The phosphorylation status of the chloroplast protein kinase STN7 of Arabidopsis affects its turnover

Abstract

The chloroplast serine-threonine protein kinase STN7 of Arabidopsis (Arabidopsis thaliana) is required for the phosphorylation of the light-harvesting system of photosystem II and for state transitions, a process that allows the photosynthetic machinery to balance the light excitation energy between photosystem II and photosystem I and thereby to optimize the photosynthetic yield. Because the STN7 protein kinase of Arabidopsis is known to be phosphorylated at four serine-threonine residues, we have changed these residues by site-directed mutagenesis to alanine (STN7-4A) or aspartic acid (STN7-4D) to assess the role of these phosphorylation events. The corresponding mutants were still able to phosphorylate the light-harvesting system of photosystem II and to perform state transitions. Moreover, we noticed a marked decrease in the level of the STN7 kinase in the wild-type strain under prolonged state 1 conditions that no longer occurs in the STN7-4D mutant. The results suggest a possible role of phosphorylation of the STN7 kinase in regulating its turnover.

Figure 1.

Phosphorylation sites in STN7. Top part: Structure of STN7 with the transmembrane domain (TM) and the kinase domain. Bottom part: The 50 C-terminal amino acids of STN7 from Arabidopsis and rice are compared. The four phosphorylation sites of Arabidopsis Ser-526, Thr-537, Thr-539, and Thr-541 are indicated and the changes in STN7-4A and STN7-4D are shown. The two conserved Cys in the N-terminal part are indicated.

Figure 2.

Expression of STN7 in wild-type and STN7 mutant lines. A, RNA-blot analysis of STN7 with 10 μg of total RNA from stn7, Col-0, and two STN7-4A lines (4A1, 4A2) and three STN7-4D lines (4D1, 4D2, 4D3). The arrow indicates the STN7 RNA that is absent in the stn7 mutant. The nature of the lower band that is also detected in the stn7 mutant is not known. Actin mRNA was used as a loading control. Size markers are indicated in nucleotides. B, Ethidium bromide (EB) staining of the gel used in A with size markers. C, Immunoblot analysis of STN7 in stn7, Col-0, four STN7-4A lines, and four STN7-4D lines. Equal loading was verified with D1 antibodies. Size marker is indicated in kD.

Figure 3.

A, Phosphorylation of LHCII is not affected in the STN7 mutant lines during state transitions. Two-week-old plants grown under 8-h-light/16-h-dark cycles were illuminated with far-red light for 40 min (FR) and then with blue light for 15 (B15) and 30 min (B30). Total proteins from Col-0, STN7-4A, and STN7-4D were examined by SDS-PAGE and immunoblotting with anti-P-Thr and STN7 antibodies. Size markers are indicated in kD. B, Transition from state 1 to state 2 occurs normally in STN7-4A and STN7-4D. Two- or 3-week-old plants were first illuminated with far-red light for 30 min (state 1). They were subsequently illuminated with blue light for 30 min (state 2). Seedlings were quick frozen in liquid nitrogen and their fluorescence emission spectra were determined in state 1 and state 2. WT, Col-0; F, fluorescence; a.u., arbitrary units. The spectra were normalized for the emission peak at 680 nm.

Figure 4.

The level of STN7 decreases under state 1 conditions. Two-week-old plants grown under continuous light were illuminated with blue light (B) for 40 min (state 2) and subsequently subjected to far-red light (FR) for 24 h (state 1). The plants were returned to blue light for 2 and 24 h. At indicated time intervals proteins were extracted and analyzed by SDS-PAGE and immunoblotting with STN7 and DET3 antibodies. The latter is a cytoplasmic protein that was used as loading control. Appropriate dilutions of the sample obtained after 24 h of blue light were performed to estimate the decrease of STN7 under FR light.

Figure 5.

The decrease of STN7 under state 1 conditions is blocked in the STN7-4D lines. Two-week-old seedlings grown under continuous light were illuminated with blue light (B) and transferred to far-red light (FR) as indicated and subsequently returned to blue light. Two STN7-4A and three STN7-4D lines were used. At indicated time intervals proteins were extracted and analyzed by SDS-PAGE and immunoblotting with STN7 and DET3 antibodies. Extracts from stn7 were used to test the specificity of the STN7 antiserum. A similar analysis was performed with the pph1 mutant line.