Synthetic Strigolactone Research Articles

Strigolactone GR24 upregulates target genes of the cytoprotective transcription factor Nrf2 in skeletal muscle

GR24 is a synthetic strigolactone analog, demonstrated to regulate the development of plants and arbuscular mycorrhizal fungi. GR24 possesses anti-cancer and anti-apoptotic properties, enhances insulin sensitivity and mitochondrial biogenesis in skeletal myotubes, inhibits adipogenesis, decreases inflammation in adipocytes and macrophages and downregulates the expression of hepatic gluconeogenic enzymes. Transcription factor Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) is a master regulator of antioxidant response, regulating a multitude of genes involved in cellular stress responses and anti-inflammatory pathways, thus maintaining cellular redox homeostasis. Nrf2 activation reduces the deleterious effects of mitochondrial toxins and has multiple roles in promoting mitochondrial function and dynamics. We studied the role of GR24 on gene expression in rat L6 skeletal muscle cells which were differentiated into myotubes. The myotubes were treated with GR24 and analyzed by microarray gene expression profiling. GR24 upregulated the cytoprotective transcription factor Nrf2 and its target genes, activating antioxidant defences, suggesting that GR24 may protect skeletal muscle from the toxic effects of oxidative stress.

Structural analysis of HTL and D14 proteins reveals the basis for ligand selectivity in Striga

HYPOSENSITIVE TO LIGHT (HTL) and DWARF14 (D14) mediate the perception of karrikin and strigolactone, which stimulates germination of the parasitic weed Striga. However, their role in parasitic seeds is poorly understood, and the basis for their differing responsiveness remains unclear. Here, we show that Striga hermonthica HTL proteins (ShHTLs) in ‘conserved’ and ‘intermediate’ clades are able to bind karrikin. The ‘divergent’ clade is able to hydrolyze strigolactone. Unexpectedly, we find that ShD14 is also capable of hydrolyzing strigolactone. Through comparative analysis of ShHTLs and ShD14 crystal structures, we provide insights into the basis for their selectivity. Moreover, we show that both ShD14 and divergent clade ShHTLs, but not conserved and intermediate clade ShHTLs, can interact with the putative downstream signaling component ShMAX2 in the presence of the synthetic strigolactone, rac-GR24. These findings provide insight into how strigolactone is perceived and how ligand specificity is determined.

Strigolactone GR24 upregulates Nrf2 target genes and may protect against oxidative stress in skeletal muscle

GR24 is a synthetic strigolactone analog, demonstrated to regulate the development of plants and arbuscular mycorrhizal fungi. GR24 possesses anti-cancer and anti-apoptotic properties, enhances insulin sensitivity and mitochondrial biogenesis in skeletal myotubes, inhibits adipogenesis, decreases inflammation in adipocytes and macrophages and downregulates the expression of hepatic gluconeogenic enzymes. Transcription factor Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) is a master regulator of antioxidant response, regulating a multitude of genes involved in cellular stress responses and anti-inflammatory pathways, thus maintaining cellular redox homeostasis. Nrf2 activation reduces the deleterious effects of mitochondrial toxins and has multiple roles in promoting mitochondrial function and dynamics. We studied the role of GR24 on gene expression in rat L6 skeletal muscle cells which were differentiated into myotubes. The myotubes were treated with GR24 and analyzed by microarray gene expression profiling. GR24 upregulated the cytoprotective transcription factor Nrf2 and its target genes, activating antioxidant defences, suggesting that GR24 may protect skeletal muscle from the toxic effects of oxidative stress.

Engineering plant architecture via CRISPR/Cas9-mediated alteration of strigolactone biosynthesis

BackgroundPrecision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering).ResultsWe used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a reduced height, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography–mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient.ConclusionsTaken together, our results show the potential and feasibility of the use of the CRISPR/Cas9 system for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.

Strigolactones-a novel class of phytohormones as anti-cancer agents.

