Lyase Pathway Research Articles

Organizing the Chaos: Novel Insights into the Regulation of Z-3-Hexenal Production in Damaged Maize Leaves

Green leaf volatiles (GLVs) are important signaling compounds that help to regulate plant defenses against pests and pathogens. Made through the hydroperoxide lyase (HPL) pathway, they are rapidly produced upon damage and can signal to other parts of the same plant or even plants nearby, where they can induce rapid defense responses directly or prime them against impending danger. In this primed state, plants can respond faster and/or stronger should pests or pathogens attack. However, while all proteins and genes involved in the biosynthesis of GLVs have been identified, little is still known about how the first two steps in the pathway, e.g., oxygenation by a lipoxygenase (LOX) and subsequent cleavage by HPL, are facilitated within the damaged tissue, resulting in the production of Z-3-hexenal (Z3al) as the first committed product of the pathway. Here, we provide evidence that several factors might be involved in the production of Z3al, including pH, Ca2+, and an environment that is highly hydrophobic. We present a model in which the extraordinary circumstances that are present at the site of Z3al production are considered, and shine new light on potential regulatory mechanisms.

Disentangling the Regulatory Response of Agrobacterium tumefaciens CHLDO to Glyphosate for Engineering Whole-Cell Phosphonate Biosensors.

Phosphonates (PHTs), organic compounds with a stable C-P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as a host and the source of genetic parts for constructing PHT biosensors. In this bacterial species, the phn gene cluster, encoding the C-P lyase pathway, is regulated by the PhnF transcriptional repressor. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise genetic optimization of the transcriptional cascade, we created a whole-cell biosensor capable of detecting GP in the 0.25-50 μM range in various samples, including soil and water.

Regulatory mechanism of methyl jasmonate and methyl dihydrojasmonate in enhancing aroma in 'Cabernet Gernischt' via the lipoxygenase pathway.

The present study explored the effects of pre-harvest methyl jasmonates spraying on the volatiles of 'Cabernet Gernischt' grapes through the lipoxygenase pathway. Headspace solid-phase microextraction combined with gas chromatography-mass spectrometry and an enzyme-linked immunosorbent assay were utilized to analyze volatile metabolites and key enzyme activities following methyl jasmonates application. Total RNA extraction and cDNA library construction were followed by transcriptome sequencing. The application of 0.1 mmol L-1 methyl jasmonate and 5 mmol L-1 methyl dihydrojasmonate significantly increased the levels of C6 compounds in 'Cabernet Gernischt' berries, and also enhanced the utilization of unsaturated fatty acids as precursors in the lipoxygenase-hydroperoxides lyase pathway. The up-regulated expression of VvADH1, VvADH2, VvHPL and VvLOX2S genes led to modulations in enzyme activity, thereby enhancing the berries's aromatic profile. There was a significant correlation between linoleic and linolenic acids (i.e. the precursors of the lipoxygenase pathway) and the activities and metabolites of key enzymes. The optimal concentration of methyl jasmonates treatment favorably influences the accumulation of green leaf volatiles in 'Cabernet Gernischt' grape. © 2024 Society of Chemical Industry.

Cathodic poised potential stimulated the electron-sensitive C-P lyase pathway in glyphosate biodegradation

Glyphosate, the most widely used herbicide globally, is accumulating in the environment and poses significant potential eco- and bio-toxicity risks. While natural attenuation of glyphosate has been reported, the efficacy varies considerably and the dominant metabolite, aminomethylphosphonic acid (AMPA), is potentially more persistent and toxic. This study investigated the bioelectrochemical system (BES) for glyphosate degradation under anaerobic, reductive conditions. Atomistic simulations using density functional theory (DFT) predicted increased thermodynamic favorability for the non-dominant C-P lyase degradation pathway under external charge, which suppressed AMPA production. Experimental results confirmed that cathodic poised potential (-0.4 V vs. Ag/AgCl) enhanced glyphosate degradation (75 % in BES vs. ∼40 % in the control conditions after 37 days), and lowered the AMPA yield (0.52 mol AMPA yield per mol glyphosate removed in BES vs. 0.77–0.86 mol mol-1 in the control conditions). Geobacter lovleyi was likely the active species driving the C-P lyase pathway, as evidenced by the increase of its relative abundance, the upregulation of its extracellular electron transfer genes (most notably mtr) and the up-regulation of its phnJ and hcp genes (encoding C-P layse and hydroxylamine reductase respectively).

