Acetosyringone Research Articles

The Only Chemoreceptor Encoded by che Operon Affects the Chemotactic Response of Agrobacterium to Various Chemoeffectors.

Chemoreceptor (also called methyl-accepting chemotaxis protein, MCP) is the leading signal protein in the chemotaxis signaling pathway. MCP senses and binds chemoeffectors, specifically, and transmits the sensed signal to downstream proteins of the chemotaxis signaling system. The genome of Agrobacterium fabrum (previously, tumefaciens) C58 predicts that a total of 20 genes can encode MCP, but only the MCP-encoding gene atu0514 is located inside the che operon. Hence, the identification of the exact function of atu0514-encoding chemoreceptor (here, named as MCP514) will be very important for us to understand more deeply the chemotaxis signal transduction mechanism of A. fabrum. The deletion of atu0514 significantly decreased the chemotactic migration of A. fabrum in a swim plate. The test of atu0514-deletion mutant (Δ514) chemotaxis toward single chemicals showed that the deficiency of MCP514 significantly weakened the chemotactic response of A. fabrum to four various chemicals, sucrose, valine, citric acid and acetosyringone (AS), but did not completely abolish the chemotactic response. MCP514 was localized at cell poles although it lacks a transmembrane (TM) region and is predicted to be a cytoplasmic chemoreceptor. The replacement of residue Phe328 showed that the helical structure in the hairpin subdomain of MCP514 is a direct determinant for the cellular localization of MCP514. Single respective replacements of key residues indicated that residues Asn336 and Val353 play a key role in maintaining the chemotactic function of MCP514.

Efficient plant regeneration and genetic transformation system of the precious fast-growing tree Toona ciliata

Toona ciliata has considerable economic value, providing high-quality materials and medicines. However, the growth and sustainable development of T. ciliata is limited due to environmental changes, poor natural regeneration ability, and excessive logging. Therefore, this study used sterile seedling leaves from a good provenance of T. ciliata as explants to establish a highly efficient regeneration and genetic transformation system. The effects of different hormones and concentrations on leaf callus induction were investigated. A genetic transformation system using kanamycin (Kan) as the selective agent was established based on the regeneration system. The results showed that Murashige and Skoog (MS) medium supplemented with 3 mg/L N-(Phenylmethyl)-9H-purin-6-amine (6-BA), 1 mg/L kinetin (KT) and 0.05 mg/L 1-naphthlcetic acid (NAA) (T1 medium) were suitable for callus formation and adventitious bud production from whole leaves at 45 days. In addition, pCAMBIA2301-EYFP was introduced into competent cells of Agrobacterium tumefaciens strain GV3101 and used for genetic transformation. Healthy leaves pre-cultured on T1 medium for three days were directly infected with A. tumefaciens infection solution (OD600 = 0.6) for 20 min through the addition of 50 mmol/L of Acetosyringone (AS). Thereafter, the infected leaves were co-cultivated for one day on T1 medium supplemented with 150 mmol/L AS, and the infected leaves were transferred to fresh T1 medium containing 10 mg/L Kan and 100 mg/L Cefotaxime (Cef). Twenty-four positive transformed plants were selected through histological staining and PCR, laser confocal microscopy of EYFP from 259 putative transformants was conducted, and the transformed plants were obtained after approximately three months.

Development of an Agrobacterium tumefaciens-mediated transformation system for Tilletia controversa Kühn

Dwarf bunt of wheat caused by Tilletia controversa Kühn has been identified an international quarantine disease, which replace the grain material into millions of teliospores. Agrobacterium tumefaciens-mediated transformation (ATMT) system is a powerful tool for fungi transformation with significant advantages of simple operation, high efficiency, and genetic stability of transformants. In this study, we constructed ATMT system for T. controversa. All the transformants were tested using Acetosyringone (AS) concentration at 150 μmol/l, hygromycin B at 25 μg/ml, 1 × 106 T. controversa hypha cells/ml, A. tumefaciens with OD600 of 0.5 co-cultivation at 16 °C for 48 h and culture was incubated at 16 °C for 20 days. Using the ATMT method, we cultivated 8 generations of transformants on complete medium (CM) containing hygromycin B antibiotic and validated by PCR, which indicate that T-DNA had been successfully inserted into each of T. controversa transformants. In addition, thermal asymmetric interlaced PCR (TAIL-PCR) evaluated the Ti element inserts were at random sites in the fungal genome. Thus, ATMT approach is an efficient tool for insertional mutagenesis of T. controversa.

