Structure-activity relationships of flavonoids in the cellular antioxidant activity assay

Chemical antioxidant activity assays are used extensively to evaluate the potential bioactivity of plant foods and their phytochemical constituents, but they do not mimic the complexity of biological systems. The cellular antioxidant activity (CAA) activity assay was developed to be a more biologically relevant model to measure antioxidant activity. Structure-activity relationships of flavonoids have been determined in many chemistry antioxidant activity assays, and they vary with the protocols. The objective of this study was to determine structure-activity relationships of selected flavonoids in the CAA assay. The structures that conferred flavonoids with the most antioxidant activity in the CAA assay were a 3',4'- o-dihydroxyl group in the B-ring, a 2,3-double bond combined with a 4-keto group in the C-ring, and a 3-hydroxyl group. Isoflavones had no cellular antioxidant activity. Flavanols with a galloyl moiety had higher antioxidant activity than those without, and a B-ring 3',4',5'-trihydroxyl group further improved their efficacy. ORAC values for flavonoids were not related to their CAA values. Knowledge of structure-activity relationships in the CAA assay may be helpful in assessing potential in vivo antioxidant activity of flavonoids.     

Structure-activity relationships of derivatives of fusapyrone, an antifungal metabolite of Fusarium semitectum

Fusapyrone (1) and deoxyfusapyrone (2) are two 3-substituted-4-hydroxy-6-alkyl-2-pyrones isolated from Fusarium semitectum that have considerable antifungal activity against molds. Because of their low zootoxicity and selective action they are potentially utilizable along with biocontrol yeasts for control of postharvest crop diseases. Seven derivatives of 1 (3 and 5-10) and one derivative of 2 (4) were obtained by chemical modifications of the glycosyl residue, the 2-pyrone ring, the aliphatic chain, or a combination thereof, and a structure-activity correlation study was carried out with regard to their zootoxicity and antifungal activity. Derivatives 7-10, as well as 1, were slightly zootoxic in Artemia salina (brine shrimp) bioassays, whereas pentaacetylation of 1 into 3, 5, and 6 resulted in a strong increase in toxicity. Compound 4, the tetraacetyl derivative of 2, was as toxic as 2. Because the structural changes of 1 that resulted in an increase of biological activity in A. salina bioassay were those that affected mainly the water solubility of the molecule, it appears that toxicity is related to hydrophobicity. Compounds 1 and 2 showed strong antifungal activity toward Botrytis cinerea, Aspergillus parasiticus, and Penicilliun brevi-compactum (minimum inhibitory concentration at 24 h = 0.78-6.25 microg/mL). Among derivatives 3-10, only compounds 7, 9, and 10 retained some activity, limited to B. cinerea and at high concentration (25-50 microg/mL). None of the compounds 1-10 inhibited the growth of the biocontrol yeasts Pichia guilliermondii and Rhodotorula glutinis at the highest concentration tested (50 microg/mL).     

拟南芥泛素家族的全基因组分析

泛素家族是一类含有保守性泛素结构域的蛋白质统称,主要通过ATP依赖性的泛素-蛋白水解酶复合体通路选择性降解细胞蛋白的各种生理活动。本研究基于HMM模型,已知泛素氨基酸序列作为训练集,搜索拟南芥信息资源数据库并鉴定AtUBQ家族成员,然后对这些基因编码的蛋白质序列进行基因结构分析、染色体定位、多序列联配、系统发育树构建和组织差异表达分析。结果表明,AtUBQ家族中共有13个推定的AtUBQ基因,命名为AtUBQ01~AtUBQ13,均属无内含子基因且结构基本相同,非均匀分布于拟南芥5条染色体;AtUBQ家族可划分为A、B和C3个亚家族,其中76.92%的基因编码蛋白质属于A亚家族;EST搜索发现除AtUBQ02和AtUBQ07基因无EST数据支持外,余下的AtUBQ基因在拟南芥根、芽和叶等7个组织中呈现差异表达,仅见AtUBQ08和AtUBQ10基因在上述7个组织中均表达,而AtUBQ03基因仅表达于叶中。本研究结果可为进一步开展该家族的生物学功能和分子进化机制的研究提供基础资料。     

拟南芥低氮耐性突变体的初步筛选

拟南芥是十字花科拟南芥属植物,分布广,有许多生态型.它具有体积小、生长期短、基因组小等特点.近年来,作为植物研究的重要模式材料,在植物研究的突破性进展中扮演着重要角色.科学家们利用拟南芥诱导突变产生氮、磷、钾以及对铝、镉敏感的突变体,对研究植物营养机理和遗传控制起了很大的促进作用.但植物营养界利用拟南芥的工作还很少.     

