Overexpression of FAX1 and ABCA9 in camelina
To investigate the function of FAX1 and ABCA9 in camelina, we overexpressed Arabidopsis FAX1 and ABCA9 genomic DNAs under the control of the cauliflower mosaic virus (CaMV)-35S promoter (Fig. 1a). The constitutive promoter was used because it was reported to promote plant growth in Arabidopsis in addition to lipid production [5, 8]. FAX1 and ABCA9 were fused with a HA-tag and a Flag-tag at the C-terminus, respectively. The production of FAX1-HA and ABCA9-Flag in camelina was confirmed by immunoblotting using anti-HA and anti-Flag antibodies, respectively (Fig. 1b, upper panel). The homozygous OE plants producing FAX1-HA and ABCA9-Flag were identified. In addition, we generated camelina lines overexpressing both FAX1 and ABCA9 by transforming the FAX1-HA construct into homozygous ABCA9-OE plants. The production of FAX1-HA and ABCA9-Flag in FAX1/ABCA9- single and double OEs were confirmed by immunoblotting (Fig. 1b, lower panel). Camelina has 3 homeologs of each of FAX1 and ABCA9 (Fig. 1c), and in WT camelina the transcript levels of the three FAX1s were relatively high in seedlings and developing seeds, but low in leaves, roots, and stems (Fig. 1c, left panel). The transcript levels of the three ABCA9 were relatively high in 4-week-old developing seeds (Fig. 1c, right panel).
FAX1 and ABCA9 increase seed and oil yield
FAX1-OEs, ABCA9-OEs, FAX1/ABCA9-OEs, and WT plants were grown side by side to determine the effect of FAX1 and ABCA9 on camelina growth and production. The OE lines had similar flowering time, plant height, and branch number as WT (Additional file 1: Fig. S1). However, the thousand-seed weight (TSW) of FAX1-OEs and ABCA9-OEs were 21% and 22% higher on average than that of WT, respectively, and FAX1/ABCA9-OEs were 40% higher on average than that of WT (Fig. 2a). Interestingly, FAX1-OEs and ABCA9-OEs had distinctive effect on seed length and seed width. FAX1-OEs increased seed length by 14%, whereas ABCA9-OEs increased seed width by 25%, while the seed length and width of FAX1/ABCA9-OEs were increase by 13% and 32%, respectively (Fig. 2a and 2b). The seed width of FAX1-OEs and seed length of ABCA9-OEs were similar with those of WT (Fig. 2b). The above results indicate that FAX1 and ABCA9 increase seed length and width, respectively, to increase seed weight.
To determine the effect of FAX1 and ABCA9 on oil production in camelina, we grew the single and double OE plants together with WT side by side with multiple replicates (n=18). Compared to WT, the seed yield per plant on average was 14% higher for FAX1-OE, 38% higher for ABCA9-OE, and 45% higher for FAX1/ABCA9-OEs (Fig. 2c). Moreover, the average oil content of WT seeds was 28%, whereas that FAX1-OE and ABCA9-OE seeds was 29% and 30%, respectively, and that of FAX1/ABCA9-OE seeds was 32% (Fig. 2c). The average increase of seed oil content over WT was 4% for FAX1-OE, 6% for ABCA9-OE, and 13% for FAX1/ABCA9-OE seeds. The results indicate that FAX1 and ABCA9 increase seed oil content and seed yield simultaneously and these two genes had an additive effect on seed oil content and seed yield, leading a substantial improvement on overall oil yield. Combining the increases in seed oil content and seed yield, the oil production per plant of FAX1-OEs and ABCA9-OEs was 22% and 55% higher than that of WT, respectively, whereas that of FAX1/ABCA9-OEs was 75% higher than that of WT (Fig. 2c).
The FAX1-OE and ABCA9-OE seeds displayed altered FA composition from WT seeds. Compared with that of WT seeds, the level of C16:0 of FAX1-OEs and ABCA9-OEs was decreased by 6% and 4%, respectively, whereas that of FAX1/ABCA9-OEs was decreased by 14% (Additional file 2: Fig. S2). Conversely, the level of C18:0 was increased by 5% for FAX1-OEs and ABCA9-OEs, and 12% for FAX1/ABCA9-OEs. The level of C18:1 was increased by 13% and 16% for FAX1-OEs and ABCA9-OEs, respectively, and 29% for FAX1/ABCA9-OEs. The level of C18:2 was increased by 6% and 7% for FAX1-OEs and ABCA9-OEs, respectively, and 10% for FAX1/ABCA9-OEs whereas that of C18:3 was decreased by 4% for FAX1-OEs and ABCA9-OEs and 10% for FAX1/ABCA9-OEs comparing to that of WT. In addition, the level of C20:1 was decreased by 4% and 11% in FAX1-OE and ABCA9-OE seeds, and 10% in FAX1/ABCA9-OE seeds (Additional file 2: Fig. S2). The above results indicate that increased FAX1 and ABCA9 expression positively affect the level of C18:0, C18:1 and C18:2, but negatively affect the level of C16:0, C18:3 and C20:1.
