Lipid composition of the Amazonian ‘Mountain Sacha Inchis’ including Plukenetia carolis-vegae Bussmann, Paniagua & C.Téllez

Several Amazonian species of Plukenetia are remarkably rich sources of polyunsaturated fatty acids, in particular α-linolenic acid. The lipid composition of the large-seeded, recently described ‘Mountain Sacha Inchi’ Plukenetia carolis-vegae is reported here for the first time, and compared with Plukenetia huayllabambana, two cultivars of Plukenetia volubilis, and a newly developed hybrid between P. volubilis and P. carolis-vegae. All species and cultivars had a very high content of polyunsaturated fatty acids, 82.6–86.7% of total fatty acids, and at least 46.6% α-linolenic acid of total fatty acids. The highest content was found in P. carolis-vegae which had 57.4%. The exceptionally high α-linolenic acid content suggests that P. carolis-vegae may be an important plant-derived dietary source of this essential fatty acid and that the species has considerable potential for further domestication and commercialisation of its seeds and seed oil. A TAG analysis was carried out for the two P. volubilis cultivars, in which LLnLn and LnLL were most prevalent, and for P. huayllabambana, in which LLnLn constituted the largest fraction, followed by LnLnLn, indicating that this large-seeded species also has interesting dietary properties.

. Comparison of the content of saturated, monounsaturated and polyunsaturated (including linoleic acid, C18:2 n-6, ω-6, and α-linolenic acid, C18:3 n-3, ω-3) fatty acids between four of the five large-seeded Plukenetia species known to have been cultivated. Data adapted from 7,14,15,18 . Image of Plukenetia conophora adapted from 13  www.nature.com/scientificreports/ carolis-vegae, and a study of the fatty acid composition of the seed oil of this species seems very relevant. Also, the current authors have demonstrated compatibility between P. carolis-vegae and P. volubilis, lending support to hybridisation events occurring within the species complex, and a study of the seed oil of the artificial hybrid P. volubilis × P. carolis-vegae may also aid in better understanding the nutritional qualities of the 'Mountain Sacha Inchis' . The current study aims to analyse the oil composition of P. carolis-vegae and P. huayllabambana using P. volubilis as a reference. Furthermore, the oil composition of the newly developed hybrid between P. volubilis and P. carolis-vegae will be studied. We hypothesise that P. carolis-vegae will have a similar lipid composition to P. huayllabambana, but with a higher fraction of ALA, and that P. volubilis × P. carolis-vegae will have a composition within the range of the parents' lipid composition.
Oil extraction. The testae of the seeds was disrupted using a mortar and pestle and carefully removed, following which the seeds were milled using a coffee grinder.
Supercritical fluid extraction was performed on a laboratory scale unit (Spe-ed SFE, Applied Separations, Allentown, PA, U.S.A.) using 10 mL extraction tubes. Oil from milled seeds (2 g) was extracted in triplicates, with exception of the P. volubilis × P. carolis-vegae sample for which material was only available for two extractions. The samples were subjected to supercritical carbon dioxide (99.7% purity, Air Liquide SA, Taastrup, Denmark) for 30 min at 500 bar and 60 °C at a flow rate of 2.5 L min −1 .
Triacylglycerol analysis. The triacylglycerol (TAG) profile of the oils were analysed by enhanced liquid chromatography (EFLC) on a SFE/HPLC hybrid system (Agilent Infinity 1260, Santa Clara, U.S.A.) according to the method of Buskov et al. 28 . A BDS hypersil C18 column (250 × 4.6 mm, 5 µm particle size, Thermo Fisher Scientific) was used and isocratic elution was performed with acetonitrile:2-propanol:CO 2 (56:14:30) at a flow rate of 1.5 mL min −1 . The column temperature was set to 40 °C, and the column back pressure was 200 bar. The TAGs were detected with UV detection at 210 nm, and with ELSD (evaporative light scattering detection, Agilent 1900 Infinity, Santa Clara, U.S.A). The ELSD conditions were as follows: nebulizer temperature of 68 °C, evaporator temperature of 40 °C, and nitrogen pressure of 0.15 MPa. A 5 µL injection loop was used for analysing the TAG profile. For identification of the individual TAG composition preparative EFLC was applied in combination with GC-FAME analysis of each fraction. For the preparative EFLC a 200 µL injection loop was used and fractions collected every minute. Each fraction was then subjected to the FAME (fatty acid methyl esterification) procedure, and the fatty acid identified using a Supelco 37 Component FAME mix (Sigma-Aldrich, Denmark) and with spiking of individual fatty acid methyl ester standards (Sigma-Aldrich, Denmark).
