Photosystem II Core Phosphorylation Heterogeneity, Differential Herbicide Binding, and Regulation of Electron Transfer in Photosystem II Preparations from Spinach

Abstract

The effect of photosystem 11 core phosphorylation on the secondary quinone acceptor of photosystem 11 (QB) domain environment was analyzed by comparative herbicide-binding studies with photosystem 11 preparations from spinach (Spinacia oleracea L. leads to a decreased rate of PSII electron transfer at saturating light intensities (17-19). Further, it has been proposed that protein phosphorylation increases the ability of the QB pocket to bind herbicides (29), which may confer protection against photoinhibition (20). However, the inhibition of the Hill reaction by herbicides in phosphorylated membranes has been related to a secondary effect on the reduction of PQ rather than to an increased affinity for herbicides (17,18). The effect of protein phosphorylation on the function of the QB site, therefore, is not clear.The presence of heterogeneous PSII core populations has been recently reported (11). These PSII cores have been shown to have different capacities to bind dinoseb (9), suggesting that protein phosphorylation may reduce the ability of the QB pocket to bind herbicides.The work presented here was designed to clarify the effect of phosphorylation on the herbicide-PQ binding domain and to distinguish the effects due to the reduction of the PQ pool. The results indicate that the activity of the most PSII-directed herbicides as well as of some synthetic quinones appears to be significantly reduced in phosphorylated membranes. MATERIALS AND METHODS Phosphorylation and Isolation of MembranesPhosphorylation of spinach (Spinacia oleracea L.) thylakoids was performed as previously described (20) with a slight modification. Thylakoids were resuspended in the suspension buffer containing 50 mm Tricine (pH 7.5), 150 mm KCI, 15 mn-m NaCl, 5 mM MgCl2, and 0.1 M sucrose. After the addition of 0.1 mm ATP, they were illuminated (50 tiE m-2 sI) for 10 min. The phosphorylation profile of polypeptides was examined in thylakoids incubated in the presence of (1 mCi, 3000 Ci/mmol) and 0.1 mi ATP. Control thylakoids were illuminated in the absence of ATP and MgCl2, washed with the suspension buffer, dark-adapted for 2 h, and finally NaF was added to a concentration of 5 mm. PSII membranes were isolated (3), solubilized in 1% DM (0.5 mg of Chl mnL1), and then applied to the cathode region of a flat-bed of granulated gel (9). The bed (2.5 x 22 cm) of Ultrodex (LKB) was prepared in 0.1 M glycine, 0. 1% DM, and 2% ampholine carrier ampholytes (pH range 3.5-5.5) and left at 40C in the dark. Isoelectrofocusing was carried out at a constant power of 3 W/tray for 16 h in an LKB Multiphor 1948 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: Phosphorylation of the D1 protein in various plant groups

“…4-6) is possibly due to a conformational change in the protein; insertion of the phosphate group alone cannot cause the observed change of 1-2 kDa in the size of the molecule. Secondly, Giardi et al (1992) have shown that the binding affinity of PSII herbicides to the QB niche decreased markedly in the PSII core populations with an increasing phosphorylation stage of the D1 protein. The loss of binding affinity was especially notable with i-Dinoseb and ioxynil (Giardi et al 1992), the herbicides which most efficiently suppressed the degradation of D1 protein under physiological light conditions (Jansen et al 1993).…”

“…Secondly, Giardi et al (1992) have shown that the binding affinity of PSII herbicides to the QB niche decreased markedly in the PSII core populations with an increasing phosphorylation stage of the D1 protein. The loss of binding affinity was especially notable with i-Dinoseb and ioxynil (Giardi et al 1992), the herbicides which most efficiently suppressed the degradation of D1 protein under physiological light conditions (Jansen et al 1993). These results suggest that the phosphorylation of the N-terminal domain may influence the orientation of those domains in the D1 protein that are involved in the formation of the QB niche.…”

Long-term drought stress induces structural and functional reorganization of photosystem II

“…The light-harvesting complexes were collected as a green band at the position of 0.4 M sucrose while the PSII core preparation was recovered as a pellet at the bottom of the tube. Alternatively, the PSII membranes were gently solubilized with 0.3% DM and applied to an isoelectrofocusing bed of Ultrodex gel (pH range 3.5-5; LKB, Bromma, Sweden) to separate light-harvesting complex protein (LHCP) from the remaining PSII cores (Giardi et al 1992). The gel bed was cut into sections 1 cm wide and the polypeptide profile was assessed by SDS-PAGE and immunoblotting as described by Barbato et al (1991).…”

“…Herbicide binding was performed on isolated thylakoids as previously reported, using radiolabelled ioxynil (3.3" 1011 Bq.mmol-i; Giardi et al 1992;Giardi 1993b), a kind gift from May and Baker Ltd. (London, UK). The QB content, the dissociation constant and the number of herbicide-binding sites were obtained from double reciprocal plots of free versus bound herbicide concentration.…”

Phosphorylation and disassembly of the photosystem II core as an early stage of photoinhibition

“…These PSII cores show different QB pocket functioning with respect to plastoquinone (PQ) binding (Giardi et al 1992a). In the present work, the effect of photoinhibitory conditions on the distribution of phosphorylated PSII core populations is studied.…”