Aliphatic Glucosinolate Content Research Articles

A novel putative 2-oxoglutarate-dependent dioxygenase gene (BoaAOP-like) regulates aliphatic glucosinolate biosynthesis in Chinese kale

Glucosinolates are important secondary metabolites in Brassica vegetables, and the side-chain modifications of aliphatic glucosinolates are regulated by 2-oxoglutarate-dependent dioxygenases (AOPs). However, how the side-chain modifications of aliphatic glucosinolates are regulated in Chinese kale remains unclear. Here, we show that the novel gene BoaAOP-like affects the biosynthesis of aliphatic glucosinolates in Chinese kale. BoaAOP-like was localized in the cytoplasm of Chinese kale. The expression levels of BoaAOP-like increased with plant growth; among different organs, BoaAOP-like was the highest in the roots. BoaAOP-like was introduced into Chinese kale using Agrobacterium-mediated transient overexpression system, of which the expression levels were 2.20- to 49.56-fold higher in transiently overexpressed plants than in wild-type plants; the content of total aliphatic glucosinolates in transiently overexpressed plants was up to 70.26-fold higher than in wild-type plants. The content of each aliphatic glucosinolate was significantly accumulated in BoaAOP-like transiently overexpressed plants, with the gluconapin content increasing the greatest when compared to the wild-type plants. The expression levels of aliphatic glucosinolate-related genes, especially MYB28 and AOP2.1 were significantly up-regulated in transiently overexpressed plants. Overall, our results indicated that BoaAOP-like plays a critical role in regulating the biosynthesis of aliphatic glucosinolate in Chinese kale.

Tumorous Stem Development of Brassica Juncea: A Complex Regulatory Network of Stem Formation and Identification of Key Genes in Glucosinolate Biosynthesis.

Stem mustard is a stem variety of mustard, an important Brassica vegetable. The formation and development of the tumorous stem, which is the key organ for the direct yield and quality, is a complex biological process involving morphogenesis, material accumulation and gene regulation. In this study, we demonstrated through anatomical studies that stem swelling is mainly dependent on the increase in the number of cells and the volume of parenchyma cells in the cortex and pith. To further understand transcript and metabolic changes during stem swelling, we obtained 27,901 differentially expressed genes, of which 671 were specifically detected using transcriptome sequencing technology in all four stages of stem swelling. Functional annotation identified enrichment for genes involved in photosynthesis, energy metabolism, cell growth, sulfur metabolism and glucosinolate biosynthesis. Glucosinolates are a group of nitrogen- and sulfur-containing secondary metabolites, which largely exist in the Cruciferous vegetables. HPLC analysis of the contents and components of glucosinolates in four different stem development stages revealed eight glucosinolates, namely, three aliphatic glucosinolates (sinigrin, glucoalyssin and gluconapin), four indole glucosinolates (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin) and one aromatic glucosinolate (gluconasturtiin). All these types of glucosinolates showed a significant downward trend during the stem swelling period. The content of aliphatic glucosinolates was the highest, with sinigrin being the main component. In addition, qPCR was used to validate the expression of nine genes involved in glucosinolate biosynthesis. Most of these genes were down-regulated during stem swelling in qPCR, which is consistent with transcriptome data. These data provide a basic resource for further molecular and genetic research on Brassica juncea.

Modulation of Glucosinolate Composition in Brassicaceae Seeds by Germination and Fungal Elicitation.

