Gibberellins Promote Seed Conditioning by Up-Regulating Strigolactone Receptors in the Parasitic Plant Striga hermonthica.

Dormant seeds of the root parasitic plant Striga hermonthica sense strigolactones from host plants as environmental cues for germination. This process is mediated by a diversified member of the strigolactone receptors encoded by HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE2 genes. It is known that warm and moist treatment during seed conditioning gradually makes dormant Striga seeds competent to respond to strigolactones, although the mechanism behind it is poorly understood. In this report, we show that plant hormone gibberellins increase strigolactone competence by up-regulating mRNA expression of the major strigolactone receptors during the conditioning period. This idea was supported by a poor germination phenotype in which gibberellin biosynthesis was depleted by paclobutrazol during conditioning. Moreover, live imaging with a fluorogenic strigolactone mimic, yoshimulactone green W, revealed that paclobutrazol treatment during conditioning caused aberrant dynamics of strigolactone perception after germination. These observations revealed an indirect role of gibberellins in seed germination in Striga, which contrasts with their roles as dominant germination-stimulating hormones in non-parasitic plants. We propose a model of how the role of gibberellins became indirect during the evolution of parasitism in plants. Our work also highlights the potential role for gibberellins in field applications, for instance, in elevating the sensitivity of seeds toward strigolactones in the current suicidal germination approach to alleviate the agricultural threats caused by this parasite in Africa.

Conferring resistance to parasitic witchweed by shifting strigolactone biosynthesis.

Strigolactones from the exudates of maize root induce germination of the parasitic witchweed Striga. Recently, Li et al. characterized the biosynthesis pathway of two strigolactones, zealactol and zealactonoic acid, which induce less Striga germination than the major maize strigolactone, zealactone. This study provides a promising strategy for plant protection against parasitic witchweed.

Organosulfur Compound Identified from Striga angustifolia (D. Don) C.J. Saldanha Inhibits Lung Cancer Growth and Induces Apoptosis via p53/mTOR Signaling Pathway.

The Striga angustifolia (D. Don) C.J. Saldanha was used as an Ayurvedic and homeopathic medicine for cancer by the tribal peoples of the Maruthamalai Hills, Coimbatore, India. Hence, the traditional use that has been proven to be effective lacks convincing scientific references. This present study was conducted to investigate the presence of potentially bioactive compounds from S. angustifolia and provides a scientific basis for the ethnobotanical utility. The organosulfur compound 5,5'-dithiobis(1-phenyl-1H-tetrazole) (COMP1) was isolated from S. angustifolia extracts, and the structures of COMP1 were elucidated and characterized by using 13C and 1H nuclear magnetic resonance (NMR) and single crystal X-ray powder diffraction (XRD). Our findings showed that COMP1 significantly reduced cell proliferation of breast and lung cancer cells, but not that of non-malignant epithelial cells. Further analysis revealed that COMP1 promoted cell cycle arrest and apoptosis of lung cancer cells. Mechanistically, COMP1 facilitates p53 activity and inhibits mammalian target of rapamycin (mTOR) signaling, thereby inducing cell cycle arrest and apoptosis of lung cancer cells by inhibiting cell growth. Our findings suggest that COMP1 may serve as a potential drug for lung cancer through the regulation of p53/mTOR pathways.

Cytokinins Induce Prehaustoria Coordinately with Quinone Signals in the Parasitic Plant Striga hermonthica.

Orobanchaceae parasitic plants are major threats to global food security, causing severe agricultural damage worldwide. Parasitic plants derive water and nutrients from their host plants through multicellular organs called haustoria. The formation of a prehaustorium, a primitive haustorial structure, is provoked by host-derived haustorium-inducing factors (HIFs). Quinones, including 2,6-dimethoxy-p-benzoquinone (DMBQ), are of the most potent HIFs for various species in Orobanchaceae, but except non-photosynthetic holoparasites, Phelipanche and Orobanche spp. Instead, cytokinin (CK) phytohormones were reported to induce prehaustoria in Phelipanche ramosa. However, little is known about whether CKs act as HIFs in the other parasitic species to date. Moreover, the signaling pathways for quinones and CKs in prehaustorium induction are not well understood. This study shows that CKs act as HIFs in the obligate parasite Striga hermonthica but not in the facultative parasite Phtheirospermum japonicum. Using chemical inhibitors and marker gene expression analysis, we demonstrate that CKs activate prehaustorium formation through a CK-specific signaling pathway that overlaps with the quinone HIF pathway at downstream in S. hermonthica. Moreover, host root exudates activated S. hermonthica CK biosynthesis and signaling genes, and DMBQ and CK inhibitors perturbed the prehaustorium-inducing activity of exudates, indicating that host root exudates include CKs. Our study reveals the importance of CKs for prehaustorium formation in obligate parasitic plants.

