Strigolactone deficiency induces jasmonate, sugar and flavonoid phytoalexin accumulation enhancing rice defense against the blast fungus Pyricularia oryzae.

Strigolactones (SLs) are carotenoid-derived phytohormones that regulate plant growth and development. While root-secreted SLs are well-known to facilitate plant symbiosis with beneficial microbes, the role of SLs in plant interactions with pathogenic microbes remains largely unexplored. Using genetic and biochemical approaches, we demonstrate a negative role of SLs in rice (Oryza sativa) defense against the blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae). We found that SL biosynthesis and perception mutants, and wild-type (WT) plants after chemical inhibition of SLs, were less susceptible to P. oryzae. Strigolactone deficiency also resulted in a higher accumulation of jasmonates, soluble sugars and flavonoid phytoalexins in rice leaves. Likewise, in response to P. oryzae infection, SL signaling was downregulated, while jasmonate and sugar content increased markedly. The jar1 mutant unable to synthesize jasmonoyl-l-isoleucine, and the coi1-18 RNAi line perturbed in jasmonate signaling, both accumulated lower levels of sugars. However, when WT seedlings were sprayed with glucose or sucrose, jasmonate accumulation increased, suggesting a reciprocal positive interplay between jasmonates and sugars. Finally, we showed that functional jasmonate signaling is necessary for SL deficiency to induce rice defense against P. oryzae. We conclude that a reduction in rice SL content reduces P. oryzae susceptibility by activating jasmonate and sugar signaling pathways, and flavonoid phytoalexin accumulation.

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.

Strigolactone mimic 2-nitrodebranone is highly active in Arabidopsis growth and development.

Strigolactones play crucial roles in regulating plant architecture and development, as endogenous hormones, and orchestrating symbiotic interactions with fungi and parasitic plants, as components of root exudates. rac-GR24 is currently the most widely used strigolactone analog and serves as a reference compound in investigating the action of strigolactones. In this study, we evaluated a suite of debranones and found that 2-nitrodebranone (2NOD) exhibited higher biological activity than rac-GR24 in various aspects of plant growth and development in Arabidopsis, including hypocotyl elongation inhibition, root hair promotion and senescence acceleration. The enhanced activity of 2NOD in promoting AtD14-SMXL7 and AtD14-MAX2 interactions indicates that the molecular structure of 2NOD is a better match for the ligand perception site pocket of D14. Moreover, 2NOD showed lower activity than rac-GR24 in promoting Orobanche cumana seed germination, suggesting its higher ability to control plant architecture than parasitic interactions. In combination with the improved stability of 2NOD, these results demonstrate that 2NOD is a strigolactone analog that can specifically mimic the activity of strigolactones and that 2NOD exhibits strong potential as a tool for studying the strigolactone signaling pathway in plants.

Transcriptional Analysis in the Arabidopsis Roots Reveals New Regulators that Link rac-GR24 Treatment with Changes in Flavonol Accumulation, Root Hair Elongation and Lateral Root Density.

The synthetic strigolactone (SL) analog, rac-GR24, has been instrumental in studying the role of SLs as well as karrikins because it activates the receptors DWARF14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2) of their signaling pathways, respectively. Treatment with rac-GR24 modifies the root architecture at different levels, such as decreasing the lateral root density (LRD), while promoting root hair elongation or flavonol accumulation. Previously, we have shown that the flavonol biosynthesis is transcriptionally activated in the root by rac-GR24 treatment, but, thus far, the molecular players involved in that response have remained unknown. To get an in-depth insight into the changes that occur after the compound is perceived by the roots, we compared the root transcriptomes of the wild type and the more axillary growth2 (max2) mutant, affected in both SL and karrikin signaling pathways, with and without rac-GR24 treatment. Quantitative reverse transcription (qRT)-PCR, reporter line analysis and mutant phenotyping indicated that the flavonol response and the root hair elongation are controlled by the ELONGATED HYPOCOTYL 5 (HY5) and MYB12 transcription factors, but HY5, in contrast to MYB12, affects the LRD as well. Furthermore, we identified the transcription factors TARGET OF MONOPTEROS 5 (TMO5) and TMO5 LIKE1 as negative and the Mediator complex as positive regulators of the rac-GR24 effect on LRD. Altogether, hereby, we get closer toward understanding the molecular mechanisms that underlay the rac-GR24 responses in the root.

