Assessing Seed Germination Response of Parasitic Plant Striga hermonthica with Small-Molecule Probes.

Seed germination of a parasitic plant Striga hermonthica is elicited by strigolactones which are exuded from roots of host plants. Here, we describe a high-throughput germination assay and a method for visualizing in vivo strigolactone receptor functions with a fluorogenic probe.

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.

Enantioselective Synthesis of Polycyclic γ-Lactams with Multiple Chiral Carbon Centers via Ni(0)-Catalyzed Asymmetric Carbonylative Cycloadditions without Stirring.

γ-Lactam derivatives with multiple contiguous stereogenic carbon centers are ubiquitous in physiologically active compounds. The development of straightforward and reliable synthetic routes to such chiral structural motifs in a stereocontrolled manner should thus be of importance. Herein, we report a strategy to construct polycyclic γ-lactam derivatives that contain more than two contiguous stereogenic centers in an enantioselective as well as atom-economic manner. Moreover, we have achieved the first enantioselective synthesis of strigolactam derivative GR-24, a racemic variant of which is a potential seed germination stimulator and plant-growth regulator. A key of the procedure presented here is a nickel(0)/chiral phosphoramidite-catalyzed asymmetric [2+2+1] carbonylative cycloaddition between readily accessible ene-imines and carbon monoxide, which proceeded enantioselectively to furnish up to 90% ee (>99% ee after recrystallization). The results of mechanistic studies, including the isolation of a chiral heteronickelacycle, support that the enantioselectivity on the two contiguous carbon atoms of the γ-lactams is determined during the oxidative cyclization on nickel(0).

A femtomolar-range suicide germination stimulant for the parasitic plant Striga hermonthica.

The parasitic plant Striga hermonthica has been causing devastating damage to the crop production in Africa. Because Striga requires host-generated strigolactones to germinate, the identification of selective and potent strigolactone agonists could help control these noxious weeds. We developed a selective agonist, sphynolactone-7, a hybrid molecule originated from chemical screening, that contains two functional modules derived from a synthetic scaffold and a core component of strigolactones. Cooperative action of these modules in the activation of a high-affinity strigolactone receptor ShHTL7 allows sphynolactone-7 to provoke Striga germination with potency in the femtomolar range. We demonstrate that sphynolactone-7 is effective for reducing Striga parasitism without impinging on host strigolactone-related processes.

Small Molecule Toolbox for Strigolactone Biology.

Strigolactones (SLs) are plant hormones associated with diverse developmental processes including plant architecture and stress responses. SLs are exuded to the soil as an ecological signal to attract symbiotic arbuscular-mycorrhizal fungi. This ecological mechanism is also used by parasitic plants to detect the presence of host plants and initiate germination. The functional diversity of SLs makes SL biology so extensive that a single methodology is not sufficient to comprehend it. This review describes the theoretical and practical aspects of the design of small molecule probes that have been used to elucidate the functions of SLs. The lessons from the development of small molecules to tackle the unique questions in SL biology might be instructive in the extending field of chemical biology in plants.

Discovery of Shoot Branching Regulator Targeting Strigolactone Receptor DWARF14.

DWARF14 (D14) is a strigolactone receptor that plays a central role in suppression of shoot branching, and hence is a potential target to increase crop productions and biomass. Recently, we reported a fluorescence turn-on probe, Yoshimulactone Green (YLG), which generates a strong fluorescence upon the hydrolysis by D14-type strigolactone receptors. Herein, we applied a YLG-based in vitro assay to a high-throughput chemical screening and identified a novel small molecule DL1 as a potent inhibitor of D14. DL1 competes with endogenous strigolactones, thereby increasing the number of shoot branching in a model plant Arabidopsis as well as in rice. Thus, DL1 is expected to be useful not only as a tool to understand the biological roles of D14 receptors in plant growth and development, but also as a potent agrochemical to improve the crop yield.

The dynamics of strigolactone perception in Striga hermonthica: a working hypothesis.

Plant-derived strigolactones have diverse functions at ecological scale, including effects upon the growth of plants themselves. The parasitic plants from the family Orobanchaceae interfere with the ecological and hormonal functions of strigolactones to generate unique germination abilities based on the sensing of host-derived strigolactones. Although the recent discovery of strigolactone receptors has enabled us to begin elucidating the mechanism of strigolactone perception, how perception relates to plant parasitism is still a mystery. In this review, we explore emerging questions by introducing recent advances in strigolactone research in parasitic plants. We also attempt to construct a conceptual framework for the unique in planta dynamics of strigolactone perception uncovered through the use of fluorescent probes for strigolactone receptors. Understanding the mechanisms of strigolactone-related processes is essential for controlling the parasitic plant Striga hermonthica, which has caused devastating damage to crop production in Africa.

Small-molecule antagonists of germination of the parasitic plant Striga hermonthica.

