影响决明无菌苗子叶原生质体分离和培养因素的研究

以决明(Cassia obtusi folia)无菌苗子叶为材料,对酶组合、无菌苗日龄,植物激素组合和培养方法对其原生质体的分离和培养的影响进行了研究。结果表明:用3％的纤维素酶和0.2％Pectinase Y-23的酶组合处理决明无菌苗子叶块8小时可以高效分离出有活力的原生质体;约14日龄的决明无菌苗子叶比较适合于原生质体的分离;适当浓度的2,4-D 有利于原生质体的分离。促进原生质体分裂的理想的植物激素组合为0.4 mg/L 2,4-D,1.0 mg/L NAA and 0.1 mg/L KT;漂浮培养法最有利于原生质体的分裂和发育。找出了适合于决明无菌苗子叶原生质体的分离和培养的酶组合、植物激索组合、有效培养方法和决明无菌苗子叶日龄。这为有效地从决明无菌苗子叶原生质体再生植株奠定了基础。     

植物苯丙烷代谢途径

众所周知,固着生长的植物经常受到环境中各种生物和非生物胁迫的威胁。所以在漫长的进化过程中,植物必须将多样的环境信号整合到其发育过程中,以实现适应性形态的发生和代谢途径的精确调控,最终使植物完成整个生长周期。研究显示,苯丙烷代谢作为植物重要的次级代谢途径之一,其代谢产物,例如木质素、孢粉素、花青素和有机酸等,在调控植物适应性生长的过程中发挥着重要功能。特别是在药用植物中,苯丙烷代谢还与众多药用活性成分的合成息息相关,几乎所有包含苯丙烷骨架的天然药效成分均由苯丙烷代谢途径直接或间接合成,例如黄酮类、萜类和酚类等。此外,经苯丙烷代谢途径产生的一些次级代谢产物还能由植物根系外泌到周际土壤中,通过改变根系微生物的菌群生态,而影响植物生长和抵抗生物或非生物胁迫的能力。同时,苯丙烷代谢介导的这种植物-微生物互作也与药用植物的道地品质密不可分。本文综述了近年来植物苯丙烷代谢途径的最新研究进展,重点对该代谢途径中代谢产物的生理功能及表达调控机制进行了介绍,以期更深入地理解药用植物苯丙烷代谢与药材性状之间的潜在关系,旨在指导优良中草药的遗传育种,以进一步促进我国中医药事业的蓬勃发展。     

Cadinane sesquiterpenoids of Phomopsis cassiae, an endophytic fungus associated with Cassia spectabilis (Leguminosae)

Five cadinane sesquiterpenes derivatives were isolated by bioassay-guided fractionation from Phomopis cassiae, an endophytic fungus isolated from Cassia spectabilis. The structures of the two diastereoisomeric 3,9,12-trihydroxycalamenenes (1, 2); 3,12-dihydroxycalamenene (3); 3,12-dihydroxycadalene (4) and 3,11,12-trihydroxycadalene (5) were established on the basis of analyses of 1D and 2D NMR and HRTOFMS experiments. Antifungal activity of the isolates was evaluated against Cladosporium sphaerospermum and Cladosporium cladosporioides, revealing 5 as the most active compound.     

Absorption,translocation, and metabolism of flurtamone in sicklepod (Cassia obtusifolia) and peanut (Arachis hypogea)

The basis of peanut tolerance to the bleaching herbicide flurtamone was examined. The absorption, translocation, and metabolism of ¹⁴C-flurtamone were examined in peanut at 6, 24, and 48 h after root application. Differences in ¹⁴C-flurtamone uptake over time were not detected. Approximately 40% of the absorbed ¹⁴C-flurtamone was in the leaves at 6 h after treatment; 60% was metabolized to polar products 41% of absorbed ¹⁴C in 6 h; 40% of this moved from roots to shoots; and 60% of this did not co-chromatograph with the parent; 9.8% of applied ¹⁴C-flurtamone was altered in leaf tissue. The levels of metabolized flurtamone increased with time after treatment (75% and 83% of applied ¹⁴C-flurtamone metabolized at 24 and 48 h, respectively). Parent ¹⁴C-flurtamone was detectable with R_t of 7 min and unknown metabolites with an R_t of 3.3, 4.4, and 5.6 min, respectively, was detected in leaf tissue at 6, 24, and 48 h after treatment.     

