Effects of editing DFR genes on flowers, leaves, and roots of tobacco
BMC plant biology(2023)
Abstract
Background DFR is a crucial structural gene in plant flavonoid and polyphenol metabolism, and DFR knockout ( DFR -KO) plants may have increased biomass accumulation. It is uncertain whether DFR -KO has comparable effects in tobacco and what the molecular mechanism is. We employed the CRISPR/Cas9 method to generate a knockout homozygous construct and collected samples from various developmental phases for transcriptome and metabolome detection and analysis. Results DFR -KO turned tobacco blossoms white on homozygous tobacco (Nicotiana tabacum) plants with both NtDFR1 and NtDFR2 knockout. RNA-seq investigation of anthesis leaf (LF), anthesis flower (FF), mature leaf (LM), and mature root (RM) variations in wild-type (CK) and DFR -KO lines revealed 2898, 276, 311, and 101 differentially expressed genes (DEGs), respectively. DFR -KO primarily affected leaves during anthesis. According to KEGG and GSEA studies, DFR -KO lines upregulated photosynthetic pathway carbon fixation and downregulated photosystem I and II genes. DFR -KO may diminish tobacco anthesis leaf photosynthetic light reaction but boost dark reaction carbon fixation. DFR -KO lowered the expression of pathway-related genes in LF, such as oxidative phosphorylation and proteasome, while boosting those in the plant–pathogen interaction and MAPK signaling pathways, indicating that it may increase biological stress resistance. DFR -KO greatly boosted the expression of other structural genes involved in phenylpropanoid production in FF, which may account for metabolite accumulation. The metabolome showed that LF overexpressed 8 flavonoid metabolites and FF downregulated 24 flavone metabolites. In DFR -KO LF, proteasome-related genes downregulated 16 amino acid metabolites and reduced free amino acids. Furthermore, the DEG analysis on LM revealed that the impact of DFR -KO on tobacco growth may progressively diminish with time. Conclusion The broad impact of DFR -KO on different phases and organs of tobacco development was thoroughly and methodically investigated in this research. DFR -KO decreased catabolism and photosynthetic light reactions in leaves during the flowering stage while increasing carbon fixation and disease resistance pathways. However, the impact of DFR -KO on tobacco growth steadily declined as it grew and matured, and transcriptional and metabolic modifications were consistent. This work offers a fresh insight and theoretical foundation for tobacco breeding and the development of gene-edited strains.
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Key words
DFR,Gene editing,CRISPR,Cas9,Flavonoid metabolism
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