Purification and Characterization of a Thermo- and pH-Stable Laccase From the Litter-Decomposing Fungus Gymnopus luxurians and Laccase Mediator Systems for Dye Decolorization.

An extracellular laccase (GLL) was purified from fermentation broth of the litter-decomposing fungus Gymnopus luxurians by four chromatography steps, which resulted in a high specific activity of 118.82 U/mg, purification fold of 41.22, and recovery rate of 42.05%. It is a monomeric protein with a molecular weight of 64 kDa and N-terminal amino acid sequence of AIGPV TDLHI, suggesting that GLL is a typical fungal laccase. GLL demonstrated an optimum temperature range of 55°C–65°C and an optimum pH 2.2 toward 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). It displayed considerably high thermostability and pH stability with about 63% activity retained after 24 h at 50°C, and 86% activity retained after 24 h at pH 2.2, respectively. GLL was significantly enhanced in the presence of K+, Na+, and Mg2+ ions. It demonstrated Km of 539 μM and kcat/Km of 140 mM–1⋅s–1 toward ABTS at pH 2.2 and 37°C. Acetosyringone (AS) and syringaldehyde (SA) were the optimal mediators of GLL (0.4 U/ml) for dye decolorization with decolorization rates of about 60%–90% toward 11 of the 14 synthetic dyes. The optimum reaction conditions were determined to be mediator concentration of 0.1 mM, temperature range of 25°C –60°C, and pH 4.0. The purified laccase was the first laccase isolated from genus Gymnopus with high thermostability, pH stability, and effective decolorization toward dyes, suggesting that it has potentials for textile and environmental applications.

Optimization of Agrobacterium Mediated Genetic Transformation in Paspalum scrobiculatum L. (Kodo Millet)

An efficient and reproducible protocol for Agrobacterium tumefaciens mediated genetic transformation was developed for kodo millet (Paspalum scrobiculatum L.) by optimizing various parameters. Agrobacterium strains EHA 105 and LBA 4404 harboring plasmids pCNL 56 and pCAMBIA 2300, respectively, provided the highest transformation efficiency. Addition of acetosyringone (AS) in infection medium (200 µM-EHA 105, 250 µM-LBA 4404) and co-cultivation medium (50 µM) increased the transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of transformed plants, respectively. The best GUS response was obtained by pretreatment of callus with an antinecrotic mixture (10 mg/L Cys + 5 mg/L Ag + 2.5 mg/L As) at infection time of 20 min followed by co-cultivation for 3 days (EHA 105) and 5 days (LBA 4404) in dark. Regenerated transgenic plants were obtained after 8 to 10 weeks of selection on callus induction medium (NAA 0.5 mg/L, BAP 1 mg/L) containing 50 mg/L Kan + 250 mg/L Cef and were rooted for 2 weeks on MS medium containing PAA (1 mg/L) and phytagel. The plantlets established in greenhouse showed normal growth. Therefore, the protocol developed in the present study can be used for development of improved varieties of kodo millet.