Glucosinolates (GSLs) are of interest for potential antimicrobial activity of their degradation products and exclusive presence in Brassicaceae. Compositional changes of aliphatic, benzenic, and indolic GSLs of Sinapis alba, Brassica napus, and B. juncea seeds by germination and fungal elicitation were studied. Rhizopus oryzae (nonpathogenic), Fusarium graminearum (nonpathogenic), and F. oxysporum (pathogenic) were employed. Thirty-one GSLs were detected by reversed-phase ultrahigh-performance liquid chromatography photodiode array with in-line electrospray ionization mass spectrometry (RP-UHPLC-PDA-ESI-MSn). Aromatic-acylated derivatives of 3-butenyl GSL, p-hydroxybenzyl GSL, and indol-3-ylmethyl GSL were for the first time tentatively annotated and confirmed to be not artifacts. For S. alba, germination, Rhizopus elicitation, and F. graminearum elicitation increased total GSL content, mainly consisting of p-hydroxybenzyl GSL, by 2–3 fold. For B. napus and B. juncea, total GSL content was unaffected by germination or elicitation. In all treatments, aliphatic GSL content was decreased (≥50%) in B. napus and remained unchanged in B. juncea. Indolic GSLs were induced in all species by germination and nonpathogenic elicitation.

Natural Variation of Glucosinolates and Their Breakdown Products in Broccoli (Brassica oleracea var. italica) Seeds.

Seeds of 32 pure lines and 6 commercial broccoli cultivars were used to investigate variation in glucosinolates and their breakdown products. The aliphatic glucosinolate content was 54.5-218.7 μmol/g fresh weight, accounting for >90% of the total glucosinolates. The major glucosinolates found were glucoraphanin and glucoerucin in 27 samples and progoitrin in 7 samples. A gas chromatography-flame ionization detector (GC-FID) method was used to identify glucosinolate breakdown products; nine products were directly determined using standards. Using Arabidopsis thaliana lines myb28myb29 and Landsberg erecta to hydrolyze each reference glucosinolate, seven products were tentatively identified. 4-(Methylsulfinyl)butyl isothiocyanate and 5-(methylsulfinyl)pentanenitrile contents were 2.6-91.1 μmol/g fresh weight and 0-35.4 μmol/g fresh weight, respectively, with epithionitriles being more common than nitriles in accessions rich in alkenyl glucosinolate. Additionally, (S)-5-vinyl-1,3-oxazolidine-2-thione was detected in accessions rich in progoitrin. Specific lines with altered glucosinolate profiles and breakdown products were obtained and discussed according to the putative glucosinolate metabolism pathway.

Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea

Key messageOverexpression of BoMYB29 gene up-regulates the aliphatic glucosinolate pathway in Brassica oleracea plants increasing the production of the anti-cancer metabolite glucoraphanin, and the toxic and pungent sinigrin.Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties.

A naturally occurring variation in the BrMAM-3 gene is associated with aliphatic glucosinolate accumulation in Brassica rapa leaves

Glucosinolate profiles significantly vary among Brassica rapa genotypes. However, the molecular basis of these variations is largely unknown. In this study, we investigated a major quantitative trait locus (QTL) controlling aliphatic glucosinolate accumulation in B. rapa leaves. The QTL, which encompasses three tandem MAM genes and two MYB genes, was detected in two BC2DH populations. Among the five-candidate genes, only the expression level of BrMAM-3 (Bra013007) was significantly correlated with the accumulation of aliphatic glucosinolates in B. rapa leaves. We identified a naturally occurring insertion within exon 1 of BrMAM-3, which is predicted to be a loss-of-function mutation, as confirmed by qRT-PCR. We determined that the loss of function was associated with the low glucosinolate content in B. rapa accessions. Furthermore, overexpressing the BrMAM-3 gene resulted in an increase in total aliphatic glucosinolates in Arabidopsis transgenic lines. Our study provides insights into the molecular mechanism underlying the accumulation of aliphatic glucosinolates in B. rapa leaves, thereby facilitating in the manipulation of total aliphatic glucosinolate content in Brassica crops.

Plant growth-promoting bacteria Kosakonia radicincitans mediate anti-herbivore defense in Arabidopsis thaliana.

