Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from squash roots

Abstract

Boron is an essential micronutrient for plant growth and the boron content of plants differs greatly, but the mechanism(s) of its uptake into cells is not known. Boron is present in the soil solution as boric acid and it is in this form that it enters the roots. We determined the boron permeability coefficient of purified plasma membrane vesicles obtained from squash (Cucurbita pepo) roots and found it to be 3 x 10(-7) +/-1.4 x 10(-8) cm s(-1), six times higher than the permeability of microsomal vesicles. Boric acid permeation of the plasma membrane vesicles was partially inhibited (30%-39%) by mercuric chloride and phloretin, a non-specific channel blocker. The inhibition by mercuric chloride was readily reversible by 2-mercaptoethanol. The energy of activation for boron transport into the plasma membrane vesicles was 10.2 kcal mol(-1). Together these data indicate that boron enters plant cells in part by passive diffusion through the lipid bilayer of the plasma membrane and in part through proteinaceous channels. Expression of the major intrinsic protein (MIP) PIP1 in Xenopus laevis oocytes resulted in a 30% increase in the boron permeability of the oocytes. Other MIPs tested (PIP3, MLM1, and GlpF) did not have this effect. We postulate that certain MIPs, like those that have recently been shown to transport small neutral solutes, may also be the channels through which boron enters plant cells.

Figure 1

Profile of the diameter of vesicles isolated from squash roots and used to determine the permeability of boric acid. The diameter was determined with dynamic light scattering. The preparation consisted of a single population of vesicles allowing accurate measurements of the permeability.

Figure 2

Change in light scattering intensity of microsomal vesicles isolated from squash roots as a result of the exposure to a transmembrane osmotic gradient following the addition of 400 mm boric acid to the external solution. The second part of the curve was fitted into a single exponential (r² = 0.99).

Figure 3

Change in light scattering intensity of microsomal vesicles isolated from squash roots as a result of the exposure to a transmembrane osmotic gradient following the addition of 200 mm Suc to the external solution. The data were fitted into a single exponential (r² = 0.97).

Figure 4

Effect of temperature on the permeability of boric acid across plasma membranes isolated from squash roots. The Ea calculated from the slope of the curve multiplied by 1.986 as described by Agre et al. (1999) and was 10.239 kcal mol⁻¹. The experiment was repeated twice and five replications were used in each treatment.

Figure 5

P_f of oocytes injected with cRNA from the MIP proteins PIP1, PIP3 (from maize), NLM1 (from Arabidopsis), and GlpF (from E. coli) and also injected with water (diethylprocarbonate treated). The values of P_f were calculated from volume changes of individual oocytes when they were exposed to hypotonic solution. The experiment was repeated three times and six to eight replicates were used in every treatment.

Figure 6

Boron uptake by oocytes injected with cRNAs from the MIP proteins PIP1, PIP3 (from maize), NLM1 (from Arabidopsis), and GlpF (from E. coli) and also injected with water (diethylprocarbonate treated). The experiment was repeated three times and four replicates were used in every treatment. Ten oocytes were used in each replicate. Double asterisk indicates statistically significant difference between the water-injected oocytes and the PIP1.