Strigolactone Analogue Research Articles

Nitrate Modulates Lateral Root Formation by Regulating the Auxin Response and Transport in Rice.

Nitrate () plays a pivotal role in stimulating lateral root (LR) formation and growth in plants. However, the role of in modulating rice LR formation and the signalling pathways involved in this process remain unclear. Phenotypic and genetic analyses of rice were used to explore the role of strigolactones (SLs) and auxin in -modulated LR formation in rice. Compared with ammonium (), stimulated LR initiation due to higher short-term root IAA levels. However, this stimulation vanished after 7 d, and the LR density was reduced, in parallel with the auxin levels. Application of the exogenous auxin α-naphthylacetic acid to -treated rice plants promoted LR initiation to levels similar to those under at 7 d; conversely, the application of the SL analogue GR24 to -treated rice inhibited LR initiation to levels similar to those under supply by reducing the root auxin levels at 10 d. D10 and D14 mutations caused loss of sensitivity of the LR formation response to . The application of and GR24 downregulated the transcription of PIN-FORMED 2 (PIN2), an auxin efflux carrier in roots. LR number and density in pin2 mutant lines were insensitive to treatment. These results indicate that LR formation by affecting the auxin response and transport in rice, with the involvement of 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.

Strigolactone analog (rac-GR24) enhances chilling tolerance in mung bean seedlings

Phytohormones, such as strigolactones, can ameliorate abiotic stress by regulating and augmenting physiological processes tailored towards stress tolerance. We investigated the physiological and biochemical responses of mung bean [Vigna radiata (L.) R. Wilczek] seedlings to treatments with different doses (0, 1, and 10 μM) of rac-GR24, a synthetic strigolactone analog, and subjected them to chilling stress. The results show that GR24 inhibited the chilling-induced decrease in photosystem II quantum efficiencies and yield while also limiting superoxide anion and H2O2 accumulation in the seedlings. Additionally, GR24 significantly lowered lipid peroxidation levels, although total antioxidant capacities were not significantly affected while radical scavenging activity and phenolic contents were diminished. Chilling-induced dehydration and disruption of water status were ameliorated by GR24 treatment via increased proline content, by 1 μM GR24 concentration, and soluble sugar accumulation. The activities of lipoxygenase and phenylpropanoid pathway enzymes (phenylalanine ammonia-lyase and tyrosine ammonia-lyase) were significantly increased by 10 μM GR24 treatment in mung bean seedlings under chilling stress. Our findings provide direct evidence for strigolactone-mediated enhancement of chilling tolerance in mung bean seedlings.

SPL14/17 act downstream of strigolactone signalling to modulate rice root elongation in response to nitrate supply

Nitrogen (N) is an essential major nutrient for food crops. Although ammonium (NH4 + ) is the primary N source of rice, nitrate (NO3 - ) can also be absorbed and utilized. Rice responds to NO3 - application by altering its root morphology, such as root elongation. Strigolactones (SLs) are important modulators of root length. However, the roles of SLs and their downstream genes in NO3 - -induced root elongation remain unclear. Here, the levels of total N and SL (4-deoxyorobanchol), and the responses of seminal root (SR) lengths to NH4 + and NO3 - were investigated in rice plants. NO3 - -promoted SR elongation, possibly due to short-term signal perception and long-term nutrient function. Compared with NH4 + condition, higher SL signalling/levels and less D53 protein were recorded in roots of NO3 - -treated rice plants. In contrast to wild-type (WT) plants, SR lengths of d mutants were less responsive to NO3 - condition, and application of rac-GR24 (SL analogue) restored SR length in d10 (SL-biosynthesis mutant) but not in d3,d14 and d53 (SL-responsive mutants), suggesting that higher SL signalling/levels participated in NO3 - -induced root elongation. D53 interacted with SPL17, and inhibited SPL17-mediated transactivation from PIN1b promoter. Mutation of SPL14/17 and PIN1b caused insensitivity of root elongation response to NO3 - and rac-GR24 applications. Therefore, we presented that perception of SLs by D14 led to degradation of D53 via the proteasome system, which released the suppression of SPL14/17-modulated the transcription of PIN1b, and resultedin root elongation under NO3 - supply.

