De novo transcriptome assembly and identification of the gene conferring a "pandan-like" aroma in coconut (Cocos nucifera L.).

Thailand's aromatic coconut (Cocos nucifera L.) is a special type of green dwarf coconut, the liquid endosperm of which is characterized by a pleasant "pandan-like" aroma due to the presence of 2-acetyl-1-pyrroline (2AP). The aim of this study was to perform a de novo assembly of transriptome from C. nucifera endosperm and to identify the gene responsible for 2AP biosynthesis. CnAMADH2 was identified as an ortholog of the rice aromatic gene and a G-to-C substitution found in exon 14 was associated with 2AP content in the aromatic green dwarf coconut accessions. The base substitution caused an amino-acid change, alanine-to-proline, at position 442 (P442A). The presence of P at this position might alter the steric conformation at the loop region and subsequently result in an unstabilized dimer conformation that could lower AMADH enzyme activity. Among AMADH/BADH protein sequences in different plant species, the P442A mutation was found exclusively in aromatic coconut. The PCR marker developed based on this sequence variation can perfectly detect the aromatic and non-aromatic alleles of the gene. This study confirms the hypothesis that plants may share a mechanism of 2AP biosynthesis. This is the first identification of the gene associated with 2AP biosynthesis in a tree plant.

Gene discovery and functional marker development for fragrance in sorghum (Sorghum bicolor (L.) Moench).

KEY MESSAGE: Sequence analysis and genetic mapping revealed that a 1,444 bp deletion causes a premature stop codon in SbBADH2 of sorghum IS19912. The non-function of SbBADH2 is responsible for fragrance in sorghum IS19912. 2-acetyl-1-pyrroline (2AP) is a potent volatile compound causing fragrance in several plants and foods. Seeds of some varieties of rice, sorghum and soybean possess fragrance. The genes responsible for fragrance in rice and soybean are orthologs that correspond to betaine aldehyde dehydrogenase 2 (BADH2). Genotypes harboring fragrance in rice and soybean contain a premature stop codon in BADH2 which impairs the synthesis of full length functional BADH2 protein leading to the accumulation of 2AP. In this study, we reported an association between the BADH2 gene and fragrance in sorghum. An F2 population of 187 plants developed from a cross between KU630 (non-fragrant) and IS19912 (fragrant) was used. Leaves of F2 and F3 progenies were evaluated for fragrance by organoleptic test, while seeds of F2 plants were analyzed for 2AP. The tests consistently showed that the fragrance is controlled by a single recessive gene. Gene expression analysis of SbBADH1 and SbBADH2 in leaves of KU630 and IS19912 at various stages revealed that SbBADH1 and SbBADH2 were expressed in both accessions. Sequence comparison between KU630 and IS19912 revealed a continuous 1,444 bp deletion encompassing exon 12 to 15 of SbBADH2 in IS19912 which introduces a frameshift mutation and thus causes a premature stop codon. An indel marker was developed to detect polymorphism in SbBADH2. Bulk segregant and QTL analyses confirmed the association between SbBADH2 and fragrance.

Quantification of flavonoids in black rice by liquid chromatography-negative electrospray ionization tandem mass spectrometry.

Systematic identification and structural characterization of flavonoids and their glycosides in bran extracts of seven Thai black rice varieties were performed by sequential uses of reversed-phase HPLC with a photodiode array detector and a combined electrospray ionization tandem mass spectrometer. Eleven flavonoids were detected, and six of these were found for the first time in rice bran. These were taxifolin-7-O-glucoside, myricetin-7-O-glucoside, isorhamnetin-3-O-acetylglucoside, isorhamnetin-7-O-rutinoside, 5,6,3',4',5'-pentahydroxyflavone-7-O-glucoside, and 5,3',4',5'-tetrahydroxyflavanone-7-O-glucoside. The quantitative results revealed that different rice varieties possessed flavonoids in different concentrations. The most abundant glycoside derivative of flavonoids widely distributed among the rice varieties was monoglucoside, such as quercetin-3-O-glucoside, isorhamnetin-3-O-glucoside, and isorhamnetin-3-O-glucoside.

Assessment of genotoxicity and antigenotoxicity of an aqueous extract of Cleistocalyx nervosum var. paniala in in vitro and in vivo models.

