Reetta Kettunen

ATP and light regulate D1 protein modification and degradation Role of D1* in photoinhibition

We have recently shown that during in vivo photoinhibition the D1 protein is degraded via a modified form, designated D1*. Depending on light conditions, the amount of D1* varies in leaves between 0 and 50% of total D1 content. By isolating thylakoids from leaves acclimated to different light levels, and performing photoinhibition experiments on these thylakoids, the following results on D1 protein degradation were obtained: (1) the protease involved in D1 degradation requires activation by light; (ii) neither acceptor nor donor side photoinhibition of PSII induces formation of D1* in vitro; (iii) in isolated thylakoids, the transformation of D1 to D1* can be induced in low light in the presence of ATP, which suggests that D1* is a phosphorylated form of the D1 protein; (iv) D1*, induced either in vivo or in vitro, is much less susceptible to degradation during illumination of isolated thylakoids than the original D1 protein. We suggest that the modification to D1* is a means to prevent disassembly of photodamaged photosystem II complex in appressed membranes.

D1 protein degradation during photoinhibition of intact leaves a modification of the D1 protein precedes degradation

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high‐light modified form that was subsequently degraded.

Degradation Pattern of Photosystem II Reaction Center Protein D1 in Intact Leaves (The Major Photoinhibition-Induced Cleavage Site in D1 Polypeptide Is Located Amino Terminally of the DE Loop)

Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. This cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves.

The rate constant of photoinhibition in vitro is independent of the antenna size of Photosystem II but depends on temperature

Abstract Photoinhibition of Photosystem (PS) II was studied in thylakoid membranes, inside-out and rightside-out thylakoid vesicles derived from appressed and non-appressed membrane regions, respectively, in detergent fractionated PS II membranes and in oxygen evolving PS II core complexes. The preparations were illuminated without added electron acceptors, and care was taken to keep the oxidizing side of PS II in a functional condition during the experiments. The first-order rate constant of photoinhibition, measured under given photon flux density, was similar in all preparations derived from appressed thylakoid regions and independent of the antenna size. This antenna size independence indicates that under photoinhibitory conditions in vitro, when most PS II traps are closed, the probability of finding an exciton in the reaction centre is not much larger than the probability of finding it in the antenna. Spillover of excitation energy from PS II to PS I may be an important factor protecting the PS IIβ of stroma thylakoids from photoinhibition. Photoinhibition in vitro is a first-order reaction even at low temperatures where the degradation of the D1 protein is slow, which demonstrates that the photoinhibited PS II centres do not protect the remaining active ones from photoinhibition, at least not in vitro. The activation energy of photoinhibition in pumpkin thylakoids, as measured between 6 and 25°C, was 15 kJ/mol; the rate constant of photoinhibition in pumpkin thylakoids increased both below 6 and above 25°C.