The continued high rates of using antibiotics in healthcare and livestock, without sufficient new compounds reaching the market, has led to a dramatic increase in antimicrobial resistance, with multidrug-resistant bacteria emerging as a major public health threat worldwide. Because the vast majority of antiinfectives are natural products or have originated from them, we assessed the predictive power of plant molecular phylogenies and species distribution modeling in the search for clades and areas which promise to provide a higher probability of delivering new antiinfective compound leads. Our approach enables taxonomically and spatially targeted bioprospecting and supports the battle against the global antimicrobial crisis.

Dalbergia monetaria is an Amazonian plant whose bark is widely used to treat urinary tract infections. This paper describes a bio-guided study of ethanolic extracts from the bark and leaves of D. monetaria, in a search for metabolites active against human pathogenic bacteria. In vitro assays were performed against 10 bacterial strains, highlighting methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Fractioning of the extracts was performed using instrumental and classical techniques, and samples were characterized by UHPLC-HRMS/MS. Ethyl acetate fractions from bark and leaves showed similar antibacterial activities. EAFB is enriched in isoflavone C-glucosides and EAFL enriched in proanthocyanidins. Subfractions from EAFL presented higher activity and showed a complex profile of proanthocyanidins constructed by (epi)-cassiaflavan and (epi)-catechin units, including dimers, trimers and tetramers. The fragmentation pattern emphasized the neutral loss of cassiaflavan units by quinone-methide fission. Fraction SL7-6, constituted by (ent)-cassiaflavan-(ent)-cassiaflavan-(epi)-catechin isomers, showed the lowest MIC against the S. aureus and P. aeruginosa with values corresponding to 64 and 32 µg/mL, respectively. Cassiaflavan-proanthocyanidins have not been found previously in another botanical genus, except in Cassia, and the traditional medicinal use of D. monetaria might be related to the antibacterial activity of proanthocyanidins characterized in the species.

AbstractThree previously undescribed natural products, phomopsinin A – C (1 – 3), together with three known compounds, namely, cis-hydroxymellein (4), phomoxanthone A (5) and cytochalasin L-696,474 (6), were isolated from the solid culture of Phomopsis sp. CAM212, an endophytic fungus obtained from Garcinia xanthochymus. Their structures were determined on the basis of spectroscopic data, including IR, NMR, and MS. The absolute configurations of 1 and 2 were assigned by comparing their experimental and calculated ECD spectra. Acetylation of compound 1 yielded 1a, a new natural product derivative that was tested together with other isolated compounds on lipopolysaccharide-stimulated RAW 264.7 cells. Cytochalasin L-696,474 (6) was found to significantly inhibit nitric oxide production, but was highly cytotoxic to the treated cells, whereas compound 1 slightly inhibited nitric oxide production, which was not significantly different compared to lipopolysaccharide-treated cells. Remarkably, the acetylated derivative of 1, compound 1a, significantly inhibited nitric oxide production with an IC50 value of 14.8 µM and no cytotoxic effect on treated cells, thereby showing the importance of the acetyl group in the anti-inflammatory activity of 1a. The study of the mechanism of action revealed that 1a decreases the expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory cytokine IL-6 without an effect on IL-1β expression. Moreover, it was found that 1a exerts its anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells by downregulating the activation of ERK1/2 and by preventing the translocation of nuclear factor κB. Thus, derivatives of phomopsinin A (1), such as compound 1a, could provide new anti-inflammatory leads.

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In nature, plants must respond to multiple stresses simultaneously, which likely demands cross-talk between stress-response pathways to minimize fitness costs. Here we provide genetic evidence that biotic and abiotic stress responses are differentially prioritized in Arabidopsis thaliana leaves of different ages to maintain growth and reproduction under combined biotic and abiotic stresses. Abiotic stresses, such as high salinity and drought, blunted immune responses in older rosette leaves through the phytohormone abscisic acid signaling, whereas this antagonistic effect was blocked in younger rosette leaves by PBS3, a signaling component of the defense phytohormone salicylic acid. Plants lacking PBS3 exhibited enhanced abiotic stress tolerance at the cost of decreased fitness under combined biotic and abiotic stresses. Together with this role, PBS3 is also indispensable for the establishment of salt stress- and leaf age-dependent phyllosphere bacterial communities. Collectively, our work reveals a mechanism that balances trade-offs upon conflicting stresses at the organism level and identifies a genetic intersection among plant immunity, leaf microbiota, and abiotic stress tolerance.

Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in Arabidopsis. Arabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.

Heteromannan (HM) is one of the most ancient cell wall polymers in the plant kingdom, consisting of β-(1–4)-linked backbones of glucose (Glc) and mannose (Man) units. Despite the widespread distribution of HM polysaccharides, their biosynthesis remains mechanistically unclear. HM is elongated by glycosyltransferases (GTs) from the cellulose synthase-like A (CSLA) family. MANNAN-SYNTHESIS RELATED (MSR) putative GTs have also been implicated in (gluco)mannan synthesis, but their roles have been difficult to decipher in planta and in vitro. To further characterize the products of the HM synthases and accessory proteins, we chose a synthetic biology approach to synthesize plant HM in yeast. The expression of a CSLA protein in Pichia pastoris led to the abundant production of plant HM: up to 30% of glycans in the yeast cell wall. Based on sequential chemical and enzymatic extractions, followed by detailed structural analyses, the newly produced HM polymers were unbranched and could be larger than 270 kDa. Using CSLAs from different species, we programmed yeast cells to produce an HM backbone composed exclusively of Man or also incorporating Glc. We demonstrate that specific MSR cofactors were indispensable for mannan synthase activity of a coffee CSLA or modulated a functional CSLA enzyme to produce glucomannan instead of mannan. Therefore, this powerful platform yields functional insight into the molecular machinery required for HM biosynthesis in plants.

The plant hormone auxin activates primary response genes by facilitating proteolytic removal of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)-inducible repressors, which directly bind to transcriptional AUXIN RESPONSE FACTORS (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼6.4 μM) were determined by isothermal titration calorimetry. In silico protein–protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein–protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.

Wound responses in plants have to be coordinated between organs so that locally reduced growth in a wounded tissue is balanced by appropriate growth elsewhere in the body. We used a JASMONATE ZIM DOMAIN 10 (JAZ10) reporter to screen for mutants affected in the organ-specific activation of jasmonate (JA) signaling in Arabidopsis thaliana seedlings. Wounding one cotyledon activated the reporter in both aerial and root tissues, and this was either disrupted or restricted to certain organs in mutant alleles of core components of the JA pathway including COI1, OPR3, and JAR1. In contrast, three other mutants showed constitutive activation of the reporter in the roots and hypocotyls of unwounded seedlings. All three lines harbored mutations in Novel Interactor of JAZ (NINJA), which encodes part of a repressor complex that negatively regulates JA signaling. These ninja mutants displayed shorter roots mimicking JA-mediated growth inhibition, and this was due to reduced cell elongation. Remarkably, this phenotype and the constitutive JAZ10 expression were still observed in backgrounds lacking the ability to synthesize JA or the key transcriptional activator MYC2. Therefore, JA-like responses can be recapitulated in specific tissues without changing a plant’s ability to make or perceive JA, and MYC2 either has no role or is not the only derepressed transcription factor in ninja mutants. Our results show that the role of NINJA in the root is to repress JA signaling and allow normal cell elongation. Furthermore, the regulation of the JA pathway differs between roots and aerial tissues at all levels, from JA biosynthesis to transcriptional activation.

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co2+ or Mn2+ as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg2+. In reaction with Co2+, PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg2+, PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co2+, Mn2+, or Mg2+ in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.