Transcriptomic Characterization of Nitrate-Enhanced Stevioside Glycoside Synthesis in Stevia (Stevia rebaudiana) Bertoni

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.

Effective Glycosylation of Cucurbitacin Mediated by UDP-Glycosyltransferase UGT74AC1 and Molecular Dynamics Exploration of Its Substrate Binding Conformations

Cucurbitacins, a group of diverse tetracyclic triterpenes, display a variety of biological effects. Glycosylation mediated by glycosyltransferases (UGTs) plays a vital role in structural and functional diversity of natural products and influences their biological activities. In this study, GT-SM, a mutant of UGT74AC1 from Siraitia grosvenorii, was chosen as a potential catalyst in glycosylation of cucurbitacins, and its optimal pH, temperature, and divalent metal ions were detected. This enzyme showed high activity (kcat/Km, 120 s−1 µM−1) toward cucurbitacin F 25-O-acetate (CA-F25) and only produced CA-F25 2-O-β-d-glucose which was isolated and confirmed by 1D and 2D nuclear magnetic resonance. A pathway for uridine diphosphate glucose (UDP-Glc) regeneration and cucurbitacin glycoside synthesis was constructed by combing GT-SM and sucrose synthase to cut down the costly UDP-Glc. The molar conversion of CA-F25 was 80.4% in cascade reaction. Molecular docking and dynamics simulations showed that CA-F25 was stabilized by hydrophobic interactions, and the C2-OH of CA-F25 showed more favorable catalytic conformation than that of C3-OH, explaining the high regioselectivity toward the C2-OH rather than the ortho-C3-OH of CA-F25. This work proved the important potential application of UGT74AC1 in cucurbitacins and provided an understanding of glycosylation of cucurbitacins.

A flavin-dependent monooxygenase catalyzes the initial step in cyanogenic glycoside synthesis in ferns

Cyanogenic glycosides form part of a binary plant defense system that, upon catabolism, detonates a toxic hydrogen cyanide bomb. In seed plants, the initial step of cyanogenic glycoside biosynthesis—the conversion of an amino acid to the corresponding aldoxime—is catalyzed by a cytochrome P450 from the CYP79 family. An evolutionary conundrum arises, as no CYP79s have been identified in ferns, despite cyanogenic glycoside occurrence in several fern species. Here, we report that a flavin-dependent monooxygenase (fern oxime synthase; FOS1), catalyzes the first step of cyanogenic glycoside biosynthesis in two fern species (Phlebodium aureum and Pteridium aquilinum), demonstrating convergent evolution of biosynthesis across the plant kingdom. The FOS1 sequence from the two species is near identical (98%), despite diversifying 140 MYA. Recombinant FOS1 was isolated as a catalytic active dimer, and in planta, catalyzes formation of an N-hydroxylated primary amino acid; a class of metabolite not previously observed in plants.