The genetics underlying metabolic signatures in a brown rice diversity panel and their vital role in human nutrition

Brown rice (Oryza sativa) possesses various nutritionally dense bioactive phytochemicals exhibiting a wide range of antioxidant, anti-cancer, and anti-diabetic properties known to promote various human health benefits. However, despite the wide claims made about the importance of brown rice for human nutrition the underlying metabolic diversity has not been systematically explored. Non-targeted metabolite profiling of developing and mature seeds of a diverse genetic panel of 320 rice cultivars allowed quantification of 117 metabolites. The metabolite genome-wide association study (mGWAS) detected genetic variants influencing diverse metabolic targets in developing and mature seeds. We further interlinked genetic variants on chromosome 7 (6.06–6.43 Mb region) with complex epistatic genetic interactions impacting multi-dimensional nutritional targets, including complex carbohydrate starch quality, the glycemic index, antioxidant catechin, and rice grain color. Through this nutrigenomics approach rare gene bank accessions possessing genetic variants in bHLH and IPT5 genes were identified through haplotype enrichment. These variants were associated with a low glycemic index, higher catechin levels, elevated total flavonoid contents, and heightened antioxidant activity in the whole grain with elevated anti-cancer properties being confirmed in cancer cell lines. This multi-disciplinary nutrigenomics approach thus allowed us to discover the genetic basis of human health-conferring diversity in the metabolome of brown rice.     

GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling

The established GC-EI-TOF-MS method for the profiling of soluble polar metabolites from plant tissue was employed for the kinetic metabolic phenotyping of higher plants. Approximately 100 typical GC-EI-MS mass fragments of trimethylsilylated and methoxyaminated metabolite derivatives were structurally interpreted for mass isotopomer analysis, thus enabling the kinetic study of identified metabolites as well as the so-called functional group monitoring of yet non-identified metabolites. The monitoring of isotope dilution after (13)CO(2) labelling was optimized using Arabidopsis thaliana Col-0 or Oryza sativa IR57111 plants, which were maximally labelled with (13)C. Carbon isotope dilution was evaluated for short (2h) and long-term (3 days) kinetic measurements of metabolite pools in root and shoots. Both approaches were shown to enable the characterization of metabolite specific partitioning processes and kinetics. Simplifying data reduction schemes comprising calculation of (13)C-enrichment from mass isotopomer distributions and of initial (13)C-dilution rates were employed. Metabolites exhibited a highly diverse range of metabolite and organ specific half-life of (13)C-label in their respective pools ((13)C-half-life). This observation implied the setting of metabolite specific periods for optimal kinetic monitoring. A current experimental design for the kinetic metabolic phenotyping of higher plants is proposed.     

Clarification of pathway-specific inhibition by Fourier transform ion cyclotron resonance/mass spectrometry-based metabolic phenotyping studies

We have developed a metabolic profiling scheme based on direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). The scheme consists of: (1) reproducible data collection under optimized FT-ICR/MS analytical conditions; (2) automatic mass-error correction and multivariate analyses for metabolome characterization using a newly developed metabolomics tool (DMASS software); (3) identification of marker metabolite candidates by searching a species-metabolite relationship database, KNApSAcK; and (4) structural analyses by an MS/MS method. The scheme was applied to metabolic phenotyping of Arabidopsis (Arabidopsis thaliana) seedlings treated with different herbicidal chemical classes for pathway-specific inhibitions. Arabidopsis extracts were directly infused into an electrospray ionization source on an FT-ICR/MS system. Acquired metabolomics data were comprised of mass-to-charge ratio values with ion intensity information subjected to principal component analysis, and metabolic phenotypes from the herbicide treatments were clearly differentiated from those of the herbicide-free treatment. From each herbicide treatment, candidate metabolites representing such metabolic phenotypes were found through the KNApSAcK database search. The database search and MS/MS analyses suggested dose-dependent accumulation patterns of specific metabolites including several flavonoid glycosides. The metabolic phenotyping scheme on the basis of FT-ICR/MS coupled with the DMASS program is discussed as a general tool for high throughput metabolic phenotyping studies.     

