Dimesulfazet, a novel rice paddy herbicide, is an inhibitor of very long-chain fatty acid biosynthesis.

Dimesulfazet can control annual and perennial sedges in rice paddies. Here we assessed its mode of action. We performed a phenotype assay of Arabidopsis, conducted a metabolomic analysis of Echinochloa crus-galli, and analyzed the endogenous concentration of very long-chain fatty acids (VLCFAs) in Schoenoplectiella juncoides. Dimesulfazet treatment caused curling and greening symptoms in the leaves and fiddlehead-like symptoms in the inflorescences of Arabidopsis. These symptoms were visually indistinguishable from those caused by flufenacet and benfuresate, which belong to Herbicide Resistance Action Committee (HRAC) Group 15. We performed GC-MS/MS analysis of primary metabolites and LC-MS analysis of lipids in the herbicide-treated E. crus-galli, followed by Orthogonal Partial Least Squares Discriminant Analysis clustering. The results showed that dimesulfazet belongs to the HRAC Group 15 cluster. The endogenous concentrations of C24:0, C26:0, and C28:0 decreased in dimesulfazet-treated plants as compared to those in the control. Overall, the mode of action of dimesulfazet involves the inhibition of VLCFA biosynthesis.

A Rationally Designed Agonist Defines Subfamily IIIA Abscisic Acid Receptors As Critical Targets for Manipulating Transpiration.

Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates Pyrabactin Resistance 1 (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin's arylnitrile mimics ABA's cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA's carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin's compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce Arabidopsis whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its in vitro potency translates to ABA-like activity in vivo. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration.

Chemical screening and development of novel gibberellin mimics.

Gibberellin (GA) plays versatile roles in the regulation of plant growth and development and therefore is widely used as a regulator in agriculture. We performed a chemical library screening and identified a chemical, named 67D, as a stimulator of seed germination that was suppressed by paclobutrazol (PAC), a GA biosynthesis inhibitor. In vitro binding assays indicated that 67D binds to the GID1 receptor. Further studies on the structure-activity relationship identified a chemical, named chemical 6, that strongly promoted seed germination suppressed by PAC. Chemical 6 was further confirmed to promote the degradation of RGA (for repressor of ga1-3), a DELLA protein, and suppress the expression levels of GA3ox1 in the same manner as GA does. 67D and its analogs are supposed to be agonists of GID1 and are expected to be utilized in agriculture and basic research as an alternative to GA.

Substituted Phthalimide AC94377 Is a Selective Agonist of the Gibberellin Receptor GID1.

Gibberellin (GA) is a major plant hormone that regulates plant growth and development and is widely used as a plant growth regulator in agricultural production. There is an increasing demand for function-limited GA mimics due to the limitations on the agronomical application of GA to crops, including GA's high cost of producing and its leading to the crops' lodging. AC94377, a substituted phthalimide, is a chemical that mimics the growth-regulating activity of GAs in various plants, despite its structural difference. Although AC94377 is widely studied in many weeds and crops, its mode of action as a GA mimic is largely unknown. In this study, we confirmed that AC94377 displays GA-like activities in Arabidopsis (Arabidopsis thaliana) and demonstrated that AC94377 binds to the Arabidopsis GIBBERELLIN INSENSITIVE DWARF1 (GID1) receptor (AtGID1), forms the AtGID1-AC94377-DELLA complex, and induces the degradation of DELLA protein. Our results also indicated that AC94377 is selective for a specific subtype among three AtGID1s and that the selectivity of AC94377 is attributable to a single residue at the entrance to the hydrophobic pocket of GID1. We conclude that AC94377 is a GID1 agonist with selectivity for a specific subtype of GID1, which could be further developed and used as a function-limited regulator of plant growth in both basic study and agriculture.

Does the brassinosteroid signal pathway in photomorphogenesis overlap with the gravitropic response caused by auxin?

