Ming-Luan Chen

ABI4 regulates primary seed dormancy by regulating the biogenesis of abscisic acid and gibberellins in arabidopsis.

Seed dormancy is an important economic trait for agricultural production. Abscisic acid (ABA) and Gibberellins (GA) are the primary factors that regulate the transition from dormancy to germination, and they regulate this process antagonistically. The detailed regulatory mechanism involving crosstalk between ABA and GA, which underlies seed dormancy, requires further elucidation. Here, we report that ABI4 positively regulates primary seed dormancy, while negatively regulating cotyledon greening, by mediating the biogenesis of ABA and GA. Seeds of the Arabidopsis abi4 mutant that were subjected to short-term storage (one or two weeks) germinated significantly more quickly than Wild-Type (WT), and abi4 cotyledons greened markedly more quickly than WT, while the rates of germination and greening were comparable when the seeds were subjected to longer-term storage (six months). The ABA content of dry abi4 seeds was remarkably lower than that of WT, but the amounts were comparable after stratification. Consistently, the GA level of abi4 seeds was increased compared to WT. Further analysis showed that abi4 was resistant to treatment with paclobutrazol (PAC), a GA biosynthesis inhibitor, during germination, while OE-ABI4 was sensitive to PAC, and exogenous GA rescued the delayed germination phenotype of OE-ABI4. Analysis by qRT-PCR showed that the expression of genes involved in ABA and GA metabolism in dry and germinating seeds corresponded to hormonal measurements. Moreover, chromatin immunoprecipitation qPCR (ChIP-qPCR) and transient expression analysis showed that ABI4 repressed CYP707A1 and CYP707A2 expression by directly binding to those promoters, and the ABI4 binding elements are essential for this repression. Accordingly, further genetic analysis showed that abi4 recovered the delayed germination phenotype of cyp707a1 and cyp707a2 and further, rescued the non-germinating phenotype of ga1-t. Taken together, this study suggests that ABI4 is a key factor that regulates primary seed dormancy by mediating the balance between ABA and GA biogenesis.

Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine in Genomic DNA from Hepatocellular Carcinoma Tissues by Capillary Hydrophilic-Interaction Liquid Chromatography/Quadrupole TOF Mass Spectrometry

BACKGROUND 5-Methylcytosine (5-mC) is an important epigenetic modification involved in development and is frequently altered in cancer. 5-mC can be enzymatically converted to 5-hydroxymethylcytosine (5-hmC). 5-hmC modifications are known to be prevalent in DNA of embryonic stem cells and neurons, but the distribution of 5-hmC in human liver tumor and matched control tissues has not been rigorously explored. METHODS We developed an online trapping/capillary hydrophilic-interaction liquid chromatography (cHILIC)/in-source fragmentation/tandem mass spectrometry system for quantifying 5-mC and 5-hmC in genomic DNA from hepatocellular carcinoma (HCC) tumor tissues and relevant tumor adjacent tissues. A polymer-based hydrophilic monolithic column was prepared and used for the separation of 12 nucleosides by cHILIC coupled with an online trapping system. Limits of detection and quantification, recovery, and imprecision of the method were determined. RESULTS Limits of detection for 5-mC and 5-hmC were 0.06 and 0.19 fmol, respectively. The imprecision and recovery of the method were determined, with the relative SDs and relative errors being <14.9% and 15.8%, respectively. HCC tumor tissues had a 4- to 5-fold lower 5-hmC content compared to tumor-adjacent tissues. In addition, 5-hmC content highly correlated with tumor stage (tumor-nodes-metastasis, P = 0.0002; Barcelona Clinic liver cancer, P = 0.0003). CONCLUSIONS The marked depletion of 5-hmC may have profound effects on epigenetic regulation in HCC and could be a potential biomarker for the early detection and prognosis of HCC.

Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC-ESI-Q-TOF-MS analysis.

In current study, we developed a highly sensitive method for the quantitative profiling of acidic phytohormones. Tandem solid-phase extraction (SPE) and liquid-liquid extraction (LLE) was employed to efficiently purify acidic phytohormones, which were further derived by 3-bromoactonyltrimethylammonium bromide (BTA) to increase the ionization efficiency in electrospray ionization-mass spectrometry detection. Additionally, fifteen BTA-derived acidic phytohormones, including ten gibberellins (GAs), were well separated with a salt gradient on poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolithic column. By employing online trapping system, the signal intensities of the analytes were significantly improved. The limits of detection (LODs, Signal/Noise=3) of targeted phytohormones ranged from 1.05 to 122.4 pg/mL, which allowed the highly sensitive determination of low abundant acidic phytohormones with tiny amount plant sample. Good reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 10.9 and 11.9%, respectively. Recoveries of the target analytes from spiked rice leave samples ranged from 88.3 to 104.3%. By employing the method developed here, we were able to simultaneously determine 11 endogenous acidic phytohormones from only 5mg of rice leave sample, which dramatically decreased the required sample amount (three orders of magnitude lower) for the profiling of low abundant acidic phytohormones compared to previous reports. Taken together, the method provided a good solution for the highly sensitive and quantitative profiling of endogenous acidic phytohormones.