Cleistocalyx nervosum var. paniala, an edible fruit found in Northern Thailand, contains high amounts of phenolic compounds with in vitro antioxidant activity. The aqueous extract of the ripe fruit was evaluated for its safety and beneficial effects using genotoxicity and toxicity tests. The C. nervosum extract was not only non-mutagenic in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of metabolic activation, but exhibited also moderate antimutagenic effects against aflatoxin B1 and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline-induced mutagenesis. Electrospray ionization-mass spectrometric analysis revealed the major anthocyanins, which included cyanidin-3,5-diglucoside, cyanidin-3-glucoside and cyanidin-5-glucoside. The administration of C. nervosum at concentration of 5,000 mg/kg bw did not induce acute toxicity in rats. A liver micronucleus test was performed to detect clastogenicity and anticlastogenicity. The extract in the dose of 1,000 mg/kg did not cause micronucleus formation in the liver of rats. Furthermore, in rats administered 100-1,000 mg/kg of the extract, no anticlastogenic effect against diethylnitrosamine-induced hepatic micronucleus formation was observed. These studies provide data concerning the safety and antimutagenic potency of an aqueous extract of C. nervosum fruit.

Adaptogenic-active components from Kaempferia parviflora rhizomes.

Kaempferia parviflora rhizome extracts obtained by maceration with hexane, chloroform, methanol, and ethanol were screened for their adaptogenic activities using swimming tests of mice. The effective adaptogenic extract dose was 500mg/kg of body weight and was given orally once a day. Crude hexane extract showed significantly shorter mouse immobilisation time than those of the other and control extracts. This crude hexane extract was separated into three fractions by column chromatography. Among these fractions, the fraction rich in terpenoids possessed the highest adaptogenic activity and was comparable to that of the crude ginseng root powder used as a reference control. Therefore, terpenes contained in this fraction could be attributed to the decrease in exhaustion during the swimming of mice. There was no effect on body weight, heart, liver, kidneys, and adrenal glands of the mice. Chemical characterisation of this adaptogenic-active fraction by NMR and GC-MS showed germacene D, ß-elemene, α-copaene, and E-caryophyllene as major constituents. Accordingly, these terpenes are considered the adaptogenic agents of K. parviflora rhizomes.

Investigation of plant hormone level changes in shoot tips of longan (Dimocarpus longan Lour.) treated with potassium chlorate by liquid chromatography-electrospray ionization mass spectrometry.

The endogenous levels of indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA) and cytokinins (CKs) and their changes were investigated in shoot tips of ten longan (Dimocarpus longan Lour.) trees for off-season flowering until 60 days after potassium chlorate treatment in comparison with those of ten control (untreated) longan trees. These analytes were extracted and interfering matrices removed with a single mixed-mode solid phase extraction under optimum conditions. The recoveries at three levels of concentration were in the range of 72-112%. The endogenous plant hormones were separated and quantified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Detection limits based on the signal-to-noise ratio ranged from 10 ng mL(-1) for gibberellin A4 (GA4) to 200 ng mL(-1) for IAA. Within the first week after potassium chlorate treatment, dry weight (DW) amounts in the treated longan shoot tips of four gibberellins, namely: gibberellin A1(GA1), gibberellic acid (GA3), gibberellin A19 (GA19) and gibberellin A20 (GA20), were found to increase to approximately 25, 50, 20 and 60 ng g(-1) respectively, all of which were significantly higher than those of the controls. In contrast, gibberellin A8 (GA8) obtained from the treated longan was found to decrease to approximately 20 ng g(-1)DW while that of the control increased to around 80 ng g(-1)DW. Certain CKs which play a role in leaf bud induction, particularly isopentenyl adenine (iP), isopentenyl adenosine (iPR) and dihydrozeatin riboside (DHZR), were found to be present in amounts of approximately 20, 50 and 60 ng g(-1)DW in the shoot tips of the control longan. The analytical results obtained from the two-month off-season longan flowering period indicate that high GA1, GA3, GA19 and GA20 levels in the longan shoot tips contribute to flower bud induction while high levels of CKs, IAA and ABA in the control longan contribute more to the vegetative development.

Deficiency in the amino aldehyde dehydrogenase encoded by GmAMADH2, the homologue of rice Os2AP, enhances 2-acetyl-1-pyrroline biosynthesis in soybeans (Glycine max L.).