Activation Mechanism of Strigolactone Receptors and Its Impact on Ligand Selectivity between Host and Parasitic Plants.

Parasitic weeds such as Striga have led to significant losses in agricultural productivity worldwide. These weeds use the plant hormone strigolactone as a germination stimulant. Strigolactone signaling involves substrate hydrolysis followed by a conformational change of the receptor to a "closed" or "active" state that associates with a signaling partner, MAX2/D3. Crystal structures of active and inactive AtD14 receptors have helped elucidate the structural changes involved in activation. However, the mechanism by which the receptor activates remains unknown. The ligand dependence of AtD14 activation has been disputed by mutagenesis studies showing that enzymatically inactive receptors are able to associate with MAX2 proteins. Furthermore, activation differences between strigolactone receptor in Striga, ShHTL7, and AtD14 could contribute to the high sensitivity to strigolactones exhibited by parasitic plants. Using molecular dynamics simulations, we demonstrate that both AtD14 and ShHTL7 could adopt an active conformation in the absence of ligand. However, ShHTL7 exhibits a higher population in the inactive apo state as compared to the AtD14 receptor. We demonstrate that this difference in inactive state population is caused by sequence differences between their D-loops and interactions with the catalytic histidine that prevent full binding pocket closure in ShHTL7. These results indicate that ligand hydrolysis would enhance the active state population by destabilizing the inactive state in ShHTL7 as compared to AtD14. We also show that the mechanism of activation is more concerted in AtD14 than in ShHTL7 and that the main barrier to activation in ShHTL7 is closing of the binding pocket.

A novel strigolactone receptor antagonist provides insights into the structural inhibition, conditioning, and germination of the crop parasite Striga.

Crop parasites of the Striga genera are a major biological deterrent to food security in Africa and are one of the largest obstacles to poverty alleviation on the continent. Striga seeds germinate by sensing small-molecule hormones, strigolactones (SLs), that emanate from host roots. Although SL receptors (Striga hermonthica HYPOSENSITIVE TO LIGHT [ShHTL]) have been identified, discerning their function has been difficult because these parasites cannot be easily grown under laboratory conditions. Moreover, many Striga species are obligate outcrossers that are not transformable, hence not amenable to genetic analysis. By combining phenotypic screening with ShHTL structural information and hybrid drug discovery methods, we discovered a potent SL perception inhibitor for Striga, dormirazine (DOZ). Structural analysis of this piperazine-based antagonist reveals a novel binding mechanism, distinct from that of known SLs, blocking access of the hormone to its receptor. Furthermore, DOZ reduces the flexibility of protein-protein interaction domains important for receptor signaling to downstream partners. In planta, we show, via temporal additions of DOZ, that SL receptors are required at a specific time during seed conditioning. This conditioning is essential to prime seed germination at the right time; thus, this SL-sensitive stage appears to be critical for adequate receptor signaling. Aside from uncovering a function for ShHTL during seed conditioning, these results suggest that future Ag-Biotech Solutions to Striga infestations will need to carefully time the application of antagonists to exploit receptor availability and outcompete natural SLs, critical elements for successful parasitic plant invasions.

Role of substrate recognition in modulating strigolactone receptor selectivity in witchweed.

Witchweed, or Striga hermonthica, is a parasitic weed that destroys billions of dollars' worth of crops globally every year. Its germination is stimulated by strigolactones exuded by its host plants. Despite high sequence, structure, and ligand-binding site conservation across different plant species, one strigolactone receptor in witchweed, ShHTL7, uniquely exhibits a picomolar EC50 for downstream signaling. Previous biochemical and structural analyses have hypothesized that this unique ligand sensitivity can be attributed to a large binding pocket volume in ShHTL7 resulting in enhanced ability to bind substrates, but additional structural details of the substrate-binding process would help explain its role in modulating the ligand selectivity. Using long-timescale molecular dynamics simulations, we demonstrate that mutations at the entrance of the binding pocket facilitate a more direct ligand-binding pathway to ShHTL7, whereas hydrophobicity at the binding pocket entrance results in a stable "anchored" state. We also demonstrate that several residues on the D-loop of AtD14 stabilize catalytically inactive conformations. Finally, we show that strigolactone selectivity is not modulated by binding pocket volume. Our results indicate that while ligand binding is not the sole modulator of strigolactone receptor selectivity, it is a significant contributing factor. These results can be used to inform the design of selective antagonists for strigolactone receptors in witchweed.