Metabolic profiling of root exudates of Arabidopsis thaliana

In addition to accumulating biologically active chemicals, plant roots continuously produce and secrete compounds into their immediate rhizosphere. However, the mechanisms that drive and regulate root secretion of secondary metabolites are not fully understood. To enlighten two neglected areas of root biology, root secretion and secondary metabolism, an in vitro system implementing root-specific elicitation over a 48-day time course was developed. After roots of Arabidopsis thaliana had been elicited with salicylic acid, jasmonic acid, chitosan, and two fungal cell wall elicitors, the secondary metabolites subsequently secreted were profiled. High-performance liquid chromatography was used to metabolically profile compounds in the root exudates, and 289 possible secondary metabolites were quantified. The chemical structures of 10 compounds were further characterized by (1)H and (13)C NMR: butanoic acid, trans-cinnamic acid, o-coumaric acid, p-coumaric acid, ferulic acid, p-hydroxybenzamide, methyl p-hydroxybenzoate, 3-indolepropanoic acid, syringic acid, and vanillic acid. Several of these compounds exhibited a wide range of antimicrobial activity against both soil-borne bacteria and fungi at the concentration detected in the root exudates.     

Structure-activity relationships of tea compounds against human cancer cells

The content of the biologically active amino acid theanine in 15 commercial black, green, specialty, and herbal tea leaves was determined as the 2,4-dinitrophenyltheanine derivative (DNP-theanine) by a validated HPLC method. To define relative anticarcinogenic potencies of tea compounds and teas, nine green tea catechins, three black tea theaflavins, and theanine as well as aqueous and 80% ethanol/water extracts of the same tea leaves were evaluated for their ability to induce cell death in human cancer and normal cells using a tetrazolium microculture (MTT) assay. Compared to untreated controls, most catechins, theaflavins, theanine, and all tea extracts reduced the numbers of the following human cancer cell lines: breast (MCF-7), colon (HT-29), hepatoma (liver) (HepG2), and prostate (PC-3) as well as normal human liver cells (Chang). The growth of normal human lung (HEL299) cells was not inhibited. The destruction of cancer cells was also observed visually by reverse phase microscopy. Statistical analysis of the data showed that (a) the anticarcinogenic effects of tea compounds and of tea leaf extracts varied widely and were concentration dependent over the ranges from 50 to 400 microg/mL of tea compound and from 50 to 400 microg/g of tea solids; (b) the different cancer cells varied in their susceptibilities to destruction; (c) 80% ethanol/water extracts with higher levels of flavonoids determined by HPLC were in most cases more active than the corresponding water extracts; and (d) flavonoid levels of the teas did not directly correlate with anticarcinogenic activities. The findings extend related observations on the anticarcinogenic potential of tea ingredients and suggest that consumers may benefit more by drinking both green and black teas.     

转防治软腐病基因拟南芥的获得及其抗病性研究

AHLs是细菌群体感应中的关键信号分子,参与包括导致作物软腐病的胡萝卜软腐欧文氏菌在内的许多植物病原菌致病因子的表达。AHL内酯酶能降解AHLs使其达不到临界浓度,从而阻断了病原菌的致病过程。将克隆到的AHL内酯酶基因SS10置于CaMV 35S启动子下构建双元载体,通过根癌农杆菌介导转化拟南芥。通过抗性筛选和分离比获得纯合转基因植物种子,进行了PCR及RT-PCR鉴定,证明目的基因在转基因拟南芥中整合并表达。初步抗病性实验表明,部分转基因植株具有显著的抗软腐病能力。     

