Unregulated Sphingolipid Biosynthesis in Gene-Edited Arabidopsis ORM Mutants Results in Nonviable Seeds with Strongly Reduced Oil Content
Unregulated Sphingolipid Biosynthesis in Gene-Edited Arabidopsis ORM Mutants Results in Nonviable Seeds with Strongly Reduced Oil Content
复制标题
DOI：
10.1105/tpc.20.00015
复制
发表时间：
2020-08-01
期刊：
影响因子：
11.6
通讯作者：
Cahoon, Edgar B.
中科院分区：
文献类型：
--
作者：
Gonzalez-Solis, Ariadna;Han, Gongshe;Cahoon, Edgar B.
通过完全敲除Orosomucoid样蛋白（ORM）基因来消除鞘脂生物合成的调节，导致神经酰胺过度积累和含油量大幅降低的无活力种子。类卵粘蛋白（ORM）与丝氨酸棕榈酰转移酶（SPT）相互作用以负调节鞘脂的生物合成，这是一个可逆过程，对于平衡生长和程序性细胞死亡所需的细胞内鞘脂水平至关重要。在这里，我们表明，ORM 1和ORM 2是必不可少的生命周期完成拟南芥（拟南芥）。来自orm 1（-/-）orm 2（-/-）突变体的种子，通过交叉CRISPR/Cas9敲除每个基因的突变体产生，积累了高水平的神经酰胺，表明鞘脂生物合成不受调节。orm 1（-/-）orm 2（-/-）种子是不能存活的，显示出异常的胚发育，并且与野生型种子相比具有>80%降低的油含量。这种表型在表达SPT亚基LCB 1的拟南芥种子中被模拟，LCB 1缺乏其第一个跨膜结构域，这对于ORM介导的SPT调节至关重要。我们确定了突变体ORM 1缺乏一个氨基酸（Met-51）附近的第二个跨膜结构域，保留其膜拓扑结构。在orm 2背景下表达该等位基因产生的植物没有提前超过苗期，超积累神经酰胺，并显示改变细胞器结构和增加衰老和发病相关的基因表达。这些幼苗还表现出鞘脂分解代谢酶的基因表达上调，指出了维持鞘脂稳态的其他机制。缺乏Met-51的ORM 1在酵母（酿酒酵母）模型中与LCB 1的相互作用严重受损，为ORM和SPT之间的调控相互作用提供了结构线索。
Removing the regulation of sphingolipid biosynthesis by completely knocking out Orosomucoid-like protein (ORM) genes results in ceramide hyperaccumulation and nonviable seeds with strongly reduced oil content. Orosomucoid-like proteins (ORMs) interact with serine palmitoyltransferase (SPT) to negatively regulate sphingolipid biosynthesis, a reversible process critical for balancing the intracellular sphingolipid levels needed for growth and programmed cell death. Here, we show that ORM1 and ORM2 are essential for life cycle completion in Arabidopsis (Arabidopsis thaliana). Seeds from orm1(-/-)orm2(-/-) mutants, generated by crossing CRISPR/Cas9 knockout mutants for each gene, accumulated high levels of ceramide, indicative of unregulated sphingolipid biosynthesis. orm1(-/-)orm2(-/-) seeds were nonviable, displayed aberrant embryo development, and had >80% reduced oil content versus wild-type seeds. This phenotype was mimicked in Arabidopsis seeds expressing the SPT subunit LCB1 lacking its first transmembrane domain, which is critical for ORM-mediated regulation of SPT. We identified a mutant for ORM1 lacking one amino acid (Met-51) near its second transmembrane domain that retained its membrane topology. Expressing this allele in the orm2 background yielded plants that did not advance beyond the seedling stage, hyperaccumulated ceramides, and showed altered organellar structures and increased senescence- and pathogenesis-related gene expression. These seedlings also showed upregulated expression of genes for sphingolipid catabolic enzymes, pointing to additional mechanisms for maintaining sphingolipid homeostasis. ORM1 lacking Met-51 had strongly impaired interactions with LCB1 in a yeast (Saccharomyces cerevisiae) model, providing structural clues about regulatory interactions between ORM and SPT.