Strigolactone and Ethylene Inhibitor Suppressing Dark-induced Leaf Senescence in Perennial Ryegrass Involving Transcriptional Downregulation of Chlorophyll Degradation

Accelerated or premature leaf senescence induced by dark conditions could be associated with chlorophyll degradation and regulated by hormones. To study the effects of strigolactone (SL) on dark-induced leaf senescence and to examine the interaction effects of SL and ethylene on regulating dark-induced leaf senescence, plants of perennial ryegrass (Lolium perenne) exposed to darkness for 8 days were treated with a synthetic SL analogue (GR24), aminoethoxyvinyl glycine [AVG (an ethylene biosynthesis inhibitor)], or SL and AVG by foliar spray. Chlorophyll content, photochemical efficiency, electrolyte leakage, and ethylene production were measured. Expressions of genes associated with leaf senescence, SL biosynthesis and signaling, ethylene biosynthesis and signaling, and chlorophyll biosynthesis and degradation were determined. Foliar application of GR24 promoted leaf senescence in perennial ryegrass grown in darkness, and the intensity of action increased with the GR24 concentration. SL-accelerated leaf senescence was associated with the downregulation of four chlorophyll biosynthesis-associated genes and upregulation of four chlorophyll degradation-associated genes. AVG had functions counteractive to SL, suppressing dark-induced leaf senescence by downregulating chlorophyll degradation genes and SL synthesis genes. Our results suggested that SL and ethylene interactively regulated leaf senescence, mainly by controlling chlorophyll degradation induced by darkness in perennial ryegrass.

Comparative transcriptome analysis reveals molecular regulators underlying pluripotent cell induction and callus formation in Anthurium andraeanum “Alabama”

Shoot regeneration from pluripotent cell masses is an important process for genetic improvement of Anthurium andraeanum through Agrobacterium-mediated transformation. However, the mechanisms underlying pluripotent cell induction are not well understood. Here, three half-leaf explant samples collected after 0-, 2-, and 30-d culture on callus-inducing medium were used for RNA isolation and transcriptome profiling. In total, 225,752 non-redundant unigenes were assembled with a mean length of 1299 bp. Differentially expressed genes among the three samples were identified by comparative transcriptome analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed the greatest number of differentially expressed genes in the comparison between 0- and 2-d cultures were related to the “ribosome.” Many differentially expressed genes in the comparison between 2- and 30-d cultures were involved in “plant hormone signal transduction,” “starch and sucrose metabolism,” and “phenylpropanoid biosynthesis.” Further investigation was focused on genes related to transcription factors, epigenetic regulation, nitrous oxide metabolism, and homeostasis of plant hormones auxin, cytokinin, brassinolide, and strigolactone. Experiments were designed to confirm the effects of nitrous oxide and strigolactone on callus formation in Anthurium andraeanum. Callus formation was enhanced by exogenous application of sodium nitroprusside, which produces nitrous oxide, but was inhibited under rac-GR24 (a strigolactone analogue) treatment. Our comparative transcriptome analysis revealed that the signal molecule nitrous oxide and plant hormones auxin, cytokinin, and brassinolide act as positive regulators in promoting callus formation, while strigolactone may inhibit callus formation. Transcriptional and epigenetic regulations also play important roles in pluripotent cell induction in Anthurium andraeanum.

Synthesis of Analogs of Strigolactones and Evaluation of Their Stability in Solution.

Strigolactones (SLs) are new plant hormones that play an important role in the control development of plants. They are germination stimulants for seed of parasitic weeds, are the branching factor of arbuscular mycorrhizal fungi and inhibitors for bud outgrowth and shoot branching. Natural SLs contain an annulated system of three rings (ABC scaffold) connected to a furanone (the D-ring) by an enol ether unit. The natural distribution of strigolactones is low, and their synthesis is long and difficult. Therefore, SL analogs are designed to have the same bioactiphore as natural SLs and an appreciable bioactivity. For the design a model is used based on the natural bioactiphore. Typical SL analogs are GR24, Nijmegen-1, and EM1 (derived from ethyl 2-phenylacetate). The synthesis of these SL analogs is reported together with their stability in aqueous solution.

Evaluation of the Effect of Strigolactones and Synthetic Analogs on Fungi.

