Natália Reis de Almeida¹, Brena Rodrigues Mota Ikehara², Frederico Garcia Pinto², Gabriel Mascarenhas Maciel¹, Ana Carolina Silva Siquieroli¹

Hidden hunger refers to malnutrition characterized by a lack of essential nutrients, despite sufficient food availability. An efficient approach to fight this condition is genetic biofortification, which aims to nutritionally enrich the edible parts of plants, promoting the production of functional foods. Lettuce (Lactuca sativa L.) is the most consumed leafy vegetable in Brazil, valued for its accessibility, diversity, and high micronutrients absorption efficiency. Given its nutritional importance, biofortification strategies for this crop can bring significant benefits to the health of the population, increasing the nutritional value of an easy-to-prepare and low-cost food. During the biofortification process, changes can occur in the primary and secondary metabolism of plants, modifying compounds such as sugars, amino acids, and organic acids, with possible effects on nutritional quality, sensory attributes, and biotic and abiotic stress resistances. In this context, metabolomics has emerged as a fundamental analytical tool to monitor metabolic changes in biofortified lines, allowing detailed evaluation of chemical profiles and ensuring maintenance or improvement of desired nutritional characteristics. Thus, this study focused on the evaluation of the metabolite profile differences between a biofortified parental lettuce line (P) and a descendant line (D) from the Genetic Improvement Program of the Federal University of Uberlândia (Monte Carmelo, Brazil). The analysis was performed on macerated lettuce leaves processed with extractant solution and cell disruptor. The extracted samples were dried, subjected to derivatization, and analyzed by gas chromatography-mass spectrometry (GC-MS). Data processing was performed using MS-Dial 5 and MetaboAnalyst 6.0 software, with statistical significance set at p < 0.05. Metabolomic analysis identified metabolic differences between lines P and D. Multivariate analysis demonstrated the separation of reduced groups that the selection of lines within biofortified plants promotes relevant metabolic variations. Line D showed a higher abundance of primary metabolites, including simple sugars and organic acids, associated with improved metabolic efficiency and enhancement of desirable nutritional characteristics. Also, a greater abundance of flavonoids and phenolic acids, which are compounds related to antioxidant activity and impair resistance to environmental stresses, was observed in D. These findings demonstrate that, even among biofortified materials, metabolic variability can be explored to develop lines with desirable chemical profiles. The use of metabolomics allowed the detection of metabolic differences that, although subtle, are relevant for the selection of genotypes with improved nutritional characteristics.

Agradecimentos: The authors thank the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG/Brazil), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil), Universidade Federal de Uberlândia, Universidade Federal de Viçosa, Grupo de Estudos em Melhoramento Genético de Hortaliças (GEN-HORT), and Grupo de Pesquisa em Análise Metabolômica da Universidade Federal de Viçosa (GPAM)