Ligand diversity contributes to the full activation of the jasmonate pathway in Marchantia polymorpha.

In plants, jasmonate signaling regulates a wide range of processes from growth and development to defense responses and thermotolerance. Jasmonates, such as jasmonic acid (JA), (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), 12-oxo-10,15(Z)-phytodienoic acid (OPDA), and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), are derived from C18 (18 Carbon atoms) and C16 polyunsaturated fatty acids (PUFAs), which are found ubiquitously in the plant kingdom. Bryophytes are also rich in C20 and C22 long-chain polyunsaturated fatty acids (LCPUFAs), which are found only at low levels in some vascular plants but are abundant in organisms of other kingdoms, including animals. The existence of bioactive jasmonates derived from LCPUFAs is currently unknown. Here, we describe the identification of an OPDA-like molecule derived from a C20 fatty acid (FA) in the liverwort Marchantia polymorpha (Mp), which we term (5Z,8Z)-10-(4-oxo-5-((Z)-pent-2-en-1-yl)cyclopent-2-en-1-yl)deca-5,8-dienoic acid (C20-OPDA). This molecule accumulates upon wounding and, when applied exogenously, can activate known Coronatine Insensitive 1 (COI1) -dependent and -independent jasmonate responses. Furthermore, we identify a dn-OPDA-like molecule (Δ4-dn-OPDA) deriving from C20-OPDA and demonstrate it to be a ligand of the jasmonate coreceptor (MpCOI1-Mp Jasmonate-Zinc finger inflorescence meristem domain [MpJAZ]) in Marchantia. By analyzing mutants impaired in the production of LCPUFAs, we elucidate the major biosynthetic pathway of C20-OPDA and Δ4-dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ4-dn-OPDA and dn-OPDA, we demonstrate the additive nature of these molecules in the activation of jasmonate responses. Taken together, our data identify a ligand of MpCOI1 and demonstrate LCPUFAs as a source of bioactive jasmonates that are essential to the immune response of M. polymorpha.

(3R,7S)-12-Hydroxy-jasmonoyl-l-isoleucine is the genuine bioactive stereoisomer of a jasmonate metabolite in Arabidopsis thaliana.

The oxylipin plant hormone (3R,7S)-jasmonoyl-l-isoleucine [or (+)-7-iso-jasmonoyl-l-isoleucine, JA-Ile] is widely recognized as a plant defense hormone against pathogens and chewing insects. The metabolism of JA-Ile into 12-OH-JA-Ile and 12-COOH-JA-Ile is the central mechanism for the inactivation of JA signaling. Recently, 12-OH-JA-Ile was reported to function as a ligand for the JA-Ile co-receptor COI1-JAZ. However, in previous studies, '12-OH-JA-Ile' used was a mixture of four stereoisomers, the naturally occurring cis-isomer (3R,7S)-12-OH-JA-Ile and the trans-isomer (3R,7R)-12-OH-JA-Ile, and the unnatural cis-isomer (3S,7R)-12-OH-JA-Ile and the trans-isomer (3S,7S)-12-OH-JA-Ile. Thus, the genuine bioactive form of 12-OH-JA-Ile has not yet been identified. In the present study, we prepared pure stereoisomers of 12-OH-JA-Ile and identified (3R,7S)-12-OH-JA-Ile as the naturally occurring bioactive form of 12-OH-JA-Ile and found that it binds to COI1-JAZ9 as effectively as (3R,7S)-JA-Ile. In addition, we revealed that the unnatural trans-isomer (3S,7S)-12-OH-JA-l-Ile functions as another bioactive isomer. The pure (3R,7S)-12-OH-JA-Ile causes partial JA-responsive gene expression without affecting the expression of JAZ8/10, which is involved in the negative feedback regulation of JA-signaling. Thus, (3R,7S)-12-OH-JA-Ile could cause weak and sustainable expression of certain JA-responsive genes until the catabolism of (3R,7S)-12-OH-JA-Ile into (3R,7S)-12-COOH-JA-Ile occurs. The use of chemically pure (3R,7S)-12-OH-JA-Ile confirmed the genuine biological activities of '12-OH-JA-Ile' by excluding the possible effects of other stereoisomers. A chemical supply of pure (3R,7S)-12-OH-JA-Ile with an exact bioactivity profile will enable further detailed studies of the unique role of 12-OH-JA-Ile in planta.

A comprehensive in vitro fluorescence anisotropy assay system for screening ligands of the jasmonate COI1-JAZ co-receptor in plants.

The phytohormone (+)-7-iso-jasmonoyl-l-isoleucine regulates many developmental and stress responses in plants and induces protein-protein interactions between COI1, the F-box component of E3 ubiquitin ligase, and jasmonate ZIM domain (JAZ) repressors. These interactions cause JAZ degradation and activate jasmonate (JA), leading to plant defense responses, growth inhibition, and senescence. Thirteen JAZ subtypes are encoded in the Arabidopsis thaliana genome, but a detailed understanding of the physiological functions of these JAZ subtypes remains unclear, partially because of the genetic redundancy of JAZ genes. One strategy to elucidate the complex JA signaling pathways is to develop a reliable and comprehensive binding assay system of the ligands with all combinations of the co-receptors. Herein, we report the development of a fluorescence anisotropy-based in vitro binding assay system to screen for the ligands of the COI1-JAZ co-receptors. Our assay enabled the first quantitative analysis of the affinity values and JAZ-subtype selectivity of various endogenous JA derivatives, such as coronatine, jasmonic acid, and 12-hydroxyjasmonoyl-l-isoleucine. Because of its high signal-to-noise ratio and convenient mix-and-read assay system, our screening approach can be used in plate reader-based assays of both agonists and antagonists of COI1-JAZ co-receptors.

