Functional identification of genes responsible for the biosynthesis of 1-methoxy-indol-3-ylmethyl-glucosinolate in Brassica rapa ssp. chinensis.

BACKGROUND: Brassica vegetables contain a class of secondary metabolites, the glucosinolates (GS), whose specific degradation products determine the characteristic flavor and smell. While some of the respective degradation products of particular GS are recognized as health promoting substances for humans, recent studies also show evidence that namely the 1-methoxy-indol-3-ylmethyl GS might be deleterious by forming characteristic DNA adducts. Therefore, a deeper knowledge of aspects involved in the biosynthesis of indole GS is crucial to design vegetables with an improved secondary metabolite profile. RESULTS: Initially the leafy Brassica vegetable pak choi (Brassica rapa ssp. chinensis) was established as suitable tool to elicit very high concentrations of 1-methoxy-indol-3-ylmethyl GS by application of methyl jasmonate. Differentially expressed candidate genes were discovered in a comparative microarray analysis using the 2 × 104 K format Brassica Array and compared to available gene expression data from the Arabidopsis AtGenExpress effort. Arabidopsis knock out mutants of the respective candidate gene homologs were subjected to a comprehensive examination of their GS profiles and confirmed the exclusive involvement of polypeptide 4 of the cytochrome P450 monooxygenase subfamily CYP81F in 1-methoxy-indol-3-ylmethyl GS biosynthesis. Functional characterization of the two identified isoforms coding for CYP81F4 in the Brassica rapa genome was performed using expression analysis and heterologous complementation of the respective Arabidopsis mutant. CONCLUSIONS: Specific differences discovered in a comparative microarray and glucosinolate profiling analysis enables the functional attribution of Brassica rapa ssp. chinensis genes coding for polypeptide 4 of the cytochrome P450 monooxygenase subfamily CYP81F to their metabolic role in indole glucosinolate biosynthesis. These new identified Brassica genes will enable the development of genetic tools for breeding vegetables with improved GS composition in the near future.

Induced production of 1-methoxy-indol-3-ylmethyl glucosinolate by jasmonic acid and methyl jasmonate in sprouts and leaves of pak choi (Brassica rapa ssp. chinensis).

Pak choi plants (Brassica rapa ssp. chinensis) were treated with different signaling molecules methyl jasmonate, jasmonic acid, linolenic acid, and methyl salicylate and were analyzed for specific changes in their glucosinolate profile. Glucosinolate levels were quantified using HPLC-DAD-UV, with focus on induction of indole glucosinolates and special emphasis on 1-methoxy-indol-3-ylmethyl glucosinolate. Furthermore, the effects of the different signaling molecules on indole glucosinolate accumulation were analyzed on the level of gene expression using semi-quantitative realtime RT-PCR of selected genes. The treatments with signaling molecules were performed on sprouts and mature leaves to determine ontogenetic differences in glucosinolate accumulation and related gene expression. The highest increase of indole glucosinolate levels, with considerable enhancement of the 1-methoxy-indol-3-ylmethyl glucosinolate content, was achieved with treatments of sprouts and mature leaves with methyl jasmonate and jasmonic acid. This increase was accompanied by increased expression of genes putatively involved in the indole glucosinolate biosynthetic pathway. The high levels of indole glucosinolates enabled the plant to preferentially produce the respective breakdown products after tissue damage. Thus, pak choi plants treated with methyl jasmonate or jasmonic acid, are a valuable tool to analyze the specific protection functions of 1-methoxy-indole-3-carbinole in the plants defense strategy in the future.

Recent progress in the use of 'omics technologies in brassicaceous vegetables.

Continuing advances in 'omics methodologies and instrumentation is enhancing the understanding of how plants cope with the dynamic nature of their growing environment. 'Omics platforms have been only recently extended to cover horticultural crop species. Many of the most widely cultivated vegetable crops belong to the genus Brassica: these include plants grown for their root (turnip, rutabaga/swede), their swollen stem base (kohlrabi), their leaves (cabbage, kale, pak choi) and their inflorescence (cauliflower, broccoli). Characterization at the genome, transcript, protein and metabolite levels has illustrated the complexity of the cellular response to a whole series of environmental stresses, including nutrient deficiency, pathogen attack, heavy metal toxicity, cold acclimation, and excessive and sub-optimal irradiation. This review covers recent applications of 'omics technologies to the brassicaceous vegetables, and discusses future scenarios in achieving improvements in crop end-use quality.

