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