Herbicide safening in maize (Zea mays)

Abstract

Safeners are agrochemicals used in conjunction with herbicides to enhance selectivity in large grained cereals, by reducing crop damage. The safening effect is associated with an increase in herbicide detoxification, via increased expression of enzymes in the xenobiotic detoxification pathway, known as the xenome. In this biosystem, cytochrome P450 (CYP) enzymes catalyse oxidation/reduction reactions, and glutathione-S-transferase (GST) enzymes catalyse conjugation reactions, leading to reduced toxicity and vacuolar sequestration. Despite their extensive use, the basis of safener specificity is largely unknown. The objective of this research was to identify, characterise and understand the mechanism of safener selectivity, and to determine the effect on specific mechanisms of herbicide metabolism, focusing on maize (Zea mays L.). A hydroponic growth system was developed to allow rapid, controlled analyses to be performed, and to test its validity as a potential replacement of large scale metabolic studies. Two safeners, metcamifen and benoxacor, are known to enhance the detoxification of triketone and chloroacetanilide classes of herbicides, respectively. The effects of these safeners on the metabolism of a triketone (mesotrione) and a chloroacetanilide (Smetolachlor) herbicide were investigated in maize to characterise their chemical specificity. The effects of the safeners on the metabolic rate and route of the radiolabelled herbicides were studied using thin layer chromatography. This study identified an enhanced rate of mesotrione metabolism by both safeners, with metcamifen causing significantly faster metabolism than benoxacor. The metabolism of S-metolachlor was unaffected by either safener. To identify if uptake rate was responsible for this selectivity, the translocation of the radiolabelled safeners was analysed through phosphorimaging. Benoxacor underwent more rapid uptake than metcamifen. The GST superfamily was then analysed at the genome level through in silico phylogenetic analyses, to further understand their involvement in metabolism. 65 proteins from 56 genes were identified and characterised using a novel nomenclature system. Investigation of GST enzyme structures and catalytic sites, using sequence alignments, and homology modelling, provided an insight into their functional properties. To determine if differential regulation of xenome enzymes was involved in safening, the expression of transcripts encoding GSTs and CYPs by the safeners was investigated by

quantitative real time polymerase chain reaction (qPCR). Safener-inducible candidate GSTs and CYPs genes were identified by next generation sequencing analysis. This study showed an early induction of these genes in maize stems with benoxacor, while metcamifen caused a later induction of these genes in the leaf, providing time and tissue specific effects that could explain the observed specificity. In addition, both Safeners caused greater induction of GSTs than CYPs. To investigate the effect of the safeners on the regulation of important GST enzymes, western blotting was performed using antibodies recognising ZmGSTU1.2-1.2 and ZmGSTF2.0-2.3. Both safeners increased the levels of the proteins, with benoxacor showing the greatest effect, and the stem displaying larger increases than the leaf. ZmGSTF2.0, ZmGSTF2.3 and ZmGSTU1.2 enzymes were expressed in E. coli, allowing for verification and characterisation of conjugating activity. In order to understand the complex signalling pathways regulating GSTs, co-expression analyses were performed using a database of maize experiments, to determine the effect of abiotic stresses on their expression. This study identified similarities between the effects of a phytohormone (12-OPDA), submergence stress, and safener treatment, potentially indicating shared signalling systems. These analyses also investigated the tissue-specific and developmental profile of GST expression, generating a comprehensive understanding of how the GST superfamily is regulated. Overall, this research has determined that the safeners, metcamifen and benoxacor, selectively enhance the metabolic rate of different herbicide classes through increased expression of specific GST and CYP enzymes involved in xenobiotic detoxification, at both the transcript and protein level. The safener-effect is both tissue and time specific, and occurs without long term alteration to the final metabolic profiles of the herbicides. The GST family has been extensively characterised, providing a better understanding of their safener selectivity. For regulation, new safeners may be subjected to such analyses, allowing for predictions of specificity and suitability, before expensive and time-consuming field trials are performed.