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.

植物脂氧合酶（lipoxygenases, LOXs）能够催化多不饱和脂肪酸的氧化反应，该过程会对谷物的耐储性产生不利影响。尽管对LOXs进行基因工程操作在改善谷物储藏品质方面具有巨大潜力，但该方法在大麦中仍未得到广泛探索。本研究从大麦基因组中鉴定出5个LOX基因，分别为HvLOXA、HvLOXB以及HvLOXC1–3。其中，HvLOXC1在发育早期籽粒、根与幼芽中表达量最高；HvLOXA主要在胚中特异性表达，而HvLOXB与HvLOXC3在各组织中均呈低水平表达。本研究借助CRISPR/Cas9介导的基因组编辑技术，成功获得了loxB、loxC1以及loxAloxC1的无转基因纯合大麦突变体。与野生型对照相比，所有突变体的株高、分蘖数及籽粒大小均未出现显著异常，但loxC1和loxAloxC1突变体的千粒重显著降低。值得注意的是，成熟籽粒中的总LOX活性在loxC1突变体中下降了36%~42%，在loxAloxC1突变体中下降了94%，而loxB突变体的总LOX活性无显著变化。此外，loxAloxC1双突变体的丙二醛含量显著低于野生型，脂肪酸积累量较野生型提升了10%~21%。人工老化处理实验结果显示，loxAloxC1突变体的谷物耐储性得到显著增强，具体表现为发芽率显著提升、脂质过氧化程度降低以及幼苗生长状况得到改善。本研究结果表明，靶向敲除LOX基因——尤其是同时敲除HvLOXA与HvLOXC1——是一种极具应用前景的遗传策略，可用于改良大麦的谷物耐储性与营养价值。