Fig 5. Conserved function of spPLD.
A. Hd3a with mutated PC binding sites (Hd3aM) did not promote flowering time of Arabidopsis as Hd3a. Transgenic Arabidopsis overexpressing Hd3a showed early flowering under long-day condition while those overexpressing Hd3aM did not (left). Number of rosette leaves and flowering time of plants were calculated (right) and statistically analyzed by Tukey’s test (lower, *, p < 0.05; ***, p < 0.001, compared to Col-0). NS, no significance. Representative images were shown (left, bar = 1 cm) and data were shown as mean ± SD (n = 15). Protein sequence of Arabidopsis FT and rice Hd3a were shown, and amino acids for PC binding of Arabidopsis FT, which is conserved in rice Hd3a and used for mutation analysis, were highlighted by red color (upper). B. Phylogenetic analysis of spPLDs in different plant species. Sequence alignment and the rectangular cladogram were generated with ClustalX 2.0 or TreeView respectively. Amino acid pairwise identity (percentage) between different protein sequences was shown. Scale bar represents 0.05 amino acid substitutions per site. C. spPLD delays flowering time of Arabidopsis. Transgenic Arabidopsis overexpressing spPLD showed delayed flowering under long-day condition. Number of rosette leaves and flowering time of plants were calculated and statistically analyzed using Tukey’s test (right, ***, p < 0.001, compared to Col-0). Representative images were shown (left, bar = 1 cm) and data were shown as mean ± SD (n = 15). D. A hypothetical model (tissue model and cellular model respectively) illustrating how secretory spPLD functions in suppressing rice heading time. spPLD showing a diurnally rhythmic and JA-upregulated expression, is secreted and hydrolyzes phosphatidylcholine (PC) at apoplast to reduce the levels of light period predominant PC species (less unsaturated PCs) in shoot apical meristem (SAM), which may lead to the reduced activity of primary pump (H+-ATPase) at plasma membrane, resulting in the less driving force for Hd3a/RFT1 entering cell at SAM, hence the decreased Hd3a/RFT1 activity and delayed heading. Other phospholipids may involve in heading time regulation through binding with and regulating the activity of Hd3a/RFT1.