Functional characterization of the PP2C family reveals NtPP2C171 as a negative regulator of drought tolerance in tobacco

Protein phosphatase class 2Cs (PP2Cs) are key regulators in the abscisic acid (ABA) signaling pathway and plant stress responses. Although genome-wide analyses of PP2C families have been conducted in several species, systematic characterization and functional validation in allotetraploid crops, such as tobacco, remain limited. This study identified 183 NtPP2C genes in the genome of cultivated tobacco. Phylogenetic analysis categorized them into 12 distinct subfamilies, with significant expansion in subfamilies D and E. This expansion was primarily attributed to segmental and tandem duplication, consistent with allopolyploid origin and whole-genome duplication of tobacco. Cis-element analysis identified numerous stress- and hormone-responsive motifs, particularly ABA-responsive elements. Expression profiling under drought stress highlighted dynamic responses, with several members showing early induction. CRISPR/Cas9-mediated knockout of NtPP2C171 , a gene belonging to the less-characterized E subfamily, significantly enhanced drought tolerance. The knockout plants exhibited improved water retention, increased activities of SOD and POD, and reduced MDA accumulation. The mutants exhibited increased sensitivity to ABA, confirming its role in ABA signaling. Comparative transcriptomics revealed that the NtPP2C171 mutation altered the drought-responsive transcriptome, enriching differentially expressed genes in ABA signaling, flavonoid biosynthesis, and MAPK pathways. Notably, NtDOX , NtCNGC13 , and NtXA21 were significantly unregulated in mutants compared to WT under drought stress, indicating them as potential downstream factors within this regulatory network. In conclusion, this study provides a systematic characterization of the NtPP2C gene family and establishes NtPP2C171 as a novel negative regulator of drought tolerance and ABA signaling in tobacco. Functional evidence suggests that the mutation of NtPP2C171 influences the downstream stress-responsive genes. These findings provide valuable insights into the molecular mechanisms of industrial plant drought tolerance and offer potential targets for molecular breeding. • A total of 183 NtPP2Cs were identified in tobacco genome, with evolutionary expansion driven by segmental duplication. • NtPP2C171 was identified as a key negative regulator of drought tolerance and ABA signaling via CRISPR-Cas9. • Knockout of NtPP2C171 significantly enhanced drought tolerance, improving water retention and antioxidant capacity. • Transcriptome analysis revealed NtPP2C171 modulates stress-responsive pathways and secondary metabolite biosynthesis.