Novel flavonol 3-sulfotransferase. Purification, kinetic properties, and partial amino acid sequence.

A flavonol sulfotransferase (EC 2.8.2.-), which catalyzes the transfer of the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the 3-hydroxyl group of flavonol aglycones, has been purified to apparent homogeneity from Flaveria chloraefolia. The specific activity of flavonol 3-sulfotransferase was enriched 2000-fold, as compared with the homogenate, with a recovery of 9%. The molecular mass of the native and denatured enzyme was found to be 34.5 kDa, suggesting that the active from of the enzyme is a monomer. The enzyme exhibited expressed specificity for position 3 of flavonol aglycones, showed two activity optima at pH 6.0 and 8.5, did not require divalent cations, and was not inhibited by either EDTA or sulfhydryl group reagents. The results of substrate interaction kinetics and product inhibition are consistent with an Ordered Bi Bi mechanism where 3'-phosphoadenosine 5'-phosphosulfate is the first substrate to bind to the enzyme and 3'-phosphoadenosine 5'-phosphate is the final product to be released. The amino acid sequence of two peptides representing 17 and 33 amino acids showed no significant sequence similarity with the amino acid sequences reported for animal sulfotransferases. Antibodies raised against F. chloraefolia 3-sulfotransferase were found to cross-react with the 3'- and 4'-sulfotransferase activities of the same plant, suggesting that the three enzymes are structurally related.     

3'-Phosphoadenosine 5'-phosphosulfate binding site of flavonol 3-sulfotransferase studied by affinity chromatography and 31P NMR

The function of Lys-59, Arg-141, and Arg-277 in PAPS binding and catalysis of the flavonol 3-sulfotransferase was investigated. Affinity chromatography of conservative mutants with PAPS analogues allowed us to determine that Lys-59 interacts with the 5' portion of the nucleotide, while Arg-141 interacts with the 3' portion, confirming assignments deduced from the crystal structure of mouse estrogen sulfotransferase [Kakuta, Y., Pedersen, L. G., Carter, C. W. , Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908]. The affinity chromatography method could be used to characterize site-directed mutants for other types of enzymes that bind nucleoside 3',5'- or 2',5'-diphosphates. 31P NMR spectra of enzyme-PAP complexes were recorded for the wild-type enzyme and K59R and K59A mutants. The results of these experiments suggest that Lys-59 is involved in the determination of the proper orientation of the phosphosulfate group for catalysis.     

Fusicoccin action in cell-suspension cultures of Corydalis sempervirens Pers.

Mid-log-phase cell suspensions of Corydalis sempervirens Pers., when incubated in micromolar or submicromolar concentrations of fusicoccin, strongly acidified the culture medium. High-affinity fusicoccin-binding sites were found in microsomes prepared from these cells using the radioligand [³H]-9-norfusicoccin-8-alcohol. Binding was saturable with an apparent dissociation constant (K _d) of 2.8 nM, a pH optimum of 6.0, a temperature optimum of 35° C and was rapid (t_1/2 = 8 min). The site abundance was 0.76±0.17 pmol · (mg of protein)^–1. In the same membrane preparations, the K⁺, Mg²⁺-ATPase (EC 3.6.1.3) was characterized. The enzyme was highly vanadate-sensitive (IC₅₀=6.5 M) and nucleotide-specific (ATPNTP), had a pH optimum of 6.2, an apparent K _m for ATP of 0.23±0.12 mM, and V _max of 10.6±1.8 nkat (mg of protein)^–1. Fusicoccin doubled V _max and lowered, by a factor of 2, the apparent K _m for ATP of the enzyme when the cells were incubated with the toxin for 30 min prior to homogenization of the cells. The stimulation of the enzyme was also pronounced when fusicoccin was added to the homogenization medium just prior to homogenization of the cells, but was slight to zero when the toxin was added at the microsomal stage. The pronounced stimulatory effect of fusicoccin on the ATPase was seen at pH 7.1, i.e. at a pH typical for the cytoplasmic compartment, but was not detectable at pH 6.2, the pH optimum of the enzyme. The implications of these findings for an understanding of fusicoccin action are discussed.Abbreviations [³H]ABE-FC 9-nor-8-(4-azido-3,5-[³H]-benzoyl-diaminoethyl)-fusicoccin - FC fusicoccin - FCol 9-norfusicoc-cin-8-alcohol - Mes 2(N-morpholino)ethanesulfonic acid This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG and the Fonds der Chemischen Industrie, Frankfurt, FRG (literature provision).     

