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

Forrest Wold-McGimsey; Caitlynd Krosch; Rocío Alarcón-Reverte; Karl Ravet; Andrew Katz; John Stromberger; Richard Esten Mason; Stephen Pearce

doi:10.3389/fpls.2023.1247680

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

Front Plant Sci. 2023 Sep 15:14:1247680. doi: 10.3389/fpls.2023.1247680. eCollection 2023.

Authors

Forrest Wold-McGimsey¹, Caitlynd Krosch¹, Rocío Alarcón-Reverte^{1

2}, Karl Ravet¹, Andrew Katz¹, John Stromberger¹, Richard Esten Mason¹, Stephen Pearce^{1

2}

Affiliations

¹ Department of Soil and Crop Sciences, Colorado State University, Fort Collins, CO, United States.
² Sustainable Soils and Crops, Rothamsted Research, Harpenden, Hertfordshire, United Kingdom.

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 crop breeding programs by inducing novel variation in loci inaccessible to recombination.

Keywords: CRISPR/Cas9; multi-target editing; polyphenol oxidase; reverse genetics; wheat.

Grants and funding

This project was supported by Competitive Grant 2022-68013-36439 (WheatCAP) from the USDA National Institute of Food and Agriculture and by funding from the Colorado Wheat Administrative Commission and the Colorado Wheat Research Foundation.