Anthocyanin biosynthetic genes in Brassica rapa

Background

Anthocyanins are a group of flavonoid compounds. As a group of important secondary metabolites, they perform several key biological functions in plants. Anthocyanins also play beneficial health roles as potentially protective factors against cancer and heart disease. To elucidate the anthocyanin biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses between Arabidopsis thaliana and B. rapa on a genome-wide level.

Results

In total, we identified 73 genes in B. rapa as orthologs of 41 anthocyanin biosynthetic genes in A. thaliana. In B. rapa, the anthocyanin biosynthetic genes (ABGs) have expanded and most genes exist in more than one copy. The anthocyanin biosynthetic structural genes have expanded through whole genome and tandem duplication in B. rapa. More structural genes located upstream of the anthocyanin biosynthetic pathway have been retained than downstream. More negative regulatory genes are retained in the anthocyanin biosynthesis regulatory system of B. rapa.

Conclusions

These results will promote an understanding of the genetic mechanism of anthocyanin biosynthesis, as well as help the improvement of the nutritional quality of B. rapa through the breeding of high anthocyanin content varieties.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-426) contains supplementary material, which is available to authorized users.     

Chemical Variability and Biological Activities of Brassica rapa var. rapifera Parts Essential Oils Depending on Geographic Variation and Extraction Technique

In the present work, the Brassica rapa var. rapifera parts essential oils and their antioxidant and antimicrobial activities were investigated for the first time depending on geographic origin and extraction technique. Gas‐chromatography (GC) and GC/mass spectrometry (MS) analyses showed several constituents, including alcohols, aldehydes, esters, ketones, norisoprenoids, terpenic, nitrogen and sulphur compounds, totalizing 38 and 41 compounds in leaves and root essential oils, respectively. Nitrogen compounds were the main volatiles in leaves essential oils and sulphur compounds were the main volatiles in root essential oils. Qualitative and quantitative differences were found among B. rapa var. rapifera parts essential oils collected from different locations and extracted by hydrodistillation and microwave‐assisted hydrodistillation techniques. Furthermore, our findings showed a high variability for both antioxidant and antimicrobial activities. The highlighted variability reflects the high impact of plant part, geographic variation and extraction technique on chemical composition and biological activities, which led to conclude that we should select essential oils to be investigated carefully depending on these factors, in order to isolate the bioactive components or to have the best quality of essential oil in terms of biological activities and preventive effects in food.     

Variation of glucosinolates in vegetable crops of Brassica rapa

Glucosinolate levels in leaves were determined in a collection of 113 varieties of turnip greens (Brassica rapa L.) from northwestern Spain grown at two sites. Sensorial attributes were also assessed by a consumer panel. The objectives were to determine the diversity among varieties in total glucosinolate content and glucosinolate profile and to evaluate their sensory attributes in relation to glucosinolate content for breeding purposes. Sixteen glucosinolates were identified, being the aliphatic glucosinolates, gluconapin and glucobrassicanapin the most abundant. Other aliphatic glucosinolates, such as progoitrin, glucoalyssin, and gluconapoleiferin were relatively abundant in varieties with a different glucosinolate profile. Indolic and aromatic glucosinolate concentrations were low and showed few differences among varieties. Differences in total glucosinolate content, glucosinolate profile and bitterness were found among varieties, with a total glucosinolate content ranging from 11.8 to 74.0micromolg(-1) dw at one site and from 7.5 to 56.9micromolg(-1) dw at the other site. Sensory analysis comparing bitterness with variation in glucosinolate, gluconapin and glucobrassicanapin concentrations suggested that these compounds and their breakdown products are not the only determinants of the characteristic flavour of this vegetable. Other phytochemicals are probably involved on the characteristic bitter flavour. The varieties MBG-BRS0132, MBG-BRS0082, MBG-BRS0173, and MBG-BRS0184 could be good candidates for future breeding programs since they had high total glucosinolate content and good agronomic performance. The presence of glucoraphanin in some varieties should be studied more extensively, because this aliphatic glucosinolate is the precursor of sulforaphane, a potent anti-cancer isothiocyanate.     

