The Jasmonic Acid Pathway Positively Regulates the Polyphenol Oxidase-Based Defense against Tea Geometrid Caterpillars in the Tea Plant (Camellia sinensis).

Polyphenol oxidases (PPOs) as inducible defense proteins, contribute to tea (Camellia sinensis) resistance against tea geometrid larvae (Ectropis grisescens), and this resistance has been associated with the jasmonic acid (JA) signaling by testing geometrid performance in our previous work. However, the regulation of PPO-based defense by JA and other hormone signaling underlying these defense responses is poorly understood. Here, we investigated the role of phytohormones in regulating the PPO response to tea geometrids. We profiled levels of defense hormones, PPO activity and CsPPO genes in leaves infested with tea geometrids. Then, hormone levels were manipulated by exogenous application of methyl jasmonate (MeJA), gibberellin acid (GA3), abscisic acid (ABA), JA biosynthesis inhibitors (sodium diethyldithiocarbamate trihydrate, DIECA and salicylhydroxamic acid, SHAM) and GA inhibitor (uniconazole, UNI). Upon geometrid attack, JA levels significantly increased, whereas GA levels notably decreased and ABA level was slightly decreased. And the PPO activity significantly increased in line with the transcript levels of CsPPO2 and CsPPO4 but not CsPPO1. There were an obvious antagonistic cross-talk between JA and GA signals and an association among JA signals, PPO response and herbivore resistance in tea plants. Pretreatment with MeJA increased PPO activity by activating the transcripts of CsPPO2 and CsPPO4, whereas application of JA inhibitor DIECA suppressed PPO activity. GA3 strongly enhanced PPO activity, but ABA did not alter PPO activity. These findings strongly suggest that JA is a central player in PPO-mediated tea resistance against tea geometrids in a manner that prioritizes defense over growth.

Transcriptional landscape of soybean (Glycine max) embryonic axes during germination in the presence of paclobutrazol, a gibberellin biosynthesis inhibitor.

Gibberellins (GA) are key positive regulators of seed germination. Although the GA effects on seed germination have been studied in a number of species, little is known about the transcriptional reprogramming modulated by GA during this phase in species other than Arabidopsis thaliana. Here we report the transcriptome analysis of soybean embryonic axes during germination in the presence of paclobutrazol (PBZ), a GA biosynthesis inhibitor. We found a number of differentially expressed cell wall metabolism genes, supporting their roles in cell expansion during germination. Several genes involved in the biosynthesis and signaling of other phytohormones were also modulated, indicating an intensive hormonal crosstalk at the embryonic axis. We have also found 26 photosynthesis genes that are up-regulated by PBZ at 24 hours after imbibition (HAI) and down-regulated at 36 HAI, which led us to suggest that this is part of a strategy to implement an autotrophic growth program in the absence of GA-driven mobilization of reserves. Finally, 30 transcription factors (mostly from the MYB, bHLH, and bZIP families) were down-regulated by PBZ and are likely downstream GA targets that will drive transcriptional changes during germination.

Mediation of Flower Induction by Gibberellin and its Inhibitor Paclobutrazol: mRNA and miRNA Integration Comprises Complex Regulatory Cross-Talk in Apple.

Guaranteeing successful flowering is very important in economic plant species, especially apple (Malus domestica Borkh.), which is difficult to induce to flower. However, the gene expression and networks involved in flowering have not been totally characterized. Here, we employed mRNA and microRNA (miRNA) sequencing to understand the different responses to gibberellin- and its inhibitor paclobutrazol- (PAC) mediated flower induction. Significant opposite cytological and morphological changes were observed in treated terminal buds, which led to a reduced flowering rate under gibberellin and an increased flowering rate under PAC. We also found that the differentially expressed mRNAs, miRNAs and miRNA target genes participated in different biological networks including hormones, photosynthesis, redox state and other metabolic processes, which provided important clues to understand the complex networks involved in apple flower induction. Additionally, we subsequently focused on one important candidate, MdSPL3, which is one of 31 apple SPL gene family members and whose transcription was inhibited by gibberellin but promoted by PAC. Functional investigation showed that MdSPL3 was located in the nucleus, and ectopic MdSPL3 activated floral meristem identity genes, promoted the formation of floral primordia and led to an earlier flowering phenotype in Arabidopsis. Our research identified critical mRNA and miRNA responsive to gibberellin or PAC, and provided a candidate framework for flower induction. This carefully orchestrated regulatory cross-talk highlighted potential targets for developing regulatory techniques and genetic improvement of flower induction in apple.

