5.5 Optimization of wax ester composition in the seeds of C. sativa
5.5.2 Crossing wax ester producing lines with a high oleic line
In parallel to the attempts to generate high oleic lines using artificial microRNAs in the background of C. sativa suneson, I got an existing high oleic line (HO line) generated by the group of Prof. E. Cahoon (UNL, NE, USA). In order to change the unfavorable fatty acid composition of C. sativa seed oil, the C.
sativa wild-type plants were transformed with the gene construct AtFAD2-RNAi+CsFAD3-RNAi+CsFAE1-RNAi to block the elongation and desaturation of C18:1 acyl-CoA. The seed oil of this Atfad3/Csfad2/Csfae1 line contains around 65% 18:1 in total fatty acids (Nguyen et al., 2013).
To optimize the composition of wax esters accumulating in seeds of C. sativa, six MaFAR/ScWS lines with high wax ester content were crossed with the HO line (mother line). The seeds of individual heterozygous plants resulting from the six crosses were germinated on steril filter papers. One of the two cotyledons of the individual seedlings were cut off and their wax ester contents were analyzed applying TLC (Supplementary Material 17), the seedlings with relatively high amounts of wax esters were planted on soil to propragate seeds of the next generation. Then, the resulting seeds of six independent MaFAR/ScWS & HO crosses were analyzed by GC-FID for the total wax ester content, and by ESI-MS/MS for the molecular species of wax esters.
The total yields of wax esters in seeds of the six MaFAR/ScWS & HO crosses ranged from 13 mg g-1 seeds to 44 mg g-1 seeds (Figure 5.5.5 A). L4 MaFAR/ScWS & HO and L5 MaFAR/ScWS & HO lines resulted in the highest wax ester accumulation up to the similar level found in seeds of the parental MaFAR/ScWS line (over 40 mg g-1 seeds). But the amounts of wax esters produced by L13 MaFAR/ScWS & HO and L26 MaFAR/ScWS & HO lines were significantly lower than that of the MaFAR/ScWS lines (Figure 5.5.5 A).
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Figure 5.5.5 Wax ester and TAG accumulation in seeds of C. sativa containing high levels of oleic acid (HO), transformed with MaFAR/ScWS (FW), six crosses of MaFAR/ScWS with the HO line (FW/HO). (A) Absolute quantification of wax esters in mg g-1 seeds. *means significantly different from MaFAR/ScWS, p<= 0.05. (B) Absolute quantification of TAG in mg g-1 seeds. (C) The relative quantification of total neutral lipids (WE, wax ester;
TAG, triacylglycerol) in mass% are calculated according to the absolute quantification of each lipid class. The data
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shown is an average of three individual heterozygous lines for each independent cross with two extraction replicates for each individual line. FW is the abbreviation of MaFAR/ScWS.
The TAG amounts in seeds of six MaFAR/ScWS & HO crosses ranged from 171 mg g-1 seeds to 251 mg g-1 seeds, which were lower than that of HO line. But these were not significantly higher than the 160 mg g-1 seeds of the MaFAR/ScWS lines (Figure 5.5.5 B). As consequence, the wax ester to TAG proportion of the six MaFAR/ScWS & HO crosses ranged from 5% to 20%, with the best performing cross (L4 MaFAR/ScWS & HO) displaying a similar percentage of wax esters in the total neutral lipids compared with the MaFAR/ScWS lines (Figure 5.5.5 C).
Figure 5.5.6 Alcohol and acyl moieties of wax esters in seeds of six C. sativa MaFAR/ScWS & HO cross lines. (A) Relative abundance of alcohol moieties in mol%. (B) Relative abundance of acyl moieties in mol%. The data shown is an average of three individual heterozygous lines for each independent cross with two extraction replicates for each individual line. *means significantly different from MaFAR/ScWS, p <= 0.05. FW is the abbreviation of MaFAR/ScWS.
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The compositions of wax esters produced by the six MaFAR/ScWS & HO crosses were obviously distinct from the MaFAR/ScWS lines (Figure 5.5.6). When MaFAR and ScWS were expressed in a high oleic background, the predominant alcohol species incorporated into wax esters was 18:1-OH, accounting for over 60 mol% of all fatty alcohol moeities, which was significantly higher than that in the MaFAR/ScWS lines. Additionally, less than 20 mol% 20:1-OH was utilized by the six MaFAR/ScWS &
HO crosses, while the other fatty acids were not significantly different from that of the MaFAR/ScWS lines (Figure 5.5.6 A).