Chemotherapy shows some promising results in the inhibition of cancer, but resistance to chemotherapy and its severe side effects may occur in due course, resulting in only restricted and narrow benefits. Therefore, there is a pressing need to find alternative chemotherapeutic drugs for combating cancers. Plants have been used since ages in medicine, and by the dawn of 19th century, various potent and promising anti-cancer products have been derived from plants. Strigolactones (SLs) are a novel class of phytohormones involved in regulating the branching of shoots. Recently, many novel synthesized SL analogues have been found to be effective against solid and non-solid tumours. These hormones have been reported to have a unique mechanism of inhibiting cancer cells by lowering their viability and promoting apoptosis and cell death at micromolar concentrations. Therefore, synthetic SL analogues could be future potent anti-cancer drug candidates. Further research is needed to identify and deduce the significance of these synthetic SL analogues.

Non-dormant Axillary Bud 1 regulates axillary bud outgrowth in sorghum.

Tillering contributes to grain yield and plant architecture and therefore is an agronomically important trait in sorghum (Sorghum bicolor). Here, we identified and functionally characterized a mutant of the Non-dormant Axillary Bud 1 (NAB1) gene from an ethyl methanesulfonate-mutagenized sorghum population. The nab1 mutants have increased tillering and reduced plant height. Map-based cloning revealed that NAB1 encodes a carotenoid-cleavage dioxygenase 7 (CCD7) orthologous to rice (Oryza sativa) HIGH-TILLERING DWARF1/DWARF17 and Arabidopsis thaliana MORE AXILLARY BRANCHING 3. NAB1 is primarily expressed in axillary nodes and tiller bases and NAB1 localizes to chloroplasts. The nab1 mutation causes outgrowth of basal axillary buds; removing these non-dormant basal axillary buds restored the wild-type phenotype. The tillering of nab1 plants was completely suppressed by exogenous application of the synthetic strigolactone analog GR24. Moreover, the nab1 plants had no detectable strigolactones and displayed stronger polar auxin transport than wild-type plants. Finally, RNA-seq showed that the expression of genes involved in multiple processes, including auxin-related genes, was significantly altered in nab1. These results suggest that NAB1 functions in strigolactone biosynthesis and the regulation of shoot branching via an interaction with auxin transport.

Stereospecific reduction of the butenolide in strigolactones in plants

Reductive metabolism of strigolactones (SLs) in several plants was investigated. Analysis of aquaculture filtrates of cowpea and sorghum each fed with four stereoisomers of GR24, the most widely used synthetic SL, revealed stereospecific reduction of the double bond at C-3′ and C-4′ in the butenolide D-ring with preference for an unnatural 2′S configuration. The cowpea metabolite converted from 2′-epi-GR24 and the sorghum metabolite converted from ent-GR24 had the methyl group at C-4′ in the trans configuration with the substituent at C-2′, different from the cis configuration of the synthetic H2-GR24 reduced with Pd/C catalyst. The plants also reduced the double bond in the D-ring of 5-deoxystrigol isomers with a similar preference. The metabolites and synthetic H2-GR24 stereoisomers were much less active than were the GR24 stereoisomers in inducing seed germination of the root parasitic weeds Striga hermonthica, Orobanche crenata, and O. minor. These results provide additional evidence of the importance of the D-ring for bioactivity of SLs.

Regulation of Root Development and Architecture by Strigolactones under Optimal and Nutrient Deficiency Conditions.

Strigolactones (SLs) constitute a group of plant hormones which are involved in multiple aspects of plant growth and development. Beside their role in shoot and root development and plant architecture in general, SLs are also involved in plant responses to nutrient deficiency by promoting interactions with symbiotic organisms and via promotion of root elongation. Recent observations on the cross talk between SLs and other hormones demonstrate that the inhibition of adventitious root formation by ethylene is independent of SLs. Additionally, it was shown that root exposure to SLs leads to the accumulation of secondary metabolites, such as flavonols or antioxidants. These data suggest pleiotropic effects of SLs, that influence root development. The discovery that the commonly used synthetic SL analogue racGR24 might also mimic the function of other plant growth regulators, such as karrikins, has led us to consider the previously published publications under the new aspects. This review summarizes present knowledge about the function of SLs in shaping root systems under optimal and nutrient deficiency conditions. Results which appear inconsistent with the various aspects of root development are singled out.

