Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells.

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.

Archaeal mevalonate pathway in the uncultured bacterium Candidatus Promineifilum breve belonging to the phylum Chloroflexota.

The archaeal mevalonate pathway is a recently discovered modified version of the eukaryotic mevalonate pathway. This pathway is widely conserved in archaea, except for some archaeal lineages possessing the eukaryotic or other modified mevalonate pathways. Although the pathway seems almost exclusive to the domain Archaea, the whole set of homologous genes of the pathway is found in the metagenome-assembled genome sequence of an uncultivated bacterium, Candidatus Promineifilum breve, of the phylum Chloroflexota. To prove the existence of the archaea-specific pathway in the domain Bacteria, we confirmed the activities of the enzymes specific to the pathway, phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, because only these two enzymes are absent in closely related Chloroflexota bacteria that possess a different type of modified mevalonate pathway. The activity of anhydromevalonate phosphate decarboxylase was evaluated by carotenoid production via the archaeal mevalonate pathway reconstituted in Escherichia coli cells, whereas that of phosphomevalonate dehydratase was confirmed by an in vitro assay using the recombinant enzyme after purification and iron-sulfur cluster reconstruction. Phylogenetic analyses of some mevalonate pathway-related enzymes suggest an evolutionary route for the archaeal mevalonate pathway in Candidatus P. breve, which probably involves horizontal gene transfer events.IMPORTANCEThe recent discovery of various modified mevalonate pathways in microorganisms, such as archaea and Chloroflexota bacteria, has shed light on the complexity of the evolution of metabolic pathways, including those involved in primary metabolism. The fact that the archaeal mevalonate pathway, which is almost exclusive to the domain Archaea, exists in a Chloroflexota bacterium provides valuable insights into the molecular evolution of the mevalonate pathways and associated enzymes. Putative genes probably involved in the archaeal mevalonate pathway have also been found in the metagenome-assembled genomes of Chloroflexota bacteria. Such genes can contribute to metabolic engineering for the bioproduction of valuable isoprenoids because the archaeal mevalonate pathway is known to be an energy-saving metabolic pathway that consumes less ATP than other mevalonate pathways do.

Advances in the investigation of N6-isopentenyl adenosine i6A RNA modification.

Prenylation (isopentenylation), a key post-transcriptional modification with a hydrophobic prenyl group onto the biomacromolecules such as RNA and proteins, influences their localization and function. Prenyltransferases mediate this process, while cytokinin oxidases degrade the prenylated adenosine in plants. This review summarizes current progress in detecting prenylation modifications in RNA across species and their effects on protein synthesis. Advanced methods have been developed to label and study these modifications in vitro and in vivo, despite challenges posed by the inert chemical properties of prenyl groups. Continued advancements in bioorthogonal chemistry promise new tools for understanding the precise biological functions of prenylated RNA modifications and other related proteins.

The utility of Streptococcus mutans undecaprenol kinase for the chemoenzymatic synthesis of diverse non-natural isoprenoids.

Isoprene chemoenzymatic cascades (ICCs) overcome the complexity of natural pathways by leveraging a streamlined two-enzyme cascade, facilitating efficient synthesis of C5-isoprene diphosphate precursors from readily available alcohol derivatives. Despite the documented promiscuity of enzymes in ICCs, exploration of their potential for accessing novel compounds remains limited, and existing methods require additional enzymes for generating longer-chain diphosphates. In this study, we present the utility of Streptococcus mutans undecaprenol kinase (SmUdpK) for the chemoenzymatic synthesis of diverse non-natural isoprenoids. Using a library of 50 synthetic alcohols, we demonstrate that SmUdpK's promiscuity extends to allylic chains as small as four carbons and benzylic alcohols with various substituents. Subsequently, SmUdpK is utilized in an ICC with isopentenyl phosphate kinase and aromatic prenyltransferase to generate multiple non-natural isoprenoids. This work provides evidence that, with proper optimization, SmUdpK can act as the first enzyme in these ICCs, enhancing access to both valuable and novel compounds.

The combination of a microbial and a non-microbial biostimulant increases yield in lettuce (Lactuca sativa) under salt stress conditions by up-regulating cytokinin biosynthesis.

