CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline

2-acetyl-1-pyrroline (2AP) is a highly effective volatile compound that gives fragrance to numerous plant species and food. Mutation(s) in the betaine aldehyde dehydrogenase 2 (BADH2) gene results in the accumulation of 2AP. However, the function of BADH genes in tobacco (Nicotiana tabacum L.) remains poorly understood. In this study, we successfully obtained four betaine aldehyde dehydrogenase (BADH) genes from tobacco. Phylogenetic analysis of the protein sequences showed that two of the four BADH genes were closely related to the wolfberry (Lycium barbarum) BADH gene (LbBADH1), so we named them NtBADH1a and NtBADH1b, respectively. The other two BADH genes were orthologues of the tomato (Solanum lycopersicum) aminoaldehyde dehydrogenase 2 (SlAMADH2) gene, and were named NtBADH2a and NtBADH2b, respectively. Expression analysis revealed that the biological functions of NtBADH1a and NtBADH1b were different from those of genes NtBADH2a and NtBADH2b. We introduced mutations into NtBADH1a, NtBADH1b, NtBADH2a and NtBADH2b in tobacco using the CRISPR/Cas9 system and identified transgenic Ntbadh mutant tobacco lines. Single mutants (Ntbadh1a, Ntbadh1b, Ntbadh2a and Ntbadh2b) and double mutants (Ntbadh1a-Ntbadh1b and Ntbadh2a-Ntbadh2b) harbored deletion or insertion of nucleotides, both of which led to the production of a frameshift, preventing protein accumulation. A popcorn-like scent was noticeable in tobacco leaves from the Ntbadh2a-Ntbadh2b double mutant, but not from any single mutant or the Ntbadh1a-Ntbadh1b double mutant or the wild type. Consistent with this observation, we only detected 2AP in fresh leaves from the Ntbadh2a-Ntbadh2b double mutant. These findings indicate that only the combined inactivation of NtBADH2a and NtBADH2b results in 2AP accumulation in tobacco, which was not related to NtBADH1.