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Biochemical and structural characterization of quizalofop-resistant wheat acetyl-CoA carboxylase.

ABSTRACT: A novel nucleotide mutation in ACC1 resulting in an alanine to valine amino acid substitution in acetyl-CoA carboxylase (ACCase) at position 2004 of the Alopecurus myosuroides reference sequence (A2004V) imparts quizalofop resistance in wheat. Genotypes endowed with the homozygous mutation in one or two ACC1 homoeologs are seven- and 68-fold more resistant to quizalofop than a wildtype winter wheat in greenhouse experiments, respectively. In vitro ACCase activities in soluble protein extracts from these varieties are 3.8- and 39.4-fold more resistant to quizalofop with the homozygous mutation in either one or two genomes, relative to the wildtype. The A2004V mutation does not alter the specific activity of wheat ACCase, suggesting that this resistance trait does not affect the catalytic functions of ACCase. Modeling of wildtype and quizalofop-resistant wheat ACCase demonstrates that the A2004V amino acid substitution causes a reduction in the volume of the binding pocket that hinders quizalofop's interaction with ACCase. Docking studies confirm that the mutation reduces the binding affinity of quizalofop. Interestingly, the models suggest that the A2004V mutation does not affect haloxyfop binding. Follow up in vivo and in vitro experiments reveal that the mutation, in fact, imparts negative cross-resistance to haloxyfop, with quizalofop-resistant varieties exhibiting higher sensitivity to haloxyfop than the wildtype winter wheat line.

SUBMITTER: Bough R

PROVIDER: S-EPMC8758669 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPolyadenylation is a crucial process that terminates mRNA molecules at their 3'-ends. It has been observed that alternative polyadenylation (APA) can generate multiple transcripts from a single gene locus, each with different polyadenylation sites (PASs). This leads to the formation of several 3' untranslated regions (UTRs) that vary in length and composition. APA has a significant impact on approximately 60-70% of eukaryotic genes and has far-reaching implications for cell proliferation, differentiation, and tumorigenesis.ResultsIn this study, we conducted long-read, single-molecule sequencing of mRNA from peanut seeds. Our findings revealed that over half of all peanut genes possess over two PASs, with older developing seeds containing more PASs. This suggesting that the PAS exhibits high tissue specificity and plays a crucial role in peanut seed maturation. For the peanut acetyl-CoA carboxylase A1 (AhACCA1) gene, we discovered four 3' UTRs referred to UTR1-4. RT-PCR analysis showed that UTR1-containing transcripts are predominantly expressed in roots, leaves, and early developing seeds. Transcripts containing UTR2/3 accumulated mainly in roots, flowers, and seeds, while those carrying UTR4 were constitutively expressed. In Nicotiana benthamiana leaves, we transiently expressed all four UTRs, revealing that each UTR impacted protein abundance but not subcellular location. For functional validation, we introduced each UTR into yeast cells and found UTR2 enhanced AhACCA1 expression compared to a yeast transcription terminator, whereas UTR3 did not. Furthermore, we determined ACC gene structures in seven plant species and identified 51 PASs for 15 ACC genes across four plant species, confirming that APA of the ACC gene family is universal phenomenon in plants.ConclusionOur data demonstrate that APA is widespread in peanut seeds and plays vital roles in peanut seed maturation. We have identified four 3' UTRs for AhACCA1 gene, each showing distinct tissue-specific expression patterns. Through subcellular location experiment and yeast transformation test, we have determined that UTR2 has a stronger impact on gene expression regulation compared to the other three UTRs.

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Biochemical and structural characterization of quizalofop-resistant wheat acetyl-CoA carboxylase.

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