To test the seed performance, we monitored seed germination rates of the above lines. The seed germination rate at 12-hrs after imbibition of FAX1- and ABCA9- single and double OEs was slightly faster than that of WT while the final seed germination rate at 60-hrs after imbibition of all the lines tested was about 99% (Additional file 3: Fig. S3). In addition, we examined the vegetative growth of the above lines by growing the FAX1-OEs, ABCA9-OEs, and FAX1/ABCA9-OEs with WT side by side. The plant size appeared to be bigger in the single and double OE plants compared with WT (Additional file 4: Fig. S4a). The overground fresh weight of 3-week-old FAX1-OE and ABCA9-OE plants was 24% and 27% higher on average than that of WT, respectively, whereas that of FAX1/ABCA9-OEs was 57% higher on average than WT (Additional file 4: Fig. S4b). Also, FAX1-OEs had one more, ABCA9-OEs had two more, FAX1/ABCA9-OEs had 2-3 more leaves of 3-week-old plants compared with WT (Additional file 4: Fig. S4b).
FAX1 and ABCA9 affect membrane glycerolipid composition
We further examined the effect of FAX1-OE and ABCA9-OE on membrane glycerolipid composition in seed pods containing developing seeds. Whole pods, instead of dissected seeds, were used because seed dissections would lead to wounding and activation of lipolytic activities. Total lipids were extracted from developing pods of 2- and 4-weeks after flowering from WT, and FAX1-1, ABCA9-1, and FAX1/ABCA9-1 OE lines, and analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). In 2-week-old pods (WOP), which was considered as the outburst stage of lipid synthesis characterized by rapid lipid synthesis and oil production [34], compared to WT, the amount of phosphatidylcholine (PC) was increased 13%, 28%, and 33% in FAX1-1, ABCA9-1, and FAX1/ABCA9-1, respectively (Fig. 3). The amount of phosphatidylethanolamine (PE) was increased 60%, 104%, and 127% in FAX1-1, ABCA9-1, and FAX1/ABCA9-1. The total membrane glycerolipid level was 9%, 24%, and 31% higher in FAX1-1, ABCA9-1, and FAX1/ABCA9-1 than WT at the early stage of developing pods. However, the amount of phosphatidylglycerol (PG) was decreased approximately 45% in all three OE lines, and the level of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) had no obvious changes (Fig. 3). When the lipid data were calculated as mol% of total lipids analyzed, PE in FAX1-1, ABCA9-1, and FAX1/ABCA9-1 was 6, 12, and 13 mol% higher, respectively, than that of WT. However, the major plastidic lipids, phosphatidylglycerol (PG), MGDG, and DGDG were all lower in three OE lines than WT (Additional file 5: Fig. S5). Phosphatidic acid (PA) constituted less than 0.2 mol% in the developing pod of all lines tested and the PA mol% in FAX1-OE, ABCA9-OE, and FAX1/ABCA9-OE lines was all lower than that of WT (Additional file 5: Fig. S5).
In 4-WOP, which was considered as the plateau stage of lipid synthesis [34], the level of PC in FAX1-1, ABCA9-1, and FAX1/ABCA9-1 was 14%, 14%, and 17% higher than that of WT (Fig. 3); The level of PE in FAX1-1, ABCA9-1, and FAX1/ABCA9-1 was 12%, 19%, and 25% higher than that of WT. The total membrane glycerolipid content in FAX1-1, ABCA9-1, and FAX1/ABCA9-1 was 3%, 5%, and 8% higher than that of WT (Fig. 3). However, the level of major plastidic lipids, PG and DGDG was decreased in FAX1-1 whereas was comparable among WT, ABCA9-1 and FAX1/ABCA9-1 at this stage (Fig. 3). PC in FAX1-1, ABCA9-1, and FAX1/ABCA9-1 was 2, 3, and 4 mol% higher than WT whereas mol% of MGDG and DGDG in the three OE lines was slightly lower than that of WT. The mol% of PE and PG was comparable between WT and OE lines at this stage (Additional file 5: Fig. S5). Also, the PA mol% was lower all OE lines than WT (Additional file 5: Fig. S5).
