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.

Insect herbivores depend on their host plants to acquire macro- and micronutrients. Here we asked how a specialist herbivore and damaging maize pest, the western corn rootworm, finds and accesses plant-derived micronutrients. We show that the root-feeding larvae use complexes between iron and benzoxazinoid secondary metabolites to identify maize as a host, to forage within the maize root system, and to increase their growth. Maize plants use these same benzoxazinoids for protection against generalist herbivores and, as shown here, for iron uptake. We identify an iron transporter that allows the corn rootworm to benefit from complexes between iron and benzoxazinoids. Thus, foraging for an essential plant-derived complex between a micronutrient and a secondary metabolite shapes the interaction between maize and a specialist herbivore.

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.

BackgroundPhytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Among them, jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding). These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. Understanding of underlying mechanisms is of principle interest for scientists working in various areas of plant biology. However, highly sensitive, precise and high-throughput methods for quantification of these phytohormones in small samples of plant tissues are still missing.ResultsHere we present an LC-MS/MS method for fast and highly sensitive determination of jasmonates, abscisic and salicylic acids. A single-step sample preparation procedure based on mixed-mode solid phase extraction was efficiently combined with essential improvements in mobile phase composition yielding higher efficiency of chromatographic separation and MS-sensitivity. This strategy resulted in dramatic increase in overall sensitivity, allowing successful determination of phytohormones in small (less than 50 mg of fresh weight) tissue samples. The method was completely validated in terms of analyte recovery, sensitivity, linearity and precision. Additionally, it was cross-validated with a well-established GC-MS-based procedure and its applicability to a variety of plant species and organs was verified.ConclusionThe method can be applied for the analyses of target phytohormones in small tissue samples obtained from any plant species and/or plant part relying on any commercially available (even less sensitive) tandem mass spectrometry instrumentation.