NtPNRF1, a novel gene regulating photosynthesis and flavonoid biosynthesis in Nicotiana tabacum: Evidence from transcriptome, metabolome and physiology

Although the transcriptional regulators of photosynthesis and flavonoid biosynthesis have been extensively studied, whether common genes affect both processes in Nicotiana species remains largely unknown. We characterized Nicotiana tabacum Photosynthesis Negative Regulation Factor 1 ( NtPNRF1) as a novel gene that affects both photosynthetic efficiency and flavonoid biosynthesis in tobacco ( Nicotiana tabacum ). CRISPR/Cas9-mediated knockout (Mut) and gene overexpression (OE) lines were generated for NtPNRF1 . Transcriptomic and metabolomic analyses were performed to identify pathway associations, which were validated through photosynthetic parameter quantification using a LI-6400XT system and flavonoid profiling via spectrophotometry. Transcriptome KEGG enrichment analysis revealed that both the overexpression and knockout of NtPNRF1 primarily affected photosynthesis and the biosynthesis of secondary metabolites. Metabolomic analysis indicated that NtPNRF1 was associated with flavonoid synthesis. Based on these findings, the photosynthetic rates of the wild type (WT), OE, and Mut lines were measured. The results showed that overexpressing NtPNRF1 resulted in a reduced photosynthetic rate, whereas knockout resulted in the opposite effect, suggesting that NtPNRF1 negatively regulated photosynthesis. Additionally, the total flavonoid content and 18 detectable flavonoid components were reduced in both knockout and overexpression lines, indicating that NtPNRF1 was involved in a complex flavonoid synthesis pathway. Expression analysis showed that both the gene knockout and overexpression of NtPNRF1 significantly affected the expression of the NtDFR , NtCHI , and NtFLS genes. Our results not only provided genetic resources for tobacco breeding aimed at enhancing light efficiency and quality but also established a framework for analyzing the functions of unknown genes.