Free calcium ion concentration in the sieve-tube sap of Ricinus communis L. seedlings

Abstract

Changes in free Ca2+ in sieve-tube sap have been proposed to be important in the regulation of phloem transport, and Ca2+-activated protein kinase activity has been described in phloem exudate (S.A. Avdiushko et al. 1997 J Plant Physiol 150: 552–559). Using atomic absorption spectrometry, we have determined that the total Ca2+ concentration in sieve-tube sap from Ricinus seedlings containing the endosperm is about 100 μM (range 80–150 μM). We used three independent methods to determine the free calcium ion concentration in the phloem sap ([Ca2+]p). The first method was to calculate [Ca2+]p from the total Ca2+ concentration, in combination with the binding constants and concentrations of the ionic solutes in phloem sap. The resultant estimate of [Ca2+]p was 63 μM. The second method used the Ca-specific fluorescent dye 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic-acid (FURAPTRA) on exuded sieve-tube sap. Although the sap interfered severely with the fluorescence properties of the dye, Ca2+ titrations enabled a value of [Ca2+]p = 20 μM to be deduced. The third method used Ca2+-selective microelectrodes on exuded sap samples, which gave an average value for [Ca2+]p = 13 μM. No significant change in this value was observed during the sap exudation period. The Ca2+ buffer capacity was determined and the result of about 0.6 mmol · l−1 · pCa−1 displayed excellent agreement with the measured values of free and total Ca2+ concentration in sieve-tube sap. Since the measured values for free Ca2+ are 20- to 100-fold higher than those usually reported for the cytosol of a range of plant cells in resting conditions, it is concluded that either regulation of [Ca2+]p is of limited physiological importance, or that the Ca2+-dependent proteins respond only to relatively high [Ca2+]p. The implications for regulation of cytosolic free Ca2+ in symplastically connected companion cells is discussed.