Suppression of carotenoid cleavage dioxygenase 1 (NtCCD1) increases carotenoid contents and attenuates reactive oxygen species (ROS) in Tobacco Leaves

Despite the nutritional and economic importance of carotenoids and their products, regulation of carotenoid content in leaves remains to be fully elucidated. Recent findings indicate that carotenoid content are determined, at least in part, by the activity of carotenoid cleavage dioxygenase 1 (CCD1). This study examined whether NtCCD1 affects leaf carotenoids in the model plant Nicotiana tabacum . Three NtCCD1s, NtCCD1a, NtCCD1b, and NtCCD1c, were investigated including their phylogenetic relationships, conserved motifs, and exon-intron architecture. Of the three transcripts, NtCCD1c exhibited the highest expression level in various tissue as determined by real-time PCR. Confocal microscopy indicated that all NtCCD1s are located in the cytoplasm. The enzymatic activity of the NtCCD1c protein was also studied by co-expressing NtCCD1c in Escherichia coli engineered to accumulate β-carotene or lycopene. SPME-GC-MS indicated that NtCCD1c cleaves β-carotene and lycopene specifically at the 9–10/9′-10′ site to produce β-ionone and pseudoionone, respectively. Virus-induced gene silencing (VIGS) of NbCCD1 increased carotenoid contents including β-carotene, α-carotene, zeaxanthin, phytoene, lutein, violaxanthin, neoxanthin, and β-cryptoxanthin, along with a decline in reactive oxygen species (ROS). RNA-seq of silenced plants suppressed NbCCD1 but enhanced expression of essential genes encoding antioxidant enzymes including NtGST, NtCuZnSOD, NtAAO, and NtPOD, along with a decrease in expression of genes related to carbon fixation such as NtRCA1, NtRCA2, and NtRBCS. These observations suggest that NtCCD1 is a negative regulator of carotenoid content and plays an essential role in the regulation of ROS levels in tobacco leaves.