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Rice (Oryza sativa L.) is a chilling-sensitive staple crop that originated in subtropical regions of Asia. Introduction of the chilling tolerance trait enables the expansion of rice cultivation to temperate regions. Here we report the cloning and characterization of HAN1, a quantitative trait locus (QTL) that confers chilling tolerance on temperate japonica rice. HAN1 encodes an oxidase that catalyzes the conversion of biologically active jasmonoyl-L-isoleucine (JA-Ile) to the inactive form 12-hydroxy-JA-Ile (12OH-JA-Ile) and fine-tunes the JA-mediated chilling response. Natural variants in HAN1 diverged between indica and japonica rice during domestication. A specific allele from temperate japonica rice, which gained a putative MYB cis-element in the promoter of HAN1 during the divergence of the two japonica ecotypes, enhances the chilling tolerance of temperate japonica rice and allows it to adapt to a temperate climate. The results of this study extend our understanding of the northward expansion of rice cultivation and provide a target gene for the improvement of chilling tolerance in rice.

Intraerythrocytic malaria parasites can obtain nearly their entire amino acid requirement by degrading host cell hemoglobin. The sole exception is isoleucine, which is not present in adult human hemoglobin and must be obtained exogenously. We evaluated two compounds for their potential to interfere with isoleucine utilization. Mupirocin, a clinically used antibacterial, kills Plasmodium falciparum parasites at nanomolar concentrations. Thiaisoleucine, an isoleucine analog, also has antimalarial activity. To identify targets of the two compounds, we selected parasites resistant to either mupirocin or thiaisoleucine. Mutants were analyzed by genome-wide high-density tiling microarrays, DNA sequencing, and copy number variation analysis. The genomes of three independent mupirocin-resistant parasite clones had all acquired either amplifications encompassing or SNPs within the chromosomally encoded organellar (apicoplast) isoleucyl-tRNA synthetase. Thiaisoleucineresistant parasites had a mutation in the cytoplasmic isoleucyl-tRNA synthetase. The role of this mutation in thiaisoleucine resistance was confirmed by allelic replacement. This approach is generally useful for elucidation of new targets in P. falciparum. Our study shows that isoleucine utilization is an essential pathway that can be targeted for antimalarial drug development.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity.

INTRODUCTION: 4-hydroxy isoleucine (4-HIL), a potent glucose-lowering agent and insulin secretagogue, is widely available in nutraceutical market as fenugreek seed extract formulations. This study aims to elucidate the oral pharmacokinetics (PK) of 4-HIL in healthy human volunteers to standardize its dose and dosing regimen, ensuring its potential for effective diabetes management. METHODOLOGY: Twelve healthy volunteers received a single oral administration of 150 mg of 4-HIL as fenugreek seed extract tablets. Caplillary blood samples were collected at various time points within 24 h and plasma levels of 4-HIL were quantified using liquid chromatography-tandem mass spectrometry. In vitro studies on 4-HIL pharmacodynamics, derived from the literature, were used to calculate the half-minimal effective concentration (EC50). PK assessments based on compartmental modelling and dosage simulation studies were conducted using PKsolver and ModVizPOP, respectively. The PK simulation included three distinct dosage regimens (150 mg thrice daily, 225 mg twice daily, or 450 mg once daily) to evaluate EC50 level attainment. RESULTS: The best-fit was observed with a two-compartmental model, with maximum 4-HIL plasma concentration (Concentration maximum, 2.42 ± 0.61 µg/mL) observed at 0.5 h (Time maximum). The derived mean EC50 of 4-HIL, needed to reduce blood glucose, was 1.50 ± 0.31 µg/mL. The PK simulation study indicated that daily intake of 450 mg 4-HIL in all three tested dosing regimens had maintained EC50 levels more than 18 h for glucose-lowering effects. CONCLUSION: The optimal 4-HIL dose of 450 mg/day up to three divided dosing regimens has proven effective and hence may be considered for future diabetic trials.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl- l -isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways 1 , 2 , 3 – 4 . Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown 1 , 2 . Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and l -Ile in plants 5 . This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions—Jar1 producing a compound essential for plant development 4 , 5 , and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications. A family of enzymes—coronafacic acid ligases, involved in the synthesis of bacterial phytotoxins—are found to catalyse amide bond formation with a wide range of substrates.

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3 R ,7 S )-jasmonoyl- l -isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs. Three-part receptor for jasmonate plant hormones The receptors for several important plant hormones have been identified in recent years, including those for auxin, the gibberellins and abscisic acid, and structure–function studies have revealed their mechanisms of action. Now the mechanism by which plant cells recognize the jasmonate phytohormones — key players in growth regulation, development and defence responses — is reported. The jasmonate receptor is a three-molecule complex consisting of the F-box protein COI1, a JAZ (JASMONATE ZIM DOMAIN) transcriptional repressor, and inositol pentakisphosphate. All three receptor components are required for high-affinity hormone binding. This system for jasmonate perception involves mechanisms that are distinct from those of the other plant hormones studied so far, although all depend on hormone-mediated protein interactions. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of the JASMONATE ZIM DOMAIN (JAZ) family of transcriptional repressors. These authors elucidate the mechanism of jasmonate perception. They present structural and pharmacological data to show that the true jasmonate receptor is a complex of both COI1 and JAZ. In addition, inositol pentakisphosphate functions as a critical component of the hormone receptor complex.

