Factors affecting the glucosinolate content of kale (Brassica oleracea acephala group)

Kales (Brassica oleracea acephala group) are important vegetable crops in traditional farming systems in the Iberian Peninsula. They are grown throughout the year to harvest their leaves and flower buds. The glucosinolate content of kales is dependent upon the environmental factors, plant part examined, phenological stage of plant growth, and level of insect damage. The objectives of this study were to evaluate the changes in the total and individual glucosinolate concentrations during plant development and to determine if significant variation of glucosinolate levels can be explained by insect pests attack and other environmental factors in four locations in northwestern Spain. The total glucosinolate concentration in leaves of B. oleracea increased with plant age from seedling to early flowering stages. At that stage, the aliphatic glucosinolate content in leaves of B. oleracea declined drastically over time as the content in the flower buds increased. The highest contents of indolyl glucosinolate (glucobrassicin) and of the aromatic glucosinolate occurred in leaves harvested at the optimum consumption stage while flower buds contained the highest concentration of aliphatic glucosinolates, especially sinigrin. Sinigrin is reported to have anticarcinogenic properties. There appears to be a loss of total and individual glucosinolate concentrations related to pest attack. Leaves damaged by lepidopterous pests contained a lower total glucosinolate content (25.8 micromol g-1 dw) than undamaged leaves (41 micromol g-1 dw). The amounts of sinigrin, glucoiberin, and glucobrassicin were also lowest in insect-damaged leaves. Environmental factors such as soil properties and temperature appear to influence the glucosinolate content in leaves although more research on this subject is needed.     

Using kale (Brassica oleracea var. acephala) as a phytoremediation plant species for lead (Pb) and cadmium (Cd) removal in saline soils

Ornamental kale (Brassica oleracea var. Acephala) is usually planted from early autumn until late winter. Since most of the plants used for phytoremediation cannot be grown during this time, kale can be a suitable option for phytoremediation and utilized during autumn and winter in urban landscape, especially in metropolitan areas where high levels of cadmium (Cd) and lead (Pb) pollutions exist. Kale growth in saline soil at different growth stages (germination and vegetative growth stages) was studied in this investigation. A factorial experiment based on completely randomized design (CRD) with four replications was used in this study. Treatments included three levels of sodium chloride (NaCl) (0, 30, and 60 mg/kg), four levels of Cd (0, 4, 8, and 16 mg/kg), and four levels of Pb (0, 1, 5, and 10 mg/kg). Results indicated that increase in Cd and Pb concentrations in the soil decreased fresh and dry weights of the plants. The results of the various growth stages revealed that under salinity stress, kale plants were able to absorb more Pb than Cd and effectively remediate Pb in polluted and saline lands. Cd accumulation in control treatment was 6.2% more than that in the saline treatments, whereas, Pb accumulation in the highest NaCl level, 60 mg/kg salinity treatment was 7.64% more than that of the control condition. Also, proline content of the plants was significantly increased under Cd and Pb stress. From the results of this study, it was concluded that using kale plant is recommended for phytoremediation of saline soils with 10 and 16 mg/kg Pb and Cd contents, respectively.     

Biochemical characterization of cystine lyase from broccoli (Brassica oleracea var. italica)

Cystine lyase is the enzyme responsible for off-aroma deterioration in fresh unblanched broccoli. In this research, cystine lyase purification from broccoli has been optimized. Only one protein peak with cystine lyase activity was found during purification. Broccoli cystine lyase was purified 100-fold to homogeneity. L-Cystine, L-cysteine-S-sulfate, L-djenkolic acid, and some S-alkyl-L-cysteines and their sulfoxides are substrates, but the enzyme had negligible activity with L-cystathionine. A K(m) value of 81.2 microM was found for L-cystine. Inhibition and K(i) determinations indicated that L-cysteine is a linear noncompetitive inhibitor with a K(i) of 5 mM and DL-homocysteine is a competitive inhibitor with a K(i) of 1.5 mM. The molecular weight of cystine lyase was determined to be 100 kDa by three methods, with two subunits of 48 kDa each and a carbohydrate content of 3%. Further characterization included cofactor quantification, the effects of temperature and pH on activity and stability, and amino acid composition.     

