Identification of an indigo precursor from leaves of Isatis tinctoria (Woad).

Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-beta-D-glucoside). We suggest the name "isatan C" for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.     

Quantitative analysis of indigo and indigo precursors in leaves of Isatis spp. and Polygonum tinctorium

Analysis of extracts from two woad species (Isatis tinctoria and Isatis indigotica) and Polygonum tinctorium revealed that only one indigo precursor (indican) was present in Polygonum, but two precursors were found in Isatis spp. This was done using high performance liquid chromatography (HPLC), coupled to an evaporative light scattering detector (ELSD). In Isatis spp., the indigo precursors indican and a fraction representing isatan B were identified. The proportion of indican and isatan B was different between the two Isatis spp. tested. For the first time, it was possible to quantify the precursors in woad plant species, and the results were found to be in good agreement with those made from total indigo quantification using two different spectrophotometric methods or a derivatization technique.     

The elusive indigo precursors in woad (Isatis tinctoria L.)--identification of the major indigo precursor, isatan A, and a structure revision of isatan B

A metabolite-profiling study of shock-frozen leaves of Isatis tinctoria L., an old indigo dye plant and medicinal herb, revealed a complex pattern of indigo-forming compounds with higher polarities than the known indigo precursors isatan B and indican. These highly unstable compounds underwent rapid post-harvest transformation and were not detected in air-dried leaves. The major indigo precursor, named isatan A (4), was isolated by rapid normal-phase and gel chromatography, along with isatan B (3). A full spectral data set of 3 showed that the previous structure assignment as 'indoxyl-5-ketogluconate' has to be revised to 1H-indol-3-yl beta-D-ribohex-3-ulopyranoside. Isatan A (4) was identified as 1H-indol-3-yl 6'-O-(carboxyacetyl)-beta-D-ribohex-3'-ulopyranoside. In aqueous solution, glycosides 3 and 4 occur as hydrates and undergo rapid hydrolysis under very mild acidic or basic conditions.     

Seasonal variation of indigo precursors in Isatis tinctoria L. and Polygonum tinctorium Ait. as affected by water deficit

In order to identify new crop suitable for indigo production in Italy, seasonal variation in productivity of indigo precursors was studied in woad (Isatis tinctoria L., Family Cruciferae) and in dyer's knotweed (Polygonum tinctorium Ait., Family Polygonaceae), grown in central Italy under temperate climate. Indigo precursors, indoxyl-3-ketogluconate (isatan B) and indoxyl-β-d-glucoside (indican), were measured in leaves by HPLC analysis under well watered versus rain fed field conditions, and the amount of indigo derived by stoichiometric calculations.Woad showed lower indigo potential than dyer's knotweed, evaluated as either amount of indigo either per leaf weight or per plant. However, in water stress conditions, woad appeared to be drought tolerant as opposed to dyer's knotweed revealed very sensitive. In fact, in dyer's knotweed leaf yield was over 50% reduced in water stress field conditions, characterizing central and southern Italy during July and August, as compared to some 30% in woad. Dyer's knotweed appears to be more productive, providing water is supplied appropriately, thus making proper irrigation plans necessary to achieve sustainable high yields.     

Experimental Identification of “Songlan” (Isatis indigotica) and Woad (I.tinctoria) Introduced into China

“Song Lan” is a source of Chinese drugs such as “Daqingye”, “Banlangen” or “Qingdai”. We have discovered that the two species, “Song Lan” (Isatis indigotica Fort.) and woad (I. tinctoria L.), were mistakenly described in the literature due to their morphological polymorphism. In order to clarify the two species, cytology examination, pollen analysis, electrophoretic analysis of isoenzymes and soluble protein were performed. The results show that previous non-trichiferous type of woad is a “Song Lan”. As in woad, “Song Lan” is also morphologically of great variability. The base of canline leaves in this species may be sagittate or auriculate. We have not found the non-trichiferous type of woad in our country. It is reported for the first time that the chromosome number for “Song Lan” is 2n=14. The content of the indole glucoside in fresh leaves of “Song Lan” is about five timeshigher than in woad. For medicine cultivation of “Song Lan” is favorable.     

