Fig 3. Altered PC species under spPLD overexpression or deficiency.

A. Fat-western immunoblot analysis revealed the binding of rice Hd3a (left) and Hd3a with mutated PC binding sites (Hd3a^M, right) to phospholipids. The phospholipid type of each dot is indicated. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PI, phosphatidylinositol; PI3P, PI 3-monophosphate; PI4P; PI5P; PI(3,4)P₂, PI 3,4-bisphosphate; PI(3,5)P₂; PI(4,5)P₂; PI(3,4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate. B. Liposome binding assay (left) and quantitative analysis (right) confirmed the preferential binding of Hd3a to PC. His-tag fused Hd3a and Hd3a with mutated PC binding sites (Hd3a^M) were purified and incubated with liposomes containing different PC:PA or PE:PA ratios. After collecting the liposomes, the portion of proteins bound to liposomes was detected by western blotting using anti-His antibody. Nonbinding protein was detected in the supernatant (bottom). Band density is measured by Image J and relative density was calculated by setting the intensity under PC:PA ratio 1:1 as 1.0. Data were presented as means ± SD (n = 3, right). C-D. Relative content of predominant PCs with different saturation status, PC (32:1), PC (36:2), PC (36:3) and PC (38:3) (C); PC (36:5), PC (36:6) and PC (38:6) (D), in ZH11 and various lines with altered spPLD expressions. Ten rice shoot apical meristem before bolting were collected and used for lipids extraction. Phospholipids were profiled by a lipidomic approach using mass spectrometry. Experiments were biologically repeated three times and data were shown as mean ± SD (n = 3). Statistical analysis was performed by Tukey’s test (*, p <0.05; **, p < 0.01 ***, p < 0.001, compared to ZH11).