Tung tree stearoyl-acyl carrier protein Δ9 desaturase improves oil content and cold resistance of Arabidopsis and Saccharomyces cerevisiae

Abstract

The seed oil of tung tree is rich in a-eleostearic acid (ESA), which endows tung oil with the characteristic of an excellently dry oil. The stearoyl-acyl carrier protein δ9 desaturase (SAD) is a rate-limiting enzyme that converts the stearic acid to the oleic acid, the substrate for the production of the α-ESA. However, the function of the two predicted VfSAD1 and VfSAD2 genes in the tung tree has not been determined. In this study, quantitative real-time PCR (qRT-PCR) analysis showed that VfSAD1 and VfSAD2 were expressed in multiple organs of tung tree but were highly expressed in the seed during the oil rapid accumulation period. Heterologous expression of VfSAD1 and VfSAD2 could promote the production of oleic acid and its derivatives in Arabidopsis thaliana and yeast BY4741, indicating that VfSAD1 and VfSAD2 possess the stearoyl-ACP desaturases function. Furthermore, both VfSAD1 and VfSAD2 could significantly improve seed oil accumulation in Arabidopsis. VfSAD1 could also significantly promote the oil accumulation in the yeast BY4741 strain. In addition, overexpression of VfSAD1 and VfSAD2 enhanced the tolerance of yeast and Arabidopsis seedlings to low temperature stress. This study indicates that the two VfSAD genes play a vital role in the process of oil accumulation and fatty acid biosynthesis in the tung tree seed, and both of them could be used for molecular breeding in tung tree and other oil crops.

Keywords: oil accumulation; oleic Acid; stearoyl-acyl carrier prote in Δ9 desaturase (SAD); the α-eleostearic acid; tung tree (Vernicia fordii).

Figure 1

The characteristic analysis of VfSAD1 and VfSAD2 protein. (A) Subcellular localization of VfSAD1 and VfSAD2 proteins. (B) Phylogenetic tree analysis of SAD proteins from tung tree and other species.

Figure 2

Oil content in the developing seed and the expression level of the two SAD genes in the multiple tissues of tung tree. (A) Oil content in the seeds from multiples development stages; (B–F) percentage of unsaturated fatty acid (B), stearic acid (C), oleic acid (D), linoleic acid (E), and α-eleostearic (F) in the seeds from multiples development stages; (G, H) the relative expression level of VfSAD1 (G) and VfSAD2 (H) in the different tissues of tung tree. The error bar represents the standard deviation (± SD) for setting three biological replicates.

Figure 3

The VfSAD1/2 improves oil content and alters the percentage of the fatty acid component in the S. cerevisiae BY4741. (A) GC-FID analysis of fatty acid methyl esters derived from neutral lipids isolated from S. cerevisiae BY4741. (B) The percentage of fatty acid components in the wild-type BY4741 containing the empty plastid and transgenic BY4741 with the overexpression of VfSAD1 or VfSAD2. (C) Oil content in the wild-type BY4741 and transgenic BY4741 containing VfSAD1 or VfSAD2. The error bar represents the standard deviation (± SD) for setting three biological replicates. Significant difference was detected by t-test (* indicates p< 0.05; ** indicates p< 0.01). A comparison between groups was conducted by one-way ANOVA test (FA component), and the different letters above the bar chart indicate significant differences (p< 0.05).

Figure 4

The overexpression of VfSAD1/2 altered the percentage of fatty acid components and oil content in the seeds of Arabidopsis. (A) Fatty acid composition in seeds of SAD1-OE line, SAD2-OE line, and fad3fae1 line. (B) The 1,000-grain weight in seeds of SAD1-OE line, SAD2-OE line, and fad3fae1 line. (C) Oil content in seeds of SAD1-OE lines and fad3fae1 line. (D) Oil content in seeds of SAD2-OE lines and fad3fae1 line. (E) The expression pattern of VfSAD1 in the SAD1-OE line and fad3fae1 line. (F) The expression pattern of VfSAD2 in the SAD2-OE line and fad3fae1 line. (G) The expression pattern of AtWRI1 in the SAD1-OE line and fad3fae1 line. (H) The expression pattern of AtWRI1 in the SAD2-OE line and fad3fae1 line. (I) The expression pattern of AtDGAT1 in the SAD1-OE line and fad3fae1 line. (J) The expression pattern of AtDGAT1 in the SAD2-OE line and fad3fae1 line. The error bar represents the standard deviation (± SD) for setting three biological replicates. Significant difference was detected by t-test (* indicates p< 0.05; ** indicates p< 0.01). A comparison between groups was conducted by one-way ANOVA test (FA component), and the different letters above the bar chart indicate significant differences (p< 0.05).

Figure 5

VfSAD1 and VfSAD2 enhance the tolerance of yeast to low temperature stress. (A, B) Yeast colonies at 30°C (A) and 17°C (B); (C, D) OD values of yeast liquid cultures treated at 30°C (C) and 17°C (D); (E) fatty acid fractions in the BY4741 treated at 17°C; (F) conversion of unsaturated fatty acids in the BY4741 treated at 17°C. The error bar represents the standard deviation (± SD) for setting three biological replicates. Significant difference was detected by t-test (* indicates p< 0.05; ** indicates p< 0.01). A comparison between groups was conducted by one-way ANOVA test (FA component), and the different letters above the bar chart indicate significant differences (p< 0.05).

Figure 6

VfSAD1 and VfSAD2 enhance the tolerance of Arabidopsis seedlings to low temperature stress. (A, B) Phenotypes (A) and survival rates (B) of the VfSAD1-overexpressing and WT Arabidopsis plants under normal and freezing treatments (−8°C for 90 min); (C, D) phenotypes (C) and survival rates (D) of the VfSAD2-overexpressing and WT Arabidopsis plants under normal and freezing treatments (−8°C for 120 min). The error bar represents the standard deviation (± SD) for setting three biological replicates.