In regard to the fatty acyl moieties in wax esters, the six MaFAR/ScWS & HO crosses were different from the almost 40 mol% 20:1-FA found in the MaFAR/ScWS lines, incorporating significantly decreased levels of 20:1 (18 mol% - 20 mol%). However, 18:1 was predominantly utilized for wax ester production by the six MaFAR/ScWS & HO crosses, accounting for 30 mol% - 37 mol% of all fatty acyl moieties (Figure 5.5.6 B). In conclusion, high levels of C18:1 substrates were incorporated into wax esters by the six MaFAR/ScWS & HO crosses.
The molecular species of wax esters in seeds of the six MaFAR/ScWS & HO crosses were analyzed by ESI-MS/MS. In general, high levels of 18:1/18:1 were accumulated in the seeds of all tested indvidual lines resulting from the six MaFAR/ScWS & HO crosses (Figure 5.5.7). The six MaFAR/ScWS & HO crosses resulted in a much higher accumulation of 18:1/18:1 in seeds, compared with MaFAR/ScWS lines, which only accumulated 4.7 mol% 18:1/18:1 in all wax ester species (Figure 5.5.7; Iven et al., 2015). Importantly, the major wax ester species is 18:1/18:1 for all six MaFAR/ScWS & HO crosses, with the highest level of 49% for the L25 MaFAR/ScWS & HO line, and the lowest level of 32% for the L26 MaFAR/ScWS & HO line (Figure.5.5.3 E and F). Furthermore, the MaFAR/ScWS combination accumulated large amounts of very long-chain wax esters (C38 - C40), with 17.7 mol% oleyl - gondonate (18:1/20:1) and 10 mol% gondoyl - gondonate (20:1/20:1). However, in the wax esters produced by the six MaFAR/ScWS & HO crosses, the level of 18:1/20:1 decreased to 8 mol% - 13 mol%, and the amount of 20:1/20:1 decreased to around 7 mol%. In contrary, more wax esters with shorter chain length (C34 - C36) were accumulated in the seeds of MaFAR/ScWS & HO crosses, with the levels of oleyl - palminate (18:1/16:0) increased up to around 16 mol% of all wax ester spesies (Figure 5.5.7).
In conclusion, expression of MaFAR and ScWS in a HO background of C. sativa resulted in an increased level of 18:1/18:1 up to around 40 mol% of all wax ester species; meanwhile, the total amounts of wax esters and TAG accumulated in seeds of C. sativa were not negatively affected.
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Figure 5.5.7 Wax ester profiles of six C. sativa MaFAR/ScWS &HO cross lines. (A) L4 FW&HO line; (B) L5 FW&HO line; (C) L9 FW&HO line; (D) L13 FW&OH line; (E) L25 FW&HO line; (F) L26 MA&HO line. Wax ester compositions were determined by nano-ESI-MS/MS. The relative abundance of the top ten wax ester molecular species (alcohol moiety/acyl moiety) are shown. The data shown is an average of ten individual heterozygous transgenic lines resulting from the six independent cross lines with two extraction replicates for each individual line. FW is the abbreviation of MaFAR/ScWS.
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6 DISCUSSION
Overall, this study tested four different strategies: (i) trying to co-localize heterologous enzymes, (ii) identifying WSs with better substrate specificities, (iii) down-regulating competing pathway, and (iv) optimizing the substrate pool for the wax synthesis pathway. In this study, the abilities to produce wax esters of different combinations of WSs in combination with the FAR from Marinobacter aqualeolei VT8 were tested in yeast and A. thaliana. These combinations showed differences in the biosynthetic performance and composition of wax esters. Moreover, the catalytic activities of WSs were shown to be heavily affected upon expression in different hosts. Furthermore, down-regulation of a single enzyme on TAG biosynthesis was insufficient for blocking this competing pathway and thus promoting the biosynthesis of wax esters as demonstrated in C. sativa. However, producing wax esters in a high oleic background in C. sativa led to an increase in the formation of 18:1/18:1 as the composition of wax esters was closely related to the fatty acid profile of the seed oil.