Striga spp. (witchweed) is an obligate parasitic plant that attaches to host roots to deplete them of nutrients. In Sub-Saharan Africa, the most destructive Striga species, Striga hermonthica, parasitizes major food crops affecting two-thirds of the arable land and over 100 million people. One potential weakness in the Striga infection process is the way it senses the presence of a host crop. Striga only germinates in the presence of the plant hormone strigolactone, which exudes from a host root. Hence small molecules that perturb strigolactone signaling may be useful tools for disrupting the Striga lifecycle. Here we developed a chemical screen to suppress strigolactone signaling in the model plant Arabidopsis. One compound, soporidine, specifically inhibited a S. hermonthica strigolactone receptor and inhibited the parasite's germination. This indicates that strigolactone-based screens using Arabidopsis are useful in identifying lead compounds to combat Striga infestations.

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.

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.

Detection of parasitic plant suicide germination compounds using a high-throughput Arabidopsis HTL/KAI2 strigolactone perception system.

Strigolactones are terpenoid-based plant hormones that act as communication signals within a plant, between plants and fungi, and between parasitic plants and their hosts. Here we show that an active enantiomer form of the strigolactone GR24, the germination stimulant karrikin, and a number of structurally related small molecules called cotylimides all bind the HTL/KAI2 α/ß hydrolase in Arabidopsis. Strigolactones and cotylimides also promoted an interaction between HTL/KAI2 and the F-box protein MAX2 in yeast. Identification of this chemically dependent protein-protein interaction prompted the development of a yeast-based, high-throughput chemical screen for potential strigolactone mimics. Of the 40 lead compounds identified, three were found to have in planta strigolactone activity using Arabidopsis-based assays. More importantly, these three compounds were all found to stimulate suicide germination of the obligate parasitic plant Striga hermonthica. These results suggest that screening strategies involving yeast/Arabidopsis models may be useful in combating parasitic plant infestations.

HY5 is involved in strigolactone-dependent seed germination in Arabidopsis.

Strigolactones (SLs) function as plant hormones that mediate a myriad of developmental responses in higher plants. SLs also act as an environmental signal to stimulate seed germination of parasitic plant species of genera Striga and Orobanche. In contrast to their hormonal roles, genetic mechanisms of how SLs stimulate parasitic seed germination are largely not known. Recently, we established a method to monitor the germination-stimulating activity of SLs in Arabidopsis using temperature as environmental constraint (thermoinhibition). Here, we show that SLs require HY5, a key transcription factor for light signal transduction, to stimulate Arabidopsis seed germination during thermoinhibiton. Genetic analysis suggests the HY5 dependent signaling pathway is independent of other known SL signaling pathways. Thermoinhibibed seeds expressed low level of HY5 while GR24 increase the level at both mRNA and protein level. A role of SLs on activating crucial light signaling components such as HY5 may hint the evolution of parasitism associated with SL usage.

The embryonic leaf identity gene FUSCA3 regulates vegetative phase transitions by negatively modulating ethylene-regulated gene expression in Arabidopsis.

BACKGROUND: The embryonic temporal regulator FUSCA3 (FUS3) plays major roles in the establishment of embryonic leaf identity and the regulation of developmental timing. Loss-of-function mutations of this B3 domain transcription factor result in replacement of cotyledons with leaves and precocious germination, whereas constitutive misexpression causes the conversion of leaves into cotyledon-like organs and delays vegetative and reproductive phase transitions. RESULTS: Herein we show that activation of FUS3 after germination dampens the expression of genes involved in the biosynthesis and response to the plant hormone ethylene, whereas a loss-of-function fus3 mutant shows many phenotypes consistent with increased ethylene signaling. This FUS3-dependent regulation of ethylene signaling also impinges on timing functions outside embryogenesis. Loss of FUS3 function results in accelerated vegetative phase change, and this is again partially dependent on functional ethylene signaling. This alteration in vegetative phase transition is dependent on both embryonic and vegetative FUS3 function, suggesting that this important transcriptional regulator controls both embryonic and vegetative developmental timing. CONCLUSION: The results of this study indicate that the embryonic regulator FUS3 not only controls the embryonic-to-vegetative phase transition through hormonal (ABA/GA) regulation but also functions postembryonically to delay vegetative phase transitions by negatively modulating ethylene-regulated gene expression.

Thermoinhibition uncovers a role for strigolactones in Arabidopsis seed germination.

Strigolactones are host factors that stimulate seed germination of parasitic plant species such as Striga and Orobanche. This hormone is also important in shoot branching architecture and photomorphogenic development. Strigolactone biosynthetic and signaling mutants in model systems, unlike parasitic plants, only show seed germination phenotypes under limited growth condition. To understand the roles of strigolactones in seed germination, it is necessary to develop a tractable experimental system using model plants such as Arabidopsis. Here, we report that thermoinhibition, which involves exposing seeds to high temperatures, uncovers a clear role for strigolactones in promoting Arabidopsis seed germination. Both strigolactone biosynthetic and signaling mutants showed increased sensitivity to seed thermoinhibition. The synthetic strigolactone GR24 rescued germination of thermoinbibited biosynthetic mutant seeds but not a signaling mutant. Hormone analysis revealed that strigolactones alleviate thermoinhibition by modulating levels of the two plant hormones, GA and ABA. We also showed that GR24 was able to counteract secondary dormancy in Arabidopsis ecotype Columbia (Col) and Cape Verde island (Cvi). Systematic hormone analysis of germinating Striga helmonthica seeds suggested a common mechanism between the parasitic and non-parasitic seeds with respect to how hormones regulate germination. Thus, our simple assay system using Arabidopsis thermoinhibition allows comparisons to determine similarities and differences between parasitic plants and model experimental systems for the use of strigolactones.