Temporally resolved GC-MS-based metabolic profiling of herbicide treated plants treated reveals that changes in polar primary metabolites alone can distinguish herbicides of differing mode of action

We conducted a comprehensive metabolic phenotyping of primary metabolism of photosynthetic tissue of Arabidopsis thaliana following spray treatment with a number of commercially used herbicides using a well established gas-chromatography mass-spectrometry profiling method. Applying this technique we were able to identify and quantify in excess of 80 polar metabolites and based on a combination of co-elution with standards and prediction from the mass spectra a similar number of lipophillic components within two chromatographic runs. The herbicides selected were glufosinate, sulcotrione, AE944 [N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine], foramsulfuron, benfuresate and glyphosate. We determined causal changes in the metabolite profiles by following their time-dependent changes using a serial sampling strategy. The resultant profiles were compared both by looking at the largest changes in a metabolite by metabolite manner and by performance of statistical analyses. These data revealed that analysis of the polar metabolites allows clear separation of the compounds under test. This finding is discussed in the context of current strategies for agrochemical discovery.     

Phosphoglucomutase-1 (PGM1) W23 and W26 variants detected in the Taiwanese Chinese population

Phosphoglucomutase-1 (PGM1) phenotyping among Taiwanese Chinese was carried out on thin layer agarose gel using isoelectric focusing techniques. During routine paternity testing, two new PGM1 variants not previously observed in Taiwanese Chinese were detected. These are PGM1 W23 and PGM1 W26.     

水稻抗草品种选育研究初报

利用PI312777抗草稻种材料,与本单位优质稻核心种质及其衍生系统材料广泛杂交配组,创建一批以PI312777衍生系统为主的新型育种材料。建立水稻抗草育种圃,重点针对抗草特性和综合农艺性状,通过田间抑草效应筛选,培育水稻抗草苗头株系,在稳定世代(F4代以上)应用以水稻特征次生物质为标记评价水稻品种(系)化感潜力的方法,辅助选育水稻抗草新品种(系)。初步提出适合华南稻区的以田间筛选和评价为主,以特征次生物质标记评价水稻品种(系)化感潜力的方法为辅,田间与实验室相结合,操作性强、简便有效的水稻抗草育种方法。     

Affinity chromatography as a method for sample preparation in gas chromatography/mass spectrometry.

Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometry (MS) technology, e.g., GC/MS and LC/MS and their variants, GC/tandem MS and LC/tandem MS, are best suited both for qualitative and quantitative analyses. GC/MS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GC/MS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentrations in complex matrices require further chromatographic techniques, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for sample purification. In recent years, affinity chromatography (e.g., boronate and immunoaffinity chromatography) has been developed to a superior technique for sample preparation of numerous classes of compounds in GC/MS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GC/MS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins.     

Analysis of tamoxifen and its metabolites in human plasma by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM)

A method for the analysis of tamoxifen and its metabolites in plasma from tamoxifen treated breast cancer patients, by capillary GC-MS using selected ion monitoring has been developed. Metabolite extraction was carried out on a Sep-pak C18 cartridge and metabolite purification by selective ion exchange chromatographic steps. Satisfactory recovery of radioactive standards through the extraction and purification steps was obtained. The method was shown to be accurate and precise with precision coefficient of variation values ranging from 4.3-11% for tamoxifen and its metabolites. Tamoxifen, 4-hydroxytamoxifen, metabolite Y and N-desmethyltamoxifen were identified with certainty in patient plasma on the basis of GC relative retention times and mass spectral comparison with authentic standards; because of their low abundance in plasma cis-metabolite E and 3,4-dihydroxytamoxifen could only be tentatively identified but identical GC behaviour and a satisfactory comparison of the abundance of key fragment ions was achieved. The tamoxifen and metabolite concentration ranges (ng X ml-1) in the group of patients who received 40 or 80 ng tamoxifen for 14 days were tamoxifen, 307-745; N-desmethyltamoxifen, 185-491; 4-hydroxytamoxifen, 1.4-2.5; 3,4-dihydroxytamoxifen, 0.7-2.0; metabolite Y, 19.0-112; and metabolite E1, 0.9-2.0.     

