Selenocysteine methyltransferase SMT catalyzed the synthesis of Se-methylselenocysteine to regulate the accumulation of glucosinolates and sulforaphane in broccoli

Selenocysteine methyltransferase(SMT)is a key enzyme involved in the Se metabolism pathway,and it is responsible for the catalysis of Se-methylselenocysteine(SeMSC)compound formation.Previous studies showed that selenium treatment activated SMT expression and promoted the accumulation of glucosinolates(GSLs)and sulforaphane,but the roles and functional mechanisms of SMT in mediating GSLs and sulforaphane synthesis remain unclear.In this study,we identified the BoSMT gene in broccoli and uncovered its roles in mediating GSLs biosynthesis.Transgenic assays revealed that BoSMT is involved in SeMSC biosynthesis in broccoli.More importantly,the contents of GSLs and sulforaphane were significantly increased in the BoSMT-overexpressing broccoli lines but decreased in the knockdown lines,suggesting that BoSMT played a positive role in regulating GSLs and sulforaphane synthesis.Further evidence indicated that BoSMT-mediated overaccumulation of GSLs and sulforaphane might be due to the increase in the endogenous SeMSC content.Compared with the mock(water)treatment,selenite-induced significantly increases of the SeMSC content in the BoSMT-knockdown plants partially compensated the phenotype of GSLs and sulforaphane loss.Compared with the mock treatment,exogenous SeMSC treatment significantly increased the contents of GSL and sulforaphane and activated GSL synthesis-related gene expression,suggesting that SeMSC acted as a positive regulator for GSL and sulforaphane production.Our findings provided novel insights into selenium-mediated GSLs and sulforaphane accumulation.The genetic manipulation of BoSMT might be a useful strategy for improving the dietary nutritional values of broccoli.

Transcriptomics integrated with metabolomics reveals the mechanism of CaCl_(2)-HCl electrolyzed water-induced glucosinolate biosynthesis in broccoli sprouts

Glucosinolates are important phytochemicals in Brassicaceae.We investigated the effect of CaCl_(2)-HCl electrolyzed water(CHEW)on glucosinolates biosynthesis in broccoli sprouts.The results showed that CHEW treatment significantly decreased reactive oxygen species(ROS)and malondialdeh yde(MDA)contents in broccoli sprouts.On the the 8^(th)day,compared to tap water treatment,the the total glucosinolate content of broccoli sprouts with CHEW treatment increased by 10.6%and calcium content was dramatically enhanced from 14.4 mg/g DW to 22.7 mg/g DW.Comparative transcriptome and metabolome analyses revealed that CHEW treatment activated ROS and calcium signaling transduction pathways in broccoli sprouts and they interacted through MAPK cascades.Besides,CHEW treatment not only promoted the biosynthesis of amino acids,but also enhanced the expression of structural genes in glucosinolate synthesis through transcription factors(MYBs,bHLHs,WRKYs,etc.).The results of this study provided new insights into the regulatory network of glucosinolates biosynthesis in broccoli sprouts under CHEW treatment.

Effects of fresh-cut and storage on glucosinolates profile using broccoli as a case study

Glucosinolates(GLS) contribute to the unique flavour, nutrition, and plant defence of the Cruciferous vegetables. Understanding the GLS changes through postharvest processing is essential for defined preservation. In this study, four different fresh-cut types, whole flower(W),floret(F), quarterly cut floret(QF) and shredded floret(FS) of broccoli, were stored for 0, 1, 2 and 3 day(s) to explore GLS responses to postharvest treatments. As a result, seven GLS were identified, mainly including glucoraphanin(RAA), neoglucobrassicin(NEO), and glucobrassicin(GBC)and accounting for 52.69%, 20.12% and 14.99% of the total GLS(21.92 ± 0.48) μmol · g ^(-1 )DW, respectively. FS had the sharpest decrease in GLS after three days of storage(6.55 ± 0.37) μmol · g-1DW, while QF had the least(10.16 ± 0.33) μmol · g ^(-1 )DW. All GLS components decreased over storage, except for 4-methoxyglucobrassicin(4 ME) in FS and QF, suggesting its key role in serious wound defence. The results suggested certain postharvest approaches influenced the flavour and nutrition of broccoli.