Ancestral sequence reconstruction of the CYP711 family reveals functional divergence in strigolactone biosynthetic enzymes associated with gene duplication events in monocot grasses.

The strigolactone (SL) class of phytohormones shows broad chemical diversity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the diversification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several diversifying oxidation reactions on the structure. Evidence was obtained for functional divergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone diversification in early land plants, which may have extended to the diversification of strigol-type SLs.

Transcriptome analysis reveals the effects of strigolactone on shoot regeneration of apple.

KEY MESSAGE: We have demonstrated that strigolactone inhibitor, Tis108, could be used to improve shoot regeneration of apple, and provided insights into the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation. Lack of an efficient transformation system largely stagnated the application of transgenic and CRISPR technology in apple rootstock. High shoot regeneration ability is an important basis for establishing an effective transformation system. In this study, we first demonstrated the inhibitory effects of strigolactones on the adventitious shoot formation of apple rootstock M26. Next, we successfully verified that strigolactone-biosynthesis inhibitor, Tis108, could be used to improve the shoot regeneration of woody plants. Our results also suggest strigolactone-biosynthesis gene, MdCCD7, can be a target gene for biotechnological improvements of shoot regeneration capacity. Furthermore, we have employed transcriptome analysis to reveal the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation. Differentially expressed genes associated with photosynthesis, secondary growth, and organ development were identified. WGCNA suggests SLs might affect shoot regeneration through interaction with other hormones, especially, auxin, cytokinin, and ethylene. We were able to identify important candidate genes mediating the cross-talk between strigolactone and other hormones during the process of adventitious shoot formation. Overall, our findings not only propose a useful chemical for improving shoot regeneration in practice but also provide insights into the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation.

Strigolactones Modulate Salicylic Acid-Mediated Disease Resistance in Arabidopsis thaliana.

Strigolactones are low-molecular-weight phytohormones that play several roles in plants, such as regulation of shoot branching and interactions with arbuscular mycorrhizal fungi and parasitic weeds. Recently, strigolactones have been shown to be involved in plant responses to abiotic and biotic stress conditions. Herein, we analyzed the effects of strigolactones on systemic acquired resistance induced through salicylic acid-mediated signaling. We observed that the systemic acquired resistance inducer enhanced disease resistance in strigolactone-signaling and biosynthesis-deficient mutants. However, the amount of endogenous salicylic acid and the expression levels of salicylic acid-responsive genes were lower in strigolactone signaling-deficient max2 mutants than in wildtype plants. In both the wildtype and strigolactone biosynthesis-deficient mutants, the strigolactone analog GR24 enhanced disease resistance, whereas treatment with a strigolactone biosynthesis inhibitor suppressed disease resistance in the wildtype. Before inoculation of wildtype plants with pathogenic bacteria, treatment with GR24 did not induce defense-related genes; however, salicylic acid-responsive defense genes were rapidly induced after pathogenic infection. These findings suggest that strigolactones have a priming effect on Arabidopsis thaliana by inducing salicylic acid-mediated disease resistance.

Contribution of strigolactone in plant physiology, hormonal interaction and abiotic stresses.

Strigolactones (SLs) are carotenoid-derived molecules, which regulate various developmental and adaptation processes in plants. These are engaged in different aspects of growth such as development of root, leaf senescence, shoot branching, etc. Plants grown under nutrient-deficient conditions enhance SL production that facilitates root architecture and symbiosis of arbuscular mycorrhizal fungi, as a result increases nutrient uptake. The crosstalk of SLs with other phytohormones such as auxin, abscisic acid, cytokinin and gibberellins, in response to abiotic stresses indicates that SLs actively contribute to the regulatory systems of plant stress adaptation. In response to different environmental circumstances such as salinity, drought, heat, cold, heavy metals and nutrient deprivation, these SLs get accumulated in plant tissues. Strigolactones regulate multiple hormonal responsive pathways, which aids plants to surmount stressful environmental constraints as well as reduce negative impact on overall productivity of crops. The external application of SL analog GR24 for its higher bioaccumulation can be one of the possible approaches for establishing various abiotic stress tolerances in plants.

