Primary inhibition of hypocotyl growth and phototropism depend differently on phototropin-mediated increases in cytoplasmic calcium induced by blue light

Abstract

The phototropin photoreceptors transduce blue-light signals into several physiological and developmental responses in plants. A transient rise in cytoplasmic calcium (Ca2+) that begins within seconds of phototropin 1 (phot1) excitation is believed to be an important element in the transduction pathways leading to one or more of the phot1-dependent responses. The goal of the present work was to determine whether the Ca2+ response was necessary for (a). the inhibition of hypocotyl elongation that develops within minutes of the irradiation, and (b). hypocotyl phototropism (curved growth of the stem in response to asymmetric illumination). After determining that pulses of light delivering photon fluences of between 1 and 1000 micromol m-2 induced growth inhibition mediated by phot1 without significant interference from other photosensory pathways, the effect of blocking the Ca2+ rise was assessed. Treatment of seedlings with a Ca2+ chelator prevented the rise in cytoplasmic Ca2+ and prevented phot1-mediated growth inhibition. However, the same chelator treatment did not impair phot1-mediated phototropism. Thus, it appears that the early, transient rise in cytoplasmic Ca2+ is an important intermediary process in at least one but not all phot1-signaling pathways.

Figure 1.

The phot1 receptor mediated primary growth inhibition. A, The growth rates in response to a single pulse of blue light (10³ μmol m^-2 delivered in 10 s) in etiolated wild-type (white circles), phot1 (white squares), and phot1phot2 (black triangles) mutant seedlings are presented. The dotted line represents the growth rate of seedlings in continuous darkness. The growth kinetics of wild-type seedlings treated with continuous irradiation (10² μmol m^-2 s^-1) are shown for comparison (black circles). All growth rates are normalized to the dark growth rate (set to “1”). Each data point represents the average growth rate of many (>15) independent seedlings. Error bars have been omitted for clarity and do not exceed 0.06. B, The average normalized growth rate 15 min after a single 50 μmol m^-2 blue-light pulse in cryptochrome and phot mutants is shown. The dark growth rate for all genotypes is set to “1” (dashed line). Data points represent the mean growth rate of at least 10 independent seedlings, and error bars represent se.

Figure 2.

The fluence-response characteristics of phot1-mediated growth inhibition. Dark-grown wild-type seedlings (black circles) were irradiated with a single pulse of blue light ranging in fluence from 10^-1 to 10⁴ μmol m^-2 or a mock pulse (D). Treatments were delivered in 10 s or less, except of the 10⁴ treatment which was delivered in 100 s. The inhibition measured in phot1 (white triangles) and phot1phot2 (black triangles) seedlings after treatment with 50, 10³, and 10⁴ μmol m^-2 blue light is presented for comparison. The results are reported as percent inhibition, calculated from the equation (1 - [growth rate at 15 min after blue-light treatment/growth rate at 15 min after a mock pulse] × 100). At least 10 seedlings were measured per fluence per genotype. Error bars represent se.

Figure 3.

BAPTA suppresses blue-light-induced Ca²⁺ influx. A pulse of blue light induced a transient rise in aequorin luminescence ([Ca²⁺]_cyt) that was suppressed by treating seedlings externally with the Ca²⁺ chelator BAPTA. A, The traces shown represent the averages of between 9 and 11 independent trials for each concentration. Error bars have been omitted for clarity. B, The average integrated luminescence (area under the curve in A, minus background) plotted against concentration. Error bars represent se.

Figure 4.

BAPTA specifically impairs phot1-mediated growth inhibition. The magnitude of blue-light-induced, phot1-mediated growth inhibition was assessed in the presence of different concentrations of BAPTA. The results are presented as “percent of normal inhibition,” which represents the magnitude of inhibition in BAPTA-treated seedlings relative to the inhibition measured in control (no BAPTA) seedlings 15 min after a 50 μmol m^-2 blue-light pulse. At least eight seedlings were measured for each BAPTA concentration. Error bars represent se.

Figure 5.

Growth on BAPTA does not affect phototropism. Seedlings were grown vertically on agar plates containing 0 (black circles) or 300 μm BAPTA (white circles) and then were irradiated with continuous unilateral blue light at a fluence rate of 2.3 × 10^-4 μmol m^-2 s^-1. Phototropic curvature was measured as a change in hypocotyl angle as determined from analysis of stacked images captured every 30 min for 120 min. Error bars represent se.