Temperature and Pollinators Drive Evolutionary and Plastic Variation in Leaf and Petal Epicuticular Waxes

Plants rely on epicuticular waxes to withstand stress and mediate interactions with other organisms, yet floral waxes remain largely unexplored despite their potential key role in reproduction. We examined how temperature and pollinators shaped leaf and petal waxes in Brassica rapa after six generations of experimental evolution under hot and ambient conditions with bumblebees (Bombus terrestris), Pieris rapae butterflies, acting as "pollinating herbivores" and also involving larval herbivory, or hand-pollination as a control. Leaves contained more wax but shorter aliphatic chains than petals. Heat reduced leaf waxes yet elongated chains, while petals lost wax and shortened chains, revealing opposite responses across organs. Insect-pollinated plants, especially those pollinated by bumblebees, showed reduced heat-driven wax loss in petals, mitigated reductions in chain length or even increases, and altered potential signalling compounds such as methyl-alkanes and α/β-amyrins, while in leaves exhibiting reduced wax amounts and dampened increases in chain-length. P. rapae exposure increased or maintained high levels of compounds linked to herbivore defence (long alcohols, indole-3-acetonitrile), suggesting possible herbivore-specific responses. These results uncover a leaf-petal wax trade-off and identify pollinators as key drivers of wax chemistry under heat. We show that leaf and petal waxes are both plastic and evolvable, emphasizing floral waxes as integral components at the crossroads of stress tolerance, herbivory, and pollination.