Abstract: INTRODUCTION: Sorghum (Sorghum bicolor) is an ideal crop for marginal land improvement due to its well-developed root system and high water-use efficiency. ASR proteins serve as key nodes that integrate plant growth and development with stress responses, playing important roles in fruit ripening and stress tolerance, particularly drought and salt tolerance. However, functional studies on the ASR family genes in sorghum regarding salt stress tolerance remain relatively limited, and their underlying molecular mechanisms require further investigation. 
RATIONALE: To identify salt-responsive SbASR genes in sorghum, we analyzed the expression patterns of ASR family members under salt stress and water deficit. To elucidate its biological role in salt acclimation, we further cloned and functionally characterized SbASR4 through subcellular localization and transcriptional activity assays, protein stability assessment, heterologous expression in Arabidopsis, gene silencing in sorghum, transcriptional analysis of stress-re­sponsive marker genes, and protein-protein interaction experiments.
RESULTS: SbASR4 was significantly downregulated under salt stress and natural water deficit. SbASR4 localized to the nucleus and plasma membrane and underwent rapid degradation under salt stress. Overexpression of SbASR4 significantly exacerbated the sensitivity of Arabidopsis to salt stress, as evidenced by reduced germination rate, inhibited root elongation, increased Na⁺ accumulation, and elevated H₂O₂ levels. In contrast, silencing SbASR4 in sorghum markedly enhanced plant salt tolerance. Under salt stress, SbASR4-silenced lines exhibited significant upregulation of SOS pathway-related genes, CAT, and SnRK1.1, along with notable changes in PP2C family gene expression. Protein-protein interaction experiments confirmed that SbASR4 interacts with SbSOS2.
CONCLUSION: Collectively, SbASR4 negatively regulates plant salt tolerance, potentially through modulating the antioxidant system, ABA signaling pathway, and Na⁺ homeostasis. This study provides new insights into the molecular mechanisms underlying salt tolerance in sorghum and identifies a potential genetic target for salt-tolerant sorghum breeding.



Effects of SbASR4 silencing on salt stress tolerance in sorghum (A) Phenotypes of control and SbASR4-silenced plants treated with 300 mmol·L^–1 NaCl for 7 and 10 days (bars=7 cm) (B) Relative expression level of SbASR4 in SbASR4-silenced lines. Comparison of physiological parameters between control and SbASR4-silenced plants under salt stress: (C) Survival rate (D) Relative fresh weight (E) Relative plant height (F) Leaf Na⁺/K⁺ ratio (G) Electrical conductivity (H) Leaf Na⁺ content (I) H₂O₂ content. In the figure, pTRV:00 represents the empty vector control, and pTRV:SbASR4 represents the SbASR4-silenced lines. Asterisks indicate significant differences between SbASR4-silenced lines and the control (*P<0.05, **P<0.01, ***P<0.001).

Key words: sorghum, ASR, salt stress, Na⁺ homeostasis, ABA signaling