Research Article | Open Access
Rafael Zárate, Elena Cequier-Sánchez, Covadonga Rodríguez, Roberto Dorta-Guerra, Nabil El Jaber-Vazdekis, Ángel G. Ravelo, "Improvement of Polyunsaturated Fatty Acid Production in Echium acanthocarpum Transformed Hairy Root Cultures by Application of Different Abiotic Stress Conditions", International Scholarly Research Notices, vol. 2013, Article ID 169510, 20 pages, 2013. https://doi.org/10.5402/2013/169510
Improvement of Polyunsaturated Fatty Acid Production in Echium acanthocarpum Transformed Hairy Root Cultures by Application of Different Abiotic Stress Conditions
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.
Lipids in general and fatty acids (FA) in particular are essential metabolites displaying many key biological functions, acting as structural components of cell membranes, energy sources, and known intermediates in signaling pathways, besides their broad interest due to their important roles in human health and nutrition [1–6].
Oil producing plants could be an alternative dietary ingredient source of omega or n-3 polyunsaturated fatty acids (PUFA) for the aquaculture industry; thus, it would be interesting to establish how the environmental factors modulate PUFA production in plants. These lack the ability to move to avoid possible environmental stress situations and, therefore, have to adapt to their environment in many different ways, and, similar to that described for fishes, temperature is one of the most influential environmental factors. Cold acclimation and the acquisition of freeze tolerance require the orchestration of many different seemingly disparate physiological and biochemical changes, including increasing sugar levels, soluble proteins, proline, certain organic acids, and new protein isoforms, alteration of lipidic membrane, and particularly differential expression of many genes coding for effector molecules that participate directly to alleviate stress .
Biological membranes are considered liquid crystals that behave as two-dimensional fluids. Thus, FA are able to participate in different adaptation mechanisms of plants to stress conditions, particularly through modification of the cell membrane fluidity and permeability. Furthermore, unsaturated FA facilitate the fluidity of lipids, having fewer van der Waals interactions, with the position of the double bonds in FA being more influential than their number . Therefore, the addition of one or two double bonds to the membrane FA drastically decreases the transition temperature, and apparently the addition of a third or fourth double bond does not affect it. However, trienoic FA (e.g., 16:3 or 18:3) are the most abundant in chloroplast membranes, and increasing them allows a larger plant tolerance against low temperatures . Inversely, in transgenic tobacco plants, in which the gene encoding chloroplast omega-3 fatty acid desaturase, which synthesizes trienoic FA, was silenced, displayed a lower level of trienoic FA than wild-type plants, offering a better acclimation to higher temperatures .
The PUFA dependence of membrane fluidity as well as of other physical properties including those necessary for membrane fusion events is a well-characterized phenomenon in animals including fish, fungi, bacteria, and plants [13–17], together with the changes that occur when temperature increases or decreases. Unsaturated FA are thought to aid in maintaining membranes in a fluid state necessary for an appropriate biological functioning [18, 19].
In plants, FA such as 16:0 and 18:1n-9 are used to form lipid membranes by means of two different metabolic pathways: the chloroplast pathway or the cytoplasmic pathway, occurring in the endoplasmic reticulum (ER). In the first one, the FA of 16 carbons is generally esterified in the second position (sn-2) of the glycerol backbone, while in the ER within the synthesized lipids predominate those with 18 carbons. From here on, the desaturation process continues catalyzed by different enzymes which add an unsaturation to 18:1n-9, resulting in 18:2n-6, and so forth. Although there are differences between the desaturases found in chloroplasts (FAD7, FAD8) and those found in the ER (FAD2, FAD3) or in nonphotosynthetic tissues. The expression of each of their coding genes and enzymes is regulated by a complex system involving a variety of environmental stimuli, such as light or temperature, stress (damage, salt stress, pathogen invasion, etc.), or in response to the presence of jasmonate in the medium. Transgenic plants expressing or silencing enzymes of the biosynthetic pathway of PUFA have allowed the artificial modification of membrane FA and their physical and physiological adaptation to low temperatures. For instance, in Arabidopsis mutants deficient in one or more desaturase enzymes (fad genes), the degree of FA unsaturation was extremely important in the plant response to cold temperatures , in particular in chloroplasts, where trienoic FA are important to ensure the correct biogenesis and maintenance of chloroplasts during plant growth at low temperatures .
