Molecular genetics of triazine resistance in Senecio vulgaris L

Herbicide resistance in weed species is a major problem of modem agriculture in which intensive use of herbicides is made. An understanding of the mechanism of resistance and how it is expressed is essential to both the future design of herbicides and their continued use in the field. Triazine resistance in the common weed $\textit{Senecio vulgaris}$ L. (common groundsel) has been studied in this work. The F1 populations formed in crosses between two independently isolated resistant biotypes with a susceptible biotype in two reciprocal matings were analysed for leaf structure differences, [$^{14}$C] atrazine binding, [$^{14}$C] azido-atrazine labelling and antibody labelling of isolated thylakoids compared to S1 populations from the parents. In addition physical characteristics of chloroplast DNA (cpDNA) and its inheritance were also studied. Resistance of groundsel seedlings to atrazine; [$^{14}$C] atrazine-binding; [$^{14}$C] azido-atrazine labelling and cpDNA restriction pattern were different in resistant compared to susceptible biotypes and the phenotypes thus distinguished were all maternally inherited in both crosses. Thylakoids of the susceptible biotypes bound [$^{14}$C] atrazine while those of the resistant biotypes did not. The binding was similar but variable among the several susceptible biotypes tested giving calculated binding constants K = 2.5-8.o.10-8M and stoichiometries of 1 binding site per 250-350 chlorophylls. The [$^{14}$C] atrazine binding was inherited maternally in the reciprocal crosses and was unaffected by the nuclear background. [$^{14}$C]Azido-atrazine labelled a major polypeptide of 32 kDa and two minor polypeptides of 23 kDa and 18kDa, only in susceptible thylakoids: resistant thylakoids were not labelled. The 32kDa polypeptide co-migrated with Photosystem II in sucrose density gradients. Western blot analysis of thylakoid proteins with antiserum to a psbA gene-product suggested that the absence of [$^{14}$C] atrazine binding and [ l4q azido-atrazine labelling in the resistant biotypes was not due to a quantitative loss of the target polypeptide but some qualitative alteration in its ability to bind herbicide. Differences in leaf structure between the two resistant and the susceptible biotype were of approximately the same magnitude and quality. The resistant leaves contained more mesophyll tissue and less airspace than the susceptible ones. These differences were not inherited maternally in contrast to a visible maternal effect on the overall morphology of Fl plants indicating that leaf structure organisation is probably not directly influenced by the resistance mutation. From comparisons of published psbA sequences of several higher plants, a BstXI restriction site polymorphism diagnostic of the mutation found in all triazine resistant higher plants was predicted. The analysis of cpDNA from several groundsel biotypes with the endonuclease BstXI revealed that the presence of the mutation in cpDNA of a biotype was invariably associated with a loss of [$^{14}$C] atrazine binding to isolated thylakoids of the same biotype. Another enzyme, HindIII also showed a polymorphism for one restriction site. This polymorphism was not invariably associated with either biotype but was fortuitously different between the resistant and susceptible parents of the crosses. This allowed the inheritance of the chloroplast genome types to be determined in the reciprocal crosses and showed that chloroplast DNA was inherited maternally in both crosses. Heterologous probes were used to identify restriction fragments of cpDNA containing the psbA gene and several subfragments of the gene from both biotypes were cloned and sequenced. Additional sequence information was obtained by sequencing the RNA transcript of the gene. The sequence of the gene consisted of an open reading frame of 1062 base-pairs. The nucleotide and deduced amino acid sequences were highly homologous to other published higher plant psbA sequences. Consistent with the results of the BstXI digestion results, an A -> G base change distinguished the resistant gene from the susceptible gene. The result of the substituion is that the resistant biotype has a Gly at position 264 of the deduced amino-acid sequence in place of the Ser found in the susceptible biotype. This change is identical to the mutations responsible for triazine resistance in all other higher plant species in which resistance has been studied at this level. Sequence data for the surrounding region of groundsel chloroplast DNA was also obtained and revealed that the genetic organisation of a 2.8 kbp Sma I cpDNA fragment containing the entire gene is similar to that of Nicotiana and different from that of spinach. The expression of chloroplast triazine resistance at the different levels of biological organisation of groundsel and other plant species is discussed.

Digitisation of this thesis was sponsored by Arcadia Fund, a charitable fund of Lisbet Rausing and Peter Baldwin