Simon William Jonathan Bright

Herbicide resistance caused by spontaneous mutation of the cytoskeletal protein tubulin

The dinitroaniline herbicides (such as trifluralin and oryzalin) have been developed for the selective control of weeds in arable crops. However, prolonged use of these chemicals has resulted in the selection of resistant biotypes of goosegrass, a major weed. These herbicides bind to the plant tubulin protein but not to mammalian tubulin. Here we show that the major α-tubulin gene of the resistant biotype has three base changes within the coding sequence. These base changes swap cytosine and thymine, most likely as the result of the spontaneous deamination of methylated cytosine. One of these base changes causes an amino-acid change in the protein: normal threonine at position 239 is changed to isoleucine. This position is close to the site of interaction between tubulin dimers in the microtubule protofilament. We show that the mutated gene is the cause of the herbicide resistance by using it to transform maize and confer resistance to dinitroaniline herbicides. Our results provide a molecular explanation for the resistance of goosegrass to dinitroanaline herbicides, a phenomenon that has arisen, and been selected for, as a result of repeated exposure to this class of herbicide.

Characterization of the safener-induced glutathione S-transferase isoform II from maize

The safener-induced maize (Zea mays L.) glutathione S-transferase, GST II (EC 2.5.1.18) and another predominant isoform, GST I, were purified from extracts of maize roots treated with the safeners R-25788 (N,N-diallyl-2-dichloroacetamide) or R-29148 (3-dichloroace-tyl-2,2,5-trimethyl-1,3-oxazolidone). The isoforms GST I and GST II are respectively a homodimer of 29-kDa (GST-29) subunits and a heterodimer of 29 and 27-kDa (GST-27) subunits, while GST I is twice as active with 1-chloro-2,4-dinitrobenzene as GST II, GST II is about seven times more active against the herbicide, alachlor. Western blotting using antisera raised against GST-29 and GST-27 showed that GST-29 is present throughout the maize plant prior to safener treatment. In contrast, GST-27 is only present in roots of untreated plants but is induced in all the major aerial organs of maize after root-drenching with safener. The amino-acid sequences of proteolytic fragments of GST-27 show that it is related to GST-29 and identical to the 27-kDa subunit of GST IV.

Feedback-insensitive aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine.

The regulatory properties of aspartate kinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) in two barley (Hordeum vulgare L.) mutants resistant to growth inhibition by lysine plus threonine, Rothamsted (R) 3004 and R3202, were compared with those in the normal, sensitive parent line cv. Bomi. Three forms of aspartate kinase (AKI, AKII, AKIII) were chromatographically separated and were considered to represent at least three independently regulated isoenzymes. Aspartate kinase I was inhibited by threonine; AKII and AKIII by lysine or lysine plus S-adenosylmethionine. The characteristics of AKI were unchanged in the mutants. Aspartate kinase II and AKIII from Bomi were both inhibited by lysine and by lysine plus S-adenosylmethionine. Aspartate kinase II from mutant R3202 was altered in its properties such that it was insensitive to lysine or lysine plus S-adenosylmethionine; AKII from mutant R3004 did not differ in its properties from AKII of Bomi. The concentration of lysine required to give half maximal inhibition of AKIII from R3004 was ten times that required for AKIII of Bomi; AKIII from R3202 did not differ from that of Bomi in this regard. There was no change in the properties of homoserine dehydrogenase of the mutants as compared with that of Bomi. We conclude that the lt1 and lt2 loci code for structural genes for lysine- and lysine plus S-adenosylmethionine-sensitive aspartate kinase isoenzymes. The mutant genes Lt1b and Lt2 in R3202 and R3004 respectively code for feedback-desensitized isoenzymes. The presence of one of these is sufficient to allow the synthesis of methionine to overcome the growth inhibition by lysine plus threonine.