决明子萌发过程中抗氧化活性变化研究

以决明干燥种子和不同萌发期决明子芽的干粉为材料,采用超声波提取法提取醇溶成分和水溶成分,通过总抗氧化活性、还原力、清除羟自由基活性和清除DPPH自由基活性的测定,研究决明子萌发过程中抗氧化活性的变化规律,为决明子的合理开发和有效利用提供理论依据.结果表明:(1)决明子和不同萌发期决明子芽干粉醇提物和水提物均具有抗氧化活性.(2)在决明子萌发过程中,醇提物的总抗氧化活性呈先下降后上升再下降的趋势,还原力、清除DPPH·活性、清除·OH活性均呈先上升后下降的趋势.在种子萌发后第7天,醇提物的抗氧化活性均高于干燥种子.(3)在决明子萌发过程中,水提物的总抗氧化活性和清除DPPH·活性呈下降趋势,还原力呈先下降后上升再下降的趋势,清除·OH活性呈先上升后下降的趋势.     

日光紫外线B辐射对甜荞苯丙烷代谢的影响

对甜荞(Fagopyrum esculentum Moench)苯丙烷次生代谢受紫外线B辐射的响应进行了研究.结果表明:不仅卢丁、槲皮素等黄酮类化合物含量在紫外线B辐射下显著升高,而且叶片苯丙烷单环酚类化合物,如阿魏酸含量等也大幅度升高,两类化合物的应激提高对甜荞的UV-B胁迫提供了重要的保护作用.     

O-Benzylhydroxylamine: An Inhibitor of Phenylpropanoid Metabolism in Plants

O-Benzylhydroxylamine (OBHA) is a potent inhibitor of phenylalanineammonialyase (PAL, EC 4.3.1.5 [EC] ) and phenylpropanoid metabolismas evidenced by its effects on three plant species [soybean(Glycine max (L.) Merr.), buckwheat (Fagopyrum esculentum Moench.),and mung bean (Vigna radiata L.)]. When supplied to roots, OBHA(10^–5 M) did not significantly inhibit light- or dark-growthof soybean seedlings, but reduced (25%) soluble hydroxyphenoliccompound accumulation in light-grown axes. Higher concentrations(510^–5 M) of OBHA caused reductions (25%) in axis freshweight of light-grown seedlings (72 h), but did not lower axisweight of dark-grown seedlings. Anthocyanin accumulation inhypocotyls of intact mung bean seedlings was reduced by 25%after 3 days light growth after treatment with OBHA (10^–5M) via root feeding. Anthocyanin content of excised, etiolatedbuckwheat hypocotyls floated on solutions of OBHA (10^–5M) and incubated in the light for 24 h was reduced by 40%. L-Phenylalanineand t-cinnamic acid, intermediates of phenylpropanoid metabolism,were able to partially reverse this inhibition in buckwheat.Extractable PAL activity (specific activity basis) in soybeanaxes was substantially reduced (20% in dark, 40% in light) asearly as 24 h after root feeding with OBHA (10^–5 M). Reductionof PAL activity (specific activity or per axis basis) by OBHAcompared to control levels, continued throughout a time courseof 96 h. Kinetic studies on soybean PAL revealed a K_m of 1.1mM for L-phenylalanine and an apparent K_i of 3.5 µM forOBHA. (Received May 31, 1985; Accepted August 6, 1985)     

Sicklepod Surface Wax Response to Photoperiod and S-(2,3-Dichloroallyl)diisopropylthiocarbamate (Diallate)

Sicklepod (Cassia obtusifolia L.) leaflet epicuticular fatty acid, fatty alcohol, and hydrocarbon contents were measured by gas-liquid chromatography from plants grown under 10-, 12-, 14-, or 16-hour photoperiods and treated with S-(2,3-dichloroallyl)diisopropylthiocarbamate (diallate) (0, 0.14, 0.28, 0.56, 1.12 kilograms per hectare). As diallate concentration increased, epicuticular fatty acid content decreased. Fatty alcohol content was maximal in plants treated with 0.28 kilograms per hectare diallate and was reduced from that level at herbicide concentrations above or below this rate. Hydrocarbon content patterns were similar to those of the fatty alcohols except that the hydrocarbons at 0.28 kilograms per hectare were 61% of that present in the control, whereas the concentration of fatty alcohols increased to 200% of the control in treatments of 0.28 kilograms per hectare diallate.     