Several authors have suggested that temperature is a factor capable of regulating even the expression of desaturase enzymes in several ways, such as transcription, posttranscription, and translational. In soybean seeds, low temperatures were applied and the data showed how D12 and D15 desaturases could be rapidly modulated in response to altered growth temperatures, while the enzymes for FA synthesis and elongation were not . The regulation of these enzymes by temperature was documented with Brassica napus fad3 desaturase gene expressed in yeast; an increase in 18:3n-3 and FAD3 enzyme was recorded at low temperatures, but no fad3 transcript was observed, suggesting that a posttranscriptional regulation was taking place .
Regarding the influence of osmotic stress on the FA profiles, osmotic pressure plays a crucial role as a regulator in the cellular water balance. Despite this important role, in in vitro plant culture systems employed for the production of secondary metabolites, much more attention has been applied to other factors, such as temperature or the nutrients present in the culture medium . Few studies have addressed the use of osmotic stress or high osmotic pressure to stimulate the production of secondary metabolites in plant cell cultures. The application of osmotic stress on cells suspension cultures of Catharanthus roseus resulted in an increase in the intracellular accumulation of catharanthine and other alkaloids . Other studies showed the influence of osmotic stress on anthocyanin production in cell culture of Vitis vinifera , in Populus deltoides , and in cell cultures of Panax notoginseng , and, more recently, it was applied in cell cultures of C. roseus , Taxus chinensis , and Panax ginseng , in order to induce the production of various alkaloids, paclitaxel, and saponins, respectively. In these studies, it has been confirmed that although occasionally the addition of sorbitol or mannitol can increase the production of metabolites of interest, it may also decrease the fresh weight of the cultures. Regarding the relationship between the induced stress and the production of PUFA, some studies have suggested that the hyperosmotic stress may reduce membrane fluidity similar to low temperatures. Contrary, the hypoosmotic stress effect is not well documented, but it has also been suggested that as the temperature is high, hypoosmotic stress may increase membrane fluidity.
Analogously, in the aquatic environment in general and in the field of fish aquaculture in particular, it is important to take into account environmental factors, especially temperature, a factor that directly influences the development and yield of crops. Cold affects fish growth and health and may decrease fish-farm production, even causing mortality through what is known as “winter syndrome” . In some species adapted to very low temperatures, there is an interesting effect in order to keep the fluidity of cell membranes; they can decrease the chain length or increase the number of double bonds of those FA esterified in phospholipids . Moreover, in deep-see living fish, it has been shown that high hydrostatic pressures exert the same influence as low temperatures, showing changes in certain enzyme activities, an increase in oxygen consumption, and increased membrane fluidity, achieved by increasing the unsaturation of FA esterified with phospholipids [33, 34].
In this study, we report the different strategies carried out in a novel Echium acanthocarpum transformed hairy root system, in order to particularly increase the production of the unusual PUFA, stearidonic (SDA; 18:4n-3) and γ-linolenic acids (GLA; 18:3n-6), of increasing pharmacological interest, by reducing culture temperature, applying osmotic stress, and changing the carbon source. Moreover, in order to determine the validation of the data and the effectiveness of the abiotic stress, a robust statistical approach was applied.
2. Materials and Methods
2.1. Plant Material
Seeds of E. acanthocarpum, donated by Jardín Botánico Viera y Clavijo, Gran Canaria, Spain, were first surface-sterilized by a brief immersion in 70% EtOH, followed by submersion in an aqueous solution of 5% (v/v) of commercial bleach for 25 min with gentle hand agitation. They were finally washed 5 times with sterile distilled water.
Treated seeds were then germinated in vitro on a solid B5  medium, supplemented with 3% sucrose, 3-4 mg/L GA3 (gibbereelic acid), and solidified with 0.7% agar, with the pH adjusted to 6.0 prior to autoclaving (115°C, 1 atm. pressure, 15 min.), contained in Petri dishes (90 mm diameter), and cultured in the dark until the beginning of germination. After germination, plants were transferred to the same solid nutrient medium without the addition of GA3, contained in translucent glass jars covered with a lid (175 mL capacity, Sigma-Aldrich, MO, US), which were placed under light conditions (16 h photoperiod and irradiance of 35 mmol m2s−1 supplied by cool white fluorescent tubes) and a temperature of °C to allow further plant growth.