Oxylipin biosynthesis via an unprecedented 16-hydroperoxide lyase pathway in green tissues of cucumber (Cucumis sativus L.) plants

The plant lipoxygenase cascade is a source of various regulatory oxylipins that play a role in cell signalling, stress adaptation, and immune response. Recently, we detected an unprecedented 16(S)-lipoxygenase, CsLOX3, in the leaves and fruit pericarp of cucumber (Cucumis sativus L.). In the present work, an array of products biosynthesized through the conversions of α-linolenic acid 16-hydroperoxide (16-HPOT) was detected. Firstly, a prominent 15-hydroxy-9,12-pentadecadienoic acid (Me/TMS) was detected, the product of hydroperoxide lyase (HPL) chain cleavage of 16-HPOT and further reduction of aldehyde 15-oxo-9,12-pentadecadienoic acid to alcohol. Besides, the presence of dicarboxylic acid, 3,6-pentadecadiene-1,15-dioic acid, was deduced from the detection of its catalytic hydrogenation product, pentadecane-1,15-dioic acid. Finally, 12,15-dihydroxypentadecanoic acid (Me/TMS) was detected amongst the hydrogenated products, thus indicating the presence of the parent 12,15-dihydroxy-9,13-pentadecadienoic acid. To confirm the proposed HPL chain cleavage, the 16(S)-HPOT was prepared and incubated with the recombinant cucumber HPL CYP74B6 enzyme. The CYP74B6 possessed high activity towards 16-HPOT. Chain cleavage yields the (9Z,12Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9Z,13E)-15-oxo-9,13-pentadecadienoic acid. Thus, the cucumber plants as well as the recombinant cucumber HPL CYP74B6 possessed unprecedented 16-HPL activity, cleaving 16-HPOT into a C15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway.

Acetyl-CoA-dependent ac4C acetylation promotes the osteogenic differentiation of LPS-stimulated BMSCs

The impaired osteogenic capability of bone marrow mesenchymal stem cells (BMSCs) caused by persistent inflammation is the main pathogenesis of inflammatory bone diseases. Recent studies show that metabolism is disturbed in osteogenically differentiated BMSCs in response to Lipopolysaccharide (LPS) treatment, while the mechanism involved remains incompletely revealed. Herein, we demonstrated that BMSCs adapted their metabolism to regulate acetyl-coenzyme A (acetyl-CoA) availability and RNA acetylation level, ultimately affecting osteogenic differentiation. The mitochondrial dysfunction and impaired osteogenic potential upon inflammatory conditions accompanied by the reduced acetyl-CoA content, which in turn suppressed N4-acetylation (ac4C) level. Supplying acetyl-CoA by sodium citrate (SC) addition rescued ac4C level and promoted the osteogenic capacity of LPS-treated cells through the ATP citrate lyase (ACLY) pathway. N-acetyltransferase 10 (NAT10) inhibitor remodelin reduced ac4C level and consequently impeded osteogenic capacity. Meanwhile, the osteo-promotive effect of acetyl-CoA-dependent ac4C might be attributed to fatty acid oxidation (FAO), as evidenced by activating FAO by L-carnitine supplementation counteracted remodelin-induced inhibition of osteogenesis. Further in vivo experiments confirmed the promotive role of acetyl-CoA in the endogenous bone regeneration in rat inflammatory mandibular defects. Our study uncovered a metabolic-epigenetic axis comprising acetyl-CoA and ac4C modification in the process of inflammatory osteogenesis of BMSCs and suggested a new target for bone tissue repair in the context of inflammatory bone diseases.