Vegetative propagation of Cecropia obtusifolia (Cecropiaceae)

Cecropia is a relatively well-known and well-studied genus in the Neotropics. Methods for the successful propagation of C. obtusifolia Bertoloni, 1840 from cuttings and air layering are described, and the results of an experiment to test the effect of two auxins, naphthalene acetic acid (NAA) and indole butyric acid (IBA), on adventitious root production in cuttings are presented. In general, C. obtusifolia cuttings respond well to adventitious root production (58.3% of cuttings survived to root), but air layering was the better method (93% of cuttings survived to root). The concentration of auxins used resulted in an overall significantly lower quality of roots produced compared with cuttings without auxin treatment. Future experiments using Cecropia could benefit from the use of isogenic plants produced by vegetative propagation.     

Isolation, Purification, and Identification of 2-(p-Hydroxyphenoxy)-5, 7-Dihydroxychromone: A Fungal-Induced Phytoalexin from Cassia obtusifolia

A single phytoalexin was isolated and purified from 12- to 14-day-old leaves of Cassia obtusifolia L. inoculated with Alternaria cassiae Jurair & Khan. The structure was elucidated by ¹H- and ¹³C-nuclear magnetic resonance and mass spectrometry as 2-(p-hydroxyphenoxy)-5,7-dihydroxychromone. The compound was shown to be derived in part from phenylalanine. Radial growth of A. cassiae was inhibited 50% by the compound at 0.3 millimolar. This moderate toxicity is compensated for by the relatively high levels (3 millimolar) accumulated. Phenoxychromones have been previously reported only as constitutive secondary metabolites in Artemisia capillaris Thunb., in which their function is unknown.     

Chronic Calorie Restriction Alters Glycogen Metabolism in Rhesus Monkeys

Chronic caloric restriction (CR) prevents the development of obesity and maintains health, slows aging processes, and prevents or substantially delays the development of non-insulin-dependent diabetes. Because changes in energy metabolism could be involved in all of these positive effects of CR, we examined glycogen synthase (GS) and glycogen phosphorylase (GP) activities and glucose 6-phosphate (G6P) and glycogen concentrations in skeletal muscle samples before and during a euglycemic hyperinsulinemic clamp in 6 older aged monkeys in which CR had been continued for 10.4 ± 2.1 years. Basal GS activity (fractional velocity and independent) was significantly higher in the CR monkeys than has been previously shown in normal, hyperinsulinemic and diabetic monkeys. The normal effect of insulin to activate GS was absent in the CR group due to the paradoxical finding in some of these monkeys of a reduction in GS activity by insulin. Insulin also had the unexpected effect of increasing the independent activity of GP above basal activity (p<0.05). There was an inverse relationship between the change (insulin-stimulated minus basal) in GS fractional velocity and GP activity ratio (r=-0.91, p<0.005). The basal independent activities of GS and GP were also inversely correlated (r=-0.79, p<0.05). The insulin-stimulated concentration of G6P tended to be higher than the basal concentration (p<0.06) and was significantly higher than that previously shown in normal monkeys (p<0.05). We suggest that long-term calorie restriction (1) results in alterations in glycogen metabolism that may be important to the anti-diabetogenic and anti-aging effects of CR and (2) unmasks early defects which may indicate the likelihood of ultimately developing obesity and diabetes.     