Cloning of nicotianamine synthase genes from Arabidopsis thaliana

Nicotianamine synthase (NAS) catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine (NA). NA is present in all the plants; it chelates metal cations, and is considered to play a role in metal homeostasis in plants. Moreover, in graminaceous monocotyledonous plants, NA is an essential intermediate in the biosynthesis of mugineic acid family phytosiderophores (MAs). In order to identify the gene encoding NAS in dicotyledonous plants, Arabidopsis thaliana databases were searched using the nucleotide sequence of the NAS gene from barley (HvNAS), which we have recently isolated. We found several ESTs and three genomic sequences highly homologous to HvNAS in the databases. Based on these nucleotide sequences and that of HvNAS, we designed 2 sets of primers to isolate the NAS orthologues in Arabidopsis and succeeded in obtaining three DNA clones encoding AtNAS (AtNAS1, 2, and 3). These clones were expressed in Escherichia coli and their protein products displayed the NAS activity. The expression of AtNASl was detected in both shoots and roots of A. thaliana by RT-PCR; AtNAS3 expression was only detected in the shoots. In contrast, AtNAS2 expression was not detected in any organs.     

SO2诱导拟南芥气孔保卫细胞致死效应研究

以拟南芥叶片下表皮为材料,研究了SO2体内衍生物——亚硫酸钠和亚硫酸氢钠混合液（3：1,mmol·L^-1／mmol·L^-1）对气孔保卫细胞的致死作用。结果表明,浓度0．5～4．5mmol·L^-1的SO2衍生物处理表皮3h可引起保卫细胞死亡,细胞死亡率呈浓度依赖性增高。抗坏血酸（AsA）或过氧化氢酶（CAT）与SO2衍生物共同作用时,保卫细胞死亡率显著降低。Ca^2＋合剂乙二醇双四乙酸（EGTA）或Ca^2＋通道抑制剂LaCl3与SO2衍生物共同作用时,保卫细胞死亡率亦显著降低。研究发现,一定浓度的SO2可诱导拟南芥保卫细胞死亡,胁迫可能通过诱导活性氧、激活质膜钙通道,造成胞外Ca^2＋内流,引发细胞死亡。     

Arabidopsis thaliana PRL1 is involved in low-calcium tolerance

Calcium (Ca) deficiency symptoms in plants often occur in agriculture; however, little is known about the mechanisms for adaptation to low-Ca conditions. To understand the mechanisms, we screened for Arabidopsis thaliana (L.) Heynh mutants sensitive to low Ca. Here, we describe one of the mutants, lcs1-1, isolated from the screen. The relative shoot growth of the mutant was reduced under the low-Ca conditions compared with the wild-type plants. Genetic mapping and genome resequencing revealed that lcs1-1 has one nonsynonymous mutation in the region of the chromosome responsible for the phenotype. The mutation is in Pleiotropic Regulatory Locus 1 (PRL1). An allelism test between lcs1-1 and a T-DNA inserted allele of prl1 demonstrates that the causal gene of lcs1-1 is PRL1. It has been reported that PRL1 is involved in sugar metabolism; however, the involvement of PRL1 in low-Ca tolerance has not been reported. Our results suggest a new insight connecting sugar metabolism with a mechanism for low-Ca tolerance in plants.     

Volatile compound biosynthesis by green leaves from an Arabidopsis thaliana hydroperoxide lyase knockout mutant

The degradation of polyunsaturated fatty acids through the lipoxygenase pathway is responsible for the production of volatile compounds that confer green sensory notes to the aroma of fruits and vegetables. The peroxidation of free linoleic or linolenic acid by action of lipoxygenase and then the lysis of the resulting hydroperoxides, through a reaction catalyzed by the hydroperoxide lyase, are the most determinant steps of this pathway. This work analyzes the impact of the hydroperoxide lyase depletion on the volatile composition of leaves from Arabidopsis thaliana that is used as model system. The work involves the characterization of the volatile profiles of the Arabidopsis plants followed by a study of the metabolism of radio-labeled linoleic acid and determinations of lipoxygenase activity. Hydroperoxide lyase-knockout plants show similar levels of C6 compounds, but the total amount of C5 compounds is 4-fold higher in mutant plants. The perspectives of production of vegetable products with a modified aroma by genetically engineering the lipoxygenase pathway were discussed with respect to the statistical results.     