Analysis on Composition and Content of Glucosinolates in Broccoli Flowers and Leaves

Glucosinolate composition and content were evaluated in flowers and leaves of 12 different broccoli varieties. The results indicated that there were 9 glucosinolates in broccoli,namely Glucoiberin(IBE); Progoitrin(PRO);Sinigrin(SIN);Glucoraphanin(RAA);Gluconapin(NAP);4-Hydroxyglucobrassicin(4OH);Glucobrassicin(GBC);4-Methoxyglucobrassicin(4ME);Neoglucobrassicin(NEO).Total glucosinolate content in flowers was 1-5 times higher than in leaves. The predominant glucosinolate in broccoli was glucoraphanin.

Identification of MAM1s in Regulation of 3C Glucosinolates Accumulation in Allopolyploid Brassica juncea

Allopolyploid Brassica juncea is particularly enriched in sinigrin,a kind of 3C aliphatic glucosinolates(GSLs),giving rise to characteristic taste after picking.However,the molecular mechanism underlying 3C aliphatic GSLs biosynthesis in this species remains unknown.In this study,we genome-widely identified GSLs metabolic genes,indicating different evolutionary rate of GSLs metabolic genes between subgenomes of B.juncea.Eight methythioalkylmalate synthase(MAMs)homologs were identified from B.juncea,in which six MAM1s were located in chloroplast and the other two were not detected with any expression.Furthermore,BjMAM1-4,BjMAM1-5,and BjMAM1-6 displayed higher expression levels in leaves than other tissues.Silenced expression analysis revealed that BjMAM1-4 and BjMAM1-6 function in 3C and 4C aliphatic GSLs accumulation.The specificity of the substrate selection for the second cycle reaction is much lower than that of the first cycle,suggesting these genes may preferentially catalyze 3C aliphatic GSLs biosynthesis.Our study provides insights into the molecular mechanism underlying the accumulation of 3C aliphatic GSLs,thereby facilitating the manipulation of aliphatic GSLs content in B.juncea.

The Arabidopsis P450 protein CYP82C2 modulates jasmonateinduced root growth inhibition, defense gene expression and indole glucosinolate biosynthesis

Jasmonic acid （JA） is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies have established that JA also performs a critical role in several aspects of plant development. Here, we describe the characterization of the Arabidopsis mutantjasmonic acid-hypersensitivel-1 （jah1-1）, which is defective in several aspects of JA responses. Although the mutant exhibits increased sensitivity to JA in root growth inhibition, it shows decreased expression of JA-inducible defense genes and reduced resistance to the necrotrophic fungus Botrytis cinerea. Gene cloning studies indicate that these defects are caused by a mutation in the cytochrome P450 protein CYP82C2. We provide evidence showing that the compromised resistance of thejah1-1 mutant to B. cinerea is accompanied by decreased expression of JA-induced defense genes and reduced accumulation of JA-induced indole glucosinolates （IGs）. Conversely, the enhanced resistance to B. cinerea in CYP82C2-overexpressing plants is accompanied by increased expression of JA-induced defense genes and elevated levels of JA-induced IGs. We demonstrate that CYP82C2 affects JA-induced accumulation of the IG biosynthetic precursor tryptophan （Trp）, but not the JA-induced IAA or pathogen-induced camalexin. Together, our results support a hypothesis that CYP82C2 may act in the metabolism of Trp-derived secondary metabolites under conditions in which JA levels are elevated. Thejah1-1 mutant should thus be important in future studies toward understanding the mechanisms underlying the complexity of JA-mediated differential responses, which are important for plants to adapt their growth to the ever-changing environments.