Salinization poses a significant challenge in agriculture, exacerbated by anthropogenic global warming. Biostimulants, derived from living microorganisms or natural extracts, have emerged as valuable tools for conventional and organic agriculture. However, our understanding of the molecular mechanisms underlying the effects of biostimulants is very limited, especially in crops under real cultivation conditions. In this study, we adopted an integrative approach to investigate the effectiveness of the combined application of plant growth-promoting bacterium (Bacillus megaterium strain BM08) and a non-microbial biostimulant under control conditions (normal watering) and salt stress. After confirming the yield increase under both conditions, we investigated the molecular mechanisms underlying the observed effect by measuring a number of physiological parameters (i.e., lipid peroxidation, antioxidants, chlorophylls, total phenolics and phytohormone content), as well as RNA sequencing and primary metabolite analyses. Our findings reveal that the combined effect of the microbial and non-microbial biostimulants led to a decrease in the antioxidant response and an up-regulation of genes involved in cytokinin biosynthesis under salt stress conditions. This, in turn, resulted in a higher concentration of the bioactive cytokinin, isopentenyladenosine, in roots and leaves and an increase in γ-aminobutyric acid, a non-proteic amino acid related to abiotic stress responses. In addition, we observed a decrease in malic acid, along with an abscisic acid (ABA)-independent up-regulation of SR-kinases, a family of protein kinases associated with abiotic stress responses. Furthermore, we observed that the single application of the non-microbial biostimulant triggers an ABA-dependent response under salt stress; however, when combined with the microbial biostimulant, it potentiated the mechanisms triggered by the BM08 bacterial strain. This comprehensive investigation shows that the combination of two biostimulants is able to elicit a cytokinin-dependent response that may explain the observed yield increase under salt stress conditions.

Membrane Permeant Analogs for Independent Cellular Introduction of the Terpene Precursors Isopentenyl- and Dimethylallyl-Pyrophosphate.

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the ß-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP.

Identification and functional characterization of two trans-isopentenyl diphosphate synthases and one squalene synthase involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Two trans-isopentenyl diphosphate synthase and one squalene synthase genes were identified and proved to be involved in the triterpenoid biosynthesis in Platycodon grandiflorus. Platycodon grandiflorus is a commonly used traditional Chinese medicine. The main bioactive compounds of P. grandiflorus are triterpenoid saponins. The biosynthetic pathway of triterpenoid saponins in P. grandiflorus has been preliminarily explored. However, limited functional information on related genes has been reported. A total of three trans-isopentenyl diphosphate synthases (trans-IDSs) genes (PgFPPS, PgGGPPS1 and PgGGPPS2) and one squalene synthase (SQS) gene (PgSQS) in P. grandiflorus were screened and identified from transcriptome dataset. Subcellular localization of the proteins was defined based on the analysis of GFP-tagged. The activity of genes was verified in Escherichia coli, demonstrating that recombinant PgFPPS catalysed the production of farnesyl diphosphate. PgGGPPS1 produced geranylgeranyl diphosphate, whereas PgGGPPS2 did not exhibit catalytic activity. By structural identification of encoding genes, a transmembrane region was found at the C-terminus of the PgSQS gene, which produced an insoluble protein when expressed in E. coli but showed no apparent effect on the enzyme function. Furthermore, some triterpenoid saponin synthesis-related genes were discovered by combining the component content and the gene expression assays at the five growth stages of P. grandiflorus seedlings. The accumulation of active components in P. grandiflorus was closely associated with the expression level of genes related to the synthesis pathway.

Identification and Functional Analysis of Two Novel Genes-Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)-from Aurantiochytrium limacinum Significantly Enhance De Novo ß-Carotene Biosynthesis in Escherichia coli.

Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo ß-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of ß-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in ß-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, ß-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.

Identification and functional analysis of isopentenyl pyrophosphate isomerase genes in the whiteflies Bemisia tabaci (Hemiptera: Aleyrodidae).

The juvenile hormone (JH) plays a vital role in the regulation of a number of physiological processes, including development, reproduction, and ovarian maturation. Isopentenyl pyrophosphate isomerase (IPPI) is a key enzyme in the biosynthetic pathway of JH. In this study, we identified an isopentenyl pyrophosphate isomerase protein from Bemisia tabaci and named it BtabIPPI. The open reading frame (ORF) of BtabIPPI is 768 bp and encodes a protein of 255 amino acids that contains a conserved domain of the Nudix family. The temporal and spatial expression profiles showed that BtabIPPI was highly expressed in the female adults.RNA interference (RNAi)-mediated silencing of BtabIPPI reduced JH titers and the relative expression of vitellogenin receptor (VgR) and JH signaling pathway genes, resulting in a dramatic reduction in fecundity and hatchability. These results indicate that the BtabIPPI gene plays an important role in the female fecundity of B. tabaci. This study will broaden our understanding of the function of IPPI in regulating insect reproduction and provide a theoretical basis for targeting IPPI for pest control in the future.