This study demonstrates that the application of the PGPB strain, Kosakonia radicincitans enhances a plant's resistance against phloem-feeding and chewing insects in Arabidopsis thaliana. The plant growth-promoting bacterial strain K. radicincitans DSM 16656 applied to A. thaliana reduced the number of phloem-feeding insects of both the specialist Brevicoryne brassicae and the generalist Myzus persicae. While weight gain of the generalist chewing insect Spodoptera exigua was reduced by 30% on A. thaliana plants treated with K. radicincitans, growth of the specialist caterpillar Pieris brassicae was not affected when compared with caterpillars from control plants. Since generalist and specialist chewing insects responded differentially to PGPB application, the implication of signaling pathways in PGPB mediated changes in plant defense was studied using two signaling pathway mutants impaired in their salicylic acid (npr1-1 mutant) or jasmonic acid (coi1-1 mutant) pathway. We found that the jasmonic acid pathway is relevant for upregulation of aliphatic glucosinolates and suppression of the chewing generalist S. exigua larval growth. Chewing from generalist P. brassicae increased glucosinolate content in A. thaliana leaves mediated via both signaling pathways. However, only in the npr1-1 mutant, which contains the highest aliphatic glucosinolate content, the P. brassicae induced further enrichment of glucosinolates, resulting in a reduction of larval growth. Effects of K. radicincitans on plant resistance could not be explained by changes in glucosinolate contents or composition. Our results demonstrate the distinct role played by K. radicincitans in suppressing insect performance in A. thaliana.

Heat and hypoxia stresses enhance the accumulation of aliphatic glucosinolates and sulforaphane in broccoli sprouts

In this study, aliphatic glucosinolates and sulforaphane were accumulated in broccoli sprouts under the stresses of heat, hypoxia and heat plus hypoxia. Glucoraphanin content in 7-day-old broccoli sprouts under heat, hypoxia and heat plus hypoxia stresses enhanced by 1.39-, 1.10- and 1.16-fold compared with the control, respectively; meanwhile, glucoerucin content increased by 1.87-, 4.17- and 3.24-fold, respectively. Aliphatic glucosinolate content was enhanced by heat stress due to the increasing expression of Elong, CYP83A1 and FMOGS-OX1. However, under hypoxia and heat plus hypoxia stresses, the expression levels of Elong, CYP83A1, UGT74B1, ST5b and AOP2 related to aliphatic glucosinolates biosynthesis were dramatically up-regulated. Heat, hypoxia and heat plus hypoxia stresses enhanced sulforaphane formation in 7-day-old sprouts by 153.73, 95.15 and 86.95 % compared with the control, respectively. However, heat plus hypoxia stress did not exhibit the synergistic effect of heat and hypoxia stresses. These results indicated that heat and hypoxia stresses were feasible strategies to enhance the accumulation of aliphatic glucosinolates and sulforaphane in broccoli sprouts.

Effect of refrigerated storage on the bioactive compounds and microbial quality of Brassica oleraceae sprouts

Brassica sprouts are a healthy and convenient food product. Due to a great number of species and varieties, the knowledge about the bioactive composition and stability of the quality during storage of different Brassica oleracea sprouts is still scarce. In this work, the glucosinolate and phenolic profile of four varieties of B. oleracea sprouts (red cabbage, broccoli, Galega kale and Penca cabbage) grown under different light conditions was monitored during 12 days of storage (at 4°C). Their microbial quality was assessed by monitoring the microbial load of mesophilic bacteria, total coliforms, yeasts and molds and presence of Salmonella. The presence of biogenic amines was also monitored to guaranty the safety of this product. As expected, the content of bioactive compounds suffered significant (p<0.05) changes during storage, especially after 7 days of storage. The two Portuguese varieties (Galega kale and Penca cabbage) had a high content of aliphatic glucosinolates (as glucoraphanin and sinigrin), having the sprouts grown under darkness a more stable content of these compounds. The phenolic profile was characterized by a prevalence of hydroxycinnamic acids derivatives, and in sprouts grown without light in particular. Sprouts did not show any contamination by Salmonella and were considered as microbiologically safe during the studied period.

