CRISPR/Cas9-Mediated Mutagenesis of Carotenoid Cleavage Dioxygenase 8 (CCD8) in Tobacco Affects Shoot and Root Architecture

Abstract

Strigolactones (SLs) are a class of phytohormones that regulate plant architecture. <i>Carotenoid cleavage dioxygenase</i> (<i>CCD</i>) genes are involved in the biosynthesis of SLs and are identified and characterized in many plants. However, the function of <i>CCD</i> genes in tobacco remains poorly understood. In this study, two closely related genes <i>NtCCD8A</i> and <i>NtCCD8B</i> were cloned from tobacco (<i>Nicotiana tabacum</i> L.). The two <i>NtCCD8</i> genes are orthologues of the tomato (<i>Solanum lycopersicum</i>) <i>carotenoid cleavage dioxygenase</i> 8 (<i>SlCCD8</i>) gene. <i>NtCCD8A</i> and <i>NtCCD8B</i> were primarily expressed in tobacco roots, but low expression levels of these genes were detected in all plant tissues, and their transcript levels significantly increased in response to phosphate limitation. <i>NtCCD8A</i> and <i>NtCCD8B</i> mutations were introduced into tobacco using the CRISPR/Cas9 system and transgenic tobacco lines for both <i>ntccd8</i> mutant alleles were identified. The <i>ntccd8a</i> and <i>ntccd8b</i> mutant alleles were inactivated by a deletion of three nucleotides and insertion of one nucleotide, respectively, both of which led to the production of premature stop codons. The <i>ntccd8</i> mutants had increased shoot branching, reduced plant height, increased number of leaves and nodes, and reduced total plant biomass compared to wild-type plants; however, the root-to-shoot ratio was unchanged. In addition, mutant lines had shorter primary roots and more of lateral roots than wild type. These results suggest that <i>NtCCD8</i> genes are important for changes in tobacco plant architecture.