Brassinosteroid (BR) and auxin co-regulate plant growth in a process termed cross-talking. Based on the assumption that their signal transductions are partially shared, inhibitory chemicals for both signal transductions were screened from a commercially available library. A chemical designated as NJ15 (ethyl 2-[5-(3,5-dichlorophenyl)-1,2,3,4-tetrazole-2-yl]acetate) diminished the growth promotion of both adzuki bean epicotyls and Arabidopsis seedlings, by the application of either BR or auxin. To understand its target site(s), bioassays with a high dependence on the signal transduction of either BR (BR-signaling) or auxin (AX-signaling) were performed. NJ15 inhibited the photomorphogenesis of Arabidopsis seedlings grown in the dark, which mainly depends on BR-signaling, while NJ15 also inhibited their gravitropic responses mainly depending on AX-signaling. On the study for the structure-activity relationships of NJ15 analogs, they showed strong correlations on the inhibitory profiles between BR- and AX-signalings. These correlations imply that NJ15 targets the downstream pathway after the integration of BR- and AX-signals.

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.

Screening and characterization of an inhibitory chemical specific to Arabidopsis gibberellin 2-oxidases.

The hydroxylation of gibberellin (GA) at the 2-position is known as the major cause of inactivation of GAs, whose reaction is catalyzed by 2-oxoglutarate dependent dioxygenases, also termed GA 2-oxidases (GA2oxs). To block GA catabolism in plants, a few chemicals can be used. To obtain novel inhibitors specific to GA2oxs, we performed in vitro random screenings by using (3)H-16,17-dihydro-GA(4) and recombinant Arabidopsis GA2ox2. As a result, one candidate, methyl 6-chloro-3H-1,2,3-benzodithiazole-4-carboxylate 2-oxide (CBTC), was selected from the screening, and was subjected to in-planta evaluations. CBTC promoted both the germination and elongation of Arabidopsis seedlings. This strongly suggests that CBTC inhibits GA2oxs in Arabidopsis with high specificity.

Degeneration of the locus ceruleus noradrenergic neurons in the stress-induced depression of rats.

We produced a model of depression in rats which have been exposed to 2-weeks forced walking stress. Electron microscopic observation on the locus ceruleus (LC) cells of the model rats disclosed low dense areas, destroyed membranes, aggregation of intracellular organs, and increased microglia. Density of LC axon terminals in the frontal cortex stained with dopamine beta-hydroxylase antiserum and percentage of LC cells stained with horseradish peroxidase or activated by electrical stimulation antidromically were low in the model. These indices increased in the model treated with imipramine. These findings suggest that the LC noradrenergic neurons degenerate in depression, but regenerate in remission.

Contribution of the stress-induced degeneration of the locus coeruleus noradrenergic neurons to the pathophysiology of depression: a study on an animal model.

A novel theory on the pathophysiology of depression would be expected to resolve a contradiction between therapeutic time lag and monoamine hypothesis. On the basis of the fact that a subgroup of depression appears during or after stress, we exposed rats to a long-term (2 weeks) forced walking stress and produced depression-model rats in one group and spontaneous recovery rats in another. The density of axon terminals of the locus coeruleus (LC) neurons in the frontal cortex stained by dopamine ß-hydroxylase antiserum was lower in the depression-model rats than in the spontaneous recovery rats and in the control rats without stress. The density was higher in the model rats daily treated with imipramine than in those treated with saline. Morphological projection (MP) index (a percentage of horseradish peroxidase-positive LC cells in total number of LC cells) and electrophysiological projection index (a percentage of LC neurons activated antidromically by electrical stimulation of the cerebral cortex) were lower in the depression-model rats than in the recovery and control rats. MP index was higher in the imipramine-treated rats than that in the saline-treated rats. Electron microscopic examination of the LC disclosed such degenerative changes as low-dense areas without structure, aggregation of intracellular organs, destroyed membranes around the rough endoplasmic reticulum (rER), a decreased number of deformed subsurface cisterns, glia invaginated into the LC neurons and prominent appearance of microglia containing increased number of lipofustin or lysosome in the model rats, but not in the spontaneous recovery rats. These findings suggest that the terminals and cell bodies of the LC noradrenergic neurons degenerate in the stress-induced depression-model rats and regenerate in the imipramine-treated model rats. This degenerative change may possibly contribute to the decrease in synthesis and metabolism of noradrenaline (NA), the slowing of axonal flow, the accumulation of NA in the neurons, the decrease in discharge rate of LC neurons without stress and the increase in release of NA in response to an additional stress. It may also explain the therapeutic time lag that is required to repair the noradrenergic neurons.