Submitted to: African Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/22/2021
Publication Date: 3/20/2021
Citation: Winders, J.R., Pechan, T. 2021. Comparison of homogenization methods for extraction of maize cob metabolites. African Journal of Biotechnology. 20(3):108-114. https://doi.org/10.5897/AJB2020.17246.
DOI: https://doi.org/10.5897/AJB2020.17246

Interpretive Summary: Metabolites are small molecule intermediates, or end products of protein metabolism that are the active compounds used for cellular signaling, growth, and defense. Plant metabolites are frequently changing as they are modified while they move through different pathways. Some examples of plant pathways are: converting sunlight into energy, transporting essential nutrients from the roots, and the defense response to invading pathogens. In maize, the pathogen-induced defense response is well documented in kernels. However, the defense response of the kernels' parent tissue and cob, is not well studied. One way to understand the defense mechanism is to study the changes in proteins (proteomics). A previous proteomic study has shown that cob tissue has a vital role in isolating and reducing fungi spread through the maize ear. This response is accomplished by an elaborate defense mechanism that is regulated by the plant's metabolism. Currently, there are no studies investigating the cob metabolite's role in limiting the spread of pathogens, nor has a metabolite extraction protocol been established for cob. Maize cob is a rigid tissue that only hardens as it ages, making it difficult to grind into the fine powder needed for metabolite extraction. This study investigates the reproducibility of three different grinding techniques hand grinding, mechanical ball-milling, and adapted focused acoustics methods. After each type of grinding was performed, metabolites were extracted and then examined. Among tested methods, the manual grinding with a mortar and pestle was found to be the most reproducible and efficient. All methods showed good reproducibility within but provided statistically different sets of different metabolites when compared across methods. This study provides the foundation for future maize cob metabolite extraction experiments. For the first time, maize cob metabolites have been successfully extracted and detected, establishing a reproducible method for further metabolomic profiling of cob defense metabolites.

Technical Abstract: Previous proteomic studies have shown maize cob tissue to have an essential role in pathogen defense. Currently, there are no studies published regarding maize cob metabolomic profiles nor an accepted method of metabolite extraction from cob. This study assesses the reproducibility of metabolite extraction from the cobs of fungal pathogen -resistant and -susceptible cultivars. Hand grinding, mechanical ball-milling and adapted focused acoustics methods of tissue homogenization were preformed and examined via LC-MS. Among tested methods, the manual grinding with a mortar and pestle was found to be the most reproducible and efficient. All methods showed good reproducibility within but provided statistically different sets of metabolite features when compared across methods. A suitable extraction method for future cob metabolomics experiments was ascertained, however careful results validation will be needed to distinguish between true biological phenomenon and artifacts rising from a methodology bias. For the first time, maize cob metabolites have been successfully extracted and detected, establishing a reproducible method for further metabolomic profiling of cob defense metabolites.