An Evolutionarily Diverged CCD4 Enzyme Negatively Regulates Mesocotyl Elongation in Rice

Carotenoid cleavage dioxygenases (CCDs) catalyze the formation of phytohormones and other growth regulators, yet the CCD4 subfamily remains underexplored in cereals. The rice gene LOC_Os12g24800 has often been misannotated as nine-cis-epoxycarotenoid dioxygenase 2 (NCED2), implicating it in abscisic acid (ABA) biosynthesis. Here, we provide a definitive functional characterization of LOC_Os12g24800 and demonstrate that it encodes a CCD4-type enzyme, OsCCD4b, distinct from NCEDs. Using in vitro, in vivo, transgenic assays, and CRISPR/Cas9 and overexpressing lines, we show that OsCCD4b preferentially cleaves carotenoids at the C7'–C8' double bond, producing C30 and C10 apocarotenoids, in contrast to the cleavage specificity of Arabidopsis CCD4. Moreover, we identify 3-OH-ß-cyclocitral, a major OsCCD4b-derived metabolite, as a previously unknown signaling molecule that suppresses mesocotyl elongation by reducing cell length and modulating sugar metabolism, independently of other growth regulators. These findings resolve a long-standing annotation ambiguity, reveal functional divergence of CCD4 enzymes between monocots and dicots, and uncover an independent apocarotenoid signaling pathway controlling rice mesocotyl development.