Despite being a highly nutritious and resilient cereal, pearl millet is not popular among consumers and food industries due to the short shelf-life of flour attributed to rapid rancidity development. The biochemical mechanism underlying rancidity, a complex and quantitative trait, needs to be better understood. The present study aims to elucidate the differential accumulation of metabolites in pearl millet that impact the rancidity process. Metabolite profiling was conducted on ten pearl millet genotypes with varying levels of rancidity—comprising high, low, and medium rancid genotypes—utilizing liquid chromatography and high-resolution mass spectrometry (LC-HRMS) at different accelerated ageing conditions. Through non-targeted metabolomic analysis, crucial metabolites associated with rancidity were identified across various biochemical pathways, including fatty acids, glycerophospholipids, sphingolipids, glycerol lipids, flavonoids, alkaloids, and terpenoids. Notably, metabolites such as fatty aldehydes, fatty alcohols, fatty esters, fatty acyls, fatty esters, and fatty amides were significantly elevated in high rancid genotypes, indicating their involvement in the rancidity process. These fatty acids-related metabolites further break down into saturated and unsaturated fatty acids. Four key fatty acids—stearic, palmitic, linoleic and linolenic acid—were quantified in the ten pearl millet genotypes, confirming their role in rancidity development. This investigation promises novel insights into utilizing metabolomics to understand the biochemical processes and facilitate precision breeding for developing low-rancidity pearl millet lines.

Aflatoxins are immunosuppressive and carcinogenic secondary metabolites, produced by the filamentous ascomycete Aspergillus flavus, that are hazardous to animal and human health. In this study, we show that multiplexed host-induced gene silencing (HIGS) of Aspergillus flavus genes essential for fungal sporulation and aflatoxin production (nsdC, veA, aflR, and aflM) confers enhanced resistance to Aspergillus infection and aflatoxin contamination in groundnut (<20 ppb). Comparative proteomic analysis of contrasting groundnut genotypes (WT and near-isogenic HIGS lines) supported a better understanding of the molecular processes underlying the induced resistance and identified several groundnut metabolites that might play a significant role in resistance to Aspergillus infection and aflatoxin contamination. Fungal differentiation and pathogenicity proteins, including calmodulin, transcriptional activator-HacA, kynurenine 3-monooxygenase 2, VeA, VelC, and several aflatoxin pathway biosynthetic enzymes, were downregulated in Aspergillus infecting the HIGS lines. Additionally, in the resistant HIGS lines, a number of host resistance proteins associated with fatty acid metabolism were strongly induced, including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol Δ-7 desaturase, ceramide kinase-related protein, sphingolipid Δ-8 desaturase, and phospholipase-D. Combined, this knowledge can be used for groundnut pre-breeding and breeding programs to provide a safe and secure food supply.