Mutation of Rice BC12/GDD1, Which Encodes a Kinesin-Like Protein That Binds to a GA Biosynthesis Gene Promoter, Leads to Dwarfism with Impaired Cell Elongation

The authors show that mutation of the rice gene encoding the kinesin-like protein BC12/GDD1 causes a significant reduction in the endogenous GA level and in cell elongation. This is a result of direct regulation of the expression of KO2, a key gene in GA biosynthesis, by GDD1 binding to the KO2 promoter. The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin-deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA3) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice.

Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene

Plant height is a decisive factor in plant architecture. Rice (Oryza sativa) plants have the potential for rapid internodal elongation, which determines plant height. A large body of physiological research has shown that ethylene and gibberellin are involved in this process. The APETALA2 (AP2)/Ethylene-Responsive Element Binding Factor (ERF) family of transcriptional factors is only present in the plant kingdom. This family has various developmental and physiological functions. A rice AP2/ERF gene, OsEATB (for ERF protein associated with tillering and panicle branching) was cloned from indica rice variety 9311. Bioinformatic analysis suggested that this ERF has a potential new function. Ectopic expression of OsEATB showed that the cross talk between ethylene and gibberellin, which is mediated by OsEATB, might underlie differences in rice internode elongation. Analyses of gene expression demonstrated that OsEATB restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A. Plant height is negatively correlated with tiller number, and higher yields are typically obtained from dwarf crops. OsEATB reduces rice plant height and panicle length at maturity, promoting the branching potential of both tillers and spikelets. These are useful traits for breeding high-yielding crops.

Highly sensitive profiling assay of acidic plant hormones using a novel mass probe by capillary electrophoresis-time of flight-mass spectrometry.

Plant hormones play crucial roles in plant growth and development. However, up to date, identification and quantification of acidic plant hormones with trace amount in complicated plant matrix is still a challenge. In current study, we developed a high sensitive assay for the determination of acidic plant hormones in rice by combining capillary electrophoresis and electrospray ionization-time of flight-mass spectrometry (CE-ESI-TOF-MS). To improve the detection sensitivity of acidic plant hormones, 3-bromoactonyltrimethylammonium bromide (BTA) was synthesized as a new mass probe, which can react efficiently with acidic plant hormones in acetonitrile containing triethylamine (TEA). The positively charged BTA-derivatives were separated by CE using amino-coated capillary, which provided a reversed electroosmotic flow (EOF) at low pH, as well as reduced the adsorption of BTA-derivatives on the inner wall of capillary. Using the CE-ESI-TOF-MS method developed in current study, 15 acidic plant hormones, including 10 gibberellins (GAs), were identified and quantified with good linearities from 1.3 to 850 ng/mL with linear coefficient R(2) values of >0.99. The limits of detection (LODs) were in the range of 0.34-4.59 ng/mL. Recoveries of compounds from spiked beverage samples ranged from 84.6 to 112.2%. And a good reproducibility was obtained by evaluating the intra and inter-day precisions with relative standard deviations (RSDs) less than 6.7 and 9.9%, respectively.

Tomato SlDREB gene restricts leaf expansion and internode elongation by downregulating key genes for gibberellin biosynthesis

Plants have evolved and adapted to different environments. Dwarfism is an adaptive trait of plants that helps them avoid high-energy costs under unfavourable conditions. The role of gibberellin (GA) in plant development has been well established. Several plant dehydration-responsive element-binding proteins (DREBs) have been identified and reported to be induced under abiotic and biotic stress conditions. A tomato DREB gene named SlDREB, which is a transcription factor and was cloned from cultivated tomato M82, was found to play a negative role in tomato plant architecture and enhances drought tolerance. Tissue expression profiles indicated that SlDREB was expressed mainly in the stem and leaf and could be induced by abscisic acid (ABA) but suppressed by GA and ethylene. SlDREB altered plant morphology by restricting leaf expansion and internode elongation when overexpressed, and the resulting dwarfism of tomato plants could be recovered by application of exogenous gibberellic acid (GA3). Transcriptional analysis of transgenic plants revealed that overexpression of SlDREB caused the dwarf phenotype by downregulating key genes involved in GA biosynthesis such as ent-copalyl diphosphate synthase (SlCPS) and GA 20-oxidases (SlGA20ox1, -2, and -4), thereby decreasing endogenous GA levels in transgenic plants. A yeast activity assay demonstrated that SlDREB specifically bound to dehydration-responsive element/C-repeat (DRE/CRT) elements of the SlCPS promoter region. Taken together, these data demonstrated that SlDREB can downregulate the expression of key genes required for GA biosynthesis and that it acts as a positive regulator in drought stress responses by restricting leaf expansion and internode elongation.

