Investigation of growth regulation by maize benzoxazinoid breakdown products
Authors: Jander Georg , Chamovitz Daniel 
Published: January 2022

Introduction Previous research had suggested that benzoxazinoids, a class of defensive metabolites found in maize, wheat, rye, and wild barley, are not only direct insect deterrents, but also influence other areas of plant metabolism. In particular, the benzoxazinoid 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxa- zin-3(4H)- one (DIMBOA) was implicated in: (i) altering plant growth by interfering with auxin signaling, and (ii) leading to the induction of gene expression changes and secondary plant defense responses. The overall goal of this proposal was to identify mechanisms by which benzoxazinoids influence other aspects of plant growth and defense. Specifically, the following hypotheses were proposed to be tested as part of an approved BARD proposal: Benzoxazinoid breakdown products directly interfere with auxin perception Global changes in maize and barley gene expression are induced by benzoxazinoid activation. There is natural variation in the maize photomorphogenic response to benzoxazinoids. Although the initial proposal included experiments with both maize and barley, there were some technical difficulties with the proposed transgenic barley experiments and most of the experimental results were generated with maize. Summary of major findings Previous research by other labs, involving both maize and other plant species, had suggested that DIMBOA alters plant growth by interfering with auxin signaling. However, experiments conducted in both the Chamovitz and the Jander labs using Arabidopsis and maize, respectively, were unable to confirm previously published reports of exogenously added DIMBOA effects on auxin signaling. Nevertheless, analysis of bx1 and bx2 maize mutant lines, which have almost no detectable benzoxazinoids, showed altered responses to blue light signaling. Transcriptomic analysis of maize mutant lines, variation in inbred lines, and responses to exogenously added DIMBOA showed alteration in the transcription of a blue light receptor, which is required for plant growth responses. This finding provides a novel mechanistic explanation of the trade-off between growth and defense that is often observed in plants. Experiments by the Jander lab and others had demonstrated that DIMBOA not only has direct toxicity against insect pests and microbial pathogens, but also induces the formation of callose in both maize and wheat. In the current project, non-targeted metabolomic assays of wildtype maize and mutants with defects in benzoxazinoid biosynthesis were used to identify unrelated metabolites that are regulated in a benzoxazinoid-dependent manner. Further investigation identified a subset of these DIMBOA-responsive compounds as catechol, as well as its glycosylated and acetylated derivatives. Analysis of co-expression data identified indole-3-glycerol phosphate synthase (IGPS) as a possible regulator of benzoxazinoid biosynthesis in maize. In the current project, enzymatic activity of three predicted maize IGPS genes was confirmed by heterologous expression. Transposon knockout mutations confirmed the function of the maize genes in benzoxazinoid biosynthesis. Sub-cellular localization studies showed that the three maize IGPS proteins are co-localized in the plastids, together with BX1 and BX2, two previously known enzymes of the benzoxazinoid biosynthesis pathway. Implications Benzoxazinoids are among the most abundant and effective defensive metabolites in maize, wheat, and rye. Although there is considerable with-in species variation in benzoxazinoid content, very little is known about the regulation of this variation and the specific effects on plant growth and defense. The results of this research provide further insight into the complex functions of maize benzoxazinoids, which are not only toxic to pests and pathogens, but also regulate plant growth and other defense responses. Knowledge gained through the current project will make it possible to engineer benzoxazinoid biosynthesis in a more targeted manner to produce pest-tolerant crops without negative effects on growth and yield.