Organ–specific hormonal cross-talk in phosphate deficiency

Plant interactions with the environment, including nutrient sensing and acquisition, are regulated by plant hormones. Responses to phosphate (Pi) deficiency are governed by strigolactones, in cross-talk with other hormones. Reaction of Arabidopsis thaliana plants to Pi deficiency has been characterized in shoot apices, leaves and roots at hormonal and selected transcript levels. Pi starvation was associated with elevation of abscisic acid and jasmonic acid in all followed organs, as well as of salicylic acid in roots. Stimulation of strigolactone signaling pathway was indicated by suppressed expression of strigolactone repressors SMXL6 and SMXL8. Simultaneously, Pi deficiency downregulated the levels of gibberellin GA4 and the most physiologically active cytokinin, trans-zeatin, in all studied organs, increasing less active cis-zeatin in roots. Data imply suppression of the plastid cytokinin biosynthetic pathway and promotion of the cytoplasmic one. Strigolactone function has been characterized comparing the effect of 5 μM synthetic strigolactone GR24 at Pi deficiency and full Pi supply (100 μM). GR24 promoted Pi uptake, including expression of Pi transporters PHT1;4 and PHT1;7. GR24 in Pi presence generally mimicked the impact of Pi deficiency in roots, with exception of jasmonic acid and salicylic acid, which were in full Pi medium, i.e. under conditions optimal for growth, decreased. Pi availability strongly affected expression of selected genes in apices, the major differences being found in the expression of Pi-transporters, auxin- and strigolactone-related genes. The data show highly organ-specific effects of Pi deficiency and decisive role of strigolactones, not only in roots but also in apices.

Identification and functional characterization of a MAX2 ortholog from switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is a sustainable cellulosic energy crop with high biomass yield on marginal soils. Tillering, an important agronomic characteristic related to biomass production in gramineous plants, is regulated by complex interacting factors, such as plant hormones. Strigolactones (SLs) comprise a novel class of plant hormones that inhibit shoot branching. The MORE AXILLARY GROWTH2 (MAX2)/DWARF 3 (D3)/RAMOSUS (RMS4) genes encode proteins involved in the SL signaling pathway in various plants. The switchgrass tetraploid genome likely contains two high-similarity MAX2 homologs, one of which is 6 bp longer than the other. The longest is named PvMAX2 and is the ortholog of MAX2 in Arabidopsis, D3 in rice, and RMS4 in petunia. PvMAX2 is expressed ubiquitously in switchgrass tissues, with higher expression levels observed in the stem and shoot. PvMAX2 gene expression is upregulated by GR24, a synthetic SL analog. Ectopic expression of PvMAX2 in the Arabidopsis max2 mutant rescued the dwarf and bushy phenotypes and small leaf size in the mutant, suggesting that functions of AtMAX2 in Arabidopsis are conserved in PvMAX2. Ectopic PvMAX2 expression also restored the wild-type primary root and hypocotyl length phenotypes and restored the response to GR24. These results indicate that PvMAX2 may play an important role in switchgrass tillering through the SL pathway.

The Role of Strigolactone in the Cross-Talk Between Arabidopsis thaliana and the Endophytic Fungus Mucor sp.

