Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.

Although phenylpropanoid-polyamine conjugates (PPCs) occur ubiquitously in plants, their biological roles remain largely unexplored. The two major PPCs of Nicotiana attenuata plants, caffeoylputrescine (CP) and dicaffeoylspermidine, increase dramatically in local and systemic tissues after herbivore attack and simulations thereof. We identified NaMYB8, a homolog of NtMYBJS1, which in BY-2 cells regulates PPC biosynthesis, and silenced its expression by RNA interference in N. attenuata (ir-MYB8), to understand the ecological role(s) of PPCs. The regulatory role of NaMYB8 in PPC biosynthesis was validated by a microarray analysis, which revealed that transcripts of several key biosynthetic genes in shikimate and polyamine metabolism accumulated in a NaMYB8-dependent manner. Wild-type N. attenuata plants typically contain high levels of PPCs in their reproductive tissues; however, NaMYB8-silenced plants that completely lacked CP and dicaffeoylspermidine showed no changes in reproductive parameters of the plants. In contrast, a defensive role for PPCs was clear; both specialist (Manduca sexta) and generalist (Spodoptera littoralis) caterpillars feeding on systemically preinduced young stem leaves performed significantly better on ir-MYB8 plants lacking PPCs compared with wild-type plants expressing high levels of PPCs. Moreover, the growth of M. sexta caterpillars was significantly reduced when neonates were fed ir-MYB8 leaves sprayed with synthetic CP, corroborating the role of PPCs as direct plant defense. The spatiotemporal accumulation and function of PPCs in N. attenuata are consistent with the predictions of the optimal defense theory: plants preferentially protect their most fitnessenhancing and vulnerable parts, young tissues and reproductive organs, to maximize their fitness.

A large number of plants accumulate N-acylated polyamines (phenolamides [PAs]) in response to biotic and/or abiotic stress conditions. In the native tobacco (Nicotiana attenuata), the accumulation of two major PAs, caffeoylputrescine and dicaffeoylspermidine (DCS), after herbivore attack is known to be controlled by a key transcription factor, MYB8. Using a broadly targeted metabolomics approach, we show that a much larger spectrum of PAs composed of hydroxycinnamic acids and two polyamines, putrescine and spermidine, is regulated by this transcription factor. We cloned several novel MYB8-regulated genes, annotated as putative acyltransferases, and analyzed their function. One of the novel acyltransferases (AT1) is shown to encode a hydroxycinnamoyl-coenzyme A:putrescine acyltransferase responsible for caffeoylputrescine biosynthesis in tobacco. Another gene (acyltransferase DH29), specific for spermidine conjugation, mediates the initial acylation step in DCS formation. Although this enzyme was not able to perform the second acylation toward DCS biosynthesis, another acyltransferase gene, CV86, proposed to act on monoacylated spermidines, was isolated and partially characterized. The activation of MYB8 in response to herbivore attack and associated signals required the activity of LIPOXYGENASE3, a gene involved in jasmonic acid (JA) biosynthesis in N. attenuata. These new results allow us to reconstruct a complete branch in JA signaling that defends N. attenuata plants against herbivores: JA via MYB8's transcriptional control of AT1 and DH29 genes controls the entire branch of PA biosynthesis, which allows N. attenuata to mount a chemically diverse (and likely efficient) defense shield against herbivores.