AUTHOR=Tian Yinshuai , Liu Xinanbei , Fan Caixin , Li Tingting , Qin Huan , Li Xiao , Chen Kai , Zheng Yunpu , Chen Fang , Xu Ying 

TITLE=Enhancement of Tobacco (Nicotiana tabacum L.) Seed Lipid Content for Biodiesel Production by CRISPR-Cas9-Mediated Knockout of NtAn1

JOURNAL=Frontiers in Plant Science

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.599474

DOI=10.3389/fpls.2020.599474

ISSN=1664-462X

ABSTRACT=<p>Tobacco (<italic>Nicotiana tabacum</italic> 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 <italic>TT8</italic> gene and its homologs negatively regulate seed lipid accumulation in <italic>Arabidopsis</italic> and <italic>Brassica</italic> species. We speculated that manipulating the homolog genes of <italic>TT8</italic> in tobacco could enhance the accumulation of seed lipid. In this present study, we found that the <italic>TT8</italic> homolog genes in tobacco, <italic>NtAn1a</italic> and <italic>NtAn1b</italic>, were highly expressed in developing seed. Targeted mutagenesis of <italic>NtAn1</italic> 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 <italic>NtAn1</italic> genes. Thus, the overall lipid productivity of the <italic>NtAn1</italic> knockout mutants was dramatically enhanced. The results in this present paper indicated that tobacco <italic>NtAn1</italic> genes regulate both PAs and lipid accumulation in the process of seed development and that targeted mutagenesis of <italic>NtAn1</italic> 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 <italic>de novo</italic> domestication for biodiesel feedstock production.</p>