Over the last years the role of fungal endophytes in plant biology has been extensively studied. A number of species were shown to positively affect plant growth and fitness, thus attempts have been made to utilize these microorganisms in agriculture and phytoremediation. Plant-fungi symbiosis requires multiple metabolic adjustments of both of the interacting organisms. The mechanisms of these adaptations are mostly unknown, however, plant hormones seem to play a central role in this process. The plant hormone strigolactone (SL) was previously shown to activate hyphae branching of mycorrhizal fungi and to negatively affect pathogenic fungi growth. Its role in the plant–endophytic fungi interaction is unknown. The effect of the synthetic SL analog GR24 on the endophytic fungi Mucor sp. growth, respiration, H2O2 production and the activity of antioxidant enzymes was evaluated. We found fungi colony growth rate was decreased in a GR24 concentration dependent manner. Additionally, the fungi accumulated more H2O2 what was accompanied by an altered activity of antioxidant enzymes. Symbiosis with Mucor sp. positively affected Arabidopsis thaliana growth, but SL was necessary for the establishment of the beneficial interaction. A. thaliana biosynthesis mutants max1 and max4, but not the SL signaling mutant max2 did not develop the beneficial phenotype. The negative growth response was correlated with alterations in SA homeostasis and a significant upregulation of genes encoding selected plant defensins. The fungi were also shown to be able to decompose SL in planta and to downregulate the expression of SL biosynthesis genes. Additionally, we have shown that GR24 treatment with a dose of 1 μM activates the production of SA in A. thaliana. The results presented here provide evidence for a role of SL in the plant–endophyte cross-talk during the mutualistic interaction between Arabidopsis thaliana and Mucor sp.

Strigolactones and interaction with auxin regulating root elongation in tall fescue under different temperature regimes

Strigolactones (SL) have recently been found to play roles in regulating root development. However, it remains unclear how SL may mediate root elongation of perennial grass species under different temperatures that differentially affect root growth. The objectives of this study were to examine effects of SL on root elongation of tall fescue and to examine the interactive effects of SL and auxin in regulating root growth under both non-stress and heat stress conditions. Tall fescue (cv. ‘Kentuck-31′) plants were treated with GR24 (a synthetic strigolactones), NAA (α-naphthylacetic acid), or NPA (auxin transport inhibitor N-1-naphthylphalamic acid) or their combination under non-stress control and heat stress (35/30 °C) in growth chamber. Crown root elongation was evaluated by measuring root length. Cell number and length in root tips were measured under confocal microscope. Expression levels of genes related to cell growth, SL signaling and auxin transport were determined. SL promoted crown root elongation in tall fescue under normal temperature and heat stress, and alleviated heat-inhibition of root growth. GR24-enhanced root elongation was accompanied with the increase in cell numbers, up-regulation of cell cycle-related genes, and down-regulation auxin transport-related genes in crown root tips of tall fescue. The positive effects of SL for promoting crown root elongation in tall fescue under both non-stress and heat stress could be mainly due to its regulation of cell division and involve the interference of auxin transport.

Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis.

Strigolactones (SL) fulfil important roles in plant development and stress tolerance. Here, we characterized the role of SL in the dark chilling tolerance of pea and Arabidopsis by analysis of mutants that are defective in either SL synthesis or signalling. Pea mutants (rms3, rms4, and rms5) had significantly greater shoot branching with higher leaf chlorophyll a/b ratios and carotenoid contents than the wild type. Exposure to dark chilling significantly decreased shoot fresh weights but increased leaf numbers in all lines. Moreover, dark chilling treatments decreased biomass (dry weight) accumulation only in rms3 and rms5 shoots. Unlike the wild type plants, chilling-induced inhibition of photosynthetic carbon assimilation was observed in the rms lines and also in the Arabidopsis max3-9, max4-1, and max2-1 mutants that are defective in SL synthesis or signalling. When grown on agar plates, the max mutant rosettes accumulated less biomass than the wild type. The synthetic SL, GR24, decreased leaf area in the wild type, max3-9, and max4-1 mutants but not in max2-1 in the absence of stress. In addition, a chilling-induced decrease in leaf area was observed in all the lines in the presence of GR24. We conclude that SL plays an important role in the control of dark chilling tolerance.

