Carotenoid cleavage, catalyzed by CAROTENOID CLEAVAGE DIOXYGENASEs (CCDs), provides signaling molecules and precursors of plant hormones. Recently, we showed that zaxinone, a apocarotenoid metabolite formed by the CCD ZAXINONE SYNTHASE (ZAS), is a growth regulator required for normal rice (Oryza sativa) growth and development. The rice genome encodes three OsZAS homologs, called here OsZAS1b, OsZAS1c, and OsZAS2, with unknown functions. Here, we investigated the enzymatic activity, expression pattern, and subcellular localization of OsZAS2 and generated and characterized loss-of-function CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and associated protein 9)-Oszas2 mutants. We show that OsZAS2 formed zaxinone in vitro. OsZAS2 was predominantly localized in plastids and mainly expressed under phosphate starvation. Moreover, OsZAS2 expression increased during mycorrhization, specifically in arbuscule-containing cells. Oszas2 mutants contained lower zaxinone content in roots and exhibited reduced root and shoot biomass, fewer tillers, and higher strigolactone (SL) levels. Exogenous zaxinone application repressed SL biosynthesis and partially rescued the growth retardation of the Oszas2 mutant. Consistent with the OsZAS2 expression pattern, Oszas2 mutants displayed a lower frequency of arbuscular mycorrhizal colonization. In conclusion, OsZAS2 is a zaxinone-forming enzyme that, similar to the previously reported OsZAS, determines rice growth, architecture, and SL content, and is required for optimal mycorrhization.

Summary: Carotenoid‐derived regulatory metabolites and hormones are generally known to arise through the oxidative cleavage of a single double bond in the carotenoid backbone, which yields mono‐carbonyl products called apocarotenoids. However, the extended conjugated double bond system of these pigments predestines them also to repeated cleavage forming dialdehyde products, diapocarotenoids, which have been less investigated due to their instability and low abundance. Recently, we reported on the short diapocarotenoid anchorene as an endogenous Arabidopsis metabolite and specific signaling molecule that promotes anchor root formation. In this work, we investigated the biological activity of a synthetic isomer of anchorene, iso‐anchorene, which can be derived from repeated carotenoid cleavage. We show that iso‐anchorene is a growth inhibitor that specifically inhibits primary root growth by reducing cell division rates in the root apical meristem. Using auxin efflux transporter marker lines, we also show that the effect of iso‐anchorene on primary root growth involves the modulation of auxin homeostasis. Moreover, by using liquid chromatography–mass spectrometry analysis, we demonstrate that iso‐anchorene is a natural Arabidopsis metabolite. Chemical inhibition of carotenoid biosynthesis led to a significant decrease in the iso‐anchorene level, indicating that it originates from this metabolic pathway. Taken together, our results reveal a novel carotenoid‐derived regulatory metabolite with a specific biological function that affects root growth, manifesting the biological importance of diapocarotenoids.

The rice (Oryza sativa L.) α/β-hydrolase D14 LIKE (D14L), a paralog of the strigolactone receptor D14, is essential for the establishment of arbuscular mycorrhizal (AM) symbiosis and responses to karrikins, smoke-derived compounds that regulate several developmental processes. It is thought that D14L is the receptor for an as yet unidentified endogenous growth regulator. Herein, we determined the effects of disrupting D14L on rice growth and metabolism. Our results revealed that D14L loss of function altered rice architecture, leading to a significant increase in root growth and mesocotyl elongation, while hindering shoot growth, and a notable decrease in the number of tillers, especially under phosphate-limiting conditions. Furthermore, d14l mutants exhibited significant reduction in total grain yield. Metabolomics analysis revealed a notable shift of key metabolites, such as carbohydrates and amino acids involved in energy production and growth, from shoots to roots. This redistribution is likely to reflect an adaptive strategy to enhance nutrient acquisition and increase root biomass, albeit at the cost of shoot growth and productivity. Our findings highlight the pivotal role of D14L in regulating the root-to-shoot growth ratio and in coordinating the metabolism and allocation of resources across the rice plant.

The natural growth regulator zaxinone increases the levels of the phytohormones strigolactone (SL) and abscisic acid in Arabidopsis ( Arabidopsis thaliana ) via unknown mechanisms. We demonstrate that parts of the effects of zaxinone in Arabidopsis depend on the SL receptor DWARF14 ( At D14), the karrikin receptor KARRIKIN INSENSITIVE2 ( At KAI2), and the F-Box protein MORE AXILLARY BRANCHING2 ( At MAX2) that mediates the signaling of SLs and karrikins. Binding assays and co-crystallization revealed zaxinone as an additional ligand of At D14 and an SL antagonist that interrupts the interaction of At D14 with At MAX2. Zaxinone also bound to At KAI2. These findings unveil a perception mechanism for zaxinone in Arabidopsis and demonstrate the capability of At D14 and At KAI2 to bind signaling molecules, other than strigolactones or karrikins, and mediate their transduction.

With increasing throughput in both the generation and phenotyping of mutant lines in plants, it is important to have an efficient and reliable genotyping method. Traditional workflows, still commonly used in many labs, have time-consuming and expensive steps, such as DNA purification, cloning and growing E. coli cultures. We propose an alternative workflow where these steps are bypassed, using Phire polymerase on fresh plant tissue, and ExoProStar treatment as preparation for sequencing. We generated CRISPR-Cas9 mutants for ZAS (ZAXINONE SYNTHASE) in rice with two guide RNAs. Using both a traditional workflow and our proposed workflow, we genotyped nine T1 plants. To interpret the sequencing output, which is often complex in CRISPR-generated mutants, we used free online automatic analysis systems and compared the results. Our proposed workflow produces results of the same quality as the old workflow, but in 1 day instead of 3 days and about 35 times cheaper. This workflow also consists of fewer steps and reduces the risk of cross contamination and mistakes. Furthermore, the automated sequence analysis packages are mostly accurate and could easily be used for bulk analysis. Based on these advantages, we encourage academic and commercial labs conducting genotyping to consider switching over to our proposed workflow.