Insect-Induced Synthesis of Phytoecdysteroids in Spinach, Spinacia oleracea

Spinach (Spinacia oleracea) foliage is known to synthesize and accumulate insect molting hormones, predominantly in the form of 20-hydroxyecdysone (20E). We previously demonstrated that root 20E accumulation is increased following root damage. We designed two further experiments to address root responses to both mechanical and insect damage. In plants grown hydroponically, removal of 35% or less of the root mass did not result in changes in root 20E levels. However, removal of 70% of the root mass stimulated 6.0- and 1.5-fold increases in the root and shoot 20E concentrations, respectively. The effects of insect damage on soil-grown plants were investigated by infesting plant roots with black vine weevil (BVW: Otiorhynchus sulcatus) larvae and allowing them to feed for seven days. Decreases in root mass occurred in young plants; however, no changes were detected in mature plants. In all cases, root herbivory resulted in at least a 3.0-fold increase in root 20E concentrations. Our previous experiments implicated jasmonic acid and the analog methyl jasmonate (MJ) in signaling the damage-induced accumulation of root 20E levels. We investigated the activity of other phytohormones and growth regulators (GRs) on the 20E accumulation patterns of young plants as a means of examining the significance of jasmonates in the induction response. Hydroponic additions of MJ (0.5 M) and the synthetic auxin, 1-naphthaleneacetic acid (NAA; 0.5 M), resulted in significant increases in root 20E levels. At the concentrations tested, indole-3-acetic acid (IAA), gibberellic acid (GA₃), abscisic acid (ABA), and trans-zeatin (Z) had no effects on root 20E concentrations. However, both NAA (0.5–5.0 M) and Z (5.0 M) treatments caused increases in the root/shoot dry mass ratios, indicating shifts in resource allocation to the roots. Treatments involving ABA (5.0 M) and Z (0.5–5.0 M) caused significant increases in shoot 20E concentrations. No other hormone treatments altered shoot accumulation patterns. The mechanisms underlying the root 20E induction phenomena were investigated through the incorporation of 2-¹⁴C]mevalonic acid (¹⁴C]MVA). Within one day, excised roots readily incorporated radioactivity into 20E from ¹⁴C]MVA. In intact plants, ¹⁴C]MVA absorbed by the roots was rapidly incorporated into root 20E pools following damage and MJ treatments. This implies that the wound-induced root 20E accumulation is the result of increased de novo 20E synthesis in the root.