Jasmonates play a number of diverse roles in plant defense and development. CORONATINE INSENSITIVE1 (COI1), an F-box protein essential for all the jasmonate responses, interacts with multiple proteins to form the SCFCOI¹ E3 ubiquitin ligase complex and recruits jasmonate ZIM-domain (JAZ) proteins for degradation by the 26S proteasome. To determine which protein directly binds to jasmonoyl-isoleucine (JA-Ile)/coronatine (COR) and serves as a receptor for jasmonate, we built a high-quality structural model of COI1 and performed molecular modeling of COI1-jasmonate interactions. Our results imply that COI1 has the structural traits for binding JA-Ile or COR. The direct binding of these molecules with COI1 was further examined using a combination of molecular and biochemical approaches. First, we used the immobilized jasmonate approach to show that the COI1 protein in crude leaf extracts can bind to the jasmonate moiety of JA-Ile. Second, we employed surface plasmon resonance technology with purified COI1 and JAZ1 protein to reveal the interaction among COI1, JA-Ile, and JAZ1. Finally, we used the photoaffinity labeling technology to show the direct binding of COR with purified insect-expressed COI1. Taken together, these results demonstrate that COI1 directly binds to JA-Ile and COR and serves as a receptor for jasmonate.

Background Cluster of differentiation 95 (CD95/Fas/Apo1) as part of the Tumor-necrosis factor (TNF) receptor family is a prototypic trigger of the ‘extrinsic’ apoptotic pathway and its activation by the trimeric ligand CD95L is of high interest. However, CD95L, when presented in solution, exhibits a low efficiency to induce apoptosis signaling in human cells. Results Here, we design a recombinant CD95L exhibiting an isoleucine zipper (IZ) motif at the N-terminus for stabilization of the trimerized CD95L and demonstrate its high apoptosis initiation efficiency. This efficiency is further enhanced by antibody-mediated crosslinking of IZ-CD95L.A cysteine amino acid fused behind the IZ is used as a versatile coupling site for bionanotechnological applications or for the development of biomedical assays. A fast, cheap, and efficient production of CD95L via the HEK293T secretory expression system is presented, along with CD95L affinity purification and functionalization. We verified the biological activity of the purified protein and identified a stabilized trimeric CD95L structure as the most potent inducer of apoptosis signaling. Conclusions The workflow and the findings reported here will streamline a wide array of future low- or high-throughput TNF-ligand screens, and their modification towards improving apoptosis induction efficiency and, potentially, anticancer therapy.

Plant immune responses mediated by the hormone jasmonoyl-L-isoleucine (JA-Ile) are metabolically costly and often linked to reduced growth. Although it is known that JA-Ile activates defense responses by triggering the degradation of JASMONATE ZIM DOMAIN (JAZ) transcriptional repressor proteins, expansion of the JAZ gene family in vascular plants has hampered efforts to understand how this hormone impacts growth and other physiological tasks over the course of ontogeny. Here, we combined mutations within the 13-member Arabidopsis JAZ gene family to investigate the effects of chronic JAZ deficiency on growth, defense, and reproductive output. A higher-order mutant (jaz decuple, jazD) defective in 10 JAZ genes (JAZ1–7, -9, -10, and -13) exhibited robust resistance to insect herbivores and fungal pathogens, which was accompanied by slow vegetative growth and poor reproductive performance. Metabolic phenotypes of jazD discerned from global transcript and protein profiling were indicative of elevated carbon partitioning to amino acid-, protein-, and endoplasmic reticulum body-based defenses controlled by the JA-Ile and ethylene branches of immunity. Resource allocation to a strong defense sink in jazD leaves was associated with increased respiration and hallmarks of carbon starvation but no overt changes in photosynthetic rate. Depletion of the remaining JAZ repressors in jazD further exaggerated growth stunting, nearly abolished seed production and, under extreme conditions, caused spreading necrotic lesions and tissue death. Our results demonstrate that JAZ proteins promote growth and reproductive success at least in part by preventing catastrophic metabolic effects of an unrestrained immune response.

The faithful charging of amino acids to cognate tRNAs by aminoacyl-tRNA synthetases (AARSs) determines the fidelity of protein translation. Isoleucyl-tRNA synthetase (IleRS) distinguishes tRNA Ile from tRNA Met solely based on the nucleotide at wobble position (N34), and a single substitution at N34 could exchange the aminoacylation specificity between two tRNAs. Here, we report the structural and biochemical mechanism of N34 recognition-based tRNA discrimination by Saccharomyces cerevisiae IleRS ( Sc IleRS). Sc IleRS utilizes a eukaryotic/archaeal-specific arginine as the H-bond donor to recognize the common carbonyl group (H-bond acceptor) of various N34s of tRNA Ile , which induces mutual structural adaptations between Sc IleRS and tRNA Ile to achieve a preferable editing state. C34 of unmodified tRNA Ile (CAU) (behaves like tRNA Met ) lacks a relevant H-bond acceptor, which disrupts key H-bonding interactions and structural adaptations and suspends the Sc IleRS·tRNA Ile (CAU) complex in an initial non-reactive state. This wobble nucleotide recognition-based structural adaptation provides mechanistic insights into selective tRNA aminoacylation by AARSs. The faithful charging of amino acids to tRNAs by aminoacyl-tRNA synthetases determines protein translation fidelity. Here, the authors showed how isoleucyl-tRNA synthetase distinguishes tRNA Ile from tRNA Met based on the recognition of wobble nucleotide (N34) and subsequent structural adaptation.