Metabolic regulatory network alterations in response to acute cold stress and ginsenoside intervention

Acute stress may trigger systemic biochemical and physiological changes in living organisms, leading to a rapid loss of homeostasis, which can be gradually reinstated by self-regulatory mechanisms and/or drug intervention strategy. However, such a sophisticated metabolic regulatory process has so far been poorly understood, especially from a holistic view. Urinary metabolite profiling of Sprague-Dawley rats exposed to cold temperature (-10 degrees C) for 2 h using GC/MS in conjunction with modern multivariate statistical techniques revealed drastic biochemical changes as evidenced by fluctuations of urinary metabolites and demonstrated the protective effect of total ginsenosides (TGs) in ginseng extracts on stressed rats. The metabonomics approach enables us to visualize significant alterations in metabolite expression patterns as a result of stress-induced metabolic responses and post-stress compensation, and drug intervention. Several major metabolic pathways including catecholamines, glucocorticoids, the tricarboxylic acid (TCA) cycle, tryptophan (nicotinate), and gut microbiota metabolites were identified to be involved in metabolic regulation and compensation required to restore homeostasis.     

Mapping of Genes for Cooking and Eating Qualities in Thai Jasmine Rice (KDML105)

Thai jasmine rice, KDML 105, is known as the best quality rice.It is known not only for its aroma but also for its good cookingand eating qualities. Amylose content (AC), gel consistency(GC) and gelatinization temperature (GT) are important traitsdetermining rice quality. A population of recombinant inbredlines (RIL) derived from KDML105 x CT9993 cross was used tostudy the genetic control of AC, GC and GT traits. A total of191 markers were used in the linkage map construction. The 1605.3cM linkage map covering nearly the whole rice genome was usedfor QTL (define QTL) analysis. Four QTLs for AC were detectedon chromosomes 3, 4, 6 and 7. These QTLs accounted for 80% ofphenotypic variation explained (PVE) in AC. The presence ofone major gene as well as several modifiers was responsiblefor the expression of the trait. Two QTLs on chromosome 6 andone on chromosome 7 were detected for GC, which accounts for57% of PVE. A single gene of major effect along with modifiergenes controls GC from this cross. The QTLs in the vicinityof waxy locus were major contributors in the expression of ACand GC. The finding that the position of QTLs for AC and GCwere near each other may reflect tight linkage or pleiotropy.Three QTLs were detected, one on chromosome 2 and two on chromosome6, which accounted for 67% of PVE in GT. Just like AC and GC,one major gene and modifier genes governed the variation inGT resulting from the KDML105 x CT9993 cross. Breeding for cookingand eating qualities will largely rely on the preferences ofthe end users.     

Identification of cannabichromene metabolites by mass spectrometry: identification of eight new dihydroxy metabolites in the rabbit

Metabolites of cannabichromene (CBC) produced by hepatic microsomal incubates from rabbits and mice were examined by gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) and (2H9)TMS derivatives. Most metabolites were hydroxylated compounds whose mass spectra gave very little information on metabolite structure as fragmentation was dominated by formation of the substituted chromenyl ion. This prevented charge localization and diagnostic fragmentation at the site of metabolic attack. This paper describes the identification of these metabolites by GC/MS techniques using both deuterium-exchange reactions and hydrogenation of the metabolites to tetrahydro derivatives; the latter method was used to suppress chromenyl ion formation and to enhance the relative abundance of diagnostic fragment ions. Twenty-one metabolites were identified. Metabolites were found hydroxylated in all positions of both aliphatic chains, with additional compounds formed by epoxidation and reduction of the aliphatic double bond in the methylpentenyl chain. Dihydroxy metabolites were hydoxylated in both the pentyl and methylpentenyl chains in positions common to those hydroxylated in the monohydroxy metabolites.     

Selected ion monitoring method for determination of nicotine, cotinine and deuterium-labeled analogs: absence of an isotope effect in the clearance of (S)-nicotine-3',3'-d2 in humans

A method for simultaneous determination of nicotine, its metabolite cotinine, and the stable isotope-labeled analogs nicotine-3',3'-d2 and cotinine-4',4'-d2 in human plasma has been developed. The method utilizes capillary column gas chromatography with detection by electron impact mass spectrometry and selected ion monitoring. Sensitivity is adequate for determination of nicotine and nicotine-d2 at concentrations as low as 1 ng ml-1, and cotinine and cotinine-d2 at concentrations as low as 10 ng ml-1 with good precision and accuracy. The method has been used to compare the elimination kinetics of (S)-nicotine-3',3'-d2 with natural nicotine in human subjects. Total clearance of nicotine-3',3'-d2 was virtually identical to the total clearance of natural nicotine, which validates the use of the deuterium-labeled analog in quantitative studies of nicotine metabolic disposition.     

