Auxin-Amido Synthetase Gene ThGH3.1 Regulates Auxin Levels to Suppress Root Development in Transgenic Arabidopsis and Tetrastigma hemsleyanum Hairy Roots

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is a prized Chinese medicinal plant renowned for its medicinal and economic importance. In our previous study, a key auxin-related gene ThGH3.1 (encoding amide synthetase) was identified by quantitative transcriptome sequencing. To explore ThGH3.1 function in root development, we generated ThGH3.1-overexpressing and RNA interference (RNAi) transgenic hairy root lines via Agrobacterium rhizogenes (A. rhizogenes)-mediated genetic transformation. The results showed that overexpression of ThGH3.1 significantly inhibited the total length and the lateral root number of hairy roots, accompanied by significantly increased levels of methyl indole-3-acetate (MeIAA) and indole-3-acetyl-aspartate (IAA-Asp). In contrast, ThGH3.1 knockdown displayed an opposite trend. To further confirm the function of ThGH3.1, the overexpression vector was heterologously transformed into wild-type Arabidopsis. After single-copy and homozygous line screening, three overexpressing lines (named G19, G29, and G32) were obtained. The primary root length of transgenic Arabidopsis was significantly shortened, with a significant increase in indole-3-acetonitrile (IAN) levels. Further pot experiments revealed that transgenic Arabidopsis grew more slowly, had significantly smaller leaf areas, and lower plant height. The indole-3-acetic acid (IAA) treatment suggested that ThGH3.1 responded to IAA. Collectively, these findings highlight the crucial roles of ThGH3.1 in regulating root development, which will deepen our understanding of the molecular mechanisms underlying root development in T. hemsleyanum.