入侵植物黄顶菊与本地植物的竞争

温室和田间试验条件下,对黄顶菊与4种本地植物(马唐、牛筋、反枝苋和苘麻)在3种混生比例下的竞争效应进行了研究.温室试验结果表明:黄顶菊与马唐或牛筋混生所占比例低时,其株高受到明显抑制,在混生比例相同或黄顶菊所占比例高时,其株高未受到抑制;与反枝苋或苘麻混生时,黄顶菊的株高受到明显抑制,而反枝苋和苘麻的株高未受到抑制;黄顶菊与这4种本地植物混生,相对产量总和接近1;与马唐混生时,黄顶菊在高比例下的相对产量与1接近,而在其他比例下的相对产量均<1;与牛筋、反枝苋和苘麻混生时,黄顶菊的相对产量均显著<1;与4种本地植物混生,黄顶菊的竞争攻击力系数均<0.田间试验和温室试验结果一致.研究表明,黄顶菊与马唐、牛筋、反枝苋和苘麻利用相同的营养资源,其竞争力比供试的本地植物弱.     

三种牧草植物对黄顶菊田间替代控制

在田间条件下,采用替代试验对比研究了3种1年生牧草——高丹草、紫花苜蓿和欧洲菊苣与入侵我国华北地区的一种外来植物黄顶菊之间的相对竞争表现;通过设置不同牧草与黄顶菊组合的替代比例(牧草与黄顶菊分别单种及1∶1、1.5∶1和2∶1比例混种),建立了3种牧草与黄顶菊的田间替代试验区。结果表明:随着3种牧草替代比例的增加,其盖度也逐渐上升,均对黄顶菊表现不同程度的抑制,其中高丹草出苗较黄顶菊更早且具有更强的遮阴能力,在各混种替代组合中完全抑制了黄顶菊生长,抑制率达100%;紫花苜蓿和欧洲菊苣与黄顶菊的混种种群中,黄顶菊的生物量、株高等均极显著低于对照,且在中等替代比例(1.5∶1)下对黄顶菊的抑制效果为最佳,抑制率分别为87.7%和96.2%;在与3种牧草竞争的条件下,黄顶菊相对产量均显著1.0,表明黄顶菊种间竞争显著小于种内竞争,使该外来种生长受到有效抑制。在黄顶菊已入侵和易于入侵的生境建立牧草替代种群是进行生态重建和保持当地生物多样性的有效手段。     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

The biosynthesis of acetovanillone in tobacco cell-suspension cultures

A soluble enzyme, extracted from tobacco cell-suspension cultures 24 h after treatment with 100 μM methyl jasmonate, has been shown to synthesize acetovanillone (apocynin) from feruloyl-CoA in the presence of NAD. The enzyme displayed Michaelis-Menten kinetics with apparent K_m values of 5.6 μM for feruloyl-CoA and 260 μM for NAD and exhibited very high specificity for its substrates. The increase in acetovanillone synthase activity was followed by an increase in the concentration of both acetovanillone and acetosyringone in the culture medium. No intermediate could be detected when analysing the reaction medium by HPLC during the formation of acetovanillone in cell-free extracts. The apparent molecular mass estimated by gel permeation on an FPLC column was ca. 79 kDa. To our knowledge, this is the first report of an enzymic system catalysing the synthesis of an acetophenone. This work demonstrates that the biosynthesis of acetophenones in tobacco proceeds from hydroxycinnamic acids through a CoA-dependent β-oxidation pathway. Interestingly in methyl jasmonate-treated cells, which synthesize very large amounts of hydroxycinnamoylputrescines, inhibition of the synthesis of these conjugates increased the concentration of acetovanillone and acetosyringone in the culture medium, suggesting that the two metabolic pathways can compete for their common precursors, i.e. hydroxycinnamoyl-CoA thioesters.     

