Dioxygenase Gene Research Articles

Shape-function of a novel metapyrocatechase, RW4-MPC: Metagenomics to SAXS data based insight into deciphering regulators of function

The oxygenases have attracted considerable attention in enzyme-mediated bioremediation of xenobiotic compounds due to their high specificity, cost-effectiveness, and targeted field applications. Here, we performed a functional metagenomics approach to cope with culturability limitations to isolate a novel extradiol dioxygenase. Fosmid clone harboring dioxygenase gene was sequenced and analyzed by bioinformatics tools. One ring-cleaving dioxygenase RW4-MPC (metapyrocatechase) was purified and characterized to examine its degradation efficiency. The RW4-MPC was significantly active in the temperature and pH range of 5 to 40 °C, and 7–10, respectively, with an optimum temperature of 25 °C and pH 8. To gain insight into observed differential activity, Small-Angle X-ray Scattering (SAXS) data of the protein samples were analyzed, which brought forth that the RW4-MPC molecules form tight globular tetramers in solution. This native association was stable till 35 °C, and protein started to associate at higher temperatures, explaining heat-induced loss of function. Similarly, RW4-MPC aggregated or lost globular profile below pH 7 or at pH 10, respectively. The kinetic parameters showed the six folds high catalytic efficiency of RW4-MPC towards 2,3-dihydroxy biphenyl than catechol and its derivatives. RW4-MPC molecules showed remarkable retention of functionality in hypersaline conditions with more than 70% activity in a buffer having 3 M NaCl concentration. In concordance, SAXS data analysis showed retention of functional shape profile in hypersaline conditions. The halotolerant and oxygen insensitive nature of this enzyme makes it a potential candidate for bioremediation.

Variation of the mangrove sediment microbiomes and their phenanthrene biodegradation rates during the dry and wet seasons

Mangrove sediment is a major sink for phenanthrene in natural environments. Consequently, this study investigated the effects of seasonal variation on the biodegradation rates of low (150 mg kg−1), moderate (600 mg kg−1), and high (1200 mg kg−1) phenanthrene-contaminated mangrove sediments using a microcosm study and identified potential key phenanthrene-degrading bacteria using high throughput sequencing of 16 S rRNA gene and quantitative-PCR of the PAH-ring hydroxylating dioxygenase (PAH-RHDα) genes. The biodegradation rates of phenanthrene in all treatments were higher in the wet-season sediments (11.58, 14.51, and 8.94 mg kg−1 sediment day−1) than in the dry-season sediments (3.51, 12.56, and 5.91 mg kg−1 sediment day−1) possibly due to higher nutrient accumulation caused by rainfall and higher diversity of potential phenanthrene-degrading bacteria. The results suggested that the mangrove sediment microbiome significantly clustered according to season. Although Gram-negative phenanthrene-degrading bacteria (i.e., Anaerolineaceae, Marinobacter, and Rhodobacteraceae) played a key role in both dry and wet seasons, distinctly different phenanthrene-degrading bacterial taxa were observed in each season. Halomonas and Porticoccus were potentially responsible for the degradation of phenanthrene in the dry and wet seasons, respectively. The knowledge gained from this study contributes to the development of effective and rationally designed microbiome innovations for oil removal.

Rose (Rosa hybrida) Ethylene Responsive Factor 3 Promotes Rose Flower Senescence via Direct Activation of the Abscisic Acid Synthesis-Related 9-CIS-EPOXYCAROTENOID DIOXYGENASE Gene.

During plant senescence, energy and nutrients are transferred to young leaves, fruits or seeds. However, senescence reduces flower quality, which leads to huge economic losses in flower production. Ethylene is an important factor affecting the quality of cut roses during transportation and storage. Ethylene-responsive factors (ERFs) are key nodes in ethylene signaling, but the molecular mechanism underlying ERFs regulated flower senescence is not well understood. We addressed this issue in the present study by focusing on RhERF3 from Rosa hybrida, an ERF identified in a previous transcriptome analysis of ethylene-treated rose flowers. Expression of RhERF3 was strongly induced by ethylene during rose flower senescence. Transient silencing of RhERF3 delayed flower senescence, whereas overexpression (OE) accelerated the process. RNA sequencing analysis of RhERF3 OE and pSuper vector control samples identified 13,214 differentially expressed genes that were mostly related to metabolic process and plant hormone signal transduction. Transient activation and yeast one-hybrid assays demonstrated that RhERF3 directly bound the promoter of the 9-cis-epoxycarotenoid dioxygenase (RhNCED1) gene and activated gene expression. Thus, a RhERF3/RhNCED1 axis accelerates rose flower senescence.

