Diacylglycerol Acetyltransferase Gene Isolated from Euonymus europaeus L. Altered Lipid Metabolism in Transgenic Plant towards the Production of Acetylated Triacylglycerols

Euonymus species from the Celastraceae family are considered as a source of unusual genes modifying the oil content and fatty acid composition of vegetable oils. Due to the possession of genes encoding enzyme diacylglycerol acetyltransferase (DAcT), Euonymus plants can synthesize and accumulate acetylated triacyglycerols. The gene from Euonymus europaeus (EeDAcT) encoding the DAcT was identified, isolated, characterized, and modified for cloning and genetic transformation of plants. This gene has a unique nucleotide sequence and amino acid composition, different from orthologous genes from other Euonymus species. Nucleotide sequence of original EeDAcT gene was modified, cloned into transformation vector, and introduced into tobacco plants. Overexpression of EeDAcT gene was confirmed, and transgenic host plants produced and accumulated acetylated triacylglycerols (TAGs) in immature seeds. Individual transgenic plants showed difference in amounts of synthesized acetylTAGs and also in fatty acid composition of acetylTAGs.


Introduction
Euonymus is a cosmopolitan genus of the family Celastraceae containing more than two hundred of species [1] mostly native in East Asia. Species Euonymus alatus L. is widely distributed and traditionally used as a medicinal plant in many Asian countries. More than 230 chemical compounds have been identified and isolated from it, including sesquiterpenoids, diterpenoids, triterpenoids, flavonoids, phenylpropanoids, lignans, steroids, alkaloids, and other compounds [1,2]. Generally, Euonymus species have the potential for treatment of many injuries, inflammation, and oxidative stress as well as diseases including cancer, diabetes, and others [2,3]. Euonymus europaeus L. (spindle tree, European spindle) is distributed in temperate climates from Central to Eastern Europe. It is mainly considered as an ornamental shrub and its homogenous wood is easy to work for special products. Bark, leaves, and The total RNA from E. europaeus was extracted from 1 g of plant tissues using the TRIzol reagent method (Invitrogen Corp., Carlsbad, CA, USA) from root, stem, leaf, aril, pericarp, and immature seed. Potential residues of genomic DNA were removed by DNase treatment (Fermentas, St. Leon-Rot, Germany). Concentration of isolated RNA was determined spectrophotometrically (Nanodrop 1000 Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). Quality of RNA has been verified by electrophoresis in 1.5% agarose-formaldehyde gel stained with ethidium bromide. The RevertAid First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) was used for the first-strand cDNA synthesis. Fifty nanograms of the first cDNA strands was used as templates for the second-strand synthesis by PCR at the same composition and cycling parameters as for genomic DNA. PCR primers were designed by the SnapGene software (GSL Biotech LLC, San Diego, CA, USA) ( Table 1). Completing of EeDAcT gene sequence was performed by overlapping of cDNA fragment sequences obtained by amplifications with primers F1-F4 ( Table 1). The PCR reaction mixture (25 µL) contained 1 × PCR buffer, 1.5 mM MgCl 2 , 10 pM of both primers, 0.2 mM each of dNTP and 0.5 U Platinum TM Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA, USA), and 100 ng of template cDNA. The PCR was performed in the same thermocycler using initial denaturation at 94 • C for 3 min, followed by 35 cycles, each of denaturation at 94 • C for 1 min, annealing at 58 • C for 1 min, extension at 72 • C for 1 min, and the final extension at 72 • C for 10 min. Products of PCR were electrophoretically analyzed in 1.5% (w/v) agarose gels pre-stained with ethidium bromide and extracted from gel using the Agarose Gel Extraction Kit (Roche Diagnostics GmbH, Mannheim, Germany). DNA sequencing was done by Sanger method in commercial sequencing service (Comenius University, Bratislava, Slovakia). Complete cDNA of the 1,2-diacyl-sn-glycerol:acetyl-CoA acetyltransferase from E. europaeus (EeDAcT) was submitted to the GenBank ® nucleotide database [23] (as accession no. MK637625.1). Sequence alignments of cDNA and proteins of the gene DAcT within the family Celastraceae Celestraceae were performed using the CLC Main Workbench 20.0 software (Qiagen N.V., Venlo, The Netherlands). The phylogenetic tree was constructed using the Clustal W software [24,25].

