Forward screening for herbicide resistance in Arabidopsis thaliana and the monocot species Triticum aestivum

Abstract

Cellulose is the primary structural component of plant cell walls and is composed of β-1,4-glucan chains. The cellulose synthesis complex is composed of multiple cellulose synthases (CESAs), which work together to extend the cellulose molecule. Although extensively studied, cellulose biosynthesis is not yet fully understood. One way to elucidate the role of CESAs is through their interactions with cellulose biosynthesis inhibitors (CBIs). CBI resistant Arabidopsis thaliana mutants were identified from previous forward genetic screens. Some identified CBI resistant mutants demonstrated altered cellulose crystallinity, and cell wall saccharification. In a larger crop species, these characteristics would allow for plant cell wall material to be efficiently converted into cellulosic ethanol. Therefore, a forward genetic screen has been carried out in Triticum aestivum, bread wheat. Three CBI resistant mutants have been identified in wheat, and were found to contain point mutations in the wheat ortholog for AtCESA3, TaCESA1. These CBI resistant mutants demonstrated altered cell wall saccharification, and cellulose crystallinity. The identification of CBI resistant mutants in wheat demonstrates that traits identified in A. thaliana can be reproduced in a monocot crop species. The second focus of this thesis was to develop new herbicide resistant lines in A. thaliana. Indaziflam is a potent herbicide used to control annual grasses and broadleaf weeds. To better understand the plant pathways affected by indaziflam, a forward genetic screen for indaziflam resistance was completed in A. thaliana. Approximately 750, 000 A. thaliana seeds were screened for indaziflam resistance, and only one allele, indaziflam resistant 1 (izr1), was identified to cause weak indaziflam resistance. Positional cloning coupled with next-generation sequencing revealed that a point mutation in the CULLIN1 (CUL1) encoding gene caused the decrease in indaziflam sensitivity. CUL1 is an E3 ligase involved in a variety of plant signalling pathways, including auxin response. Along with decreased sensitivity to indaziflam, izr1 seedlings demonstrated reduced sensitivity to auxin mimicking herbicides. Comparing izr1 to a known auxin resistant CUL1 mutant, cul1-6, and the known CBI resistant CESA mutant, ixr1-1, revealed that alterations to the CUL1 protein or CESA3 can affect indaziflam sensitivity.