Glucosinolate profiles in Cardamine fauriei and effect of light quality on glucosinolate concentration

Most Brassicaceae plants produce glucosinolates (GSLs), which are hydrolyzed to form isothiocyanates (ITCs) that contribute to a stimulative flavor and antibiotic activity beneficial to human health. Three glucosinolates (GSLs), gluconapin, glucoibarin and glucobrassicin, were isolated from plants of Cardamine fauriei Maxim. (Brassicaceae; Ezo-wasabi in Japanese), a wild, edible herb originating from Hokkaido, Japan. These GSLs comprise two types: aliphatic (gluconapin and glucoibarin) and indolic (glucobrassicin). Total GSL concentration in the leaf blade, petiole and root of the plant grown in natural light were 62.81, 43.94 and 17.63mgg−1 dry mass, respectively. When the plants were grown under various wavelengths of light supplied by LEDs, total GSL content in the leaf blade was highest when plants were irradiated by red+blue light. In addition, the ratio of the content of the indolic GSL to that of aliphatic GSL was greater after irradiation with by red or green alone rather than blue alone, red+blue, or white (red+blue+green). Because the last three types of irradiation include blue wavelengths, aliphatic GSL content can be increased by irradiation with blue light wavelengths, whereas the indolic GSL levels decreased. The results indicate that it is possible to control the GSL component ratio in C. fauriei by irradiating plants with particular wavelengths.

The major aliphatic glucosinolate content in Korean radish during vegetative and reproductive growth

Glucosinolates (GSLs) are secondary metabolites abundant in Brassica vegetables and the interest on this compound has been grown due to their health benefits. The amounts of major aliphatic GSLs, glucoraphasatin (GRH), and glucoraphenin (GRE) were measured in five radish cultivars during their vegetative and reproductive growth. The GRE content was higher in seed (ranging from 16.5 to 19.4 mg·g−1 dry weight) than that of leaves or roots in all five genotypes. The GRE content sharply declined during germination and remained stable at the basal level for the subsequent vegetative growth period. In contrast, the amount of GRH, an immediate precursor of the GRE, steadily increased during the early growth period up to 8 weeks and remained at a similar level thereafter. As growth of seedlings progressed, the inner leaves contained higher amounts of GRH than the outer leaves. In roots, smaller amounts of GRE were found, as compared to other tissues. However, the GRH was abundant in roots and mature inner leaves. The GRE tended to accumulate in reproductive organs, while GRH was found primarily in the roots and the inner leaves at the mature stage.

Antibiotic properties of the glucosinolates of Brassica oleracea var. acephala similarly affect generalist and specialist larvae of two lepidopteran pests

Glucosinolates may deter generalist insect feeding as their toxicity causes fitness damage, whereas insects specialized in brassicaceous plants may circumvent the toxic effect. By using no-choice leaf tests, we investigated whether larval development time, body weight, mortality and feeding rate of the generalist Mamestra brassicae (Lepidoptera, Noctuidae) and the specialist Pieris rapae (Lepidoptera, Pieridae), were affected by six genotypes of Brassica oleracea var. acephala, selected for having high or low concentration of sinigrin, glucoiberin (aliphatics) and glucobrassicin (indole). Two phenological plant stages were used. On young plants, M. brassicae most consumed the high sinigrin and low glucoiberin genotypes. Larvae weighed more on the high sinigrin plants. Development time took longer on the low glucoiberin genotype. On mature plants, consumption rate decreased on the high glucoiberin genotype. Larval weight decreased on the high sinigrin, glucoiberin and glucobrassicin genotypes, and development time increased with high glucobrassicin concentration. Pupal weight and mortality rate increased on mature plants, irrespective of the genotype. Pieris rapae fed most on young plants with high sinigrin, and larval weight increased on the high glucoiberin genotype. Mortality increased with low glucoiberin and low glucobrassicin. On mature plants, larval weight decreased with high sinigrin and glucoiberin. The high glucoiberin genotype was the less consumed and also induced a longer development time. High content of aliphatic glucosinolates offered mature plants significant antibiosis defence against both the lepidopterans, whereas the indole glucosinolate was marginally effective. Young plants were more consumed and increased larval weight likely because glucosinolate concentration was still not optimal.