Establishment of Codonopsis pilosula (Franch.) Nannf. transformed roots, influence of the culture conditions on root growth and production of essential oil

Codonopsis pilosula (Franch.) Nannf. is a species whose radix has long been used in traditional folk medicine in China, Japan and Korea. Codonopsis Radix is obtained from cultivations mainly in Gansu, Shanxi, Sichuan, and Guizhou Provinces of China. This study describes an efficient protocol for genetic transformation of C. pilosula by Rhizobium rhizogenes and the obtainment of transformed roots of this species with valuable medicinal properties. It was found that both the bacterial strain (A4 and ATCC 15834) and acetosyringone (AcS) addition influenced the efficiency of transformation. The highest frequency of root induction (69 %) was observed on the Murashige and Skoog (MS) agar medium supplemented with AcS after transformation with A4 strain. The transformation was confirmed by PCR analysis. Of the tested media (MS, Schenk and Hildebrandt - SH, Gamborg - B5, and Woody Plant - WPM) those with full macro- and microsalt concentrations elicited significantly greater C. pilosula transformed root (CP2) biomass accumulation than the half-strength media, apart from MS medium. The greatest CP2 root biomass was observed in SH medium (1.65 g flask−1 DW). Among the tested sucrose concentrations (10−70 g L−1), the best result was obtained by using 30 g L−1 sucrose. Essential oils isolated by hydrodistillation from CP2 transformed roots and commercially available raw material, C. pilosula roots, had very similar qualitative composition. The most important group of common constituents of both oils were sesquiterpene hydrocarbons. The main among twenty two compounds in this group possessed skeletons of barbatane, thujopsane and bisabolane; the predominant ones being α- and β-barbatene, followed by β-chamigrene and thujopsene. This study presented that C. pilosula transformed roots can be a potential source for the industrial production of the raw material from species of the genus Codonopsis used in traditional Chinese medicine.

Introduction of the Nicotiana protein kinase (NPK1) gene by combining Agrobacterium-mediated transformation and recurrent somatic embryogenesis to enhance salt tolerance in cauliflower

Cauliflower is exposed to various biotic and abiotic stresses, including increased salinity due to the intensive irrigation of crops. Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play important roles in regulating innate immune responses in plants. Based on involvement of tobacco MAP kinase kinase kinase (NPK1) in stress response, the effect of the expression of NPK1 transgene to NaCl salt stress tolerance in cauliflower KFRM4 lines was studied. The Agrobacterium tumefaciens-mediated transformation protocol, using EHA101(pSHX004) vector harbouring the NPK1 and phosphinothricin N-acetyltransferase (bar) genes, the cyclic somatic embryogenesis regeneration pathway, the application of acetosyringone (AS) during co-cultivation and a delayed phosphinothricine (PPT) selection procedure provided sufficient transformation efficiency of 7.33% without escapes. PCR analysis indicated the integration of both NPK1 and bar transgenes in regenerated cauliflower lines. Transgenic cauliflower lines, exposed to NaCl stress in vitro, showed higher growth rates, greater ability to retain chlorophyll and carotenoids, and increased osmotic regulation capacity compared with non-transformed control plants. The tolerance level of transformed lines correlated with the level of NPK1 gene expression estimated by RT-qPCR, and the L2 line with the highest NPK1 expression displayed the greatest tolerance to NaCl stress. None of the obtained cauliflower transformed lines grown in greenhouses showed any morphological or yield differences compared with non-transformed plants. Furthermore, the expression of the bar gene facilitated the tolerance of transformed lines to the total herbicide PPT, applied at concentrations 2–3 times higher than those routinely used for weed control in the crop field. The results underlined that constitutively expressing NPK1 can significantly contribute to enhanced salt stress tolerance in cauliflower, suggesting that this could be a promising basis for the creation of new stress tolerance cruciferous vegetable lines.

Improving T-DNA Transfer to Tamarix hispida by Adding Chemical Compounds During Agrobacterium tumefaciens Culture.