EeDAcT Gene Synthesis and Expression in Tobacco
The gene encoding the enzyme EeDAcT was synthesized (Eurofins Genomics Germany GmbH, Ebersberg, Germany) in full length. The synthetic EeDAcT gene was modified by addition of restriction endonuclease sites compatible with cloning the gene into binary transformation vector pRI 101-AN (TaKaRa Bio Inc., Dalian, China) containing the 35S promoter of cauliflower mosaic virus (CaMV). Sequences recognized by XbaI, NdeI were added in front of the 5 -end of the gene, and sequences recognized by SalI, EcoRI, SacI enzymes were added behind the 3 -end of the gene. Original positions recognizing by NdeI (position 800), XbaI (712), and EcoRI (550) inside the EeDAcT gene were eliminated, Life 2020, 10, 205 5 of 16 but the open reading frame was conserved. Chemically competent Escherichia coli cells, strain DH10B (New England Biolabs Inc., Ipswich, MA, USA), were used for transformation. Transformed bacteria were selected on LB medium [26] agar plates containing kanamycin and verified by molecular analysis of plasmid. The plasmid containing pRI-101AN-natEeDAcT construct was used for transformation of Agrobacterium tumefaciens cells by electroporation [27]. Positive transformants were selected on LB medium agar plates containing kanamycin and rifampicin and cultivated in 10 mL liquid LB medium containing 10 µg/mL of rifampicin and 50 µg/mL of kanamycin, under shaking (250 rpm) at 28 • C for [24][25][26][27][28][29][30][31][32][33][34][35][36] h. An optical density (OD 600 ) of 0.8-1.0 was obtained after 24-36 h, measured with NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Leaf discs of tobacco were transformed using Agrobacterium-mediated protocol [28]. Kanamycin at concentration 50 µg/mL was used as a selection pressure during regeneration of transformed cells and rooting of regenerated shoots. Transgenic plants were transferred from in vitro to in vivo and cultivated in greenhouse conditions. Presence of the EeDAcT transgene was detected in transgenic tobacco plants by PCR using primer pair 5 -TCGCTCCCTTGAACATCTCT-3 and 5 -GGAAAATAAGCCCAACGTGA-3 . Expected size of the PCR product was 579 bp. The PCR reaction mixture and thermocycler type were the same as previously. The PCR parameters were as follows: initial denaturation at 94 • C for 3 min, followed by 32 cycles, each consisting of a denaturation at 94 • C for 1 min, annealing at 60 • C for 25 s, extension at 72 • C for 1 min, and the final extension step at 72 • C for 10 min. PCR products were separated in 1.5% (w/v) agarose gel in 1xTBE buffer (1.1% Tris-HCl; 0.1% Na 2 EDTA; 0.55% boric acid) pre-stained with 0.10 µL/mL of ethidium bromide.

AcetylTAGs in E. europaeus
Presence of acetylTAGs was analyzed in root, stem, leaf, flower, aril, pericarp, and immature seeds of E. europaeus. Only immature seeds contained a high level of acetylTAGs. On the opposite, the aril tissue surrounding the seed produced the highest levels of long-chain triacylglycerols (lcTAGs). Other evaluated tissues and organs accumulated lcTAGs, but not acetylTAGs ( Figure 1). Leaf discs of tobacco were transformed using Agrobacterium-mediated protocol [28]. Kanamycin at concentration 50 μg/mL was used as a selection pressure during regeneration of transformed cells and rooting of regenerated shoots. Transgenic plants were transferred from in vitro to in vivo and cultivated in greenhouse conditions.