A scalable synthesis of (+)-coronafacic acid.

A facile, efficient, and scalable synthesis of optically pure coronafacic acid by resolution of racemic coronafacic acid obtained using an improved version of Watson's method has been developed. By optimizing the boron-mediated aldol reaction of Watson, we were able to prepare 2.1 g of racemic coronafacic acid. This was coupled with (S)-4-isopropyl-2-oxazolidinone to give a mixture of diastereomeric coronafacyl oxazolidinones, which were readily separable by silica-gel column chromatography to give 630 mg of optically pure (+)-coronafacic acid.

Jasmonic Acid Inhibits Auxin-Induced Lateral Rooting Independently of the CORONATINE INSENSITIVE1 Receptor.

Plant root systems are indispensable for water uptake, nutrient acquisition, and anchoring plants in the soil. Previous studies using auxin inhibitors definitively established that auxin plays a central role regulating root growth and development. Most auxin inhibitors affect all auxin signaling at the same time, which obscures an understanding of individual events. Here, we report that jasmonic acid (JA) functions as a lateral root (LR)-preferential auxin inhibitor in Arabidopsis (Arabidopsis thaliana) in a manner that is independent of the JA receptor, CORONATINE INSENSITIVE1 (COI1). Treatment of wild-type Arabidopsis with either (-)-JA or (+)-JA reduced primary root length and LR number; the reduction of LR number was also observed in coi1 mutants. Treatment of seedlings with (-)-JA or (+)-JA suppressed auxin-inducible genes related to LR formation, diminished accumulation of the auxin reporter DR5::GUS, and inhibited auxin-dependent DII-VENUS degradation. A structural mimic of (-)-JA and (+)-coronafacic acid also inhibited LR formation and stabilized DII-VENUS protein. COI1-independent activity was retained in the double mutant of transport inhibitor response1 and auxin signaling f-box protein2 (tir1 afb2) but reduced in the afb5 single mutant. These results reveal JAs and (+)-coronafacic acid to be selective counter-auxins, a finding that could lead to new approaches for studying the mechanisms of LR formation.

Stable Iron Porphyrin Intramolecularly Coordinated by Alcoholate Anion: Synthesis and Evaluation of Axial Ligand Effect of Alcoholate on Spectroscopy and Catalytic Activity.

We synthesized intramolecularly aliphatic alcoholate-coordinated iron porphyrins (1a, 1b) that retain their axial coordination in the presence of another ligand or oxidant. The electron-donative character of alcoholate was less than that of thiolate, and the coordination ability of a sixth ligand to 1a and 1b was very much lower than in the case of the thiolate-coordinated compounds. Density functional theory calculations indicated that the marked difference in coordination ability could be explained in terms of thermodynamic and steric factors. The catalytic oxidizing ability of the thiolate-coordinated compound, SR complex, was much higher than that of 1a.

Role of Thiolate Ligand in Spin State and Redox Switching in the Cytochrome P450 Catalytic Cycle.

The catalytic cycle of cytochrome P450 involves a change from the resting-state, water-bound, six-coordinated form (1, low-spin state) to a five-coordinated form (2, high-spin state) upon binding of a hydrophobic substrate. Here, we used a heme-thiolate model complex (SR complex) with THF as a model of nonionic H2O to address the question of whether or not coordination of nonionic water is sufficient to induce the low-spin state. Measurements of electronic absorption spectra and magnetic properties confirmed that five-coordinated SR complex has a high-spin state, and THF-bound, six-coordinated SR has a low-spin state in dichloromethane at ambient temperature. The redox potential E1/2 (FeII/FeIII) of THF-bound SR was 80-90 mV more negative than that of five-coordinated SR. These properties indicate SR is a good model of P450. Our results suggest that thiolate coordination plays a key role in setting the low energy barrier between the high-spin and low-spin states.

Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA.

A versatile solid-phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher-order structures of DNA.

[Tuberculous epididymitis after Bacillus Calmette-Guérin intravesical therapy : a case report].

We describe a case of tuberculous epididymitis following Bacillus Calmette-Guérin (BCG) instillation in a 79-year-old man. He had received transuretheral resection of bladder tumor in Sep 2009. Histopathological diagnosis was urothelial carcinoma, high grade, pTa and pTis. To prevent recurrence, he received maintenance therapy for Feb 2010-May 2011 after eight weekly intravesical instillation of BCG. Skin fistula with discharge in the left scrotum occurred in Sep 2011. Treatment with levofloxacin was not effective. Therefore, we performed bilateral orchiectomy (he had hormone therapy of prostate cancer) and left scrotal skin resection. Histopathological examine showed tuberculous epididymitis. He had no signs of recurrence 2 years postoperatively and has not received any anti-tuberculous chemotherapy.

Synthesis of the carbon framework of scholarisine A by intramolecular oxidative coupling.

Scholarisine A, isolated from the leaves of Alstonia scholaris, is a monoterpene indole alkaloid with an unprecedented cage-like structure. In this paper, preparation of the distinctive cage-like core skeleton of scholarisine A is described. The key feature of this synthetic strategy is an intramolecular oxidative coupling reaction at the late stage to construct a 10-oxa-tricyclo[5.3.1.0(3, 8)]undecan-9-one structure fused with indolenine. Intramolecular oxidative coupling by using N-iodosuccinimide gave the carbon framework of scholarisine A in moderate yield, which is the first example of intramolecular oxidative-coupling reaction between non-activated enolate and indole. This study lays the foundation for continued investigations towards the total synthesis of scholarisine A.