Evaluating the impact of sprouting conditions on the glucosinolate content of Brassica oleracea sprouts

The glucosinolates content of brassica plants is a distinctive characteristic, representing a healthy advantage as many of these compounds are associated to antioxidant and anti-carcinogenic properties. Brassica sprouts are still an underutilized source of these bioactive compounds. In this work, four varieties of brassica sprouts (red cabbage, broccoli, Galega kale and Penca cabbage), including two local varieties from the North of Portugal, were grown to evaluate the glucosinolate profile and myrosinase activity during the sprouting. Also the influence of light/darkness exposure during sprouting on the glucosinolate content was assessed. Glucosinolate content and myrosinase activity of the sprouts was evaluated by HPLC methods. All sprouts revealed a higher content of aliphatic glucosinolates than of indole glucosinolates, contrary to the profile described for most of brassica mature plants. Galega kale sprouts had the highest glucosinolate content, mainly sinigrin and glucoiberin, which are recognized for their beneficial health effects. Penca cabbage sprouts were particularly richer in glucoraphanin, who was also one of the major compounds in broccoli sprouts. Red cabbage showed a higher content of progoitrin. Regarding myrosinase activity, Galega kale sprouts showed the highest values, revealing that the use of light/dark cycles and a sprouting phase of 7–9days could be beneficial to preserve the glucosinolate content of this variety.

Feeding Brassica juncea or Brassica napus canola meal at increasing dietary inclusions to growing-finishing gilts and barrows

Novel Brassica (B.) juncea has a thinner seed coat and therefore lower fibre content than conventional B. napus canola meal (CM) and could potentially be fed at greater dietary inclusions to pigs. In a 2×2×3 factorial arrangement, 528 barrows and 528 gilts [33.6kg body weight] housed in 48 pens (22 barrows or gilts) were fed either B. juncea or B. napus CM at 100, 200 or 300g/kg of diet with up to 200g/kg wheat DDGS to slaughter weight (120kg). Compared with B. napus, B. juncea CM had 32g/kg greater CP, 12g/kg lower crude fat, 86g/kg lower ADF, and 91g/kg lower NDF content. However, aliphatic glucosinolate content was 2.7 times greater in B. juncea (11.76μmol/g) than B. napus CM (4.34μmol/g). For the entire trial (d 0–72), daily weight gain (ADG) was not affected by canola species, but feed disappearance (ADFI) was 45g/d lower (P=0.06) and feed efficiency 7g/g greater (P<0.05) for pigs fed B. juncea than B. napus CM. Carcass traits were not affected by canola species except dressing, which was 1% lower (P<0.05) for pigs fed B. juncea than B. napus CM. For the entire trial, increasing CM inclusion from 100 to 300g/kg of diet decreased ADFI (P<0.001) by 184g/d, decreased ADG by 32g/d (P<0.05), but increased feed efficiency (P<0.001) by 14g/g. Dietary CM inclusion level did not affect farm ship live weight to slaughter, carcass backfat thickness, lean yield, or index. Nonetheless, carcass weight was 0.9kg lower (P<0.05), dressing was 1% lower (P<0.001), loin depth was 1.3mm lower (P<0.01), and days to slaughter was 2.3 days greater for pigs fed 300 compared with those fed 100g CM/kg diet. In conclusion, growing-finishing pigs can be fed diets including B. juncea CM the same as conventional B. napus CM without decreasing growth performance or carcass traits. Feeding growing-finishing pigs a diet with 300 vs. 200 or 100g/kg CM with up to 200g/kg of wheat DDGS resulted in decreased weight gain and a minor decrease in carcass weight, dressing and loin depth, but increased feed efficiency.