Partial Purification and Some Properties of Flavonol 7-Sulfotransferase from Flaveria bidentis

A novel flavonol-specific sulfotransferase was partially purified from the shoot tips of Flaveria bidentis var. Angustifolia O.K. (Asteraceae) by chromatography on 3′-phosphoadenosine 5′-phosphate-agarose affinity column and chromatofocusing on Mono P. The latter step resulted in the separation of two isoforms, both of which exhibited expressed specificity for position 7 of quercetin 3,3′- and quercetin 3,4′-disulfate. The 7-sulfotransferase isoforms I and II had a pH optimum of 7.5 in phosphate buffer, apparent pl values of 6.5 and 6.3, and an M_r of 35,000. They had no requirement for divalent cations and were not inhibited by EDTA or SH group reagents. Their K_m values for both the sulfate donor and flavonol acceptor were of the same order of magnitude (0.20-0.46 micromolar). This enzyme, together with the recently reported flavonol 3-, 3′-, and 4′-sulfotransferases from F. chloraefolia (L Varin, RK Ibrahim [1989] Plant Physiol 90: 977-981) form the complement involved in the biosynthesis of polysulfated flavonols in this genus. A proposed sequential order for the enzymatic sulfation in both species is described.     

入侵植物黄顶菊与3种牧草竞争效应研究

以入侵植物黄顶菊和多年生黑麦草、紫花苜蓿、高丹草3种牧草为试验材料,采用盆栽取代试验方法观察了不同密度及比例条件下4种植物的竞争表现,为黄顶菊生物替代提供理论依据.结果表明:(1)在3种牧草中,高丹草对黄顶菊株高控制效果最为明显,并以苗期效果最好,在低密度比例下对黄顶菊抑制率即可达60.00%;而多年生黑麦草和紫花苜蓿的控制效果较差,在低密度比例下对黄顶菊起不到抑制作用.(2)在高丹草低密度替代组合中,黄顶菊单株生物量、分枝数比对照均明显减少,抑制率分别可达91.40%和44.87%;而多年生黑麦草和紫花苜蓿各密度替代组合中,黄顶菊单株生物量、分枝数与对照相同或大于对照.(3)在各个生育时期,除高密度高丹草替代组合外,其他组合中黄顶菊的相对产量值均极显著小于1.0,生长受明显抑制;而在各替代密度下,多年生黑麦草、紫花苜蓿与黄顶菊竞争替代效果均不明显.研究发现,高丹草的替代效果明显优于多年生黑麦草和紫花苜蓿,可以作为生物替代的材料对黄顶菊进行替代控制,且在植株比为1∶3比例下即可实现理想控制效果.     

Secretion of active recombinant phytase from soybean cell-suspension cultures.

Phytase, an enzyme that degrades the phosphorus storage compound phytate, has the potential to enhance phosphorus availability in animal diets when engineered into soybean (Glycine max) seeds. The phytase gene from Aspergillus niger was inserted into soybean transformation plasmids under control of constitutive and seed-specific promoters, with and without a plant signal sequence. Suspension cultures were used to confirm phytase expression in soybean cells. Phytase mRNA was observed in cultures containing constitutively expressed constructs. Phytase activity was detected in the culture medium from transformants that received constructs containing the plant signal sequence, confirming expectations that the protein would follow the default secretory pathway. Secretion also facilitated characterization of the biochemical properties of recombinant phytase. Soybean-synthesized phytase had a lower molecular mass than did the fungal enzyme. However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molecular mass (49 kD). Temperature and pH optima of the recombinant phytase were indistinguishable from the commercially available fungal phytase. Thermal inactivation studies of the recombinant phytase suggested that the additional protein stability would be required to withstand the elevated temperatures involved in soybean processing.     

