Physiological and Phosphoproteomic Analyses Revealed That the NtPOD63 L Knockout Mutant Enhances Drought Tolerance in Tobacco

Drought is an important environmental factor affecting plant growth and development. Tobacco is a widely cultivated and economically important crop worldwide. Drought can decrease the yield and quality of tobacco. Peroxidase III can maintain the balance of peroxy free radicals and H2O2 in plants and participate in a variety of physiological processes, including lignin biosynthesis and auxin catabolism. In this study, we first found that a CRISPR/Cas9-mediated peroxidase 63-like (NtPOD63 L) knockout mutant had high drought tolerance, such as reduced stomatal apertures, increased lignin content and interfascicular fiber number, and induced ABA-related gene expression. Then, a quantitative phosphoproteomic analysis of tobacco mutant leaves was conducted after PEG treatment. Phosphoproteomic analysis revealed 609 phosphorylated proteins, among which 266 were upregulated and 343 were downregulated. KEGG pathway analysis indicated that the cluster with “Pyruvate metabolism” contained the most upregulated pathways. The cluster with “RNA degradation”, “Cysteine and methionine metabolism,” and “Tryptophan metabolism contained the three primary downregulated pathways. Sixty-seven kinases were found to change phosphorylation levels. Our study provides new information for understanding NtPOD63 L function in drought stress and describes the molecular regulation mechanisms of the tobacco response to drought at the posttranslational level.