biostudies-arrayexpressABDUGAFFOR ABLAZOVOryza sativahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16215Carotenoid 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-β-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.biostudies-arrayexpressGrowth Protocol - The vermiculite experiment was performed as described by Patil et al. (2019) with some modifications. Initially, 250 ml of vermiculite was added to the glass vessels (6 cm diameter, 18 cm height). Then, approximately twenty (dry) seeds were placed on this layer, and following this, 500 ml of vermiculite was added on top of the seeds. Lastly, 400 mL of Milli-Q water (pH 5.8) was added to each bottle, and each was tightly closed and incubated in complete darkness above conditions for 10 daysLibrary Construction - Illumina Novaseq platform. Library preparation, sequencing, and subsequent data analysis were conducted by Novogene Technology.Sample Treatment - The vermiculite experiment was performed as described by Patil et al. (2019) with some modifications. Initially, 250 ml of vermiculite was added to the glass vessels (6 cm diameter, 18 cm height). Then, approximately twenty (dry) seeds were placed on this layer, and following this, 500 ml of vermiculite was added on top of the seeds. Lastly, 400 mL of Milli-Q water (pH 5.8) was added to each bottle, and each was tightly closed and incubated in complete darkness above conditions for 10 daysSample Collection - Total RNA was extracted from rice mesocotyls of the OsCCD4b CRSIPR mutant and the overexpression lines using TRI-Reagent with Direct-zol RNA MiniPrep Kit according to the manufacturer's instructions (Zymo Research). Seedlings that were nine days old, with 4-5 seedlings grouped together for each sample, were collected in a dark room illuminated by green lights. The quality and quantity of RNA were assessed using the NanoDrop 6000.Sequencing - Illumina Novaseq platform. Library preparation, sequencing, and subsequent data analysis were conducted by Novogene Technology.Nucleic Acid Extraction - Total RNA was extracted from rice mesocotyls of the OsCCD4b CRSIPR mutant and the overexpression lines using TRI-Reagent with Direct-zol RNA MiniPrep Kit according to the manufacturer's instructions (Zymo Research). Seedlings that were nine days old, with 4-5 seedlings grouped together for each sample, were collected in a dark room illuminated by green lights. The quality and quantity of RNA were assessed using the NanoDrop 6000.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - The clean paired-end reads were aligned to the Nipponbare reference genome (http://rice.uga.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/) utilising Hisat2 v2.0.5.Data Transformation - FPKMMetabolomicsUnknownTranscriptomicsGenomicsProteomicsDESeqIllumina Novaseq platformGrowthroomHisat2 v2.0.5Illumina HiSeq 4000RNA-seq of coding RNAOryza sativaABDUGAFFOR ABLAZOVfalseAn Evolutionarily Diverged CCD4 Enzyme Negatively Regulates Mesocotyl Elongation in RiceCarotenoid 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-β-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.2025-12-12T00:00:00Z2026-05-27T22:10:50.933Z2025-11-20T01:48:23.894ZE-MTAB-16215ERP185358EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969