Characterization and Effect of Light on the Plasma Membrane H⁺-ATPase of Bean Leaves

Abstract

Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the Km was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for I day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, Km, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however, both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves. environment and hyperpolarizing the membrane potential (19). The resulting electrochemical gradient facilitates the uptake of ions, amino acids, and sugars (19). The lightinduced proton excretion from bean leaf cells is presumed to be mediated by the PM H+-ATPase. Light might stimulate H+ excretion in at least four ways: (a) by increasing the supply of substrate (ATP); (b) by altering the cytoplasmic pH; (c) by increasing the activity of the existing enzyme (e.g. by phosphorylation of the enzyme); or (d) by increasing the amount of H+-ATPase in the PM. Both of the latter two mechanisms should result in an enhanced activity of the PM ATPase in isolated PM vesicles.Although the PM ATPase from roots and stems has been extensively studied (19), the enzyme in leaves has received only limited attention. In the study reported here, we have used the aqueous phase partitioning method (7) to isolate highly purified PM vesicles from the primary leaves of Phaseolus vulgaris L. and have characterized the ATPase in these vesicles. We have used this system to show that while light promotes the ATPase-mediated proton excretion (24), a lightinduced change in ATPase activity cannot explain this effect. MATERIALS AND METHODS

Comparison of developmental gradients for growth, ATPase, and fusicoccin-binding activity in mung bean hypocotyls.

“…In tobacco calli, the PM ATPase activity changed with time, but was not correlated with the growth rate (2). In bean leaf, the PM ATPase decreased by 50% in light-grown versus dark-grown leaves (15). In the present study, the ATPase activity was not correlated with either the in vivo elongation rate or with the capacity of the isolated sections to grow.…”

“…The upper cut was the cut that removed the cotyledons, then sequential sections were cut. Briefly, section a includes the whole hook region; section b is the region 0.0 to 7.5 mm below the hook; section c is the region cut 7.5 to 15.0 mm from the hook; section d is the region 15 (23). Goat anti-rabbit second antibody coupled to alkaline phosphatase (Bio-Rad, 1 mg/ mL concentration) was utilized at 1/5000 dilution.…”

Isolation and characterization of P-type H+-ATPase genes from potato

“…It seems therefore important to study regulatory mechanisms that control H + -ATPase expression and activity at the molecular level. H + -ATPases are highly regulated by a number of endogenous and exogenous factors, for example by hormones, phytotoxins and physical factors such as temperature and light [3,13,17,21,40]. A number of possible mechanisms for regulation at the post-translational level have been described [e.g.…”

Light-Induced Changes in Hydrogen, Calcium, Potassium, and Chloride Ion Fluxes and Concentrations from the Mesophyll and Epidermal Tissues of Bean Leaves. Understanding the Ionic Basis of Light-Induced Bioelectrogenesis1

“…Enhanced H ϩ extrusion induced by light may be an important factor promoting leaf enlargement through an increase in wall extensibility (Linnemeyer et al, 1990;Elzenga et al, 1995). Activation of the H ϩ pump by photosynthesis might also be relevant to phloem loading and the removal of photosynthate from the mesophyll cells (Marrè et al, 1989).…”