Presence of the EeDAcT transgene was detected in transgenic tobacco plants by PCR using primer pair 5′-TCGCTCCCTTGAACATCTCT-3′ and 5′-GGAAAATAAGCCCAACGTGA-3′. Expected size of the PCR product was 579 bp. The PCR reaction mixture and thermocycler type were the same as previously. The PCR parameters were as follows: initial denaturation at 94 °C for 3 min, followed by 32 cycles, each consisting of a denaturation at 94 °C for 1 min, annealing at 60 °C for 25 s, extension at 72 °C for 1 min, and the final extension step at 72 °C for 10 min. PCR products were separated in 1.5% (w/v) agarose gel in 1xTBE buffer (1.1% Tris-HCl; 0.1% Na2EDTA; 0.55% boric acid) pre-stained with 0.10 μL/mL of ethidium bromide.

AcetylTAGs in E. europaeus
Presence of acetylTAGs was analyzed in root, stem, leaf, flower, aril, pericarp, and immature seeds of E. europaeus. Only immature seeds contained a high level of acetylTAGs. On the opposite, the aril tissue surrounding the seed produced the highest levels of long-chain triacylglycerols (lcTAGs). Other evaluated tissues and organs accumulated lcTAGs, but not acetylTAGs ( Figure 1). The spot corresponding with acetylTAGs from immature seeds of E. europaeus was scraped from the TLC plate, extracted, re-analysed, and compared with the standard of acetylTAGs, the The spot corresponding with acetylTAGs from immature seeds of E. europaeus was scraped from the TLC plate, extracted, re-analysed, and compared with the standard of acetylTAGs, the synthetic  The dominant fatty acid in acetylTAGs as well as in lcTAGs of E. europaeus immature seeds was the oleic acid ( Figure 3). The fraction of acetylTAGs contained 61.3% and fraction of lcTAG 53.2% of oleic acid, respectively. Seeds accumulated also 9.0% of the essential linoleic acid in acetylTAGs and 11.8% in lcTAGs. The palmitic and stearic acids represented saturated fatty acids in acetylTAGs with a percentage 21.5% and 6.6%, respectively. Their content in lcTAGs was 26.8% and 5.5%, respectively. The cis-vaccenic acid was a minor component in both fractions of oil (1.4% and 2.7%, respectively), the heptadecanoic acid was not detected in lcTAGs and in acetylTAGs represented only 0.3%.

Diacylglycerol Acetyltransferase Gene from E. europaeus
Initial metabolomic analysis of lipids in seeds of E. europaeus confirmed the presence of 3-acetyl-1,2-diacylglycerols (acetylTAGs). Based on this prerequisite, the responsible diacylglycerol The dominant fatty acid in acetylTAGs as well as in lcTAGs of E. europaeus immature seeds was the oleic acid ( Figure 3). The fraction of acetylTAGs contained 61.3% and fraction of lcTAG 53.2% of oleic acid, respectively. Seeds accumulated also 9.0% of the essential linoleic acid in acetylTAGs and 11.8% in lcTAGs. The palmitic and stearic acids represented saturated fatty acids in acetylTAGs with a percentage 21.5% and 6.6%, respectively. Their content in lcTAGs was 26.8% and 5.5%, respectively. The cis-vaccenic acid was a minor component in both fractions of oil (1.4% and 2.7%, respectively), the heptadecanoic acid was not detected in lcTAGs and in acetylTAGs represented only 0.3%.  The dominant fatty acid in acetylTAGs as well as in lcTAGs of E. europaeus immature seeds was the oleic acid ( Figure 3). The fraction of acetylTAGs contained 61.3% and fraction of lcTAG 53.2% of oleic acid, respectively. Seeds accumulated also 9.0% of the essential linoleic acid in acetylTAGs and 11.8% in lcTAGs. The palmitic and stearic acids represented saturated fatty acids in acetylTAGs with a percentage 21.5% and 6.6%, respectively. Their content in lcTAGs was 26.8% and 5.5%, respectively. The cis-vaccenic acid was a minor component in both fractions of oil (1.4% and 2.7%, respectively), the heptadecanoic acid was not detected in lcTAGs and in acetylTAGs represented only 0.3%.

