Improvement of Polyunsaturated Fatty Acid Production in Echium acanthocarpum Transformed Hairy Root Cultures by Application of Different Abiotic Stress Conditions

Abstract

Fatty acids are of great nutritional, therapeutic, and physiological importance, especially the polyunsaturated n-3 fatty acids, possessing larger carbon chains and abundant double bonds or their immediate precursors. A few higher plant species are able to accumulate these compounds, like those belonging to the Echium genus. Here, the novel E. acanthocarpum hairy root system, which is able to accumulate many fatty acids, including stearidonic and α-linolenic acids, was optimized for a better production. The application of abiotic stress resulted in larger yields of stearidonic and α-linolenic acids, 60 and 35%, respectively, with a decrease in linoleic acid, when grown in a nutrient medium consisting of B5 basal salts, sucrose or glucose, and, more importantly, at a temperature of 15°C. The application of osmotic stress employing sorbitol showed no positive influence on the fatty acid yields; furthermore, the combination of a lower culture temperature and glucose did not show a cumulative boosting effect on the yield, although this carbon source was similarly attractive. The abiotic stress also influenced the lipid profile of the cultures, significantly increasing the phosphatidylglycerol fraction but not the total lipid neither their biomass, proving the appropriateness of applying various abiotic stress in this culture to achieve larger yields.

Figure 1

Fresh weight (g) variation of Echium acanthocarpum transformed hairy roots cell line E1.5 cultured in three different growth media and temperatures. Values represent the mean of three replicates (n = 3)  ±  SD. Depending on the culture temperature 25°C (culture B1) or 15°C (cultures C1–C4), the sampling points varied as T1 = 5 days for culture B1 and 15 days for cultures C1–C4; T2 = 10 days for culture B1 and 25 days for cultures C1–C4; T3 = 15 days for culture B1 and 35 days for cultures C1–C4; T4 = 20 days for culture B1 and 45 days for cultures C1–C4; T5 = 25 days for culture B1 and 55 days for cultures C1–C4; and T6 = 30 days for culture B1 and 65 days for cultures C1–C4.

Figure 2

Total lipid (TL) content (mg/g DW) of E1.5 Echium acanthocarpum transformed hairy root cell line cultured in different culture media and temperatures. Values represent the mean of three independent replicates (n = 3) ± SD. Depending on the growth temperature, the sampling points varied as T1, 5 days for culture B1 and 15 days for cultures C1–C4; T2, 10 days for culture B1 and 25 days for cultures C1–C4; T3, 15 days for culture B1 and 35 days for cultures C1–C4; T4, 20 days for culture B1 and 45 days for cultures C1–C4; T5, 25 days for culture B1 and 65 days for cultures C1–C4; and T6, 35 days for culture B1 and 75 days for cultures C1–C4.

Figure 3

Factor loading plots of the principal component analyses of the percentages of lipid classes in Echium acanthocarpum E1.5 cell line hairy roots, growing in different media at 15°C (C1–C4 cultures). PC = phosphatidylcholine; PS + PI = phosphatidylserine and phosphatidylinositol; PG = phosphatidylglycerol; and PE = phosphatidylethanolamine.

Figure 4

REGR factor score for PC1 depending on sampling point 4 or 5 and categorized by type of culture (C1–C4), showing the interaction effect of abiotic stress and time.

Figure 5

(a), (b) Plot of Echium acanthocarpum hairy root samples in terms of the principal components PC1 and PC2 stratified according to (a) cultures (C1–C4) or (b) sampling points.

Figure 6

Double bond index (DBI) in Echium acanthocarpum hairy roots cultured in different growth media at two temperatures. DBI was calculated as [(% 18:1n) + 2 × (% 18:2n) + 3 × (% 18:3n) + 4 × (18:4n)]/100.

Figure 7

Absolute amounts of different fatty acids (μg/gDW) present in Echium acanthocarpum hairy roots grown in B5 medium, 3% sucrose, 1% PVP at 15°C (culture C1). Each value is the mean ± standard deviation of three replicates.

Figure 8

Absolute amounts of different fatty acids (μg/gDW) present in Echium acanthocarpum hairy roots grown in B5 medium, 3% sucrose, 1% PVP at 25°C (control culture B1). Each value is the mean ± standard deviation of three replicates.

Figure 9

Absolute amounts of different fatty acids (μg/gDW) present in Echium acanthocarpum hairy roots grown in B5 medium, 3% sucrose, 0.2 M sorbitol, 1% PVP, at 15°C (culture C2). Each value is the mean ± standard deviation of three replicates expressed.

Figure 10

Absolute amounts of different fatty acids (μg/gDW) present in Echium acanthocarpum hairy roots grown in B5 medium, 3% glucose, 1% PVP at 15°C (culture C3). Each value is the mean ± standard deviation of three replicates.

Figure 11

Absolute amounts of different fatty acids (μg/gDW) present in Echium acanthocarpum hairy roots grown in B5 medium, 3% glucose, 0.2 M sorbitol, 1% PVP at 15°C (culture C4). Each value is the mean ± standard deviation of three replicates.

Figure 12

Factor loadings for the percentages of fatty acids in Echium acanthocarpum hairy root cultures B1 and C1 grown at 25°C and 15°C, obtained after principal component analysis.

Figure 13

Factor loadings obtained after principal component analysis, for the percentages of fatty acids in Echium acanthocarpum hairy root cultures C1–C4 grown at 15°C.