Sulfur dioxide alleviates programmed cell death in barley aleurone by acting as an antioxidant.

Sulfur dioxide (SO2), a gaseous signaling molecule in animal cells, has recently been found to play a physiological role in plants. Here we studied the role of SO2 in gibberellic acid (GA3)-induced programmed cell death (PCD) in barley (Hordeum vulgare L.) aleurone layers. The application of the SO2 donor (NaHSO3/Na2SO3, 1:3 M/M) effectively alleviated PCD in barley aleurone layers in a dose-dependent manner with an optimal concentration of 50 µM. Further investigations showed that SO2 reduced the accumulation of hydrogen peroxide (H2O2), superoxide anion (⋅O2-) and malondialdehyde (MDA) in aleurone layers. Moreover, the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and guaiacol peroxidase (POD) were enhanced by SO2 donor treatment. Meanwhile, lipoxygenase (LOX) activity was attenuated by SO2 donor treatment. Furthermore, an induction of endogenous H2S and NO were also observed in SO2-treated aleurone layers, suggesting interactions of SO2 with other well-known signaling molecules. Taken together, we show that SO2 negatively regulated PCD by acting as an antioxidant to scavenge excessive reactive oxygen species (ROS) generated during PCD.

Abscisic acid regulates seed germination of Vellozia species in response to temperature.

The relationship between the phytohormones, gibberellin (GA) and abscisic acid (ABA) and light and temperature on seed germination is still not well understood. We aimed to investigate the role of the ABA and GA on seed germination of Vellozia caruncularis, V. intermedia and V. alutacea in response to light/dark conditions on different temperature. Seeds were incubated in GA (GA3 or GA4 ) or ABA and their respective biosynthesis inhibitors (paclobutrazol - PAC, and fluridone - FLU) solutions at two contrasting temperatures (25 and 40 °C). Furthermore, endogenous concentrations of active GAs and those of ABA were measured in seeds of V. intermedia and V. alutacea during imbibition/germination. Exogenous ABA inhibited the germination of Vellozia species under all conditions tested. GA, FLU and FLU + GA3 stimulated germination in the dark at 25 °C (GA4 being more effective than GA3 ). PAC reduced seed germination in V. caruncularis and V. alutacea, but did not affect germination of V. intermedia at 40 °C either under light or dark conditions. During imbibition in the dark, levels of active GAs decreased in the seeds of V. intermedia, but were not altered in those of V. alutacea. Incubation at 40 °C decreased ABA levels during imbibition in both V. caruncularis and V. alutacea. We conclude that the seeds of Vellozia species studied here require light or high temperature to germinate and ABA has a major role in the regulation of Vellozia seed germination in response to light and temperature.

Enterobacter sp. I-3, a bio-herbicide inhibits gibberellins biosynthetic pathway and regulates abscisic acid and amino acids synthesis to control plant growth.

