Red light and calmodulin regulate the expression of the psbA binding protein genes in Chlamydomonas reinhardtii

Abstract

In the unicellular green alga Chlamydomonas reinhardtii, translation of the chloroplast-encoded psbA mRNA is regulated by the light-dependent binding of a nuclear-encoded protein complex (RB38, RB47, RB55 and RB60) to the 5'-untranslated region of the RNA. Despite the absence of any report identifying a red light photoreceptor within this alga, we show that the expression of the rb38, rb47 and rb60 genes, as well as the nuclear-encoded psbO gene that directs the synthesis of OEE1 (oxygen evolving enhancer 1), is differentially regulated by red light. Further elucidation of the signal transduction pathway shows that calmodulin is an important messenger in the signaling cascade that leads to the expression of rb38, rb60 and psbO, and that a chloroplast signal affects rb47 at the translational level. While there may be several factors involved in the cascade of events from the perception of red light to the expression of the rb and psbO genes, our data suggest the involvement of a red light photoreceptor. Future studies will elucidate this receptor and the additional components of this red light signaling expression pathway in C. reinhardtii.

Fig. 1

Accumulation of rb38, rb47, rb60 and psbO RNA in dark- or light-grown wt and y-1 cells. The steady-state levels of total (Total) and polyribosome-associated (Poly) RNA were examined by Northern blot analysis after exposure of cells to constant white light (L) or constant darkness (D).

Fig. 2

Accumulation of rb38, rb47, rb60 and psbO mRNA in response to different light qualities. Wt C. reinhardtii cells were grown in the dark and exposed to white, blue or red light for 0, 20, 40 or 60 min. A 15 μg aliquot of total RNA (A) or 2 μg of polysomal RNA (B) was evaluated using Northern blot analysis.

Fig. 3

Northern blot analysis of rb38, rb47, rb60 and psbO accumulation in y-1 cells after exposure to red light for 0, 20, 40 or 60 min. A 15 μg aliquot of total RNA was assessed.

Fig. 4

Accumulation of RB38, RB47, RB60 and OEE1 protein in response to different light qualities. Wt C. reinhardtii cells were grown in the dark and exposed to white, blue or red light for 0, 20, 40 or 60 min. Equal amounts of soluble protein were separated by size using SDS–PAGE and transferred to nitrocellulose. Blots were decorated with a primary and secondary antibody, followed by alkaline phosphatase staining.

Fig. 5

Accumulation of RB38, RB47, RB60, OEE1 and OEE2 protein in y-1 cells under white light. Cells were grown in the dark and exposed to white light for 0, 20, 40, 60, 120 or 240 min. Equal amounts of soluble protein were separated by size using SDS–PAGE and transferred to nitrocellulose. Blots were decorated with a primary and secondary antibody, followed by alkaline phosphatase staining.

Fig. 6

Accumulation of rb38, rb47, rb60 and psbO after treatment with the CaM antagonist W7. Wt C. reinhardtii cells were grown in the dark and then treated with 50, 25, 11 or 5 μM W7 for 30 min in the dark (D) or in red (R) light. Total RNA accumulation was evaluated using Northern analysis.

Fig. 7

Accumulation of RB38, RB47, RB60 and OEE1 protein after treatment with the CaM antagonist W7. Wt C. reinhardtii cells were grown in the dark and then treated with 50, 25, 11 or 5 μM W7 for 30 min in the dark (D) or in red (R) light. Equal amounts of soluble protein were separated by size using SDS–PAGE and transferred to nitrocellulose. Blots were decorated with a primary and secondary antibody, followed by alkaline phosphatase staining.

Fig. 8

Morphology of C. reinhardtii in the presence and absence of the CaM antagonist W7. Wt C. reinhardtii cells were exposed to red light for 30 min without (A) or with 50 μM (B), 25 μM (C), 11 μM (D) or 5 μM (E) W7. A 50 μM W7 treatment was also carried out for 120 min (F). Cells were viewed using an Olympus BX-60 optical microscope with ×60 magnification. Pictures were taken from each sample using a Hamamatsu digital camera.