Under sterile conditions, 50–60-day-old plants were employed for guided infection with Agrobacterium rhizogenes strain LBA1334 harboring a pBIN19-gus intron plasmid by repeatedly stabbing the internodal stem areas with a fine needle containing bacteria [35, 36]. After 25–30 days, hairy roots of 3-4 mm in length had developed and were aseptically excised and transferred to B5 liquid medium, containing the antibiotic cefotaxime (100 mg/L) as well as 1% of the antioxidant polyvinylpyrrolidone (PVP) for several subcultures. Finally, actively growing bacterium-free hairy roots were cut into small segments and routinely cultured and refreshed in Erlenmeyer flasks (250 mL), containing 30 mL of sterile B5 liquid medium supplemented with 3% sucrose and 1% of PVP (standard nutrient medium), sealed with a double layer of aluminum foil, and placed on an orbital shaker at 95 rpm at °C in the dark.
For culture growth and FA production and analysis, different hairy root culture media of the established E1.5 cell line were investigated, each providing a particular abiotic stress and culture conditions (Table 1). In order to cover the entire growth period for each culture, sampling times were different since the kinetics of growth differed due mainly to the culture temperature; thus, the sampling points were as follows 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.
PVP: polyvinyl pyrrolidone.|
2.2. Lipid Extraction and Transesterification of Lipids
Hairy roots were separated from the liquid nutrient medium by vacuum filtration, weighed, and lyophilised at −80°C for 24 h using a freeze-dryer (Christ Alpha 2-4, Osterode, Germany). Each sample was powdered using a mortar and pestle with liquid nitrogen. After homogenisation, total lipid was extracted following the method previously described [36–38].
Lipid aliquots (2 mg) were subjected to acid-catalyzed transesterification by dissolving the sample in 1 mL toluene, employed to ensure that the neutral lipids got properly dissolved, plus 2 mL of a mixture of MeOH/1% H2SO4, and incubated in a capped glass test tube at 50°C for 16 h . Prior to transmethylation, heneicosae-noic acid (21:0) (2.5% of the total lipid analysed, 50 μg), was added as internal standard to the lipid extracts. Transesterification was conducted as previously described [36–38]. Preparative thin layer chromatography employing silica gel G-25 glass sheets (Macherey-Nagel, Germany), developed with a solvent system composed of hexane/diethyl ether/acetic acid 97.7% (90 : 10 : 1, by vol) and visualized after brief sublimation of iodine with slight heat, was used for the isolation and purification of the fatty acids methyl esters (FAMEs). These ran close to the solvent front and were then scrapped off the glass sheet, extracted with 10 mL hexane/ethyl ether (1 : 1, v/v), and dried under nitrogen. Finally, the samples were dissolved in 0.5–1.0 mL hexane and kept under nitrogen in sealed glass vials at −20°C until analysis.
2.3. Gas Chromatography of FAMEs
Analysis and quantification of FAMEs were conducted by GC employing a Shimadzu GC-14A apparatus (Shimadzu, Japan) equipped with a flame ionization detector (250°C), a Supelcowax 10 fused silica capillary column (30 m × 0.32 mm ID), employing helium as carrier gas. Samples (0.6 μL) were injected into the system by an on-column autoinjector (Shimadzu AOC-17) at 50°C. A temperature program of 180°C for the first 10 min, followed by an increase of 2.5°C/min until reaching 215°C, was employed for separation of the compounds.
FAMEs were identified according to their RT compared with standards of individual commercial FAMEs (linoleic acid methyl ester, methyl γ-linolenate, methyl oleate, stearidonic acid methyl ester, and heneicosanoic acid) and a well-characterized fish oil mix (Sigma-ref LUPE). They were quantified according to the amount of 21:0 added as internal standard prior to transmethylation and by comparison with a calibration curve created with the individual standards.
2.4. Statistical Analysis
Results are present as the means and standard deviations of three replicates for each sampling time for each of the treatments and cultures. The data were checked for normal distribution by one-sample Kolmogorov-Smirnov test as well as for homogeneity of the variance with the Levene test, and, when necessary, Bartlett test was also applied. When variance was not homogeneous, Kruskal-Wallis and Games-Howele tests were conducted to assess statistical differences. The effects of culture conditions and FA levels were firstly determined using one-way ANOVA test . The percentages and total amounts of FA, particularly the contents of GLA and SDA in the different cultures, were included as variables in a principal component analysis (PCA). Principal components were subsequently analysed by two-way ANOVA to study the combined effects of both factors, FA profiles and stress conditions, as well as their interconnections. Statistical analyses were performed employing the SPSS software (versions 15.0 and 17.0, SPSS Inc., IL, USA).
3. Results and Discussion
Studies of growth conditions and type of stress applied to cultures were conducted in order to achieve FA profiles richer in 6-desaturated products, such as SDA and GLA.