Quantitative trait loci for glucosinolate accumulation in Brassica rapa leaves

Glucosinolates and their breakdown products have been recognized for their effects on plant defense, human health, flavor and taste of cruciferous vegetables. Despite this importance, little is known about the regulation of the biosynthesis and degradation in Brassica rapa. Here, the identification of quantitative trait loci (QTL) for glucosinolate accumulation in B. rapa leaves in two novel segregating double haploid (DH) populations is reported: DH38, derived from a cross between yellow sarson R500 and pak choi variety HK Naibaicai; and DH30, from a cross between yellow sarson R500 and Kairyou Hakata, a Japanese vegetable turnip variety. An integrated map of 1068 cM with 10 linkage groups, assigned to the international agreed nomenclature, is developed based on the two individual DH maps with the common parent using amplified fragment length polymorphism (AFLP) and single sequence repeat (SSR) markers. Eight different glucosinolate compounds were detected in parents and F(1)s of the DH populations and found to segregate quantitatively in the DH populations. QTL analysis identified 16 loci controlling aliphatic glucosinolate accumulation, three loci controlling total indolic glucosinolate concentration and three loci regulating aromatic glucosinolate concentrations. Both comparative genomic analyses based on Arabidopsis-Brassica rapa synteny and mapping of candidate orthologous genes in B. rapa allowed the selection of genes involved in the glucosinolate biosynthesis pathway that may account for the identified QTL.     

Influence of enhanced UV-B radiation on biomass allocation and pigment concentrations in leaves and reproductive structures of greenhouse-grown Brassica rapa

We assessed the effects of enhanced ultraviolet-B radiation (UV-B; 280–320 nm) on biomass allocation to roots, shoots, leaves and flowers in the annual Brassica rapa. In addition, we investigated how concentrations of chlorophyll and UV-B-absorbing compounds in leaves, ovaries and pollen changed in response to enhanced UV-B. Plants were grown for 38 d in a greenhouse under lampbanks providing daily biologically effective UV-B doses equivalent to those under ambient mid-March stratospheric ozone levels or 16% (low-enhanced UV-B) or 32% (high-enhanced UV-B) ozone depletion levels for Morgantown, WV, USA. Total and aboveground biomass of plants was less under low-enhanced UV-B, but similar to ambient controls under high-enhanced UV-B. Concentrations of UV-B-absorbing compounds in leaves (area basis) increased under high-enhanced UV-B by about 20%, but were similar to ambient controls under low-enhanced UV-B. More effective protection due to higher screening-compound concentrations in plants under high-enhanced UV-B may explain why biomass production was not reduced. Plants under high-enhanced UV-B also had more reproductive biomass and produced more flowers, and had less root mass, than plants under ambient or low-enhanced UV-B. Concentrations of leaf total chlorophyll were not affected by UV-B treatment. While UV-B treatment had no affect on concentrations of UV-B-absorbing compounds in ovaries, concentrations in pollen from plants under both enhanced-UV-B treatments were >40% greater than ambient controls.     

扫帚菜-白菜轮作对白菜镉吸收的影响

通过盆栽和大田试验研究扫帚菜Kochia scoparia对土壤中镉(Cd)的富集效率,并利用盆栽试验将其与4个白菜品种进行轮作试验,验证扫帚菜对土壤Cd污染的修复效果。结果表明,扫帚菜各部位富集能力表现为叶>根>茎,富集系数分别为15.07、5.44和2.96;种植扫帚菜后土壤总Cd降低6.02%-13.60%;土壤脲酶和酸性磷酸酶活性也有所提高。盆栽轮作结果表明,扫帚菜-白菜轮作系统中白菜地上部Cd含量与未轮作的对照组相比平均降低17.21%,生物量有略微增加,地上部对Cd的转运系数无明显变化。结果显示,通过扫帚菜与白菜轮作不仅可以增加白菜产量,而且可以有效降低白菜可食部Cd含量,实现边生产边治理的绿色农业理念。     