Pronounced effects of slug herbivory on seedling recruitment of Brassica cultivars and accessions, especially those with low levels of aliphatic glucosinolates

In demographic studies on Brassica cultivars and accessions we observed large genotypic variation in the ability of seedlings to establish. Here we quantify the role of slugs during establishment by including and excluding slug herbivory.Slug exclusion during the first 10 days after germination led to 26.5 times more surviving seedlings in modern canola varieties of Brassica napus, 4.3 times more in feral B. napus and 1.9 times more in wild Brassica rapa. While seed and seedlings of canola were low in aliphatic glucosinolates (AGS), feral B. napus and wild B. rapa had much higher AGS levels. Consequently, we find a positive correlation between the survival of young seedlings and AGS concentration. Concentrations of indole glucosinolates (IGS) in seeds were much lower than those of AGS and did not correlate significantly with survival. Subsequently, we exposed 10-day-old seedlings to herbivory. In the 4 days following exposure, slug herbivory was negatively correlated to AGS concentration but not to IGS.In choice experiments in the lab, the preference of the slug Arion lusitanicus also correlated negatively with AGS content. A. lusitanicus preferred to feed on B. napus rather than on wild B. rapa, but had no significant preference when presented with plants similar in AGS content.Slugs can be a limiting factor for seedling recruitment in populations of B. napus, especially for modern canola cultivars with a low AGS content.

CML42-mediated calcium signaling coordinates responses to Spodoptera herbivory and abiotic stresses in Arabidopsis.

In the interaction between Arabidopsis (Arabidopsis thaliana) and the generalist herbivorous insect Spodoptera littoralis, little is known about early events in defense signaling and their link to downstream phytohormone pathways. S. littoralis oral secretions induced both Ca²⁺ and phytohormone elevation in Arabidopsis. Plant gene expression induced by oral secretions revealed up-regulation of a gene encoding a calmodulin-like protein, CML42. Functional analysis of cml42 plants revealed more resistance to herbivory than in the wild type, because caterpillars gain less weight on the mutant, indicating that CML42 negatively regulates plant defense; cml42 also showed increased aliphatic glucosinolate content and hyperactivated transcript accumulation of the jasmonic acid (JA)-responsive genes VSP2 and Thi2.1 upon herbivory, which might contribute to increased resistance. CML42 up-regulation is negatively regulated by the jasmonate receptor Coronatine Insensitive1 (COI1), as loss of functional COI1 resulted in prolonged CML42 activation. CML42 thus acts as a negative regulator of plant defense by decreasing COI1-mediated JA sensitivity and the expression of JA-responsive genes and is independent of herbivory-induced JA biosynthesis. JA-induced Ca²⁺ elevation and root growth inhibition were more sensitive in cml42, also indicating higher JA perception. Our results indicate that CML42 acts as a crucial signaling component connecting Ca²⁺ and JA signaling. CML42 is localized to cytosol and nucleus. CML42 is also involved in abiotic stress responses, as kaempferol glycosides were down-regulated in cml42, and impaired in ultraviolet B resistance. Under drought stress, the level of abscisic acid accumulation was higher in cml42 plants. Thus, CML42 might serve as a Ca²⁺ sensor having multiple functions in insect herbivory defense and abiotic stress responses.

Glucosinolates and other anti-nutritive compounds in canola meals from varieties cultivated in Egypt and Japan

