Induction and Regulation of Expression of a Low-CO2-Induced Mitochondrial Carbonic Anhydrase in Chlamydomonas reinhardtii

Abstract

The time course of and the influence of light intensity and light quality on the induction of a mitochondrial carbonic anhydrase (CA) in the unicellular green alga Chlamydomonas reinhardtii was characterized using western and northern blots. This CA was expressed only under low-CO2 conditions (ambient air). In asynchronously grown cells, the mRNA was detected 15 min after transfer from air containing 5% CO2 to ambient air, and the 21-kD polypeptide was detected on western blots after 1 h. When transferred back to air containing 5% CO2, the mRNA disappeared within 1 h and the polypeptide was degraded within 3 d. Photosynthesis was required for the induction in asynchronous cultures. The induction increased with light up to 500 mumol m-2 s-1, where saturation occurred. In cells grown synchronously, however, expression of the mitochondrial CA was also detected in darkness. Under such conditions the expression followed a circadian rhythm, with mRNA appearing in the dark 30 min before the light was turned on. Algae left in darkness continued this rhythm for several days.

Figure 1

Time-course experiments of the induction and degradation of mtCA. A, Northern analysis of the relative amounts of the mtCA mRNA. High-CO₂-grown cells were transferred to low-CO₂ conditions at time 0. After 24 h, the culture was transferred back to high-CO₂ conditions. The triangle represents cells left on low CO₂ for 1 week. All lanes on the northern blot contained 5 μg of total RNA. B, Western blot of the induction of mtCA. Numbers represent hours after transfer to low CO₂. All lanes contained 0.5 μg of protein. C, Western blot of the degradation of mtCA. Numbers represent hours after transfer from low CO₂ to high CO₂. All lanes contained 0.5 μg of protein.

Figure 2

Western-blot analysis of the induction and degradation of mtCA under mixotrophic and phototrophic growth. A, Amount of mtCA in cells adapted to low-CO₂ conditions for 4 or 8 h in the absence (−Ac) or presence (+Ac) of acetate in the growth medium. All lanes contained 0.5 μg of protein. B, Amount of mtCA in low-CO₂-adapted phototrophic cells after transfer to three different growth conditions for 3 d.

Figure 3

Induction of mtCA under different light intensities. A, The relative amount of mtCA mRNA after transfer to low-CO₂ conditions for 15 (▴), 30 (•), and 60 (▪) min under different light intensities. All lanes on the northern blot contained 5 μg of total RNA. B, Western blot of mtCA after 2 h of induction to low-CO₂ conditions under different light intensities. The light intensities were: lane 1, 25; lane 2, 75; lane 3, 150; lane 4, 300; lane 5, 450; and lane 6, 1200 μmol m⁻² s⁻¹. All lanes contained 0.5 μg of protein.

Figure 4

The induction of mtCA under red light. Western-blot analysis of samples withdrawn from the culture before (5% CO₂) and after (air) 4 h of induction under red light (684 nm) of 10 μmol m⁻² s⁻¹. Both lanes contained 0.5 μg of protein.

Figure 5

Northern-blot analyses of mtCA mRNA levels in synchronously grown cells. After three light/dark cycles, the culture was transferred to low CO₂ at the beginning of the fourth dark period. A, Cells left under a light/dark regime. B, Cells transferred to continuous darkness. Samples for mRNA analyses were withdrawn 1, 6, and 11 h into the light period and 1, 6, and 11.5 h into the dark period. All lanes on the northern blot contained 5 μg of total RNA.