黄顶菊入侵对土壤养分和酶活性的影响

比较了外来植物黄顶菊不同入侵程度土壤养分和土壤酶活性变化规律,探讨了外来植物入侵对土壤生态的影响机制。结果表明,与裸土和本地植物土壤相比,黄顶菊入侵显著提高了有机质、全氮、硝态氮和铵态氮的含量,而全磷和速效磷的含量有所下降,且随着入侵程度增强趋势更为明显。重度入侵土壤有机质较本地植物土壤提高5.7%,全氮提高23.4%;而重度入侵土壤全磷含量只有本地植物的85%,土壤速效磷含量则下降了50%。黄顶菊重度入侵土壤和轻度入侵土壤脲酶含量分别为0.04和0.03mg.g-1.24h-1,均显著高于裸土和本地植物土壤,土壤磷酸酶活性变化规律与之类似,而多酚氧化酶无明显的变化。黄顶菊入侵可以改变土壤养分和土壤酶活性,创造对自身生长有利的土壤环境,并借此增强其竞争能力,实现种群的进一步扩张。     

自然入侵条件下黄顶菊丛枝菌根定殖及发育的研究

为探讨丛枝菌根(arbuscular mycorrhiza,AM)真菌在黄顶菊Flaveria bidentis入侵过程中可能发挥的作用,首先调查和研究了黄顶菊AM的侵入和生长表现。在黄顶菊入侵严重的河北省选择采样地点,分别从衡水地区的滨湖新区小北田村、冀州市漳下村、枣强县北王庄村、桃城区八里庄村、滨湖新区顺民庄村和滨湖新区刘家台村6个地点按黄顶菊重、中、轻3种盖度和不同生育期(苗期、花期和结籽期)采集根系和根区土样。分离根区土壤中AM真菌孢子、观察AM发育特征、测定孢子密度、AM真菌侵染率、丛枝着生率等,并与土壤理化性质进行了相关性分析。结果在各采样地点均观察到AM典型结构,不同样地菌丝侵染率、I型丛枝密度以及孢子密度的最大值均出现在八里庄村,泡囊密度和A型丛枝密度的最大值分别出现在小北田村和刘家台村;不同盖度下AM真菌侵染率最高值均出现在重度侵染区;不同生长时期黄顶菊的AM也有差异,除了丛枝密度最高值出现在花期和菌丝侵染率差异不显著外,菌丝侵染率、泡囊密度和孢子密度均在结籽期出现最大值。土壤理化特性也显著影响黄顶菊AM的发育,菌丝侵染率与土壤有机质呈显著正相关,与p H值呈显著负相关,与速效P含量呈极显著负相关;A型丛枝密度与p H含量呈显著负相关;I型丛枝密度与土壤全N含量呈极显著正相关;孢子密度与有机质含量和全N含量均呈显著正相关。因此,AM真菌侵染可能会促进黄顶菊的入侵。     

不同盐胁迫对外来植物黄顶菊生理特征的影响

以外来植物黄顶菊幼苗为材料,通过室内盆栽实验比较不同盐度中性盐(NaCl)和碱性盐(Na2CO3)胁迫对其生长和生理指标的影响。结果表明:(1)两种盐胁迫均能不同程度引起黄顶菊叶片细胞膜的完整性损伤和叶片电解质外渗率升高,且Na2CO3的伤害更严重;黄顶菊植株叶内丙二醛含量在各盐度NaCl胁迫处理下无显著变化,而在Na2CO3处理下却显著升高,且150 mmol/L Na2CO3处理叶内达到最高值(2.284×10-2μmol.g-1FW)。(2)黄顶菊的生长在低盐度的NaCl(50 mmol/L)处理下受到一定的促进,而相同盐度的Na2CO3下却受到明显抑制;经NaCl处理的黄顶菊地上部分和地下部分日相对生长率均大于相同盐度的Na2CO3处理。(3)黄顶菊叶内可溶性糖和游离脯氨酸含量在NaCl胁迫处理中变化不显著,而它们在Na2CO3处理植株中却变化显著,两者在200mmol/L Na2CO3处理植株叶内分别达到177.3μmol.g-1FW和4.21 mg.g-1DW;NaCl处理的黄顶菊地上部分含水量明显高于相同盐度下的Na2CO3处理,而两种盐对黄顶菊地下部分相对含水量的影响不显著。研究发现,黄顶菊对于中性盐渍土具有较强的耐性和抗性,而在碱性盐渍土上的侵入和发展均受到一定的抑制。     

Control of diferulate formation in dicotyledonous and gramineous cell-suspension cultures

