Abstract: INTRODUCTION: Submergence tolerance is one of the most crucial stress resistances among garden plants. Willow is a plant that has strong tolerance to submergence. The objective of this study is to clone the TTF family gene SmTTF59 of willow and construct overexpression and silencing vectors in order to explore its role in responding to submergence stress in willow. 

RATIONALE: Studies have demonstrated that TTF family transcription factors play critical regulatory roles in plant responses to abiotic stresses.?Salix matsudana, a species with strong submergence tolerance, serves as an ideal model for investigating submergence stress adaptation mechanisms. By silencing?SmTTF59 in Salix matsudana and conducting hypoxia stress experiments on?SmTTF59-transgenic Arabidopsis, this study provides theoretical foundations for understanding the complex regulatory mechanisms of Trihelix transcription factors and lays a groundwork for genetic breeding of submergence-tolerant?Salix matsudana. 

RESULTS: The results showed that the coding sequence (CDS) of the SmTTF59 gene in Salix matsudana is 834 bp in length, encoding 277 amino acid residues with a protein molecular weight of 33,044.40 Da. Through PCR experiments, the 834-bp CDS of SmTTF59 was successfully amplified and used to construct recombinant vectors pWM101 and pYL156. Hypoxic stress experiments on transgenic Arabidopsis demonstrated that SmTTF59-overexpressing lines exhibited enhanced hypoxia tolerance compared to wild-type plants, characterized by longer root lengths and higher fresh weights. Physiological analyses revealed higher levels of antioxidant enzymes, such as peroxidase (POD) and catalase (CAT), in transgenic lines. Virus-induced gene silencing (VIGS) experiments in willow plants further indicated that SmTTF59 knockdown lines displayed poorer growth under submergence stress compared to controls, including severe leaf decay and abscission. Quantitative measurements showed that silenced plants had lower root fresh weight, root dry weight, and POD activity, along with higher relative electrical conductivity, compared to control plants. These results collectively confirm that SmTTF59 knockdown significantly reduces willow tolerance to submergence stress. 

CONCLUSION: The better anoxic tolerance of SmTTF59 transgenic Arabidopsis thaliana proved that this gene might be related to the response of willow to submergence stress. Through the VIGS experiment on willow, it was verified that this gene played a positive regulatory role in submergence. The results of this study are of great significance for the subsequent breeding work of submergence-tolerant willow varieties.