Evaluation of granular formulated strigolactone analogs for Striga suicidal germination.

BACKGROUND: Striga hermonthica, an obligate root parasitic weed, poses significant threat to cereal production in sub-Saharan Africa. Lowering Striga seed bank in infested soils is a promising strategy to mitigate infestation levels. The dependency of Striga seed germination on strigolactones opens up the possibility of a "suicidal germination" approach, where synthetic germination stimulants induce lethal germination in the absence of a host. Implementing this approach requires active germination stimulants with a suitable formulation for field application. Here, we describe the development of slow-releasing granular formulation of two potent germination stimulants 'Methyl Phenlactonoate 3' and 'Nijmegen-1' and the assessment of their activity under Lab, greenhouse, mini-field, and field conditions. RESULTS: Under laboratory conditions, the granular formulation of either of the two germination stimulants (1.25 mg per plate, corresponding to 0.09 mg a.i.) induced Striga seed germination at a rate of up to 43%. With 10 mg granular product (0.75 mg a.i.) per pot, we observed 77-83% reduction in Striga emergence under greenhouse pot conditions. Application of the formulated stimulants under artificially or naturally infested fields resulted in approximately 56%, 60%, and 72% reduction in Striga emergence in maize, sorghum, and millet fields in Kenya and Burkina Faso, respectively. CONCLUSION: Our findings on the newly designed granular formulation of Methyl Phenlactonoate 3 and Nijmegen-1 reveal encouraging prospects for addressing the Striga problem in Africa. These findings underscore several significant advantages of the formulated stimulants, including suitability for the African agricultural context, and, most importantly, their effectiveness in reducing Striga infection. This article is protected by copyright. All rights reserved.

Distinguishing the functions of canonical strigolactones as rhizospheric signals.

Strigolactones (SLs) act as regulators of plant architecture as well as signals in rhizospheric communications. Reduced availability of minerals, particularly phosphorus, leads to an increase in the formation and release of SLs that enable adaptation of root and shoot architecture to nutrient limitation and, simultaneously, attract arbuscular mycorrhizal fungi (AMF) for establishing beneficial symbiosis. Based on their chemical structure, SLs are designated as either canonical or non-canonical; however, the question of whether the two classes are also distinguished in their biological functions remained largely elusive until recently. In this review we summarize the latest advances in SL biosynthesis and highlight new findings pointing to rhizospheric signaling as the major function of canonical SLs.

Strigolactone biosynthesis in rice can occur via a 9-cis-3-OH-10'-apo-ß-carotenal intermediate.

Strigolactones (SLs) play a crucial role in regulating plant architecture and mediating rhizosphere interactions. They are synthesized from all-trans-ß-carotene converted into the intermediate carlactone (CL) via the intermediate 9-cis-ß-apo-10'-carotenal. Recent studies indicate that plants can also synthesize 3-OH-CL from all-trans-ß-zeaxanthin via the intermediate 9-cis-3-OH-ß-apo-10'-carotenal. However, the question of whether plants can form bioactive SLs from 9-cis-3-OH-ß-apo-10'-carotenal remains elusive. In this study, we supplied the 13 C-labeled 9-cis-3-OH-ß-apo-10'-carotenal to rice seedlings and monitored the synthesis of SLs using liquid chromatography-mass spectrometry (LC-MS) and Striga bioassay. We further validated the biological activity of 9-cis-3-OH-ß-apo-10'-carotenal-derived SLs using the ccd7/d17 SL-deficient mutant, which demonstrated increased Striga seed-germinating activity and partial rescue of tiller numbers and plant height. Our results establish 9-cis-3-OH-ß-apo-10'-carotenal as a significant SL biosynthetic intermediate with implications for understanding plant hormonal functions and potential applications in agriculture.

Integration of rice apocarotenoid profile and expression pattern of Carotenoid Cleavage Dioxygenases reveals a positive effect of ß-ionone on mycorrhization.

