With the high demand for a healthy diet, it is necessary and important to explore natural sweeteners used in food that enhance palatability but minimize calories. Citrus is considered a good potential source of noncaloric sweeteners, but to date, only one sweetness modulator has been found in this most common fruit crop. Herein, an efficient strategy based on an in-house database and the untargeted and targeted metabolomics analyses was proposed to screen sweeteners or sweet-enhancing compounds from citrus. Eight sweeteners or sweetness-enhancing compounds were screened out, seven of which were newly identified from the genus Citrus. Surprisingly, we identified naturally occurring oxime V, which previously was only known as a synthetic compound. The contents of five compounds, in 11 citrus cultivars or unreleased selections across two production years, were compared. Successful identification of these natural sweeteners and sweetness-enhancing compounds in citrus fruit indicated the potential to identify the relevant biosynthetic pathways and to breed new citrus cultivars containing these compounds that provided both palatability and lower sugar consumption. This study also demonstrated that the proposed metabolomics-based screening strategy could greatly boost the identification of taste modulators with low contents in natural resources.

Huanglongbing (HLB) is one of the most destructive citrus diseases, mainly caused by the Gram-negative bacteria Candidatus Liberibacter asiaticus. Aiming at unraveling the mechanisms of different scion/rootstock combinations on improving HLB-affected orange juice quality, the effects of rootstocks on the metabolites of HLB-affected sweet orange juices were investigated using a combined strategy of untargeted metabolomics and machine learning. A total of 2531 ion features were detected using UHPLC-Q-Orbitrap high-resolution electrospray ionization mass spectrometry, and 54 metabolites including amino acids, amines, flavonoids, coumarins, fatty acids, and glycosides were definitely or tentatively identified as the differential markers based on the random forest algorithm. Furthermore, 24 metabolites were verified and semi-quantified using authentic standards. Notably, the presence of specific amino acids and amines, especially polyamines, indicated that different rootstocks might affect glutamate, aspartate, proline, and arginine metabolism to regulate the physiological response against HLB. Meanwhile, the production of flavonoids and prenylated coumarins suggested that rootstocks influenced phenylalanine and phenylpropanoid metabolism. The possible metabolic pathways were proposed, and the important intermediates were verified by authentic standards. These results provide new insights on the effects of rootstocks on the metabolites of HLB-affected sweet orange juices.

Mandarins have many unique flavonoids with documented health benefits and that help to prevent chronic human diseases. Flavonoids are difficult to measure and cannot be phenotyped without the use of specialized equipment; consequently, citrus breeders have not used flavonoid contents as selection criteria to develop cultivars with increased benefits for human health or increased tolerance to diseases. In this study, peel, pulp, and seed samples collected from many mandarin accessions and their hybrids were analyzed for the presence of selected flavonoids with documented human health benefits. A genome-wide association study (GWAS) was used to identify SNPs associated with biosynthesis of flavonoids in these mandarin accessions, and there were 420 significant SNPs were found to be associated with 28 compounds in peel, pulp, or seed samples. Four candidate genes involved in flavonoid biosynthesis were identified by enrichment analysis. SNPs that were found to be associated with compounds in pulp samples have the potential to be used as markers to select mandarins with improved phytonutrient content to benefit human health. Mandarin cultivars bred with increased flavonoid content may provide value to growers and consumers.