Diacylglycerol Acetyltransferase Gene from E. europaeus
Initial metabolomic analysis of lipids in seeds of E. europaeus confirmed the presence of 3-acetyl-1,2-diacylglycerols (acetylTAGs). Based on this prerequisite, the responsible diacylglycerol acetyltransferase-encoding gene was identified using high homology of this gene within the family Celastraceae Celestraceae. Primers for detection of DAcT in E. europaeus (EeDAcT) were designed

Diacylglycerol Acetyltransferase Gene from E. europaeus
Initial metabolomic analysis of lipids in seeds of E. europaeus confirmed the presence of 3-acetyl-1,2-diacylglycerols (acetylTAGs). Based on this prerequisite, the responsible diacylglycerol acetyltransferase-encoding gene was identified using high homology of this gene within the family  Determination of cDNA sequence of EeDAcT gene was performed using a series of amplifications of cDNA and followed overlapping of amplified fragments ( Figure 5). All fragments obtained from cDNA were sequenced, and the resulted nucleotide sequence of the natural EeDAcT gene has been submitted into the GenBank database (accession number MK637625.1).  Determination of cDNA sequence of EeDAcT gene was performed using a series of amplifications of cDNA and followed overlapping of amplified fragments ( Figure 5). All fragments obtained from cDNA were sequenced, and the resulted nucleotide sequence of the natural EeDAcT gene has been submitted into the GenBank database (accession number MK637625.1).  Figure 4, lanes 10, 11). Subsequently, a total RNA from individual organs and tissues (root, stem, leaf, aril, pericarp, immature seed) of E. europaeus was isolated and transcribed to cDNA. Primers derived from the EaDAcT gene of E. alatus provided positive amplifications and fragments of expected size only in immature seeds ( Figure 4, lanes 6, 7). Determination of cDNA sequence of EeDAcT gene was performed using a series of amplifications of cDNA and followed overlapping of amplified fragments ( Figure 5). All fragments obtained from cDNA were sequenced, and the resulted nucleotide sequence of the natural EeDAcT gene has been submitted into the GenBank database (accession number MK637625.1). Comparison of complete cDNA sequences of the DAcT genes with other species of the Celastraceae family declared the originality of the natural EeDAcT gene at six nucleotide positions (555, 558, 717, 718, 720, 804) ( Figure 6). The highest degree of identity (98.53%) of cDNA sequence was of the natural EeDAcT gene with the homologous gene from E. atropurpureus (MF061249.1). High identity match was also observed with the homologous gene from E. alatus (GU594061.1). The lowest degree of similarity of EeDAcT was with the relevant cDNA sequence from Celastrus scandens L. (MF061248.1). This was also supported by the phylogenetic analysis (Figure 7). The phylogenetic tree created according to cDNA sequences of DAcT genes (Figure 7) resembled clustering of species of EeDAcT was with the relevant cDNA sequence from Celastrus scandens L. (MF061248.1). This was also supported by the phylogenetic analysis (Figure 7). The phylogenetic tree created according to cDNA sequences of DAcT genes (Figure 7) resembled clustering of species of the genus Euonymus L. established by chemical composition of 3-acetyl-1,2-diacyl-sn-glycerols from seeds of mature fruits [10]

Expression of EeDAcT Gene in Tobacco
Natural EeDAcT gene isolated from E. europaeus contained sequences recognized with the same restriction endonucleases that were used to clone this gene into a plasmid vector. Therefore, these sites were eliminated during the design process of synthetic EeDAcT gene. The open reading frame

