The molecular pathways leading to GABA and lactic acid accumulation in florets of organic broccoli rabe (<i>Brassica rapa</i> subsp. <i>sylvestris</i>) stored as fresh or as minimally processed product

In the context of organic farming, the introduction of a local product to wider markets and an evaluation of storage effects, metabolic and transcriptomic variations in two broccoli rabe genotypes from production cycles of two different years were studied by comparing florets of stored fresh (SF) and packaged (P) for 4 days with those harvested fresh from the field (H). Twenty-five hydrosoluble compounds, including amino acids, carbohydrates, and organic acids, were quantified by untargeted nuclear magnetic resonance (NMR). Principal component analysis produced a neat separation among the three commodity statuses with P being the most divergent and SF closer to H. In the packaged florets, carbohydrate levels dropped significantly (over -52%), while the levels of amino acids and organic acids varied. There was an increase in stress-responsive phenylalanine and valine (over 30%) and succinic and α-ketoglutaric acids (over 75%). Compound correlation analyses indicated a carbohydrate sink towards γ-aminobutyric acid (GABA) and lactic acid (LA) metabolism under hypoxic conditions in packaged florets. RNA-seq analysis revealed that over 4000 genes were differentially expressed in SF vs H and 8000 in P vs H. Several CAR and AA pathways were significantly enriched in S and even more significantly in P, when compared to H. A map of gene expression (175 genes) and metabolite contents (14 compounds) was constructed to elucidate the gene routes that lead to accumulation of GABA and LA, known for healthy properties, in P. WGCNA and promoter binding site analyses enabled the identification of transcription factors (bZIP, WRKY, ERF types), interactions, and targeted genes encoding key enzymes in GABA and LA accumulation.