Primary cell wall polysaccharides of some plants carry ester-linked feruloyl groups that can be oxidatively dimerised both within the protoplast and after secretion into the apoplast. Apoplastic dimerisation has been postulated to form inter-polysaccharide cross-links, contributing to wall assembly, but this role remains conjectural. By feeding cultured cells with [¹⁴C]cinnamate, we monitored the kinetics of polysaccharide-binding and subsequent dimerisation of ¹⁴C-labelled feruloyl groups. Cultured maize and spinach cells took up [¹⁴C]cinnamate more rapidly than barley, Arabidopsis, Acer, tomato and rose cultures. Maize and spinach cells rapidly formed [¹⁴C]feruloyl-polysaccharides and, simultaneously, low-M_r [¹⁴C]feruloyl esters. When all free [¹⁴C]cinnamate had been consumed, there followed a gradual recruitment of radiolabel from the low-M_r pool into the polysaccharide fraction. A proportion of the [¹⁴C]feruloyl-polysaccharides was sloughed into the culture medium, the rest remaining wall-bound. Some of the polysaccharide-bound [¹⁴C]feruloyl groups were coupled to form dehydrodiferulates. At least six putative isomers of [¹⁴C]dehydrodiferulate were formed both rapidly (thus intra-protoplasmically) and gradually (thus mainly apoplastically). These data do not support the hypothesis that intra-protoplasmic dimerisation yields predominantly one isomer (8–5′-dehydrodiferulate). In maize, apoplastic coupling was much more extensive in 7-day old than in 2-day-old cultures; indeed, in 2-day-old cultures apoplastic coupling could not be evoked even by exogenous H₂O₂, suggesting strong control of peroxidase action by apoplastic factors. When apoplastic coupling was minimised by exogenous application of peroxidase-blockers (iodide, dithiothreitol and cysteine), a higher proportion of the secreted [¹⁴C]feruloyl-polysaccharides was sloughed into the medium. This observation lends support to the hypothesis that feruloyl coupling contributes to wall assembly.     

Cyanidin 3-glucoside accumulation in plane tree (Platanus acerifolia) cell-suspension cultures

The accumulation of only one anthocyanin, cyanidin 3-glucoside, in cell-suspension cultures of plane tree (Platanus aceriflia) is reported for the first time. During a time span of 6 years, no new anthocyanin was detected and cyanidin 3-glucoside was maintained at about 35 mg l^–1 cell culture medium. This stable cell culture system could therefore be used for the biotechnological production of cyanidin 3-glucoside.     

黄顶菊的大孢子发生、雌配子体与胚胎发育

用常规石蜡制片对黄顶菊(Flaveria bidentis(L.) Kuntze)大孢子发生、雌配子体和胚胎的发育过程进行了观察.黄顶菊雌蕊柱头二裂,2心皮,1室,单胚珠,基生胎座,单珠被,薄珠心,倒生胚珠,具发达的珠被绒毡层.珠心表皮下分化出孢原细胞,孢原细胞直接发育为大孢子母细胞,大孢子母细胞减数分裂形成直列四分体...     

外来入侵种黄顶菊及其伴生植物光合特性初步研究

以不同生育期的外来入侵植物黄顶菊及其4种伴生杂草为材料,在实验网室条件下测定比较了它们的基本光合和叶绿素荧光特性,从而探讨黄顶菊具有高生物量及较强入侵性的原因,为其防控提供理论依据.结果表明:(1)5种植物的净光合速率日变化均呈单峰曲线,黄顶菊净光合速率以现蕾期最高,且大于其他4种伴生杂草;(2)3个生长时期的黄顶菊水分利用效率低于棒头草,高于齿果酸模和曼陀罗,而与反枝苋相当,显示较高的抗旱性;(3)影响5种植物光合速率主要因子为非气孔因素,影响黄顶菊以及棒头草、反枝苋等的主要生态因子是光合有效辐射,显示出其较强的喜光特性;(4)5种植物在午间较强的光照条件下均发生了不同程度光抑制,处于现蕾期和开花期的黄顶菊以及其余3种植物午间的Fv/Fm有轻微下降,其PSⅡ实际光化学效率维持在较高的水平.可见,黄顶菊具有较强的光合能力,更适应于在夏季高温干旱的环境下生长,从而表现出较强的入侵性.