A high degree of genetic diversity is revealed in Isatis spp. (dyer's woad) by amplified fragment length polymorphism (AFLP)

Genetic diversity in 38 genotypes, representing 28 individual genotypes from five landraces of Isatis tinctoria (three German: Tubingen, Potsdam and Erfurt, one Swiss and one English), five genotypes of Isatis indigotica (Chinese woad) and five genotypes of Isatis glauca, were investigated using AFLP analysis. Five primer combinations detected a total of 502 fragments of which 436 (86.9%) were polymorphic. The level of polymorphism recorded within each species was 29.8, 86.9 and 35.8% for I. indigotica, I. tinctoria and I. glauca, respectively. Clearly, genetic diversity within I. tinctoria was greater than that observed in I. indigotica or I. glauca. Cluster analyses of the AFLP data using UPGMA and PCO revealed the complete separation of the genotypes of each species into distinct groups. I. indigotica separated as an entirely independent group, whereas I. glauca formed a separate cluster within the I. tinctoria group. Indeed, I. tinctoria and I. glauca are more closely related to each other than either is to I. indigotica. In addition, the genotypes of each landrace, apart from one from the English group, were clearly discriminated. However, the anomalous genotype did associate with the rest of its group when it was linked with the Erfurt group. These results provide new and useful information about the make-up of the Isatis genome, which has not previously been evaluated. They will be useful in the selection of plant material for variety development and conservation of the gene-pool.     

A new HPLC-ELSD method to quantify indican in Polygonum tinctorium L. and to evaluate beta-glucosidase hydrolysis of indican for indigo production

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase 相似文献   

Influence of light on natural indigo production from woad (Isatis tinctoria)

In woad (Isatis tinctoria), field observations indicated, that after periods of dry sunny weather, indigo yields increased significantly, suggesting that light intensity and quality affected indigo precursor production. Therefore, woad was grown under different light intensities and in the presence or absence of supplementary UV light. In general, plants supplied with more light produced more indigo than those given lesser light. When plants under greater light regimes were transferred to lesser light conditions, then indigo production declined. The opposite was also true, indicating that greater indigo production can be obtained from plants harvested after periods of increased sunlight.     

冻伤蓝变对广义虾脊兰属植物中4种吲哚基衍生物含量的影响

以6种广义虾脊兰属植物和2种树兰亚科植物为材料,利用液相色谱 串联三重四极杆质谱仪(LCMS QQQ)测定了冻伤处理前后花和叶片中靛苷、靛红、靛蓝和靛玉红4种吲哚基衍生物的含量,分析广义虾脊兰属植物吲哚基衍生物的生成及种属间含量的差异。结果显示：(1)4种吲哚基衍生物在所测定的6种广义虾脊兰属植物中均被检出,但在2种树兰亚科植物五唇兰和足茎毛兰中均未被发现。(2)在所测定的6种广义虾脊兰属植物花和叶片中,冻伤处理后的靛蓝、靛玉红和靛红含量均显著上升,而靛苷含量显著下降,同时花中的吲哚基衍生物含量均高于叶片。(3)6种广义虾脊兰属植物花和叶中吲哚基衍生物总含量以黄兰花最高,三褶虾脊兰叶最低。研究表明,冻伤处理引起靛苷向靛蓝的大量转化是导致冻伤后广义虾脊兰属植物组织中呈现出蓝色的主要原因,推测吲哚基衍生物可能也是一类与植物防御相关的化合物,在植物抵御逆境中扮演着重要的角色。     

Indole Compounds Related to Auxins and Goitrogens of Woad (Isatis tinctoria L.)

Five conspicuous indole derivatives are present in leaves and other tissues of woad (Isatis tinctoria L.). They were identified as tryptophan, isatan B, glucobrassicin, neoglucobrassicin, and glucobrassicin-1-sulfonate. The latter three indole glucosinolates are present at levels of at least 260, 69, and 200 milligrams per kilogram fresh weight and were isolated as crystalline salts. Comparison of physical and chemical properties, particularly NMR spectral analysis, confirms that the 1-methoxyglucobrassicin structure suggested for neoglucobrassicin is correct, whereas further evidence for the even more unusual sulfonation of the ring nitrogen in glucobrassicin-1-sulfonate was obtained. Glucobrassicin-1-sulfonate has an enzymic degradation pattern identical to that of glucobrassicin. As it too releases thiocyanate, it must be added to the list of known plant goitrogens. These studies and the techniques described establish woad as exceptionally suitable higher plant material for metabolic studies of indoles related to goitrogens and auxins.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

