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.

类粘蛋白样蛋白 (ORM) 与丝氨酸棕榈酰转移酶 (SPT) 相互作用，负向调节鞘脂生物合成，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，我们证明 ORM1 和 ORM2 对于拟南芥 ( Arabidopsis thaliana ) 生命周期的完成至关重要。通过每个基因的 CRISPR/Cas9 敲除突变体杂交产生的orm1 ^{– / –} orm2 ^{– / –}突变体的种子积累了高水平的神经酰胺，表明鞘脂生物合成不受调节。 orm1 ^{– / –} orm2 ^{– / –}种子无法存活，胚胎发育异常，并且与野生型种子相比，含油量降低了 80% 以上。这种表型在表达 SPT 亚基 LCB1 的拟南芥种子中被模仿，该亚基缺乏第一个跨膜结构域，这对于 ORM 介导的 SPT 调节至关重要。我们鉴定了 ORM1 的突变体，其第二个跨膜结构域附近缺少一个氨基酸 (Met-51)，但保留了其膜拓扑结构。在orm2背景中表达该等位基因产生的植物不会超出幼苗阶段，神经酰胺超积累，并显示出细胞器结构的改变以及衰老和发病机制相关基因表达的增加。这些幼苗还表现出鞘脂分解代谢酶基因表达上调，表明维持鞘脂稳态的其他机制。 在酵母（酿酒酵母）模型中，缺乏 Met-51 的 ORM1 与 LCB1 的相互作用受到严重损害，这为 ORM 和 SPT 之间的调节相互作用提供了结构线索。