Variation of glucosinolates on position orders of flower buds in turnip rape (Brassica rapa)

To glucosinolate (GSL) contents on flower buds depending on their position orders in turnip rape (Brassica rapa), three Japanese ‘Nabana’ cultivars such as cv. No. 21 (Brassica rapa, early type), cv. Husanohana (B. rapa, late type) and cv. Norin No. 20 (B. napus) were investigated using HPLC analysis. Ten GSLs including glucoraphanin, sinigrin, glucoalyssin, napoleiferin, gluconapin, 4-hydroxyglucobrassicin, glucobrassicanapin, glucobrassicin, and gluconasturtiin were detected. Differences in individual and total GSL contents were found between two plant varieties, which are also depending on various developmental stages. Among the GSLs, gluconapin (mean 23.11 μmole/g dry weight (DW) and glucobrassicanapin (mean 13.41 μmole/g DW) documented the most abundant compounds and contributed average 39 and 27% of the total GSLs, but indolyl and aromatic GSLs together accounted >10% of the total GSLs. The presence of significant quantities of gluconapin in the cultivars should be studied more extensively, since the GSL is mainly responsible for the bitter taste.     

Isolation and characterization of microsatellites in Brassica rapa L

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.     

Alternative management tactics for control of Phyllotreta cruciferae and Phyllotreta striolata (Coleoptera: Chrysomelidae) on Brassica rapa in Massachusetts

The flea beetles Phyllotreta cruciferae (Goeze) and Phyllotreta striolata (F.) (Coleoptera: Chrysomelidae: Alticinae) are significant pests of crops in the Brassicaceae family. From 2001 to 2003, the efficacy of both new and commonly used treatments for the control of flea beetles in brassicas, Brassica rapa L., were evaluated in three small plot, randomized complete block design trials. Row cover and carbaryl (applied as a weekly foliar spray) were found to be the most consistent at reducing damage in comparison with untreated controls in all trials. Two new products that may provide adequate flea beetle control are spinosad (in either conventional or organic formulations) and thiamethoxam. The plant-derived compounds azidiractin and pyrethrin did not protect treated plants from flea beetle feeding. Treatment of plants with kaolin, or removal of the beetles with a vacuum, also did not reduce the level of crop damage. The level of damage at harvest was found to be correlated with population size of flea beetles in each plot, as measured by captures on yellow sticky traps and direct visual counts. Removal of the outer two leaves of individual B. rapa plants reduced the total number of holes per plant by 40%, while only removing 15% of the leaf area.     

Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens

Summary Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1–9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus.     

白菜型油菜自交亲和性变异分析

对不同来源的85份白菜型油菜品种的自交亲和指数研究表明,白菜型油菜的自交亲和性存在较大幅度的变异,自交亲和指数在0．00～9．28之间,这种变异不仅存在于品种间,而且存在于品种内不同个体间。85份参试材料中,自交亲和指数小于1的有50个品种;35个品种自交亲和指数大于1,自交亲和指数在1．00～9．28之间,其中亲和指数大于4的材料有28、44、45、55x、75、119、123、139号品种等。依据自交亲和指数,可将参试材料分为4种类型:高自交亲和类型(自交亲和指数>4．00);自交亲和类型(自交亲和指数1．00～3．99);自交不亲和类型(0．00< 自交亲和指数<1．00);高自交不亲和类型(自交亲和指数=0．00)。自交亲和性因地区而异,一般西部地区的品种自交亲和性高于其它地区的品种。     