NADPH oxidase-dependent H2O2 production mediates salicylic acid-induced salt tolerance in mangrove plant Kandelia obovata by regulating Na+/K+ and redox homeostasis.

Salicylic acid (SA) is known to enhance salt tolerance in plants. However, the mechanism of SA-mediated response to high salinity in halophyte remains unclear. Using electrophysiological and molecular biological methods, we investigated the role of SA in response to high salinity in mangrove species, Kandelia obovata, a typical halophyte. Exposure of K. obovata roots to high salinity resulted in a rapid increase in endogenous SA produced by phenylalanine ammonia lyase pathway. The application of exogenous SA improved the salt tolerance of K. obovata, which depended on the NADPH oxidase-mediated H2O2. Exogenous SA and H2O2 increased Na+ efflux and reduced K+ loss by regulating the transcription levels of Na+ and K+ transport-related genes, thus reducing the Na+/K+ ratio in the salt-treated K. obovata roots. In addition, exogenous SA-enhanced antioxidant enzyme activity and its transcripts, and the expressions of four genes related to AsA-GSH cycle as well, then alleviated oxidative damages in the salt-treated K. obovata roots. However, the above effects of SA could be reversed by diphenyleneiodonium chloride (the NADPH oxidase inhibitor) and paclobutrazol (a SA biosynthesis inhibitor). Collectively, our results demonstrated that SA-induced salt tolerance of K. obovata depends on NADPH oxidase-generated H2O2 that affects Na+/K+ and redox homeostasis in response to high salinity.

The beneficial effects of the tobacco hydroperoxide lyase pathway in whitefly host adaptation

Plants respond to herbivore attack by emitting complex mixtures of volatile compounds to repel herbivores or attract predators and parasitoids. In this study, however, we revealed that tobacco green leaf volatiles (GLVs) were beneficial compounds for whitefly adaptation to host plants. Our study indicates that GLVs are closely associated with plant susceptibility to whiteflies. Whitefly infestation elevated the transcript of the hydroperoxide lyase (HPL) gene, one gene responsible for catalyzing the synthesis of C6 or C9 aldehydes from fatty acid hydroperoxides. Overexpression of HPL increased the emission of GLVs (hexanal, 1- hexanal, trans-2- hexanal and cis-3- hexanal) and resulted in improved performance for whiteflies. Exogenous application of GLVs promoted whitefly survival and fecundity and increased the plant's attraction to whiteflies. Thus, our study provides new insights into the role of the HPL pathway and GLVs in influencing herbivore adaptation to host plants.

Persicaria minor F-box Gene Pmf-box1 Indirectly Affects Arabidopsis thaliana LOX-HPL Pathway for Green Leaf Volatile Production

Green leaf volatiles (GLVs) play an essential role in plant defence, plant-plant interaction and plant-insect interaction. The plant releases GLVs and inhibits the growth and propagation of plant pathogens. In this study, overexpression of PmF-box1 in wild type A. thaliana showed the downregulation of genes involved in the lipoxygenase-hydroperoxide lyase (LOX-HPL) pathway, which contributes to the biosynthesis of GLVs. It resulted in a marked reduction of hexanal production in the PmF-box1-overexpressing plant. The expression pattern of LOX-HPL branch genes in the kelch-repeat modified PmF-box1 (KMF)-overexpressing plant showed a pattern much closer to the expression of LOX-HPL branch genes in the vector control (VC) plant. It was shown that the functional KMF protein sequence was not responsible for the significant reduction of all GLVs including hexanal, 1-hexanol, (Z)-3-hexen-1-ol, and the carbon 5 (C5) volatile, 1-penten-3-ol, in plants overexpressing KMF. Furthermore, this study also showed that the relative proportion of production of 1-penten-3-ol to hexanal was higher in the PmF-box1-overexpressing plant. Based on the current comparative literature search, PmF-box1 does not appear to interact directly with the proteins or transcription factors of the LOX-HPL pathway. On the other hand, PmF-box1 interacts with SAMS1, which subsequently influences the HPL pathway enzyme genes. Thus, this study highlights the potential roles of PmF-box1 in the manipulation of GLV productions.

