Benzoxazinoids (BXs) are secondary metabolites present in many Poaceae including the major crops maize, wheat, and rye. In contrast to other potentially toxic secondary metabolites, BXs have not been targets of counter selection during breeding and the effect of BXs on insects, microbes, and neighbouring plants has been recognised. A broad knowledge about the mode of action and metabolisation in target organisms including herbivorous insects, aphids, and plants has been gathered in the last decades. BX biosynthesis has been elucidated on a molecular level in crop cereals. Recent advances, mainly made by investigations in maize, uncovered a significant diversity in the composition of BXs within one species. The pattern can be specific for single plant lines and dynamic changes triggered by biotic and abiotic stresses were observed. Single BXs might be toxic, repelling, attractive, and even growth-promoting for insects, depending on the particular species. BXs delivered into the soil influence plant and microbial communities. Furthermore, BXs can possibly be used as signalling molecules within the plant. In this review we intend to give an overview of the current data on the biosynthesis, structure, and function of BXs, beyond their characterisation as mere phytotoxins.

In the plant kingdom beta-glucosidases (BGLUs) of the glycosidase hydrolase family 1 have essential function in primary metabolism and are particularly employed in secondary metabolism. They are essential for activation in two-component defence systems based on stabilisation of reactive compounds by glycosylation. Based on de novo assembly we isolated and functionally characterised BGLUs expressed in leaves of Lamium galeobdolon (LgGLUs). LgGLU1 could be assigned to hydrolysis of the benzoxazinoid GDIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one glucoside). Within the Lamiaceae L. galeobdolon is distinguished by the presence GDIBOA in addition to the more common iridoid harpagide. Although LgGLU1 proved to be promiscuous with respect to accepted substrates, harpagide hydrolysis was not detected. Benzoxazinoids are characteristic defence compounds of the Poales but are also found in some unrelated dicots. The benzoxazinoid specific BGLUs have recently been identified for the grasses maize, wheat, rye and the Ranunculaceae Consolida orientalis. All enzymes share a general substrate ambiguity but differ in detailed substrate pattern. The isolation of the second dicot GDIBOA glucosidase LgGLU1 allowed it to analyse the phylogenetic relation of the distinct BGLUs also within dicots. The data revealed long periods of independent sequence evolution before speciation.

Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ¹³C) during photosynthesis in the C₄ crop maize. Changes in Δ¹³C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ¹³C through a single gene with potential applications in maize breeding.

Plant specialised metabolites constitute a layer of chemical defence. Classes of the defence compounds are often restricted to a certain taxon of plants, e.g. benzoxazinoids (BX) are characteristically detected in grasses. BXs confer wide-range defence by controlling herbivores and microbial pathogens and are allelopathic compounds. In the crops maize, wheat and rye high concentrations of BXs are synthesised at an early developmental stage. By transfer of six Bx-genes (Bx1 to Bx5 and Bx8) it was possible to establish the biosynthesis of 2,4-dihydroxy-1,4-benzoxazin-3-one glucoside (GDIBOA) in a concentration of up to 143 nmol/g dry weight in Arabidopsis thaliana. Our results indicate that inefficient channeling of substrates along the pathway and metabolisation of intermediates in host plants might be a general drawback for transgenic establishment of specialised metabolite biosynthesis pathways. As a consequence, BX levels required for defence are not obtained in Arabidopsis. We could show that indolin-2-one (ION), the first specific intermediate, is phytotoxic and is metabolised by hydroxylation and glycosylation by a wide spectrum of plants. In Arabidopsis, metabolic stress due to the enrichment of ION leads to elevated levels of salicylic acid (SA) and in addition to its intrinsic phytotoxicity, ION affects plant morphology indirectly via SA. We could show that Bx3 has a crucial role in the evolution of the pathway, first based on its impact on flux into the pathway and, second by C3-hydroxylation of the phytotoxic ION. Thereby BX3 interferes with a supposedly generic detoxification system towards the non-specific intermediate.

The sustainability of maize cultivation would benefit tremendously from early sowing, but is hampered by low temperatures during early development in temperate climates. We show that allelic variation within the gene encoding subunit M of the NADH-dehydrogenase-like (NDH) complex (ndhm1) in a European maize landrace affects several quantitative traits that are relevant during early development in cold climates through NDH-mediated cyclic electron transport around photosystem I, a process crucial for photosynthesis and photoprotection. Beginning with a genome-wide association study for maximum potential quantum yield of photosystem II in dark-adapted leaves (Fv/Fm), we capitalized on the large phenotypic effects of a hAT transposon insertion in ndhm1 on multiple quantitative traits (early plant height [EPH], Fv/Fm, chlorophyll content, and cold tolerance) caused by the reduced protein levels of NDHM and associated NDH components. Analysis of the ndhm1 native allelic series revealed a rare allele of ndhm1 that is associated with small albeit significant improvements of Fv/Fm, photosystem II efficiency in light-adapted leaves (ΦPSII), and EPH compared with common alleles. Our work showcases the extraction of favorable alleles from locally adapted landraces, offering an efficient strategy for broadening the genetic variation of elite germplasm by breeding or genome editing.

Both deficiency and toxicity of the micronutrient boron lead to severe reductions in crop yield. Despite this agricultural importance, the molecular basis underlying boron homeostasis in plants remains unclear. To identify molecular players involved in boron homeostasis in maize (Zea mays L.), we measured boron levels in the Goodman-Buckler association panel and performed genome-wide association studies. These analyses identified a benzoxazinless (bx) gene, bx3, involved in the biosynthesis of benzoxazinoids, such as DIMBOA, which are major defense compounds in maize. Genes involved in DIMBOA biosynthesis are all located in close proximity in the genome, and benzoxazinoid biosynthesis mutants, including bx3, are all DIMBOA deficient. We determined that leaves of the bx3 mutant have a greater boron concentration than those of B73 control plants, which corresponded with enhanced leaf tip necrosis, a phenotype associated with boron toxicity. By contrast, other DIMBOA-deficient maize mutants did not show altered boron levels or the leaf tip necrosis phenotype, suggesting that boron is not associated with DIMBOA. Instead, our analyses suggest that the accumulation of boron is linked to the benzoxazinoid intermediates indolin-2-one (ION) and 3-hydroxy-ION. Therefore, our results connect boron homeostasis to the benzoxazinoid plant defense pathway through bx3 and specific intermediates, rendering the benzoxazinoid biosynthesis pathway a potential target for crop improvement under inadequate boron conditions.