Agropine-type rolA modulates ROS homeostasis in an auxin-dependent manner in rolA-expressing cell cultures of Rubia cordifolia L

Abstract

Long-term cultured calli may experience a biosynthetic shift due to the IAA-dependent expression of the rolA gene, which also affects ROS metabolism. The "hairy root" syndrome is caused by the root-inducing Ri-plasmid of Rhizobium rhizogenes, also known as Agrobacterium rhizogenes. The Ri-plasmid contains genes known as rol genes or root oncogenic loci, which promote root development. The important implications of the rolA gene from the T-DNA include reduced plant size, resistance to infections, and the activation of specialised metabolism. Nevertheless, rolA does not belong to the plast gene group because its function is still uncertain. Recent investigations have shown two important effects of the rolA gene. First, the production of secondary metabolites has changed in long-term cultivated rolA-transgenic calli of Rubia cordifolia L. Second, the expression of both the rolA and rolB genes has a strong auxin-dependent antagonistic effect on reactive oxygen species (ROS) homeostasis. In this work, we attempted to elucidate two rolA gene phenomena: what caused the secondary metabolism of long-term cultured calli to change? How does the individual expression of the rolA gene affect ROS homeostasis? We analysed SNPs in the 5' untranslated region and coding region of the rolA gene. These mutations do not affect the known essential amino acids of the RolA proteins. Notably, in the promoter of the rolA gene, an ACTTTA motif for auxin-mediated transcription factors was identified. Using two separate cell cultures, we demonstrated the strong auxin dependence of rolA gene expression. The expression of genes involved in ROS metabolism decreased in response to an auxin-mediated increase in rolA gene expression. Two assumptions can be made. The long-term cultivation of calli may cause changes in the hormonal state of the culture over time, which may modulate the action of the RolA protein. Moreover, auxin-dependent expression of the rolA gene led to a decrease in ROS metabolism. It can be assumed that the antagonistic interaction between rolA and rolB prevents strong rolB-induced auxin sensitivity and oxidative bursts to balance the cell state.

Keywords: Rhizobium rhizogenes; RolA oncogene; Anthraquinone; Auxins; Callus culture; Hairy root”; Reactive oxygen species.