Marine picocyanobacterial PhnD1 shows specificity for various phosphorus sources but likely represents a constitutive inorganic phosphate transporter

Despite being fundamental to multiple biological processes, phosphorus (P) availability in marine environments is often growth-limiting, with generally low surface concentrations. Picocyanobacteria strains encode a putative ABC-type phosphite/phosphate/phosphonate transporter, phnDCE, thought to provide access to an alternative phosphorus pool. This, however, is paradoxical given most picocyanobacterial strains lack known phosphite degradation or carbon-phosphate lyase pathway to utilise alternate phosphorus pools. To understand the function of the PhnDCE transport system and its ecological consequences, we characterised the PhnD1 binding proteins from four distinct marine Synechococcus isolates (CC9311, CC9605, MITS9220, and WH8102). We show the Synechococcus PhnD1 proteins selectively bind phosphorus compounds with a stronger affinity for phosphite than for phosphate or methyl phosphonate. However, based on our comprehensive ligand screening and growth experiments showing Synechococcus strains WH8102 and MITS9220 cannot utilise phosphite or methylphosphonate as a sole phosphorus source, we hypothesise that the picocyanobacterial PhnDCE transporter is a constitutively expressed, medium-affinity phosphate transporter, and the measured affinity of PhnD1 to phosphite or methyl phosphonate is fortuitous. Our MITS9220_PhnD1 structure explains the comparatively lower affinity of picocyanobacterial PhnD1 for phosphate, resulting from a more limited H-bond network. We propose two possible physiological roles for PhnD1. First, it could function in phospholipid recycling, working together with the predicted phospholipase, TesA, and alkaline phosphatase. Second, by having multiple transporters for P (PhnDCE and Pst), picocyanobacteria could balance the need for rapid transport during transient episodes of higher P availability in the environment, with the need for efficient P utilisation in typical phosphate-deplete conditions.

Identification of the key genes contributing to the LOX-HPL volatile aldehyde biosynthesis pathway in jujube fruit

Jujube (Ziziphus jujuba Mill.) is a traditional popular fruit widely grown in China. The volatiles in jujube determine its unique flavor and the high fruit quality required by consumers. However, the biosynthesis of volatiles in jujube were remain unknown. By using gas chromatography–mass spectrometry, there were 46 volatile compounds were identified and determined from three representative jujube fruit types at six developmental stages, including the dry-used (Z. jujuba cv. ‘Junzao’), the fresh-used (Z. jujuba cv. ‘Dongzao’), and wild jujube (Z. jujuba var. spinosa Hu. cv. ‘Qingjiansuanzao’). The aldehydes were identified as major volatile contributors to flavor, of which (E)-2-hexenal was the primary volatile in jujube fruit. Then LOX and HPL gene family were identified in jujube, which were involved in aldehyde biosynthesis through the lipoxygenase-hydroperoxide lyase (LOX-HPL) pathway. Gene expression analysis suggested that ZjLOX3, ZjLOX4, and ZjHPL1 were highly correlated with the accumulation of (E)-2-hexenal, and their proteins were localized to the nucleus and cytoplasm. Transient over-expression of ZjLOX3, ZjLOX4, and ZjHPL1 in jujube fruit significantly enhanced the accumulation of (E)-2-hexenal. Our study provides valuable information on the major volatiles and their biosynthesis in different types of jujube fruit. These results will help determine flavor improvements for future breeding.