The Effect of Epimedium Isopentenyl Flavonoids on the Broiler Gut Health Using Microbiomic and Metabolomic Analyses.

Epimedium (EM), also known as barrenwort, is a traditional medicinal plant rich in isopentenyl flavonols, which have beneficial biological activities and can improve human and animal health, but its mechanism is still unclear. In this study, ultra-high-performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry (UHPLC-Q-TOF/MS) and ultra-high-performance liquid chromatography triple-quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) were used to analyse the main components of EM, and isopentenyl flavonols such as Epimedin A, B, and C as well as Icariin were the major components of EM. Meanwhile, broilers were selected as model animals to illuminate the mechanism of Epimedium isopentenyl flavonols (EMIE) on gut health. The results showed that supplementation with 200 mg/kg EM improved the immune response, increased cecum short-chain fatty acids (SCFAs) and lactate concentrations, and improved nutrient digestibility in broilers. In addition, 16S rRNA sequencing showed that EMIE altered the composition of cecal microbiome, increasing the relative abundance of beneficial bacteria (Candidatus Soleaferrea and Lachbospiraceae NC2004 group and Butyricioccus) and reducing that of harmful bacteria (UBA1819, Negativibacillus, and Eisenbergiella). Metabolomic analysis identified 48 differential metabolites, of which Erosnin and Tyrosyl-Tryptophan were identified as core biomarkers. Erosnin and tyrosyl-tryptophan are potential biomarkers to evaluate the effects of EMIE. This shows that EMIE may regulate the cecum microbiota through Butyricicoccus, with changes in the relative abundance of the genera Eisenbergiella and Un. Peptostreptococcaceae affecting the serum metabolite levels of the host. EMIE is an excellent health product, and dietary isopentenyl flavonols, as bioactive components, can improve health by altering the microbiota structure and the plasma metabolite profiles. This study provides the scientific basis for the future application of EM in diets.

The gibberellic acid derived from the plastidial MEP pathway is involved in the accumulation of Bamboo mosaic virus.

A gene upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) infection was revealed as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (NbDXR). DXR is the key enzyme in the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway that catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol-4-phosphate. Knockdown and overexpression of NbDXR followed by BaMV inoculation revealed that NbDXR is involved in BaMV accumulation. Treating leaves with fosmidomycin, an inhibitor of DXR function, reduced BaMV accumulation. Subcellular localization confirmed that DXR is a chloroplast-localized protein by confocal microscopy. Furthermore, knockdown of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, one of the enzymes in the MEP pathway, also reduced BaMV accumulation. The accumulation of BaMV increased significantly in protoplasts treated with isopentenyl pyrophosphate. Thus, the metabolites of the MEP pathway could be involved in BaMV infection. To identify the critical components involved in BaMV accumulation, we knocked down the crucial enzyme of isoprenoid synthesis, NbGGPPS11 or NbGGPPS2. Only NbGGPPS2 was involved in BaMV infection. The geranylgeranyl pyrophosphate (GGPP) synthesized by NbGGPPS2 is known for gibberellin synthesis. We confirmed this result by supplying gibberellic acid exogenously on leaves, which increased BaMV accumulation. The de novo synthesis of gibberellic acid could assist BaMV accumulation.

Enhancement of linalool production in Saccharomyces cerevisiae by utilizing isopentenol utilization pathway.

BACKGROUND: Linalool is a monoterpenoid, also a vital silvichemical with commercial applications in cosmetics, flavoring ingredients, and medicines. Regulation of mevalonate (MVA) pathway metabolic flux is a common strategy to engineer Saccharomyces cerevisiae for efficient linalool production. However, metabolic regulation of the MVA pathway is complex and involves competition for central carbon metabolism, resulting in limited contents of target metabolites. RESULTS: In this study, first, a truncated linalool synthase (t26AaLS1) from Actinidia arguta was selected for the production of linalool in S. cerevisiae. To simplify the complexity of the metabolic regulation of the MVA pathway and increase the flux of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), we introduced the two-step isopentenyl utilization pathway (IUP) into S. cerevisiae, which could produce large amounts of IPP/DMAPP. Further, the S. cerevisiae IDI1 (ecoding isopentenyl diphosphate delta-isomerase) and ERG20F96W-N127W (encoding farnesyl diphosphate synthase) genes were integrated into the yeast genome, combined with the strategies of copy number variation of the t26AaLS1 and ERG20F96W-N127W genes to increase the metabolic flux of the downstream IPP, as well as optimization of isoprenol and prenol concentrations, resulting in a 4.8-fold increase in the linalool titer. Eventually, under the optimization of carbon sources and Mg2+ addition, a maximum linalool titer of 142.88 mg/L was obtained in the two-phase extractive shake flask fermentation. CONCLUSIONS: The results show that the efficient synthesis of linalool in S. cerevisiae could be achieved through a two-step pathway, gene expression adjustment, and optimization of culture conditions. The study may provide a valuable reference for the other monoterpenoid production in S. cerevisiae.

