WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds

Abstract

Previous attempts at engineering Arabidopsis (Arabidopsis thaliana) to produce seed oils containing hydroxy fatty acids (HFA) have resulted in low yields of HFA compared with the native castor (Ricinus communis) plant and caused undesirable effects, including reduced total oil content. Recent studies have led to an understanding of problems involved in the accumulation of HFA in oils of transgenic plants, which include metabolic bottlenecks and a decrease in the rate of fatty acid synthesis. Focusing on engineering the triacylglycerol assembly mechanisms led to modest increases in the HFA content of seed oil, but much room for improvement still remains. We hypothesized that engineering fatty acid synthesis in the plastids to increase flux would facilitate enhanced total incorporation of fatty acids, including HFA, into seed oil. The transcription factor WRINKLED1 (WRI1) positively regulates the expression of genes involved in fatty acid synthesis and controls seed oil levels. We overexpressed Arabidopsis WRI1 in seeds of a transgenic line expressing the castor fatty acid hydroxylase. The proportion of HFA in the oil, the total HFA per seed, and the total oil content of seeds increased to an average of 20.9%, 1.26 µg, and 32.2%, respectively, across five independent lines, compared with 17.6%, 0.83 µg, and 27.9%, respectively, for isogenic segregants. WRI1 and WRI1-regulated genes involved in fatty acid synthesis were up-regulated, providing for a corresponding increase in the rate of fatty acid synthesis.

Figure 1.

FA analysis of fae1 RcFAH12 WRI1 transgenics. Primary transformants were grown to maturity, then T2 seed from individual T1 plants was separated into transgenic seeds expressing the DsRed marker (red) and nontransgenic siblings (brown) and analyzed by GC. A, Percentage HFA, sorted by highest level. B, HFA per seed, sorted by highest level. One to three replicates were analyzed per line. Error bars represent sd.

Figure 2.

Transcriptional analysis of WRI1 gene expression. WRI1 transcript level was measured by qRT-PCR in 11- to 12-DAF developing seeds of fae1 and fae1 RcFAH12 WRI1 T3 lines 13, 42, and 22. n = 4, and error bars represent se. Two-tailed Student’s t test, P ≤ 0.05 for all lines compared with fae1.

Figure 3.

Extended analysis of FA from T4 seeds of fae1 RcFAH12 WRI1. Seed samples were analyzed from fae1, fae1 RcFAH12, and T4 seeds of fae1 RcFAH12 WRI1 line 13. A, Percentage HFA. B, HFA per seed. C, Total FA per seed. D, Seed oil content. Horizontal bars represent average values of 10 to 11 individuals. Each symbol represents an individual plant, and error bars represent se. One-way ANOVA, different letters above each average indicates values that are significantly different.

Figure 4.

Characterization of TAG species. A, HFA-containing molecular species of TAG. TAG molecular species from fae1 RcFAH12 and fae1 RcFAH12 WRI1 line 13 were separated by thin-layer chromatography (TLC) and quantified by GC. Columns indicate TAG molecular species containing zero, one, or two HFA (0-H, 1-H, and 2-H). n = 3, and error bars represent se. B, Regiochemical analysis of 1-HFA-TAG. n ≥ 3, and error bars represent se. C, Regiochemical analysis of 2-HFA-TAG. n ≥ 3, and error bars represent se.

Figure 5.

Analysis of gene expression in developing seeds. Transcript levels of genes involved in FA synthesis and TAG accumulation were analyzed by qRT-PCR in 11- to 12-DAF developing seeds of fae1, fae1 RcFAH12, and fae1 RcFAH12 WRI1 T3 line 13. n = 4 biological replicates, and error bars represent se. One-tailed Student’s t test, columns with different letters are significantly different.

Figure 6.

Comparison of FA synthesis rate in developing seeds. Seeds of fae1, fae1 RcFAH12, and fae1 RcFAH12 WRI1 line 13 were harvested 11 to 12 DAF and incubated in tritiated water for 30 min. n = 5, and error bars represent se. One-way ANOVA, columns with different letters are significantly different.

Figure 7.

Analysis of germination rate. Seeds from plants of fae1, fae1 RcFAH12, and fae1 RcFAH12 WRI1 line 13 were plated onto 1× Linsmaier and Skoog plates containing 2% Suc. Each data point represents an average of seven replicates of 59 to 75 seeds per genotype. n = 7, and error bars represent se.

Figure 8.

Plant growth and seed production. Plants of fae1, fae1 RcFAH12, and fae1 RcFAH12 WRI1 line 13 were grown side by side, and seeds were collected at maturity. A, Plant height. B, Seed yield per plant. C, Average seed weight. n = 5 to 11, and error bars represent se. One-way ANOVA, columns with different letters are significantly different.