2-Acetyl-1-pyrroline (2AP), the volatile compound that provides the 'popcorn-like' aroma in a large variety of cereal and food products, is widely found in nature. Deficiency in amino aldehyde dehydrogenase (AMADH) was previously shown to be the likely cause of 2AP biosynthesis in rice (Oryza sativa L.). In this study, the validity of this mechanism was investigated in soybeans (Glycine max L.). An assay of AMADH activity in soybeans revealed that the aromatic soybean, which contains 2AP, also lacked AMADH enzyme activity. Two genes, GmAMADH1 and GmAMADH2, which are homologous to the rice Os2AP gene that encodes AMADH, were characterized. The transcription level of GmAMADH2 was lower in aromatic varieties than in nonaromatic varieties, whereas the expression of GmAMADH1 did not differ. A double nucleotide (TT) deletion was found in exon 10 of GmAMADH2 in all aromatic varieties. This variation caused a frame-shift mutation and a premature stop codon. Suppression of GmAMADH2 by introduction of a GmAMADH2-RNAi construct into the calli of the two nonaromatic wild-type varieties inhibited the synthesis of AMADH and induced the biosynthesis of 2AP. These results suggest that deficiency in the GmAMADH2 product, AMADH, plays a similar role in soybean as in rice, which is to promote 2AP biosynthesis. This phenomenon might be a conserved mechanism among plant species.

A SNP in GmBADH2 gene associates with fragrance in vegetable soybean variety "Kaori" and SNAP marker development for the fragrance.

Fragrance in soybean is due to the presence of 2-acetyl-1-pyrroline (2AP). BADH2 gene coding for betaine aldehyde dehydrogenase has been identified as the candidate gene responsible for fragrance in rice (Oryza sativa L.). In this study, using the RIL population derived from fragrant soybean cultivar "Kaori" and non-fragrant soybean cultivar "Chiang Mai 60" (CM60), STS markers designed from BADH2 homolog were found associating with 2AP production. Genetic mapping demonstrated that QTL position of fragrance and 2AP production coincides with the position of GmBADH2 (Glycine max betaine aldehyde dehydrogenase 2). Sequence comparison of GmBADH2 between Kaori and non-fragrant soybeans revealed non-synonymous single-nucleotide polymorphism (SNP) in exon 10. Nucleotide substitution of G to A in the exon results in an amino acid change of glycine (GGC; G) to aspartic acid (GAC; D) in Kaori. The amino acid substitution changes the conserved EGCRLGPIVS motif of GmBADH2, which is essential for functional activity of GmBADH2 protein, to EGCRLDPIVS motif, suggesting that the SNP in GmBADH2 is responsible for the fragrance in Kaori. Five single nucleotide-amplified polymorphism (SNAP) markers which are PCR-based allele specific SNP markers were developed for fragrance based on the SNP in GmBADH2. Two markers specific to A allele produced a band in only Kaori, while three markers specific to G alleles produced a band in only CM60. The simple PCR-based allele specific SNAP markers developed in the present study are useful in marker-assisted breeding of fragrant soybean.

Comprehensive two-dimensional gas chromatography-mass spectrometry analysis of volatile constituents in Thai vetiver root oils obtained by using different extraction methods.

INTRODUCTION: Vetiver root oil is known as one of the finest fixatives used in perfumery. This highly complex oil contains more than 200 components, which are mainly sesquiterpene hydrocarbons and their oxygenated derivatives. Since conventional GC-MS has limitation in terms of separation efficiency, the comprehensive two-dimensional GC-MS (GC x GC-MS) was proposed in this study as an alternative technique for the analysis of vetiver oil constituents. OBJECTIVE: To evaluate efficiency of the hyphenated GC x GC-MS technique in terms of separation power and sensitivity prior to identification and quantitation of the volatile constituents in a variety of vetiver root oil samples. METHODOLOGY. Dried roots of Vetiveria zizanioides were subjected to extraction using various conditions of four different methods; simultaneous steam distillation, supercritical fluid, microwave-assisted, and Soxhlet extraction. Volatile components in all vetiver root oil samples were separated and identified by GC-MS and GC x GC-MS. The relative contents of volatile constituents in each vetiver oil sample were calculated using the peak volume normalization method. RESULTS: Different techniques of extraction had diverse effects on yield, physical and chemical properties of the vetiver root oils obtained. Overall, 64 volatile constituents were identified by GC-MS. Among the 245 well-resolved individual components obtained by GC x GC-MS, the additional identification of 43 more volatiles was achieved. CONCLUSION: In comparison with GC-MS, GC x GC-MS showed greater ability to differentiate the quality of essential oils obtained from diverse extraction conditions in terms of their volatile compositions and contents.

Rapid method for quantitative analysis of the aroma impact compound, 2-acetyl-1-pyrroline, in fragrant rice using automated headspace gas chromatography.