Strigolactones (SLs) are components of root exudates as a consequence of active release from the roots into the soil. Notably, they have been described as stimulants of seed germination in parasitic plants and of the presymbiotic growth in arbuscular mycorrhizal (AM) fungi, which are a crucial component of the plant root beneficial microbiota. SLs have therefore the potential to influence other microbes that proliferate in the soil around the roots and may interact with plants. A direct effect of SL analogs on the in vitro growth of a number of saprotrophic or plant pathogenic fungi was indeed reported.Here we describe a standardized method to evaluate the effect of SLs or their synthetic analogs on AM and filamentous fungi. For AM fungi, we propose a spore germination assay since it is more straightforward than the hyphal branching assay and it does not require deep expertise and skills. For filamentous fungi that can grow in axenic cultures, we describe the assay based on SLs embedded in the solid medium or dissolved in liquid cultures where the fungus is inoculated to evaluate the effect on growth, hyphal branching or conidia germination. These assays are of help to test the activity of natural SLs as well as of newly designed SL analogs for basic and applied research.

A Protoplast-Based Bioassay to Quantify Strigolactone Activity in Arabidopsis Using StrigoQuant.

Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors may be also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, the genetically encoded, ratiometric sensor StrigoQuant was developed and allowed the quantification of the activity of a wide concentration range of SLs. StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL analogue GR24. We then show the recording of the luminescence signal and how the obtained data are processed and used to assess/determine SL perception.

Methods for Phenotyping Shoot Branching and Testing Strigolactone Bioactivity for Shoot Branching in Arabidopsis and Pea.

Shoot branching is a highly variable trait that evolves during plant development and is influenced by environmental and endogenous cues such as hormones. In particular, strigolactones (SLs) are hormones that play a key role in the control of shoot branching. Branch primordia, axillary buds formed in the leaf axils, display differential growth depending on their position in the plant and also respond to hormone signaling. In this chapter, we will describe how to quantify the degree of shoot branching in two plant model species, Arabidopsis and pea, commonly used to decipher the control of this complex trait. We will also propose several methods to perform treatments of SL or SL analogs, to investigate their bioactivity and effect on the shoot branching patterns of plants of different genotypes.

Evaluation of Bioactivity of Strigolactone-Related Molecules by a Quantitative Luminometer Bioassay.

The binding of strigolactones to their receptor, the α/β hydrolase DWARF14 (D14), leads to the modulation of transcriptional activity by destabilization of specific transcriptional corepressors via proteasomal degradation. Subsequently, strigolactones also promote D14 degradation by the same pathway. Here we describe an innovative quantitative bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to the firefly luciferase, developed to identify new strigolactone analogs capable to activate the strigolactone signaling.

Unraveling the MAX2 Protein Network in Arabidopsis thaliana: Identification of the Protein Phosphatase PAPP5 as a Novel MAX2 Interactor

AbstractThe F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke-derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/β-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. In addition, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14, and KAI2 protein network by tandem affinity purification in Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were copurified, among which were general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than in D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination under suboptimal conditions and seedling development. In addition, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic SL analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14, and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL-related phenotypes and, hence, provide another link with the light pathway.

Chemotropic Assay for Testing Fungal Response to Strigolactones and Strigolactone-Like Compounds.

Current knowledge on the mechanism of strigolactones (SLs) as signaling molecules during specific interactions in the rhizosphere is mainly related to the control of germination of parasitic weed seeds and hyphal branching of arbuscular mycorrhizal fungi. Thus, the role of plant secreted SLs in regulating the growth and development of root-colonizing fungi still remains controversial. Fusarium oxysporum can sense and respond to extracellular signals through oriented germ tube emergence and redirectioning of hyphal growth toward gradients of nutrients, sex pheromones, or plant root exudates. However, chemoattractant activity of SLs against microorganisms living in the soil has not been tested so far. Here we propose a quantitative chemotropic assay to understand if and how soil fungi could sense gradients of SLs and SLs-like sources. In the example case of F. oxysporum, hyphae of fungal representative mutants preferentially grow toward the synthetic SL analog GR24; and this chemotropic response requires conserved elements of the fungal invasive growth mitogen-activated protein kinase (MAPK) cascade.

