The mode of action of some herbicides, viz., DCMU, simetone, and diquat, was investigated by studying their effects upon oxygen evolution and cyclic photophosphorylation in the unicellular green alga, Scenedesmus
Oxygen evolution was measured with the aid of the WARBURG technique, the suspension medium mostly was WARBURG buffer no 9, the gas phase was air, in general, the temperature 25°C.
Cyclic photophosphorylation was determined by measuring inorganic phosphate uptake by the algae during a 90 minutes illumination period under a nitrogen atmosphere.
In Chapter III, it is shown that phosphate uptake under the conditions described is saturated at much lower light intensity than oxygen evolution (fig. 4). In far-red light, preferentially absorbed by PS I, there is almost no oxygen evolution (table 2), while the fixation of phosphate proceeds equally well as in white light (fig. 5). This demonstrates that the latter process represents cyclic photophosphorylation in vivo.
In Chapter IV, it is shown that DCMU, at light saturation and at 5 mm 3
cells/ml, inhibits oxygen evolution for 50% at a concentration of 2 x 10 -7
M (fig. 6). This concentration has no effect on dark O 2
-uptake (fig. 11). The degree of inhibition of oxygen evolution by DCMU decreases with increasing suspension density (section IV. 1.2.). Moreover, the degree of inhibition depends on light intensity (section IV. 1.4.), but not on temperature (section IV. 1.5.). By washing the cells, the inhibiting effect of DCMU can be removed (section IV. 1.3.). DCMU has no effect on the photosynthetic quotient (table 4).
Phosphate fixation in white light is much less sensitive to DCMU than oxygen evolution; in far-red light phosphate fixation is more strongly inhibited than in white light, but less than oxygen evolution (fig. 36).
In Chapter V, the effect of simetone is studied. Simetone has effects, qualitatively similar to those of DCMU on oxygen evolution and phosphate fixation. It differs from DCMU only in being less effective, not accumulated by the algae, and washed out more easily.
In Chapter VI, experiments on the influence of diquat on oxygen evolution and phosphate fixation are presented. The inhibition of O 2
-evolution by diquat increases with time; treatment in light for one hour with 2 x 10 -5
M gives an inhibition of about 50% (fig. 27). The degree of inhibition decreases with increasing suspension density (fig. 29) which points to an accumulation of diquat by the cells.
The inhibiting effect cannot be washed out, but when diquat is added in the absence of oxygen or in the presence of 10 -5
M simetone, the inhibition after washing is decreased (section VI.1.4.). It is concluded that diquat can be removed to a large extent by washing, and that oxygen is required to bring about the inhibiting effect.
Diquat initially stimulates dark O 2
-uptake which subsequently changes into inhibition (section VI.1.2.). The degree of inhibition of O 2
-evolution increases with light intensity. Inhibition was observed both in the light-limited and in the light-saturated part of the photosynthesis-light curve. The degree of inhibition increases with temperature (section VI.1.5.).
During the first 45 minutes after addition of diquat (with air as a gas phase), the photosynthetic quotient is increased (table 10). This shows that during this time diquat acts as a HILL-oxidant for O 2
-evolution, while CO 2
-uptake is decreased by lack of reduced NADP.
Diquat was found to inhibit phosphate fixation to about 50 % (in the complete absence of oxygen) (fig. 35).
The results are consistent with the hypothesis that diquat is reduced to a free radical in the photosynthetic process and also, to a smaller extent, in respiration. The reaction of this free radical with water and oxygen leads to the formation of toxic peroxide radicals or hydrogen peroxide (fig. 40). These peroxides are assumed to disrupt cellular organization, structure and function.
In Chapter VII, the mode of action of the various herbicides studied, and the implications of the results for the mechanism of photosynthesis are discussed. The results obtained give rise to the following interpretation of the mode of action of DCMU and simetone: both herbicides affect the oxidized state of a substance X, which may represent, or is very close to, the primary electron acceptor of PS II. Moreover, X takes part in the cyclic electron transport chain (fig. 40). X might be plastoquinone; the binding with the herbicides probably occurs via hydrogen bonds (figs. 38, 39).
With respect to the mechanism of photosynthesis it is concluded that cyclic photophosphorylation occurs also in vivo
. The cyclic electron transport chain probably contacts the non-cyclic one at the level of plastoquinone. Evidence suggests that there are two phosphorylation sites in the cyclic electron transport chain: one between cytochrome 559 and cytochrome 553, and another one in the chain of PS I between Z and plastoquinone. For more details regarding these considerations, cf. section VII.2.3.