Multiple caleosins have overlapping functions in oil accumulation and embryo development

Caleosins are lipid droplet- and endoplasmic reticulum-associated proteins. To investigate their functions in oil accumulation, expression levels of caleosins in developing seeds of Arabidopsis thaliana were examined and four seed-expressed caleosins (CLO1, CLO2, CLO4, and CLO6) were identified. The four single mutants showed similar minor changes of fatty acid composition in seeds. Two double mutants (clo1 clo2 and clo1×clo2) demonstrated distinct changes of fatty acid composition, a 16-23% decrease of oil content, and a 10-13% decrease of seed weight. Moreover, a 40% decrease of oil content, further fatty acid changes, and misshapen membranes of smaller lipid droplets were found in seeds of quadruple CLO RNAi lines. Notably, ~40% of quadruple CLO RNAi T1 seeds failed to germinate, and deformed embryos and seedlings were also observed. Complementation experiments showed that CLO1 rescued the phenotype of clo1 clo2. Overexpression of CLO1 in seedlings and BY2 cells increased triacylglycerol content up to 73.6%. Transcriptome analysis of clo1 clo2 developing seeds showed that expression levels of some genes related to lipid, embryo development, calcium signaling, and stress responses were affected. Together, these results suggest that the major seed-expressed caleosins have overlapping functions in oil accumulation and show pleiotropic effects on embryo development.