Strigolactones affect the translocation of nitrogen in rice

Strigolactones (SLs) are involved in the nutrient-dependent control of plant root and shoot architecture. The total sufficient uptake of nitrogen (N), and also its appropriate distribution, is essential for the normal growth and development of plants; however, the effect of SLs on N translocation in plants remains unknown. Here, the SL-signaling mutant dwarf 3 (d3), the biosynthesis mutant dwarf 10 (d10), and wild-type (WT) rice (Oryza sativa ssp. Japonica cv. Nipponbare) were used to investigate the relationship between N nutrition and the regulatory role of SLs. Relative to WT, the d10 mutant had a higher N concentration in older leaves but a lower N concentration in younger leaves, while the d3 mutant showed a considerably lower N concentration, especially in its younger leaves under normal N levels. By contrast, both d3 and d10 mutants contained higher N in their leaves under N-deficient conditions. The 15N uptake and distribution analysis revealed that the significantly different N concentrations among the d3, d10, and WT plants only occurred in their leaves, not in their roots. Moreover, when provided with an external supply of GR24, the synthetic SLs altered the leaf N distribution of the d10 mutant but not those of the d3 mutant and WT. Together, these results suggested that the effect of SLs on plant growth and development may be linked to N translocation to different shoot tissues.

Synthetic strigolactone analogues reveal anti-cancer activities on hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) remains one of the leading causes of death worldwide. The complex etiology is attributed to many factors like heredity, cirrhosis, hepatitis infections or the dysregulation of the different molecular pathways. Nevertheless, the current treatment regimens have either severe side effects or tumors gradually acquire resistance upon prolonged use. Thus, developing a new selective treatment for HCC is the need of the hour. Many anticancer agents derived from plants have been evaluated for their cytotoxicity towards many human cancer cell lines. Strigolactones (SLs)-a newly discovered class of phytohormones, play a crucial role in the development of plant-root and shoot. Recently, many synthetic analogues of SLs have demonstrated pro-apoptotic effects on different cancer cell lines like prostate, breast, colon and lung. In this study, we tested synthetic SLs analogues on HCC cell line-HepG2 and evaluated their capability to induce cell proliferation inhibition and apoptosis. Primary WST-1 assays, followed by annexin-V/7AAD staining, demonstrated the anti-proliferative effects. The SLs analogues TIT3 and TIT7 were found to significantly reduce HepG2 cell viability in a dose- and time-dependent manner and induce apoptosis. Interestingly, though TIT3 and TIT7 strongly affected cancer cell proliferation, both compounds showed moderate anti-proliferative effect on normal cells. Further, migration of cancer cells was suppressed upon treatment with TIT3 and TIT7 in a wound healing assay. In summary, these findings suggest that two SLs analogues TIT3 and TIT7 exert selective inhibitory effects on cancer cells most likely through targeting microtubules. SLs analogues could be used in future as potential anti-cancer candidates in chemotherapy.

The elusive ligand complexes of the DWARF14 strigolactone receptor.

Strigolactones, a group of terpenoid lactones, control many aspects of plant growth and development, but the active forms of these plant hormones and their mode of action at the molecular level are still unknown. The strigolactone protein receptor is unusual because it has been shown to cleave the hormone and supposedly forms a covalent bond with the cleaved hormone fragment. This interaction is suggested to induce a conformational change in the receptor that primes it for subsequent interaction with partners in the signalling pathway. Substantial efforts have been invested into describing the interaction of synthetic strigolactone analogues with the receptor, resulting in a number of crystal structures. This investigation combines a re-evaluation of models in the Protein Data Bank with a search for new conditions that may permit the capture of a receptor-ligand complex. While weak difference density is frequently observed in the binding cavity, possibly due to a low-occupancy compound, the models often contain features not supported by the X-ray data. Thus, at this stage, we do not believe that any detailed deductions about the nature, conformation, or binding mode of the ligand can be made with any confidence.

Exogenous strigolactone interacts with abscisic acid-mediated accumulation of anthocyanins in grapevine berries.