Agrobacterium tumefaciens-mediated gene transfer is the most commonly used method for plant genetic engineering. However, during the period of A. tumefaciens culture, the effects of Agrobacterium culture before inoculation on genetic transformation are poorly understood. In the present study, we investigated the factors that affect the genetic transformation efficiency during Agrobacterium culture using Tamarix hispida as transgenic plant material. Agrobacterium treatment with spermidine (Spe), azacitidine (5-AzaC), dithiothreitol (DTT), or acetosyringone (AS) alone all significantly improved the efficiency of T-DNA transfer. Treatment with 5-AzaC reduced DNA methylation in Agrobacterium to induce the expression of virulence (vir) family genes, including virA, virB1, virC1, virD2, virD4 virE2, and virG. Spe treatment significantly induced the expression of all the studied genes, including virA, virB1, virC1, virD1, virD2, virD4, virE2, and virG. DTT treatment decreased reactive oxygen species accumulation. AS treatment activated the expression of the genes virA, virB1, virC1, virD1, virD2, virD4 and virG. All these effects resulted in increased T-DNA transfer. Additionally, combined Spe, 5-AzaC, DTT, and AS treatment improve Agrobacterium infection to a greater extent compared with their use alone, increasing T-DNA transfer by more than 8-fold relative to no treatment. Therefore, to improve genetic transformation, pretreatment of Agrobacterium during the culture period is important for improving genetic transformation efficiency.

Ionic Strength Effect Alters the Heterogeneous Ozone Oxidation of Methoxyphenols in Going from Cloud Droplets to Aerosol Deliquescent Particles.

Methoxyphenols are one of the most abundant classes of biomarker tracers for atmospheric wood smoke pollution. The reactions of atmospheric oxidants (ozone, OH) with methoxyphenols can contribute to the formation of secondary organic aerosols (SOA). Here, for the first time, we use the well-established vertical wetted wall flow tube (VWWFT) reactor to assess the effect of ionic strength (I), pH, temperature, and ozone concentration on the reaction kinetics of ozone with acetosyringone (ACS), as a representative methoxyphenol compound. At fixed pH 3, typical for acidic atmospheric deliquescent particles, and at I = 0.9 M adjusted by Na2SO4, the uptake coefficient (γ) of O3 increases by 2 orders of magnitude from γ = (5.0 ± 0.8) × 10-8 on neat salt solution (Na2SO4) to γ = (6.0 ± 0.01) × 10-6 on a mixture of ACS and Na2SO4. The comparison of the uptake coefficients of O3 at different pH values indicates that the reaction kinetics strongly depends on the acidity of the phenolic group of ACS. The observed different reactivity of gas-phase ozone with ACS has implications for ozone uptake by the dilute aqueous phase of cloud droplets and by aerosol deliquescent particles loaded with inorganic salts, and it can affect the formation of SOA in the atmosphere.

Inhibition kinetics of acetosyringone on xylanase in hydrolysis of hemicellulose.

Many phenolic compounds, derived from lignin during the pretreatment of lignocellulosic biomass, could obviously inhibit the activity of cellulolytic and hemicellulolytic enzymes. Acetosyringone (AS) is one of the phenolic compounds produced from lignin degradation. In this study, we investigated the inhibitory effects of AS on xylanase activity through kinetic experiments. The results showed that AS could obviously inhibit the activity of xylanase in a reversible and noncompetitive binding manner (up to 50% activity loss). Inhibitory kinetics and constants of xylanase on AS were conducted by the HCH-1model (β=0.0090±0.0009mM-1). Furthermore, intrinsic and 8-anilino-1-naphthalenesulfonic (ANS)-binding fluorescence results showed that the tertiary structure of AS-mediated xylanase was altered. These findings provide new insights into the role of AS in xylanase activity. Our results also suggest that AS was an inhibitor of xylanase and targeting AS was a potential strategy to increase xylose production.

A New Laccase of Lac 2 from the White Rot Fungus Cerrena unicolor 6884 and Lac 2-Mediated Degradation of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH4)2SO4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1 detoxification. AFB1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2′ -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1 degradation by Lac 2. The product of AFB1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1 (AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1 present in food and feeds.