Metabolism of the insecticide metofluthrin in cabbage (Brassica oleracea)

The metabolic fate of metofluthrin [2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (E,Z)-(1R,3R)-2,2-dimethyl-3-(prop-1-enyl)cyclopropanecarboxylate] separately labeled with (14)C at the carbonyl carbon and the α-position of the 4-methoxymethylbenzyl ring was studied in cabbage ( Brassica oleracea ). An acetonitrile solution of (14)C-metofluthrin at 431 g ai ha(-1) was once applied topically to cabbage leaves at head-forming stage, and the plants were grown for up to 14 days. Each isomer of metofluthrin applied onto the leaf surface rapidly volatilized into the air and was scarcely translocated to the untreated portion. On the leaf surface, metofluthrin was primarily degraded through ozonolysis of the propenyl side chain to produce the secondary ozonide, which further decomposed to the corresponding aldehyde and carboxylic acid derivatives. In the leaf tissues, the 1R-trans-Z isomer was mainly metabolized to its dihydrodiol derivative probably via an epoxy intermediate followed by saccharide conjugation in parallel with the ester cleavage, whereas no specific metabolite was dominant for the 1R-trans-E isomer. Isomerization of metofluthrin at the cyclopropyl ring was negligible for both isomers. In this study, the chemical structure of each secondary ozonide derivative was fully elucidated by the various modes of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy together with cochromatography with the synthetic standard, and their cis/trans configuration was examined by the nuclear Overhauser effect (NOE) difference NMR spectrum.     

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.)

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).     

Neglected landraces of collard (Brassica oleracea L. var. viridis) from the Carolinas (USA)

A common garden crop grown in the coastal plain region of North and South Carolina (United States) is the non-heading, leafy green type of Brassica oleracea L. known as collard (B. oleracea L. subsp. oleracea convar. acephala (DC.) Alef. var. viridis L.). Predominantly a fall and winter vegetable in this region, collard is often the only green planting to be found in the yard or garden of a rural home during these cool seasons. Historically, the traditional collard patch and even commercial fields were planted with unique varieties perpetuated by individual seed savers, and collectively, the regional diversity for this crop was probably very significant for well over a century. Genetic erosion of this collard germplasm pool has been severe in recent decades as commercial hybrids have been adopted by both large-scale producers and home gardeners. Although a significant number of collard landraces are being perpetuated to this day, existing diversity among landraces still grown in the region is now in the hands of an aging population of seed savers who maintain germplasm through on-farm preservation. From 2003 to 2006, we explored the coastal plain region of North and South Carolina in search of collard gardens containing traditional landraces. Exploration trips were conducted mid-winter to early spring. About 90 samples of collard were obtained from seed savers during the course of this exploration. Observations of morphological differences of these landraces indicate that significant diversity exists in this group. Obtained landraces are being deposited into the U.S. plant introduction collection and will be available for future use. This preserved collection could prove to be an important new source of genes for B. oleracea improvement.     

Protein from red cabbage (Brassica oleracea) seeds with antifungal, antibacterial, and anticancer activities

A 30 kDa antifungal protein was purified from red cabbage ( Brassica oleracea ) seeds. It exhibited a molecular mass and N-terminal amino acid sequence disinct from those of previously isolated Brassica antifungal proteins. The protocol used entailed ion exchange chromatography on Q-Sepharose and SP-Sepharose followed by fast protein liquid chromatography on Mono S. The protein hindered mycelial growth in Mycosphaerella arachidicola (with an IC50=5 μM), Setospaeria turcica, and Bipolaris maydis. It also inhibited the yeast Candida albicans with an IC50=96 μM. It exerted its antifungal action by permeabilizing the fungal membrane as evidenced by staining with Sytox green. The antifungal activity was stable from pH 3 to 11 and from 0 to 65 °C. It manifested antibacterial activity against Pseudomonas aeruginosa (IC50=53 μM). Furthermore, after 48 h of culture, it suppressed proliferation of nasopharyngeal cancer and hepatoma cells with IC50=50 and 90 μM, respectively.     