Determination of benzimidazole residues in animal tissue samples by combination of magnetic solid-phase extraction with capillary zone electrophoresis

Benzimidazole drugs (BZDs) comprise a large number of synthetic anthelmintics, which are widely used in food-producing animals for prophylactic and therapeutic purposes. To protect consumers from the risks related to BZDs residues, a simple, rapid, and efficient method for simultaneous determination of ten BZDs in animal tissues samples was developed. This analytical procedure involved extracting samples with magnetic solid-phase extraction (MSPE) using magnetite/silica/poly (methacrylic acid-co-ethylene glycol dimethacrylate) (Fe(3)O(4)/SiO(2)/poly (MAA-co-EGDMA)) magnetic microspheres, and determination by capillary zone electrophoresis (CZE). To improve the sensitivity of the method, we employed the electrokinetic injection with field-amplified sample stacking technique (FASS). Berbine solution was used as internal standard to minimize the fluctuation of analytical results. Under the optimized extraction conditions, good linearities were obtained for the ten BZDs with the correlation coefficients (R(2)) above 0.9920. The limits of detections (LODs) for ten BZDs were 1.05-10.42 ng/g in swine muscle and 1.06-12.61 ng/g in swine liver, respectively. The intra- and inter-day relative standard deviations (RSDs) of the developed method were less than 13.6%. The recoveries of the ten BZDs for the spiked samples ranged from 81.1% to 105.4% with RSDs less than 9.3%.

Preparation of methacrylate-based monolith for capillary hydrophilic interaction chromatography and its application in determination of nucleosides in urine.

A novel poly(N-acryloyltris(hydroxymethyl)aminomethane-co-pentaerythritol triacrylate) (NAHAM-co-PETA) monolith was prepared in the 100 μm i.d. capillary and investigated for capillary liquid chromatography (cLC). The polymer monolith was synthesized by in situ polymerization of NAHAM and PETA in the presence of polyethylene glycol (PEG) in dimethyl sulfoxide (DMSO) as the porogen. The porous structure of monolith was optimized by changing the ratio of NAHAM to PETA, the molecular weight and amount of PEG. To evaluate the separation performance of the resultant polymer monolith, several groups of model compounds (including nucleosides, benzoic acids and anilines) were selected to perform cLC separation. Our results showed that these model compounds can be baseline separated on the resultant poly(NAHAM-co-PETA) monolithic column with the optimized mobile phases. The column efficiency was estimated to be 87,000 plates/m for acrylamide. In addition, this monolithic column was coupled with on-line solid-phase microextraction (SPME) for the analysis of four nucleosides (uridine, adenosine, cytidine, guanosine) in urine. The limit of detection of the proposed method was in the range from 40 to 52 ng/mL. The method reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 8.3% and 10.2%, respectively. Recoveries of the target analytes from spiked urine samples were ranged from 86.5% to 106.8%.

Preparation of organic–inorganic hybrid silica monolith with octyl and sulfonic acid groups for capillary electrochromatograhpy and application in determination of theophylline and caffeine in beverage

An organic-inorganic hybrid silica monolithic column with octyl and sulfonic acid groups has been prepared by sol-gel technique for capillary electrochromatograhpy. The structure of hybrid monolith was optimized by changing the composition of tetraethoxysilane (TEOS), octyltriethoxysilane (C(8)-TEOS) and 3-mercaptopropyltrimethoxysilane (MPTMS) in the mixture of precursors. Then, the obtained hybrid monolith was oxidized using hydrogen peroxide (30%, w/w) to yield sulfonic acid groups. The sulfonic acid group, which served as strong cation-exchanger, dominated the charge on the surface of the capillary column and generated stable electroosmotic flow (EOF) in a wide range of pH. The monolithic column was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and elemental analysis (EA), and the performance of column was evaluated in detail by separating different kinds of compounds with column efficiency up to 155,000 plates/m for thiourea. In addition, this monolithic column was also applied in the analysis of theophylline (TP) and caffeine (CA) in beverages. The detection limits were 0.39 and 0.48 microg/mL for theophylline and caffeine, respectively. The method reproducibility was tested by evaluating the intra- and inter-day precisions, and relative standard deviations of less than 3.9 and 8.4%, respectively, were obtained. Recoveries of compounds from spiked beverage samples ranged from 87.2 to 105.2%.