3.1. Effect of Stress on Echium acanthocarpum Hairy Roots Growth
In order to carry out these experiments, hairy roots from line E1.5 were used taking into account the previously described results [36, 40]. The data of growth were recorded under different nutrient media and conditions (cultures B1, C1, C2, C3, and C4).
A typical growth curve was achieved for all cultures, with a lag phase, which tended to be more pronounced in cultures C2–C4 than culture C1, at sampling points 1 and 2 (Figure 1). Then, the cultures started their exponential growth phase. Finally, the cultures described a stationary phase (points 4 and 5), and from that point on, cell death took place, in which cultures lost their hairy root morphology, showing also vitrification and browning.
In culture B1, a maximum fresh weight of 1.38 g was registered at sampling point 6, whereas the maximum of culture C1 was 2.76 g at sampling point 5. Statistical analysis of fresh weight of both cultures showed only significant difference for sampling points 1 and 2 (Figure 1).
In relation to the carbon source used, when glucose was used instead of sucrose, it would be expected that glucose would be able to induce a faster growth, particularly in the lag phase, since sucrose would require to be hydrolyzed into the monomers glucose and fructose to then be taken up by the tissues. However, both carbon sources displayed comparable growth rates (Figure 1). Thus, when fresh weight variation of hairy roots cultured at 15°C (cultures C1–C4) was compared, no significant differences were observed in any of the analyzed points, except for the first one (Figure 1). In culture C2, containing 0.2 M sorbitol as osmotic pressure inducing agent, fresh weight reached 3.04 g at sampling point 5. Similarly, in cultures C3 and C4, the maximum values were recorded at sampling point 5, 2.81 and 2.00 g, respectively, although no significant differences were observed in any of the sampling points (Figure 1). Interestingly, it has been described that the application of osmotic stress tends to decrease the fresh weight of in vitro cultured rice or wheat callus; this is mainly due to the accumulation of metabolites or osmolytes in the cytosol, such as glycinebetaine, proline, or soluble carbohydrates, which balance the effect of the osmotic pressure produced by the extracellular solute concentration, avoiding the massive loss of intracellular water [41–43]. This fact would not be present in E. acanthocarpum hairy roots, since no reduction in fresh weight was observed, but a rather slight increase. Taking into account the final fresh weight versus the initial fresh weight, culture C2 consisting of B5 mineral salts, 3% sucrose, 1% PVP and 0.2 M sorbitol was the most effective, with a 12-fold increase, whereas fresh weight of C1, C3, and C4 increased in the range of 8–11 folds. Nonetheless, a negative interaction of sorbitol on the growth of certain in vitro cultures, such as ginseng roots, with 3% sucrose was published. The data showed a considerable growth reduction when sorbitol (0.2–0.3 M) was added . However, a beneficial growth effect upon the addition of sorbitol (54.97 mM) to a certain varieties of rice in vitro cultures has also been described . It might be possible that sorbitol, a polyhydric alcohol sugar, could be metabolized by tissues when sucrose amounts get finished. In our study, the addition of sorbitol does not show any detrimental effect on growth but a rather slight nonsignificant increase.
3.2. Effect of Stress on Echium acanthocarpum Hairy Roots Lipid Composition: Principal Component Analyses
In general, the total lipid (TL) extracted from culture C1 hairy roots grown at 15°C was statistically similar to that of culture B1 grown at 25°C (Figure 2). The temperature did not significantly affect TL, and apparently E. acanthocarpum hairy roots are able to maintain homeostasis under these conditions. The data also indicate the possibility of further reducing the culture temperature to likely boosts its effect, on the production of desired 6-desaturated fatty acids, GLA, and SDA, as has been published for B. napus cultures grown at 4°C  or Arabidopsis grown at 12°C .
Analogously, the addition of sorbitol and/or glucose or both (cultures C2, C3, C4) did not seem to have a direct influence on TL since mostly no statistical differences were recorded except for sampling point 4 (Figure 2), although some reports showed that under water stress, total lipid clearly dropped in leaves of A. thaliana and Cocos nucifera [47, 48]. The decrease in TL is often associated with the richness of saturated FA in the leaf, as they are a more accessible substrate for hydrolysis and peroxidation reactions  and also because the leaves contain chiefly monogalactosyl diacylglycerides (MGDG) and digalactosyl diacylglycerids (DGDG), which are mainly esterified with saturated FA under stress conditions. Therefore, some authors claim that chloroplasts and their membranes are the most sensitive plant organelle to water stress [50, 51] and low temperatures [18, 52, 53]. Given the nature of hairy roots, in which tissue cells with chlorophyll are not present, and therefore are not photosynthetic organs, could be the reason why E. acanthocarpum hairy roots seem not to be much affected by water or osmotic stress conditions, and the increment of TL at the initial sampling times might be due to a larger cell division rate in these meristematic tissues, accompanied by a major formation of cytoplasmic membranes and production of PUFA .
