This study discusses the genetic mutations that have a significant association with economically important traits that would benefit tea breeders. The purpose of this study was to analyze the leaf quality and SNPs in quality-related genes in the tea plant collection of 20 mutant genotypes growing without nitrogen fertilizers. Leaf N-content, catechins, L-theanine, and caffeine contents were analyzed in dry leaves via HPLC. Additionally, the photochemical yield, electron transport efficiency, and non-photochemical quenching were analyzed using PAM-fluorimetry. The next generation pooled amplicon–sequencing approach was used for SNPs-calling in 30 key genes related to N metabolism and leaf quality. The leaf N content varied significantly among genotypes (p ≤ 0.05) from 2.3 to 3.7% of dry mass. The caffeine content varied from 0.7 to 11.7 mg g⁻¹, and the L-theanine content varied from 0.2 to 5.8 mg g⁻¹ dry leaf mass. Significant positive correlations were detected between the nitrogen content and biochemical parameters such as theanine, caffeine, and most of the catechins. However, significant negative correlations were observed between the photosynthetic parameters (Y, ETR, Fv/Fm) and several biochemical compounds, including rutin, Quercetin-3-O-glucoside, Kaempferol-3-O-rutinoside, Kaempferol-3-O-glucoside, Theaflavin-3′-gallate, gallic acid. From our SNP-analysis, three SNPs in WRKY57 were detected in all genotypes with a low N content. Moreover, 29 SNPs with a high or moderate effect were specific for #316 (high N-content, high quality) or #507 (low N-content, low quality). The use of a linear regression model revealed 16 significant associations; theaflavin, L-theanine, and ECG were associated with several SNPs of the following genes: ANSa, DFRa, GDH2, 4CL, AlaAT1, MYB4, LHT1, F3′5′Hb, UFGTa. Among them, seven SNPs of moderate effect led to changes in the amino acid contents in the final proteins of the following genes: ANSa, GDH2, 4Cl, F3′5′Hb, UFGTa. These results will be useful for further evaluations of the important SNPs and will help to provide a better understanding of the mechanisms of nitrogen uptake efficiency in tree crops.

For the first time, we used an in vitro vs. in vivo experimental design to reveal core pathways under nitrogen deficiency (ND) in an evergreen tree crop. These pathways were related to lignin biosynthesis, cell redox homeostasis, the defense response to fungus, the response to Karrikin, amino acid transmembrane transport, the extracellular region, the cellular protein catabolic process, and aspartic-type endopeptidase activity. In addition, the mitogen-activated protein kinase pathway and ATP synthase (ATP)-binding cassette transporters were significantly upregulated under nitrogen deficiency in vitro and in vivo. Most of the MAPK downstream genes were related to calcium signaling (818 genes) rather than hormone signaling (157 genes). Moreover, the hormone signaling pathway predominantly contained auxin- and abscisic acid-related genes, indicating the crucial role of these hormones in ND response. Overall, 45 transcription factors were upregulated in both experiments, 5 WRKYs, 3 NACs, 2 MYBs, 2 ERFs, HD-Zip, RLP12, bHLH25, RADIALIS-like, and others, suggesting their ND regulation is independent from the presence of a root system. Gene network reconstruction displayed that these transcription factors participate in response to fungus/chitin, suggesting that nitrogen response and pathogen response have common regulation. The upregulation of lignin biosynthesis genes, cytochrome genes, and strigalactone response genes was much more pronounced under in vitro ND as compared to in vivo ND. Several cell wall-related genes were closely associated with cytochromes, indicating their important role in flavanols biosynthesis in tea plant. These results clarify the signaling mechanisms and regulation of the response to nitrogen deficiency in evergreen tree crops.

Nitrogen (N) availability strongly influences tea growth and the accumulation of quality-related metabolites, but long-term responses to withdrawal of external N can vary among cultivars. In this study, we characterized phenotypic, spectral, biochemical, and transcriptomic responses of four Camellia sinensis accessions—‘Kolkhida’, ‘Karatum’, and γ-irradiation-derived mutant forms #582 and #619—during two, four, or six months of complete withdrawal of exogenous NH₄NO₃ in greenhouse sand culture. Phenotypic, spectral, and biochemical measurements included all four accessions at all three time points; RNA-seq data were available for all four accessions were represented at four and six months, whereas two-month point were available only for ‘Kolkhida’ and ‘Karatum’. Nitrogen withdrawal decreased L-theanine and caffeine and generally increased simple and gallated catechins, consistent with a change in carbon/nitrogen balance towards phenolic metabolism. The four cultivars exhibited markedly distinct responses. ‘Karatum’ showed the optimum resilience, with minimal phenotypic alteration and a neutral transcriptional response, while #582 demonstrated a stronger stress-associated phenotype and metabolic response. Within the available RNA-seq comparisons, DEG counts were lower at four months than at six months, while the two-month datasets for ‘Kolkhida’ and ‘Karatum’ contained the largest DEG sets for those two cultivars, suggesting a phased acclimation process. CsELIP1, CsSRG1, CsHHO2/4, CsNRT2.4, and CsTAT2 were identified as potential candidate genes associated with nitrogen limitation, flavonoid regulation, and amino acid metabolism. Our findings show that N-deficiency reactions in tea are highly cultivar- and time-dependent, making single-time-point evaluations insufficient. We propose ‘Karatum’ as a promising candidate for further evaluation in low-nitrogen dose–response and field trials based on our combined biochemical and transcriptional findings.

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.