SummaryHerbivore attack leads to resource conflicts between plant defensive strategies. Photoassimilates are required for defensive compounds and carbon storage below ground and may therefore be depleted or enriched in the roots of herbivore-defoliated plants. The potential role of belowground tissues as mediators of induced tolerance-defense trade-offs is unknown.We evaluated signaling and carbohydrate dynamics in the roots of Nicotiana attenuata following Manduca sexta attack. Experimental and natural genetic variability was exploited to link the observed metabolite patterns to plant tolerance and resistance.Leaf-herbivore attack decreased sugar and starch concentrations in the roots and reduced regrowth from the rootstock and flower production in the glasshouse and the field. Leafderived jasmonates were identified as major regulators of this root-mediated resource-based trade-off: lower jasmonate levels were associated with decreased defense, increased carbohydrate levels and improved regrowth from the rootstock. Application and transport inhibition experiments, in combination with silencing of the sucrose non-fermenting (SNF) -related kinase GAL83, indicated that auxins may act as additional signals that regulate regrowth patterns.In conclusion, our study shows that the ability to mobilize defenses has a hidden resourcebased cost below ground that constrains defoliation tolerance. Jasmonate-and auxin-dependent mechanisms may lead to divergent defensive plant strategies against herbivores in nature.

Benzoxazinoids are important defense compounds in grasses. Here, we investigated the biosynthesis and biological roles of the 8-O-methylated benzoxazinoids, DIM 2 BOA-Glc and HDM 2 BOA-Glc. Using quantitative trait locus mapping and heterologous expression, we identified a 2-oxoglutarate-dependent dioxygenase (BX13) that catalyzes the conversion of DIMBOA-Glc into a new benzoxazinoid intermediate (TRIMBOA-Glc) by an uncommon reaction involving a hydroxylation and a likely ortho-rearrangement of a methoxy group. TRIMBOA-Glc is then converted to DIM 2 BOA-Glc by a previously described O-methyltransferase BX7. Furthermore, we identified an O-methyltransferase (BX14) that converts DIM 2 BOA-Glc to HDM 2 BOA-Glc. The role of these enzymes in vivo was demonstrated by characterizing recombinant inbred lines, including Oh43, which has a point mutation in the start codon of Bx13 and lacks both DIM 2 BOA-Glc and HDM 2 BOA-Glc, and Il14H, which has an inactive Bx14 allele and lacks HDM 2 BOA-Glc in leaves. Experiments with near-isogenic maize lines derived from crosses between B73 and Oh43 revealed that the absence of DIM 2 BOA-Glc and HDM 2 BOA-Glc does not alter the constitutive accumulation or deglucosylation of other benzoxazinoids. The growth of various chewing herbivores was not significantly affected by the absence of BX13-dependent metabolites, while aphid performance increased, suggesting that DIM 2 BOA-Glc and/or HDM 2 BOA-Glc provide specific protection against phloem feeding insects.

Highly adapted herbivores can phenocopy two-component systems by stabilizing, sequestering and reactivating plant toxins. However, whether these traits protect herbivores against their enemies is poorly understood. We demonstrate that the western corn rootworm Diabrotica virgifera virgifera, the most damaging maize pest on the planet, specifically accumulates the root-derived benzoxazinoid glucosides HDMBOA-Glc and MBOA-Glc. MBOA-Glc is produced by D. virgifera through stabilization of the benzoxazinoid breakdown product MBOA by N-glycosylation. The larvae can hydrolyze HDMBOA-Glc, but not MBOA-Glc, to produce toxic MBOA upon predator attack. Accumulation of benzoxazinoids renders D. virgifera highly resistant to nematodes which inject and feed on entomopathogenic symbiotic bacteria. While HDMBOA-Glc and MBOA reduce the growth and infectivity of both the nematodes and the bacteria, MBOA-Glc repels infective juvenile nematodes. Our results illustrate how herbivores combine stabilized and reactivated plant toxins to defend themselves against a deadly symbiosis between the third and the fourth trophic level enemies.

SummaryJasmonates regulate plant secondary metabolism and herbivore resistance. How they influence primary metabolites and how this may affect herbivore growth and performance are not well understood.We profiled sugars and starch of jasmonate biosynthesis-deficient and jasmonate-insensitive Nicotiana attenuata plants and manipulated leaf carbohydrates through genetic engineering and in vitro complementation to assess how jasmonate-dependent sugar accumulation affects the growth of Manduca sexta caterpillars.We found that jasmonates reduce the constitutive and herbivore-induced concentration of glucose and fructose in the leaves across different developmental stages. Diurnal, jasmonatedependent inhibition of invertase activity was identified as a likely mechanism for this phenomenon. Contrary to our expectation, both in planta and in vitro approaches showed that the lower sugar concentrations led to increased M. sexta growth. As a consequence, jasmonate-dependent depletion of sugars rendered N. attenuata plants more susceptible to M. sexta attack.In conclusion, jasmonates are important regulators of leaf carbohydrate accumulation and this determines herbivore growth. Jasmonate-dependent resistance is reduced rather than enhanced through the suppression of glucose and fructose concentrations, which may contribute to the evolution of divergent resistance strategies of plants in nature.

Maize and wheat independently evolved the capacity to control plant defenses through O-methylation of regulatory benzoxazinoids.