In order to study the effect of both decreasing the culture temperature from 25 to 15°C and the presence of other stress agents in E. acanthocarpum hairy roots, in lipid composition, sampling points 4 and 5 were taken as the most representative ones by making a compromise between biomass production and the rate of FA production . Lipid class composition was affected by both, the temperature and the presence of sorbitol and glucose in the nutrient medium, although generally no significant differences were observed between the values of lipid classes PC, PS + PI, and PE and total polar and neutral lipids (Tables 2(a) and 2(b)). Among polar lipids, phosphatidylcholine (PC), phosphatidylserine, and phosphatidylinolsitol (PS + PI) ranged from 8.53 to 9.13% and 4.51 to 6.17% of the total lipids for PC and PS + PI, respectively, in cultures B1 and C1 (Table 2(a)). Different authors propose that under water stress conditions, free FA are produced most abundantly by the action of lipases over polar lipids and subsequently stored in esterified triacylglycerols (TAG), increasing thereby the neutral lipids. Furthermore, the polar lipids rise may be explained because water stress increases the synthesis of new membranes . In the same fashion, it has been observed in roots grown at low temperatures how total content of polar lipids was augmented twice its initial value at room temperature [56, 57].
|(a) Data of sampling point 4|
|(b) Data of sampling point 5|
|Significant differences () between cultures given by Student’s t-test when comparing the mean of percentages of the lipid classes for cultures B1 and C1.|
Significant differences of TL values, also showing homogeneous subclusters as indicated by Tuckey's test when comparing the mean of TL values.
TL: total lipid; PC: phosphatidylcholine; PS + PI: sum of phosphatidylserine and phosphatidylinositol; PG: phosphatidylglycerol; PE: phosphatidylethanolamine.
Percentage values were transformed by the arcsin transformation prior to statistical analysis.
Values represent the mean of three independent experiments () ± SD.
Entry of culture B1 for sampling point 5 is empty because data were collected only for sampling 4.
There was a clear increasing tendency of phosphatidylglycerol (PG) in E. acanthocarpum cultures grown at 15°C, going from an average of 3.48% of TL at 25°C (culture B1, Table 2(a)) to 8,25% of the TL at 15°C (culture C1, Table 2(a)). The rise of PG in E. acanthocarpum hairy roots in response to a reduction of temperature is consistent with several studies where the content of PG in roots of Avicennia germinans was higher than in those plants more resistant to low temperatures . In another study carried out on the plasma membrane of wheat seedlings, a rise of all classes of phospholipids, including PG, was recorded when the culture temperature was 2°C . It has also been speculated that the direct relationship between temperature and richness of PG in plant tissues is due to the glycerol-3P-acyltransferase specificity, responsible for the esterification of certain FA, usually 18:0, 18:1, and 16:0 at sn-1 position of PG and also due to its response to temperature [60, 61]. No significant changes were observed in PE in B1 and C1 cultures, with percentages ranging from 10.75 to 12.18% of TL. Finally, when calculating the total values of polar and neutral lipids, no drastic changes were observed, with values of 29.70 to 33.39% and from 59.59 to 64.05%, respectively (Table 2(a)). After studying the values for each lipid class in the other cultures (C2–C4), and compared to those profiles obtained in C1, no significant changes were detected, except for the case of PG, reaching 13.77% of TL in culture C2 (Tables 2(a) and 2(b)). There are very few studies addressing the distribution of lipid classes in roots, although one describes the PG content in the thylakoid membranes and its relative increase, together with unsaturation enrichment of FA esterified in PG against low temperatures, both in monocotyledons and dicotyledons [61, 62]. Also, it was reported that there is an enrichment of the saturated 16:0, 18:0 FA, constituting about 40% of those esterified FA in PG, and the monoene trans-16:1, in plant species sensitive to low temperatures, while the wealth of saturated FA in resistant species to low temperatures decreased to 20% . These data suggest a direct relationship of this phospholipid with plant resistance to low temperatures, although other factors providing such feature might also exist .