Critical Levels of Boron in Soils for Cauliflower (Brassica oleracea Var. Botrytis)

Influence of boron (B) application to cauliflower (Brassica oleracea var. botrytis) was investigated in a pot experiment taking 15 Inceptisols with four levels of B. The critical levels of B for deficiency, adequacy and toxicity in soil and in cauliflower plant were also determined. Hot-calcium chloride (CaCl₂) extractable B in these soils varied from 0.33 to 0.78 mg kg^-1 and its content for deficiency to cauliflower was 0.48 mg kg^-1. Boron application significantly increased cauliflower yield, plant B concentration and uptake of B. The critical plant B concentrations for deficiency, sufficiency and toxicity varied with the growth stages and the values being 26, 31 and 48 mg kg^-1 at 50 days of growth and 17, 24 and 35.5 mg kg^-1 at harvest, respectively. The study also recommends application of fertilizer B at the rate 0.9–4.5 kg ha^-1 for optimum B nutrition to cauliflower in Inceptisols of the Gangetic plains of India.     

Purification and partial characterization of broccoli (Brassica oleracea Var. Italica) peroxidases

Three peroxidase (POD) isoenzymes were purified from a soluble extract of broccoli stems. The acidic and neutral PODs were purified to homogeneity by using ion exchange and hydrophobic chromatography. The basic POD was purified by cation exchange and gel filtration chromatography. The neutral and basic PODs had molecular masses of approximately 43 kDa, and the acidic POD had a molecular mass of 48 kDa by SDS-PAGE. pI was approximately 4, 5, and 8 for acidic, neutral, and basic PODs, respectively. Optimum activity using guaiacol as the H donor was obtained at pH approximately 6 for both neutral and basic PODs and at pH approximately 4 for acidic POD. All three of the purified isoenzymes are glycosylated. Reaction rates with various substrates including guaiacol, guaiacol/MBTH, DMAB/MBTH, and ferulic acid/MBTH were different among the isoenzymes. K(m) and amino acid composition were also determined.     

Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea

An extraction and preparative HPLC method has been devised to simultaneously purify sulforaphane and sulforaphane nitrile from the seed of Brassica oleracea var. italica cv. Brigadier. The seed was defatted with hexane, dried, and hydrolyzed in deionized water (1:9) for 8 h. The hydrolyzed seed meal was salted and extracted with methylene chloride. The dried residue was redissolved in a 5% acetonitrile solution and washed with excess hexane to remove nonpolar contaminants. The aqueous phase was filtered through a 0.22-microm cellulose filter and separated by HPLC using a Waters Prep Nova-Pak HR C-18 reverse-phase column. Refractive index was used to detect sulforaphane nitrile, and absorbance at 254 nm was used to detect sulforaphane. Peak identification was confirmed using gas chromatography and electron-impact mass spectrometry. Each kilogram of extracted seed yielded approximately 4.8 g of sulforaphane and 3.8 g of sulforaphane nitrile. Standard curves were developed using the purified compounds to allow quantification of sulforaphane and sulforaphane nitrile in broccoli tissue using a rapid GC method. The methodology was used to compare sulforaphane and sulforaphane nitrile content of autolyzed samples of several broccoli varieties.     