Expression of EeDAcT Gene in Tobacco
Natural EeDAcT gene isolated from E. europaeus contained sequences recognized with the same restriction endonucleases that were used to clone this gene into a plasmid vector. Therefore, these sites were eliminated during the design process of synthetic EeDAcT gene. The open reading frame

Expression of EeDAcT Gene in Tobacco
Natural EeDAcT gene isolated from E. europaeus contained sequences recognized with the same restriction endonucleases that were used to clone this gene into a plasmid vector. Therefore, these Life 2020, 10, 205 10 of 16 sites were eliminated during the design process of synthetic EeDAcT gene. The open reading frame has not been changed, and changes in codon usage were minimized. Transformation vector pRI 101-AN contains constitutive promoter CaMV 35S and the 5 -untranslated region that should provide a higher expression of the gene of interest [29]. Resulted plasmid pRI 101-AN-EeDAcT was transformed into competent Escherichia coli cells DH10B and its sequence was verified by the Sanger sequencing. Agrobacterium tumefaciens, strain EHA105, was transformed with plasmid vector pRI 101-AN-EeDAcT, and transformed cells were selected using kanamycin (50 µg/mL) and rifampicin (10 µg/mL) and again verified by PCR analysis.
Presence of the EeDAcT transgene in transformed tobacco was confirmed by PCR analysis. Five regenerated and analyzed plants generated amplicons relevant to the presence of EeDAcT transgene (Figure 9, lanes 6-10). has not been changed, and changes in codon usage were minimized. Transformation vector pRI 101-AN contains constitutive promoter CaMV 35S and the 5′-untranslated region that should provide a higher expression of the gene of interest [29]. Resulted plasmid pRI 101-AN-EeDAcT was transformed into competent Escherichia coli cells DH10B and its sequence was verified by the Sanger sequencing. Agrobacterium tumefaciens, strain EHA105, was transformed with plasmid vector pRI 101-AN-EeDAcT, and transformed cells were selected using kanamycin (50 μg/mL) and rifampicin (10 μg/mL) and again verified by PCR analysis. Presence of the EeDAcT transgene in transformed tobacco was confirmed by PCR analysis. Five regenerated and analyzed plants generated amplicons relevant to the presence of EeDAcT transgene (Figure 9, lanes 6-10). Expression of the EeDAcT transgene was monitored using cDNA from individual parts of transgenic tobacco plants (root, stem, leaf, seed), and differences were detected ( Figure 10). Transgenic plants T3, T4, and T5 expressed EeDAcT transgene in leaves, stems, and immature seeds, not in roots. The T1 transgenic plant expressed transgene in immature seeds, stems, and roots, not in leaves, and the T2 plant di not express the transgene in any organ.
Analysis of EeDAcT transgene at the level of DNA as well as cDNA exhibited presence of PCR products with length 579 bp, corresponding with the expected length using designed primers (Figures 9 and 10).  Expression of the EeDAcT transgene was monitored using cDNA from individual parts of transgenic tobacco plants (root, stem, leaf, seed), and differences were detected ( Figure 10). Transgenic plants T3, T4, and T5 expressed EeDAcT transgene in leaves, stems, and immature seeds, not in roots. The T1 transgenic plant expressed transgene in immature seeds, stems, and roots, not in leaves, and the T2 plant di not express the transgene in any organ.
