Nitrogen availability modulates carotene biosynthesis, chromoplast biogenesis, and cell wall composition in carrot...

Carrot callus grown on a medium with increased nitrogen have reduced carotenoid accumulation, changed gene expression, high amount of vesicular plastids and altered cell wall composition. Carotenoid biosynthesis is vital for plant development and quality, yet its regulation under varying nutrient conditions remains unclear. To explore the effects of nitrogen (N) availability, we used carrot (Daucus carota L.) model callus cultures in vitro as a controlled system for studying nutrient-regulated metabolic processes. Two mineral media differing in N content and NO₃⁻/NH₄⁺ ratios were used. Comprehensive analyses, HPLC, transmission electron microscopy, immunochemistry, and RNA sequencing, revealed notable cellular and molecular responses to N treatments. The results demonstrated that N supplementation reduced carotenoid content by 50%, particularly β-carotene and α-carotene. The composition of chromoplast types shifted, with vesicular chromoplasts dominating (55%), followed by a globular type (23%), while in the control callus, globular and crystalline types predominated (57% and 33%, respectively). Immunohistochemistry showed increased presence of high-esterified pectins and arabinogalactan proteins in N-treated cells. Transcriptomic analysis identified 1704 differentially expressed genes (DEGs), including only two in the carotenoid biosynthesis pathway: phytoene synthase 2 (PSY2) and zeaxanthin epoxidase (ZEP). PSY2, which encodes the carotenoid rate-limiting enzyme, showed expression levels that corresponded with reduced carotene content. Other DEGs included 15 involved in nitrogen transport, 1 in nitrogen assimilation, 40 in cell wall biosynthesis and modification, and 9 in phenylpropanoid/flavonoid pathways. N-treated callus exhibited altered expression of MADS-box, NLP, bZIP, and ethylene-responsive transcription factors. These findings reveal how nitrogen availability disrupts carotenoid biosynthesis and triggers extensive chromoplast and cell wall remodeling, providing a cellular framework for understanding nutrient-regulated metabolic shifts.