Genetic variation at bx1 controls DIMBOA content in maize

The main hydroxamic acid in maize (Zea mays L.) is 2-4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to leaf-feeding by several corn borers. Most genes involved in the DIMBOA metabolic pathway are located on the short arm of chromosome 4, and quantitative trait loci (QTLs) involved in maize resistance to leaf-feeding by corn borers have been localized to that region. However, the low resolution of QTL linkage mapping does not allow convincing proof that genetic variation at bx loci was responsible for the variability for resistance. This study addressed the following objectives: to determine the QTLs involved in DIMBOA synthesis across genetically divergent maize inbreds using eight RIL families from the nested association mapping population, to check the stability of QTLs for DIMBOA content across years by evaluating two of those RIL families in 2 years, and to test the involvement of bx1 by performing association mapping with a panel of 281 diverse inbred lines. QTLs were stable across different environments. A genetic model including eight markers explained approximately 34% of phenotypic variability across eight RIL families and the position of the largest QTL co-localizes with the majority of structural genes of the DIMBOA pathway. Candidate association analysis determined that sequence polymorphisms at bx1 greatly affects variation of DIMBOA content in a diverse panel of maize inbreds, but the specific causal polymorphism or polymorphisms responsible for the QTL detected in the region 4.01 were not identified. This result may be because the causal polymorphism(s) were not sequenced, identity is masked by linkage disequilibrium, adjustments for population structure reduce significance of causal polymorphisms or multiple causal polymorphisms affecting bx1 segregate among inbred lines.     

The role of fungicides in the physiology of higher plants: Implications for defense responses

Plants react to pathogen attack through a variety of active and passive defense mechanisms primarily related to the metabolism of phenolic compounds and oxidative metabolism. Thus the activation of defensive reactions is associated with the increased expression of a great number of genes that encode enzymes involved in the biosynthetic pathway of phenolic compounds. Similarly, the activation of oxidative metabolism precedes the expression of defense genes during plant-pathogen interactions, so both metabolic processes must exert a major function in directing the mechanisms to resist disease. Similarly, it has been suggested that certain fungicides used to mitigate or prevent pathogen attack may be involved in activating certain defensive responses of plants. However, the fact that such substances may influence the key steps of the phenolic and oxidative processes has scarcely been studied. Our work confirms the results proposed by other authors, who suggest that certain wide-spectrum fungicides, in addition to their antibiotic action against pathogens, may be involved in the activation of some defensive responses of plants.     

Hydroxamic Acid glucosyltransferases from maize seedlings

Hydroxamic acids occur in several forms in maize (Zea mays L.) with 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) being the predominant form and others including 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) being found at lower concentrations. Two enzymes capable of glucosylating hydroxamic acids were identified in maize protein extracts and partially purified and characterized. The total enzyme activity per seedling increased during the first 4 days of germination and was concurrent with the accumulation of DIMBOA. Purification of the enzymes by ammonium sulfate precipitation followed by Sephadex G-200 and Q-Sepharose gel chromatography resulted in a 13-fold increase in specific activity. The enzymes are initially separated into two peaks (peak 1 and peak 2) of activity by Q-Sepharose gel chromatography. The peak 1 glucosyltransferase had 3.6% of the DIMBOA glucosylating activity when DIBOA was used as substrate, whereas this percentage increased to 57% for the peak 2 enzyme. The enzyme in peak 2 has a K_m of 174 micromolar for DIMBOA and a K_m of 638 micromolar for DIBOA; the enzyme in peak 1 has a K_m of 217 micromolar for DIMBOA and its activity on DIBOA was too low to determine a K_m. The identification of two glucosyltransferases capable of glucosylating hydroxamic acids in vitro serves as an initial step in the characterization of the enzymes involved in production of hydroxamic acids in maize.     

A role for cyclic hydroxamates in aluminium resistance in maize?

Hydroxamate siderophores have been found to alleviate Al toxicity in bacteria. In Poaceae plants cyclic hydroxamates, like DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and its derivatives have mostly been studied in relation to either defence against insects or allelopathy. In this study the influence of Al on concentrations of these benzoxazinoids (Bx) in root tips, whole roots and root xylem exudates of Zea mays L. varieties differing in Al resistance was analyzed by HPLC-MS. Aluminium resistant maize variety Sikuani maintained considerably higher Bx levels in root tips than the Al sensitive variety Bakero. In vitro binding of Al to DIMBOA was shown by fluorescence quenching. Addition of DIMBOA to Al-containing nutrient solution protected the sensitive maize against Al toxicity as shown by bioassays using callose and haematoxylin staining of root tips as stress indicators. This is the first study showing that Bx can detoxify Al in solution. Tissue analysis data provide first, circumstantial, support for a role of Bx in defence against Al toxicity also in planta.     

Protein networks reveal organ-specific defense strategies in maize in response to an aboveground herbivore

Many of the proteins and defense pathways in maize that are activated in an organ-specific manner in leaves and roots during aboveground caterpillar attack have not yet been identified. In this study, we examined systemic and organ-specific defenses in the insect-resistant maize genotype, Mp708, when infested aboveground with fall armyworm (FAW, Spodoptera frugiperda). We used proteomic and network biology analyses and then integrated these data with known FAW resistance QTL to create a protein abundance QTL (pQTL) subnetwork. Using 10-plex tandem mass spectrometry tags (TMT) proteomics technique, we identified a total of 4675 proteins in leaves and roots of control and FAW-infested plants. Among the identified proteins, 794 had significant differences in abundance in response to FAW herbivory. Proteins that were upregulated in leaves during FAW infestation included jasmonic acid biosynthetic enzymes, cysteine proteases, protease inhibitors, REDOX-related proteins, and peroxidases. In roots, highly abundant proteins were involved in ET biosynthesis, DNA expression regulation, and pyruvate biosynthesis. We found many proteins that possibly contribute different defense functions to FAW resistance in Mp708. One potential resistance mechanism identified was that trade-offs between growth and defense responses were reduced in Mp708. Some of the proteins involved in this trade-off that were found within the pQTL subnetwork were the Kinesin-like protein (GRMZM2G046186_P01) and Pi starvation-induced protein (GRMZM2G118037_P01). We proposed other mechanisms contributing to resistance that suggest that jasmonic acid and ethylene control the local accumulation of insecticidal cysteine protease (MIR1-CP) in leaves, while ethylene controlled the systemic accumulation of MIR1-CP in roots. Finally, we hypothesized that receptor kinases such as receptor protein kinase 1 (GRMZM2G055678) could be involved in the activation of root-specific defense responses during aboveground insect infestation.