Fatty acid analysis. The FAME procedure was performed according to the AOAC Official Method 969.33 29 , with modifications as follows; 20 mg oil was added to 1 mL methanolic NaOH (0.5 M) with 200 µL internal standard (heptanoic acid, decanoic acid, and heptadecanoic acid; 10 mg/mL) (Sigma-Aldrich, Denmark), and vortex mixed with 1.5 mL boron-trifluoride methanol (BF 3 ) solution. The mixture was heated at 70 °C for 2 min, where after a saturated NaCl solution was added and the FAMEs extracted into cyclohexane (1 mL).
The fatty acid profile was determined using gas chromatography (GC) with flame ionization detection (FID) using the Agilent Technologies GC System 7820A (Santa Clara, California, USA). A Supelco SP™-2380 capillary column (L × I.D. 30 m × 0.25 mm, d f 0.20 μm) was used. Supelco 37 Component FAME mix (Sigma-Aldrich, Denmark) was used as a standard. The temperature program was as follows: initial temperature of 60 °C hold 2 min, ramp to 200 °C hold 0 min, and ramp to 240 °C hold 7 min. Helium was used as the carrier gas with a flow of 2.477 mL min −1 , the injection volume was 1 mL with a split ratio of 52:1, and cyclohexane was used as a solvent.
The samples collected from preparative EFLC were evaporated to dryness before the esterification process, and a few variations were made to the procedures described above. Less solvents were used; 100 µL methanolic NaOH, 150 µL BF 3 solution, and 200 µL cyclohexane. In the GC analysis, injection was changed to splitless, but otherwise no changes were made to the GC method. Data analysis. An analysis of variance (ANOVA) test followed by a Tukey HSD test was used to determine significant differences in the fatty acid composition of the different species and cultivars of Plukenetia.
Plukenetia huayllabambana. Plukenetia huayllabambana is one of the more recently described species of Plukenetia 2 and have very large seeds; estimated as 6627 mm 3 per seed compared with the approx. 997 mm 3 of P. volubilis 4 . According to a recently published revised classification of the Plukenetia genus, P. huayllabambana is a putative hybrid between P. volubilis and the newly described P. sylvestris, a large-seeded species of the high elevation species complex sister to P. volubilis 3 . Our analysis shows that seed oil from P. huayllabambana has a significantly higher content of ALA (56.6 ± 0.2%) than P. volubilis from both Ecuador and Peru (51.5 ± 3.3 and 46.6 ± 1.2%, respectively), while the content of LA is also significantly lower (26.8 ± 0.1%, Fig. 2). This content corresponds well with the values previously reported for P. huayllabambana, which range from 51.3 to 58.2% ALA and from 25.8 to 29.3% LA; an ALA content generally exceeding that of P. volubilis 12,15,17 .
Plukenetia carolis-vegae. The oil composition of P. carolis-vegae was analysed for the first time in the current study. Plukenetia carolis-vegae has been hypothesised to be a cultivated and fully or semi-domesticated species derived from wild, naturally occurring populations of P. sylvestris. Further, P. carolis-vegae is a part of the high elevation 'Mountain Sacha Inchi' species complex sister to P. volubilis also consisting of P. huayllabambana and P. sylvestris 3 . The oil composition of P. carolis-vegae is of particular interest since the seeds are the largest in the species complex; approx. 7069 mm 3 per seed 4 .