MYB34, MYB51, and MYB 122 Distinctly Regulate Indolic Glucosinolate Biosynthesis in Arabidopsis thaliana

The MYB34, MYB51, and MYB122 transcription factors are known to regulate indolic glucosinolate （IG） biosynthesis in Arabidopsis thaliana. To determine the distinct regulatory potential of MYB34, MYB51, and MYB122, the accumulation of IGs in different parts of plants and upon treatment with plant hormones were analyzed in A. thaliana seedlings. It was shown that MYB34, MYB51, and MYB122 act together to control the biosynthesis of 13M in shoots and roots, with MYB34 controlling biosynthesis of IGs mainly in the roots, MYB51 regulating biosynthesis in shoots, and MYB122 having an accessory role in the biosynthesis of IGs. Analysis of glucosinolate levels in seedlings of myb34, myb51, myb122, myb34 myb51 double, and myb34 myb51 myb122 triple knockout mutants grown in the presence of abscisic acid （ABA）, salicylic acid （SA）, jasmonate （JA）, or ethylene （ET） revealed that： （1） MYB51 is the central regulator of IG synthesis upon SA and ET signaling, （2） MYB34 is the key regulator upon ABA and JA signaling, and （3） MYB122 plays only a minor role in JA/ET-induced glucosinolate biosynthesis. The myb34 myb51 myb122 triple mutant is devoid of IGs, indicating that these three MYB factors are indispensable for IG production under standard growth conditions.

Purification of a Sinapine-Glucoraphanin Salt from Broccoli Seeds

A sinapine (sinapoylcholine)-glucoraphanin salt has been isolated from broccoli seeds and characterized by NMR and mass spectrometry. This salt extraction method can be used to purify glucoraphanin free from contamination by glucoiberin.

Effect of CO_2 enrichment on the glucosinolate contents under different nitrogen levels in bolting stem of Chinese kale (Brassica alboglabra L.)

The effects of CO2 enrichment on the growth and glueosinolate （GS） concentrations in the bolting stem of Chinese kale （Brassica alboglabra L.） treated with three nitrogen （N） concentrations （5, 10, and 20 mmol/L） were investigated. Height, stem thickness, and dry weights of the total aerial parts, bolting stems, and roots, as well as the root to shoot ratio, significantly increased as CO2 concentration was elevated from 350 to 800 μl/L at each N concentration. In the edible part of the bolting stem, 11 individual GSs were identified, including 7 aliphatic and 4 indolyl GSs. GS concentration was affected by the elevated CO2 concentration, N concentration, and CO2×N interaction. At 5 and 10 mmol N/L, the concentrations of aliphatic GSs and total GSs significantly increased, whereas those ofindolyl GSs were not affected, by elevated atmospheric CO2. However, at 20 mmol N/L, elevated CO2 had no significant effects on the concentrations of total GSs and total indolyl GSs, but the concentrations of total aliphatic GSs significantly increased. Moreover, the bolting stem carbon （C） content increased, whereas the N and sulfur （S） contents decreased under elevated CO2 concentration in the three N treatments, resulting in changes in the C/N and N/S ratios. Also the C/N ratio is not a reliable predictor of change of GS concentration, while the changes in N and S contents and the N/S ratio at the elevated CO2 concentration may influence the GS concentration in Chinese kale bolting stems. The results demonstrate that high nitrogen supply is beneficial for the growth of Chinese kale, but not for the GS concentration in bolting stems, under elevated CO2 condition.

Effects of different cooking methods on health-promoting compounds of broccoli

The effects of five domestic cooking methods,including steaming,microwaving,boiling,stir-frying,and stir-frying followed by boiling(stir-frying/boiling),on the nutrients and health-promoting compounds of broccoli were investigated.The results show that all cooking treatments,except steaming,caused significant losses of chlorophyll and vitamin C and significant decreases of total soluble proteins and soluble sugars.Total aliphatic and indole glucosinolates were significantly modified by all cooking treatments but not by steaming.In general,the steaming led to the lowest loss of total glucosinolates,while stir-frying and stir-frying/boiling presented the highest loss.Stir-frying and stir-frying/boiling,the two most popular methods for most homemade dishes in China,cause great losses of chlorophyll,soluble protein,soluble sugar,vitamin C,and glucosinolates,but the steaming method appears the best in retention of the nutrients in cooking broccoli.