Synthesis, Characterization and In vitro Antibacterial Activity of A Novel Strigolactones Analogues TIT3

Traditional antibiotic abuse caused increased the incidence of bacteria resistance to treatment. Strigolactones (SLs) are phytohormones that play a vital role in the plant growing root, shoot and flowering. We previously reported that, SLs analogues exert pro-apoptotic effects on HepG2 cell lines. The current study investigated the antimicrobial activity of selected SLs analogue TIT3 against different strains of bacteria including gram positive and gram negative bacteria: Staphylococcus aureus, Salmonella typhimuriu, Escherichia coli, Klebsiella pneumoni and B. subtilis. The results obtained were compared with 100 µg amoxicillin. Results obtained showed that, TIT3 inhibited the growth of S. cereus (50 µg), Salmonella typhimuriu (20 µg), Escherichia coli (30µg), Klebsiella pneumoni (50 µg) and B. subtilis (50 µg) with mean inhibition zones diameter being 12.1 mm , 13.2 mm, 12.5, 8.9 and 12.9 mm respectively. It was concluded that, TIT3 is promising effective antibacterial agents compared with amoxicillin for different strains of bacteria that resistant to different antibiotics.

ζ-Carotene Isomerase Suppresses Tillering in Rice through the Coordinated Biosynthesis of Strigolactone and Abscisic Acid

Rice tillering is an important agronomic trait affecting grain yield. Here, we identified a high-tillering mutant tillering20 (t20), which could be restored to the wild type by treatment with the strigolactone (SL) analog rac-GR24. T20 encodes a chloroplast ζ-carotene isomerase (Z-ISO), which is involved in the biosynthesis of carotenoids and their metabolites, SL and abscisic acid (ABA). The t20 mutant has reduced SL and ABA, raising the question of how SL and ABA biosynthesis is coordinated, and whether they have overlapping functions in tillering. We discovered that rac-GR24 stimulated T20 expression and enhanced all-trans-β-carotene biosynthesis. Importantly, rac-GR24 also stimulated expression of Oryza sativa 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (OsNCED1) through induction of Oryza sativa HOMEOBOX12 (OsHOX12), promoting ABA biosynthesis in shoot base. On the other hand, ABA treatment significantly repressed SL biosynthesis and the ABA biosynthetic mutants displayed elevated SL biosynthesis. ABA treatment reduced the number of basal tillers in both t20 and wild-type plants. Furthermore, while ABA-deficient mutants aba1 and aba2 had the same number of basal tillers as wild type, they had more unproductive upper tillers at maturity. This work demonstrates complex interactions in the biosynthesis of carotenoid, SLs and ABA, and reveals a role for ABA in the regulation of rice tillering.

Strigolactone Analogues Derived from Dihydroflavonoids as Potent Seed Germinators for the Broomrapes.

The broomrapes (Orobanche and Phelipanche spp.) and witchweeds (Striga spp.) are a class of parasitic weeds, which are distributed widely in the tropical, subtropical, and temperate areas of the globe. Since they have completely consistent lifecycles with the host plants, it is difficult to control them selectively through using the conventional herbicides. Inducing suicidal germination of these weed seeds by small molecular signaling agents proved to be a promising strategy for the management of parasitic weeds. As a class of naturally occurring terpenoid metabolites, strigolactones (SLs) show significant biological activities including stimulation germination of weed seeds, inhibition of shoot-branching, and so on. However, the widespread application of these natural SLs is greatly limited by their extremely low natural abundance and complex molecular structures. Design and synthesis of the simplified analogues as natural SLs alternatives provide a viable avenue for the efficient control of these parasitic weeds. We herein disclose the development of a novel class of SLs analogues derived from dihydroflavonoids as potent seed germinators of parasitic weeds. It was shown that one of them displayed a higher potential toward the seed germination of the broomrapes than the positive control GR24. The structure-activity relationship of these SLs analogues was further validated on the basis of the binding affinity experiment to strigolactone receptor protein HTL7 by using a YLG fluorescent probe method.