The seed oil of P. carolis-vegae was found to contain 57.4 ± 0.0% ALA (Fig. 2), which was the highest measured value in the study and was significantly different from the values of ALA measured in the seed oil of both P. volubilis cultivars and the hybrid P. volubilis × P. carolis-vegae. It was also higher than the value measured for www.nature.com/scientificreports/ P. huayllabambana, albeit not significantly. Conversely, the value of LA was the lowest measured, 25.2 ± 0.0% of the seed oil, which was significantly lower than the values measured in the oil of the P. volubilis cultivars and the hybrid, and also lower, though not significantly, than the value measured for P. huayllabambana seed oil. The very high ALA content of P. carolis-vegae may be caused by genetic factors or may be related to altitude and temperature; the material of P. carolis-vegae was collected at the highest altitude of the 'Mountain Sacha Inchis' in this study (1610 m), and Cai et al. (2012) observed that the ALA content of P. volubilis generally increased with higher altitude and decreasing temperatures 30 . However, studies of the oil composition of P. carolis-vegae cultivated at lower altitudes need to be conducted to assess whether the high ALA content is due to environment, genetics or both. The amount of OA in P. carolis-vegae seed oil was 10.5 ± 0.0%, which was significantly higher than in both P. volubilis cultivars, P. huayllabambana, and the hybrid P. volubilis × P. carolis-vegae. The content of eicosenoic and palmitic acid in P. carolis-vegae seed oil (0.6 ± 0.0 and 4.9 ± 0.0%, respectively) was found to be mostly similar to the content in the seed oil of the other analysed species. In contrast, the content of eicosanoic and stearic acid (0.3 ± 0.0 and 1.0 ± 0.1%, respectively) was significantly lower than in both the P. volubilis cultivars and P. huayllabambana but similar to the levels in the hybrid. Overall, the most striking difference between the oils was the very high ALA content of P. carolis-vegae seed oil, a property which may be promising for further cultivation and domestication of the species.
Plukenetia volubilis × Plukenetia carolis-vegae. The ALA content of the P. volubilis × P. carolis-vegae hybrid seed oil is 46.8%, which is similar to that of the two P. volubilis cultivars, but lower than that of P. huayllabambana and P. carolis-vegae. Conversely, the LA content of the oil is 39.9%, which is significantly higher than all other samples except P. volubilis from Peru (Fig. 2). The OA lipid fraction is 7.1%, which is similar to that of the two P. volubilis cultivars and P. huayllabambana, but significantly different from that of P. carolis-vegae. Since the P. volubilis × P. carolis-vegae hybrid is a cross between P. volubilis and P. carolis-vegae, it is to be expected that fatty acid composition of the oil will be in between the composition of these two. While the values are not significantly different from those of P. volubilis from Peru, they are not very similar to the values of P. carolis-vegae, although the fruit morphology of the hybrid shows similarity with that of P. carolis-vegae. This dissimilarity may be due to either the genetic composition of this specific cross or may be an effect of environmental conditions. The hybrid was cultivated in a nursery at a lower altitude than the collection altitudes of either of the parents, and if altitude and temperature is indeed an essential driver of conversion from ALA to LA 30 , this might have influenced the seed oil composition of the hybrid.

Triacylglycerol (TAG) profile. The distribution of fatty acids in the TAG molecules varies between species
and cultivars and is responsible for the chemical, physical, and biological properties of oils and fats 36 . In P. volubilis from Ecuador, P. volubilis from Peru, and P. huayllabambana, 15 different TAGs were identified, containing six different fatty acids ( Table 1).
Genetic control of the fatty acid composition. Across all the examined species and cultivars, the SFA content is relatively low, ranging from 5.7% in P. carolis-vegae × P. volubilis to 8.4% in P. huayllabambana. Similarly, the MUFA content ranges from 7.6% in P. carolis-vegae × P. volubilis to 11.1% in P. carolis-vegae (Fig. 3). The remainder of the fatty acids is PUFA, comprised of ALA and LA, in total 84% of the seed oil in P. volubilis from Ecuador, 83.1% in P. volubilis from Peru, 83.4% in P. huayllabambana, 82.6% in P. carolis-vegae, and 86.7% in P. carolis-vegae × P. volubilis. Comparatively, linseed and chia oil contain approx. 74 and 80% PUFA, respectively 32 . The total PUFA content observed in the current study is largely similar across the species and cultivars. However, the relative fractions of ALA and LA vary considerably (Fig. 3), with P. carolis-vegae seed oil containing the highest amount of ALA (57.4%) and the P. carolis-vegae × P. volubilis hybrid containing the lowest (46.8%) although it has the highest total amount of PUFA. The differences in the composition of the PUFA fraction might be a result of genetic differences between the species and cultivars in combination with environmental factors.