Gibberellin Oxidase Gene Family in L. chinense: Genome-Wide Identification and Gene Expression Analysis.

GAox is a key enzyme for the transformation of gibberellins, and belongs to the 2-ketoglutarate dependent dioxygenase gene family (2ODD). However, a systematic analysis of GAox in the angiosperm L. chinense has not yet been reported. Here, we identified all LcGAox gene family members in L. chinense, which were classified into the three subgroups of GA20ox, C19GA2ox, and C20GA2ox. Comparison of the gene structure, conserve motifs, phylogenetic relationships, and syntenic relationships of gibberellin oxidase gene families in different species indicated that the gene functional differences may be due to the partial deletion of their domains during evolution. Furthermore, evidence for purifying selection was detected between orthologous GAox genes in rice, grape, Arabidopsis, and L. chinense. Analysis of the codon usage patterns showed that mutation pressure and natural selection might have induced codon usage bias in angiosperms; however, the LcGAox genes in mosses, lycophytes, and ambarella plants exhibited no obvious codon usage preference. These results suggested that the gibberellin oxidase genes were more primitive. The gene expression pattern was analyzed in different organs subjected to multiple abiotic stresses, including GA, abscisic acid (ABA), and chlormequat (CCC) treatment, by RNA-seq and qRT-PCR, and the stress- and phytohormone-responsive cis-elements were counted. The results showed that the synthesis and decomposition of GA were regulated by different LcGAox genes in the vegetative and reproductive organs of L. chinense, and only LcGA2ox1,4, and 7 responded to the NaCl, polyethylene glycol, 4 °C, GA, ABA, and CCC treatment in the roots, stems, and leaves of seedlings at different time periods, revealing the potential role of LcGAox in stress resistance.

Effects of polycyclic aromatic hydrocarbon structure on PAH mineralization and toxicity to soil microorganisms after oxidative bioremediation by laccase

While bioremediation using soil microorganisms is considered an energy-efficient and eco-friendly approach to treat polycyclic aromatic hydrocarbon (PAH)-contaminated soils, a variety of polar PAH metabolites, particularly oxygenated ones, could increase the toxicity of the soil after biodegradation. In this study, a typical bio-oxidative transformation of PAH into quinones was investigated in soil amended with laccase using three PAHs with different structures (anthracene, benzo[a]anthracene, and benzo[a]pyrene) to assess the toxicity after oxidative bioremediation. The results show that during a 2-month incubation period the oxidation process promoted the formation of non-extractable residues (NERs) of PAHs, and different effects on mineralization were observed among the three PAHs. Oxidation enhanced the mineralization of the high-molecular-weight (HMW) PAHs (benzo[a]anthracene and benzo[a]pyrene) but inhibited the mineralization of the low-molecular-weight (LMW) PAH (anthracene). The inhibition of anthracene suggests increased toxicity after oxidative bioremediation, which coincided with a decrease in soil nitrification activity, bacterial diversity and PAH-ring hydroxylating dioxygenase gene copies. The analysis of PAH metabolites in soil extract indicated that oxidation by laccase was competitive with the natural transformation processes of PAHs and revealed that intermediates other than quinone metabolites increased the toxicity of soil during subsequent degradation. The different metabolic profiles of the three PAHs indicated that the toxicity of soil after PAH oxidation by laccase was strongly affected by the PAH structure. Despite the potential increase in toxicity, the results suggest that oxidative bioremediation is still an eco-friendly method for the treatment of HMW PAHs since the intermediates from HMW PAHs are more easily detoxified via NER formation than LMW PAHs.

Novel clades of soil biphenyl degraders revealed by integrating isotope probing, multi-omics, and single-cell analyses

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used 13C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein–stable isotope probing. The detection of 13C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.

High expression of ring-hydroxylating dioxygenase genes ensure efficient degradation of p-toluate, phthalate, and terephthalate by Comamonas testosteroni strain 3a2.