Carotenoids are susceptible to degrading processes initiated by oxidative cleavage reactions mediated by Carotenoid Cleavage Dioxygenases that break their backbone, leading to products called apocarotenoids. These carotenoid-derived metabolites include the phytohormones abscisic acid and strigolactones, and different signaling molecules and growth regulators, which are utilized by plants to coordinate many aspects of their life. Several apocarotenoids have been recruited for the communication between plants and arbuscular mycorrhizal (AM) fungi and as regulators of the establishment of AM symbiosis. However, our knowledge on their biosynthetic pathways and the regulation of their pattern during AM symbiosis is still limited. In this study, we generated a qualitative and quantitative profile of apocarotenoids in roots and shoots of rice plants exposed to high/low phosphate concentrations, and upon AM symbiosis in a time course experiment covering different stages of growth and AM development. To get deeper insights in the biology of apocarotenoids during this plant-fungal symbiosis, we complemented the metabolic profiles by determining the expression pattern of CCD genes, taking advantage of chemometric tools. This analysis revealed the specific profiles of CCD genes and apocarotenoids across different stages of AM symbiosis and phosphate supply conditions, identifying novel reliable markers at both local and systemic levels and indicating a promoting role of ß-ionone in AM symbiosis establishment.

Abscisic acid inhibits germination of Striga seeds and is released by them likely as a rhizospheric signal supporting host infestation.

Seeds of the root parasitic plant Striga hermonthica undergo a conditioning process under humid and warm environments before germinating in response to host-released stimulants, particularly strigolactones (SLs). The plant hormone abscisic acid (ABA) regulates different growth and developmental processes, and stress response; however, its role during Striga seed germination and early interactions with host plants is under-investigated. Here, we show that ABA inhibited Striga seed germination and that hindering its biosynthesis induced conditioning and germination in unconditioned seeds, which was significantly enhanced by treatment with the SL analog rac-GR24. However, the inhibitory effect of ABA remarkably decreased during conditioning, confirming the loss of sensitivity towards ABA in later developmental stages. ABA measurement showed a substantial reduction of its content during the early conditioning stage and a significant increase upon rac-GR24-triggered germination. We observed this increase also in released seed exudates, which was further confirmed by using the Arabidopsis ABA-reporter GUS marker line. Seed exudates of germinated seeds, containing elevated levels of ABA, impaired the germination of surrounding Striga seeds in vitro and promoted root growth of a rice host towards germinated Striga seeds. Application of ABA as a positive control caused similar effects, indicating its function in Striga/Striga and Striga/host communications. In summary, we show that ABA is an essential player during seed dormancy and germination processes in Striga and acts as a rhizospheric signal likely to support host infestation.

Disruption of the rice 4-DEOXYOROBANCHOL HYDROXYLASE unravels specific functions of canonical strigolactones.

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.

Zaxinone mimics (MiZax) efficiently promote growth and production of potato and strawberry plants under desert climate conditions.

Climate changes and the rapid expanding human population have become critical concerns for global food security. One of the promising solutions is the employment of plant growth regulators (PGRs) for increasing crop yield and overcoming adverse growth conditions, such as desert climate. Recently, the apocarotenoid zaxinone and its two mimics (MiZax3 and MiZax5) have shown a promising growth-promoting activity in cereals and vegetable crops under greenhouse and field conditions. Herein, we further investigated the effect of MiZax3 and MiZax5, at different concentrations (5 and 10 µM in 2021; 2.5 and 5 µM in 2022), on the growth and yield of the two valuable vegetable crops, potato and strawberry, in the Kingdom of Saudi of Arabia. Application of both MiZax significantly increased plant agronomic traits, yield components and total yield, in five independent field trials from 2021 to 2022. Remarkably, the amount of applied MiZax was far less than humic acid, a widely applied commercial compound used here for comparison. Hence, our results indicate that MiZax are very promising PGRs that can be applied to promote the growth and yield of vegetable crops even under desert conditions and at relatively low concentrations.

New Series of Zaxinone Mimics (MiZax) for Fundamental and Applied Research.

The apocarotenoid zaxinone is a recently discovered regulatory metabolite required for proper rice growth and development. In addition, zaxinone and its two mimics (MiZax3 and MiZax5) were shown to have a remarkable growth-promoting activity on crops and a capability to reduce infestation by the root parasitic plant Striga through decreasing strigolactone (SL) production, suggesting their potential for application in agriculture and horticulture. In the present study, we developed a new series of MiZax via structural modification of the two potent zaxinone mimics (MiZax3 and MiZax5) and evaluated their effect on plant growth and Striga infestation. In general, the structural modifications to MiZax3 and MiZax5 did not additionally improve their overall performance but caused an increase in certain activities. In conclusion, MiZax5 and especially MiZax3 remain the likely most efficient zaxinone mimics for controlling Striga infestation.