Development and optimization of a metabolomic method for analysis of adherent cell cultures

In this investigation, a gas chromatography/mass spectrometry (GC/MS)-based metabolomic protocol for adherent cell cultures was developed using statistical design of experiments. Cell disruption, metabolite extraction, and the GC/MS settings were optimized aiming at a gentle, unbiased, sensitive, and high-throughput metabolomic protocol. Due to the heterogeneity of the metabolome and the inherent selectivity of all analytical techniques, development of unbiased protocols is highly complex. Changing one parameter of the protocol may change the response of many groups of metabolites. In this investigation, statistical design of experiments and multivariate analysis also allowed such interaction effects to be taken into account. The protocol was validated with respect to linear range, precision, and limit of detection in a clonal rat insulinoma cell line (INS-1 832/13). The protocol allowed high-throughput profiling of metabolites covering the major metabolic pathways. The majority of metabolites displayed a linear range from a single well in a 96-well plate up to a 10 cm culture dish. The method allowed a total of 47 analyses to be performed in 24 h.     

Metabolite profiling of maize grain: differentiation due to genetics and environment

A comparative metabolite profiling approach based on gas chromatography-mass spectrometry (GC/MS) was applied to investigate the impact of genetic background, growing location and season on the chemical composition of maize grain. The metabolite profiling protocol involved sub-fractionation of the metabolites and allowed the assessment of about 300 distinct analytes from different chemical classes (polar to lipophilic), of which 167 could be identified. A comparison, over three consecutive growing seasons, of the metabolite profiles of four maize cultivars which differed in their maturity classification, was carried out using principal component analysis (PCA). This revealed a strong separation of one cultivar in the first growing season, which could be explained by the immaturity of the kernels of this cultivar compared with others in the field trial. Further evaluations by pair-wise comparison using Student’s t-test and analysis of variance (ANOVA) showed that the growing season was the most prominent impact factor driving variation of the metabolite pool. An increased understanding of metabolic variation was achieved by analysis of a second sample set comprising one cultivar grown for 3 years at four locations. The applied GC/MS-based metabolite profiling demonstrated the natural variation in maize grain metabolite pools resulting from the interplay of environment, season, and genotype.     

水、旱条件下稻米蒸煮和营养品质性状与土壤水分环境互作分析及其QTL定位

以旱稻品种IRAT109与水稻品种越富杂交构建的DH群体的116个株系及其亲本为材料,在水、旱2种栽培条件下种植,研究了稻米蒸煮和营养品质性状的变化规律,在水、旱2个土壤水分环境下对直链淀粉含量(AC)、胶稠度(GC)、碱消值(GT)和蛋白质含量(PC)4个蒸煮和营养品质性状进行QTL定位及QTLs与环境互作分析。结果表明,以上4个品质性状在水、旱2种不同栽培条件下差异较大,说明这些性状受水分条件影响较大,旱栽条件下稻米蒸煮和营养各品质性状均有不同程度的升高,其中蛋白质含量平均提高37.9%。QTL分析结果表明,4个稻米品质性状在2个环境中的表现型值都为连续分布,均存在超亲遗传类型,共检测到7个加性效应QTL与稻米蒸煮和营养品质性状4项指标有关,分别位于第1、2、3、6、8、11染色体上,单个QTLs对性状的贡献率在1.91%～19.77%之间。位于第3染色体上控制碱消值的QGt3,第6染色体上控制直链淀粉含量的QAc6,在2个不同土壤水分条件下均与环境存在显著互作,对环境互作的贡献率分别为8.99%和47.86%。控制直链淀粉含量的2对上位性QTLs与土壤水分环境显著互作,贡献率较大,分别为32.54%和11.82%。并筛选到5个主效QTL(QGt6b、QGt8、QGt11、QGc1和QPc2)在抗旱育种中可用于蒸煮和营养各品质性状MAS改良。     