Para-toluic acid, a major pollutant in industrial wastewater, is hazardous to human health. It has been demonstrated that Gram-negative bacteria are among the most effective degraders of para-toluic acid. In this study, the ability of Comamonas testosteroni strain 3a2, isolated from a petrochemical industry wastewater, to degrade para-toluic acid was investigated. The effect of different carbon (glucose and ethylene glycol) and nitrogen sources (urea, yeast extract, peptone, NaNO3, NH4NO3) on the biodegradation of para-toluic acid by the isolate 3a2 was evaluated. Furthermore, ring hydroxylating dioxygenase genes were amplified by PCR and their expression was evaluated during the biodegradation of para-toluic acid. The results indicated that strain 3a2 was able to degrade up to 1000mg/L of para-toluic acid after 14h. The highest degradation yield was recorded in the presence of yeast extract as nitrogen source. However, the formation of terephthalic acid and phthalic acid was noted during para-toluic acid degradation by the isolate 3a2. Toluate 1,2-dioxygenase, terephthalate 1,2 dioxygenase, and phthalate 4,5 dioxygenase genes were detected in the genomic DNA of 3a2. The induction of ring hydroxylating dioxygenase genes was proportional to the concentration of each hydrocarbon. This study showed that the isolate 3a2 can produce terephthalate and phthalate during the para-toluic acid biodegradation, which were also degraded after 24h.

Influence of polycyclic aromatic hydrocarbon pollution on the diversity and function of bacterial communities in urban wetlands.

Human disturbance has become the primary driving factor behind declining urban wetland ecological health due to rapid urbanization. Sediment microbial communities are critical for wetland ecosystem functioning but experience a range of natural and anthropogenic stressors due to rapid urbanization and land use changes, especially in developing countries. Polycyclic aromatic hydrocarbons (PAHs) released into the environment primarily come from anthropogenic sources like industrial activities and traffic emissions. Environmental PAH contamination is accelerating due to rapid urbanization, which also increases potential PAH-related dangers to human health. However, PAHs are widely distributed and not easy to centrally control. Microorganisms are the primary mediators of wetland purification, with most PAH-degrading microorganisms being bacteria. To better understand the influence of PAH contamination on urban wetland microbial communities, bacterial community compositions within sediments of urban wetlands in three land use types were investigated using high-throughput DNA sequencing and bioinformatics analyses. Statistical analyses revealed significant differences in overall microbial compositions among the three land use types, although γ-proteobacteria was the dominant phyla across all samples. Among the potential PAH-degrading bacterial taxa in sediments, Sphingomonas was the most prevalent. The distributions of PAH-degrading taxa were primarily affected by variance in organic compound abundances in addition to various physico-chemical variables, among which high-ring PAH content was a key parameter associated with bacterial distributions, except in the riverine wetlands. Functional inference via phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) indicated that 30 of the 43 genes related to PAH metabolism were predicted to be present within the genomes of bacteria among the three land use type. In particular, dioxygenase and dehydrogenase genes involved in PAH degradation were inferred to be prevalent, indicating that the host urban wetlands exhibited strong potential for organic pollutant degradation.

A leucoanthocyanidin dioxygenase gene (RtLDOX2) from the feral forage plant Reaumuria trigyna promotes the accumulation of flavonoids and improves tolerance to abiotic stresses.

Reaumuria trigyna, a Tamaricaceae archaic recretohalophyte, is an important feral forage plant in the desert steppe of northwestern China. We identified two significantly differentially expressed leucoanthocyanidin dioxygenase genes (RtLDOX/RtLDOX2) and investigated the function and characteristics of RtLDOX2. RtLDOX2 from R. trigyna was rapidly upregulated by salt, drought, and abscisic acid, consistent with the stress-related cis-regulatory elements in the promoter region. Recombinant RtLDOX2 converted dihydrokaempferol to kaempferol in vitro, and was thus interchangeable with flavonol synthase, a dioxygenase in the flavonoid pathway. Transgenic plants overexpressing RtLDOX2 accumulated more anthocyanin and flavonols under abiotic stresses, speculating that RtLDOX2 may act as a multifunctional dioxygenase in the synthesis of anthocyanins and flavonols. Overexpression of RtLDOX2 enhanced the primary root length, biomass accumulation, and chlorophyll content of salt-, drought-, and ultraviolet-B-stressed transgenic Arabidopsis. Antioxidant enzyme activity; proline content; and expression of antioxidant enzyme, proline biosynthesis, and ion-transporter genes were increased in transgenic plants. Therefore, RtLDOX2 confers tolerance to abiotic stress on transgenic Arabidopsis by promoting the accumulation of anthocyanins and flavonols. This in turn increases reactive oxygen species scavenging and activates other stress responses, such as osmotic adjustment and ion transport, and so improves tolerance to abiotic stresses.

