Measurement of calcium fluxes in plants using ⁴⁵Ca

Abstract

This paper deals with the effect of calcium binding in the cell wall on the measured ⁴⁵Ca influx in Chara corallina Klein ex Will. esk. R.D. Wood. Calcium in the cell wall was in the range 687–1197 (μmol · m⁻² compared to the sap which contained only 144–256 μmol · m⁻². In dilute culture solutions the calcium content of the cell wall was relatively independent of external calcium at concentrations above about 0.1 mol · m⁻³. The half-times for exchange of calcium from ⁴⁵Ca-labelled cell walls varied from 45 min at 0.05 mol · m⁻³ to less than 2 min at 2 mol · m⁻³. The effectiveness of other cations in displacing calcium from cell walls was in the order La > Zn > Co > Ni > Mg. Rinsing of ⁴⁵Ca-labelled cell walls in 2 mol · m⁻³ LaCl₃ for 20 min removed more than 99% of the bound ⁴⁵Ca. However, the residual ⁴⁵Ca activity in isolated cell walls following La³⁺ rinsing was similar to that in whole cells. It is concluded that in whole cells ⁴⁵Ca influx cannot normally be distinguished from extracellular binding of calcium. Methods are described for the measurement of ⁴⁵Ca fluxes in charophyte cells by isolation of intracellular ⁴⁵Ca after the uptake period using techniques which avoid contamination from the large amount of tracer bound in the cell wall. At an external calcium concentration of 1 mol · m⁻³, the plasmalemma influx was approx. 0.2 nmol · m⁻² · s⁻¹ of which about half entered the vacuole and half was effluxed back into the external solution. The cytoplasm filled with calcium with a half-time of 40–50 min with an ‘apparent’ pool size of 50 mmol · m⁻³. After 2 h the net flux to the cell was almost the same as the vacuolar flux. The fluxes reported are an order of magnitude lower than previously reported calcium fluxes in plants.