Very few bacterial species were identified as bio-herbicides for weed control. The present research was focused to elucidate the plant growth retardant properties of Enterobacter sp. I-3 during their interaction by determining the changes in endogenous photosynthetic pigments, plant hormones and amino acids. The two bacterial isolates I-4-5 and I-3 were used to select the superior bacterium for controlling weed seeds (Echinochloa crus-galli L. and Portulaca oleracea L.) germination. The post-inoculation of I-3 (Enterobacter sp. I-3) significantly inhibited the weeds seed germination than their controls. The mechanism of bacterium induced plant growth reduction was identified in lettuce treated with I-3 bacterium and compared their effects with known chemical herbicide, trinexapac-ethyl (TE). The treatment of I-3 and TE showed a significant inhibitory effect on shoot length, leaf number, leaf length, leaf width, shoot weight, root weight and chlorophyll content in lettuce seedlings. The endogenous gibberellins (GAs) and abscisic acid (ABA) analysis showed that Enterobacter sp. I-3 treated plants had lower levels of GAs (GA12, GA19, GA20 and GA8) and GAs/ABA ratio and then, the higher level of ABA when compared to their controls. Indeed, the individual amino acids ie., aspartic acid, glutamic acid, glycine, threonine, alanine, serine, leucine, isoleucine and tyrosine were declined in TE and I-3 exposed plants. Our results suggest that the utilization of Enterobacter sp. I-3 inhibits the GAs pathway and amino acids synthesis in weeds to control their growth can be an alternative to chemical herbicides.

Gibberellin-Regulation and Genetic Variations in Leaf Elongation for Tall Fescue in Association with Differential Gene Expression Controlling Cell Expansion.

Leaf elongation rate (LER) is an important factor controlling plant growth and productivity. The objective of this study was to determine whether genetic variation in LER for a fast-growing ('K-31'), and a dwarf cultivar ('Bonsai') of tall fescue (Festuca arundinacea) and gibberellic acid (GA) regulation of LER were associated with differential expression of cell-expansion genes. Plants were treated with GA3, trinexapac-ethyl (TE) (GA inhibitor), or water (untreated control) in a hydroponic system. LER of 'K-31' was 63% greater than that of 'Bonsai', which corresponded with 32% higher endogenous GA4 content in leaf and greater cell elongation and production rates under the untreated control condition. Exogenous application of GA3 significantly enhanced LER while TE treatment inhibited leaf elongation due to GA3-stimulation or TE-inhibition of cell elongation and production rate in leaves for both cultivars. Real-time quantitative polymerase chain reaction analysis revealed that three α-expansins, one ß-expansin, and three xyloglucan endotransglycosylase (XET) genes were associated with GA-stimulation of leaf elongation, of which, the differential expression of EXPA4 and EXPA7 was related to the genotypic variation in LER of two cultivars. Those differentially-expressed expansin and XET genes could play major roles in genetic variation and GA-regulated leaf elongation in tall fescue.

Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions.

Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis.

Chemical Promotion of Endogenous Amounts of ABA in Arabidopsis thaliana by a Natural Product, Theobroxide.

Plant hormones are a group of structurally diverse small compounds that orchestrate the cellular processes governing proper plant growth and environmental adaptation. To understand the details of hormonal activity, we must study not only their inherent activities but also the cross-talk among plant hormones. In addition to their use in agriculture, plant chemical activators, such as probenazole and uniconazole, have made great contributions to understand hormonal cross-talk. However, the use of plant chemical activators is limited due to the lack of activators for certain hormones. For example, to the best of our knowledge, there are only a few chemical activators previously known to stimulate the accumulation of ABA in plants, such as absinazoles and proanthocyanidins. In many cases, antagonistic effects have been examined in experiments using exogenously applied ABA, although these studies did not account for biologically relevant concentrations. In this report, it was found that a natural product, theobroxide, had potential as a plant chemical activator for stimulating the accumulation of ABA. Using theobroxide, the antagonistic effect of ABA against GAs was proved without exogenously applying ABA or using mutant plants. Our results suggest that ABA levels could be chemically controlled to elicit ABA-dependent biological phenomena.

CbCBF from Capsella bursa-pastoris enhances cold tolerance and restrains growth in Nicotiana tabacum by antagonizing with gibberellin and affecting cell cycle signaling.