Role of the Alternaria alternata Blue-Light Receptor LreA (White-Collar 1) in Spore Formation and Secondary Metabolism

Alternaria alternata is a filamentous fungus that causes considerable loss of crops of economically important feed and food worldwide. It produces more than 60 different secondary metabolites, among which alternariol (AOH) and altertoxin (ATX) are the most important mycotoxins. We found that mycotoxin production and spore formation are regulated by light in opposite ways. Whereas spore formation was largely decreased under light conditions, the production of AOH was stimulated 2- to 3-fold. ATX production was even strictly dependent on light. All light effects observed could be triggered by blue light, whereas red light had only a minor effect. Inhibition of spore formation by light was reversible after 1 day of incubation in the dark. We identified orthologues of genes encoding the Neurospora crassa blue-light-perceiving white-collar proteins, a cryptochrome, a phytochrome, and an opsin-related protein in the genome of A. alternata. Deletion of the white-collar 1 (WC-1) gene (lreA) resulted in derepression of spore formation in dark and in light. ATX formation was strongly induced in the dark in the lreA mutant, suggesting a repressing function of LreA, which appears to be released in the wild type after blue-light exposure. In addition, light induction of AOH formation was partially dependent on LreA, suggesting also an activating function. A. alternata ΔlreA was still able to partially respond to blue light, indicating the action of another blue-light receptor system.     

Phenylpropanoid glycosides of Mimulus guttatus (yellow monkeyflower)

Yellow monkeyflower [Mimulus guttatus DC., (Phyrmaceae)] has long been a model plant species for studies in genetics, evolution, and ecology, including plant–animal interactions. Nonetheless, exceedingly little is known about its secondary chemistry. We have discovered that the foliage of yellow monkeyflower contains a diverse suite of phenylpropanoid glycosides (PPGs); a class of compounds with many known biological activities. Using ¹H and ¹³C NMR and UV and MS chromatography techniques, we positively identified five PPGs from the leaves of yellow monkeyflower. Four of these compounds occur in other species and one is previously undescribed. We also present UV and high-resolution tandem MS data that putatively identify 11 additional foliar compounds as PPGs. This initial discovery and elucidation of yellow monkeyflower's secondary chemistry will be important for continued study of the genetics and ecology of this model species.     

Root-Zone Salinity Alters Raffinose Oligosaccharide Metabolism and Transport in Coleus

Exposure of variegated coleus (Coleus blumei Benth.) plants to a saline root-zone environment (60 mM NaCl:12 mM CaCl2) resulted in a significant decline in elongation growth rate over the 30-d experimental period. During the initial 5 to 10 d of exposure, mature source leaves showed strongly diminished rates of photosynthesis, which gradually recovered to close to the control rates by the end of the experiment. In green leaf tissues, starch levels showed the same transient decline and recovery pattern. Low starch levels were accompanied by the appearance of several novel carbohydrates, including high-molecular-weight raffinose family oligosaccharides (RFOs) with a degree of polymerization (DP) of 5 to 8, and an O-methylated inositol (OMI). New enzyme activities, including galactan:galactan galactosyltransferase, for the synthesis of high-DP RFOs and myo-inositol 6-O-methyltransferase for O-methylation of myo-inositol, were induced by salinity stress. Phloem-sap analysis showed that in the stressed condition substantially more sucrose than RFO was exported, as was the OMI. In white sink tissues these phloem sugars were used to synthesize high-DP RFOs but not OMIs. In sink tissues galactan:galactan galactosyltransferase but not myo-inositol 6-O-methyltransferase was induced by salinity stress. Models reflecting the changes in carbohydrate metabolism in source and sink tissues in response to salinity stress are presented.     

Phenylpropanoid glucosides from leaves of Coussarea hydrangeifolia (Rubiaceae)

Phenylpropanoid glycosides, 1'-O-benzyl-alpha-L-rhamnopyranosyl-(1'-->6')-beta-D-glucopyranoside (1) and alpha-L-xylopyranosyl-(4'-->2')-(3-O-beta-D-glucopyranosyl)-1'-O-E-caffeoyl-beta-D-glucopyranoside (2), together with the known derivatives, 1,6-di-O-caffeoyl-beta-D-glucopyranoside (3), 1-O-(E)-caffeoyl-beta-D-glucopyranoside (4) and 1-O-(E)-feruloyl-beta-D-glucopyranoside (5), were isolated from leaves of Coussarea hydrangeifolia. Their structures were determined by IR, HRESIMS, and 1D and 2D NMR experiments, and their antioxidant activities, evaluated by assaying the free radical scavenging capacity using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical as substrate. The antioxidant activities of 3 and 4 (IC50 values of 15.0 and 19.2 microM, respectively) were comparable to that of the standard positive control caffeic acid, whilst 2 and 5 were only weakly active and 1 was inactive.     

