Transcriptomic and Biochemical Responses of Camellia sinensis (L.) Kuntze Callus Cultures to Nitrogen Deficiency In Vitro

Nitrogen deficiency is one of the key factors limiting the growth and raw-material quality of tea plants; however, cell-autonomous mechanisms underlying the response of Camellia sinensis (L.) Kuntze to reduced nitrogen availability remain insufficiently studied. This study characterized biochemical and transcriptomic changes in callus cultures of three tea cultivars (‘Kolkhida’, ‘Karatum’, and cultivar #582) after two months of cultivation on Murashige–Skoog medium lacking nitrogen-containing components. Nitrogen withdrawal caused visible stress symptoms, reduced total nitrogen in ‘Kolkhida’ and ‘Karatum’ (total nitrogen was not determined in cultivar #582), and altered the accumulation of L-theanine, caffeine, and catechins in a cultivar-dependent manner. ‘Kolkhida’ showed decreases in simple catechins, L-theanine, and caffeine; ‘Karatum’ showed increases in simple and gallated catechins together with a decrease in L-theanine; and cultivar #582 showed smaller changes in most measured biochemical traits. RNA-seq identified 1898 differentially expressed genes (DEGs) in ‘Kolkhida’, 4551 in ‘Karatum’, and 9869 in cultivar #582. The enriched Gene Ontology term “response to karrikin” (GO:0080167) was common to all three cultivars, whereas cultivar-specific profiles differed substantially. In the flavonoid biosynthesis pathway, key phenylpropanoid genes were predominantly downregulated in cultivar #582 but induced in ‘Kolkhida’ and ‘Karatum’. RT-qPCR confirmed the direction of change for selected genes. Under the tested in vitro conditions, ‘Kolkhida’ showed the strongest biochemical deterioration, whereas cultivar #582 combined the greatest biochemical stability with the most extensive transcriptomic remodeling. Because total nitrogen was not measured in cultivar #582, its apparent tolerance remains provisional. Tea callus cultures therefore provide a controlled discovery system for cell-autonomous candidate responses, but cultivar rankings and candidate markers require validation in independent callus lines and whole plants.