Molecular aspects of herbicide binding in chloroplasts = [Molekulaire aspekten van herbicide binding in chloroplasten]

Many weed-controlling agents act by inhibiting the process of photosynthesis. Their mode of action is a displacement of the secondary quinone electron acceptor of photosystem II from its proteinaceous binding environment. This results in a blocking of the electron transport. Consequently plants are no longer able to produce ATP and to reduce NADP +, which eventually leads to starvation and death.In this thesis an attempt is made to characterize the interactions of herbicides with their binding environment. Several herbicides were used to measure binding kinetics in a wildtype and a triazine -resistant biotype of Common Lambsquarters, Chenopodium album L.In Chapter 1 a general introduction is given on photosynthesis, chloroplast structure and function, the chloroplast genome, and herbicide action and resistances.Chapter 2 describes the methods used to isolate chloroplasts and to measure oxygen production. Also the fluorescence induction measurements are described, which can be used as a rapid method to discriminate between herbicide-sensitive and -resistant plants. Procedures for the simultaneous isolation of total plant nucleic acids, for the separation of DNA and RNA and for the respective sequence analyses are outlined.In Chapter 3 a model is described which can be used to simulate flash- induced oxygen production by isolated thylakoids. In the literature a model was described before, which explains the observed 4-step oscillation in flash-induced oxygen evolution by assuming the existence of 4 different S-states of the oxygen evolving complex. This model is refined here by assuming the values of the miss parameters αto be dependent on both the redox state of the quinone acceptor complex (Q A .Fe.Q B ) of photosystem II and on the S-state transition involved. The best fit between theoretical and experimental oxygen evolution patterns is obtained when 4 different miss parameters are distinguished, corresponding with the 4 S-state transitions of the oxygen evolving complex. The values of two of these αparameters, notably those for the 2 ndand 4 thflash, are found to approach 0, while the other two have values of about 0.2 and 0.4. The value for the double hit parameter βis found to be about 0.06. The oxygen evolving complexes of thoroughly dark adapted chloroplasts are found to be for almost 100 % in the most stable single oxidized state S 1 . A fraction of 30 % of the reaction centers is assumed to be connected to a oneelectron donor D, which is able to reduce the S 2 - or S 3 -state of the oxygen evolving complex with a half time of 2-3 seconds.In Chapter 4 the derived model is used to determine the exchange ,parameters of herbicides with the secondary acceptor Q B . These exchanges, which influence the oxygen evolution patterns, can be determined by comparing experimental and theoretical patterns for various herbicide concentrations and flash frequencies. The I 50 -values derived from the measurements are in agreement with values measured with other methods. The resistance to triazine compounds proved to be caused by an increase in the herbicide release rather than by a lower binding rate. Configuration around a chiral carbon atom, present in two pairs of isomers of cyanoacrylate inhibitors, has a strong influence on inhibition properties. The differences are largely due to alterations in the release kinetics. This observation suggests that herbicide binding is determined mainly by physical properties, like e.g. hydrophobicity. A stationary binding, resulting in a significant electron transport inhibition, requires a strict molecular shape.In Chapter 5 partial sequence analyses from DNA and mRNA isolated from a Chenopodium album wildtype and a triazine -resistant biotype are presented. The only difference found was an adenine to guanine point mutation resulting in a serine to glycine alteration at position 264 in the D1 (herbicide binding) protein.The thesis is concluded with Chapter 6, the general discussion, in which an alternative explanation for redox reactions at the photosystem II acceptor side is presented. In this model the non-heme iron, located between the primary and the secondary acceptor of photosystem II, is proposed to play an active role in in vivo electron transport