Plasma membrane phylloquinone biosynthesis in nonphotosynthetic parasitic plants.

Nonphotosynthetic holoparasites exploit flexible targeting of phylloquinone biosynthesis to facilitate plasma membrane redox signaling. Phylloquinone is a lipophilic naphthoquinone found predominantly in chloroplasts and best known for its function in photosystem I electron transport and disulfide bridge formation of photosystem II subunits. Phylloquinone has also been detected in plasma membrane (PM) preparations of heterotrophic tissues with potential transmembrane redox function, but the molecular basis for this noncanonical pathway is unknown. Here, we provide evidence of PM phylloquinone biosynthesis in a nonphotosynthetic holoparasite Phelipanche aegyptiaca. A nonphotosynthetic and nonplastidial role for phylloquinone is supported by transcription of phylloquinone biosynthetic genes during seed germination and haustorium development, by PM-localization of alternative terminal enzymes, and by detection of phylloquinone in germinated seeds. Comparative gene network analysis with photosynthetically competent parasites revealed a bias of P. aegyptiaca phylloquinone genes toward coexpression with oxidoreductases involved in PM electron transport. Genes encoding the PM phylloquinone pathway are also present in several photoautotrophic taxa of Asterids, suggesting an ancient origin of multifunctionality. Our findings suggest that nonphotosynthetic holoparasites exploit alternative targeting of phylloquinone for transmembrane redox signaling associated with parasitism.

ShHTL7 requires functional brassinosteroid signaling to initiate GA-independent germination.

In the model plant Arabidopsis thaliana, two mutually antagonistic hormones regulate germination: abscisic acid (ABA) which promotes dormancy and gibberellins (GA) which breaks dormancy. Mutants auxotrophic for or insensitive to GA do not germinate. However, changes in the signaling flux through other hormone pathways will permit GA-independent germination. These changes include increased brassinosteroid (BR) signaling and decreased ABA signaling. Recently, strigolactone (SL) was also shown to enable GA-independent germination, provided the seeds express the SL receptor ShHTL7 from the parasitic plant Striga hermonthica. Here we show that a mutation which reduces sensitivity to BR (bri1-6) prevents ShHTL7 from promoting GA-independent germination. Further, we show that neither ShHTL7 nor the constitutive karrikin signaling mutant smax1-2 confer insensitivity to ABA. These results suggest ShHTL7 requires functional BR perception to bypass the GA requirement for germination.

Comparative Studies of Potential Binding Pocket Residues Reveal the Molecular Basis of ShHTL Receptors in the Perception of GR24 in Striga.

Root parasitic weeds such as Striga spp. have caused significant losses in agriculture production worldwide. The seed germination of the weeds depends on strigolactones (SLs) that target a series of HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE2 in Striga hermonthica (ShHTL) proteins. In the present study, 60 ShHTL7 mutants were constructed, and the equilibrium dissociation constants for GR24 (a synthetic SL analogue, commonly used as a standard in SL germination studies) against these mutants were measured by surface plasmon resonance. Based on these data, the SL binding pocket residues were distinguished. Of them, some specific residues for ShHTL7 were found, such as T142, T190, and M219. A model showing quite well internal and external predictive abilities was established by the mutation-dependent biomacromolecular quantitative structure-activity relationship method. It provided an expanded understanding for GR24 binding to a series of ShHTL receptors and should help design broad-spectrum agrochemicals with cross interactions with several members of SL receptors.

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.

Crystal structure and biochemical characterization of Striga hermonthica HYPO-SENSITIVE TO LIGHT 8 (ShHTL8) in strigolactone signaling pathway.

Striga is a parasitic weed that disperses easily, and its seeds can persist in the soil for many years, presenting long-term threats to food security. If SLs stimulate the seed germination of root parasitic weeds before planting, weeds will wither due to no host. Therefore, it is necessary to determine the mechanism of strigolactone (SL) signaling in Striga to reduce the impacts of this parasitic weed. Striga has eleven different kinds of HYPO-SENSITIVE to LIGHT (ShHTL) hydrolases. Different ShHTL hydrolases exhibit distinct responses to SLs, despite these ShHTLs exhibiting more than 60% sequence identity. Currently, structural information is available for only five ShHTL proteins, and more structural information is needed to design Striga germination stimulants or inhibitors. In this paper, we report the crystal structure of ShHTL8, which is determined at a resolution of 1.4 Å. Scanning fluorimetry and HPLC assays indicate that L125, M147, M154 and I194 are important binding sites, and of which L125 may act as a key holder involved in the catalytic reaction. Additionally, the corresponding residue, Y124 of ShHTL1 and F135 of ShHTL2 also play a significant role in the substrate recognition.

