Blue light-induced kinetics of H+ and Ca2+ fluxes in etiolated wild-type and phototropin-mutant Arabidopsis seedlings

Abstract

Ion flux kinetics associated with blue light (BL) treatment of two wild types (WTs) and the phot1, phot2 and phot1/phot2 mutants of Arabidopsis were studied by using the MIFE noninvasive ion-selective microelectrode technique. BL induced significant changes in activity of H(+) and Ca(2+) transporters within the first 10 min of BL onset, peaking between 3 and 5 min. In all WT plants and in phot2 mutants, BL induced immediate Ca(2+) influx. In phot1 and phot1/phot2 mutants, net Ca(2+) flux remained steady. It is suggested that PHOT1 regulates Ca(2+) uptake into the cytoplasm from the apoplast. Changes in ion fluxes were measured from cotyledons of intact seedlings and from the cut top of the hypocotyl of decapitated seedlings. Thus the photoreceptors mediating BL-induced Ca(2+) and H(+) fluxes are present in the rest of the decapitated seedling and probably in the cotyledons as well. The H(+) and Ca(2+) flux responses to BL appear not to be linked because, (i) when changes were observed for both ions, Ca(2+) flux changed almost immediately, whereas H(+) flux lagged by about 1.5 min; (ii) in the Wassilewskija ecotype, changes in H(+) fluxes were small. Finally, wave-like changes in Ca(2+) and H(+) concentrations were observed along the cotyledon-hook axis regardless of its orientation to the light.

Figure 1

Three types of experiments used in this study. (A) H⁺ and Ca²⁺ ion selective microelectrodes were located in either the hook or the cotyledon region. Net ion fluxes were measured as described in Materials and Methods. (B) Similar measurements carried out on decapitated seedlings. (C) Three identical electrodes (either H⁺ or Ca²⁺) were separated by about 0.4 mm along the seedling surface (open hook) to study apparent pH and Ca²⁺ waves in response to BL.

Figure 2

Net Ca²⁺ (A) and H⁺ (B) fluxes (influx positive) and pH changes (C) measured from decapitated hypocotyls in response to BL of 15 mmol m⁻² (given as a 10-min pulse at 5 min). One of five to eight representative examples is shown for each WT ecotype Col (closed diamonds) and WS (closed squares), phot1(closed circles), phot2 (open circles), and phot1/phot2 (closed triangles) mutants. Each point represents the ion flux averaged over a 10-s interval. In A, ΔCa²⁺ is the change (shown for phot2) from initial flux to the maximum flux at about 3 min after BL onset. In B, ΔH⁺ is the change for phot1 from initial flux to the minimum at about 4 min after BL onset. In C, ΔpH is the change (shown for phot1/phot2) from the initial pH to the maximum pH value within the first 5 min after BL onset.

Figure 3

Magnitude of changes in net Ca²⁺ (A) and H⁺ (B) flux and external pH (C) measured from cotyledon region (filled columns) and decapitated hypocotyl (open columns) in response to BL given as described in Fig. 2. Error bars are SEM (n = 5–8)

Figure 4

Two typical examples of sequential time delays in changes of Ca²⁺ and H⁺ concentrations in Col seedlings. (Upper) Changes in external pH and Ca²⁺ concentration measured at three different spots, separated by about 0.4 mm in response to unilateral BL from the hook side. (Lower) Responses from other plants having BL given from the cotyledon side. The light treatment was given between 5 and 15 min.