Abscisic acid induced a negative geotropic response in dark-incubated Chlamydomonas reinhardtii

Abstract

The phytohormone abscisic acid (ABA) plays a role in stresses that alter plant water status and may also regulate root gravitropism and hydrotropism. ABA also exists in the aquatic algal progenitors of land plants, but other than its involvement in stress responses, its physiological role in these microorganisms remains elusive. We show that exogenous ABA significantly altered the HCO₃^- uptake of Chamydomonas reinhardtii in a light-intensity-dependent manner. In high light ABA enhanced HCO₃^- uptake, while under low light uptake was diminished. In the dark, ABA induced a negative geotropic movement of the algae to an extent dependent on the time of sampling during the light/dark cycle. The algae also showed a differential, light-dependent directional taxis response to a fixed ABA source, moving horizontally towards the source in the light and away in the dark. We conclude that light and ABA signal competitively in order for algae to position themselves in the water column to minimise photo-oxidative stress and optimise photosynthetic efficiency. We suggest that the development of this response mechanism in motile algae may have been an important step in the evolution of terrestrial plants and that its retention therein strongly implicates ABA in the regulation of their relevant tropisms.

Figure 1

Exogenous abscisic (ABA) acid differentially altered HCO₃⁻ uptake by C. reinhardtii in a light-intensity-dependent manner. Mid log phase (A₇₅₀ = 0.3) algal cultures were immobilised in alginate gel beads containing the concentrations of ABA indicated and in (A) were incubated for 1 h under different light intensities (7.6 to 223.2 µmoles photons m⁻² s⁻¹) with bicarbonate indicator buffer in TAP media. Depletion of HCO₃⁻ in the media was monitored as the change in absorbance in the indicator buffer at 550 nm after the incubation period with conversion to the change in the concentration of HCO₃⁻ in the TAP media by reference to a standard calibration. Shown are the mean δHCO₃⁻ (mM) h⁻¹ of n = 5 replicates with errors as the 95% confidence intervals around the means in each case. In (B) mid log phase (A₇₅₀ = 0.3) algal cultures were sampled 1 h into the photoperiod and subsequently exposed for 1 h to the different light intensities indicated. Algal cells were pelleted from n = 3 replicate 25 mL aliquots of culture. Pellets were extracted and assessed for ABA content by competitive ELISA (MyBioSource Inc.). Data are shown as the mean ABA content cell⁻¹ with error bars shown as +/− the 95% confidence interval around the mean in each case.

Figure 2

Exogenous abscisic acid (ABA) induced a negative geotropic movement response in C. reinhardtii CC-1021. Algal cultures were grown under a 16 h photoperiod to the mid log phase (A₇₅₀ = 0.3) growth stage and were sampled at the time points indicated over a 24 h period where the 16 h photoperiod commenced at time = 0 h. Fully dispersed algal samples were placed in measuring cylinders +/−50 µM ABA to form a 10 cm vertical water column and for 50 min were either illuminated from above with high-light (319.8 µmoles photons m⁻² s⁻¹) (A,B) or placed in the dark (C,D). Shown is a representative image of n = 5 replicate experiments indicating the positions attained by the algae immediately following the period of incubation in either the light or dark.

Figure 3

Exogenous abscisic acid (ABA) induced a negative geotropic movement response in C. reinhardtii CC-1021. Algal cultures were grown under a 16 h photoperiod to the mid log phase (A₇₅₀ = 0.3) growth stage and were sampled at (a) 1 h, (b) 8 h, (c) 15 h, (d) 20 h and (e) 23 h during a 24 h period where the 16 h photoperiod commenced at time = 0 h. Fully dispersed algal samples were placed in measuring cylinders +/−50 µM ABA to form 10 cm vertical culture columns and for 50 min were either illuminated from above with high-light (319.8 µmoles photons m⁻² s⁻¹) or placed in the dark. After incubation the A₇₅₀ at the depths indicated was measured and the relative cell density ratios at each depth were determined within each treatment. Shown are the mean data of n = 5 replicate experiments +/− the 95% confidence intervals around the mean in each case.

