CRISPR/Cas9-mediated editing of barley lipoxygenase genes promotes grain fatty acid accumulation and storability

Plant lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids, which can adversely affect grain storability. Although the genetic engineering of <i>LOXs</i> holds great potential for improving grain storage quality, this approach remains largely unexplored in barley. In this study, we identified five <i>LOX</i> genes in the barley genome: <i>HvLOXA</i>, <i>HvLOXB</i>, and <i>HvLOXC1–3</i>. <i>HvLOXC1</i> exhibited the highest expression in early developing grains, roots, and shoots; <i>HvLOXA</i> was predominantly expressed in embryos, whereas <i>HvLOXB</i> and <i>HvLOXC3</i> were weakly expressed across tissues. Transgene-free homozygous barley mutants of <i>loxB</i>, <i>loxC1</i>, and <i>loxAloxC1</i> were generated using CRISPR/Cas9-mediated genome editing. Compared to the wild-type, all mutants displayed normal plant height, tiller number, and grain size, although the <i>loxC1</i> and <i>loxAloxC1</i> mutants exhibited significantly lower thousand grain weights. Notably, the total LOX activity in mature grains decreased by 36–42% in <i>loxC1</i> mutants and by 94% in <i>loxAloxC1</i> mutants, with no significant change observed in <i>loxB</i> mutants. Additionally, the <i>loxAloxC1</i> double mutants had a significantly lower malondialdehyde content and accumulated 10–21% more fatty acids than the wild-type. Artificial aging treatment experiments revealed that <i>loxAloxC1</i> mutants had enhanced grain storability, demonstrated by significantly higher germination rates, reduced lipid peroxidation, and improved seedling growth. Our findings highlight that the targeted knockout of <i>LOX</i> genes, particularly the double mutation of <i>HvLOXA</i> and <i>HvLOXC1</i>, represents a promising genetic strategy for improving grain storability and nutritional value in barley.