Abstract
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.
Highlights
2" Sphingolipids are essential, abundant endomembrane and plasma membrane lipids that 3" contribute to membrane function, vesicular trafficking, and the mediation of cellular 4" processes in eukaryotes (Coursol et al, 2003; Liang et al, 2003; Chen et al, 2006; 5" Markham et al, 2011)
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
We show that ORM1 and ORM2 are essential for lifecycle completion in Arabidopsis thaliana
Summary
2" Sphingolipids are essential, abundant endomembrane and plasma membrane lipids that 3" contribute to membrane function, vesicular trafficking, and the mediation of cellular 4" processes in eukaryotes (Coursol et al, 2003; Liang et al, 2003; Chen et al, 2006; 5" Markham et al, 2011). The majority of LCBs occur in ceramides. 28" sphingolipids for growth and membrane function while limiting the accumulation of LCBs. 29" and ceramides until needed to trigger specific cellular functions, such as PCD-mediated. 39" and Orm2p suppress SPT activity in response to elevated sphingolipid levels through a. Sphingolipid-responsive regulation of the ORM-SPT interaction in S. cerevisiae. 57" DNA mutant yielded fertile plants with increased accumulation of LCBs and ceramides. 64" plants has precluded assessment of SPT regulation in the absence of ORM proteins. 69" the first transmembrane domain of LCB1, which is known to interact with ORM for SPT. 74" domain of ORM, strongly reduced the ORM-LCB1 interaction. 76" are associated with their regulatory interaction with the LCB1 subunit of SPT
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