Daminozide Alters Anthocyanin Metabolism in Ray Florets of Bronze Chrysanthemum (Chrysanthemum morifolium Ramat.)

Daminozide is a well-known chemical inhibitor of the gibberellic acid biosynthesis pathway regulating the vegetative growth of potted chrysanthemums (Chrysanthemum morifolium Ramat.). However, the precise mechanism underlying daminozide-related floral color loss is unknown. To investigate the latter, in two separate greenhouse experiments, bronze flowering chrysanthemum cultivars ‘Baton Rouge’ and ‘Pelee’ were treated weekly with consecutive (0 or 5,000 mg l^?1) foliar daminozide spray applications at early, intermediate, and late stages during the short-day photoperiod. The ray florets of both cultivars were sampled, and the effect of daminozide application on anthocyanins and their biosynthetic precursors were determined by HPLC. Daminozide applied to ‘Baton Rouge’ plants at early developmental stages was correlated with partial loss of red color, and HPLC analysis determined that this was associated with a 75 % reduction in ray floret anthocyanins. Conversely, a near complete loss of red coloration in daminozide-sprayed ‘Pelee’ relative to control plants was associated with as much as a 98 % decline in anthocyanins, irrespective of the time of application. HPLC analysis determined that daminozide application was associated with a 22–50 % increase in the flavones apigenin 7-O-rutinoside, acacetin 7-O-rutinoside, diosmetin 7-O-rutinoside, and eupatorin, and a 68 % increase in the flavonol quercetin 3-O-glucoside, in ray florets of ‘Pelee’ relative to control plants. There was no relative change in ‘Baton Rouge’ flavone and flavonol levels. The accumulation of bronze C. morifolium flavones and flavonols following foliar daminozide application suggests that red color loss is associated with inhibition of anthocyanidin synthase of ‘Pelee’ ray florets.     

Phenylpropanoid glycosyltransferases from osage orange (Maclura pomifera) fruit

Flavonoids and isoflavonoids are well known for their beneficial effects on human health and their anti-insect and anti-microbial activities in plants. Osage orange fruit is rich in prenylated isoflavones and dihydrokaempferol and its glucoside. Four glycosyltransferases were identified from a collection of osage orange fruit expressed sequence tags. Biochemical characterization suggested that the glycosyltransferase UGT75L4 might be responsible for glucosylation of dihydrokaempferol in vivo, although this enzyme exhibited broad substrate recognition toward isoflavonoids and flavonoids in vitro. UGT88A4 was active on coumarin substrates. Identification of highly active phenylpropanoid glycosyltransferases will facilitate the metabolic engineering of glycosylated natural products in plants.     

Rapid Activation of Phenylpropanoid Metabolism in Elicitor-Treated Hybrid Poplar (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) Suspension-Cultured Cells

Elicitor induction of phenylpropanoid metabolism was investigated in suspension-cultured cells of the fast-growing poplar hybrid (Populus trichocarpa Torr. & Gray × Populus deltoides Marsh) H11-11. Treatment of cells with polygalacturonic acid lyase or two fungal elicitors resulted in rapid and transient increases in extractable l-phenylalanine ammonia lyase and 4-coumarate:coenzyme A ligase enzyme activities. The substrate specificity of the inducible 4-coumarate:coenzyme A ligase enzyme activity appeared to differ from substrate specificity of 4-coumarate:coenzyme A ligase enzyme activity in untreated control cells. Large and transient increases in the accumulation of l-phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase mRNAs preceded the increases in enzyme activities and were detectable by 30 minutes after the start of elicitor treatment. Chalcone synthase, cinnamyl alcohol dehydrogenase, and coniferin β-glucosidase enzyme activities were unaffected by the elicitors, but a large and transient increase in β-glucosidase activity capable of hydrolyzing 4-nitrophenyl-β-glucoside was observed. Subsequent to increases in l-phenylalanine ammonialyase and 4-coumarate:coenzyme A ligase enzyme activities, cell wall-bound thioglycolic acid-extractable compounds accumulated in elicitor-treated cultures, and these cells exhibited strong staining with phloroglucinol, suggesting the accumulation of wall-bound phenolic compounds.