Life 2020, 10, x FOR PEER REVIEW 10 of 16 has not been changed, and changes in codon usage were minimized. Transformation vector pRI 101-AN contains constitutive promoter CaMV 35S and the 5′-untranslated region that should provide a higher expression of the gene of interest [29]. Resulted plasmid pRI 101-AN-EeDAcT was transformed into competent Escherichia coli cells DH10B and its sequence was verified by the Sanger sequencing. Agrobacterium tumefaciens, strain EHA105, was transformed with plasmid vector pRI 101-AN-EeDAcT, and transformed cells were selected using kanamycin (50 μg/mL) and rifampicin (10 μg/mL) and again verified by PCR analysis. Presence of the EeDAcT transgene in transformed tobacco was confirmed by PCR analysis. Five regenerated and analyzed plants generated amplicons relevant to the presence of EeDAcT transgene (Figure 9, lanes 6-10). Expression of the EeDAcT transgene was monitored using cDNA from individual parts of transgenic tobacco plants (root, stem, leaf, seed), and differences were detected ( Figure 10). Transgenic plants T3, T4, and T5 expressed EeDAcT transgene in leaves, stems, and immature seeds, not in roots. The T1 transgenic plant expressed transgene in immature seeds, stems, and roots, not in leaves, and the T2 plant di not express the transgene in any organ.
Analysis of EeDAcT transgene at the level of DNA as well as cDNA exhibited presence of PCR products with length 579 bp, corresponding with the expected length using designed primers (Figures 9 and 10).   Analysis of EeDAcT transgene at the level of DNA as well as cDNA exhibited presence of PCR products with length 579 bp, corresponding with the expected length using designed primers (Figures 9 and 10).

AcetylTAGs in Transgenic Tobacco
The content of oil in immature seeds (approximately 12 days after pollination) of nontrangenic tobacco plant was 15.8% of fresh weight. Lipids (lcTAGs, acetylTAGs, free fatty acids (FFAs), free and esterified sterols, and polar lipids) were isolated and separated from immature seeds of transgenic and control tobacco plants by TLC coupled with densitometry. Nontransgenic control plants did not produce any acetylated TAGs, as they do not have the necessary genetic and thus no enzymatic background. New lipid structures were identified only in the three transgenic lines (T3, T4, T5). They were identified as a fraction of acetylTAGs, using the di-18:1-acetylTAG synthetic standard (Figure 11a). AcetylTAGs were the dominant lipids in immature seeds with content in the range of 29.5-54.8% and content of fatty acids in acetylTAGs was different in individual transgenic tobacco lines (Figure 11c). Lines were different also in the amount of accumulated lcTAGs as well as in content of fatty acids in lcTAGs (Figure 11b). T3 and T5 plants contained similar values (4.73% and 7.05%), but the T4 plant accumulated a very high content (53.9%) of lcTAGs.
Life 2020, 10, x FOR PEER REVIEW 11 of 16

AcetylTAGs in Transgenic Tobacco
The content of oil in immature seeds (approximately 12 days after pollination) of nontrangenic tobacco plant was 15.8% of fresh weight. Lipids (lcTAGs, acetylTAGs, free fatty acids (FFAs), free and esterified sterols, and polar lipids) were isolated and separated from immature seeds of transgenic and control tobacco plants by TLC coupled with densitometry. Nontransgenic control plants did not produce any acetylated TAGs, as they do not have the necessary genetic and thus no enzymatic background. New lipid structures were identified only in the three transgenic lines (T3, T4, T5). They were identified as a fraction of acetylTAGs, using the di-18:1-acetylTAG synthetic standard (Figure 11a). AcetylTAGs were the dominant lipids in immature seeds with content in the range of 29.5-54.8% and content of fatty acids in acetylTAGs was different in individual transgenic tobacco lines (Figure 11c). Lines were different also in the amount of accumulated