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. Carotenoid cleavage dioxygenase (CCD) genes are involved in the biosynthesis of SLs and are identified and characterized in many plants. However, the function of CCD genes in tobacco remains poorly understood. In this study, two closely related genes NtCCD8A and NtCCD8B were cloned from tobacco (Nicotiana tabacum L.). The two NtCCD8 genes are orthologues of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase 8 (SlCCD8) gene. NtCCD8A and NtCCD8B 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. NtCCD8A and NtCCD8B mutations were introduced into tobacco using the CRISPR/Cas9 system and transgenic tobacco lines for both ntccd8 mutant alleles were identified. The ntccd8a and ntccd8b 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 ntccd8 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 NtCCD8 genes are important for changes in tobacco plant architecture.