Vegetable oils are mainly stored in the form of triacylglycerol (TAG) in oilseeds. Fatty acids (FAs), one of the building blocks for TAG assembly, are synthesized in plastids and then exported to the endoplasmic reticulum for storage oil synthesis. A recent study demonstrated that the export of FAs from plastids was mediated by a FAX (FA export) family protein. However, the significance of FAs export from plastid during seed oil accumulation has not been investigated. In this study, we found that FAX2 was highly expressed in developing Arabidopsis seeds and the expression level was consistent with FAs synthesis activity. FAX2 mutant seeds showed an approximately 18% reduction of lipid levels compared with wild-type seeds. By contrast, overexpression of FAX2 enhanced seed lipid accumulation by up to 30%. The FAs export activity of FAX2 was confirmed by yeast mutant cell complementation analysis. Our results showed that FAX2 could interact with other proteins to facilitate FAs transport. Taken together, these results indicate that FAX2-mediated FA export from plastids is important for seed oil accumulation, and that FAX2 can be used as a target gene for increasing lipid production in oilseeds.

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Background: Tobacco seed oil could be used as an appropriate feedstock for biodiesel production. However, the high linoleic acid content of tobacco seed oil makes it susceptible to oxidation. Altering the fatty acid profile by increasing the content of oleic acid could improve the properties of biodiesel produced from tobacco seed oil. Results: Four FAD2 genes, NtFAD2-1a, NtFAD2-1b, NtFAD2-2a, and NtFAD2-2b, were identified in allotetraploid tobacco genome. Phylogenetic analysis of protein sequences showed that NtFAD2-1a and NtFAD2-2a originated from N. tomentosiformis, while NtFAD2-1b and NtFAD2-2b from N. sylvestris. Expression analysis revealed that NtFAD2-2a and NtFAD2-2b transcripts were more abundant in developing seeds than in other tissues, while NtFAD2-1a and NtFAD2-1b showed low transcript levels in developing seed. Phylogenic analysis showed that NtFAD2-2a and NtFAD2-2b were seed-type FAD2 genes. Heterologous expression in yeast cells demonstrated that both NtFAD2-2a and NtFAD2-2b protein could introduce a double bond at the Δ 12 position of fatty acid chain. The fatty acid profile analysis of tobacco fad2-2 mutant seeds derived from CRISPR-Cas9 edited plants showed dramatic increase of oleic acid content from 11% to over 79%, whereas linoleic acid decreased from 72 to 7%. In addition, the fatty acid composition of leaf was not affected in fad2-2 mutant plants. Conclusion: Our data showed that knockout of seed-type FAD2 genes in tobacco could significantly increase the oleic acid content in seed oil. This research suggests that CRISPR-Cas9 system offers a rapid and highly efficient method in the tobacco seed lipid engineering programs.

Tobacco (Nicotiana tabacum L.) seed lipid is a promising non-edible feedstock for biodiesel production. In order to meet the increasing demand, achieving high seed lipid content is one of the major goals in tobacco seed production. The TT8 gene and its homologs negatively regulate seed lipid accumulation in Arabidopsis and Brassica species. We speculated that manipulating the homolog genes of TT8 in tobacco could enhance the accumulation of seed lipid. In this present study, we found that the TT8 homolog genes in tobacco, NtAn1a and NtAn1b, were highly expressed in developing seed. Targeted mutagenesis of NtAn1 genes was created by the CRISPR-Cas9-based gene editing technology. Due to the defect of proanthocyanidin (PA) biosynthesis, mutant seeds showed the phenotype of a yellow seed coat. Seed lipid accumulation was enhanced by about 18 and 15% in two targeted mutant lines. Protein content was also significantly increased in mutant seeds. In addition, the seed yield-related traits were not affected by the targeted mutagenesis of NtAn1 genes. Thus, the overall lipid productivity of the NtAn1 knockout mutants was dramatically enhanced. The results in this present paper indicated that tobacco NtAn1 genes regulate both PAs and lipid accumulation in the process of seed development and that targeted mutagenesis of NtAn1 genes could generate a yellow-seeded tobacco variety with high lipid and protein content. Furthermore, the present results revealed that the CRISPR-Cas9 system could be employed in tobacco seed de novo domestication for biodiesel feedstock production.