Programming adaptive laboratory evolution of 4-hydroxyisoleucine production driven by a lysine biosensor in Corynebacterium glutamicum

4-Hydroxyisoleucine (4-HIL) is a promising drug for treating diabetes. In our previous study, 4-HIL was synthesized from self-produced L-isoleucine (Ile) in Corynebacterium glutamicum by expressing an Ile dioxygenase gene. Although the 4-HIL production of recombinant strain SZ06 increased significantly, a by-product, L-lysine (Lys) was accumulated because of the share of the first several enzymes in Ile and Lys biosynthetic pathways. In this study, programming adaptive laboratory evolution (ALE) was designed and conducted in SZ06 to promote 4-HIL biosynthesis. At first, a programming evolutionary system pMK was constructed, which contains a Lys biosensor LysG-PlysE and an evolutionary actuator composed of a mutagenesis gene and a fluorescent protein gene. The evolutionary strain SZ06/pMK was then let to be evolved programmatically and spontaneously by sensing Lys concentration. After successive rounds of evolution, nine mutant strains K1 − K9 with significantly increased 4-HIL production and growth performance were obtained. The maximum 4-HIL titer was 152.19 ± 14.60 mM, 28.4% higher than that in SZ06. This titer was higher than those of all the metabolic engineered C. glutamicum strains ever constructed. The whole genome sequencing of the nine evolved strains revealed approximately 30 genetic mutations in each strain. Only one mutation was directly related to the Lys biosynthetic pathway. Therefore, programming ALE driven by Lys biosensor can be used as an effective strategy to increase 4-HIL production in C. glutamicum.

Transcriptome and proteome analyses of the molecular mechanisms underlying changes in oil storage under drought stress in Brassica napus L.

AbstractRapeseed (Brassica napus L.) is the second most important oilseed crop in edible vegetable oil and bioenergy; however, drought stress generally causes a decrease in rapeseed yield and oil content, especially during the reproductive stage. In our study, we measured the oil and protein contents and gibberellic acid (GA) and abscisic acid (ABA) levels in seeds that were acquired on the 30th, 40th, and 50th days after flowering under control and drought treatments. RNA and protein libraries were constructed from the stressed seeds to perform transcriptome and proteome analyses, respectively. Our results demonstrated that the oil content decreased due to four primary mechanisms: downregulation of fatty acid biosynthesis‐associated genes and proteins; upregulation of fatty acid degradation‐associated genes and proteins; enhancement of protein storage due to changes in the abundances of relevant genes and proteins; and upregulation of Gly‐Asp‐Ser‐Leu (GDSL) gene expression, potentially as the result of upregulating the GA biosynthesis gene GA20ox3 and downregulating the GA inactivating gene GA2ox3 and thus an increase in GA content. During seed maturation, oil storage change may also relate to increasing ABA content as the upregulation of two members of NCED6 (9‐cis‐epoxycarotenoid dioxygenase) gene family involved in ABA biosynthesis, and the upregulation of genes involved in ABA signal transduction. These results will help to establish a foundation for breeding excellent varieties of rapeseed with high oil content for areas with frequent droughts to promote the supply of edible vegetable oil and biofuel.

Pathway-based analysis of anthocyanin diversity in diploid potato.