Synthetic strigolactone (rac-GR24) alleviates the adverse effects of heat stress on seed germination and photosystem II function in lupine seedlings

There is increasing experimental evidence that strigolactones, a class of carotenoid-derived sesquiterpenoid hormones, and their downstream signal components play a role in plant resilience to abiotic stress. Strigolactones positively influence plant coping mechanisms in response to abiotic stressors like drought and high salinity. In this study, we examined the effects of rac-GR24 (a synthetic strigolactone analog) and strigolactone inhibitors on the physiological and molecular responses associated with thermotolerance during seed germination and seedling development in Lupinus angustifolius under heat stress. Photosystem I & II functions were also evaluated via Chl a fluorescence transient analysis in heat stressed lupine seedlings. Our results suggest a putative role for GR24 in mediating tolerance to heat stress during seed germination and seedling development albeit these responses appeared independent of D14-mediated signalling. Seeds primed with GR24 had the highest of all germination indices, enhanced proline content and reduced peroxidation of lipids. GR24 also enhanced the activities of enzymes of the antioxidant and glyoxalase systems in lupine seedlings. The JIP-test indicated that GR24 conferred resistance to heat stress-induced damage to the oxygen evolution complex while also preventing the inactivation of PSII reaction centres thus ensuring PSII thermotolerance.

Rational Design of Novel Fluorescent Enzyme Biosensors for Direct Detection of Strigolactones.

Strigolactones are plant hormones and rhizosphere signaling molecules with key roles in plant development, mycorrhizal fungal symbioses, and plant parasitism. Currently, sensitive, specific, and high-throughput methods of detecting strigolactones are limited. Here, we developed genetically encoded fluorescent strigolactone biosensors based on the strigolactone receptors DAD2 from Petunia hybrida, and HTL7 from Striga hermonthica. The biosensors were constructed via domain insertion of circularly permuted GFP. The biosensors exhibited loss of cpGFP fluorescence in vitro upon treatment with the strigolactones 5-deoxystrigol and orobanchol, or the strigolactone analogue rac-GR24, and the ShHTL7 biosensor also responded to a specific antagonist. To overcome biosensor sensitivity to changes in expression level and protein degradation, an additional strigolactone-insensitive fluorophore, LSSmOrange, was included as an internal normalization control. Other plant hormones and karrikins resulted in no fluorescence change, demonstrating that the biosensors report on compounds that specifically bind the SL receptors. The DAD2 biosensor likewise responded to strigolactones in an in vivo protoplast system, and retained strigolactone hydrolysis activity. These biosensors have applications in high-throughput screening for agrochemical compounds, and may also have utility in understanding strigolactone mediated signaling in plants.

Translation of Strigolactones from Plant Hormone to Agriculture: Achievements, Future Perspectives, and Challenges.

Strigolactones (SLs) control plant development, enhance symbioses, and act as germination stimulants for some of the most destructive species of parasitic weeds, making SLs a potential tool to improve crop productivity and resilience. Field trials demonstrate the potential use of SLs as agrochemicals or genetic targets in breeding programs, with applications in improving drought tolerance, increasing yields, and controlling parasitic weeds. However, for effective translation of SLs into agriculture, understanding and exploiting SL diversity and the development of economically viable sources of SL analogs will be critical. Here we review how manipulation of SL signaling can be used when developing new tools and crop varieties to address some critical challenges, such as nutrient acquisition, resource allocation, stress tolerance, and plant-parasite interactions.

Initiation of arbuscular mycorrhizal symbiosis involves a novel pathway independent from hyphal branching.

The arbuscular mycorrhizal symbiosis is a very common association between plant roots and soil fungi, which greatly contributes to plant nutrition. Root-exuded compounds known as strigolactones act as symbiotic signals stimulating the fungus prior to root colonization. Strigolactones also play an endogenous role in planta as phytohormones and contribute to the regulation of various developmental traits. Structure-activity relationship studies have revealed both similarities and differences between the structural features required for bioactivity in plants and arbuscular mycorrhizal fungi. In the latter case, bioassays usually measured a stimulation of hyphal branching on isolated fungi of the Gigaspora genus, grown in vitro. Here, we extended these investigations with a bioassay that evaluates the bioactivity of strigolactone analogs in a symbiotic situation and the use of the model mycorrhizal fungus Rhizophagus irregularis. Some general structural requirements for bioactivity reported previously for Gigaspora were confirmed. We also tested additional strigolactone analogs bearing modifications on the conserved methylbutenolide ring, a key element of strigolactone perception by plants. A strigolactone analog with an unmethylated butenolide ring could enhance the ability of R. irregularis to colonize host roots. Surprisingly, when applied to the isolated fungus in vitro, this compound stimulated germ tube elongation but inhibited hyphal branching. Therefore, this compound was able to act on the fungal and/or plant partner to facilitate initiation of the arbuscular mycorrhizal symbiosis, independently from hyphal branching and possibly from the strigolactone pathway.