The distribution of lipid classes in E. acanthocarpum hairy roots was similar to that of a nonphotosynthetic tissue, exhibiting high polar lipid values, mainly PE and PG, not recording the typical photosynthetic tissue lipids, such as monogalactosyl diacylglycerides (MGDG) and digalactosye diacylglycerides (DGDG), present in chloroplasts.
In order to carry out the statistical analysis of the data, variables for cultures B1 and C1, sharing the same culture medium, were analysed to observe how temperature (25 and 15°C) affects the distribution of lipid classes. Only significant differences for PG were observed, whose percentage was the highest when grown at 15°C (Tables 2(a) and 2(b)). Secondly, in order to study the influence of the use of glucose and 0.2 M sorbitol in the nutrient medium and the time of sampling, a principal component analysis (PCA) to the percentages of the obtained lipid classes for C1–C4 cultures was performed (Table 3). We obtained two principal components (PC1 and PC2), which explained 85.68% of the variance. PC2 (19.78% of variance) was positively correlated with the PG, while PC1 (65.91% of variance) was positively related to PC, PS + PI, PE, and total polar lipids, while it was negatively correlated with the neutral lipids (Table 3, Figure 3).
Factor loading of the variable (correlations) with PC1 are shown in bold, and those correlated with PC2 are shown in italic. |
PC: phosphatidylcholine; PS + PI: phosphatidylserine and phosphatidylinositol; PG: phosphatidylglycerol; PE: phosphatidylethanolamine.
Rotation method: Varimax with Kaiser.
Accordingly, it could be assumed that PC1 is correlated with the richness of polar lipids and PC2 reflects the amount of PG in hairy root tissues.
After removal of the principal components PC1 and PC2, we conducted a two-way ANOVA test in order to determine whether the studied factors, that is, stressing factors and sampling points and the interaction between them, had influence on the extracted new variables PC1 and PC2 (Table 4).
Sign.: significance ().|
Only when both factors interacted for PC1, the level of significance was less than 0.05 (Table 4, Figure 4); that is, polar lipids usually remained constant. Conversely, the PC2 variable, reflecting the content of PG in the sample, (Figure 3, Table 3) was influenced by the presence of sorbitol or glucose in the nutrient medium (Table 4). Interestingly, it was observed that PG content was significantly higher in C2–C4 cultures, those in which 0.2 M sorbitol or 3% glucose was present. These data are consistent to those observed in A. thaliana leaves reporting a nonsignificant increase of this class of lipids; moreover, when water stress conditions became more severe, a PG decrease and a pronounced increase of typical lipid classes of green tissues (MGDG and DGDG) were observed as well as ALA (18:3n-3) in PC lipid class and DGDG . Likewise, it was reported the same PG increase in Pachyrhizus ahipa leaves ; furthermore, in barley roots grown in the presence of 100 mM NaCl, PG reached around 5% of TL  and, in Carthamus tinctorius leaves, where PG and the set of polar lipids especially PC rose under smooth water stress conditions but decreased significantly when water conditions became more acute . On the other hand, PG content was not influenced by time or sampling point or by the interaction of both in the culture (Table 4).
Therefore, when samples were plotted as a function of the main components and stratified according to the presence of stressing factors, a clear separation between the control group, culture C1, or the other cultures (C2–C4), also grown at 15°C, was observed (Figure 5(a)). When these samples were stratified according to the sampling points, an overlap between the groups was observed (Figure 5(b)).
3.3. Effect of Stress on the Unsaturation Degree of the Fatty Acids
The FA detected in E. acanthocarpum hairy roots were the same as those reported previously , but the percentages of each FA changed considerably. Thus, the following saturated FA were detected: palmitic acid (16:0) and stearic acid (18:0), along with other minor FA like 20:0, 22:0, and 24:0 (Tables 5 and 6). Similarly, high percentages of the monounsaturated FA 18:1n-9 and 18:1n-7 were measured, with a total of approximately 28% of saturated FA, with 6% of monoenes in all samples. The more characteristic polyunsaturated n-6 FA, LA and GLA, represented 50–55% of the total FA. Furthermore, the n-3 PUFA, ALA and SDA, represented 6–9% of all FA, depending on the cultures (Tables 5 and 6).
ND: Not detected. n-6 and n-3 6-desaturation indexes were calculated as 18:3n-6/(18:3n-6 + 18:2n-6) and 18:4n-3/(18:4n-3 + 18:3n-3), respectively.|
Double bond index (DBI) was calculated as [(%18:1n) + 2 (%18:2n) + 3 (%18:3n) + 4 (18:4n)]/100. Values represent the mean of three replicates () SD.