The tonoplast V-type H
+-ATPase (V-ATPase; EC 3.6.1.34) of higher plants
responds by its activity and molecular-subunit fine structure to environmental
parameters, such as temperature and salinity, or to the mode of photosynthesis
in C3–CAM intermediate plants. To test the
hypothesis that the enzyme is responding to varied mineral nutrition in
similar ways, tobacco plants were grown at low (2 mM) and high (40 mM) nitrate
supply, and a group of plants was also transferred from the low to the high
nitrate concentration for 48 h before analysis. Direct correlation of V-ATPase
properties with nitrate regimes and leaf nitrate content were not found, but
indirect effects related to nitrate-affected growth were obtained. Although
the ATP-hydrolysis of isolated tonoplast vesicles was similar under all three
conditions, H + -transport activity and hence the
apparent H + -transport activity/ATP-hydrolysis
coupling ratio of the V-ATPase in native tonoplast vesicles was high at low
growth-limiting nitrate supply and after growth acceleration following the
transfer of plants from low to high nitrate. Immunological differences of the
V-ATPase suggested that the membrane-integral proteolipid subunit
c showed structural differences related to nitrate
nutrition. These differences were revealed by differential cross-reactions
with two polyclonal antisera directed against the V-ATPase of
Kalanchoë daigremontiana, respectively. The
immunological differences could possibly be due to the expression of different
forms of subunit c correlated with actual growth rates
of leaves, which were high at 40 mM and after the transfer from 2 mM to 40 mM,
and low at 2 mM nitrate. These complex responses to variation in nitrogen
nutrition of plants are consistent with the notion in the literature that
nitrate is both a nutrient and a signal for plant growth.
The model plant tobacco (Nicotiana tabacum L.) was chosen for a survey of the subunit composition of the V-ATPase at the protein level. V-ATPase was purified from tobacco leaf cell tonoplasts by solubilization with the nonionic detergent Triton X-100 and immunoprecipitation. In the purified fraction 12 proteins were present. By matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and amino acid sequencing 11 of these polypeptides could be identified as subunits A, B, C, D, F, G, c, d and three different isoforms of subunit E. The polypeptide which could not be identified by MALDI analysis might represent subunit H. The data presented here, for the first time, enable an unequivocal identification of V-ATPase subunits after gel electrophoresis and open the possibility to assign changes in polypeptide composition to variations in respective V-ATPase subunits occurring as a response to environmental conditions or during plant development.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.