Transcriptomic Analysis of the Regulatory Mechanism of Tea Polyphenol Biosynthesis in Chionanthus retusus and Functional Characterization of CrHSP70-14 in Terms of Its Effect on Tea Polyphenols

<b>Background:</b><i>Chionanthus retusus Lindl. et Paxt.</i>, a deciduous tree of the genus Chionanthus (Oleaceae), represents a significant native species and a widely cultivated ornamental. Its tender leaves can be processed into tea, traditionally consumed in southern China under the common name "Nuomi Cha". <b>Methods:</b> Our team quantified the tea polyphenol content across 150 individual trees of <i>C. retusus</i> and selected three low-polyphenol (ZB_D_14, AQ_2, AQ_1) and three high-polyphenol (SX_3, SXG_D_8, TS_D_13) lines for transcriptome sequencing of their young leaves. The resulting data were analyzed to screen for candidate genes. Subsequently, transgenic plants were constructed, and their tea polyphenol content was determined. <b>Results:</b> A significant difference in tea polyphenol content was confirmed between the high- and low-polyphenol lines. Weighted Gene Co-expression Network Analysis (WGCNA) pinpointed a key module strongly associated with tea polyphenol synthesis, encompassing 432 DEGs, which were predominantly enriched in pathways like phenylpropanoid biosynthesis. A comparative transcriptomic analysis further yielded 84 DEGs (40 up- and 44 down-regulated). Enrichment analysis showed these were primarily involved in flavonoid and phenylpropanoid biosynthesis pathways. Expression profiling of genes in the tea polyphenol biosynthetic pathway indicated that several key genes (e.g., <i>4CL</i>, <i>CHS</i>, <i>DFR</i>) were highly expressed in the high-content lines. A gene interaction network related to this synthesis identified 20 hub genes (e.g., <i>CrHSP70-14</i>, <i>CrMYB44</i>, <i>CrbHLH92</i>). Functional validation of four hubs (<i>CrMYB44</i>, <i>CrHSP70-14</i>, <i>CrCDC6B</i>, <i>CrRAE1</i>) via tobacco transient transformation assays demonstrated that all four significantly elevated tea polyphenol levels, with <i>CrHSP70-14</i> overexpression yielding the highest content. Furthermore, stable <i>CrHSP70-14</i> overexpression transgenic tobacco lines were generated, exhibiting significantly higher leaf tea polyphenol content versus controls. <b>Conclusions:</b> This study identifies multiple regulatory genes involved in <i>C. retusus</i> tea polyphenol biosynthesis, provides initial mechanistic insights, and establishes a molecular foundation for breeding specialized tea cultivars of this species.