RESPONSE IN THE INHIBITOR EFFICIENCY OF SUBSTITUTED PHENOLS ON PS-II ACTIVITY IN 6 MUTANTS OF THE D1 PROTEIN SUBUNIT IN CHLAMYDOMONAS-REINHARDTII

The D1 protein subunit of the reaction centre of photosystem II is als o a herbicide-binding protein. Blockage of the Q(B) binding site in th e D1 protein is the mode of action of many herbicide grouped as classi cal diuron atrazine type or as phenol group. Tolerance of photosynthet ic electron flow in PS II to classical herbicides like diuron or atraz ine is observed in several mutants of higher plants and algae with ami no acid substitutions in the D1 protein. But in most of these mutants, the few phenol-type inhibitors tested were not tolerated, some of the m even displaying supersensitivity. This seemed to indicate a substant ial change of orientation of the phenolic inhibitors in the Q(B) site when compared to the classical herbicides. However, we report here on the appreciable tolerance of mutants to phenol-type inhibitors of the D1 protein. We investigated the effect of 47 derivatives of 6-alkyl-su bstituted 2-halogeno-4-nitrophenols and of 2,4-dinitrophenols on photo synthetic electron flow in six mutants of Chlamydomonas reinhardtii. T he mutants were obtained either by site-selected screening towards met ribuzin or diuron or by site-directed mutagenesis. With the exception of an asn266thr mutant, all the mutants responded toward the phenol in hibitors either by decreased or enhanced sensitivity compared with the wild type. Greatest effects were observed with a val2l9ile mutant. To lerance ranged from zero to 1.7 pI50 units depending on the substituen t at position 6 of the phenol ring. Similarly, but somewhat lower, is the tolerance in a phe255tyr mutant. A leu275phe and an ala251val muta nt show a mixed behaviour of tolerance to some, and supersensitivity t o other derivatives. Many phenols display supersensitivity in the ser2 64ala mutant, up to 100-fold, and there is never tolerance. It is poss ible to rationalize the cross resistance of phenol-type inhibitors of PS II with their substitution pattern, particularly in the va1219ile m utant. The extent of tolerance depends entirely on the size and shape of the substituent in position 6. The maximum is found with 2-bromo-4- nitro-6 benzylphenol with a 50-fold decrease in inhibitor potency in t his mutant. The data allow us to orient now also the phenol-type inhib itors in the herbicide binding niche in the D1 protein in a way not to o dissimilar from that of the classical inhibitors. The concept that n ot only the classical herbicides but also phenol-type inhibitors act b y binding to the D1 protein is now greatly supported by the observatio n of tolerance if the sequence of the D1 protein is changed. The princ iple difference of the phenols to the classical herbicides can now be specified clearly: it is the response to the ser264ala mutant where th ere is no tolerance of phenol-type inhibitors in contrast to the class ical herbicides. This is not invalidated by observations that some phe nols may bind to other subunits of PS II as well, nor that many of the m are uncouplers. These are to be considered as secondary effects not contributing to the high affinity binding to the D1 protein.