Plant cells respond to cold stress via a regulatory mechanism leading to enhanced cold acclimation accompanied by growth retardation. The C-repeat binding factor (CBF) signaling pathway is essential for cold response of flowering plants. Our previously study documented a novel CBF-like gene from the cold-tolerant Capsella bursa-pastoris named CbCBF, which was responsive to chilling temperatures. Here, we show that CbCBF expression is obviously responsive to chilling, freezing, abscisic acid, gibberellic acid (GA), indoleacetic acid or methyl jasmonate treatments and that the CbCBF:GFP fusion protein was localized to the nucleus. In addition, CbCBF overexpression conferred to the cold-sensitive tobacco plants enhanced tolerance to chilling and freezing, as well as dwarfism and delayed flowering. The leaf cells of CbCBF overexpression tobacco lines attained smaller sizes and underwent delayed cell division with reduced expression of cyclin D genes. The dwarfism of CbCBF transformants can be partially restored by GA application. Consistently, CbCBF overexpression reduced the bioactive gibberellin contents and disturbed the expression of gibberellin metabolic genes in tobacco. Meanwhile, cold induced CbCBF expression and cold tolerance in C. bursa-pastoris are reduced by GA. We conclude that CbCBF confers cold resistance and growth inhibition to tobacco cells by interacting with gibberellin and cell cycle pathways, likely through activation of downstream target genes.

BRAHMA ATPase of the SWI/SNF chromatin remodeling complex acts as a positive regulator of gibberellin-mediated responses in arabidopsis.

SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways.

Chemical screening of an inhibitor for gibberellin receptors based on a yeast two-hybrid system.

We applied a yeast two-hybrid (Y2H) system to the high-throughput monitoring of two proteins' interaction, a receptor for phytohormone gibberellin (GA) and its direct signal transducer DELLA. With this system, we screened inhibitors to the interaction. As a result, we discovered a chemical, 3-(2-thienylsulfonyl)pyrazine-2-carbonitrile (TSPC), and we confirmed that TSPC is an inhibitor for GA perception by in vitro and in planta evaluations.

High levels of jasmonic acid antagonize the biosynthesis of gibberellins and inhibit the growth of Nicotiana attenuata stems.

Hormones play pivotal roles in regulating plant development, growth, and stress responses, and cross-talk among different hormones fine-tunes various aspects of plant physiology. Jasmonic acid (JA) is important for plant defense against herbivores and necrotic fungi and also regulates flower development; in addition, Arabidopsis mutants over-producing JA usually have stunted stems and wound-induced jasmonates suppress Arabidopsis growth, suggesting that JA is also involved in stem elongation. Gibberellins (GAs) promote stem and leaf growth and modulate seed germination, flowering time, and the development of flowers, fruits, and seeds. However, little is known about the interaction between the JA and GA pathways. Two calcium-dependent protein kinases, CDPK4 and CDPK5, are important suppressors of JA accumulation in a wild tobacco species, Nicotiana attenuata. The stems of N. attenuata silenced in CDPK4 and CDPK5 (irCDPK4/5 plants) had dramatically increased levels of JA and exhibited stunted elongation and had very high contents of secondary metabolites. Genetic analysis indicated that the high JA levels in irCDPK4/5 stems accounted for the suppressed stem elongation and the accumulation of secondary metabolites. Supplementation of GA(3) to irCDPK4/5 plants largely restored normal stem growth to wild-type levels. Measures of GA levels indicated that over-accumulation of JA in irCDPK4/5 stems inhibited the biosynthesis of GAs. Finally, we show that JA antagonizes GA biosynthesis by strongly inhibiting the transcript accumulation of GA20ox and possibly GA13ox, the key genes in GA production, demonstrating that high JA levels antagonize GA biosynthesis in stems.

Opening of Iris flowers is regulated by endogenous auxins.

