Natural overexpression of carotenoid cleavage dioxygenase 4 in tomato alters carotenoid flux

Authors: YOO, HEE JO - Kyungpook National University; CHUNG, MI-YOUNG - Suncheon National University; LEE, HYUN-AH - Yonam College; LEE, SOO-BIN - Kyungpook National University; GRANDILLO, SILVANA - Istituto Di Bioscienze E Biorisorse-Uos Portici-cnr; Giovannoni, James; LEE, JEMIN - Kyungpook National University

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/26/2022
Publication Date: 1/30/2023
Citation: Yoo, H., Chung, M., Lee, H., Lee, S., Grandillo, S., Giovannoni, J.J., Lee, J. 2023. Natural overexpression of carotenoid cleavage dioxygenase 4 in tomato alters carotenoid flux. Plant Physiology. 192(2):1289-1306. https://doi.org/10.1093/plphys/kiad049.
DOI: https://doi.org/10.1093/plphys/kiad049

Interpretive Summary: Carotenoids are integral for light harvesting and photosynthesis, as well as photoprotection from photooxidative damage under excess light conditions. Carotenoids provide sources of pigment variation in reproductive organs to attract seed dispersers and pollinators. Carotenoids also play important roles in human health as antioxidants, reducing the risk of various diseases such as cancers and eye diseases. The oxidative breakage of carotenoids results in the formation of a diverse family of essential metabolites, known as apocarotenoids. Apocarotenoid volatiles are vital flavor components in economically important crops. The contents of flavor volatiles are typically substantially lower in cultivated tomato varieties than in heirloom varieties. Loss of flavor in cultivated tomato (Solanum lycopersicum) is associated with diminished production of sugars and volatile precursors, including fatty acids, amino acids, and carotenoids. In this study, the overexpression of the ShCCD4b gene from the tomato wild relative S. habrochaites was identified as a genetic determinant of dark orange fruit color and apocarotenoid flavors whihc could be used to enhance quality of lower quality production tomato varieties.

Technical Abstract: Carotenoids and apocarotenoids function as pigments and flavor volatiles in plants that enhance consumer appeal and offer health benefits. Tomato (Solanum lycopersicum.) fruit, especially those of wild species, exhibit a high degree of natural variation in carotenoid and apocarotenoid contents. Using positional cloning and an introgression line (IL) of Solanum habrochaites “LA1777', IL8A, we identified carotenoid cleavage dioxygenase 4 (CCD4) as the factor responsible for controlling the dark orange fruit color. CCD4b expression in ripe fruit of IL8A plants was ~8,000 times greater than that in the wild type, presumably due to 5' cis-regulatory changes. The ShCCD4b-GFP fusion protein localized in the plastid. Phytoene, '-carotene, and neurosporene levels increased in ShCCD4b-overexpressing ripe fruit, whereas trans-lycopene, ß-carotene, and lutein levels were reduced, suggestive of feedback regulation in the carotenoid pathway by an unknown apocarotenoid. Solid-phase microextraction–gas chromatography–mass spectrometry analysis showed increased levels of geranylacetone and ß-ionone in ShCCD4b-overexpressing ripe fruit coupled with a ß-cyclocitral deficiency. In carotenoid-accumulating Escherichia coli strains, ShCCD4b cleaved both '-carotene and ß-carotene at the C9–C10 (C9'–C10') positions to produce geranylacetone and ß-ionone, respectively. Exogenous ß-cyclocitral decreased carotenoid synthesis in the ripening fruit of tomato and pepper (Capsicum annuum), suggesting feedback inhibition in the pathway. Our findings will be helpful for enhancing the aesthetic and nutritional value of tomato and for understanding the complex regulatory mechanisms of carotenoid and apocarotenoid biogenesis.