Apoplast redox metabolism: Effect of acetovanillone (apocynin) and acetosyringone, on their co-oxidation and redox properties

A common response of plant leaves and roots to stress, biotic or abiotic, is the oxidative burst. In leaves, this results in reactive oxygen species being produced within the apoplast, the space surrounding the responding cells. A second response that occurs in the tobacco apoplast during this period is the production and accumulation of phenolic metabolites. The interaction of these redox active metabolites should have major effects on the redox potential of the apoplast and subsequently affect the outcome of the plant tissue. To begin to understand the effects of these responses on the redox potential, two induced phenolics, acetovanillone (AV) and acetosyringone (AS) were studied in vitro with peroxidase and H2O2, all of which are present in the tobacco apoplast. While both phenolics were observed to be substrates for peroxidase, AV was found to react 10x faster than AS. The oxidation of AV resulted in an increase of the redox potential to a maximum of 0.35 V. The oxidation of AS resulted in an increase to a maximum of 0.55 V. Interestingly, the redox potential was not proportional to the oxidation of either phenolic. It was found that when present together, co-oxidation occurs with the faster acting AV as a catalyst to oxidize AS. This has an immediate effect on the redox potential. The rate of AS oxidation is dependent on the AV concentration and thus so is the redox potential reaching 4.5 V. The results suggest that these phenolics could play a role in the regulation of the apoplast redox potential during the early stages of stress.

Identification of the genes involved in growth characters of medicinal fungus Ophiocordyceps sinensis based on Agrobacterium tumefaciens-mediated transformation.

Ophiocordyceps sinensis, one of the well-known and precious fungal species in the world, parasitizes soil-dwelling larvae of ghost moths on the Tibetan Plateau. The genetic intractability of this extremely psychrophilic and slow-growing O. sinensis fungus is a major limitation on the molecular study. In this study, an Agrobacterium tumefaciens-mediated genetic transformation (ATMT) system for this fungus was established. ATMT procedure was optimized based on the fungal recipient, Agrobacterium strains, and different co-cultivation conditions. Blastospores were ideal recipients for this system. Acetosyringone (AS) was not essential for the transformation of O. sinensis. Sixty to 100 hygromycin B-resistant transformants per 1 × 106 blastospores were obtained. Southern blot analysis indicated the presence of a random single-copy integration of T-DNA into the O. sinensis genome. The insertional transformants with altered growth characters such as colony, blastospore, and fruiting body production were selected to identify the T-DNA flanking sequences by modified hiTAIL-PCR and FPNI-PCR techniques. Eight genes, including genes for an MFS transporter, a 2-oxoglutarate dehydrogenase, a DNA-directed RNA polymerase III complex subunit Rpc37, a cytochrome oxidase subunit I, a mitochondrial import inner membrane translocase subunit tim54, a cytidine deaminase, a phosphoribosylaminoimidazole carboxylase, and a histone H3-like centromeric protein cse-4 were identified. This ATMT system provides a useful tool for gene discovery and characterization of O. sinensis and contributes to the better understanding of the mysterious life cycle of O. sinensis and the molecular interaction between this fungus and its host insects.

An optimized Agrobacterium tumefaciens-mediated transformation system for random insertional mutagenesis in Fonsecaea monophora

Chromoblastomycosis (CBM) is a chronic cutaneous or subcutaneous mycosis that is prevalent worldwide. Though CBM tends not to be fatal, it is difficult to treat and complications can include chronic, marked lesions, lymphatic damage, and neoplastic transformation. Fonsecaea monophora, as a new species segregated from Fonsecaea pedrosoi, is the predominant causative pathogen of CBM in southern China. However, research about F. monophora has been limited, which may be due to a lack of an effective genetic manipulation system for F. monophora. In this study, we successfully established a random insertional mutagenesis system by Agrobacterium tumefaciens-mediated transformation (ATMT) in F. monophora for the first time. In order to improve the efficiency of ATMT, various co-culture conditions were optimized, including: acetosyringone (AS) concentrations, co-culture duration, ratio of bacteria to conidia, and the A. tumefaciens strains. In addition, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to identify the transferred DNA (T-DNA) flanking sequences of the F. monophora transformants. The valuable transformants obtained in this study will be used for research in the future.

