Chemical and genetic basis of orange flavor

Authors: FAN, ZHEN - University Of Florida; Jeffries, Kristen; Sun, Xiuxiu; OLMEDO, GABRIELA - Orise Fellow; Zhao, Wei; Mattia, Matthew; STOVER, EDDY - Retired ARS Employee; MANTHEY, JOHN - Retired ARS Employee; BALDWIN, ELIZABETH - Retired ARS Employee; LEE, SEONGHEE - University Of Florida; GMITTER, FREDERICK - University Of Florida; Plotto, Anne; Bai, Jinhe

Submitted to: Science Advances
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2023
Publication Date: 2/28/2024
Citation: Fan, Z., Jeffries, K.A., Sun, X.N., Olmedo, G., Zhao, W., Mattia, M.R., Stover, E., Manthey, J., Baldwin, E., Lee, S., Gmitter, F., Plotto, A., Bai, J. Chemical and genetic basis of orange flavor. Science Advances. 10(9). 2024. https://doi.org/10.1126/sciadv.adk2051.
DOI: https://doi.org/10.1126/sciadv.adk2051

Interpretive Summary: Citrus greening disease, or Huanglongbing (HLB), has devastated citrus orchards in Florida since 2005. One of the solutions to maintain the orange juice industry alive is to plant varieties tolerant to the disease. Recently, it was observed that some mandarin genotypes and some citrus introgressed with Poncirus trifoliata, a distant relative of citrus, presented some degree of tolerance to HLB. However, the flavor of these hybrids has not been described. This work presents a comprehensive analysis of 179 genotype/harvest combinations to understand the underlying chemical components of orange flavor. It showed that esters were a necessary component to orange flavor, relative to mandarin flavor. Further genetic analysis revealed a novel alcohol acyl transferase enzyme responsible for ester production in citrus. This work presents an important step in the understanding of flavor formation in citrus.

Technical Abstract: Sweet orange, Citrus sinensis, has limited genetic diversity and is highly susceptible to Huanglongbing (HLB). New orange-like hybrids tolerant to HLB are strong candidates to be alternatives to C. sinensis. However, the knowledge about key compounds for orange flavor and their genetic control is still elusive. A flavor and chemical evaluation of juice samples over 179 harvest/genotype combinations, including 58 genotypes of oranges, mandarins, Poncirus trifoliata, and mandarins introgressed with P. trifoliata revealed distinct volatile compositions for each species/hybrid. A random forest model not only showed 78% prediction accuracy (R2) for untrained samples from a new season, but also identified 26 compounds important for orange flavor. Among them, seven esters, including methyl hexanoate, ethyl hexanoate, ethyl 3-hydroxyhexanoate, ethyl octanoate, methyl butanoate, ethyl butanoate and ethyl 2-methylbutanoate, were determined to be key compounds to distinguish orange from mandarin. Further chemical cluster analysis revealed at least six of seven esters that were under the same genetic control. Differential gene expression analysis between ester producers and non-producers identified a novel alcohol acyltransferase (CsAAT1) responsible for ester production in orange. Its biosynthetic activity for both straight- and branched- chain esters was validated via transient overexpression assays in citrus. A SNP-based DNA marker within the CDS region showed 100% sensitivity for 28 tested cultivars. Our work facilitated the understanding of important compounds for orange flavor, their biosynthetic pathways and greatly expanded the breeders’ toolbox to develop orange-like cultivars in a time of need.