甘蓝S位点受体激酶基因（SRK）编码区的甲基化分析

克隆了位于自交不亲和型甘蓝(Brassica oleracea)S位点受体激酶基因(SRK)上第一编码区中包含两个CCGG位点的目的片段,通过甲基化敏感限制性内切酶-PCR法,利用对甲基化敏感性不同的Msp Ⅰ和HpaⅡ分别对柱头乳突和花药基因组DNA及其PCR产物进行交叉组合式的酶切与电泳,首次对SRK基因编码区的特定DNA区段进行了甲基化分析.使用相同的SRK基因特异性引物时,柱头乳突和花药基因组DNA作为模板均可以扩增出清晰目标谱带,且目标谱带经Msp Ⅰ/Hpa Ⅱ完全酶切后可产生预期的谱带类型;而经Msp Ⅰ/Hpa Ⅱ完全酶切后的此二基因组DNA再作为模板进行PCR,均无特异性的目标谱带扩出.这些结果初步表明,自交不亲和型甘蓝花粉的SRK基因可能不存在甲基化封闭.     

甘蓝S-位点受体激酶（SRK）激酶结构域编码区分子特性及其与类硫氧还蛋白（THL1）相互作用检测

以甘蓝(Brassica oleracea L)E1为材料,采用PCR和RT-PCR技术,扩增S-位点受体激酶(SRK)激酶结构域(SRKE1)基因组DNA和cDNA序列,得到片段长度分别为1711和1 241 bp.序列分析表明,该基因组DNA序列由5个内含子和6个外显子组成,编码407个氨基酸;将sRKE1编码序列克隆到原核表达质粒pET43.1和真核表达质粒pPIC9K中,分别在表达宿主菌大肠杆菌(Eschedchia coli)BL21和毕赤酵母(Pichia pastoris)GS115中进行表达.利用表达的类硫氧还蛋白(THL1)与SRKE1孵育,结果显示SRKE1可与THL1结合并形成稳定的复合体.     

Protective effect of selenium in Broccoli (Brassica oleracea) plants subjected to cadmium exposure

The protective effect of selenium against the cadmium-induced oxidative effect in broccoli ( Brassica oleracea) plants was studied. Plants grown in hydroponic culture were supplied with selenium [as Se(IV)] and cadmium [as Cd(II)], individually or simultaneously. Cadmium accumulation in roots was noticeably higher than in the aerial parts of the plants, and this effect was even more acute when selenium was simultaneously added. Cadmium phytotoxicity was evidenced by an increase in the malondialdehyde (MDA) concentration in the roots and a decrease of photosynthetic pigment and tocopherol concentration in the aerial parts of the plant. The simultaneous addition of selenium alleviated cadmium-induced stress in the roots after 40 days of exposition. In the leaves, a more remarkable decrease of tocopherol and chlorophyll concentration was observed in the cadmium-enriched plants after 10 days of exposure. The results provided evidence that selenium supplementation helps the plant to minimize the cadmium oxidant effect. Tocopherol concentration in broccoli fruit of cadmium-supplied plants was not affected in comparison to control. However, the proportion of alpha-tocopherol increases with the addition of selenium. This response is important not only for the protective effect against oxidative damage in the plant but also in terms of human nutrition.     

Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the anticancer agent sulforaphane

In some cruciferous plants, epithiospecifier protein (ESP) directs myrosinase (EC 3.2.3.1)-catalyzed hydrolysis of alkenyl glucosinolates toward epithionitrile formation. Here, for the first time, we show that ESP activity is negatively correlated with the extent of formation of the health-promoting phytochemical sulforaphane in broccoli (Brassica oleracea L. ssp. italica). A 43 kDa protein with ESP activity and sequence homology to the ESP of Arabidopsis thaliana was cloned from the broccoli cv. Packman and expressed in Escherichia coli. In a model system, the recombinant protein not only directed myrosinase-dependent metabolism of the alkenyl glucosinolate epi-progoitrin [(2S)-2-hydroxy-3-butenyl glucosinolate] toward formation of an epithionitrile but also directed myrosinase-dependent hydrolysis of the glucosinolate glucoraphanin [4-(methylsulfinyl)butyl glucosinolate] to form sulforaphane nitrile, in place of the isothiocyanate sulforaphane. The importance of this finding is that, whereas sulforaphane has been shown to have anticarcinogenic properties, sulforaphane nitrile has not. Genetic manipulation designed to attenuate or eliminate expression of ESP in broccoli could increase the fractional conversion of glucoraphanin to sulforaphane, enhancing potential health benefits.     