Disruption of the cytochrome CYP711A5 gene reveals MAX1 redundancy in rice strigolactone biosynthesis.

Strigolactones (SLs) inhibit shoot branching/tillering and are secreted by plant roots as a signal to attract symbiotic mycorrhizal fungi in the rhizosphere, particularly under phosphate starvation. However, SLs are also hijacked by root parasitic weeds as inducer for the germination of their seeds. There are around 35 natural SLs divided, based on their structures, into canonical and non-canonical SLs. Cytochrome P450 enzymes of the 711 clade, such as MORE AXILLARY GROWTH1 (MAX1) in Arabidopsis, are a major driver of SL structural diversity. Monocots, such as rice, contain several MAX1 homologs that participate in SL biosynthesis. To investigate the function of OsMAX1-1900 in planta, we generated CRISPR/Cas9 mutants disrupted in the corresponding gene. Characterizing of the generated mutants at metabolite and phenotype level suggests that OsMAX1-1900 loss-of-function does neither affect the SL pattern nor rice architecture, indicating functional redundancy among rice MAX1 homologs.

Biomimetic Mineralization for Smart Biostimulant Delivery and Crop Micronutrients Fortification.

Sustainable and precise fortification practices are necessary to ensure food security for the increasing human population. Precision agriculture aims to minimize the use of fertilizers and pesticides by developing smart materials for real-life agricultural practices. Here, we show that biomimetic mineralization can be efficiently employed to encapsulate and controllably release plant biostimulants (MiZax-3) to improve the quality and yield of capsicum (Capsicum annum) crops in field experiments. ZIF-8 encapsulation of MiZax-3 (MiZIFs) could significantly enhance its stability up to around 679 times (6p value = 0.0072) at field conditions. Our results demonstrate that the coordinating Zn ions and the MiZax-3 play a vital role in improving Zn content in the produced fruits by 2-fold, which is the first report of this nature on Zn content in fruits. We envision this platform as a starting point to investigate other biocompatible coordination-based platforms for micronutrient delivery in precision agriculture.

Does zaxinone counteract strigolactones in shaping rice architecture?

The cleavage of plant carotenoids leads to apocarotenoids, a group of metabolites including precursors of the hormones strigolactones (SLs) and abscisic acid, regulatory and signaling molecules. Zaxinone is a recently discovered apocarotenoid growth regulator that improves growth and suppress SL biosynthesis in rice (Oryza sativa). To test if zaxinone also counteracts the growth regulatory effects of SLs in rice, we co-supplied zaxinone and the synthetic SL analog rac-GR24 to the rice SL-deficient DWARF17 (d17) mutant. Results showed that co-application of GR24 and zaxinone still rescued d17 phenotype, indicating that zaxinone and GR24 act independently in regulating root and shoot growth and development in rice.

The Arabidopsis D27-LIKE1 is a cis/cis/trans-ß-carotene isomerase that contributes to Strigolactone biosynthesis and negatively impacts ABA level.

The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-ß-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-ß-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely a result of its formation of 9-cis-ß-carotene that was present at higher levels in AtD27-like1 overexpressing lines. By contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis-ß-carotene isomerase that affects the content of both cis-carotenoid-derived plant hormones, ABA and SLs.

ZAXINONE SYNTHASE 2 regulates growth and arbuscular mycorrhizal symbiosis in rice.

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.

Perspectives on the metabolism of strigolactone rhizospheric signals.

Strigolactones (SLs) are a plant hormone regulating different processes in plant development and adjusting plant's architecture to nutrition availability. Moreover, SLs are released by plants to communicate with beneficial fungi in the rhizosphere where they are, however, abused as chemical cues inducing seed germination of root parasitic weeds, e.g. Striga spp., and guiding them towards host plants in their vicinity. Based on their structure, SLs are divided into canonical and non-canonical SLs. In this perspective, we describe the metabolism of root-released SLs and SL pattern in rice max1-900 mutants, which are affected in the biosynthesis of canonical SLs, and show the accumulation of two putative non-canonical SLs, CL+30 and CL+14. Using max1-900 and SL-deficient d17 rice mutants, we further investigated the metabolism of non-canonical SLs and their possible biological roles. Our results show that the presence and further metabolism of canonical and non-canonical SLs are particularly important for their role in rhizospheric interactions, such as that with root parasitic plants. Hence, we proposed that the root-released SLs are mainly responsible for rhizospheric communications and have low impact on plant architecture, which makes targeted manipulation of root-released SLs an option for rhizospheric engineering.