Canola meals from six varieties cultivated in Egypt (Seru4 and Pactol) and Japan (Kirariboshi, Tohoku95, Oominantane and Kizakinonatane) were investigated regarding anti-nutritive compounds, namely glucosinolates, phytic acid, sinapine and total phenols. All varieties except Kirariboshi contained a high level of total glucosinolates. A profile of 12 glucosinolates in all varieties was identified; aliphatic glucosinolates, progoitrin, epiprogoitrin, gluconapin, glucobrassicanapin and glucoberin were the most abundant. Other aliphatic glucosinolates, including sinigrin and gluconapoliferin, were of low level. Indolic and aromatic glucosinolates (4-Hydroxybrassicin, Glucotropeolin, Glucobrassicin, Gluconasturtin and4-Methoxybrassicin) were low and showed few differences among varieties. The anti-nutritional effects are associated, to a large degree, with the content of aliphatic glucosinolates rather than indole glucosinolates, and the total aliphatic glucosinolates are present at a higher level in all varieties than total aromatic glucosinolate (including indole glucosinolate). Therefore, canola meal from all varieties could cause many anti-nutritive effects. Also it was found that Seru4, Pactol, Tohoku95, Oominantane and Kizakinonatane are high glucosinolates varieties because they contained more than 30 μmol g-1 however, only Kirariboshi was a low glucosinolate variety. The differences in the content of individual and total glucosinolates among the varieties may be attributed to genetic and environmental factors. Phytic acid was at a high level in all varieties with few differences. Oominatane contained the lowest level and Pactol contained the highest one. No remarkable differences were observed among Kirariboshi, Kizakinonatane and Tohoku. For total phenols, Tohoku contained the lowest level of total phenols while Pactol and Seru4 contained the highest level. Also, insignificant differences were observed among Kirariboshi,Oominatane and Kizakinonatane in total phenols. Sinapine was found in all varieties with significant differences among varieties. Tohoku and Oominatane contained the lowest levels of sinapine, while Seru4 contained the highest one. Insignificant differences were observed among Kirariboshi, Kizakinonatane and Pactol. Thus canola meals from all varieties must be treated to remove or reduce the aliphatic glucosinolates and other anti-nutritive compounds.Key words: Canola, glucosinolates, phytate, sinapine, phenols

Glucosinolates within a collection of white head cabbages (Brassica oleracea var. capitata sub.var. alba) from Turkey

Glucosinolates of a population of white head cabbages collected from different geographical regions of Turkey were determined at two different plant developmental stages (early and late development). The glucosinolates detected were glucoiberin, glucoraphanin, sinigrin of aliphatics and glucobrassicin, neoglucobrassicin, 4-methoxyglucobrassicin and 4-hydroxyglucobrassicin of indoles. Variation were observed in terms of glucosinolate profile and levels. However, the most abundant glucosinolate observed was glucobrassicin synthesized at significantly high levels. While total aliphatic glucosinolate content of genotypes ranged from 0.33 - 5.63 imolg-1 dw and 0.46 - 16.14 imolg-1 dw at early and late developmental stages, respectively, in the first year, the levels ranged from 0.05 - 7.72 imolg-1 dw and 0.12 - 14.60 imolg-1 dw at early and late developmental stages, respectively, in the second experimental year. In general, aliphatic glucosinolate content of cabbages were low compared to indoles; glucobrassicin in particular. The presence of glucoiberin and glucoraphanin in almost all cabbage genotypes is promising. Potential individuals synthesizing aliphatic glucosinolates together with glucobrassicin at relatively higher levels were selected for future breeding purposes aimed to develop novel lines with improved health benefits. Results suggested that both aliphatic and indole glucosinolate levels were higher at later stages of development.

