淹水耐受性是木本园林植物关键抗逆性状之一。旱柳(Salix matsudana)作为典型的强耐淹树种, 其对淹水胁迫的适应机制具有重要研究价值。TTF (Trihelix)转录因子是植物中特有的一类转录因子, 在应对各种非生物胁迫(如淹水、盐和低温)中发挥重要作用。本研究聚焦旱柳Trihelix家族基因SmTTF59, 系统解析其在淹水胁迫中的分子功能。通过PCR扩增获得SmTTF59全长序列, 基于同源重组技术构建过表达载体pWM101-SmTTF59及病毒诱导基因沉默(VIGS)载体pYL156-SmTTF59, 并通过遗传转化技术实现SmTTF59在拟南芥(Arabidopsis thaliana)中的过表达及在旱柳中的特异性沉默。SmTTF59过表达拟南芥在缺氧胁迫下的根长、鲜重和抗氧化酶活性显著高于野生型, 提示该基因在植物响应缺氧胁迫过程中可能通过增强抗氧化防御系统来提升胁迫耐受性。进一步通过旱柳的病毒诱导基因沉默(VIGS)实验证实, 沉默植株的淹水耐受性显著降低, 表明其作为正调控因子参与柳树淹水胁迫应答通路。研究结果为解析木本植物耐淹机制及定向选育高抗性柳树品种提供了关键的分子靶点。

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.

Phenotypic differences between pre- and post-flooding and phenotypic comparison between WT and SmTTF59 transgenic Arabidopsis thaliana under normoxic and hypoxic conditions. Elucidated the function of the SmTTF59 gene in response to submergence stress.