Anthocyanin biosynthesis is one of the most studied pathways in plants due to the important ecological role played by these compounds and the potential health benefits of anthocyanin consumption. Given the interest in identifying new genetic factors underlying anthocyanin content we studied a diverse collection of diploid potatoes by combining a genome-wide association study and pathway-based analyses. By using an expanded SNP dataset, we identified candidate genes that had not been associated with anthocyanin variation in potatoes, namely a Myb transcription factor, a Leucoanthocyanidin dioxygenase gene and a vacuolar membrane protein. Importantly, a genomic region in chromosome 10 harbored the SNPs with strongest associations with anthocyanin content in GWAS. Some of these SNPs were associated with multiple anthocyanin compounds and therefore could underline the existence of pleiotropic genes or anthocyanin biosynthetic clusters. We identified multiple anthocyanin homologs in this genomic region, including four transcription factors and five enzymes that could be governing anthocyanin variation. For instance, a SNP linked to the phenylalanine ammonia-lyase gene, encoding the first enzyme in the phenylpropanoid biosynthetic pathway, was associated with all of the five anthocyanins measured. Finally, we combined a pathway analysis and GWAS of other agronomic traits to identify pathways related to anthocyanin biosynthesis in potatoes. We found that methionine metabolism and the production of sugars and hydroxycinnamic acids are genetically correlated to anthocyanin biosynthesis. The results contribute to the understanding of anthocyanins regulation in potatoes and can be used in future breeding programs focused on nutraceutical food.

Genetically blocking HPD via CRISPR-Cas9 protects against lethal liver injury in a pig model of tyrosinemia type I

Hereditary tyrosinemia type I (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury (LLI). Therapeutic options for HT1 remain limited. The FAH−/− pig, a well-characterized animal model of HT1, represents a promising candidate for testing novel therapeutic approaches to treat this condition. Here, we report an improved single-step method to establish a biallelic (FAH−/−) mutant porcine model using CRISPR-Cas9 and cytoplasmic microinjection. We also tested the feasibility of rescuing HT1 pigs through inactivating the 4-hydroxyphenylpyruvic acid dioxygenase (HPD) gene, which functions upstream of the pathogenic pathway, rather than by directly correcting the disease-causing gene as occurs with traditional gene therapy. Direct intracytoplasmic delivery of CRISPR-Cas9 targeting HPD before intrauterine death reprogrammed the tyrosine metabolism pathway and protected pigs against FAH deficiency-induced LLI. Characterization of the F1 generation revealed consistent liver-protective features that were germline transmissible. Furthermore, HPD ablation ameliorated oxidative stress and inflammatory responses and restored the gene profile relating to liver metabolism homeostasis. Collectively, this study not only provided a novel large animal model for exploring the pathogenesis of HT1, but also demonstrated that CRISPR-Cas9-mediated HPD ablation alleviated LLI in HT1 pigs and represents a potential therapeutic option for the treatment of HT1.

Integrated Omics Strategy Reveals Cyclic Lipopeptides Empedopeptins from Massilia sp. YMA4 and Their Biosynthetic Pathway.

Empedopeptins—eight amino acid cyclic lipopeptides—are calcium-dependent antibiotics that act against Gram-positive bacteria such as Staphylococcus aureus by inhibiting cell wall biosynthesis. However, to date, the biosynthetic mechanism of the empedopeptins has not been well identified. Through comparative genomics and metabolomics analysis, we identified empedopeptin and its new analogs from a marine bacterium, Massilia sp. YMA4. We then unveiled the empedopeptin biosynthetic gene cluster. The core nonribosomal peptide gene null-mutant strains (ΔempC, ΔempD, and ΔempE) could not produce empedopeptin, while dioxygenase gene null-mutant strains (ΔempA and ΔempB) produced several unique empedopeptin analogs. However, the antibiotic activity of ΔempA and ΔempB was significantly reduced compared with the wild-type, demonstrating that the hydroxylated amino acid residues of empedopeptin and its analogs are important to their antibiotic activity. Furthermore, we found seven bacterial strains that could produce empedopeptin-like cyclic lipopeptides using a genome mining approach. In summary, this study demonstrated that an integrated omics strategy can facilitate the discovery of potential bioactive metabolites from microbial sources without further isolation and purification.

