In Vitro vs. In Vivo Transcriptomic Approach Revealed Core Pathways of Nitrogen Deficiency Response in Tea Plant (Camellia sinensis (L.) Kuntze)

For the first time, we used an <i>in vitro</i> vs. <i>in vivo</i> 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 <i>WRKYs</i>, 3 <i>NACs</i>, 2 <i>MYBs</i>, 2 <i>ERFs</i>, <i>HD-Zip</i>, <i>RLP12</i>, <i>bHLH25</i>, <i>RADIALIS</i>-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 <i>in vitro</i> ND as compared to <i>in vivo</i> 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.