Chemical fractions and response of cauliflower (Brassica oleracea var. botrytis) to soil applied boron

The present study was conducted with an objective to estimate the distribution of boron (B) application in various soil fractions and their plant response for assessing the availability in the soil. Two soils (alluvial and red soil) and five levels of B (0, 0.5, 1, 2 and 3?mg B kg^?1 soil) were applied in the pot experiment, and pots were sown with cauliflower (Sabour Agrim) arranged in a completely randomized block design (CRD) with three replications. Result showed that the curd yield of cauliflower increased significantly upto 2?mg B kg^?1 soil irrespective of soils. The percent yield increase was 14.78 and 15.01 in alluvial and red soil over the control, respectively. The initial total B content was 35.88 (alluvial soil) and 15.51 (red soil) mg·kg^?1. The mean content of Fraction I, II, III, IV and V in alluvial soil was 1.11, 1.54, 0.65, 1.49, and 95.18% and in red soil was 2.68, 4.47, 6.62, 2.50, and 83.59% of the total soil B, respectively. For changes in amount of B fractions due to B applications there was significant effect on all the fractions except Fraction II. The increase in apparent B uptake was 0.43?mg B kg^?1 in alluvial soil and 0.25?mg B kg^?1 in red soil over the control (0?mg B kg^?1 soil). Regression equation of yield and B fractions showed the relationship between first four fractions to the yield. Residual fraction was found to be collinear during calculation. Overall the study predicted the bioavailability and dynamics of B in the two distinct soils.     

Antioxidant capacity of different broccoli (Brassica oleracea) genotypes using the oxygen radical absorbance capacity (ORAC) assay

Antioxidant capacity of hydrophilic and lipophilic extracts from eight broccoli genotypes was compared using the oxygen radical absorbance capacity (ORAC) assay. Each genotype was analyzed for carotenoid, tocopherol, ascorbic acid, and flavonoid content. Results indicate that the antioxidant capacity of hydrophilic extracts ranged from 65.8 to 121.6 micromol trolox equivalents (TE)/g of tissue, and the capacity of lipophilic extracts ranged from 3.9 to 17.5 micromol TE/g. Ascorbic acid and flavonoid content of the hydrophilic extracts did not explain the total variation in antioxidant capacity of those extracts, suggesting either the presence of other antioxidant components that have yet to be identified or that the known antioxidants are producing synergistic effects. The carotenoids did correlate with antioxidant capacity of the lipophilic extracts and accounted for the majority of the variability in that fraction. The variability in hydrophilic and lipophilic antioxidant capacity found among these genotypes suggests that potential efficacy from antioxidants will vary considerably from genotype to genotype.     

甘蓝花粉特异表达的多聚半乳糖醛酸酶基因BoMF9的克隆与特征分析

根据白菜花粉特异的多聚半乳糖醛酸酶基因BcMF9的全长序列设计引物，通过PCR直接扩增的方法从结球甘蓝（Brassica. oleracea var. capitata）和芥蓝（B. oleracea var. alboglabra）中克隆得到BcMF9的同源基因BoMF9（BoMF9c和BoMF9l）。生物信息学分析和系统树构建发现BoMF9c和BoMF9l具有花粉特异表达的多聚半乳糖醛酸酶基因的序列特征。不同组织的表达特征分析发现它们分别在结球甘蓝和芥蓝的花蕾中特异表达。推测BoMF9c和BoMF9l可能在花粉萌发和花粉管伸长中起作用。     

effect of dose size on bioavailability of acylated and nonacylated anthocyanins from red cabbage (Brassica oleracea L. Var. capitata)