Phenol and p-nitrophenol biodegradations by acclimated activated sludge: Influence of operational conditions on biodegradation kinetics and responding microbial communities

This study aims to evaluate the influence of operational factors (i.e. substrate concentration, activated sludge concentration and acclimation concentration) and the microbial communities of activated sludge on the aerobic biodegradation of phenol and p-nitrophenol (PNP). During the biodegradation process, the phenol or PNP biodegradation and activated sludge growth kinetics were determined and the interaction effects of the operational factors on the kinetics were assessed. The phenol biodegradation kinetics was well described by zero-order model (R2 > 0.813). At low phenol concentration (< 200 mg/L), the biodegradation rate can be enhanced by increasing activated sludge concentration and acclimation concentration. From the specific oxygen uptake rate (SOUR) analysis, lower SOUR values for PNP relative to phenol suggests higher toxicity of PNP, with inhibition detected above 50 mg/L for both phenol and PNP. In view of the inadequacy of zero-order equation for PNP biodegradation, the kinetic data was fitted into Haldane equation that incorporate the inhibitory effect. From the results, the value of critical PNP concentration was observed to increase at higher acclimation concentration, indicating the attenuation of inhibitory effects on PNP-acclimated activated sludge. For microbial growth, only the factor of acclimation concentration greatly affected the growth kinetics, in which the Ks/Ki ratio was greatly reduced when the acclimation concentration was doubled. Lastly, potential degrading strains of Acinetobacter sp. USM, Rhodococcus sp. USM1 and Caballeronia sp. USM2, were successfully isolated from the acclimated activated sludge. From the results of dioxygenase genes amplification, the phenol degradation mechanism was identified following ortho fission pathway.

Drought Intensity-Responsive Salicylic Acid and Abscisic Acid Crosstalk with the Sugar Signaling and Metabolic Pathway in Brassica napus.

The aim of this study was to characterize hormonal crosstalk with the sugar signaling and metabolic pathway based on a time course analysis of drought intensity. Drought intensity-responsive changes in the assimilation of newly fixed carbon (C) into soluble sugar, the content of sugar and starch, and expression of genes involved in carbohydrate metabolism were interpreted as being linked to endogenous abscisic acid (ABA) and salicylic acid (SA) levels and their signaling genes. The ABA and SA levels in the drought-stressed leaves increased together during the early drought period (days 0–6), and additional ABA accumulation occurred with depressed SA during the late period (days 6–14). Although drought treatment decreased the assimilation of newly fixed C into soluble sugar, representing a 59.9%, 33.1%, and 62.9% reduction in 13C-glucose, 13C-fructose, and 13C-sucrose on day 14, respectively, the drought-responsive accumulation of soluble sugars was significant. During the early period, the drought-responsive accumulation of hexose and sucrose was concurrent with the upregulated expression of hexokinase 1 (HXK1), which, in turn, occurred parallel to the upregulation of ABA synthesis gene 9-sis-epoxycarotenoid dioxygenase (NCED3) and SA-related genes (isochorismate synthase 1 (ICS1) and non-expressor of pathogenesis-related gene (NPR1)). During the late period, hexose accumulation, sucrose phloem loading, and starch degradation were dominant, with a highly enhanced expression of the starch degradation-related genes β-amylase 1 (BAM1) and α-amylase 3 (AMY3), which were concomitant with the parallel enhancement of sucrose non-fermenting−1 (Snf1)-related protein kinase 2 (SnRK2).2 and ABA-responsive element binding 2 (AREB2) expression in an ABA-dependent manner. These results indicate that the drought-responsive accumulation of sugars (especially SA-mediated sucrose accumulation) is part of the acclamatory process during the early period. Conversely, ABA-responsive hexose accumulation and sucrose phloem loading represent severe drought symptoms during the late drought period.

NCED expression is related to increased ABA biosynthesis and stomatal closure under aluminum stress

Aluminum (Al)-induced decrease in leaf hydration has been associated with low gas exchange, especially stomatal conductance (gs). However, the mechanisms explaining these responses are unclear. Citrus limonia was exposed to 0 and 1480 μM Al in nutrient solution for 90 days to test whether the low gs and leaf hydration in plants exposed to Al is associated with increased 9-cis-epoxycarotenoid dioxygenase (NCED) gene expression and abscisic acid (ABA) biosynthesis. Relative leaf water content (RWC), water potential (Ψw) and gas exchange in the leaves, as well as leaf and root ClNCED3, ClNCED1 and ClNCED5 expression and accumulation of ABA and its metabolites (phaseic acid, dihydrophaseic acid, (+)-7′-hydroxy-ABA and ABA-β-d-glucosyl ester) were measured. Aluminum up-regulated ClNCED3 and induced ABA accumulation in the roots before impairments in leaf water status (low Ψw, RWC and gs) could be observed. Leaf ABA concentration increased from 7 to 90 days and this could be partially explained by the up-regulation of ClNCED3, ClNCED1 and ClNCED5 in this organ. Stomatal closure occurred concomitantly with the increase of ABA concentration, and this result provides further evidence of the role of ABA modulation of plant hydration under Al stress.