水杨酸处理对采后西兰花褪绿转黄及营养品质的影响

本实验探讨了水杨酸(salicylic acid,SA)处理对贮藏期间西兰花色泽以及营养品质的保护作用。结果表明,SA处理明显推迟了西兰花褪绿转黄进程,处理样本表现出较高的叶绿素含量、-a^(*)/b^(*)值、量子产量和荧光下降比,以及较低的L^(*)值和黄化指数。同时,与对照组相比,处理样品萝卜硫苷、芸薹葡糖硫苷、萝卜硫素、吲哚-3-甲醇及抗坏血酸(ascorbic acid,AsA)的损失显著受到抑制,总酚和总黄酮含量的积累明显增加;此外,处理组样本抗氧化能力明显提高,丙二醛含量显著降低。多变量统计分析直观地展示了感官变量及营养变量在两组样本中的分布,进一步证明SA处理对萝卜硫苷和AsA的保护作用更强,且可有效诱导抗氧化能力的提高。

外源谷胱甘肽对青花菜硫代葡萄糖苷合成的影响

为探究谷胱甘肽对硫代葡萄糖苷(GSLs)生物合成的影响,以现蕾期青花菜品种耐寒优秀为试验材料,研究不同浓度的还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)及丁硫堇(BSO)对青花菜花球中硫代葡萄糖苷及其相关底物含量、酶活性和基因表达的影响。结果表明,与CK(蒸馏水)相比,5 mg·L^(-1) GSH在处理48 h显著提高了青花菜花球中总硫苷、脂肪族硫苷含量,在24~48 h显著提高半胱氨酸(Cys)含量,在6~24 h显著提高了谷胱甘肽含量,在3~12 h显著提高了硫苷合成相关基因的表达量;45 mg·L^(-1) GSH处理在48 h显著降低了青花菜花球中总硫苷、脂肪族硫苷含量,在3~48 h则显著提高了半胱氨酸含量,在6~48 h则显著提高了谷胱甘肽含量,在3~12 h显著抑制了硫苷合成相关基因的表达。与CK相比,5 mg·L^(-1) GSSG处理下青花菜花球中总硫苷、脂肪族硫苷含量显著升高,而25、45及65 mg·L^(-1) GSSG处理则对总硫苷、脂肪族硫苷含量没有产生显著影响。综上所述,外源谷胱甘肽对硫苷含量的影响具有浓度效应,5 mg·L^(-1) GSH促进硫苷合成,45 mg·L^(-1) GSH抑制硫苷合成。

硫酸锌处理对西兰花芽苗关键活性成分影响及硫苷变化分子机制初步探讨

为探究喷施不同浓度硫酸锌对西兰花芽苗活性成分积累和抗氧化活性的影响,并从分子水平探讨硫酸锌对西兰花芽苗中硫苷的调控机制,本文以7 d西兰花芽苗为试验材料,采用不同浓度(0、1、2、4、8 mmol/L)硫酸锌(Zinc sulphate,ZnSO_(4))处理西兰花芽苗,测定西兰花芽苗叶绿素、抗坏血酸、总黄酮、总酚、硫苷含量、黑芥子酶活以及DPPH自由基清除能力,研究不同浓度ZnSO_(4)处理组对西兰花芽苗活性成分的影响,并且利用荧光定量PCR技术分析硫苷合成关键基因表达量的变化。结果表明,各处理组叶绿素含量均低于对照;抗坏血酸含量随硫酸锌浓度的增大呈上升趋势,各处理组均高于对照组。总酚含量在ZnSO_(4)浓度为8 mmol/L时含量最高且显著高于对照组(P<0.05);总黄酮、总硫苷和RAA含量、黑芥子酶活性及DPPH自由基清除率在ZnSO_(4)浓度为2 mmol/L时达最大值。在ZnSO_(4)浓度为2 mmol/L时CYP83A1、CYP79F1与FMO-GSOX2基因表达量显著提高(P<0.05);FMO-GSOX2与总硫苷、RAA、脂肪族硫苷、黑芥子酶呈极显著正相关(P<0.01);CYP79F1、CYP83A1与总硫苷、RAA、脂肪族硫苷呈显著性正相关(P<0.05)。说明FMO-GSOX2、CYP79F1和CYP83A1表达量的上调促进了总硫苷、脂肪族硫苷和RAA的合成。本研究结果表明,外源喷施适量硫酸锌能促进硫苷合成相关基因的表达,其中2 mmol/L ZnSO_(4)可有效增加西兰花芽苗中活性物质含量及抗氧化活性。

