Sequence analyses of eight metribuzin-resistant mutants of photoautotrophic Chenopodium rubrum cell cultures revealed new mutations in the psbA gene coding for the 32 kDa herbicide binding protein. Mutants were found to possess either two or three changes in the amino acid sequence of the Di-protein between positions 219 and 272.

Chenopodium rubrum photoautotrophic cell suspensions were grown in plastic tissue culture dishes under photoautotrophic conditions. Growth was monitored by measuring cell number, packed cell volume, chlorophyll content and oxygen production. Such microtiter dishes are suitable systems for the serial assay of growth inhibition and various physiological effects (i.e. chlorophyll fluorescence, cell viability, oxygen production) of photoautotrophic cells as caused by herbicides and fungal phytotoxins. The applicability of the test system is discussed.

For establishing metribuzin-resistant, photoautotrophic Chenopodium rubrum cell cul­tures plated cells, callus cultures or suspension cultures were subjected to selection proce­ dures. The most effective procedure was the stepwise increase in the concentration of the herbicide from 0.01 μᴍ to 10 μᴍ in suspension cultures, which resulted in the isolation of eight different metribuzin-resistant photoautotrophic cell lines. Conjugation metabolism or a decrease in the uptake and translocation of the selective agent were not responsible for resist­ ance, which was stable in the absence of the inhibitor over numerous growth cycles. Meas­ urements of the photosynthetic electron transport, analyses of fluorescence induction kinetics and determination of the binding properties of 14C-labelled metribuzin to isolated thylakoids indicated that resistance of the cell lines is based on an alteration in the photosystem II her­ bicide-binding protein (D 1 protein). RFLP analysis of the psbA gene of the eight resistant cell lines demonstrated that none of them possess an amino acid exchange in position 264 of the D1 protein leading to altered herbicide-binding properties.

Eight photoautotrophic cell cultures of Chenopodium rubrum, which are resistant against the photosystem II inhibitor metribuzin, were characterized for their growth parameters, chlorophyll content and photosynthetic capacity. Herbicide resistance of the eight lines results from different mutations in the D 1 protein of photosystem II, which is the target for different photosystem II inhibitors. In the presence of 10-5 ᴍ metribuzin the eight lines showed substantial growth reduction depending on the degree of resistance, and this effect is explained by a reduced electron transport in photosystem II. The impaired photosynthetic capacity of the green cells in the presence of high metribuzin concentrations, leads to compensation effects similar to shade accommodation of plants. Adaptation includes an increase of the chlorophyll content, a decrease of the chlorophyll a/b ratios as well as an increase of thylakoid stacking and cell number per unit fresh weight. In the absence of the herbicide photosynthetic electron transport is not impaired, as indicated by measurements of electron transfer rates in photosystem II and flash-induced reduction kinetics of P-700+. In summary the alterations of the D 1 protein of the eight cell lines do not result in a reduced electron transport in photosystem II.