Alterações no fluxo metabólico em rotas associadas ao metabolismo energético de células-guarda e mesofílicas sob condições de escuro e luz

Abstract

Climate change, intensified by global warming, has pressured agriculture to search for plants with improved water use efficiency (WUE) under more extreme conditions. WUE is regulated by stomata, which consist of a stomatal pore surrounded by two guard cells (GC). The metabolism of these cells and its regulation are the main targets of studies aimed at developing cultivars more resistant to stress conditions. In this context, this study aimed to compare the metabolic changes in GC and mesophyll cells (MC) of cowpea during darkness and after illumination to observe how guard cell metabolism is regulated. Metabolic flux experiments were conducted using ¹³C-NaHCO₃, ¹³C-glucose, and ¹³C-succinate in GC and leaf discs (LD) under continuous darkness or after the transition to light. Samples were frozen in liquid N₂ after 60 minutes in plates with the ¹³C-labeled substrates, they were then macerated and used for metabolic extraction and derivatization. After derivatization, the samples were analyzed by gas chromatography coupled with mass spectrometry, and the ¹³C enrichment values were obtained following established protocols in the literature. The relative enrichment results showed a higher labeling of metabolites in GC, particularly organic acids and sugars related to the Krebs cycle and the glycolytic and gluconeogenic pathways, respectively. Our results demonstrated that aspartate was preferentially labeled in guard cells, regardless of the analyzed substrate, and that citrate labeling from ¹³C-succinate was higher in these cells compared to MC. These results suggest a high CO₂ fixation mediated by the phosphoenolpyruvate carboxylase (PEPc) enzyme in GC, a characteristic similar to that observed in C₄ plants, and a high carbon flux through the Krebs cycle, respectively. In contrast, alanine labeling was higher in illuminated MC labelled by 13C-bicarbonate. Therefore, while MC exhibited more evident changes after exposure to light, the metabolism of GC demonstrated less drastic metabolic responses to light for its functioning, with a more intense flux through the gluconeogenesis, Krebs cycle, and associated pathways.