al., 1996, Bot Acta 109, 382-387).
immunofluorescence could be detected in Elodea stems, whereas the
leaves were obviously labelled by H+ATPase antibodies, markedly
more immunofluorescence was seen in the layer of smaller cells on the lower
side of the leaves (Bauer et al. 1996, Bot Acta 109, 382-387). In
parallel experiments using microsomes isolated separately from elodea leaves
and stems, only the leaf microsomes contained ATPase activity with
properties of the plasma membrane H+-ATPase, while microsomes
derived from Elodea stems hardly any activity of this type was
detected. In western blots of leaf microsome preparations a 100 kDa band was
detected by anti-ATPase antibody, whereas only a very faint band was
detected in microsome preparations from Elodea stems.
2. Polypeptides detected by anti-plasma
membrane H+-ATPase antibodies in lower plants
antibodies directed to maize plasma membrane H+-ATPase and
antiserum directed to oat plasma membrane H+-ATPase were used to
detect antigenic molecules in several species of algae. Mikrosomes prepared
from algae were screened for the presence of H+-ATPase antigen
using SDS-PAGE and western blotting. In none of the microsomal preparations
from the algae (20-40 g protein per lane) was a signal of comparable
strength encountered, as in Cucurbita hypocotyl membrane vesicles (4
g per lane) used as positive control. Polypeptides with a molecular weight
around 100 kDa were detected by anti-ATPase antiserum and monoclonal
anti-ATPase antibodies in microsomes from the green algae Spirogyra,
Mougeoutia, and Zygnema.
Polypeptides with a molecular weight at 66 kDa were labelled in microsomes
from Chara with monoclonal anti-ATPase antibodies as well as with
anti-ATPase antiserum. This polypeptide was however of extreme abundance
(strong Coomassie stain!) in the Chara microsomes, so the labelling
by anti-ATPase antibodies was not of very high specificity. Even carefully
monitored inhibition of proteases in Chara microsomes did not yield a
labelled polypetide in the range of 100 kDa. Polypeptides in microsome
preparations from other green as well as brown and red algae species did
show no, or very weak and variable labelling with anti-ATPase antibodies.
Microsomes from Lunularia (a liverworth) did show clearly a 100 kDa
polypeptide labelled, but again distinctly weaker as in the Cucurbita
The experimental work in this report has been financially supported by the Land
Ratajczak R, Luetzelschwab M, Michalke W, Waechter R, Fischer-Schliebs E,
Ullrich CI (2001) Immunolocalization of plasma-membrane H+-ATPase and
tonoplast-type pyrophosphatase in the plasma membrane of the sieve
element-companion cell complex in the stem of Ricinus communis L.
Planta 213: 11-19.
Himly M, Gehwolf R, Kriechbaumer R, Strasser D, Michalke W, Richter K,
Ferreira F, Obermeyer G (2001) Molecular and physiological characterisation
of a 14-3-3 protein from lily pollen grains regulating the activity of the
plasma membrane H+ ATPase during pollen grain germination and tube growth.
Planta 213: 132-141.
J, Michalke W, Hertel R (2001) An auxin transport inhibitor interferes with
unicellular gravitropism in protonemata of the moss Ceratodon purpureus.
Plant Biol. 3: 357-363.
Michalke W, Schikora A. (2002) Proton pumping by tomato roots. Effect of Fe
deficiency and hormones on the activity and distribution of plasma membrane
H+-ATPase in rhizodermal cells. Plant Cell Env 26: 361-370.
Rober-Kleber N, Albrechtov JTP, Fleig S, Huck N, Michalke W, Wagner E,
Speth V, Neuhaus G, Fischer-Iglesias C. (2003) Plasma membrane H+-ATPase is
involved in auxin mediated cell elongation during wheat embryo development.
Plant Physiology 131: 1302-1312.
Sibole, Catalina Cabot, Wolfgang Michalke et al. (2005) Relationship between
expression of the PM H+-ATPase, growth and ion partitioning in
the leaves of salt treated Medicago species. Planta (online