lcTAGs as well as in content of fatty acids in lcTAGs (Figure 11b). T3 and T5 plants contained similar values (4.73% and 7.05%), but the T4 plant accumulated a very high content (53.9%) of lcTAGs. Polar lipids, free fatty acids, and free sterols were minor lipids in transgenic tobacco seeds ( Figure 12). Polar lipids, free fatty acids, and free sterols were minor lipids in transgenic tobacco seeds ( Figure 12). Proportion of acetylTAGs and lcTAGs in immature seeds of tobacco transformed with modified EeDAcT gene showed a strong effect of transgene ( Figure 13). T3 and T5 plants synthesized a high level of acetylTAGs (54.8% for T3 and 53.5% for T5). lcTAGs were a minor compound, with content of 4.7% (T3) and 7.1% (T5). The T4 plant had the opposite parameters. It contained 29.5% of acetylTAGs, but 53.9% of lcTAGs. The proportion of acetyl TAGs to lcTAGs in two tobacco transgenic lines (T3, T5) was similar, even higher than in immature seeds of E. europaeus ( Figure 13).  Proportion of acetylTAGs and lcTAGs in immature seeds of tobacco transformed with modified EeDAcT gene showed a strong effect of transgene ( Figure 13). T3 and T5 plants synthesized a high level of acetylTAGs (54.8% for T3 and 53.5% for T5). lcTAGs were a minor compound, with content of 4.7% (T3) and 7.1% (T5). The T4 plant had the opposite parameters. It contained 29.5% of acetylTAGs, but 53.9% of lcTAGs. The proportion of acetyl TAGs to lcTAGs in two tobacco transgenic lines (T3, T5) was similar, even higher than in immature seeds of E. europaeus ( Figure 13). Proportion of acetylTAGs and lcTAGs in immature seeds of tobacco transformed with modified EeDAcT gene showed a strong effect of transgene ( Figure 13). T3 and T5 plants synthesized a high level of acetylTAGs (54.8% for T3 and 53.5% for T5). lcTAGs were a minor compound, with content of 4.7% (T3) and 7.1% (T5). The T4 plant had the opposite parameters. It contained 29.5% of acetylTAGs, but 53.9% of lcTAGs. The proportion of acetyl TAGs to lcTAGs in two tobacco transgenic lines (T3, T5) was similar, even higher than in immature seeds of E. europaeus ( Figure 13).  The fatty acid composition in lcTAGs showed similarities in contents of oleic and palmitic acids, but differences in linoleic acid. T4 plant accumulated the lowest levels of palmitic (26.3%) and oleic acids (31.0%), but a significant higher level of linoleic acid (27.7%) compared with T5 (only 1.3%) and T3 (4.1%).

Discussion
Euonymus europaeus L., similarly to other Euonymus species, possesses the genetic background and biochemical pathways for the synthesis of 3-acetyl-1,2-diacyl-sn-glycerols. It accumulates acetylTAGs in mature seeds and a very limited content also in arils [9]. The studies were performed almost exclusively on a related species, E. alatus. Immature seeds of E. europaeus used in this study also contained a high content of acetylTAGs, but they were not detected in aril tissues surrounding seeds (Figures 1 and 3). As was previously published in regard to E. alatus [17,20], the sn-1 and sn-2 positions of acetylglycerides of E. europaeus are esterified with common fatty acids, predominantly with oleic acid, followed by palmitic and linoleic acids ( Figure 3). Consequently, E. europaeus could be included among the candidates from the family of Celastraceae for isolation, cloning, and utilization of the gene encoding the responsible enzyme, diacylglycerol acetyltransferase. Compared with EaDAcT, the EeDAcT gene is unique. The enzyme EeDAcT itself is different in six amino acids against EaDAcT, and there are even greater differences compared with other Euonymus DAcTs ( Figure 8). This suggests that the acetylation activity of EeDAcT could be different from that of other Euonymus DAcTs. This assumption is based on an already revealed significant variation in activity of diacylglycerol acetyltransferases originated from other Euonymus species expressed in transgenic yeast [18].