Strigolactone signaling inhibition increases adventitious shoot formation on internodal segments of ipecac.

MAIN CONCLUSION: SL inhibited adventitious shoot formation of ipecac, whereas the SL-related inhibitors promoted adventitious shoot formation. SL-related inhibitors might be useful as new plant growth regulators for plant propagation. In most plant species, phytohormones are required to induce adventitious shoots for propagating economically important crops and regenerating transgenic plants. In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), however, adventitious shoots can be formed without phytohormone treatment. Here we evaluated the effects of GR24 (a synthetic strigolactone, SL), SL biosynthetic inhibitors, and an SL antagonist on adventitious shoot formation during tissue culture of ipecac. We found that exogenously applied GR24 suppressed indole-3-acetic acid transport in internodal segments and decreased the number of adventitious shoots formed; in addition, the distribution of adventitious shoots changed from the apical to middle region of the internodal segments. In contrast, the SL-related inhibitors promoted adventitious shoot formation on both apical and middle regions of the segments. In particular, SL antagonist treatment increased endogenous cytokinin levels and induced multiple shoot development. These results indicate that SL inhibits adventitious shoot formation in ipecac. In ipecac, one of the shoots in each internodal segment becomes dominant and auxin derived from that shoot suppresses the other shoot growth. Here, this dominance was overcome by application of SL-related inhibitors. Therefore, SL-related inhibitors might be useful as new plant growth regulators to improve the efficiency of plant propagation in vitro.

Exogenous strigolactones impact metabolic profiles and phosphate starvation signalling in roots.

Strigolactones (SLs) are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also affecting plant interactions with harmful organisms. They are also plant hormones in-planta, acting as modulators of plant responses under nutrient-deficient conditions, mainly phosphate (Pi) starvation. In the present work, we investigate the potential role of SLs as regulators of early Pi starvation signalling in plants. A short-term pulse of the synthetic SL analogue 2'-epi-GR24 promoted SL accumulation and the expression of Pi starvation markers in tomato and wheat under Pi deprivation. 2'-epi-GR24 application also increased SL production and the expression of Pi starvation markers under normal Pi conditions, being its effect dependent on the endogenous SL levels. Remarkably, 2'-epi-GR24 also impacted the root metabolic profile under these conditions, promoting the levels of metabolites associated to plant responses to Pi limitation, thus partially mimicking the pattern observed under Pi deprivation. The results suggest an endogenous role for SLs as Pi starvation signals. In agreement with this idea, SL-deficient plants were less sensitive to this stress. Based on the results, we propose that SLs may act as early modulators of plant responses to P starvation.

Strigolactones and their crosstalk with other phytohormones.

BACKGROUND: Strigolactones (SLs) are a diverse class of butenolide-bearing phytohormones derived from the catabolism of carotenoids. They are associated with an increasing number of emerging regulatory roles in plant growth and development, including seed germination, root and shoot architecture patterning, nutrient acquisition, symbiotic and parasitic interactions, as well as mediation of plant responses to abiotic and biotic cues. SCOPE: Here, we provide a concise overview of SL biosynthesis, signal transduction pathways and SL-mediated plant responses with a detailed discourse on the crosstalk(s) that exist between SLs/components of SL signalling and other phytohormones such as auxins, cytokinins, gibberellins, abscisic acid, ethylene, jasmonates and salicylic acid. CONCLUSION: SLs elicit their control on physiological and morphological processes via a direct or indirect influence on the activities of other hormones and/or integrants of signalling cascades of other growth regulators. These, among many others, include modulation of hormone content, transport and distribution within plant tissues, interference with or complete dependence on downstream signal components of other phytohormones, as well as acting synergistically or antagonistically with other hormones to elicit plant responses. Although much has been done to evince the effects of SL interactions with other hormones at the cell and whole plant levels, research attention must be channelled towards elucidating the precise molecular events that underlie these processes. More especially in the case of abscisic acid, cytokinins, gibberellin, jasmonates and salicylic acid for which very little has been reported about their hormonal crosstalk with SLs.

