THE REGULATION OF BRANCHED-CHAIN ESTER PRECURSOR BIOSYNTHESIS IN RIPENING FRUITS

The aroma of fruits is an important indicator of ripeness to potential consumers. It is appreciated by humans as being essential to the organoleptic quality of fresh fruits. The two most consumed fruits in the United States, bananas and apples, have aroma profiles dominated by straight- and branched-chain esters. These compounds confer the characteristic flavors of these fruits. Despite the value of these esters to quality perception, their biosynthesis is still poorly understood. The central theme of this work is to understand how these fruits produce an ample supply of precursors for the biosynthesis of esters. The application of acetohydroxyacid synthase inhibitors demonstrated, through aroma and amino acid analyses of treated apple, banana, and ornamental quince, that the precursors to branched-chain esters are ultimately supplied through de novo synthesis and not from protein degradation. Inhibitor treatment, paired with 13C-labeled acetate, allowed further elucidation of biochemical networks. Sensory analysis of inhibitor-treated apples, supplemented with population genetics analyses, demonstrated that humans can detect the absence of branched-chain esters and, via breeding/propagation efforts, have selected for fruit that are capable of branched-chain ester synthesis. Inhibitor application revealed a potentially unique means for butyl ester biosynthesis in banana fruit and inspired a precursor feeding study in plantain to better understand the origins of these important aroma compounds in banana. The specific mechanisms underlying banana aroma precursor biosynthesis were also explored. Aroma, respiration, and metabolic intermediate data demonstrated a coordinated shift of branched-chain amino acid metabolism that is facilitated by the alternative splicing of two otherwise feedback-regulated enzymes, acetohydroxyacid synthase and isopropylmalate synthase. The incidence of splicing and fold-change of the alternative transcripts was localized to only fruit pulp and increased with ripening. Enzymatic assays and transient expression in tobacco indicated these alternative isoforms to be immune to feedback regulation and to be able to supply ample precursors for branched-chain ester synthesis. Banana fruit aroma biodiversity was also explored, identifying several promising cultivars for future studies. Finally, artificial banana flavor was also investigated, revealing that the ersatz flavor substitute in foodstuffs is not emblematic of the banana of the past, ‘Gros Michel’, nor the banana of today, ‘Cavendish’.