硫代葡萄糖苷浸种对青花菜种子萌发及生理特性的影响

以青花菜种子为试验材料,采用培养皿进行发芽试验,设置蒸馏水、200 mmol/L NaCl和15%PEG-60003种培养条件,研究不同质量浓度[0μg/mL(CK)、0.94μg/mL、1.88μg/mL、3.75μg/mL、7.50μg/mL、15.00μg/mL]硫代葡萄糖苷浸种对青花菜种子萌发指标、幼苗形态指标及生理指标的影响。研究结果表明,与CK相比,蒸馏水培养下,硫代葡萄糖苷浸种对青花菜种子的发芽率和发芽势没有显著影响,7.50μg/mL硫代葡萄糖苷浸种的青花菜根冠比显著提高;1.88~7.50μg/mL硫代葡萄糖苷浸种的青花菜POD活性显著高于对照,其中3.75μg/mL硫代葡萄糖苷浸种能显著提高青花菜的SOD和CAT活性,且MDA含量显著低于对照。盐胁迫下,7.50μg/mL硫代葡萄糖苷浸种能显著提高青花菜的SOD、POD活性,显著降低MDA含量。干旱胁迫下,硫代葡萄糖苷浸种均能显著提高青花菜种子萌发指标,显著降低青花菜MDA含量;0.94μg/mL硫代葡萄糖苷浸种能显著增加青花菜的主根长,1.88μg/mL和3.75μg/mL硫代葡萄糖苷浸种能显著提高青花菜的根冠比;15.00μg/mL硫代葡萄糖苷浸种能显著增加青花菜的POD活性。隶属函数分析结果表明,蒸馏水培养和干旱胁迫下3.75μg/mL硫代葡萄糖苷浸种及盐胁迫下7.50μg/mL硫代葡萄糖苷浸种的隶属函数值最高。综上所述,硫代葡萄糖苷浸种能有效缓解青花菜种子萌发期和幼苗期旱害和盐害,干旱胁迫下浸种最适质量浓度为3.75μg/mL,盐胁迫下浸种最适质量浓度为7.50μg/mL。

西兰花中葡萄糖异硫氰酸盐诱导人胃腺癌SGC-7901细胞凋亡的初步研究

目的探讨对西兰花中葡萄糖异硫氰酸盐(glucosinolates,GS)诱导人胃腺癌SGC-7901细胞凋亡的作用及其可能机制。方法不同质量浓度GS处理人胃腺癌SGC-7901细胞,通过SRB法、细胞形态学观察、流式细胞仪和激光共聚焦显微镜实验,观察GS对SGC-7901细胞的抑制率,对细胞凋亡和细胞周期的影响。结果1、10、100、1000μg/mL的GS作用于SGC-7901细胞72h后,抑制了SGC-7901细胞的增殖,其IC50为187.723μg/mL;300μg/mL的GS作用SGC-7901细胞24h后,细胞出现早期凋亡的形态;300、600、1200μg/mL的GS作用于SGC-7901细胞24h后,可见细胞凋亡率分别为(14.54±6.69)%、(10.11±6.25)%、(34.12±13.29)%,并且G2期细胞数目减少至0,对细胞周期有影响;100、200、300μg/mL的GS作用于SGC-7901细胞24h后,细胞内的Ca2+浓度升高,且随着GS给药剂量的增加而升高。结论GS能够促进人胃腺癌SGC-7901细胞凋亡,影响细胞周期,此作用可能是GS升高细胞内Ca2+的浓度而达到的。

反相离子对色谱法测定油菜籽(饼)中的硫苷

用反相离子对色谱法直接测定油菜籽(饼)中原始硫苷成分,在样品预处理中加入抗氧化剂以防止吲哚类硫苷的氧化分解。研究了主要色谱参数对硫苷组分保留值和选择性的影响,采用烯丙基硫苷或苯甲酸作内标,对硫苷的6种组分进行了定性定量的测定。

