Dynamic changes of oil content and FA compositions in developing SASK
The oil accumulation during SASK development showed a sigmoid pattern, exhibiting a sharp rise between 4 and 8 WAA and a maximal level 8 WAA (Figure 1). Comparative analysis indicated that the oil increase was higher in AS-80 than in AS-84, which ultimately led to a higher oil content in AS-80 (53.48 g/100 g) than in AS-84 (44.85 g/100 g). GC-MS analysis indicated that eight FA species are predominantly found in SASKs, namely 16:0, 16:1, 18:0, 18:1, 18:2 and 18:3 (polyunsaturated fatty acids, PUFAs), 20:0 and 20:1(very long-chain fatty acids, VLCFAs; C＞20) (Table 1). Among those FAs, C18:1 (oleic acid) and C18:2 (linoleic acid) represented the major compositions in SASK oil (Table 1). The FA profiles were different between AS-80 and AS-84, and the former exhibited a higher content of oleic acid and linoleic acid (Table 1). Based on these results, the SASKs from 4, 6 and 8 WAA, which respectively represent early, mid and late phases of oil accumulation, were selected as optimal samples for lipidomic analysis.
Changes to TAG molecular species during SASK development
Triglycerides, a naturally occurring ester of three FAs and glycerol, are the chief constituent of storage oil. After identifying the TAG molecular species, the two prominent species were 18:1/18:1/18:2 and 18:1/18:1/18:3, followed by 18:2/18:2/18:2, 18:1/18:1/18:1, 16:1/18:1/18:2 (Supplementary Table S1 and Figure 2). It is worth noticing that C18:1 and C18:2 are the main FA chains at all time points, in keeping with the analytic results of FA compositions (Table 1). Although these TAG molecules both in AS-80 and AS-84 showed a gradual accumulation during development, a significantly increased content (especially for 18:1/18:2/18:2 and 18:1/18:1/18:3) could be observed in AS-80 (Figure 2). The different rate of increment could explain why the SASKs from AS-80 have a higher oil content. It will be noted that those TAG molecules containing VLCFAs are only minor components (Supplementary Table S1).
Changes to DAG and PA molecules during SASK development
The main molecular species of DAG in developing SASK were 18:3/18:3, 18:1/18:1 and 18:2/18:2 (Figure 3A). The content of 18:3/18:3 showed notable decrease during oil accumulation, whereas 18:1/18:1 and 18:2/18:2 were little changed throughout development (Figure 3A and Supplementary Table S2). There were only very small amounts of DAG molecular species containing VLCFAs (Supplementary Table S2).
After removing the phosphate group, PA molecules can be invoked as the immediate precursor to DAG in the Kennedy pathway. The main molecular species of PA were 18:1/18:1 and 16:0/18:2 (Figure 3B and Supplementary Table S3). This was particularly noticeable with similarly dynamic profiles between AS-80 and AS-84. The 18:1/18:1 increase in abundance between 4 and 6 WAA, while 16:0/18:2 decreased with SASK maturity (Figure 3B). Other significant molecular species of PA were those containing PUFAs (18:1/18:2, 16:0/18:3, 18:2/18:1, 18:3/18:3, 18:2/18:3 and 18:3/18:2) or those containing VLCFAs (18:1/20:1 and 18:1/20:2). During SASK development, most of those PA species containing PUFAs and VLCFAs tended to decrease (Supplementary Table S3).
Changes to phospholipids during SASK development
In plants, PC is a central intermediate in the flux of FAs or DAGs, or both substrates into TAG. By lipidomic analysis, the two main molecular species in developing SASK were 18:1/18:1 and 18:1/18:2, and both showed a significant rise with the development of SASK (Figure 4A and Supplementary Table S4). Other significant PC molecular species were 16:0/18:1, 18:2/18:1, 16:0/18:3, 16:1/16:1, 18:3/18:3 and 18:2/18:2 (Figure 4A). Interestingly, those PC species containing PUFAs had the maximum values at 6 WAA (the mid stage of oil accumulation). There were only minor amounts of PC species containing VLCFAs (Supplementary Table S4).