A Plastidic Transporter Involved in Aliphatic Glucosinolate Biosynthesis

The distinctive flavors and anticarcinogenic properties of cruciferous vegetables are largely due to the presence of glucosinolates. These sulfur-rich metabolites are derived from one of several amino acids via three stages: (1) elongation of the amino acid side chain; (2) formation of the core glucosinolate skeleton; and (3) secondary modification of the side chain (reviewed in Grubb and Abel, 2006). Met-derived aliphatic glucosinolates constitute the majority of glucosinolates in many Brassicaceae species, including Arabidopsis (Kliebenstein et al., 2001). Biosynthesis of these glucosinolates is highly compartmentalized, with preliminary reactions and final stages occurring in the cytosol and side chain elongation in the chloroplast. Prompted by the discovery that the R2R3-MYB transcription factor High Aliphatic Glucosinolate 1 (HAG1/MYB28) is a key regulator of Met-derived glucosinolate biosynthesis in Arabidopsis (Gigolashvili et al., 2007; Hirai et al., 2007), Gigolashvili et al. (pages 1813–1829) set out to identify novel targets of HAG1. Using transcriptome coexpression analysis, they found that BAT5, which encodes a putative bile acid transporter with a hitherto uncharacterized role in plants, is coexpressed with HAG1. Cotransformation assays using a HAG1 effector and a ProBAT5-GUS reporter showed that HAG1 transactivates the BAT5 promoter, confirming that BAT5 is indeed a target of HAG1. Furthermore, the authors established that full-length BAT5-GFP localizes to chloroplasts in transiently transformed Arabidopsis leaves (see figure). Figure 1 BAT5-GFP localizes to chloroplasts. BAT5-GFP fluorescence (A) and chloroplast autofluorescence (B) in transgenic Arabidopsis leaves and the overlay (C) of (A) and (B) showing colocalization (yellow) of BAT5-GFP and chloroplasts. The authors tested whether BAT5 has a role in aliphatic glucosinolate biosynthesis by examining the effect of downregulation of BAT5 on glucosinolate content. Although a homozygous bat5 mutant with vastly reduced steady state levels of BAT5 mRNA appeared to be normal under standard conditions, metabolic analysis revealed that it had significantly reduced levels of aliphatic glucosinolates. Complementation of the bat5 mutant with an estradiol-inducible BAT5 vector rescued the glucosinolate composition to almost wild-type levels, and the degree of induced gene expression correlated with the level of aliphatic glucosinolates produced. The effect on aliphatic glucosinolate content was even more pronounced in BAT5 microRNA overexpressing lines, which have undetectable levels of BAT5 mRNA. The observation that wounding and methyl jasmonate, which are known to induce glucosinolate biosynthesis, upregulate the expression of BAT5, provides further evidence that BAT5 is involved in glucosinolate biosynthesis. Finally, the authors sought to pinpoint the function of BAT5 in aliphatic glucosinolate biosynthesis by feeding bat5 and wild-type Arabidopsis plants with various intermediates of the Met-derived glucosinolate pathway and then analyzing the glucosinolate content. The results suggest that BAT5 transports Met derivatives into the chloroplast for side chain elongation and exports elongated products back into the cytosol for conversion into glucosinolates. Thus, BAT5 plays an integral role in Met-derived glucosinolate biosynthesis in Arabidopsis.

A Systems Biology Approach Identifies a R2R3 MYB Gene Subfamily with Distinct and Overlapping Functions in Regulation of Aliphatic Glucosinolates

BackgroundGlucosinolates are natural metabolites in the order Brassicales that defend plants against both herbivores and pathogens and can attract specialized insects. Knowledge about the genes controlling glucosinolate regulation is limited. Here, we identify three R2R3 MYB transcription factors regulating aliphatic glucosinolate biosynthesis in Arabidopsis by combining several systems biology tools.Methodology/Principal Findings MYB28 was identified as a candidate regulator of aliphatic glucosinolates based on its co-localization within a genomic region controlling variation both in aliphatic glucosinolate content (metabolite QTL) and in transcript level for genes involved in the biosynthesis of aliphatic glucosinolates (expression QTL), as well as its co-expression with genes in aliphatic glucosinolate biosynthesis. A phylogenetic analysis with the R2R3 motif of MYB28 showed that it and two homologues, MYB29 and MYB76, were members of an Arabidopsis-specific clade that included three characterized regulators of indole glucosinolates. Over-expression of the individual MYB genes showed that they all had the capacity to increase the production of aliphatic glucosinolates in leaves and seeds and induce gene expression of aliphatic biosynthetic genes within leaves. Analysis of leaves and seeds of single knockout mutants showed that mutants of MYB29 and MYB76 have reductions in only short-chained aliphatic glucosinolates whereas a mutant in MYB28 has reductions in both short- and long-chained aliphatic glucosinolates. Furthermore, analysis of a double knockout in MYB28 and MYB29 identified an emergent property of the system since the absence of aliphatic glucosinolates in these plants could not be predicted by the chemotype of the single knockouts.Conclusions/SignificanceIt seems that these cruciferous-specific MYB regulatory genes have evolved both overlapping and specific regulatory capacities. This provides a unique system within which to study the evolution of MYB regulatory factors and their downstream targets.