不同类型花椰菜硫代葡萄糖苷组分与含量分析

采用HPLC法测定了3种类型15个花椰菜品种的硫代葡萄糖苷（Glucosinolates,简称硫苷）组分及含量。结果表明：供试花椰菜品种均含有9种硫苷,包括5种脂肪族硫苷：3-甲基硫氧烯丙基硫苷（Glucoiberin,IBE）、2-羟基-3-丁烯基硫苷（Progoitrin,PRO）、2-丙烯基硫苷（Sinigrin,SIN）、4-甲基硫氧丁基硫苷（Glucoraphanin,RAA）和3-丁烯基硫苷（Gluconapin,NAP）;4种吲哚族硫苷：4-羟基吲哚基-3-甲基硫苷（4-Hydroxyglucobrassicin,4OH）、3-甲基吲哚基硫苷（Glucobrassicin,GBC）、4-甲氧基吲哚基-3-甲基硫苷（4-Methoxyglucobrassicin,4ME）和1-甲氧基吲哚基-3-甲基硫苷（Neoglucobrassicin,NEO）。各品种的吲哚族硫苷总含量均高于脂肪族硫苷总含量,其中NEO和GBC是花椰菜的主要硫苷组分,分别占总硫苷含量的42.61%和35.02%。不同花椰菜品种的硫苷总含量差异较大,变异范围在0.3323~4.8728μmol·g-1（FW）之间;紫花菜的总硫苷含量最高,松花菜其次,紧花菜最低。

西兰花中硫代葡萄糖苷的提取及抑菌试验初报

采用甲醇法提取西兰花中硫代葡萄糖苷,利用重量法测定其中硫代葡萄糖苷的含量,得出硫代葡萄糖苷最佳提取条件,并对硫代葡萄糖苷提取液进行酸水解,通过抑菌圈实验,分析了西兰花中硫代葡萄糖苷水解产物异硫氰酸酯类的抑菌性。其结果表明,采用80%甲醇溶液、料液比为10:40(w/v)、80℃为提取的最佳条件,西兰花花蕾中硫代葡萄糖苷的含量最高,达到39.90μ mol/g;花茎中为18.28μ mol/g;叶中为11.86μ mol/g。实验结果表明异硫氰酸酯类对金黄色葡萄球菌(Staphylococcus aureus)、白葡萄球菌(White staphylococcus)、枯草芽孢杆菌(Bacillus subtilis)大肠杆菌(Escherichia coli)的抑菌作用均较明显。

西兰花中葡萄糖异硫氰酸盐对S_(180)小鼠抗氧化功能的影响

目的:研究西兰花中葡萄糖异硫氰酸盐(glucosinolates,GS)对S180小鼠抗氧化功能的影响。方法:以S180实体瘤小鼠为模型,考察GS对S180小鼠的抑瘤率、免疫脏器指数的影响;运用试剂盒测定S180小鼠红细胞中SOD和CAT,血清中GSH及全血中红细胞MDA含量。结果:GS可显著抑制S180实体瘤的生长(P<0.01),显著升高S180小鼠胸腺指数和脾指数(P<0.05,P<0.01);可显著提高荷瘤小鼠红细胞中SOD、CAT活力(P<0.05,P<0.01),显著地提高血清中GSH的活力(P<0.01),同时显著降低全血中红细胞MDA含量(P<0.05,P<0.01)。结论:GS对S180小鼠实体瘤的生长具有显著的抑制作用,其作用机制可能与提高荷瘤机体的抗氧化功能有关。

西兰花中葡萄糖异硫氰酸盐诱导HepG2细胞凋亡

目的:研究西兰花中葡萄糖异硫氰酸盐(glucosinolates,GS)诱导肝癌HepG2细胞凋亡作用及其机制。方法:不同浓度GS处理肝癌HepG2细胞,通过SRB法、细胞形态学观察、流式细胞仪和激光共聚焦显微镜观察GS对HepG2细胞的生长抑制作用及其对细胞凋亡的影响。结果:GS可抑制HepG2细胞的增殖,其GI50为318.4μg·mL-1;300μg·mL-1的GS作用HepG2细胞24h后,细胞出现早期凋亡的形态;300、600μg·mL-1的GS对HepG2细胞凋亡率分别为(17.22±3.45)%和(25.50±5.72)%;100、200、300μg·mL-1的GS作用于HepG2细胞24h后,细胞内的Ca2+浓度显著升高(P<0.05,P<0.01)。结论:GS能够诱导人肝癌细胞系HepG2细胞凋亡,其机制与GS升高细胞内Ca2+浓度有关。