Enhancing effect of 5-azacytidine on saline–alkaline resistance of <i>Akebia trifoliata</i> and underlying physiological and transcriptomic mechanisms

Saline-alkaline stress is a common problem in <i>Akebia trifoliata</i> cultivation. In this study, the enhancing effects of 5-azacytidine (5-AzaC) on the resistance of <i>A. trifoliata</i> to saline-alkaline stress and the underlying mechanisms were investigated. Plant height, stem diameter, biomass, root length, fresh weight of root, and root/shoot ratio of 6-month-old <i>A. trifoliata</i> seedlings were measured after saline-alkaline stress with or without 5-AzaC treatment. Moreover, the contents of photosynthetic pigments, malondialdehyde (MDA), H₂O₂, sodium, soluble sugar, and proline; activities of superoxide dismutase, peroxidase (POD), and catalase (CAT); and anatomical structures of root, stem, and leaf were assessed. Furthermore, comparative transcriptome sequencing was performed. The results demonstrated that growth and development of <i>A. trifoliata</i> were severely inhibited under saline-alkaline stress, suggesting that the seedlings were exposed to severe oxidative and osmotic stresses. Treatment with exogenous 5-AzaC could significantly relieve the symptoms of saline-alkaline stress in <i>A. trifoliata</i>. Under saline-alkaline stress, 5-AzaC could increase the stem diameter, biomass, root length, fresh weight of root, and root/shoot ratio and minimize damages to the anatomical structure. Moreover, absorption of Na⁺ was reduced; ionic balance was maintained; POD and CAT activities were significantly improved; proline and soluble sugar contents increased, and H₂O₂ and MDA contents decreased. Transcriptome analysis revealed that 5-AzaC functioned via regulating KEGG pathways such as plant hormone signal transduction, phenylpropanoid biosynthesis, photosynthesis, amino sugar and nucleotide sugar metabolism, and glutathione metabolism under saline-alkaline stress. Particularly, enhanced expression of genes from the auxin pathway in plant hormone signal transduction; the lignin synthetic pathway in phenylpropanoid biosynthesis; and photosystem II, photosystem I, photosynthetic electron transport, and F-type ATP pathway in photosynthesis may be related to 5-AzaC-induced saline-alkaline resistance. The results provided theoretical references for <i>A. trifoliata</i> cultivation in saline-alkaline soil and application of 5-AzaC to improve saline-alkaline tolerance in plants.