Flower opening in Iris (Iris×hollandica) requires elongation of the pedicel and ovary. This moves the floral bud upwards, thereby allowing the tepals to move laterally. Flower opening is requires with elongation of the pedicel and ovary. In cv. Blue Magic, we investigated the possible role of hormones other than ethylene in pedicel and ovary elongation and flower opening. Exogenous salicylic acid (SA) and the cytokinins benzyladenine (N6-benzyladenine, BA) and zeatin did not affect opening. Jasmonic acid (JA) and abscisic acid (ABA) were slightly inhibitory, but an inhibitor of ABA synthesis (norflurazon) was without effect. Flower opening was promoted by gibberellic acid (GA(3)), but two inhibitors of gibberellin synthesis (4-hydroxy-5-isopropyl-2-methylphenyltrimethyl ammonium chloride-1-piperidine carboxylate, AMO-1618; ancymidol) did not change opening. The auxins indoleacetic acid (IAA) and naphthaleneacetic acid (NAA) strongly promoted elongation and opening. An inhibitor of auxin transport (2,3,5-triodobenzoic acid, TIBA) and an inhibitor of auxin effects [α-(p-chlorophenoxy)-isobutyric acid; PCIB] inhibited elongation and opening. The data suggest that endogenous auxins are among the regulators of the pedicel and ovary elongation and thus of flower opening in Iris.

Gibberellin is required for the formation of tension wood and stem gravitropism in Acacia mangium seedlings.

BACKGROUND AND AIMS: Angiosperm trees generally form tension wood on the upper sides of leaning stems. The formation of tension wood is an important response to gravitational stimulus. Gibberellin appears to be involved in the differentiation of secondary xylem, but it remains unclear whether gibberellin plays a key role in the formation of tension wood and plant gravitropism. Therefore, a study was designed to investigate the effects of gibberellin and of inhibitors of the synthesis of gibberellin, namely paclobutrazole and uniconazole-P, on the formation of tension wood and negative stem gravitropism in Acacia mangium seedlings. METHODS: Gibberellic acid (GA(3)), paclobutrazole and uniconazole-P were applied to seedlings via the soil in which they were growing. Distilled water was applied similarly as a control. Three days after such treatment, seedlings were tilted at an angle of 45° from the vertical, and samples of stems were collected for analysis 2 weeks, 2 months and 6 months after tilting. The effects of treatments on the stem recovery degree (Rº) were analysed as an index of the negative gravitropism of seedlings, together the width of the region of tension wood in the upper part of inclined stems. KEY RESULTS: It was found that GA(3) stimulated the negative gravitropism of tilted seedling stems of A. mangium, while paclobutrazole and uniconazole-P inhibited recovery to vertical growth. Moreover, GA(3) stimulated the formation of tension wood in tilted A. mangium seedlings, while paclobutrazole and uniconazole-P strongly suppressed the formation of tension wood, as assessed 2 weeks after tilting. CONCLUSIONS: The results suggest that gibberellin plays an important role at the initial stages of formation of tension wood and in stem gravitropism in A. mangium seedlings in response to a gravitational stimulus.

Gibberellin biosynthetic inhibitors make human malaria parasite Plasmodium falciparum cells swell and rupture to death.

Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites.

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice.

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA(3)) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA(1) and real-time PCR quantitative assay analyses confirmed that the decrease of GA(1) in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice.

Epigenetic and physiological effects of gibberellin inhibitors and chemical pruners on the floral transition of azalea.

