Katherine E. Steinback

Phosphorylation of the light-harvesting chlorophyll-protein regulates excitation energy distribution between photosystem II and photosystem I.

Abstract The kinetics of thylakoid membrane protein phosphorylation in the presence of light and adenosine triphosphate is correlated to an incease in the 77 °K fluorescence emission at 735 nm (F735) relative to that at 685 nm (F685). Analysis of detergent-derived submembrane fractions indicate phosphorylation only of the polypeptides of Photosystem II, and the light-harvesting chlorophyll-protein complex serving Photosystem II (LHC-II). Although several polypeptides are phosphorylated, only the dephosphorylation kinetics of LHC-II follow the kinetics of the decrease of the F735 F685 fluorescence emission ratios. The relative quantum yield of Photosystem II was significantly lower in phosphorylated membranes compared to dephosphorylated membranes. Reversible LHC-II phosphorylation thus provides the physiological mechanism for the control of the distribution of absorbed excitation energy between the two photosystems.

Characterization of chloroplast thylakoid polypeptides in the 32-kDa region: Polypeptide extraction and protein phosphorylation affect binding of photosystem II-directed herbicides

In order to distinguish between two photosystem II proteins with apparent molecular weights of about 32 kDa, mild extraction procedures were used to remove several thylakoid membrane components. A 32-kDa protein that stained intensely with Coomassie brilliant blue could be extracted from the thylakoid membranes without removing the 32-kDa herbicide receptor protein, which stained poorly with Coomassie brilliant blue. The nonextracted protein was readily detectable after in vivo polypeptide labeling with [35S]methionine or after in vitro covalent tagging with [14C]azidoatrazine. The procedures used to extract the intensely stained, 32-kDa polypeptide resulted in changes in herbicide-binding characteristics, presumably due to conformational changes in the herbicide-binding environment. Alterations of membrane surface charge by protein phosphorylation also influenced herbicide binding.

Mapping of the triazine binding site to a highly conserved region of the QB-protein

A number of herbicide classes, including the s-triazines and ureas (atrazine, diuron) inhibit photosynthetic electron transport via a direct interaction with the QB-protein. This protein, also known as the 32-kDa protein or herbicide binding protein, is believed to bind the plastoquinone QB, which functions as the second stable electron acceptor at the reducing side of Photosystem II. The site of covalent attachment of the photoaffinity herbicide analog azido-[14C]atrazine to the QB-protein of spinach chloroplast thylakoid membranes has been determined. Two amino acid residues are labeled; one residue is methionine-214, the other lies between histidine-215 and arginine-225. Both residues are within a region of the amino acid sequence which is highly conserved between the QB-protein and the L and M reaction center proteins of Rhodopseudomonas capsulata and R. sphaeroides. This region includes the site of a mutation which results in diuron resistance in Chlamydomonas reinhardi (valine-219). However, this region is well removed from point mutations at phenylalanine-255 (which gives rise to atrazine resistance in C. reinhardi) and at serine-264, (which results in extreme atrazine resistance in C. reinhardi and naturally occurring weed biotypes). The patterns of labeling and mutation imply that the quinone and herbicide binding site is formed by at least two protein domains.

MOLECULAR CHARACTERIZATION OF THE TARGET SITE(S) FOR HERBICIDES WHICH AFFECT PHOTOSYNTHETIC ELECTRON TRANSPORT

Abstract Many commercial herbicides inhibit photosynthetic electron transport; for several chemical families these effects are the primary mode of action. In recent years our laboratories have investigated the nature of herbicide binding sites in chloroplast membranes. These studies have contributed new information to our understanding of photosynthetic electron transport processes, and have provided information which may be of value in the chemical design of herbicides acting on these processes. Polypeptides of the 32–34 Kdalton size class, which are components of the photosystem II (PS II) complex, have been proposed to be determinants of triazine herbicide binding sites. Herbicide-induced inhibition of electron transport results from non-covalent, reversible binding of one herbicide molecule per PS II complex. Recent data indicates that herbicide binding causes displacement of a native plastoquinone molecule (acting as the second electron carrier on the reducing side of PS II) from its binding site. A means of studying the biological variability in herbicide binding sites has been provided by naturally-occurring weed species which have developed triazine-herbicide resistance. A genetically determined structural alteration of the 32–34 Kdalton polypeptide species of the PS II complex has been suggested to be the mechanism giving rise to this type of resistance. In addition, mutants or developing systems of green algae have also been characterized to delineate other polypeptide determinants of the PS II complex which are critical for herbicide binding and action.