Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

A - Papers appearing in refereed journals

Wold-McGimsey, F., Krosch, C., Alarcon-Reverte, R., Ravet, K., Katz, A., Stromberger, J., Mason, R. E. and Pearce, S. 2023. Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains. Frontiers in Plant Science. 14 (15 sept), p. 1247680. https://doi.org/10.3389/fpls.2023.1247680

AuthorsWold-McGimsey, F., Krosch, C., Alarcon-Reverte, R., Ravet, K., Katz, A., Stromberger, J., Mason, R. E. and Pearce, S.
Abstract

Introduction:
Polyphenol oxidases (PPO) are dual activity metalloenzymes that catalyse the production of quinones. In plants, PPO activity may contribute to biotic stress resistance and secondary metabolism but is undesirable for food producers because it causes the discolouration and changes in flavour profiles of products during post-harvest processing. In wheat (Triticum aestivum L.), PPO released from the aleurone layer of the grain during milling results in the discolouration of flour, dough, and end-use products, reducing their value.
Loss-of-function mutations in the PPO1 and PPO2 paralogous genes on homoeologous group 2 chromosomes confer reduced PPO activity in the wheat grain. However, limited natural variation and the proximity of these genes complicates the selection of extremely low-PPO wheat varieties by
recombination. The goal of the current study was to edit all copies of PPO1 and PPO2 to drive extreme reductions in PPO grain activity in elite wheat varieties.
Results:
A CRISPR/Cas9 construct with one single guide RNA (sgRNA) targeting a conserved copper binding domain was used to edit all seven PPO1 and PPO2 genes in the spring wheat cultivar ‘Fielder’. Five of the seven edited T1 lines exhibited significant reductions in PPO activity, and T2 lines had PPO activity up to 86.7% lower than wild-type. The same construct was transformed into the elite winter wheat cultivars ‘Guardian’ and ‘Steamboat’, which have five PPO1 and PPO2
genes. In these varieties PPO activity was reduced by >90% in both T1 and T2 lines. In all three varieties, dough samples from edited lines exhibited reduced browning.
Discussion:
This study demonstrates that multi-target editing at late stages of variety development could complement selection for beneficial alleles in cropbreeding programs by inducing novel variation in loci inaccessible to recombination

KeywordsWheat; Reverse genetics; Multi-target editing; CRISPR/Cas9; Polyphenol oxidase
Year of Publication2023
JournalFrontiers in Plant Science
Journal citation14 (15 sept), p. 1247680
Digital Object Identifier (DOI)https://doi.org/10.3389/fpls.2023.1247680
Open accessPublished as ‘gold’ (paid) open access
Publisher's version
Output statusPublished
Publication dates
Online15 Sep 2023
Publication process dates
Accepted28 Aug 2023
PublisherFrontiers Media SA
ISSN1664-462X

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