Strigolactones as small molecule communicators.

Originally identified as an allelochemical involved in plant host-parasite interactions, strigolactones have more recently been shown to have much broader communication roles. Strigolactones function as a symbiotic communicator in plants and mycorrhizal fungi interactions and have also been shown to have hormonal roles in higher plants. This ability to act as both an exogenous and an endogenous signal has interesting implications with respect to the constraints on strigolactone structures. Probing the hormonal function of strigolactones using chemical biology and genetics is beginning to provide clues as to how strigolactones were co-opted as an allelochemical signal by parasitic plants.

A small-molecule screen identifies new functions for the plant hormone strigolactone.

Parasitic weeds of the genera Striga and Orobanche are considered the most damaging agricultural agents in the developing world. An essential step in parasitic seed germination is sensing a group of structurally related compounds called strigolactones that are released by host plants. Although this makes strigolactone synthesis and action a major target of biotechnology, little fundamental information is known about this hormone. Chemical genetic screening using Arabidopsis thaliana as a platform identified a collection of related small molecules, cotylimides, which perturb strigolactone accumulation. Suppressor screens against select cotylimides identified light-signaling genes as positive regulators of strigolactone levels. Molecular analysis showed strigolactones regulate the nuclear localization of the COP1 ubiquitin ligase, which in part determines the levels of light regulators such as HY5. This information not only uncovers new functions for strigolactones but was also used to identify rice cultivars with reduced capacity to germinate parasitic seed.

Strigolactones: a new hormone with a past.

The recent discovery of an endogenous hormonal activity for strigolactones in shoot branching was surprising since these molecules were thought to mostly play roles as signaling molecules between organisms. Even in the context of plant hormones, strigolactones appear to be different in that their role in plant development is quite restricted. This most probably reflects early days and new hormonal functions will most probably be found for these compounds in the future. In this respect, the exogenous role of strigolactones in parasitic plant seed germination may hint to functions of this compound in seed development. However, showing new roles for strigolactones in the seed or any other aspect of plant development for that matter will require developing assays in model genetic systems such as Arabidopsis and rice where we can take full advantage of the experimental tools that are available.

Quantitative kinetic analysis of lung nodules using the temporal subtraction technique in dynamic chest radiographies performed with a flat panel detector.

Early detection and treatment of lung cancer is one of the most effective means of reducing cancer mortality, and to this end, chest X-ray radiography has been widely used as a screening method. A related technique based on the development of computer analysis and a flat panel detector (FPD) has enabled the functional evaluation of respiratory kinetics in the chest and is expected to be introduced into clinical practice in the near future. In this study, we developed a computer analysis algorithm to detect lung nodules and to evaluate quantitative kinetics. Breathing chest radiographs obtained by modified FPD and breath synchronization utilizing diaphragmatic analysis of vector movement were converted into four static images by sequential temporal subtraction processing, morphological enhancement processing, kinetic visualization processing, and lung region detection processing. An artificial neural network analyzed these density patterns to detect the true nodules and draw their kinetic tracks. Both the algorithm performance and the evaluation of clinical effectiveness of seven normal patients and simulated nodules showed sufficient detecting capability and kinetic imaging function without significant differences. Our technique can quantitatively evaluate the kinetic range of nodules and is effective in detecting a nodule on a breathing chest radiograph. Moreover, the application of this technique is expected to extend computer-aided diagnosis systems and facilitate the development of an automatic planning system for radiation therapy.

Development of a kinetic analysis technique for PACS management and a screening examination in dynamic radiography.

The purpose of this study was to develop a method of kinetic analysis for picture archiving and communication system (PACS) management and computer-aided diagnostic application in dynamic chest radiography. The main analytical technique used in this study was a new algorithm that converts dynamic radiographs into a color-static image. The algorithm is a visualization technique for kinetic information that uses the intensity-density transformation and the direction classification in optical flow. The image made by the new algorithm was defined as a "kinetic map," and, by analysis using the kinetic map, a patient collation system and nodule detection system were constructed. By analysis that used an artificial neural network of certain feature vectors as kinetic map similarity, the collation system obtained good identification performance. Temporal subtraction processing between a current-status map with simulated nodule and previous-status map detected the region of abnormality as the simulated nodule. It is expected that our method of analysis will be useful as a screening examination for risk management and computer-aided diagnostic application in dynamic chest radiography.