Nitrogen forms (nitrate (NO₃⁻) or ammonium (NH₄⁺)) are vital to plant growth and metabolism. In stevia (Stevia rebaudiana), it is important to assess whether nitrogen forms can influence the synthesis of the high-value terpene metabolites-steviol glycosides (SGs), together with the underlying mechanisms. Field and pot experiments were performed where stevia plants were fertilized with either NO₃⁻ or NH₄⁺ nutrition to the same level of nitrogen. Physiological measurements suggested that nitrogen forms had no significant impact on biomass and the total nitrogen content of stevia leaves, but NO₃⁻-enhanced leaf SGs contents. Transcriptomic analysis identified 397 genes that were differentially expressed (DEGs) between NO₃⁻ and NH₄⁺ treatments. Assessment of the DEGs highlighted the responses in secondary metabolism, particularly in terpenoid metabolism, to nitrogen forms. Further examinations of the expression patterns of SGs synthesis-related genes and potential transcription factors suggested that GGPPS and CPS genes, as well as the WRKY and MYB transcription factors, could be driving N form-regulated SG synthesis. We concluded that NO₃⁻, rather than NH₄⁺, can promote leaf SG synthesis via the NO₃⁻-MYB/WRKY-GGPPS/CPS module. Our study suggests that insights into the molecular mechanism of how SG synthesis can be affected by nitrogen forms.

Ilex dabieshanensis is not only an important ornamental plant, but can also be used to produce Kuding tea, owing to its lipid-lowering and anti-inflammatory medicinal properties. The genetic transformation of I. dabieshanensis is currently difficult, which restricts functional gene studies and molecular breeding research on this species. Virus-induced gene silencing (VIGS) is a powerful tool for determining gene functions in plants. The present study reports the first application of VIGS mediated by a tobacco rattle virus (TRV) vector in I. dabieshanensis. We tested the efficiency of the VIGS system to silence Mg-chelatase H subunit (ChlH) gene through agroinfiltration. The agroinfiltrated leaves of I. dabieshanensis exhibited a typical yellow-leaf phenotype of ChlH gene silencing at 21 days post infiltration. Endogenous ChlH expression levels in the leaves of yellow-leaf phenotype plants were all significantly lower than that in the leaves of mock-infected and control plants. Overall, our results indicated that the TRV-based VIGS system can efficiently silence genes in I. dabieshanensis, and this system will contribute to efficient functional genomics research in I. dabieshanensis.