Phosphatidyl-ethanolamine (PE) is synthesized using the same enzyme with PE biosynthesis and DAG from the Kennedy pathway. Here, we examined PE molecular species to compare with those of PC. The main molecular species of PE were 16:0/18:3, 18:1/18:1, 16:0/18:1 and 18:2/18:2 (Figure 4B and Supplementary Table S5). It was clear from the data that the 18:2/18:2 accumulation during SASK development showed a V-pattern with a minimum value at 6 WAA, whereas 16:0/18:3, 18:1/18:1 and 16:0/18:1 represented an inverse pattern with a maximum value at 6 WAA (Figure 4B). The temporal pattern of PE molecular species was noticeably different from those of PC in developing SASK. Other noteworthy species were 16:1/16:1 and 18:1/18:2, which showed little change in proportion during SASK development (Figure 4B).
Utilizing PA from the Kennedy pathway, phosphatidylglycerol (PG) is synthesized. The main molecular species of PA included 22:0/18:1, 22:4/18:3, 22:2/18:2, 16:0/18:1 and 16:0/18:2 (Figure 4C and Supplementary Table S6). The species of 22:2/18:2 and 16:0/18:1 had the maximum values at 6 WAA, while 22:0/18:1, 16:0/18:2 and 22:4/18:3 decreased during SASK development (Figure 4C). Intriguingly, the VLCFAs were the main composition, which were significantly different from PE and PC. This, along with minor amounts of VLCFAs in PA, DAG and TAG, suggests that PG is not involved in TAG accumulation.
Acyl fluxes through PC may determine TAG FA composition
As described above, three mechanisms allow the flux of FA through PC for eventual TAG synthesis [9-11]. First, PC acyl editing (Figure 5, orange arrows). The fatty acid desaturase 2 (FAD2)  and FAD3  convert PC-bound oleate (C18:1) to linoleate (C18:2) and then linolenate (C18:3), respectively. The qRT-PCR data indicated that the FAD2 gene displayed a bell-shaped pattern of expression during the oil accumulation period (between 4 and 8 WAA), while FAD3 genes showed a low expression level (Figure 6A). This suggests, as noted for FA compositions in developing SASK (Table 1), that linoleic acid rather than linolenic acid is a major composition in SASK oil. Results also indicate that the PC acyl editing is necessary for SASK oil accumulation.
Second, acyl chains on PC can also be incorporated into the sn-1 and sn-2 positions of DAG via two known enzymatic routes (Figure 5, blue arrows). Our qRT-PCR results indicated the expression levels of PDCT and DAG-CPT both displayed a downtrend with SASK development (Figure 6A). Surprisingly, the main species of 18:3/18:3 DAG also showed a notable decrease during SASK development (Figure 3A). Considering the lack of 18:3/18:3 species in PA (Figure 3B), it seems plausible that PDCT and DAG-CPT can generate 18:3/18:3 DAG from PC.
Third, the final step in TAG synthesis can also be catalyzed by PDAT (Figure 5, purple arrows), using the sn-2 and sn-1 (at a quarter of the rate for the sn-2 position) acyl group from PC . PDATs from Arabidopsis and other plants have been shown to have high activity with PC containing unsaturated FAs [28, 29]. With this knowledge, we sought to use the lipidomic data to evaluate the possible contribution of PDAT to TAG accumulation. If the oilseeds are in the period of high oil biosynthetic rate, the flow of FAs is mostly into oil accumulation, and is not unduly influenced by other lipid metabolism . Thus, these lipidomic data from 6 WAA (high biosynthetic rate) were chosen for evaluating the PDAT role in TAG biosynthesis. Using the sn-2 and sn-1 (a quarter of rate) acyl group of PC as the acyl donor, we assumed the ratios of C18:1/ C18:2 and C18:2/ C18:3. Also, the rates of C18:1/ C18:2 and C18:2/ C18:3 from the sn-1, sn-2 and sn-3 acyl group of TAG were evaluated. As expected, the rates of C18:1/ C18:2 and C18:2/ C18:3 from the sn-1 and sn-2 of TAG had enormous differences with those from sn-2 of PC in both AS-80 and AS-84 (Figure 6B). These data suggest that PDAT does not function in sn-1 and sn-2 acylation of TAG. Although the C18:1/ C18:2 rate showed a substantial difference between sn-3 of TAG and sn-2 of PC, it was worth noting that the C18:2/ C18:3 rate from sn-3 of TAG was close to this from sn-2 of PC in both AS-80 and AS-84 (Figure 6B). Our data implicate involvement of PDAT in sn-3 acylation of TAG, and strong substrate selectivity for PUFAs by PDAT enzyme.
In summary, the above-mentioned results imply the importance of PC-mediated “PUFA trafficking” for TAG biosynthesis, which may determine the FA composition of TAG in developing SASK.