In silico analysis of NRAMP and zip genes in brassicaceae and zinc-responsive gene expression in brassica rapa and brassica juncea

Zinc (Zn) is essential for plants but becomes toxic at high concentrations. While zinc (Zn) deficiency is a widespread problem both worldwide and in Turkey, Zn toxicity arising from agricultural pollution also constitutes a significant risk in certain regions. In this thesis, the response to Zn stress in Brassica juncea (S-9) and Brassica rapa was investigated in a multilayered manner by combining experimental and in silico approaches. The focus was on Zn-associated transporter gene families NRAMP and ZIP. In silico characterization of these genes was performed, and the expression dynamics of NRAMP4 and ZIP4 under varying Zn levels were analyzed by qRT-PCR at organ- and species-specific levels. Seedlings were cultivated in vermiculite for 45 days in three replicates; subsequently, Zn accumulation in roots and leaves was determined by ICP-OES, morphological traits were assessed, and NRAMP4 and ZIP4 expression profiles were analyzed by qRT-PCR. Physiological results showed that plant viability was maintained at low-to-moderate Zn levels, whereas toxicity symptoms appeared at higher concentrations (500–1000 µM). ICP-OES revealed a dose-dependent increase in Zn accumulation, with B. juncea accumulating more in roots and B. rapa more in leaves. qRT-PCR analyses indicated that NRAMP4 expression in B. juncea roots increased at moderate Zn levels but fluctuated at higher doses, while in B. rapa leaves it showed strong dose-dependent induction, reaching ~7-fold at 1000 µM. ZIP4 expression was limited in B. juncea roots but strongly induced in B. rapa leaves, reaching 10–27-fold at 500–1000 µM. In silico analyses yielded phylogenetic trees of NRAMP and ZIP/IRT families with strong bootstrap support; BLAST confirmed reliable orthologs, qRT-PCR primer sets based on Arabidopsis from the Brassicaceae family were designed, and 3D modeling visualized transmembrane topologies. In conclusion, this thesis integrates organ–dose–species interactions under Zn stress, the expression dynamics of the NRAMP4–ZIP4 axis, and the in silico portrait of NRAMP/ZIP gene families, thereby providing a robust framework for Zn homeostasis in Brassica species; and proposes candidate genes for biotechnological and phytoremediation applications targeting Zn deficiency and toxicity.