Genome-wide identification of MST, SUT and SWEET family sugar transporters in root parasitic angiosperms and analysis of their expression during host parasitism.

BACKGROUND: Root parasitic weeds are a major constraint to crop production worldwide causing significant yearly losses in yield and economic value. These parasites cause their destruction by attaching to their hosts with a unique organ, the haustorium, that allows them to obtain the nutrients (sugars, amino acids, etc.) needed to complete their lifecycle. Parasitic weeds differ in their nutritional requirements and degree of host dependency and the differential expression of sugar transporters is likely to be a critical component in the parasite's post-attachment survival. RESULTS: We identified gene families encoding monosaccharide transporters (MSTs), sucrose transporters (SUTs), and SWEETs (Sugars Will Eventually be Exported Transporters) in three root-parasitic weeds differing in host dependency: Triphysaria versicolor (facultative hemiparasite), Phelipanche aegyptiaca (holoparasite), and Striga hermonthica (obligate hemiparasite). The phylogenetic relationship and differential expression profiles of these genes throughout parasite development were examined to uncover differences existing among parasites with different levels of host dependence. Differences in estimated gene numbers are found among the three parasites, and orthologs within the different sugar transporter gene families are found to be either conserved among the parasites in their expression profiles throughout development, or to display parasite-specific differences in developmentally-timed expression. For example, MST genes in the pGLT clade express most highly before host connection in Striga and Triphysaria but not Phelipanche, whereas genes in the MST ERD6-like clade are highly expressed in the post-connection growth stages of Phelipanche but highest in the germination and reproduction stages in Striga. Whether such differences reflect changes resulting from differential host dependence levels is not known. CONCLUSIONS: While it is tempting to speculate that differences in estimated gene numbers and expression profiles among members of MST, SUT and SWEET gene families in Phelipanche, Striga and Triphysaria reflect the parasites' levels of host dependence, additional evidence that altered transporter gene expression is causative versus consequential is needed. Our findings identify potential targets for directed manipulation that will allow for a better understanding of the nutrient transport process and perhaps a means for controlling the devastating effects of these parasites on crop productivity.

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.

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.

Found in Translation: Applying Lessons from Model Systems to Strigolactone Signaling in Parasitic Plants.

Strigolactones (SLs) are small molecules that act as endogenous hormones to regulate plant development as well as exogenous cues that help parasitic plants to infect their hosts. Given that parasitic plants are experimentally challenging systems, researchers are using two approaches to understand how they respond to host-derived SLs. The first involves extrapolating information on SLs from model genetic systems to dissect their roles in parasitic plants. The second uses chemicals to probe SL signaling directly in the parasite Striga hermonthica. These approaches indicate that parasitic plants have co-opted a family of α/ß hydrolases to perceive SLs. The importance of this genetic and chemical information cannot be overstated since parasitic plant infestations are major obstacles to food security in the developing world.

Structure-function analysis identifies highly sensitive strigolactone receptors in Striga.

Strigolactones are naturally occurring signaling molecules that affect plant development, fungi-plant interactions, and parasitic plant infestations. We characterized the function of 11 strigolactone receptors from the parasitic plant Striga hermonthica using chemical and structural biology. We found a clade of polyspecific receptors, including one that is sensitive to picomolar concentrations of strigolactone. A crystal structure of a highly sensitive strigolactone receptor from Striga revealed a larger binding pocket than that of the Arabidopsis receptor, which could explain the increased range of strigolactone sensitivity. Thus, the sensitivity of Striga to strigolactones from host plants is driven by receptor sensitivity. By expressing strigolactone receptors in Arabidopsis, we developed a bioassay that can be used to identify chemicals and crops with altered strigolactone levels.

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.

PARASITIC PLANTS. Probing strigolactone receptors in Striga hermonthica with fluorescence.

Elucidating the signaling mechanism of strigolactones has been the key to controlling the devastating problem caused by the parasitic plant Striga hermonthica. To overcome the genetic intractability that has previously interfered with identification of the strigolactone receptor, we developed a fluorescence turn-on probe, Yoshimulactone Green (YLG), which activates strigolactone signaling and illuminates signal perception by the strigolactone receptors. Here we describe how strigolactones bind to and act via ShHTLs, the diverged family of α/ß hydrolase-fold proteins in Striga. Live imaging using YLGs revealed that a dynamic wavelike propagation of strigolactone perception wakes up Striga seeds. We conclude that ShHTLs function as the strigolactone receptors mediating seed germination in Striga. Our findings enable access to strigolactone receptors and observation of the regulatory dynamics for strigolactone signal transduction in Striga.