Metabolomics of plant root exudates: From sample preparation to data analysis.

Plants release a set of chemical compounds, called exudates, into the rhizosphere, under normal conditions and in response to environmental stimuli and surrounding soil organisms. Plant root exudates play indispensable roles in inhibiting the growth of harmful microorganisms, while also promoting the growth of beneficial microbes and attracting symbiotic partners. Root exudates contain a complex array of primary and specialized metabolites. Some of these chemicals are only found in certain plant species for shaping the microbial community in the rhizosphere. Comprehensive understanding of plant root exudates has numerous applications from basic sciences to enhancing crop yield, production of stress-tolerant crops, and phytoremediation. This review summarizes the metabolomics workflow for determining the composition of root exudates, from sample preparation to data acquisition and analysis. We also discuss recent advances in the existing analytical methods and future perspectives of metabolite analysis.

Canonical strigolactones are not the major determinant of tillering but important rhizospheric signals in rice.

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.

9-cis-ß-Apo-10'-carotenal is the precursor of strigolactones in planta.

MAIN CONCLUSION: 13C-isotope feeding experiments demonstrate that the apocarotenoid 9-cis-ß-apo-10'-carotenal is the precursor of several strigolactones in rice, providing a direct, in planta evidence for its role in strigolactone biosynthesis. Strigolactones (SLs) are plant hormone that regulates plant architecture and mediates rhizospheric communications. Previous in vitro studies using heterogously produced enzymes unraveled the conversion of all-trans-ß-carotene via the intermediate 9-cis-ß-apo-10'-carotenal into the SL precursor carlactone. However, a direct evidence for the formation of SLs from 9-cis-ß-apo-10'-carotenal is still missing. To provide this evidence, we supplied rice seedlings with 13C-labeled 9-cis-ß-apo-10'-carotenal and analyzed their SLs by LC-MS. Our results show that 9-cis-ß-apo-10'-carotenal is the SL precursor in planta and reveal, for the first time, the application of labeled long-chain apocarotenoids as a promising approach to investigate apocarotenoid metabolism and the genesis of carotenoid-derived growth regulators and signaling molecules.

Zaxinone synthase controls arbuscular mycorrhizal colonization level in rice.

The Oryza sativa (rice) carotenoid cleavage dioxygenase OsZAS was described to produce zaxinone, a plant growth-promoting apocarotenoid. A zas mutant line showed reduced arbuscular mycorrhizal (AM) colonization, but the mechanisms underlying this behavior are unknown. Here, we investigated how OsZAS and exogenous zaxinone treatment regulate mycorrhization. Micromolar exogenous supply of zaxinone rescued root growth but not the mycorrhizal defects of the zas mutant, and even reduced mycorrhization in wild-type and zas genotypes. The zas line did not display the increase in the level of strigolactones (SLs) that was observed in wild-type plants at 7 days post-inoculation with AM fungus. Moreover, exogenous treatment with the synthetic SL analog GR24 rescued the zas mutant mycorrhizal phenotype, indicating that the lower AM colonization rate of zas is caused by a deficiency in SLs at the early stages of the interaction, and indicating that during this phase OsZAS activity is required to induce SL production, possibly mediated by the Dwarf14-Like (D14L) signaling pathway. OsZAS is expressed in arbuscule-containing cells, and OsPT11prom::OsZAS transgenic lines, where OsZAS expression is driven by the OsPT11 promoter active in arbusculated cells, exhibit increased mycorrhization compared with the wild type. Overall, our results show that the genetic manipulation of OsZAS activity in planta leads to a different effect on AM symbiosis from that of exogenous zaxinone treatment, and demonstrate that OsZAS influences the extent of AM colonization, acting as a component of a regulatory network that involves SLs.