Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study

Abstract

Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells.

Keywords: Arabidopsis; blue light; calcium signaling; chloroplast movements; mesophyll cells; phototropin2.; thaliana.

Fig. 1.

(A) Example X-ray spectra obtained from a selected rectangular region of the periphery of an Arabidopsis wild-type mesophyll cell (including the cell wall and a fragment of cytoplasm), which contains precipitates of calcium and antimony. The relative content (Weight %) of individual elements was calculated on the basis of the peak area (arrow) characteristic of the calcium–Kα line (Ca K) and antimony–Lα line (Sb L). The error value quoted is sigma, which is the statistical error for the calculated Wt%. (B) An example map showing calcium (yellow) and antimony (red) localization within the analyzed cells. (C) Control X-ray spectra obtained from a selected rectangular region of the periphery of a mesophyll cell (including the cell wall and a fragment of cytoplasm) not treated with potassium pyroantimonate. Chl, chloroplast; IS, intercellular space; V, vacuole.

Fig. 2.

(A–D) The localization of calcium and antimony precipitates in Arabidopsis wild-type mesophyll cells in darkness. Arrows indicate precipitates of calcium and antimony at the cell wall; the white arrowhead precipitates in the cytosol; a double arrow precipitates in the vacuole; and black arrowheads precipitates adjacent to the chloroplast envelope and the tonoplast. CW, cell wall, Chl, chloroplast; IS, intercellular space; V, vacuole.

Fig. 3.

(A, B) The localization of calcium and antimony precipitates in Arabidopsis wild-type mesophyll cells after 3min blue light treatment of 100 μmol m⁻² s⁻¹. Cross-section of the precipitates (C, D) perpendicular and (F) parallel to the cell wall plane. (E) The 3D model of the precipitate created from (D). Arrows indicate precipitates of calcium and antimony at the cell wall; white arrowheads precipitates in the middle lamella; and black arrowheads precipitates adjacent to the chloroplast envelope and the tonoplast. A long white arrow indicates precipitates on the tonoplast directed towards the vacuole interior; a short white arrow precipitates on the outer membrane of the chloroplast envelope pointing towards the cytosol; and a medium sized arrow indicates precipitates in the cytosol. CW, cell wall, Chl, chloroplast; IS, intercellular space; V, vacuole.

Fig. 4.

(A–D) The localization of calcium and antimony precipitates in Arabidopsis wild-type mesophyll cells after 3min red light treatment of 100 μmol m⁻² s⁻¹. Arrows indicate precipitates of calcium and antimony at the cell wall. Chl, chloroplast; IS, intercellular space; V, vacuole.

Fig. 5.

The localization of calcium and antimony precipitates in mesophyll cells of the Arabidopsis phototropin mutants phot2 and phot1phot2 in darkness, after 3min of blue or red light treatment of 100 μmol m⁻² s⁻¹. Chl, chloroplast; IS, intercellular space; V, vacuole.

Fig. 6.

The relative content of (A) calcium and (B) antimony in precipitates at the periphery of mesophyll cells (including the cell wall and a fragment of cytoplasm) of the Arabidopsis wild type and phot2, phot1phot2 mutants. The relative content of a given element in precipitates from dark-adapted leaves (black bars), leaves after blue light irradiation (blue bars), and leaves after red light irradiation (red bars). Each bar shows an average of 5–9 spectra of cell regions obtained from leaves harvested from 2–4 independent plant batches. Error bars indicate the SE. *P=0.01–0.05; **P=0.001–0.01; ***P <0.001.

Fig. 7.

The surface area of precipitate cross-sections outside the cell walls (A) facing the intercellular space or (B) localized in the vacuole at the tonoplast in wild-type, phot2, and phot1phot2 mutant Arabidopsis mesophyll cells. The area was measured in dark-adapted leaves (black bars), blue light-irradiated leaves (blue bars), and red light-irradiated leaves (red bars). Each bar shows an average of at least five images of mesophyll obtained from leaves harvested from 2–4 independent plant batches. In (B), the bar for the wild-type red light group is missing as no precipitates at the tonoplast were observed in these conditions. Error bars indicate the SE. *P=0.01–0.05; **P=0.001–0.01; ***P <0.001.