Metabolomics reveals the role of isopentenyl group in coumarins metabolism.

Coumarins are a group of natural compounds commonly found in the families of Rutaceae and Umbelliferae. 7-Isopentenyloxycoumarin (ISC), auraptene (AUR), and umbelliprenin (UM) belong to prenyloxycoumarins (PYCs), which link isopentenyl, geranyl, and farnesyl group at C7 position, respectively. The substituent of 7-ethoxycoumarin (ETC) is the ethyl group. In this study, UPLC-ESI-QTOF-MS (ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-MS)-based metabolomics was used to evaluate the in vivo and in vitro metabolism of PYCs. Results showed that ETC produced 10 known metabolites, and ISC was transformed into 17 metabolites in vivo and in vitro, which were undescribed compounds. A total of 35 AUR metabolites, including 34 undescribed metabolites were identified, and 21 metabolites were reported for the first time in UM. The results indicated that hydroxylation and N-acetylcysteine conjugation were the common metabolic reactions for PYCs. The metabolic rates of ETC, ISC, AUR and UM were 26%, 36%, 81%, and 38%, respectively, in human liver microsome, while they were 24%, 40%, 80%, and 37%, respectively, in mouse liver microsomes. In addition, recombinant cytochrome P450s (CYPs) screening showed that CYP1A1, 2C19, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of hydroxylation metabolites. Together, these results suggest that the isopentenyl group plays an important role in the metabolism of PYCs.

Isopentenylated Bibenzyls and Phenolic Compounds from Dendrobium chrysotoxum Lindl.

Two new isopentenyl bibenzyls, denchrysotonols A and B (1-2), along with 26 known phenolic compounds, were isolated from the stems of cultivated Dendrobium chrysotoxum Lindl. Their chemical structures were clearly elucidated by extensive spectroscopic analysis. Biological evaluation of isolated compounds revealed that phenanthrenes (14, 16-17, 20, and 22) and fluorenone 25 exhibited anti-inflammatory activities which inhibited nitric oxide (NO) production in LPS-activated RAW264.7 macrophages with the IC50 values ranging from 9.4 to 32.5 µM. Moreover, bibenzyls (1-2 and 7) showed good anti-proliferative activities against triple-negative breast cancer (TNBC) cells (HCC1806, MDA-MB-231, and MB-MB-468) with the IC50 values ranging from 8.1 to 18.6 µM, of which 1 and 2 seemed preferentially inhibit MDA-MB-231 cells.

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade.

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s-1 M-1, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s-1 M-1, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0-9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0-8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.

Lepidopteran mevalonate pathway optimization in Escherichia coli efficiently produces isoprenol analogs for next-generation biofuels.

Terpenes constitute the largest class of natural products with over 55,000 compounds with versatile applications including drugs and biofuels. Introducing structural modifications to terpenes through metabolic engineering is an efficient and sustainable way to improve their properties. Here, we report the optimization of the lepidopteran mevalonate (LMVA) pathway towards the efficient production of isopentenyl pyrophosphate (IPP) analogs as terpene precursors. First, we linked the LMVA pathway to NudB, a promiscuous phosphatase, resulting in the production of the six-carbon analog of 3-methyl-3-buten-1-ol (isoprenol), 3-ethyl-3-buten-1-ol (C6-isoprenol). Using C6-isoprenol as the final product, we then engineered the LMVA pathway by redirecting its upstream portion from a thiolase-dependent pathway to a beta-oxidation pathway. The beta-oxidation LMVA pathway transforms valeric acid, a platform chemical that can be produced from biomass, into C6-isoprenol at a titer of 110.3 mg/L, improved from 5.5 mg/L by the thiolase LMVA pathway, which used propionic acid as a feedstock. Knockout of the E. coli endogenous thiolase genes further improved the C6-isoprenol titer to 390 mg/L, implying efficient production of homo isopentenyl pyrophosphate (HIPP). The beta-oxidation LMVA-NudB pathway also converts butanoic acid and hexanoic acid into isoprenol and isoprenol's seven-carbon analog, 3-propyl-3-buten-1-ol (C7-isoprenol), respectively, suggesting the beta-oxidation LMVA pathway produces IPP and C7-IPP from the corresponding fatty acids. Fuel property tests revealed the longer chain isoprenol analogs have lower water solubilities, similar or higher energy densities, and comparable research octane number (RON) boosting effects to isopentenols. This work not only optimizes the LMVA pathway, setting the basis for homoterpene biosynthesis to expand terpene chemical space, but provides an efficient pathway to produce isoprenol analogs as next-generation biofuels from sustainable feedstocks.

Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao, interferes with cytokinin metabolism during infection of Micro-Tom tomato and promotes symptom development.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

Two isopentenyl resorcinols from Peperomia tetraphylla.

Two isopentenyl resorcinols, peperobtusin B and peperobtusin C, have been isolated from Peperomia tetraphylla. Their structures were determined on the basis of spectroscopic methods, especially 1H NMR, 13C NMR, 2D NMR, and HR-TOF-MS. Two compounds were evaluated for cytostatic activity against G2, A 549, Hela and HCT 116 cells, but cytostatic activity of both compounds is weak.

The subcellular localization of two isopentenyl diphosphate isomerases in rice suggests a role for the endoplasmic reticulum in isoprenoid biosynthesis.

KEY MESSAGE: Both OsIPPI1 and OsIPPI2 enzymes are found in the endoplasmic reticulum, providing novel important insights into the role of this compartment in the synthesis of MVA pathway isoprenoids. Isoprenoids are synthesized from the precursor's isopentenyl diphosphate (IPP) and dimethylallyl diphosphosphate (DMAPP), which are interconverted by the enzyme isopentenyl diphosphate isomerase (IPPI). Many plants express multiple isoforms of IPPI, the only enzyme shared by the mevalonate (MVA) and non-mevalonate (MEP) pathways, but little is known about their specific roles. Rice (Oryza sativa) has two IPPI isoforms (OsIPPI1 and OsIPPI2). We, therefore, carried out a comprehensive comparison of IPPI gene expression, protein localization, and isoprenoid biosynthesis in this species. We found that OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues, and its expression in de-etiolated leaves mirrored the accumulation of phytosterols, suggesting a key role in the synthesis of MVA pathway isoprenoids. We investigated the subcellular localization of both isoforms by constitutively expressing them as fusions with synthetic green fluorescent protein. Both proteins localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids, due to an N-terminal transit peptide which is not present in OsIPPI1. Despite the plastidial location of OsIPPI2, the expression of OsIPPI2 mRNA did not mirror the accumulation of chlorophylls or carotenoids, indicating that OsIPPI2 may be a redundant component of the MEP pathway. The detection of both OsIPPI isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. Our work shows that the ER plays an as yet unknown role in the synthesis of MVA-derived isoprenoids, with important implications for the metabolic engineering of isoprenoid biosynthesis in higher plants.

Analysis of the isoprenoid pathway intermediates, dimethylallyl diphosphate and isopentenyl diphosphate, from crude plant extracts by liquid chromatography tandem mass spectrometry.

OBJECTIVE: We sought to develop a sensitive and accurate analytical method for the detection and quantification of IDP and DMADP as well as their monophosphate derivatives in crude plant extracts. METHODS: A liquid chromatography method coupled to tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) was established to measure the amounts of IDP and DMADP down to low picogram levels, which was linear over at least three orders of magnitude. Extracts were enriched using an anion exchanger, and chromatographic separation was achieved using a ß-cyclodextrin column. A S-thiolodiphosphate analog of DMADP was employed as an internal standard. RESULTS: Dilution series of authentic compounds were used to determine the limits of detection and quantification for IDP, DMADP and their corresponding monophosphates. A survey of plant species producing varying amounts of isoprenoids showed a corresponding variation in IDP and DMADP with the ratio of DMADP/IDP ranging from 4:1 to 2:1. Trace levels of isopentenyl monophosphate (IP) and dimethylallyl monophosphate (DMAP) were also detected. CONCLUSION: The LC-MS/MS method described enables absolute quantification of in planta levels of IDP and DMADP for the first time. The method is also suitable for analysing bacterial and animal samples as well as enzyme assays.