Establishment of an Agrobacterium tumefaciens-mediated transformation system for Tilletia foetida

Tilletia foetida causes wheat common smut disease with severe loss of yield production and seed quality. In this study, a low-cost, rapid, and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for T. foetida mutagenesis was constructed: Transformants were screened with hygromycin B at 100 μg/ml, cefotaxime sodium concentrations with 200 μg/ml, Acetosyringone (AS) concentration at 200 μmol/l, 1 × 106 T. foetida hypha cells/ml, co-cultivation at 22 °C with 24 h and culture was incubated at 16 °C up to day 7. Fourteen transformants were randomly selected and confirmed using the specific primers to amplify the fragment of hygromycin phosphotransferase gene. At the same time, PCR analysis was performed to detect Agrobacterium tumefaciens Vir gene to eliminate false positives. The transformants were cultivated up to 8 generations on hygromycine B-containing complete medium (CM) and confirmed by PCR. The results indicated that 80% of T. foetida transformants were hygromycine B resistant. In conclusion, our analyses identified an efficient T-DNA insertion system for T. foetida and the results will be useful for further understanding the pathogenic mechanism via generation of the insertional mutants.

Enhanced transformation of sulfonamide antibiotics by manganese(IV) oxide in the presence of model humic constituents

In this study, a manganese(IV) oxide-mediator (MnO2-mediator) system for the abatement of sulfonamide antibiotics was evaluated. Two simple model humic constituents, syringaldehyde (SA) and acetosyringone (AS), could promote the transformation of sulfonamides at pH 5–8. Two additional potential mediators, tannic acid and 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonate (ABTS), had negligible enhancement on the transformation of sulfonamides by MnO2. The enhancing effect was attributed to the reaction of the oxidized mediator (i.e., phenoxy radical or benzoquinone-like compounds) produced from the oxidation of the mediators by MnO2 with SMX. Thereby cross-coupling products from sulfamethoxazole (SMX) with oxidized SA were formed in the MnO2-SA system, which was confirmed by liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry. Coexisting metal ions (i.e., Ca(II), Mg(II) and Mn(II)) showed inhibitory effects in the order of Mn(II)> Ca(II)> Mg(II). For repetitive runs of the MnO2-SA-SMX system, MnO2 lost its oxidative capacity due to the sorption of Mn(II) on the reactive sites of the MnO2 surface. A full regeneration of partially deactivated MnO2 by oxidation of the sorbed Mn(II) with Mn(VII) could be achieved.

Ionic strength effects on the photochemical degradation of acetosyringone in atmospheric deliquescent aerosol particles

A number of laboratory experimental investigations, field campaigns, and modeling results have emphasized the role of aqueous-phase photochemical reactions in the formation of secondary organic aerosols (SOA). However, investigations focused on aqueous-phase reactions under high ionic-strength conditions are scarce. Here we study the photochemical behavior of a lignin-derived compound, acetosyringone (AcS), upon addition of an inert salt (NaClO4). The increase in the ionic strength modifies the acidic constant of AcS, enhancing its deprotonation. As a consequence, the UV-VIS absorption spectra of AcS undergo modifications due to red shifts at high ionic strength of the electronic transitions n → π* (from λmax = 297 nm to λmax = 354 nm) and π → π* (from λmax = 214 nm to λmax = 247 nm). At fixed pH = 4, representative of moderately acidic atmospheric aerosol deliquescent particles, the pseudo-first-order rate constants (k1st) of AcS increased by ∼6 times from a dilute aqueous phase to a solution with an effective ionic strength Ieff. = 0.46 M. The rate constant then followed a saturation trend at elevated ionic strength up to Ieff. = 3.1 M. A similar saturation effect of the observed rate constants with ionic strength was observed in presence of NaCl and Na2SO4. Differential absorption spectroscopy (DAS) methodology was applied to examine the changes in absorption spectra of AcS upon prolonged light irradiation. The very subtle pH-induced changes of the absorption spectra of irradiated AcS are due to the formation of acidic compounds emerged upon photochemical transformation of AcS.

Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus

BackgroundLarge T-DNA fragment transfer has long been a problem for Agrobacterium-mediated transformation. Although vector systems, such as the BIBAC series, were successfully developed for the purpose, low transformation efficiencies were consistently observed.ResultsTo gain insights of this problem in monocot transformation, we investigated the T-strand accumulation of various size of T-DNA in two kinds of binary vectors (one copy vs. multi-copy) upon acetosyringone (AS) induction and explored ways to improve the efficiency of the large T-DNA fragment transfer in Agrobacterium-mediated rice transformation. By performing immuno-precipitation of VirD2-T-strands and quantitative real-time PCR assays, we monitored the accumulation of the T-strands in Agrobacterium tumeficiens after AS induction. We further demonstrated that extension of AS induction time highly significantly improved large-size T-DNA transfer to rice cells.ConclusionsOur data provide valuable information of the T-strand dynamics and its impact on large T-DNA transfer in monocots, and likely dicots as well.

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of D. lotus ( Diospyros lotus L.)

Abstract In vitro regeneration and Agrobacterium-mediated genetic transformation system of D. lotus (Diospyros lotus L.) has been achieved using leaf explants. Two-step regeneration method was developed. First, the explants cultured in callus induction medium containing 0.1 mg/L naphthalene acetic acid (NAA) and 3.0 mg/L thidiazuron (TDZ) and cultured in darkness for 10 days. Second, leaf discs were introduced on shoot induction medium containing 1 mg/L trans-zeatin (ZT) and 0.1 mg/L indole-3-acetic acid (IAA) after 15 days of inoculation, callus induction rate of Diospyros lotus L. was 100%, average number of adventitious shoots was 4.26, regeneration rate was 94.28% respectively. The highest number root formation was observed in MS(1/2 N) supplemented with 0.5 mg/L 6-benzylaminopurine (6-BA) and 1.0 mg/L indole-3-butyric acid (IBA). A protocol for genetic transformation of D. lotus via Agrobacterium was established. Agrobacterium strain EHA105 carrying vector pCAMBIA1301-GA2OX2 was used to optimize the transformation conditions, which include pre-culture period, bacterial optical density, infection time, co-culture duration, concentration of acetosyringone (AS) and concentration of hygromycin. Improved transformation rates (14.27 ± 1.32%) were achieved when explants pre-cultured for 4 days were incubated with an Agrobacterium suspension with a culture density of OD600 = 0.5 for 10 min, followed by 1-day co-cultivation. Successful transformation was confirmed by histochemical GUS activity, hpt and gus gene PCR analyses of the regeneration hygromycin resistant plantlets.

An extracellular yellow laccase from white rot fungus Trametes sp. F1635 and its mediator systems for dye decolorization

A novel extracellular laccase was purified from fermentation broth of the white rot fungus Trametes sp. F1635 by a three-step protocol including two consecutive ion-exchange chromatography steps on DEAE-Sepharose and SP-Sepharose, and a final gel-filtration on Superdex 75. The purified laccase (TsL) was a monomeric protein with the molecular mass of 64.8 kDa. It demonstrated high oxidation activity of 4.00 × 104 U/mg towards ABTS. Its N-terminal amino acid sequence was AIGPVADLTIINNAV which was unique and sharing high similarity of other fungal laccases. TsL was a yellow laccase based on absorption spectrum analysis. It demonstrated an acidic pH optimum of 2.6 and temperature optimum of 50 °C towards ABTS. The Km and Vmax values towards ABTS were estimated to 18.58 μM and 1.35 μmol/min, respectively. TsL manifested effective decolorization activity towards eriochrome black T (EBT), remazol brilliant blue R (RBBR), malachite green (MG), and eriochrome black T (EBT) (over 60%). Violuric acid (VA) and acetosyringone (AS) were the optimal mediators for the laccase in dye decolorization. Results suggest that TsL demonstrates great potential for dye decolorization and water treatment.