Integrating transcriptome, rhizosphere microbiome, and exosome microRNA analyses unravels the response of tobacco (Nicotiana tabacum L.) to atrazine stress

Atrazine is widely used to control weeds in crop production. However, residual atrazine in the soil causes phytotoxicity to subsequent tobacco ( Nicotiana tabacum L.) in the cereal crop-tobacco rotation system. The regulatory mechanisms on the response of tobacco to atrazine remain unclear, which prevents the development of effective strategies for improving the resistance of tobacco against atrazine stress. In this study, the response of tobacco to atrazine was investigated by analyzing transcriptome, exosome-like nanoparticles (ELNs) miRNAs, and rhizosphere microbial community. Atrazine with concentrations of > 0.67 mg/kg inhibited tobacco growth and caused obvious toxic symptoms. Atrazine increased malondialdehyde and hydrogen peroxide accumulations and regulated oxidative enzymes’ activities. Transcriptome analysis showed that atrazine suppressed root and leaf development, photosynthesis, chloroplast biosynthesis, and stomatal movement. Many genes encoding glutathione S-transferase, glutathione hydrolase, and thioredoxin were regulated by atrazine, which may be involved in atrazine detoxification and tolerance. Atrazine decreased the relative abundance of tobacco rhizosphere bacteria Chloroflexi, Actinobacteriota, Verrucomicrobiota, and Cyanobacteria, but increased that of Proteobacteria, Bacteroidota, Gemmatimonadota, and Armatimonadota. Atrazine suppressed the alpha diversity and richness of rhizosphere fungi. The abundance of Aspergillus and Mortierella was increased by atrazine, which may be the potential resources for identifying atrazine-degrading microbes. Atrazine enhanced expression of phenylpropanoid biosynthetic genes coupled with Proteobacteria enrichment, which may help tobacco to survive from atrazine stress. Additionally, five atrazine-responsive miRNAs, novel_17/55/60/97/99, were identified in ELNs isolated from tobacco roots. Finally, three 4-coumarate:coenzyme A ligase ( 4CL ) genes were identified by integrating transcriptome and miRNA data. It is proposed that atrazine induced 4CL genes’ expression by suppressing miRNA novel_55/97 abundance, and then activating phenylpropanoid pathways for reconstructing rhizosphere microbial community. This study provides new insights into the response of plants to atrazine, and suggests the key genes, ELNs miRNAs, and microbes involved in atrazine detoxification. • Atrazine at concentrations of > 0.67 mg/kg inhibited tobacco growth and caused obvious toxic symptoms. • Atrazine suppressed root/leaf development, photosynthesis, chloroplast biosynthesis and stomatal movement. • Enhanced phenylpropanoid biosynthesis and Proteobacteria abundance may help tobacco to survive from atrazine stress. • Five novel atrazine-responsive miRNAs in ELNs were identified. • Atrazine may induce 4CL genes’ expression by suppressing miRNA novel_55/97 abundance, and then activating phenylpropanoid pathways.