Selection and regeneration of herbicide-resistant plant cells, and resistant enzymes

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Sulfonylurea herbicides suppress plant growth by inhibiting acetolactate synthase, a key enzyme of branched-chain amino acid biosynthesis. Carrot and rice cells resistant to 10^<-7>M and 10^<-5>M bensulfuron methyl, respectively, were selected. Their branched-chain amino acid and soluble protein levels were less reduced and ALSs were less sensitive to BSM than nonselected susceptible cells.BSM-resistant carrot cells have cross-resistance to pyrazosulfuron-ethyl (PE) and imazaqwin (IQ). ALS inhibition was non-competitive (mixed) type by BSM and PE,and un-competitive by IQ to a substrate, pyruvate. Michaelis constant to pyruvate of resistant cells was a little higher than susceptible cells. Resistant ALS showed 100 times higher I_<50> (50% inhibition concentration) to BSM,but only several times to PE and IQ,showing less cross-resistances. Feed back inhibition by branched-chain amino acids were seen to same extent in both types of cells. Crude ALS extract from the cells were separated into two active peakes by gel-filtration colum chromatography. The activity of second peak was inhibited by both BSM and branched-chain amino acid, while that of resistant cells was less inhibited by BSM.Resistant cells had 1.5-several times higher branched-chain amino acid level, suggesting that the ALS was less sensitive to feed back inhibition. The composition of soluble proteins in the cells was not affected by BSM treatment, indicationg that the reduction of branched-chain amino acids level to lead to only decrease in peotein levels.These results suggest the mutation of ALS insensitive to herbicides is due to the herbicide resistance in suspension cultured plant cells.