The walnut JrVHAG1 gene is involved in cadmium stress response through ABA-signal pathway and MYB transcription regulation

Background: Vacuolar H⁺-ATPase (V-ATPase) is a vital protein complex involved in abiotic stress response in plants. The G subunit of Juglans regia (JrVHAG1) was previously identified as a drought tolerance-related gene involved in the ABA (abscisic acid)-signal pathway. Heavy metal stress is becoming a major detriment for plant growth, development, and production. In order to understand the role of JrVHAG1, the potential function mechanism of JrVHAG1 exposed to CdCl₂ stress was confirmed in this study.

Results: Transcription of JrVHAG1 was induced by ABA and increased to 58.89-fold (roots) and 7.38-fold (leaves) and by CdCl₂ to 2.65- (roots) and 11.42-fold (leaves) relative to control, respectively. Moreover, when treated simultaneously with ABA and CdCl₂ (ABA+CdCl₂), JrVHAG1 was up-regulated to 110.13- as well as 165.42-fold relative to control in the roots and leaves, accordingly. Compared to the wild type (WT) Arabidopsis plants, the transgenic plants with overexpression of JrVHAG1 (G2, G6, and G9) exhibited increased seed germination rate, biomass accumulation, proline content, and activities of superoxide dismutase (SOD) and peroxidase (POD) under ABA, CdCl₂, and ABA+CdCl₂ treatments. In contrast, the reactive oxygen species (ROS) staining, malondialdehyde (MDA) content, hydrogen dioxide (H₂O₂) content, as well as electrolyte leakage (EL) rates of transgenic seedlings were all lower than those of WT exposed to ABA, CdCl₂ and ABA+CdCl₂ stresses. Furthermore, a 1200 bp promoter fragment of JrVHAG1 was isolated by analyzing the genome of J. regia, in which the cis-elements were identified. This JrVHAG1 promoter fragment showed expression activity that was enhanced significantly when subjected to the above treatments. Yeast one-hybrid assay and transient expression analysis demonstrated that JrMYB2 specifically bound to the MYBCORE motif and shared similar expression patterns with JrVHAG1 under ABA, CdCl₂ and ABA+CdCl₂ stress conditions.

Conclusions: Our results suggested that the JrVHAG1 gene functions as a CdCl₂ stress response regulator by participating in ABA-signal pathway and MYB transcription regulation network. JrVHAG1 gene is a useful candidate gene for heavy metal stress tolerance in plant molecular breeding.