Promotion of biological H2 (Bio-H2) production by the nitrogen-fixing anaerobic microbial consortia using humin, a solid-phase humic substance

Dark fermentative biological hydrogen (Bio-H2) production is expected to be a clean and sustainable H2 production technology, and the technologies have been studied to increase in the product yield as index. This study achieved high product yields of Bio-H2 using nitrogen-fixing consortia under nitrogen-deficient conditions with glucose or mannitol as substrate and humin as the extracellular electron mediator: 4.12mol-H2/mol-glucose and 3.12mol-H2/mol-mannitol. The high Bio-H2 production was observed under the conditions where both nitrogenase and hydrogenase were active in the presence of humin. Nitrogenase activity was confirmed by acetylene reduction activity and hydrogenase activity by Bio-H2 production under nitrogenase-inhibiting conditions with NH4NO3. [Fe-Fe] hydrogenase detected by a specific PCR and acetate, butyrate, formate, lactate, and pyruvate produced as by-products suggested the involvement of both pyruvate-ferredoxin-oxidoreductase and pyruvate formate lyase pathways in Bio-H2 production. Humin promoted the Bio-H2 production beyond the capacity of the consortium, which had reached saturation with the optimum concentrations of glucose and mannitol. Carbon balance suggested the concurrent H2 consumption by hydrogenotrophic methanogenesis and acetogenesis. Bio-H2 production of the washed and starved consortium with reduced humin under conditions with or without NH4NO3 suggests that humin promoted hydrogenase and nitrogenase activity by donating extracellular electrons. Clostridium and Ruminococcus in the consortia were considered major hydrogen producers. Thus, this study demonstrated the outstanding potential of nitrogen-fixing consortia under nitrogen-deficient conditions with humin as an extracellular electron mediator for dark fermentative Bio-H2 production with high yields.

GhENODL6 Isoforms from the Phytocyanin Gene Family Regulated Verticillium Wilt Resistance in Cotton.

Verticillium wilt (VW), a fungal disease caused by Verticillium dahliae, currently devastates cotton fiber yield and quality seriously, yet few resistance germplasm resources have been discovered in Gossypium hirsutum. The cotton variety Nongda601 with suitable VW resistance and high yield was developed in our lab, which supplied elite resources for discovering resistant genes. Early nodulin-like protein (ENODL) is mainly related to nodule formation, and its role in regulating defense response has been seldom studied. Here, 41 conserved ENODLs in G. hirsutum were identified and characterized, which could divide into four subgroups. We found that GhENODL6 was upregulated under V. dahliae stress and hormonal signal and displayed higher transcript levels in resistant cottons than the susceptible. The GhENODL6 was proved to positively regulate VW resistance via overexpression and gene silencing experiments. Overexpression of GhENODL6 significantly enhanced the expressions of salicylic acid (SA) hormone-related transcription factors and pathogenicity-related (PR) protein genes, as well as hydrogen peroxide (H2O2) and SA contents, resulting in improved VW resistance in transgenic Arabidopsis. Correspondingly, in the GhENODL6 silenced cotton, the expression levels of both phenylalanine ammonia lyase (PAL) and 4-coumarate-CoA ligase (4CL) genes significantly decreased, leading to the reduced SA content mediating by the phenylalanine ammonia lyase pathway. Taken together, GhENODL6 played a crucial role in VW resistance by inducing SA signaling pathway and regulating the production of reactive oxygen species (ROS). These findings broaden our understanding of the biological roles of GhENODL and the molecular mechanisms underlying cotton disease resistance.

Developmental Stages Affect the Capacity to Produce Aldehyde Green Leaf Volatiles in Zea mays and Vigna radiata.