外源磷或有机质对板蓝根吸收转运砷的影响

采用土壤培养方法,研究了不同含砷水平土壤中添加外源磷或有机质对砷在板蓝根地下部和地上部累积与分配的影响。结果表明,在外源添加磷或者有机质的情况下,与自然土相比含砷土对板蓝根的生长有一定的促进作用;在自然土(13.4 mg/kg)中,外源磷没有明显影响板蓝根地下部对砷的累积,却显著降低了砷由地下部向地上部的转运,并且添加200 mg P2 O5/kg显著降低了砷在地上部的累积。然而,在含砷土(33.4 mg/kg)中,100 mg P2O5/kg处理显著降低了砷在地下部的累积,但随磷用量的增加反而促进了地下部砷的累积;在添加有机质试验中,10 g/kg的有机质显著降低了自然土中板蓝根地下部和地上部对砷的累积,并且砷的吸收能力也明显下降。在含砷土(23.4 mg/kg)中,添加5 g/kg的有机质不仅降低了砷在板蓝根中的富集,而且降低了其对砷的吸收能力,提高了砷由地下部向地上部的转运,但是随着有机质施用量增至10 g/kg,地下部砷含量及其吸收砷的能力均有一定程度的增大。因此,在砷水平较低的自然土壤上种植板蓝根添加200 mg P2O5/kg和10 g/kg的有机质是控制砷在该草药体内积累的适宜用量,而在砷水平较高的土壤上100 mg P2O5/kg和5 g/kg的有机质是降低板蓝根体内砷累积的适宜用量。     

不同氮素形态和配比对菘蓝根的生长及含氮成分含量和总量的影响

以来源于安徽亳州、甘肃张掖、安徽阜阳、山西运城和陕西商洛的5个种源菘蓝( Isatis indigotica Fort.)为实验材料,采用田间试验法对不施氮素(对照),仅施用NO3--N(T1)或NH4+-N(T2),混合施用氮素也n(NH4+-N)：n(NO3--N)=75：25(T3)、n(NH4+-N)：n(NO3--N)=50：50(T4)和n(NH4+-N)：n(NO3--N)=25：75(T5)页以及仅施用CO(NH2)2(T6)条件下各种源菘蓝的单株根鲜质量和干质量,根折干率,根中游离总氨基酸、可溶性蛋白质和总氮含量,以及单株根中游离总氨基酸、可溶性蛋白质和总氮总量的差异进行了比较分析。结果表明：在同一施氮条件下不同种源间菘蓝根的各项指标均有一定差异;而与各自的对照相比,不同施氮条件下同一种源菘蓝根的各项指标也有一定差异。总体来看,安徽亳州和山西运城种源菘蓝根的生长指标均在T6组中较高,甘肃张掖种源根的生长指标则在T2组中相对较高,安徽阜阳种源根的生长指标在T5组中最高,陕西商洛种源根的生长指标在T4组中较高。各施氮条件下安徽亳州种源菘蓝的单株根中游离总氨基酸、可溶性蛋白质和总氮总量均低于其对照,并在T6组中相对较高;甘肃张掖和陕西商洛种源根的上述3项指标分别在T2和T3组中最高,而安徽阜阳和山西运城种源根的上述3项指标则分别在T5和T1组中较高。研究结果显示：不同氮素形态和配比对菘蓝根生长和根中含氮成分积累的影响效应存在种源间的差异,并且对同一种源根生长和根中含氮成分积累有益的氮素形态和配比也不同。因此,为了获得高产优质的板蓝根药材,建议在菘蓝的栽培过程中针对不同种源采取适宜的施氮措施,并兼顾生长量和有效成分含量。     

菘蓝CYP83B1基因的克隆与表达分析

对菘蓝（Isatis indigotica Fort.）CYP83B1基因进行了克隆与表达模式分析。结果显示,IiCYP83B1基因全长为1652 bp,包含2个外显子和1个内含子;cDNA全长为1500 bp,编码499个氨基酸。IiCYP83B1编码的蛋白没有跨膜结构域和信号肽,主要定位于内质网膜,属于亲水性蛋白,二级结构主要由无规则卷曲螺旋和α-螺旋组成,与萝卜（Raphanus sativus Linn.）、欧洲油菜（Brassica napus L.）、甘蓝（Brassica oleracea L.）和芜菁（Brassica rapa L.）等植物的CYP83B1蛋白具有较高的同源性。qRT-PCR分析结果表明,IiCYP83B1基因在菘蓝的根、茎、叶、花和果中均有表达,且以叶中的表达量最高;在幼苗期、生长期和花期稳定表达且均显著高于萌芽期;茉莉酸甲酯（methyl jasmonate,MeJA）和葡萄糖（glucose,Glu）能够显著促进该基因的表达,而低温（4℃）和水杨酸（salicylic acid,SA）处理对其表达具有一定的抑制效应。本研究结果可为进一步探讨IiCYP83B1基因的功能提供参考。     