Figure 4

In the absence of light exogenous abscisic acid (ABA) failed to induce a negative geotropic movement in the C. reinhardtii mutant strains CC-477 and CC-2492 that lack functional flagella. Algal cultures of the mutant strains were grown under a 16 h photoperiod to the mid log phase (A₇₅₀ = 0.3) growth stage and were sampled 1 h into the photoperiod. Fully dispersed algal samples were placed in measuring cylinders +/−50 µM ABA to form 10 cm vertical culture columns and for 50 min were incubated in the dark. After incubation the A₇₅₀ at the depths indicated was measured and the relative cell density ratios at each depth were determined within each treatment. Shown in (A) are the means of data from n = 4 replicate experiments for each mutant +/− the 95% confidence intervals around the means in each case. Shown in (B) is a representative image of the positions attained by the algae immediately following the period of incubation.

Figure 5

Exogenous 1-naphthaleneacetic acid (NAA), 1-aminocyclopropane-1-carboxylic acid (ACC) and hydrogen peroxide (H₂O₂) did not significantly affect the vertical positioning of C. reinhardtii CC-1021 in the dark. Algal cultures were grown under a 16 h photoperiod to the mid log phase (A₇₅₀ = 0.3) growth stage and were sampled 8 h into the 16 h photoperiod. Fully dispersed algal samples were placed in measuring cylinders +/− either 50 µM (a) NAA, (b) H₂O₂ or (c) ACC, ABA and ACC + ABA to form 10 cm vertical culture columns and for 50 min were either illuminated from above with high-light (319.8 µmoles photons m⁻² s⁻¹) or placed in the dark. After incubation the A₇₅₀ at the depths indicated were measured and the relative cell density ratios for each depth were determined within each treatment. Shown are the mean data of either n = 5 (a) or n = 3 (b,c) replicate experiments +/− the 95% confidence intervals around the means in each case.

Figure 6

C. reinhardtii CC1021 showed opposite taxis to a fixed source of exogenous abscisic acid (ABA) in the light and dark. Algal cultures were grown under a 16 h photoperiod to the mid log phase (A₇₅₀ = 0.3) growth stage and were sampled mid photoperiod. Fully dispersed samples of the algal culture were placed in open top 1.5 × 1.5 × 12 cm light-permeable acrylic troughs with a plug of agarose containing 1 mM ABA at the end indicated and were immediately placed horizontally either in the light (45 µmoles photons m⁻² s⁻¹) or the dark for 50 min. After incubation samples were simultaneously taken at incremental distances from the agar plug and their A₇₅₀ measured. Shown in (A) is a representative image indicating the positions attained by the algae immediately following the incubation period. The treatment-dependent relative cell densities at increasing distances from the agar plugs is shown in (B). Significant (P < 0.05 by χ² tests of goodness of fit) differences in the proportion of algal cells found at various distances from the ABA-containing agar plug between the light and dark incubations of the algae are indicated^(*).

Figure 7

Endogenous abscisic acid (ABA) levels in C. reinhardtii CC-1021. Algal cultures were grown under a 16 h photoperiod to the mid log phase (A₇₅₀ = 0.3) growth stage and were sampled at the time points indicated over a subsequent 24 h period where the 16 h photoperiod commenced at time = 0 h. Algal cells were pelleted from n = 3 replicate 25 mL aliquots of culture. At each sampling point the A₇₅₀ of the cultures were determined to assess cell numbers. Pellets were extracted and assessed for ABA content by competitive ELISA (MyBioSource Inc.). Data are shown as the mean ABA content cell⁻¹ with error bars shown as +/− the 95% confidence interval around the mean in each case. Selected significant (P < 0.05 by 2 sample t-test) pairwise differences in mean ABA levels are indicated^{(a to d)}.