Metal ion-inducing metabolite accumulation in Brassica rapa

Plants face a number of biotic and abiotic environmental stress factors during growth. Among the abiotic factors, in particular, a great deal of attention has been paid to metals not only because of their increasing amounts in the environment due to rapid industrial development but also because of the variation of metal composition in soil. Cultivation of crops close to industrial areas or irrigation with contaminated water may result in both growth inhibition and tissue accumulation of metals. Brassica species are well known as metal accumulators and are being used for phytoremediation of contaminated soils. However, the metal tolerance mechanism in the plant still remains unclear. In order to investigate the metabolomic changes induced by metal ions in Brassica, plants were subjected to concentrations 50, 100, 250 and 500mmol of copper (Cu), iron (Fe) and manganese (Mn) in separate treatments. (1)H NMR and two-dimensional NMR spectra coupled with principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were applied to investigate the metabolic change in Brassica rapa (var. Raapstelen). The (1)H-NMR analysis followed by the application of chemometric methods revealed a number of metabolic consequences. Among the metabolites that showed variation, glucosinolates and hydroxycinnamic acids conjugated with malates were found to be the discriminating metabolites as were primary metabolites like carbohydrates and amino acids. This study shows that the effects of Cu and Fe on plant metabolism were larger than those of Mn and that the metabolomic changes varied not only according to the type of metal but also according to its concentration.     

Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins

The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel β-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa     

Gravity control of growth form in Brassica rapa and Arabidopsis thaliana (Brassicaceae): Consequences for secondary metabolism

How gravity influences the growth form and flavor components of plants is of interest to the space program because plants could be used for food and life support during prolonged missions away from the planet, where that constant feature of Earth's environment does not prevail. We used plant growth hardware from prior experiments on the space shuttle to grow Brassica rapa and Arabidopsis thaliana plants during 16-d or 11-d hypergravity treatments on large-diameter centrifuge rotors. Both species showed radical changes in growth form, becoming more prostrate with increasing g-loads (2-g and 4-g). In Brassica, height decreased and stems thickened in a linear relationship with increasing g-load. Glucosinolates, secondary compounds that contribute flavor to Brassica, decreased by 140% over the range of micro to 4-g, while the structural secondary compound, lignin, remained constant at ～15% (w/w) cell wall dry mass. Stem thickening at 4-g was associated with substantial increases in cell size (47%, 226%, and 33% for pith, cortex, and vascular tissue), rather than any change in cell number. The results, which demonstrate the profound effect of gravity on plant growth form and secondary metabolism, are discussed in the context of similar thigmostresses such as touch and wind.     

Metabolomic analysis of methyl jasmonate treated Brassica rapa leaves by 2-dimensional NMR spectroscopy

The metabolomic analysis of Brassica rapa leaves treated with methyl jasmonate was performed using 2-dimensional J-resolved NMR spectroscopy combined with multivariate data analysis. The principal component analysis of the J-resolved NMR spectra showed discrimination between control and methyl jasmonate treated plants by principal components 1 and 2. While the level of glucose, sucrose and amino acids showed a decrease after methyl jasmonate treatment, hydroxycinnamates and glucosinolate were highly increased. Methyl jasmonate treatment resulted in a long-term accumulation of indole glucosinolate and indole-3-acetic acid, lasting up to 14 days after treatment. Malate conjugated hydroxycinnamates also exhibited an increase until 14 days after methyl jasmonate treatment, these compounds might play an important role in plant defence responses mediated by methyl jasmonate.     

Aphid responses to volatile cues from turnip plants (Brassica rapa) infested with phloem-feeding and chewing herbivores

Herbivore-induced plant volatiles provide foraging cues for herbivores and for herbivores’ natural enemies. Aphids induce plant volatile emissions and also utilize plant-derived olfactory volatile cues, but the chemical ecology of aphids and other phloem-feeding insects is less extensively documented than that of chewing insects. Here, we characterize the volatile cues emitted by turnip plants (Brassica rapa) under attack by an aphid (Myzus persicae) or by the chewing lepidopteran larva Heliothis virescens. We also tested the behavioral responses of M. persicae individuals to the odors of undamaged and herbivore-damaged plants presented singly or in combination, as well as to the odor of crushed conspecifics (simulating predation). Gas chromatographic analysis of the volatile blend of infested turnips revealed distinct profiles for both aphid- and caterpillar-induced plants, with induced compounds including green-leaf alcohols, esters, and isothiocyanates. In behavioral trials, aphids exhibited increased activity in the presence of plant odors and positive attraction to undamaged turnip plants. However, aphids exhibited a strong preference for the odors of healthy versus plants subjected to herbivore damage, and neither aphid- or caterpillar-damaged plants were attractive compared to clean-air controls. Reduced aphid attraction to herbivore-infested plants may be mediated by changes in the volatile blend constituent composition, including large amounts of isothiocyanates and green-leaf volatiles or, in the case of aphid-infested plants, of the aphid alarm pheromone, (E)-β-farnesene.     

