Transcriptomic insight into zinc dependency of vindoline accumulation in Catharanthus roseus leaves: relevance and potential role of a CrZIP.

Foliar-applied Zn on Catharanthus roseus enhanced production of vindoline, the main impediment precursor for costly anticancer bisindoles. A leaf-abundant CrZIP was characterized for likely role in modulating vindoline metabolism. The leaf-localized Catharanthus roseus alkaloid, vindoline, is the major impediment precursor in the production of scanty and expensive anticancer bisindoles, vinblastine and vincristine. Earlier studies have hinted toward the role of micronutrients in its accumulation. Here, the effect of various micronutrients on the leaf vindoline content was analyzed. Foliar application of zinc (Zn, 100 ppm) was found to be most effective for enhancing the vindoline content. Comparison of leaf samples of Zn-treated and control plants was performed using paired-end transcriptome sequencing and promising candidates among the differentially expressed genes were identified. A zinc-regulated, iron-regulated transporter-like proteins (ZIP) family member, which was found to be upregulated in Zn-treated samples, was taken up for functional characterization. Complementation of the zrt1/zrt2 yeast mutant (ZHY3) by CrZIP suggested that it encodes a functional Zn transporter. VIGS and transient overexpression of CrZIP in C. roseus showed a direct relationship between its expression and vindoline content. GFP fusion-based subcellular localization indicated chloroplast localization of CrZIP. Previously, vindoline biosynthesis has been reported to be dependent on intact chloroplasts. The chloroplast localization of CrZIP and its direct relationship with vindoline content in this study identifies the possible role of CrZIP, in modulating zinc-induced vindoline accumulation in the plant.