Abstract
Protein phosphorylation is an important post-translational modification that can regulate the protein function. The current knowledge on the phosphorylation status of plant oil body (OB) proteins is inadequate. This present study identifies the distinct physiological substrates of Arabidopsis serine/threonine/tyrosine protein kinase (STYK) and its role in seed oil accumulation; the role of Arabidopsis OLE1, a major seed OB protein has also been elucidated. In vitro kinase assay followed by mass spectrometry identifies residue that are phosphorylated by STYK. Further, co-expression of OLE1 and STYK in yeast cells increases the cellular lipid levels and reduces the total lipid when OLE1 was replaced with OLE1T166A. Moreover, in vivo experiments with OB isolated from wild-type and styk knock-out lines show the ability of STYK to phosphorylate distinct OB proteins. OLE1T166A mutant and Arabidopsis styk mutant demonstrate the significant reduction of its substrate phosphorylation. styk mutant line significantly reduces the amount of total seed oil as compared to wild-type seeds. Together, our results provide the evidences that Arabidopsis At2G24360 (STYK) is phosphorylating oil body proteins and the phosphorylation regulates the oil content in Arabidopsis seeds.
Highlights
In plants, oil body (OB) proteins are highly conserved and are grouped into structural proteins or enzymes[1]
Recent studies have indicated that post-translational modifications (PTM) of OB proteins play a role in these interactions
The serine/ threonine/tyrosine protein kinase (STYK) activity depended on the time of the reaction (Fig. 1B), the amount of OLE1 that was used (Fig. 1C) and the amount of ATP that was used in the reaction (Supplementary Fig. S3A)
Summary
Oil body (OB) proteins are highly conserved and are grouped into structural proteins or enzymes[1]. The major seed OB proteins oleosin, caleosin and steroleosin have been shown to play an important role in regulating the OB structure and lipid accumulation[4]. The ubiquitinylation of oleosin and caleosin in germinating sesame seeds has been proposed to play a role in the interaction of OBs with glyoxysomes which is needed for breaking down the lipids to release free fatty acids to provide energy[7]. The in vivo phosphorylation of AhOLE3 was not shown These studies demonstrate the current need for the identification of phosphorylated proteins and their corresponding kinases in plant seed OBs of Arabidopsis. The present study is directed to identify the role of Arabidopsis phosphorylated OB proteins in seeds and their corresponding protein kinases. We show various OB protein phosphorylation, the role of OLE1 in regulating the lipid content and its effect upon phosphorylation has been demonstrated in detail
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