The role of phytoecdysteroids in spinach (Spinacia oleracea): Physiological responses to below ground herbivory support a plant defense hypothesis

Abstract

Polyhydroxylated steroids with insect molting hormone activity were discovered in plants over thirty years ago. The major endogenous molting hormone of insects is believed to be 20-hydroxyecdysone (20E) and interestingly, it is also the most commonly encountered phytoecdysteroid (PE) in plants. Ecdysteroids control developmental programs in both immature and adult insects however, the role of PEs in plants has not been demonstrated. PEs are hypothesized to function as either plant hormones or plant defenses against phytophagous insects. Many toxic secondary metabolites are concentrated in apical meristems where herbivory would result in the greatest reduction in plant fitness. Similarly, the highest concentrations of 20E in spinach were associated with the stems and vasculature while old leaves and roots displayed low levels. In plants, concentrations of toxic or deterrent metabolites are often rapidly induced following attack. In spinach roots, both mechanical damage and insect herbivory resulted in rapid increases in 20E concentrations. The plant wound signal, jasmonic acid was strongly implicated in signaling this response. Known plant hormones and chemical defenses are regulated differently. Pulse chase studies with [2-¹⁴C] mevalonic acid demonstrated that de novo root 20E biosynthesis occurred during the induction and, once synthesized, 20E was stable for over one month. This result is does not support the plant hormone hypothesis, as plant hormones typically undergo rapid conjugation or catabolism. The induction of root 20E concentrations occurred without similar changes in related membrane phytosterols. Simply, pathway specificity was demonstrated as increased 20E accumulation was not part of an overall increase in steroids. To empirically examine the hypothesis that PEs function as plant defenses against insects, a series of experiments were designed with the fungus gnat Bradysia impatiens. Results indicated that root herbivory by larvae induced 20E levels in roots, larval preference for diets containing induced 20E levels was reduced, larval survivorship on 20E containing diets was lower, and plants with induced root 20E levels were better protected from attack. Together, these results support the plant defense hypothesis at both the physiological and ecological level.