Calcium-dependent regulation of anthraquinone biosynthesis by the sweet potato Ib-rolb/C gene in Rubia cordifolia callus cultures

Abstract

This study investigates how the Ib-rolB/C (root locus B/C) gene from sweet potato (Ipomoea batatas), a naturally transgenic plant, influences growth and secondary metabolism in Rubia cordifolia callus cultures. Specifically, its role in regulating anthraquinone (AQ) biosynthesis via calcium-dependent signaling pathways was examined. Transgenic callus lines expressing Ib-rolB/C exhibited robust growth and up to a sixfold increase in AQ production compared to non-transformed controls, particularly when cultured on cytokinin-enriched media. Gene expression analysis revealed strong upregulation of key biosynthetic genes (RcICS, RcOSBS, RcOSBL, and RcIPPi) as well as several calcium-dependent protein kinase (CDPK) isoforms, supporting the hypothesis that Ib-rolB/C modulates calcium-responsive pathways that contribute to the activation of secondary metabolism. Experiments under calcium-deficient and calcium-excess conditions in the culture medium, together with calcium channel inhibitor treatments, confirmed that AQ biosynthesis in transgenic lines is tightly coupled to calcium levels, whereas biomass accumulation was less affected. These findings highlight the regulatory function of Ib-rolB/C in linking calcium signaling with secondary metabolism and offer new insights into the molecular mechanisms of naturally transgenic systems.