Genome, metabolic pathways and characteristics of cometabolism of dibenzothiophene and the biodiesel byproduct glycerol in Paraburkholderia sp. C3

Utilization of glycerol, a biodiesel byproduct, has not been well explored. In the present study, glycerol and the other carbon sources were studied for cometabolism of dibenzothiophene (DBT), a model chemical commonly used in bioremediation studies, by Paraburkholderia sp. C3. This study showed a direct association between rhamnolipids (RLs) biosynthesis and DBT biodegradation induced by different carbon sources in a Paraburkholderia specie. Glycerol can induce the strain C3 produce at least four RLs. The RL precursor is mainly derived from the fatty acid synthesis (FAS II) and β-oxidation pathway. The genome contained two (fabF and fabG) and four (fadA, fadE, fadB and echA) genes involved in FAS II and β-oxidation, respectively. The genome also carried the rhlA and rhlB genes involved in rhamnosyltransferase for RL biosynthesis and two DBT dioxygenase genes (nahAc and catA). The findings suggest a viable approach of using the biodiesel byproduct glycerol to remediate contaminated environments.

Carotenoid Production in Escherichia coli: Case of Acyclic Carotenoids.

Among isoprenoids, carotenoids were the first group of compounds which were synthesized from foreign genes in non-carotenogenic Escherichia coli as a heterologous host. A great variety of carotenoids have been shown to be produced in E. coli due to the introduction of combinations of carotenoid biosynthesis genes, which were isolated from carotenogenic organisms. Carotenoids that have been produced in E. coli are mostly cyclic carotenoids that retain carbon 40 (C40) basic structure, except for acyclic carotene lycopene. On the other hand, acyclic carotenoids, which can also be produced in E. coli, comprise a group of carotenoids with diverse chain lengths, i.e., with C20, C30, C40, or C50 basic skeleton. As for acyclic C30, C40, and C50 carotenoids, carotenogenic genes of bacterial origin were needed, while a cleavage dioxygenase gene of higher-plant origin was utilized for the synthesis of acyclic C20 carotenoids. The present chapter is a review on the biosynthesis of such diverse acyclic carotenoids at the gene level.

The apple gene responsible for columnar tree shape reduces the abundance of biologically active gibberellin.

Ectopic expression of the apple 2-oxoglutarate-dependent dioxygenase (DOX, 2ODD) gene, designated MdDOX-Co, is thought to cause the columnar shape of apple trees. However, the mechanism underlying the formation of such a unique tree shape remains unclear. To solve this problem, we demonstrated that Arabidopsis thaliana overexpressing MdDOX-Co contained reduced levels of biologically active gibberellin (GA) compared with wild type. In summary: (i) with biochemical approaches, the gene product MdDOX-Co was shown to metabolize active GA A4 (GA4 ) to GA58 (12-OH-GA4 ) in vitro. MdDOX-Co also metabolized its precursors GA12 and GA9 to GA111 (12-OH-GA12 ) and GA70 (12-OH-GA9 ), respectively; (ii) Of the three 12-OH-GAs, GA58 was still active physiologically, but not GA70 or GA111 ; (iii) Arabidopsis MdDOX-Co OE transformants converted exogenously applied deuterium-labeled (d2 )-GA12 to d2 -GA111 but not to d2 -GA58 , whereas transformants converted applied d2 -GA9 to d2 -GA58 ; (iv) GA111 is converted poorly to GA70 by GA 20-oxidases in vitro when GA12 is efficiently metabolized to GA9 ; (v) no GA58 was detected endogenously in MdDOX-Co OE transformants. Overall, we conclude that 12-hydroxylation of GA12 by MdDOX-Co prevents the biosynthesis of biologically active GAs in planta, resulting in columnar phenotypes.