The major PC species (34:3, 34:2, 36:5, and 36:4 PC) in FAX1-1, ABCA9-1, and FAX1/ABCA9-1 were increased in 2-WOP, and 36:5, 36:4, and 36:3 PC were increased in 4-WOP compared with those of WT (Fig. 4). Similarly, the major PE species (34:3, 34:2, 36:6, 36:5, 36:4, and 36:3 PE) of FAX1-1, ABCA9-1, and FAX1/ABCA9-1 were all increased in 2-WOP, and only 34:3, 34:2, 36:5, and 36:4 PE were increased in 4-WOP compared to those of WT (Fig. 4). Also, the effect of FAX1 and ABCA9 exhibited additive effect, especially on 34:2 and 36:4 PC, and 34:3, 34:2, 36:5, and 36:4 PE (Fig. 4). In contrast, the major PG species (32:0, 34:4, 34:3, 34:2, and 34:1 PG) of FAX1-1, ABCA9-1, and FAX1/ABCA9-1 were all decreased compared to those of WT in 2-WOP, while only 34:3 PG was decreased in FAX1-1, and 34:4 PG decreased in ABCA9-1, and FAX1/ABCA9-1 in 4-WOP (Fig. 4). Although the total amount of MGDG was similar between WT and OE lines (Fig. 3), the level of some MGDG species, especially in FAX1-1, was different from that of WT. For instance, two major MGDG spices, 34:6 and 36:6 MGDG, in FAX1-1 was decreased both in 2- and 4-WOP compared with WT (Fig. 4). For DGDG, only 36:5 DGDG in 2-WOP, and 36:6 and 36:5 DGDG in 4-WOP of FAX1-1 were lower than WT (Fig. 4). Similarly, the mol% of 34:2 PC of ABCA9-1 and FAX1/ABCA9-1 in 2-WOP, and 36:5 PC of ABCA9-1 and FAX1/ABCA9-1 in 4-WOP were higher than that of WT (Additional file 6: Fig. S6); the mol% of 34:2 PE of ABCA9-1 and FAX1/ABCA9-1 was higher than WT both in 2- and 4-WOP; whereas the mol% of 34:2 PG of FAX1-1 was lower than WT; the major MGDG species 36:6 was lower in FAX1-1 and FAX1/ABCA9-1; and the major DGDG species 36:6 was lower in FAX1-1 in 4-WOP (Additional file 6: Fig. S6). The above lipid data indicate that overexpression of FAX1 and ABCA9 also affect membrane lipid composition in developing pod, and the effect differs at different developmental stage.
FAX1 and ABCA9 alter the expression of genes in oil production in seeds
To gain insights into the enhancing oil production, we compared the transcript level of genes that are involved in oil accumulation in developing seeds of the FAX1- and ABCA9-OE lines with WT. The transcript level of AtFAX1 in 2- and 4-week-old developing seeds (WODS) was high in FAX1-OE and FAX1/ABCA9-OE lines, but was not detected in ABCA9-OE lines, as expected. Similarly, the level of AtABCA9 was high in ABCA9-OE and FAX1/ABCA9-OE lines, but was not detected in FAX1-OE lines (Additional file 7: Fig. S7). The transcript level of three DGAT1s (Acyl-CoA: diacylglycerol acyltransferase 1) was higher in ABCA9-1, and slightly lower in FAX1-1 in 2-WODS whereas that of two DGAT2s was higher in 2-WODS, and all three DGAT2 were higher in 4-WODS in all OE lines than WT (Fig. 5). The transcript of one PDAT1 (phospholipid: diacylglycerol acyltransferase 1) was detected, and its level was higher than that of WT in all OE lines at both stages. The level of three PDAT2s transcripts was higher in ABCA9-1 and FAX1/ABCA9-1 in 2-WODS, whereas that of two PDAT2s transcripts was higher in three OE lines but the other PDAT2 was lower in ABCA9-1 and FAX1/ABCA9-1 in 4-WODS. The transcript level of one WRI1 was lower in FAX1-1 and ABCA9-1 whereas another WRI1 was higher in FAX1/ABCA9-1 in 2-WODS (Fig. 5). At 4-WODS, the level of two WRI1s was lower in ABCA9-1 than WT. The transcript level of LEC1 (leaf cotyledon 1) homeologs were higher in 2- and 4-WODS in all OE lines than WT. One NPC6 (nonspecific phospholipase C 6) displayed a higher level in FAX1- and ABCA9-OEs in 2- and 4-WODS, and another had a higher level in FAX1-1 and ABCA9-1 in 2-WODS (Fig. 5).