The ability to control the timing of flowering is a key strategy in planning the production of ornamental species such as azaleas; however, it requires a thorough understanding of floral transition. DNA methylation is involved in controlling the functional state of chromatin and gene expression during floral induction pathways in response to environmental and developmental signals. Plant hormone signalling is also known to regulate suites of morphogenic processes in plants and its role in flowering-time control is starting to emerge as a key controlling step. This work investigates if the gibberellin (GA) inhibitors and chemical pinching applied in improvement of azalea flowering alter the dynamics of DNA methylation or the levels of polyamines (PAs), GAs and cytokinins (CKs) during floral transition, and whether these changes could be related to the effects observed on flowering ability. DNA methylation during floral transition and endogenous content of PAs, GAs and CKs were analysed after the application of GA synthesis inhibitors (daminozide, paclobutrazol and chlormequat chloride) and a chemical pruner (fatty acids). The application of GA biosynthesis inhibitors caused alterations in levels of PAs, GAs and CKs and in global DNA methylation levels during floral transition; also, these changes in plant growth regulators and DNA methylation were correlated with flower development. DNA methylation, PA, GA and CK levels can be used as predictive markers of plant floral capacity in azalea.

Synergism between demethylation inhibitor fungicides or gibberellin inhibitor plant growth regulators and bifenthrin in a pyrethroid-resistant population of Listronotus maculicollis (Coleoptera: Curculionidae).

In 2007-2008, the "annual bluegrass weevil," Listronotus maculicollis Kirby (Coleoptera: Curculionidae), a serious pest of Poa annua L. (Poales: Poaceae) on U.S. golf courses, was shown to be resistant to two pyrethroids, bifenthrin and lambda-cyhalothrin. In 2008, we showed that bifenthrin resistance was principally mediated by oxidase detoxification (cytochrome P450 [P450]). P450s can be inhibited by demethylation inhibitor fungicides and gibberellin inhibitor plant growth regulators, both of which are commonly used on golf courses. We tested these compounds for synergistic activity with bifenthin against a pyrethroid-resistant population of L. maculicollis. The LD50 value for bifenthrin was significantly reduced from 87 ng per insect (without synergists) to 9.6-40 ng per insect after exposure to the fungicides fenarimol, fenpropimorph, prochloraz, propiconazole, and pyrifenox and the plant growth regulators flurprimidol, paclobutrazol, and trinexapac-ethyl. Simulated field exposure with formulated products registered for use on turf revealed enhanced mortality when adult weevils were exposed to bifenthrin (25% mortality, presented alone) combined with field dosages of propiconizole, fenarimol, flurprimidol, or trinexapac-ethyl (range, 49-70% mortality).

Biosynthesis of uterotonic diterpenes from Montanoa tomentosa (zoapatle).

Montanoa tomentosa (zoapatle) is a Central American plant used in Mexico in traditional herbal medicine to ease childbirth labor and to cure certain female disorders. Recently, crude extracts of M. tomentosa have been reported to have an aphrodisiacal effect on male rats. The bioactive molecules are the uterotonic diterpenes kaurenoic acid (KA), grandiflorenic acid (GF), and monoginoic acid (MO). Roots of M. tomentosa contain all three diterpenes, whereas in leaves only kaurenoic and GF are present. However, despite the pharmacological importance of these compounds, specific information about their biosynthesis and localization in the plant is not available. In this investigation, we followed the metabolic transformation of a tritium-labeled diterpene-precursor via geranylgeranyl diphosphate into each of the three diterpenes. Inhibitors of gibberellin biosynthesis were used to elucidate the sequence of conversion of the intermediates. Our results suggest the biosynthetic conversion of KA into GF by a putative cytochrome P450-like desaturase. Partial characterization of the enzyme revealed that it requires NADPH and O2 but is inhibited by 50 microM paclobutrazol, suggesting a cytochrome P450 desaturase like enzyme (EC 1.14.14.-). Optimal reaction conditions are 32 degrees C and a pH of 7.6, respectively. Apparent kinetics parameters for KA gave a K(m,app) of 36.31 microM, and a V(max, app) of 13.6 nmol KA mg(1)protein h(-1). Based on the data presented, a putative biosynthetic pathway is proposed for the uterotonic diterpenes of M. tomentosa.