Green leaf volatiles (GLV) are essentially produced by the green parts of plants upon damage. GLV are mainly 6-carbon molecules derived from fatty acids through the hydroperoxide lyase pathway and can serve as airborne signals to other parts of the same plant and to neighboring plants and help to protect them against biotic and abiotic stresses. However, while the biosynthesis is generally well understood, little is known about how plants regulate the production of these important signaling molecules. To better understand how the developmental stage of the plant affects aldehyde GLV production, we selected Zea mays and Vigna radiata to represent mono- and dicot plants for this analysis. We show that the capacity to produce aldehyde GLV strongly depends on the developmental stage of the plant. Major differences in the quantity, and in the quality of these compounds were found, not only in leaves from different developmental stages, but also in different areas within a leaf. The results demonstrate that the capacity to produce GLV varies significantly within a plant and the potential implications of these findings are discussed.

OsLMP1, Encoding a Deubiquitinase, Regulates the Immune Response in Rice.

Lesion mimic mutants have become an effective material for understanding plant-microbe interactions and the immune mechanism in plants. Although many mechanisms responsible for the lesion mimic phenotype have been clarified in plants, the mechanism by which lesion mimic is regulated by posttranslational modification remained largely elusive, especially in rice. In this study, a mutant with the lesion mimic phenotype was obtained and named lmp1-1. Physiological measurements and quantitative real-time PCR analysis showed that the defense response was activated in the mutants. Transcriptome analysis showed that the phenylalanine ammonia lyase (PAL) pathway was activated in the mutant, causing the accumulation of salicylic acid (SA). The results of mapping based cloning showed that OsLMP1 encodes a deubiquitinase. OsLMP1 can cleave ubiquitination precursors. Furthermore, OsLMP1 epigenetically modifies SA synthetic pathway genes by deubiquitinating H2B and regulates the immune response in rice. In summary, this study deepens our understanding of the function of OsLMP1 in the plant immune response and provides further insight into the relationship between plants and pathogenic microorganisms.

Methylphosphonate Degradation and Salt-Tolerance Genes of Two Novel Halophilic Marivita Metagenome-Assembled Genomes from Unrestored Solar Salterns.

Aerobic bacteria that degrade methylphosphonates and produce methane as a byproduct have emerged as key players in marine carbon and phosphorus cycles. Here, we present two new draft genome sequences of the genus Marivita that were assembled from metagenomes from hypersaline former industrial salterns and compare them to five other Marivita reference genomes. Phylogenetic analyses suggest that both of these metagenome-assembled genomes (MAGs) represent new species in the genus. Average nucleotide identities to the closest taxon were <85%. The MAGs were assembled with SPAdes, binned with MetaBAT, and curated with scaffold extension and reassembly. Both genomes contained the phnCDEGHIJLMP suite of genes encoding the full C-P lyase pathway of methylphosphonate degradation and were significantly more abundant in two former industrial salterns than in nearby reference and restored wetlands, which have lower salinity levels and lower methane emissions than the salterns. These organisms contain a variety of compatible solute biosynthesis and transporter genes to cope with high salinity levels but harbor only slightly acidic proteomes (mean isoelectric point of 6.48).

Bioremediation potential of glyphosate-degrading microorganisms in eutrophicated Ecuadorian water bodies

Phosphonate compounds are the basis of many xenobiotic pollutants, such as Glyphosate (N-(phosphonomethyl-glycine). Only procaryotic microorganisms and the lower eukaryotes are capable of phosphonate biodegradation through C–P lyase pathways. Thus, the aim of this study was to determine the presence of C–P lyase genes in Ecuadorian freshwater systems as a first step towards assessing the presence of putative glyphosate degraders. To that end, two Nested PCR assays were designed to target the gene that codifies for the subunit J (phnJ), which breaks the C-P bond that is critical for glyphosate mineralization. The assays designed in this study led to the detection of phnJ genes in 7 out of 8 tested water bodies. The amplified fragments presented 85–100% sequence similarity with phnJ genes that belong to glyphosate-degrading microorganisms. Nine sequences were not reported previously in the GenBank. The presence of phosphonate degraders was confirmed by isolating three strains able to grow using glyphosate as a unique carbon source. According to the 16S sequence, these strains belong to the Pantoea, Pseudomonas, and Klebsiella genera. Performing a Nested PCR amplification of phnJ genes isolated from eutrophicated water bodies, prior to isolation, may be a cost-effective strategy for the bioprospection of new species and/or genes that might have new properties for biotech industries, laying the groundwork for additional research.

