The calcium rhythms of different cell types oscillate with different circadian phases

Abstract

Transgenic tobacco (Nicotiana plumbaginifolia) seedlings containing the Ca(2+)-sensitive luminescent protein aequorin have been shown to exhibit circadian variations in cytosolic calcium. Concomitant measurements of cytosolic and nuclear calcium show that circadian variations in the cytoplasm are not expressed in the nucleus. To investigate whether all cells of transgenic seedlings contribute equally to circadian variations in cytosolic calcium, different promoters eliciting different expression patterns have been placed upstream of aequorin and used for transformation. The circadian peak occurred at different times in the three transgenic lines constructed. Luminescence imaging of these transgenic lines indicated that aequorin was differentially accumulated among the main tissues and cells of the seedlings and overcoat technology with applied epidermal strips indicated that the surface cell layers contribute the vast majority of luminescent light. We conclude that the Ca(2+) rhythmicities of cells and tissues oscillate with distinct differences in phase, that this might represent different underlying cellular control mechanisms and that these observations have significant implications for our understanding and study of Ca(2+) mediated signal transduction in plant cells.

Figure 1

Variation in Ca²⁺-dependent luminescence from transgenic seedlings containing cytosolic or chloroplastic aequorin. Light emission from 10- to 14-d-old seedlings was monitored over 13 d. Individual days are marked with bars. The light-dark regime is indicated at the bottom of the figure.

Figure 2

Variation in Ca²⁺-dependent luminescence from transgenic seedlings containing cytosolic or nuclear aequorin. a, MAQ2.4 plants expressing cytoplasmic aequorin; b, untransformed; c, MAQ7.11 plants expressing nuclear aequorin. The light-dark regime is indicated at the bottom of the figure.

Figure 3

Variation in luminescence from transgenic seedlings through 1 d and inset showing multiple days. Luminescence of MAQ2.4 (a), MAQ15 (b), and MAQ16 (c) seedlings was monitored in continuous white light. The oscillation of 1 d with the mean ± se as a horizontal bar over the peak (n = 20) is shown with further cycles of each rhythm shown inset. The dotted line indicates subjective dawn (CT 0) in each case. d, Shows the effects of the addition of exogenous ABA (at the arrow) on the stomatal behavior of seedlings that have undergone a 2-d pretreatment with 100 μm ABA followed by 1 d free of ABA (●) compared with non-ABA treated controls (○). These pretreatment conditions mimic the treatment of MAQ15 plants.

Figure 4

Variation in seedling apoaequorin content throughout 1 circadian d. MAQ2.4, MAQ15, MAQ16, and wild-type tobacco seedlings were collected throughout one complete 24-h period. After homogenisation and reconstitution, the aequorin was discharged with an excess of CaCl₂.

Figure 5

Patterns of aequorin discharge in the three transgenic lines. Bright field images of MAQ2.4 (a), MAQ15 (c), and MAQ16 (e) transgenic seedlings. b, d, and f, The corresponding luminescent images of active aequorin discharged from each by a freeze-thaw cycle in the presence of 10 mm CaCl₂. Magnification ×20. The bar beneath e represents 1 mm. g and i, Bright field images of MAQ2.4 seedlings; h and j, the corresponding luminescent images at ×200 and ×400, respectively. The bars under g and i represent 100 and 50 μm, respectively. k and m, Bright field images of MAQ15 and MAQ16 plants (arrows denote positions of stomates); l and n, the corresponding luminescent images at ×400 magnification. Bar size is the same as Figure 5 (i) and 50 μm.

Figure 6

Effect of an epidermal “overcoat” on the detection of freeze-thaw aequorin luminescence from MAQ2.4 cotyledons. Bright-field image (a) and the associated luminescence image with the epidermal peel draped over the upper of the two cotyledons (b). While not visible on top of the cotyledon in the bright field picture, traces of the epidermal strip can be distinguished on either side. Bar = 0.4 mm.