High‐throughput two‐dimensional root system phenotyping platform facilitates genetic analysis of root growth and development

High‐throughput phenotyping of root systems requires a combination of specialized techniques and adaptable plant growth, root imaging and software tools. A custom phenotyping platform was designed to capture images of whole root systems, and novel software tools were developed to process and analyse these images. The platform and its components are adaptable to a wide range root phenotyping studies using diverse growth systems (hydroponics, paper pouches, gel and soil) involving several plant species, including, but not limited to, rice, maize, sorghum, tomato and Arabidopsis. The RootReader2D software tool is free and publicly available and was designed with both user‐guided and automated features that increase flexibility and enhance efficiency when measuring root growth traits from specific roots or entire root systems during large‐scale phenotyping studies. To demonstrate the unique capabilities and high‐throughput capacity of this phenotyping platform for studying root systems, genome‐wide association studies on rice (Oryza sativa) and maize (Zea mays) root growth were performed and root traits related to aluminium (Al) tolerance were analysed on the parents of the maize nested association mapping (NAM) population.     

不同波长蓝光LED对两个品种水稻秧苗生长的影响

传统的大田育秧方式容易受到自然条件的影响,如在连续的阴雨天容易导致烂秧,而采用人工可控的工厂化育秧则有助于解决此类问题。LED光源具有光质纯、光效高以及低能耗等优势,是一种有望在工厂化育秧中应用的人工光源。该研究以生长特性差异明显的水稻天优998(籼稻)和圣稻14(粳稻)为材料,在光强为(60±5)μmol·m~-2·s~-1、光周期为12 h·d~-1的条件下,以450、470和490 nm 3个波长的蓝光LED照射天优998和圣稻14秧苗,考察了不同波长蓝光对两个品种的三叶期和五叶期水稻秧苗生长的影响。结果表明:蓝光处理下两个品种、不同秧龄幼苗的壮苗指数、根系活力和根数增加,体内碳、氮代谢活性增强,抗氧化物酶活性也会增加。3个波长的蓝光对两个品种、不同秧龄水稻秧苗生长的影响存在差异,450 nm照射下水稻秧苗的各项生长指标较优,生产上育秧可根据秧龄选择450 nm或490 nm作为独立和组合光源。     

Cluster analysis as selection and dereplication tool for the identification of new natural compounds from large sample sets

Cluster analysis of gas-chromatographic (GC) data of ca. 500 bacterial isolates was used as an aid in detection and identification of new natural compounds. This approach reduces the number of GC/MS analysis (dereplication) and concomitantly improves the selection of samples with high probability to contain unknown natural products. Lipophilic bacterial extracts were derivatized and analyzed by GC under standardized conditions. A program was developed to convert chromatographic data into a two-dimensional matrix. Based on the results of hierarchical cluster analysis samples were selected for further investigation by GC/MS and NMR. This approach avoided unnecessary analysis of similar samples. By this method, the unusual oligoprenylsesquiterpenes 1 and 2 as well as new aromatic amides 7 and 8 were identified.     

Biotransformation of (-)-(R)-α-Phellandrene: Antimicrobial Activity of Its Major Metabolite

Terpene derivatives converted by microbial biotransformation constitute an important resource for natural pharmaceutical, fragrance, and aroma substances. In the present study, the monoterpene α-phellandrene was biotransformed by 16 different strains of microorganisms (bacteria, fungi, and yeasts). The transformation metabolites were initially screened by TLC and GC/MS, and then further characterized by NMR spectroscopic techniques. Among the six metabolites characterized, 6-hydroxypiperitone, α-phellandrene epoxide, cis-p-menth-2-en-1-ol, and carvotanacetone, which originated from (-)-(R)-α-phellandrene, are reported for the first time in this study. Additionally, the substrate and the metabolite 5-p-menthene-1,2-diol were subjected to in vitro antibacterial and anticandidal tests. The metabolite showed moderate-to-good inhibitory activities (MICs=0.125 to >4?mg/ml) against various bacteria and especially against Candida species in comparison with its substrate (-)-(R)-α-phellandrene and standard antimicrobial agents.