[Effect of GR24 on accumulation of diterpenoids in Tripterygium wilfordii suspension cells].

Terpenoids are main bioactive components in Tripterygium wilfordii,but the contents of some terpenoids are relatively low. In order to provide scientific evidence for the regulation of terpenoids in T. wilfordii,this research explored the effect of GR24 on accumulations of four diterpenoids( triptolide,tripterifordin,triptophenolide,and triptinin B) in T. wilfordii suspension cells by biological technology and UPLC-QQQ-MS/MS. The results indicated that 100 µmol·L-1 GR24 inhibited the accumulations of triptolide,tripterifordin,triptophenolide,and triptinin B to different degrees. Compared with the control group,the contents of 4 diterpenoids( in the induced group) were down to 96.59%,63.80%,61.02% and 33.59% in 240 h,respectively. Among them,the accumulation of triptinin B iswas significantly inhibited. In addition,the key time point of inhibitory effect was 120 h after induction with GR24 in some diterpenoids. This is the first systematic study focusing on the effect of GR24 on the accumulations of diterpenoids in T. wilfordii suspension cells. The dynamic accumulation ruleregularity of four diterpenoids after induced by GR24 was summarized,which laid a foundation for further study on the chemical response mechanism of terpenoids to GR24.

Largely additive effects of gibberellin and strigolactone on gene expression in Arabidopsis thaliana seedlings.

The phytohormones gibberellin (GA) and strigolactone (SL) are involved in essential processes in plant development. Both GA and SL signal transduction mechanisms employ α/ß-hydrolase-derived receptors that confer E3 ubiquitin ligase-mediated protein degradation processes. This suggests a common evolutionary origin of these pathways and possibly a molecular interaction between them. One such indication stems from rice, where the DELLA protein of the GA pathway was reported to interact with the SL receptor. Here, we examine the physiological interaction between both pathways through the analysis of GA (ga1) and SL biosynthesis (max1 and max3) mutants. In ga1 max double mutants, we find indications only for additive interactions when examining several phenotypic readouts. We further identify short-term transcriptional responses to GA and the synthetic SL rac-GR24 through next-generation sequencing of poly-adenylated RNAs (RNA-seq) in ga1 max1. Remarkably, both hormones lead to predominantly additive transcriptional changes of a largely overlapping set of genes. The expression of only a few genes was altered in a synergistic manner but, interestingly, these include the genes encoding the GA catabolic enzyme GA2 OXIDASE2 (GA2ox2) as well as the SL pathway regulators BRANCHED1 (BRC1) and SUPPRESSOR OF max2 1-LIKE8 (SMXL8). We conclude that GA and rac-GR24 signaling in Arabidopsis seedlings converge at the level of transcription of a common gene-set.

Identification of genes involved in fungal responses to strigolactones using mutants from fungal pathogens.

Strigolactones (SLs) as components of root exudates induce hyphal branching of arbuscular mycorrhizal (AM) fungi which is thought to favor the establishment of the beneficial symbiosis. Little is known on how AM fungi respond to SLs. Since AM fungi are poor model systems due to their obligate biotrophism and the lack of genetic transformation protocols, we took advantage of the sensitivity of several phytopathogenic fungi to GR24, a synthetic SLs analog. With the aim to identify the molecular determinants involved in SLs response in AM fungi and assuming conserved mechanisms in the fungal kingdom, we exploited the fungal pathogens Botrytis cinerea and Cryphonectria parasitica, for which mutant collections are available. Exposure of B. cinerea and C. parasitica to GR24 embedded in solid medium led to reduction of fungal radial growth. We set up the screening of a set of well-characterized gene deletion mutants to isolate genotypes with altered responses to SLs. Two B. cinerea mutants (defective of BcTrr1, a thioredoxin reductase and BcLTF1, a GATA transcription factor) turned out to be less responsive to GR24. One feature shared by the two mutants is the overproduction of reactive oxygen species (ROS). Indeed, an oxidizing effect was observed in a B. cinerea strain expressing a redox-sensitive GFP2 in the mitochondrial intermembrane space upon exposure to GR24. ROS and mitochondria are, therefore, emerging as mediators of SLs actions.