Recent studies indicate that anthocyanin intake conveys a variety of health benefits, which depend on absorption and metabolic mechanisms that deliver anthocyanins and their bioactive metabolites to responsive tissues. The anthocyanin bioavailability of red cabbage (Brassica oleracea L. var. capitata) was evaluated as reflected by urinary excretion of anthocyanins and anthocyanin metabolites. Twelve volunteers consumed 100, 200, and 300 g of steamed red cabbage (containing 1.38 micromol of anthocyanins/g of cabbage) in a crossover design. Anthocyanin concentration in cabbage extract and urine was measured by HPLC-MS/MS. Six nonacylated and 30 acylated anthocyanins were detected in red cabbage, and 3 nonacylated anthocyanins, 8 acylated anthocyanins, and 4 metabolites were present in urine. Mean 24 h excretion of intact anthocyanins increased linearly from 45 (100 g dose) to 65 nmol (300 g dose) for acylated anthocyanins and from 52 (100 g dose) to 79 nmol (300 g dose) for nonacylated anthocyanins. Urinary recovery of intact anthocyanins (percent of anthocyanin intake) decreased linearly from 0.041% (100 g dose) to 0.020% (300 g dose) for acylated anthocyanins and from 0.18% (100 g dose) to 0.09% (300 g dose) for nonacylated anthocyanins. Anthocyanin metabolites consisted of glucuronidated and methylated anthocyanins. The results show that red cabbage anthocyanins were excreted in both intact and metabolized forms and that recovery of nonacylated anthocyanins in urine was >4-fold that of acylated anthocyanins.     

The Roles of Diethylenetriamine Pentaacetate (DTPA) and Ethylenediamine Disuccinate (EDDS) in Remediation of Selenium from Contaminated Soil by Brussels Sprouts (Brassica oleracea var. gemmifera)

The objective of this study was to investigate the effects of adding different rates of diethylenetriamine pentaacetate (DTPA) at different concentrations (0, 0.5, 1, and 5 mmol kg⁻¹) and ethylenediamine disuccinate (EDDS) at 0, 5, 7.5, and 10 mmol kg⁻¹ on the capacity of Brussels sprouts plants to take up Se from soils contaminated with 0, 5, 10, and 15 mg kg⁻¹ NaSeO₄, under a greenhouse conditions. Results indicated that the application of DTPA and EDDS to Se-contaminated soils significantly affect plant Se concentration, Se uptake, and dry matter yield of plants. Se concentration in the plant leaves, stems, and roots increased with increase in DTPA and EDDS application doses, but total Se uptake increased from 0 to 1.0 and 7.5 mmol kg⁻¹ DTPA and EDDS application doses, respectively, and decreased after those levels due to toxic Se concentration for plant. Most plant available fractions and the carbonate, metal oxide, and organic matter-bound fractions increased linearly with Se application. At all DTPA and EDDS application rates, the Se concentrations in the leaves were about two to three times higher than those in the roots and about three to four times higher than those in the stems. This study suggests that the above-ground organs like leaf and shoots of Brussels sprouts can effectively be used in the removal of Se from soils contaminated with Se. Under the conditions in this experiment, Brussels sprouts were capable of removing 0.9–1.8 mg Se pot⁻¹ when harvested at maturity without any chelating agent take into consideration one growing season per year. Based on the data of present experiment, it would be necessary to approximately 57–67 growing seasons without EDDS and EDTA to remove all total Se from polluted soil. Selenium removal can be further increased 12- to 20-fold with 7.5 mmol kg⁻¹ EDDS and 1.0 mmol kg⁻¹ DTPA application, respectively.