Identification and characterization of LIM gene family in Brassica rapa

Background

LIM (Lin-11, Isl-1 and Mec-3 domains) genes have been reported to trigger the formation of actin bundles, a major higher-order cytoskeletal assembly, in higher plants; however, the stress resistance related functions of these genes are still not well known. In this study, we collected 22 LIM genes designated as Brassica rapa LIM (BrLIM) from the Brassica database, analyzed the sequences, compared them with LIM genes of other plants and analyzed their expression after applying biotic and abiotic stresses in Chinese cabbage.

Results

Upon sequence analysis these genes were confirmed as LIM genes and found to have a high degree of homology with LIM genes of other species. These genes showed distinct clusters when compared to other recognized LIM proteins upon phylogenetic analysis. Additionally, organ specific expression of these genes was observed in Chinese cabbage plants, with BrPLIM2a, b, c, BrDAR1, BrPLIM2e, f and g only being expressed in flower buds. Furthermore, the expression of these genes (except for BrDAR1 and BrPLIM2e) was high in the early flowering stages. The remaining genes were expressed in almost all organs tested. All BrDAR genes showed higher expression in flower buds compared to other organs. These organ specific expressions were clearly correlated with the phylogenetic grouping. In addition, BrWLIM2c and BrDAR4 responded to Fusarium oxysporum f. sp. conglutinans infection, while commonly two BrDARs and eight BrLIMs responded to cold, ABA and pH (pH5, pH7 and pH9) stress treatments in Chinese cabbage plants.

Conclusion

Taken together, the results of this study indicate that BrLIM and BrDAR genes may be involved in resistance against biotic and abiotic stresses in Brassica.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-641) contains supplementary material, which is available to authorized users.     

Toward unraveling the structure of Brassica rapa genome

Genomic research in any organism encompasses understanding structure of the target genome and genes, their function, and evolution. Brassica rapa , which is phylogenetically related to Arabidopsis thaliana , is an important species with respect to its uses as vegetable, oil, and fodder. The availability of suitable genetic and genomic resources is a prerequisite to undertake genomic research in B. rapa . We have developed reference mapping populations of Chinese cabbage ( B. rapa ssp. pekinensis ) comprising 78 doubled haploid lines and over 250 recombinant inbred lines. Two Bacterial Artificial Chromosome (BAC) libraries, generated by restriction enzymes Hin dIII (KBrH) and Bam HI (KBrB), comprise 56 592 and 50 688 clones, respectively. We have also constructed 22 cDNA libraries from different plant tissues consisting of 104 914 clones with an average length of 575 bp. Initial BAC-end sequence analysis of 1473 clones of the KBrH library led us to understand the structure of B. rapa genome with respect to extent of genic sequences and their annotation, and relative abundance of different types of repetitive DNAs. Full-length sequence analysis of BAC clones revealed extensive triplication of B. rapa DNA segments coupled with variable gene losses within the segments. The formulation of the 'Multinational Brassica Genome Project' has laid the foundation to sequence the complete genome of B. rapa ssp. pekinensis by the international Brassica research community. It has been proposed to undertake BAC-to-BAC sequencing of genetically mapped seed BACs. In recent years, development of bioinformatics tools in Brassica has given a boost to structural genomics research in Brassica species. The research undertaken with the availability of various genomic resources in the public domain has added to our understanding of the structure of B. rapa .