Glucosinolate Profiles of Arabidopsis thaliana in Response to Cadmium Exposure

The influence of cadmium on growth, cadmium accumulation, composition, and content of glucosinolates was investigated in Arabidopsis thaliana after 4 weeks of growth in hydroponics. Accumulation of 3,820 and 321 μg Cd g^?1 dry weight in the roots and leaves of A. thaliana, respectively, exposed to 50 μM Cd. Cadmium treatment significantly decreased the total concentration of glucosinolates both in the leaves and roots. Cd-induced alteration of total glucosinolate content in the roots was mainly due to the decrease of indolyl-glucosinolates. In the Cd treatment leaves, significant decreases were, respectively, detected for glucoibervirin and 4-methoxyglucobrassicin (P?<?0.01), while other glucosinolate levels did not decrease significantly. In response to cadmium, the three indolyl-glucosinolates all showed significant decreases in the roots. The distinctive influence of cadmium on glucosinolate profiles in Cd-sensitive A. thaliana may be of great ecological importance, decreasing the resistance to phytophage attack. Taken together, our data is discussed in relation to jasmonic acid and salicylic acid as possible molecules that modulate the alteration of glucosinolate profiles in response to cadmium. The similar effects of Cd treatment on the levels of individual glucosinolates in leaves and roots were observed at higher-concentration cadmium treatment (100 μM Cd).     

Structure-activity relationships of flavonoids as potential inhibitors of glycogen phosphorylase

Flavonoids are ubiquitous components in vegetables, fruits, tea, and wine. Therefore, they are often consumed in large quantities in our daily diet. Several flavonoids have been shown to have potential as antidiabetic agents. In the present study, we focused on inhibition of glycogen phosphorylase (GP) by flavonoids. 6-Hydroxyluteolin, hypolaetin, and quercetagetin were identified as good inhibitors of dephosphorylated GP (GPb), with IC 50 values of 11.6, 15.7, and 9.7 microM, respectively. Furthermore, a structure-activity relationship study revealed that the presence of the 3' and 4' OH groups in the B-ring and double bonds between C2 and C3 in flavones and flavonols are important factors for enzyme recognition and binding. Quercetagetin inhibited GPb in a noncompetitive manner, with a K i value of 3.5 microM. Multiple inhibition studies by Dixon plots suggested that quercetagetin binds to the allosteric site. In primary cultured rat hepatocytes, quercetagetin and quercetin suppressed glucagon-stimulated glycogenolysis, with IC 50 values of 66.2 and 68.7 microM, respectively. These results suggested that as a group of novel GP inhibitors, flavonoids have potential to contribute to the protection or improvement of control of diabetes type II.     

手性除草剂2甲4氯丙酸对映体在拟南芥中吸收转运及亚细胞分布的差异研究

为探明不同手性构型的2甲4氯丙酸(mecoprop,MCPP)在双子叶植物中的吸收、代谢等差异,以手性14C-MCPP对映体为示踪剂,野生哥伦比亚型拟南芥为受试植物,研究实验室条件下不同手性构型的MCPP在拟南芥中的吸收转运与亚细胞分布特征.结果表明,拟南芥对14C-MCPP 2种对映体的吸收效率分别为90.45％(1...     

Natural variation of Arabidopsis thaliana root architecture in response to nitrate availability

Root system architecture (RSA) plays a crucial role in nutrient and water uptake in plants. RSA can be modulated to improve nitrogen use efficiency (NUE) of crops, decreasing the need for fertilization. In this study, we analyzed 12 root parameters in 48 Arabidopsis accessions grown in media with low or high nitrogen (N). Low N repressed six root traits and enhanced six others. A principal component analysis showed that six components contributed to 90.08% of N availability. The primary component contributed to 35.64% of the variation and was associated with LR characteristics. The 48 Arabidopsis accessions were divided into five response groups. Stw-0 had the highest biomass production and a high N concentration in both roots and shoots, independent of growth conditions. H55 had numerous LRs at low N. Our results provide N-responsive parameters suitable for mapping of quantitative trait loci related with root morphology.     