Strigolactones affect tomato hormone profile and somatic embryogenesis.

MAIN CONCLUSIONS: Exogenously applied GR24 affected somatic embryo formation and morphogenesis of strigolactone-deficient tomato mutant through cross-talk with auxins and cytokinins indicating involvement of SLs in the embryogenic process. Strigolactones (SLs) mediate the regulation of plant responses to the environment through cross-talk with other plant hormones, especially auxins. Auxins play a crucial role in coordinating the morphogenesis and development of plant reproductive organs, including the signal-transduction cascade leading to the reprogramming of gene-expression patterns before embryo formation. SLs' role in these processes is unknown, in contrast to their proven involvement in auxin transport and distribution. We used tomato cv. M82 and its SL-deficient mutant SL-ORT1 to study the influence of SLs on hormone profile in tomato roots and shoots, and their involvement in somatic embryogenesis (SE) and morphogenesis (adventitious root formation). The synthetic SL GR24 had different effects on SE of M82 and SL-ORT1, indicating that SLs influence the cytokinin-to-auxin ratio in tomato SE.

The Whats, the Wheres and the Hows of strigolactone action in the roots.

MAIN CONCLUSION: Strigolactones control various aspects of plant development, including root architecture. Here, we review how strigolactones act in the root and survey the strigolactone specificity of signaling components that affect root development. Strigolactones are a group of secondary metabolites produced in plants that have been assigned multiple roles, of which the most recent is hormonal activity. Over the last decade, these compounds have been shown to regulate various aspects of plant development, such as shoot branching and leaf senescence, but a growing body of literature suggests that these hormones play an equally important role in the root. In this review, we present all known root phenotypes linked to strigolactones. We examine the expression and presence of the main players in biosynthesis and signaling of these hormones and bring together the available information that allows us to explain how strigolactones act to modulate the root system architecture.

Functional redundancy in the control of seedling growth by the karrikin signaling pathway.

MAIN CONCLUSION: SMAX1 and SMXL2 control seedling growth, demonstrating functional redundancy within a gene family that mediates karrikin and strigolactone responses. Strigolactones (SLs) are plant hormones with butenolide moieties that control diverse aspects of plant growth, including shoot branching. Karrikins (KARs) are butenolide molecules found in smoke that enhance seed germination and seedling photomorphogenesis. In Arabidopsis thaliana, SLs and KARs signal through the α/ß hydrolases D14 and KAI2, respectively. The F-box protein MAX2 is essential for both signaling pathways. SUPPRESSOR OF MAX2 1 (SMAX1) plays a prominent role in KAR-regulated growth downstream of MAX2, and SMAX1-LIKE genes SMXL6, SMXL7, and SMXL8 mediate SL responses. We previously found that smax1 loss-of-function mutants display constitutive KAR response phenotypes, including reduced seed dormancy and hypersensitive growth responses to light in seedlings. However, smax1 seedlings remain slightly responsive to KARs, suggesting that there is functional redundancy in karrikin signaling. SMXL2 is a strong candidate for this redundancy because it is the closest paralog of SMAX1, and because its expression is regulated by KAR signaling. Here, we present evidence that SMXL2 controls hypocotyl growth and expression of the KAR/SL transcriptional markers KUF1, IAA1, and DLK2 redundantly with SMAX1. Hypocotyl growth in the smax1 smxl2 double mutant is insensitive to KAR and SL, and etiolated smax1 smxl2 seedlings have reduced hypocotyl elongation. However, smxl2 has little or no effect on seed germination, leaf shape, or petiole orientation, which appear to be predominantly controlled by SMAX1. Neither SMAX1 nor SMXL2 affect axillary branching or inflorescence height, traits that are under SL control. These data support the model that karrikin and strigolactone responses are mediated by distinct subclades of the SMXL family, and further the case for parallel butenolide signaling pathways that evolved through ancient KAI2 and SMXL duplications.

Global Transcriptomic Analysis Reveals the Mechanism of Phelipanche aegyptiaca Seed Germination.

