NtRAV4 negatively regulates drought tolerance in Nicotiana tabacum by enhancing antioxidant capacity and defence system.

KEY MESSAGE: NtRAV4 is a nucleus-localised protein and no self-activation effect. ntrav4 mutants maintain the steady state of the ROS system under drought stress by enhancing antioxidant capacity and defence system. The APETALA2/ethylene response factor (AP2/ERF) transcription factor (TF) family plays an important role in plant responses to environmental stresses. In this study, we identified a novel NtRAV4 TF, a member of RAV subfamily among AP2/ERF gene family, which have AP2 and B3 domain in its N- and C-terminus, respectively. Subcellular localisation and self-activation activity analysis revealed that NtRAV4 localised in the nucleus and had no self-activation effect. The overexpression and gene editing vectors of NtRAV4 were constructed by homologous recombination and CRISPR/Cas9 gene editing methods, and transformed into tobacco by agrobacterium-mediated method. ntrav4 led to the appearance of termination codon in advance and lacked the unique B3 domain of RAV subfamily protein. Further analysis displayed that knockout of the NtRAV4 in tobacco increased drought tolerance with high relative water content, accompanied by reduced stomatal aperture, density, and stomatal opening ratio compared to overexpression lines and WT. Moreover, ntrav4 knockout plants also exhibited increased osmotic tolerance with low malondialdehyde (MDA) and ion leakage (EL), less accumulation of O2•- and H2O2, and high enzymatic antioxidant (SOD, POD, CAT) activities, non-enzymatic antioxidant (AsA-GSH cycle) contents and hormone (IAA, ABA, GA3, and ZR) levels under drought stress. Furthermore, ntrav4 mutants in tobacco improved the expression levels of ROS-related proline synthesis and stress-responsive genes under osmotic stress. Our results indicate that NtRAV4 negatively regulates plant tolerance to drought stress by reducing water loss and activating the antioxidant system and stress-related gene expression to maintain the steady state of the ROS system.