Technology Center, China Tobacco Yunnan Industrial Co. LTD, No. 181 Hongjin Road, Kunming, 650000, Yunnan Province, China.
Jiangsu Academy of Agricultural Sciences, No. 50 Zhongling Street, Nanjing, 210014, Jiangsu Province, China.
BMC Plant Biol. 2023 Jul 5;23(1):349. doi: 10.1186/s12870-023-04307-7.
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.
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.
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.
DFR 是植物类黄酮和多酚代谢中的关键结构基因，DFR 敲除（DFR-KO）植物可能会增加生物量积累。DFR-KO 是否在烟草中具有类似的效果以及其分子机制尚不确定。我们采用 CRISPR/Cas9 方法生成了一个敲除纯合构建体，并从不同发育阶段收集样品进行转录组和代谢组检测和分析。
在具有 NtDFR1 和 NtDFR2 敲除的纯合烟草（Nicotiana tabacum）植株上，DFR-KO 使烟草花朵变白。对野生型（CK）和 DFR-KO 系开花叶（LF）、开花花（FF）、成熟叶（LM）和成熟根（RM）的转录组变化进行 RNA-seq 研究，分别发现 2898、276、311 和 101 个差异表达基因（DEGs）。DFR-KO 主要影响开花期的叶片。根据 KEGG 和 GSEA 研究，DFR-KO 系上调了光合作用途径的碳固定，下调了光系统 I 和 II 基因。DFR-KO 可能会降低烟草开花叶光合作用的光反应，但会促进暗反应的碳固定。DFR-KO 降低了 LF 中与途径相关的基因的表达，如氧化磷酸化和蛋白酶体，同时上调了植物-病原体相互作用和 MAPK 信号通路中的基因表达，表明它可能增加生物应激抗性。DFR-KO 大大上调了 FF 中参与苯丙烷生物合成的其他结构基因的表达，这可能是代谢物积累的原因。代谢组学表明，LF 中 8 种类黄酮代谢物表达上调，FF 中 24 种黄酮类代谢物表达下调。在 DFR-KO LF 中，蛋白酶体相关基因下调了 16 种氨基酸代谢物，并减少了游离氨基酸。此外，对 LM 的 DEG 分析表明，DFR-KO 对烟草生长的影响随着时间的推移逐渐减弱。
本研究系统全面地研究了 DFR-KO 对烟草发育不同阶段和器官的广泛影响。DFR-KO 在开花期减少了叶片的分解代谢和光反应，同时增加了碳固定和抗病途径。然而，随着烟草的生长和成熟，DFR-KO 对烟草生长的影响逐渐减弱，转录和代谢修饰是一致的。这项工作为烟草育种和基因编辑菌株的开发提供了新的见解和理论基础。
