Abscisic acid (ABA) is an important carotenoid-derived phytohormone that plays essential roles in plant response to biotic and abiotic stresses as well as in various physiological and developmental processes. In Arabidopsis, ABA biosynthesis starts with the epoxidation of zeaxanthin by the ABA DEFICIENT 1 (ABA1) enzyme, leading to epoxycarotenoids; e.g., violaxanthin. The oxidative cleavage of 9-cis-epoxycarotenoids, a key regulatory step catalyzed by 9-CIS-EPOXYCAROTENOID DIOXYGENASE, forms xanthoxin, which is converted in further reactions mediated by ABA DEFICIENT 2 (ABA2), ABA DEFICIENT 3 (ABA3), and ABSCISIC ALDEHYDE OXIDASE 3 (AAO3) into ABA. By combining genetic and biochemical approaches, we unravel here an ABA1-independent ABA biosynthetic pathway starting upstream of zeaxanthin. We identified the carotenoid cleavage products (i.e., apocarotenoids, β-apo-11-carotenal, 9-cis-β-apo-11-carotenal, 3-OH-β-apo-11-carotenal, and 9-cis-3-OH-β-apo-11-carotenal) as intermediates of this ABA1-independent ABA biosynthetic pathway. Using labeled compounds, we showed that β-apo-11-carotenal, 9-cis-β-apo-11-carotenal, and 3-OH-β-apo-11-carotenal are successively converted into 9-cis-3-OH-β-apo-11-carotenal, xanthoxin, and finally into ABA in both Arabidopsis and rice. When applied to Arabidopsis, these β-apo-11-carotenoids exert ABA biological functions, such as maintaining seed dormancy and inducing the expression of ABA-responsive genes. Moreover, the transcriptomic analysis revealed a high overlap of differentially expressed genes regulated by β-apo-11-carotenoids and ABA, suggesting that β-apo-11-carotenoids exert ABA-independent regulatory activities. Taken together, our study identifies a biological function for the common plant metabolites, β-apo-11-carotenoids, extends our knowledge about ABA biosynthesis, and provides new insights into plant apocarotenoid metabolic networks.

Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga -resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed–germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibitor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.

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.

The obligate hemiparasite Striga hermonthica is one of the major global biotic threats to agriculture in sub-Saharan Africa, causing severe yield losses of cereals. The germination of Striga seeds relies on host-released signaling molecules, mainly strigolactones (SLs). This dependency opens up the possibility of deploying SL analogs as “suicidal germination agents” to reduce the accumulated seed bank of Striga in infested soils. Although several synthetic SL analogs have been developed for this purpose, the utility of these compounds in realizing the suicidal germination strategy for combating Striga is still largely unknown. Here, we evaluated the efficacy of three potent SL analogs (MP3, MP16, and Nijmegen-1) under laboratory, greenhouse, and farmer’s field conditions. All investigated analogs showed around a 50% Striga germination rate, equivalent to a 50% reduction in infestation, which was comparable to the standard SL analog GR24. Importantly, MP16 had the maximum reduction of Striga emergence (97%) in the greenhouse experiment, while Nijmegen-1 appeared to be a promising candidate under field conditions, with a 43% and 60% reduction of Striga emergence in pearl millet and sorghum fields, respectively. These findings confirm that the selected SL analogs appear to make promising candidates as simple suicidal agents both under laboratory and real African field conditions, which may support us to improve suicidal germination technology to deplete the Striga seed bank in African agriculture.

Strigolactones (SLs) regulate many developmental processes, including shoot-branching/tillering, and mediate rhizospheric interactions. SLs originate from carlactone (CL) and are structurally diverse, divided into a canonical and a noncanonical subfamily. Rice contains two canonical SLs, 4-deoxyorobanchol (4DO) and orobanchol (Oro), which are common in different plant species. The cytochrome P450 OsMAX1-900 forms 4DO from CL through repeated oxygenation and ring closure, while the homologous enzyme OsMAX1-1400 hydroxylates 4DO into Oro. To better understand the biological function of 4DO and Oro, we generated CRISPR/Cas9 mutants disrupted in OsMAX1-1400 or in both OsMAX1-900 and OsMAX1-1400 . The loss of OsMAX1-1400 activity led to a complete lack of Oro and an accumulation of its precursor 4DO. Moreover, Os1400 mutants showed shorter plant height, panicle and panicle base length, but no tillering phenotype. Hormone quantification and transcriptome analysis of Os1400 mutants revealed elevated auxin levels and changes in the expression of auxin-related, as well as of SL biosynthetic genes. Interestingly, the Os900/1400 double mutant lacking both Oro and 4DO did not show the observed Os1400 architectural phenotypes, indicating their being a result of 4DO accumulation. Treatment of wild-type plants with 4DO confirmed this assumption. A comparison of the Striga seed germinating activity and the mycorrhization of Os900, Os900/1400 , and Os1400 loss-of-function mutants demonstrated that the germination activity positively correlates with 4DO content while disrupting OsMAX1-1400 has a negative impact on mycorrhizal symbiosis. Taken together, our paper deciphers the biological function of canonical SLs in rice and reveals their particular contributions to establishing architecture and rhizospheric communications.

Striga hermonthica, a member of the Orobanchaceae family, is an obligate root parasite of staple cereal crops, which poses a tremendous threat to food security, contributing to malnutrition and poverty in many African countries. Depleting Striga seed reservoirs from infested soils is one of the crucial approaches to minimize subterranean damage to crops. The dependency of Striga germination on the host-released strigolactones (SLs) has prompted the development of the “Suicidal Germination” strategy to reduce the accumulated seed bank of Striga. The success of aforementioned strategy depends not only on the activity of the applied SL analogs, but also requires suitable application protocol with simple, efficient, and handy formulation for rain-fed African agriculture. Here, we developed a new formulation “Emulsifiable Concentration (EC)” for the two previously field-assessed SL analogs Methyl phenlactonoate 3 (MP3) and Nijmegen-1. The new EC formulation was evaluated for biological activities under lab, greenhouse, mini-field, and field conditions in comparison to the previously used Atlas G-1086 formulation. The EC formulation of SL analogs showed better activities on Striga germination with lower EC₅₀ and high stability under Lab conditions. Moreover, EC formulated SL analogs at 1.0 µM concentrations reduced 89–99% Striga emergence in greenhouse. The two EC formulated SL analogs showed also a considerable reduction in Striga emergence in mini-field and field experiments. In conclusion, we have successfully developed a desired formulation for applying SL analogs as suicidal agents for large-scale field application. The encouraging results presented in this study pave the way for integrating the suicidal germination approach in sustainable Striga management strategies for African agriculture.