High mutagenic activity of juice from pak choi (Brassica rapa ssp. chinensis) sprouts due to its content of 1-methoxy-3-indolylmethyl glucosinolate, and its enhancement by elicitation with methyl jasmonate.

Cruciferous vegetables have the reputation to protect against cancer, an effect attributed to glucosinolates (GLS) and their breakdown products. However, some GLS are mutagenic, an activity associated with cancer initiation rather than chemoprevention. We show that juices from steamed pak choi sprouts are strongly mutagenic in Salmonella typhimurium TA100 upon addition of fresh myrosinase. Growth of the plants in the presence of methyl jasmonate, a hormone eliciting defence factors, led to 20-fold enhanced mutagenic activity. The level of 1-methoxy-3-indolylmethyl (1-MIM)-GLS was similarly increased, whereas those of other GLS were only elevated 0.8- to 3.2-fold. 1-MIM-GLS is a potent mutagen, whose activity is further enhanced by human sulphotransferase 1A1 (hSULT1A1), an activation not observed with other GLS. The mutagenicity of the pak choi juices was increased 20-fold in bacteria expressing hSULT1A1. A tiny level of juice from elicitated sprouts, 0.04% in the mutagenicity assay, was sufficient to double the number of revertants above the spontaneous level. We conclude that pak choi juice is mutagenic, an activity that can be strongly affected by the growth conditions. It is owed essentially to a single component, 1-MIM-GLS. We recommend using cultivars, growth conditions and/or food preparations that keep the level of this GLS congener low.

Glucosinolates from pak choi and broccoli induce enzymes and inhibit inflammation and colon cancer differently.

High consumption of Brassica vegetables is considered to prevent especially colon carcinogenesis. The content and pattern of glucosinolates (GSLs) can highly vary among different Brassica vegetables and may, thus, affect the outcome of Brassica intervention studies. Therefore, we aimed to feed mice with diets containing plant materials of the Brassica vegetables broccoli and pak choi. Further enrichment of the diets by adding GSL extracts allowed us to analyze the impact of different amounts (GSL-poor versus GSL-rich) and different patterns (broccoli versus pak choi) of GSLs on inflammation and tumor development in a model of inflammation-triggered colon carcinogenesis (AOM/DSS model). Serum albumin adducts were analyzed to confirm the up-take and bioactivation of GSLs after feeding the Brassica diets for four weeks. In agreement with their high glucoraphanin content, broccoli diets induced the formation of sulforaphane-lysine adducts. Levels of 1-methoxyindolyl-3-methyl-histidine adducts derived from neoglucobrassicin were the highest in the GSL-rich pak choi group. In the colon, the GSL-rich broccoli and the GSL-rich pak choi diet up-regulated the expression of different sets of typical Nrf2 target genes like Nqo1, Gstm1, Srxn1, and GPx2. GSL-rich pak choi induced the AhR target gene Cyp1a1 but did not affect Ugt1a1 expression. Both colitis and tumor number were drastically reduced after feeding the GSL-rich pak choi diet while the other three diets had no effect. GSLs can act anti-inflammatory and anti-carcinogenic but both effects depend on the specific amount and pattern of GSLs within a vegetable. Thus, a high Brassica consumption cannot be generally considered to be cancer-preventive.

Genotypic variation of the glucosinolate profile in pak choi (Brassica rapa ssp. chinensis).

Thirteen different pak choi (Brassica rapa ssp. chinensis) cultivars were characterized regarding their glucosinolate profile analyzed by HPLC-DAD-MS. The identified glucosinolates were subjected to principal component analysis, and three distinct groups of pak choi sprouts were identified. Group differences were marked mainly by variations in the aliphatic glucosinolate profile such as differing levels of 3-butenyl glucosinolate and 2-hydroxy-3-butenyl glucosinolate as well as by their varying proportional ratios. In addition, the three groups of pak choi sprouts varied by the presence or absence of 2-hydroxy-4-pentenyl glucosinolate and in level and composition of butyl glucosinolates. This classification is reflected by relative mRNA expression level of 2-oxoacid-dependent dioxygenase. As in sprouts, the major glucosinolates in mature leaves were found to be the aliphatic glucosinolates. However, unlike in sprouts, an additional aliphatic glucosinolate, 5-methylsulfinylpentyl glucosinolate, was detected as characteristic ontogenetic variation in mature leaves in 12 of the 13 pak choi cultivars analyzed.