Metabolic Flow of C6 Volatile Compounds From LOX-HPL Pathway Based on Airflow During the Post-harvest Process of Oolong Tea.

Aroma is an essential quality indicator of oolong tea, a tea derived from the Camellia sinensis L. plant. Carboxylic 6 (C6) acids and their derivative esters are important components of fatty acid (FA)-derived volatiles in oolong tea. However, the formation and regulation mechanism of C6 acid during postharvest processing of oolong tea remains unclear. To gain better insight into the molecular and biochemical mechanisms of C6 compounds in oolong tea, a combined analysis of alcohol dehydrogenase (ADH) activity, CsADH2 key gene expression, and the FA-derived metabolome during postharvest processing of oolong tea was performed for the first time, complemented by CsHIP (hypoxia-induced protein conserved region) gene expression analysis. Volatile fatty acid derivative (VFAD)-targeted metabolomics analysis using headspace solid-phase microextraction–gas chromatography time-of-flight mass spectrometry (HS-SPEM-GC-TOF-MS) showed that the (Z)-3-hexen-1-ol content increased after each turnover, while the hexanoic acid content showed the opposite trend. The results further showed that both the ADH activity and CsADH gene expression level in oxygen-deficit-turnover tea leaves (ODT) were higher than those of oxygen-turnover tea leaves (OT). The C6-alcohol-derived ester content of OT was significantly higher than that of ODT, while C6-acid-derived ester content showed the opposite trend. Furthermore, the HIP gene family was screened and analyzed, showing that ODT treatment significantly promoted the upregulation of CsHIG4 and CsHIG6 gene expression. These results showed that the formation mechanism of oolong tea aroma quality is mediated by airflow in the lipoxygenase–hydroperoxide lyase (LOX-HPL) pathway, which provided a theoretical reference for future quality control in the postharvest processing of oolong tea.

Transcriptome analysis of Clavibacter michiganensis subsp. michiganensis-infected tomatoes: a role of salicylic acid in the host response

Bacterial canker of tomato (Solanum lycopersicon) caused by the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) is an economically important disease. To understand the host defense response to Cmm infection, transcriptome sequences in tomato cotyledons were analyzed by RNA-seq. Overall, 1788 and 540 genes were upregulated and downregulated upon infection, respectively. Gene Ontology enrichment analysis revealed that genes involved in the defense response, phosphorylation, and hormone signaling were over-represented by the infection. Induced expression of defense-associated genes suggested that the tomato response to Cmm showed similarities to common plant disease responses. After infection, many resistance gene analogs (RGAs) were transcriptionally upregulated, including the expressions of some receptor-like kinases (RLKs) involved in pattern-triggered immunity. The expressions of WRKYs, NACs, HSFs, and CBP60s encoding transcription factors (TFs) reported to regulate defense-associated genes were induced after infection with Cmm. Tomato genes orthologous to Arabidopsis EDS1, EDS5/SID1, and PAD4/EDS9, which are causal genes of salicylic acid (SA)-deficient mutants, were upregulated after infection with Cmm. Furthermore, Cmm infection drastically stimulated SA accumulation in tomato cotyledons. Genes involved in the phenylalanine ammonia lyase pathway were upregulated, whereas metabolic enzyme gene expression in the isochorismate synthase pathway remained unchanged. Exogenously applied SA suppressed bacterial growth and induced the expression of WRKYs, suggesting that some Cmm-responsive genes are regulated by SA signaling, and SA signaling activation should improve tomato immunity against Cmm.

Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.