Besides signalling to soil organisms, strigolactones (SLs) control above- and below-ground morphology, in particular shoot branching. Furthermore, SLs interact with stress responses, possibly thanks to a crosstalk with the abscisic acid (ABA) signal. In grapevine (Vitis vinifera L.), ABA drives the accumulation of anthocyanins over the ripening season. In this study, we investigated the effects of treatment with a synthetic strigolactone analogue, GR24, on anthocyanin accumulation in grape berries, in the presence or absence of exogenous ABA treatment. Experiments were performed both on severed, incubated berries, and on berries attached to the vine. Furthermore, we analysed the corresponding transcript concentrations of genes involved in anthocyanin biosynthesis, and in ABA biosynthesis, metabolism, and membrane transport. During the experiment time courses, berries showed the expected increase in soluble sugars and anthocyanins. GR24 treatment had no or little effect on anthocyanin accumulation, or on gene expression levels. Exogenous ABA treatment activated soluble sugar and anthocyanin accumulation, and enhanced expression of anthocyanin and ABA biosynthetic genes, and that of genes involved in ABA hydroxylation and membrane transport. Co-treatment of GR24 with ABA delayed anthocyanin accumulation, decreased expression of anthocyanin biosynthetic genes, and negatively affected ABA concentration. GR24 also enhanced the ABA-induced activation of ABA hydroxylase genes, while it down-regulated the ABA-induced activation of ABA transport genes. Our results show that GR24 affects the ABA-induced activation of anthocyanin biosynthesis in this non-climacteric fruit. We discuss possible mechanisms underlying this effect, and the potential role of SLs in ripening of non-ABA-treated berries.

Commentary: Synthetic Strigolactone analogues reveal anti-cancer activities on hepatocellular carcinoma cells

Commentary: Synthetic Strigolactone analogues reveal anti-cancer activities on hepatocellular carcinoma cells

Stability of strigolactone analog GR24 toward nucleophiles.

Strigolactones (SLs) are plant hormones that play various roles in plant development. The chemical stability of SLs depends on the solvent, the pH, and the presence of nucleophiles. Hydrolysis leads to detachment of the butenolide ring, and plays a crucial role in the initial stages of the signal-transduction process occurring between the receptor and the SL signaling molecule. To date, two different mechanisms have been proposed for SL hydrolysis. Results obtained from kinetic, thermodynamic, and mass spectral data for the reaction between the widely used synthetic SL analog GR24 and seven different nucleophiles demonstrate that the reaction proceeds via the Michael addition-elimination mechanism. This study provides valuable information on the chemical stability of GR24 in different plant growth media and buffers. Such information is valuable for scientists using GR24 treatments to study SL-regulated processes in plants. © 2017 Society of Chemical Industry.

Mutations in the MIT3 gene encoding a caroteniod isomerase lead to increased tiller number in rice

Carotenoids not only play important roles in light harvesting and photoprotection against excess light, but also serve as precursors for apocaroteniod hormones such as abscisic acid (ABA) and strigolactones (SLs). Although light- and ABA-associated phenotypes of the carotenoid biosynthesis mutants such as albino, leaf variegation and preharvest sprouting have been studied extensively, the SLs-related branching phenotype is rarely explored. Here we characterized four allelic rice mutants named mit3, which exhibited moderately increased tiller number, semi-dwarfism and leaf variegation. Map-based cloning revealed that MIT3 encodes a carotenoid isomerase (CRTISO), the key enzyme catalyzing the conversion from prolycopene to all-trans-lycopene in carotenoid biosynthesis. Prolycopene was accumulated while all-trans-lycopene was barely detectable in the dark-grown mit3 seedlings. Accordingly, content of lutein and β-carotene, the two most abundant carotenoids, was significantly reduced. Furthermore, content of epi-5DS, a native SL, was significantly reduced in mit3. Exogenously applied GR24, a synthetic SL, could rescue the tillering phenotype of mit3. Double mutant analysis of mit3 with the SLs biosynthesis mutant d17 revealed that MIT3 controls tiller development upstream of the SLs biosynthesis pathway. Our results reveal that the tillering phenotype of mit3 is due to SL deficiency and directly link carotenoid deficiency with SL-regulated rice tillering.