Abstract
Serine palmitoyltransferase (SPT), a pyridoxyl-5'-phosphate-dependent enzyme, catalyzes the first and rate-limiting step in sphingolipid biosynthesis. In humans and yeast, orosomucoid proteins (ORMs) negatively regulate SPT and thus play an important role in maintaining sphingolipid levels. Despite the importance of sphingoid intermediates as bioactive molecules, the regulation of sphingolipid biosynthesis through SPT is not well understood in plants. Here, we identified and characterized the Arabidopsis thaliana ORMs, ORM1 and ORM2. Loss of function of both ORM1 and ORM2 (orm1 amiR-ORM2) stimulated de novo sphingolipid biosynthesis, leading to strong sphingolipid accumulation, especially of long-chain bases and ceramides. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays confirmed that ORM1 and ORM2 physically interact with the small subunit of SPT (ssSPT), indicating that ORMs inhibit ssSPT function. We found that orm1 amiR-ORM2 plants exhibited an early-senescence phenotype accompanied by H2O2 production at the cell wall and in mitochondria, active vesicular trafficking, and formation of cell wall appositions. Strikingly, the orm1 amiR-ORM2 plants showed increased expression of genes related to endoplasmic reticulum stress and defenses and also had enhanced resistance to oxidative stress and pathogen infection. Taken together, our findings indicate that ORMs interact with SPT to regulate sphingolipid homeostasis and play a pivotal role in environmental stress tolerance in plants.
Highlights
In eukaryotes, sphingolipids make up ;40% of the lipids of the plasma membrane and are abundant in other endomembranes
Our confocal microscopy observations confirmed that ORM1 and ORM2 localized to the endoplasmic reticulum (ER) of Arabidopsis (Figure 1D)
Our results indicate that Arabidopsis orosomucoid proteins (ORMs) function to regulate sphingolipid biosynthesis in maintaining sphingolipid homeostasis and play a role in response to abiotic and biotic stresses
Summary
Sphingolipids make up ;40% of the lipids of the plasma membrane and are abundant in other endomembranes. The functions of these key lipids have been intensively investigated in mammals and yeast for decades (Hannun and Obeid, 2008), and recent work has begun to explore sphingolipid biochemistry in plants. Sphingolipids play pivotal roles as membrane structural components, as bioactive molecules involved in signal transduction and cell regulation, and in a wide range of other biological processes, including secretion, programmed cell death, autophagy, stress responses, and cell-cell interactions (Liang et al, 2003; Markham et al, 2011; Sentelle et al, 2012; Bi et al, 2014; Li et al, 2015; Wu et al, 2015).
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