Metabolic engineering reveals LUP5 as a determinant of saponin composition and insect feeding preference in Barbarea...

Plant-insect coevolution has been a major driver of specialized metabolite diversification, yet the genetic basis of natural variation in defensive chemistry remains poorly understood. The wild crucifer winter cress (Barbarea vulgaris) comprises 2 ecotypes, an insect-resistant G-type and a susceptible P-type, characterized by distinct triterpenoid saponin profiles. To investigate the causal relationship between saponin composition and insect feeding preference, we established a stable transformation system for B. vulgaris. P-type B. vulgaris accumulates lupeol-derived saponins, and expression of the G-type β-amyrin synthase gene LUP5 in the susceptible P-type conferred up to a 95% reduction in diamondback moth (Plutella xylostella) feeding, accompanied by increased accumulation of 3 hederagenin-derived monodesmosidic saponins. Comparison of LUP5 expression driven by its native promoter and by the constitutive 35S promoter revealed that the native promoter is activated in young leaves, but not in young developing shoots, and leads to increased hederagenin accumulation in leaves. This expression pattern reflects the coordinated expression of downstream pathway genes and prevents expression in developing shoots. Our results provide direct in planta evidence that LUP5 is a key determinant of natural variation in insect feeding preference in B. vulgaris, underscoring the pivotal role of the saponin backbone in herbivore deterrence. By linking promoter activity to metabolite structural diversity, this work provides mechanistic and conceptual insights into how plants coordinate specialized metabolism and defense.