The common pathway of PUFA biosynthesis in plants is initiated in the plastid with the formation of acylchains by the fatty acid synthase (FAS) complex, generating C16:0 and C18:0 fatty acids. Desaturation ensues by the action of a stearoyl-acyl carrier protein desaturase (SAD) to form OA (C18:1), which is further desaturated to LA (C18:2) by fatty acid desaturase-2 (FAD2) in the endoplasmic reticulum, and the third double bond is introduced at the ω-3 position of LA by fatty acid desaturase-3 (FAD3), also in the endoplasmic reticulum 38,39 . Until recently, the molecular mechanisms underlying the synthesis of the very high PUFA content in P. volubilis had not been elucidated, although a few studies had been published 31,40,41 . However, in a study by Yang et al. 39 two FAD genes named PvFAD2 and PvFAD3 were isolated from P. volubilis and demonstrated to catalyse the synthesis of LA and ALA, respectively, although the authors point out that their results do not fully explain the massive accumulation of PUFA in P. volubilis seeds. Nevertheless, the differences observed between the species and cultivars included in the current study may at least in part be caused by differences in the expression of PvFAD2 and PvFAD3. If so, P. carolis-vegae may have a relatively high expression of PvFAD3, leading to the synthesis of a very high amount of ALA in the seed oil, while, conversely, P. volubilis from Peru may have a lower expression of PvFAD3, yielding a lower amount of ALA in the seed oil. Furthermore, it can be speculated that the similar amounts of PUFA, but varying compositions of the PUFA fraction, observed in almost all the species and cultivars of the study could be an effect of LA being used as a substrate for PvFAD3 to produce ALA, reducing the amount of LA while increasing the amount of ALA.
Intriguingly, P. carolis-vegae × P. volubilis has the highest PUFA fraction of all the studied species and cultivars of Plukenetia, although the ALA fraction is comparably small. This characteristic could be caused by a higher expression of genes early in the biosynthetic pathway of ALA synthesis, e.g. SAD, in combination with a lower expression of PvFAD3 compared to the other samples. However, it is also possible that a lower growing temperature would have induced activity of PvFAD3 and led to a higher accumulation of ALA relatively to LA; Yang et al. 39 found that the activity of PvFAD3 was sensitive to temperature when expressed in yeast (Saccharomyces cerevisiae) cells, with low temperature (20 °C) significantly increasing biosynthesis of ALA. Accordingly, an analysis of the seed oil of all the 'Mountain Sacha Inchis' , including the new species, P. sylvestris, and P. volubilis, grown at 20 and 30 °C might be useful in better understanding the very high accumulation of PUFA, primarily ALA in the large-seeded species of Plukenetia.

Conclusion
The seed oil of the 'Mountain Sacha Inchis' P. carolis-vegae and P. huayllabambana, the two cultivars of P. volubilis, and the newly developed hybrid between P. volubilis and P. carolis-vegae contains exceptionally high amounts of PUFA, at least 82%, and a remarkably high amount of ALA (C18:3 n-3, ω-3), at least 46%. It is of particular interest that the very large-seeded P. carolis-vegae has the highest ALA content in the lipid fraction, > 57%, followed by P. huayllabambana, P. volubilis, and P. volubilis × P. carolis-vegae. This underlines that P. carolis-vegae is a highly promising underutilised crop that may provide many benefits for farmers as well as consumers. Plukenetia carolis-vegae, but also P. huayllabambana and P. volubilis, has a huge potential for further domestication and industrialisation, due to the extraordinary nutritional qualities of the seed oil. Additionally, cultivation of the 'Mountain Sacha Inchis' may provide both nutritional and economic benefits for farmers and their communities. Finally, as demonstrated by the very high PUFA content of the P. volubilis × P. carolis-vegae hybrid, hybridisation breeding using 'Mountain Sacha Inchi' germplasm might grant exciting opportunities.