Chloroplast Ca2+ Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes

Abstract

Chloroplasts require a fine-tuned control of their internal Ca²⁺ concentration, which is crucial for many aspects of photosynthesis and for other chloroplast-localized processes. Increasing evidence suggests that calcium regulation within chloroplasts also may influence Ca²⁺ signaling pathways in the cytosol. To investigate the involvement of thylakoids in Ca²⁺ homeostasis and in the modulation of chloroplast Ca²⁺ signals in vivo, we targeted the bioluminescent Ca²⁺ reporter aequorin as a YFP fusion to the lumen and the stromal surface of thylakoids in Arabidopsis (Arabidopsis thaliana). Thylakoid localization of aequorin-based probes in stably transformed lines was confirmed by confocal microscopy, immunogold labeling, and biochemical analyses. In resting conditions in the dark, free Ca²⁺ levels in the thylakoid lumen were maintained at about 0.5 μm, which was a 3- to 5-fold higher concentration than in the stroma. Monitoring of chloroplast Ca²⁺ dynamics in different intrachloroplast subcompartments (stroma, thylakoid membrane, and thylakoid lumen) revealed the occurrence of stimulus-specific Ca²⁺ signals, characterized by unique kinetic parameters. Oxidative and salt stresses initiated pronounced free Ca²⁺ changes in the thylakoid lumen. Localized Ca²⁺ increases also were observed on the thylakoid membrane surface, mirroring transient Ca²⁺ changes observed for the bulk stroma, but with specific Ca²⁺ dynamics. Moreover, evidence was obtained for dark-stimulated intrathylakoid Ca²⁺ changes, suggesting a new scenario for light-to-dark-induced Ca²⁺ fluxes inside chloroplasts. Hence, thylakoid-targeted aequorin reporters can provide new insights into chloroplast Ca²⁺ storage and signal transduction. These probes represent novel tools with which to investigate the role of thylakoids in Ca²⁺ signaling networks within chloroplasts and plant cells.

Figure 1.

Overview of the cloning strategy for the creation of expression vectors targeting YA to the thylakoid lumen and thylakoid membrane, and analysis of aequorin expression in Arabidopsis transgenic lines. A and B, The targeting sequences for the thylakoid lumen (A) and for the thylakoid membrane (B) were cloned into an expression cassette in front of YA using the restriction enzymes ApaI and NotI. The complete expression cassettes were transferred subsequently into the binary vector pBIN19 carrying Basta resistance. C and D, Analysis of aequorin expression by RT-PCR (C) and immunoblotting (D) in Arabidopsis transgenic lines stably transformed with the constructs encoding TL-YA and TM-YA. For RT-PCR analyses, actin was used as a housekeeping gene. For immunoblot analyses, total protein extracts (50 μg) were separated by 10% SDS-PAGE, transferred to PVDF, and incubated with an anti-aequorin antibody (diluted 1:10,000). Black and white arrowheads indicate the YA chimeras targeted to the thylakoid lumen and the thylakoid membrane, respectively. The black arrow indicates the YA targeted to the cytosol, excluding the nucleus, in the Arabidopsis line CPK17_G2A-NES-YA (Cyt-YA), used here as a positive control.

Figure 2.

Phenotype, chloroplast ultrastructure, and photosynthetic activity of Arabidopsis transgenic lines stably expressing TL-YA and TM-YA. A, Representative photographs of 4-week-old seedlings of TL-YA, TM-YA, and wild-type (WT) lines. B, Transmission electron microscopy (TEM) observations of chloroplast ultrastructure, showing good preservation of thylakoids. Bars = 1 µm (left column) and 250 nm (right column). C, Pulse amplitude modulation imaging analyses of Arabidopsis seedlings at 4, 5, and 6 weeks. Data are means ± se of five independent experiments. No significant differences were observed (Student’s t test).

Figure 3.

Confocal microscopy analyses, immunogold labeling, and biochemical analyses of Arabidopsis seedlings of the TL-YA and TM-YA lines. A, Fluorescence microscopy images of mesophyll cells of Arabidopsis seedling leaves with a YFP filter and a chlorophyll filter. An overlay of the two channels also is shown. Bars = 25 µm (top row) and 5 µm (bottom row). B, Immunocytochemical analyses of aequorin subcellular localization. The wild-type (WT) line and a line expressing YA in the stroma (Str-YA) were used as negative and positive controls, respectively. White arrowheads indicate gold particles. Bars = 100 nm. S, Starch granule; Str, stroma; Th, thylakoids. C, Quantitative analyses of immunogold-labeled particles. Data are means ± se of 40 different fields from three biological replicates. D, Immunoblot analyses of isolated thylakoids, incubated in the absence (−) or presence (+) of 0.1 µg µL⁻¹ thermolysin, as indicated. Isolated thylakoids corresponding to 50 µg of protein were probed with an anti-aequorin antibody (top gel). Equal loading was confirmed by Ponceau Red staining of the blot membrane (bottom gel). Black and white arrowheads indicate YA chimeras targeted to the thylakoid lumen and the thylakoid membrane, respectively.

Figure 4.

Monitoring of subchloroplast free [Ca²⁺] in Arabidopsis cell suspension cultures in response to environmental stimuli. Ca²⁺ measurements were conducted in Str-YA (blue traces), TM-YA (violet traces), and TL-YA (green traces) lines. A to C, In resting conditions. D to F, In response to 10 mm H₂O₂. G to I, In response to 0.3 m NaCl. J to L, In response to 20 µg mL⁻¹ OGs. In A, D, G, and J, data are presented as means ± se (black shading) of 15 traces obtained from 15 aliquots of suspension-cultured cells derived from five independent growth replicates. Arrows indicate the time of stimulation (100 s). B, C, E, F, H, I, K, and L show statistical analyses of Ca²⁺ levels recorded in the different transgenic lines at two different time points: after 150 s (B, E, and K) and 600 s (C, F, I, and L). In H, the maximum [Ca²⁺] (at the peak) is reported. Bars labeled with different letters differ significantly (P < 0.05, Student’s t test).

Figure 5.

Monitoring of subchloroplast free [Ca²⁺] in Arabidopsis seedlings in response to environmental stimuli. Ca²⁺ measurements were conducted in Str-YA (blue traces), TM-YA (violet traces), and TL-YA (green traces) lines. A to C, In resting conditions. D to F, In response to 10 mm H₂O₂. G to I, In response to 0.3 m NaCl. J to L, In response to 20 µg mL⁻¹ OGs. In A, D, G, and J, data are presented as means ± se (black shading) of 15 traces obtained from 15 different plants derived from five independent growth replicates. Arrows indicate the time of stimulation (100 s). In the inset in G, a magnification of the region between 150 and 400 s is shown. B, C, E, F, H, I, K, and L show statistical analyses of Ca²⁺ levels recorded after 150 s (B, E, and K), after 600 s (C, F, I, and L), and at the peak (H). Bars labeled with different letters differ significantly (P < 0.05, Student’s t test).

Figure 6.

Measurement of Ca²⁺ fluxes in response to light-to-dark transition. Ca²⁺ assays were conducted in Arabidopsis seedlings stably expressing YA chimeras in different subcellular locations: stroma (A; blue trace), thylakoid lumen (B; green trace), thylakoid membrane (C; violet trace), cytosol (D; gray trace), and cytosolic surface of the plastid outer envelope (E; brown trace). Data are presented as means ± se (black shading) of 15 traces obtained from 15 different plants derived from five independent growth replicates. In all graphs, the lights-off stimulus starts at time 0.