The nucleotide sequence of cDNA obtained by reverse transcription from RNA isolated from immature seeds and the DNA sequence of the original EeDAcT gene isolated from E. europaeus were identical. This suggests that the EeDAcT gene has a simple, intronless structure. Such genes occupy approximately about one-fifth of all protein-encoding genes within plant genomes [30]. They include housekeeping genes, for example, genes encoding key enzymes included in the primary metabolism, storage proteins, and other proteins [31,32]. The fact that the EeDAcT gene does not contain introns emphasizes its importance in plant metabolism and plant life. This also increases the interest to transfer such genes into oil-producing plant species. Due to the intronless structure of the natural EeDAcT gene, only minor in silico redesigning and synthesis of an artificial gene for transformation into the host plant was necessary. Tobacco plants were used for heterologous expression of the EeDAcT gene. Although tobacco with a modified metabolic pathway of lipid biosynthesis can be also considered as a promising non-food crop for biofuel production [33], here it was used only as a model plant species for overexpression of isolated and modified EeDAcT gene. Results of metabolomic analysis in immature seeds of transgenic tobacco have coincided with the EeDAcT gene expression detected at the transcriptomic level. Expression of EeDAcT transgene varied within individual transgenic plants, and different levels of synthesized acetylTAGs in plants were associated with this. None of the transformed plants transcribed the EeDAcT gene in all four monitored parts (immature seeds, stems, leaves, roots) ( Figure 10). There are several possible reasons, such as the positional effect of transgene, regulation sequences and flanking sequences of host DNA in the site of transgene integration, transgene copy number in the host genome, and epigenetic effects of gene silencing [34][35][36][37]. Therefore, the impact of the enzymatic activity of the transgene product was evaluated. That is the best indication of changes caused by the expression of alien gene in the metabolism of the host plant.
The natural composition of fatty acids in tobacco seed oil is very diverse and depends on the tobacco genotype. Linoleic acid is dominant in some genotypes, the oleic acid in others, and in some genotypes there is a relatively well-balanced ratio between linoleic, oleic, and palmitic acids [38]. These ratios are more or less constant also in different vegetation conditions. Serious shifts in oil content and fatty acid composition tend to be associated with overexpression of alien genes related to lipid biosynthesis. This is also a typical impact of DAcT gene transfer into the host plants [33,39,40]. However, the main aim of introduction of DAcT gene is acetylation of synthesized TAGs. Transgenic Life 2020, 10, 205 14 of 16 tobacco plants obtained by introduction of EeDAcT gene generated both changes. They synthesized acetylated TAGs, but also variation in the content of individual fatty acids in acetylTAGs appeared. The palmitic acid dominated in acetylTAGs in T3 transgenic line, while oleic acid dominated in T4 and T5 lines (Figure 11b). There was also variation in the content of lcTAGs. Two lines (T3, T5) produced a very low amount of lcTAGs, whereas the line T4 produced predominantly lcTAGs ( Figure 12). Differences in content of bounded fatty acids in lcTAGs were also found. High content of linoleic acid was present in lcTAGs in T4 transgenic line, very low in two others, but in the case of oleic acid it was vice versa.
Summarizing of these results reveals that the presence of EeDAcT transgene, along with the impact of genetic transformation and transgene integration effect itself, causes extensive changes in lipid metabolism of the host plants. In addition to the acetylation of TAGs, changes also occur in the composition and relative proportions of lipid structures and fatty acids. Experiences from transgenic yeast and Arabidopsis seeds expressing the EaDAcT from E. alatus revealed that this enzyme can acetylate a wide range of diacylglycerol substrates [16]. This can result in significant biochemical, physiological, and even morphological changes in the host organism that must be studied. This enzyme can also induce changes in the plant itself, giving the plant better resistance to environmental stresses. Overexpression of genes encoding different DGATs in a transgenic plant also changes quality parameters of oils required for either technical or nutritional use. Therefore, this approach could be extremely important for the development of new genotypes of plants advantageous for crop production.
From the point of view of possible applications of EeDAcT gene isolated from E. europeaus, the most important conclusion is that the enzyme EeDAcT encoded by this transgene was able to produce acetylate TAGs in transgenic plants. The host plants accumulated acetylTAGs not previously present, in a ratio similar to that in E. europaeus.