Structural characterization of the major flavonoid glycosides from Arabidopsis thaliana seeds

Information gains from the seed of the model plant Arabidopsis thaliana (Brassicaceae) have greatly contributed to a better understanding of flavonoid synthesis and may be used for crop improvement. However, exhaustive identification of the flavonoid accumulated in Arabidopsis seed was still lacking. Complementary investigations of seed flavonoids by LC-ESI-MS, LC-ESI-MS-MS, and NMR spectroscopy in Arabidopsis led to full characterization of monoglycosides, namely, quercetin 3-O-alpha-rhamnopyranoside and kaempferol 3-O-alpha-rhamnopyranoside, and diglycosides, namely, quercetin and kaempferol 3-O-beta-glucopyranoside-7-O-alpha-rhamnopyranoside and quercetin and kaempferol 3,7-di-O-alpha-rhamnopyranoside. Interestingly, the tt7 mutant that lacks flavonoid-3'-hydroxylase and consequently accumulates only kaempferol derivatives was shown to contain three additional products, kaempferol 3-O-beta-glucopyranoside, kaempferol 3-O-alpha-rhamnopyranoside-7-O-beta-glucopyranoside, and the triglycoside kaempferol 3-O-beta-[alpha-rhamnopyranosyl(1-->2)-glucopyranoside]-7-O-alpha-rhamnopyranoside. Taken together these results allow a scheme for flavonol glycosylation to be proposed.     

STMP在拟南芥分枝发育过程中的功能鉴定

拟南芥(Arabidopsis thaliana)中含有脂/固醇结合的相关脂转移蛋白结构域(steroidogenic acute regulatory related lipid transfer,START)的膜相关蛋白(START domain containing-membrane related protein,STMP),STMP是START保守域(第84～295位)功能未知的蛋白质,由440个氨基酸组成、具有跨膜片段、属于磷脂酰胆碱转运蛋白.为明确STMP在拟南芥发育调控过程中的作用,深入了解拟南芥发育调控的分子机理,本研究用35S强启动子启动START结构域,构建START结构域的过表达载体,利用农杆菌(Agrobacterium tumefaciens)侵染花序法把STMP的过表达载体转入野生型拟南芥,获得过表达STMP的转基因拟南芥,并用qRT-PCR技术进行验证;分析转基因拟南芥分枝发育状况,明确STMP在拟南芥分枝调控过程中的作用;用qRT-PCR技术分析STMP的时空表达特性;构建STMP和绿色荧光蛋白(green fluorescent protein,GFP)的融合蛋白载体,把此载体转入野生型拟南芥中,对转基因拟南芥中GFP进行荧光观察,从而对STMP进行亚细胞定位.本研究获得了过表达STMP的转基因株系;过表达STMP的转基因拟南芥分枝比野生型明显增多,突变体stmp的分枝减少;STMP的时空表达特性分析表明,茎中STMP的表达量最高,STMP定位在细胞质膜和细胞质中.结果表明STMP可以促进拟南芥的分枝发育,本研究对完善拟南芥分枝调控机制具有重要的意义,可以为植物株型的定向设计提供理论依据.     

Selective one-step extraction of Arabidopsis thaliana seed oleosins using organic solvents

Oleosins are hydrophobic proteins from oleaginous seeds, surrounding and stabilizing oil bodies. They are known to display interesting interfacial properties. Specific sera were raised against four different A. thaliana oleosins and used in dot-blot assays for oleosin quantification. These assays were used to set up extraction of oleosins from A. thaliana seeds. One mixture of chloroform/methanol gave optimal oleosin extraction. Extracted proteins represented 9% of seed proteins and were identified by immunoblot and proteomic analyses. Oleosins accounted for 79% of the extracted proteins. This simple one-step procedure allows selective extraction and concentration of oleosins from seeds without tedious oil body purification. Oleosin extract was indeed used to demonstrate the presence of the rare oleosin S5 in mature seeds. Moreover, this method will be useful to investigate the potential use of oleosins as emulsifier and to question their possible allergenicity.