Phelipanche aegyptiaca is one of the most destructive root parasitic plants of Orobanchaceae. This plant has significant impacts on crop yields worldwide. Conditioned and host root stimulants, in particular, strigolactones, are needed for unique seed germination. However, no extensive study on this phenomenon has been conducted because of insufficient genomic information. Deep RNA sequencing, including de novo assembly and functional annotation was performed on P. aegyptiaca germinating seeds. The assembled transcriptome was used to analyze transcriptional dynamics during seed germination. Key gene categories involved were identified. A total of 274,964 transcripts were determined, and 53,921 unigenes were annotated according to the NR, GO, COG, KOG, and KEGG databases. Overall, 5324 differentially expressed genes among dormant, conditioned, and GR24-treated seeds were identified. GO and KEGG enrichment analyses demonstrated numerous DEGs related to DNA, RNA, and protein repair and biosynthesis, as well as carbohydrate and energy metabolism. Moreover, ABA and ethylene were found to play important roles in this process. GR24 application resulted in dramatic changes in ABA and ethylene-associated genes. Fluridone, a carotenoid biosynthesis inhibitor, alone could induce P. aegyptiaca seed germination. In addition, conditioning was probably not the indispensable stage for P. aegyptiaca, because the transcript level variation of MAX2 and KAI2 genes (relate to strigolactone signaling) was not up-regulated by conditioning treatment.

From lateral root density to nodule number, the strigolactone analogue GR24 shapes the root architecture of Medicago truncatula.

In the rhizosphere, strigolactones not only act as crucial signalling molecules in the communication of plants with parasitic weeds and arbuscular mycorrhiza, but they also play a key role in regulating different aspects of the root system. Here we investigated how strigolactones influence the root architecture of Medicago truncatula. We provide evidence that addition of the synthetic strigolactone analogue GR24 has an inhibitory effect on the lateral root density. Moreover, treatment with GR24 of Sinorhizobium meliloti-inoculated M. truncatula plants affects the nodule number both positively and negatively, depending on the concentration. Plants treated with 0.1 µM GR24 had a slightly increased number of nodules, whereas concentrations of 2 and 5 µM strongly reduced it. This effect was independent of the autoregulation of nodulation mechanism that is controlled by SUPER NUMERIC NODULE. Furthermore, we demonstrate that GR24 controls the nodule number through crosstalk with SICKLE-dependent ethylene signalling. Additionally, because the expression of the nodulation marker EARLY NODULATION11 was strongly reduced in GR24-treated plants, we concluded that strigolactones influence nodulation at a very early stage of the symbiotic interaction.

Regioselective and stereospecific hydroxylation of GR24 by Sorghum bicolor and evaluation of germination inducing activities of hydroxylated GR24 stereoisomers toward seeds of Striga species.

Bioconversion of GR24, the most widely used synthetic strigolactone (SL), by hydroponically grown sorghum (Sorghum bicolor) and biological activities of hydroxylated GR24 stereoisomers were studied. Analysis of extracts and exudates of sorghum roots previously fed with a racemic and diastereomeric mixture of GR24, using liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (MRM), confirmed uptake of GR24 and suggested its conversion to mono-hydroxylated products. Two major GR24 metabolites, 7-hydroxy-GR24 and 8-hydroxy-GR24, were identified in the root extracts and exudates by direct comparison of chromatographic behavior with a series of synthetic mono-hydroxylated GR24 analogues. Separate feeding experiments with each of the GR24 stereoisomers revealed that the hydroxylated products were derived from 2'-epi-GR24, an evidence of sterical recognition of the GR24 molecule by sorghum. Trans-4-hydroxy-GR24 isomers derived from all GR24 stereoisomers were detected in the exudates as minor metabolites. The synthetic hydroxy-GR24 isomers induced germination of Striga hermonthica in decreasing order of C-8>C-7>C-6>C-5>C-4. In contrast the stereoisomers having the same configuration of orobanchol, irrespective of position of hydroxylation, induced germination of Striga gesnerioides. The results confirm previous reports on structural requirements of SLs and ascribe a critical role to hydroxylation, but not to the position of the hydroxyl group in the AB part of the molecule, in induction of S. gesnerioides seed germination.