A rapid method employing static headspace gas chromatography (HS-GC) has been developed and validated for quantitative analysis of the impact aroma compound, 2-acetyl-1-pyrroline (2AP), in grains of fragrant rice. This developed method excludes wet extraction, and the rice headspace volatiles are brought directly and automatically to GC analysis. The conditions of the static HS autosampler were optimized to achieve high recovery and sensitivity. The most effective amount of rice sample used was 1 g, which provided 51% recovery and a linear multiple headspace extraction (MHE) plot of the peak area of 2AP. The sensitivity of the method was enhanced by utilizing a megabore fused silica capillary column in conjunction with a nitrogen-phosphorus detector (NPD). Method validations performed for both static HS-GC-FID and HS-GC-NPD demonstrated linear calibration ranges of 20-10 000 (r(2) = 0.9997) and 5-8000 (r(2) = 0.9998) ng of 2AP/g of rice sample, respectively. The limits of detection for both systems were 20 and 5 ng of 2AP, and the limits of quantitation were 0.30 and 0.01 g of brown rice sample, respectively. Reproducibility calculated as intraday and interday coefficients of variation were 3.25% RSD (n = 15) and 3.92% RSD (n = 35), respectively, for SHS-GC-FID and 1.87% RSD (n = 15) and 2.85% RSD (n = 35), respectively, for SHS-GC-NPD. The method was found to be effective when applied to the evaluation of aroma quality, based on 2AP concentrations, of some fragrant rice samples.

Highly volatile constituents of Vetiveria zizanioides roots grown under different cultivation conditions.

Roots of Vetiveria zizanioides Nash (Mae Hae; Thai ecotype) planted in three different cultivation systems (normal soil, normal soil with added microbes and semi- hydroponically) were extracted using a simultaneous steam distillation and solvent extraction (SDE) apparatus. Yields of the essential oils obtained were 0.27, 0.18 and 0.06%, respectively. The separation profiles obtained by comprehensive two-dimensional gas chromatography (GCxGC) and solid phase microextraction (SPME)-GCxGC analyses of the crude essential oils showed a total of 156 and 48 well-resolved components, respectively. The highly volatile fractions isolated from the three essential oils by SPME were subjected to analysis by GC-MS and 42 compounds were identified in total. Volatile component profiles of the oils obtained by normal soil and semi-hydroponic cultivation were similar, whereas a quantitative difference was noted in some major volatiles when the cultivation system containing microbes was utilized.

Identification and quantitation of the rice aroma compound, 2-acetyl-1-pyrroline, in bread flowers (Vallaris glabra Ktze).

The aroma impact compound, 2-acetyl-1-pyrroline (2AP), has been identified for the first time in headspace of fresh bread flowers (Vallaris glabra Ktze) in which volatile components were extracted by solid-phase microextraction (SPME) at room temperature prior to analysis by gas chromatography-mass spectrometry. A total of 50 volatiles were detected. Among these, 23 volatiles were identified, predominantly in a group of terpenes. More volatiles were found in the extract of fresh bread flowers obtained by continuous steam distillation and solvent extraction (SDS). Of the 40 volatiles identified, the additional components were mainly straight-chain saturated hydrocarbons. 2AP was found in the extracts obtained by both SPME (0.37%) and SDS (2.71% relative proportion). Quantitative analyses of 2AP in bread flowers and other plant materials were performed by solvent extraction employing acidic solutions and capillary GC with flame ionization detection. The highest concentration of 2AP was found in dried flowers of V. glabra at 26.1 mg/kg. By comparison with other plant sources, fresh leaves of Pandanus amaryllifolius Roxb contain 2AP at 10.3 mg/kg and Thai fragrant rice, Khao Dawk Mali 105, at 3.0 mg/kg.

Enhanced sensitivity and selectivity in the detection of polycyclic aromatic hydrocarbons using sequential simplex optimization, the addition of an organic modifier and wavelength programming.

Optimized separation of a mixture of 16 priority pollutant polycyclic aromatic hydrocarbons (PAHs) by high performance liquid chromatography (HPLC) using the sequential simplex optimization method was accomplished by varying the starting and ending compositions of acetonitrile and water, linear gradient time, mobile phase flow rate, column temperature and holding time of the final mobile phase composition. Focusing on the two sets of difficult-to-separate pairs (acenaphthene-fluorene and benzo[g,h,i]perylene-indeno[1,2,3-c,d]pyrene), analysis time was reduced by about ten percent through the use of an organic modifier (isopropanol or methanol), under both optimum and near-optimum conditions, while maintaining good separation of the remaining PAHs. High sensitivity for all of the 16 PAHs was achieved by wavelength programming during elution using five wavelengths (224, 235, 254 270 and 296 nm), depending upon the molar absorptivities of the individual compounds. Detection limits (DLs) ranging from 0.002 (benzo[a]pyrene) to 0.140 mug ml(-1) (acenaphthene) were achieved for this set of 16 standard compounds.