Synthesis of some O-, S- and N-glycosides of hept-2-ulopyranosonamides

(O-Peracylated α-d-gluco- and -galacto-hept-2-ulopyranosylbromide)onamides gave the corresponding (alkyl β-d-glyco-hept-2-ulopyranoside)onamides under Koenigs-Knorr conditions, and similar aryl glycosides were obtained with sodium phenolates; (aryl and hetaryl 2-thio-β-d-gluco-hept-2-ulopyranoside)onamides were formed with thiophenols in the presence of K₂CO₃ in acetone, and reactions with aniline in CH₂Cl₂ furnished (N-phenyl β-d-glyco-hept-2-ulopyranosylamine)onamides. Some deprotected derivatives of d-gluco configuration obtained by the Zemplén protocol showed no significant inhibition against rabbit muscle glycogen phosphorylase b.     

菘蓝IiLEA基因的克隆及胁迫表达分析

胚胎发育晚期丰富蛋白(LEA蛋白),是指胚胎发育后期种子中大量积累的一系列蛋白质,大量研究表明这些蛋白质的积累与渗透胁迫耐受性密切相关.该实验以250 mmol/L NaCl溶液处理9 h的菘蓝幼苗为材料,经RT-PCR方法扩增得到LEA基因,命名为IiLEA.经测序确认该基因含有648 bp的开放阅读框,编码由215个氨基酸组成的亲水性蛋白质.对培养30 d的菘蓝无菌幼苗进行自然干旱处理和盐胁迫处理,Northern杂交结果显示,正常生长条件下,IiLEA基因在菘蓝幼苗体内不表达,随着胁迫时间的延长其表达量逐渐增加,到9 h时表达量达到峰值,干旱处理和盐处理有相似结果,表明该基因可能受逆境胁迫诱导表达.     

Transmission of Pandora neoaphidis in the presence of co-occurring arthropods

Transmission of the entomopathogenic fungus Pandora neoaphidis to the nettle aphid Microlophium carnosum was assessed in the presence of arthropods that co-exist with the fungus within the habitat but do not compete for aphid hosts. The presence of a parasitoid significantly enhanced transmission, and transmission rates were similar for both enemy and non-enemy parasitoids. Although herbivory of nettle leaves by Peacock butterfly (Inchis io) caterpillars indirectly reduced the number of M. carnosum by >30% due to a reduction in leaf area for feeding, the addition of I. io significantly increased transmission of P. neoaphidis in the remaining aphids. It is likely that enhanced transmission in the presence of A. rhopalosiphii and I. io is due to disturbance and subsequent movement of the aphid, resulting in contact with conidia deposited on the leaf surface. The presence and impact of co-occurring arthropods should be taken into consideration when assessing the transmission of fungal entomopathogens.     

Quassinoid glucosides from seeds of Brucea amarissima

Quassinoid glucosides, javanicosides I, J, K and L, were isolated from the seeds of Brucea amarissima (Lour.) Desv. ex B. A. Gomes (Simaroubaceae), along with two known quassinoids, i.e. bruceins D and E, and seven known quassinoid glucosides, yadanziosides B, C, E, I and K, bruceoside B and yadanzigan. Their structures were elucidated by analysis of the spectral data and chemical evidence.     

黄土高原樟子松和落叶松与其他树种枯落叶混合分解对土壤的影响

通过采集树木枯落叶与土壤进行室内混合分解培养试验,研究了黄土高原常见的樟子松和落叶松与其他树种枯落叶混合分解对土壤性质的影响及存在的相互作用,从而为不同树木种间关系的探索和该地区人工纯林的混交改造提供科学指导。结果表明:12种枯落叶单一分解均明显提高了土壤脲酶(54%—110%)、脱氢酶(85%—288%)和磷酸酶(81%—301%)活性以及有机质(29%—55%)和碱解N(12%—49%)含量,但对土壤速效P含量和CEC的影响存在较大差异。综合而言,樟子松分别与白桦、刺槐、白榆、柠条和落叶松枯落叶混合分解在对土壤性质的影响中存在相互促进作用,而分别与小叶杨、沙棘、紫穗槐、侧柏和辽东栎枯落叶混合分解在对土壤性质的影响中存在相互抑制作用;落叶松分别与刺槐、白桦、小叶杨和紫穗槐枯落叶混合分解在对土壤性质的影响中存在相互促进作用,而分别与柠条、侧柏、辽东栎、沙棘、油松和白榆枯落叶混合分解在对土壤性质的影响中存在相互抑制作用。     

Molecular cloning and characterization of rat LC3A and LC3B--two novel markers of autophagosome

Rat microtubule-associated protein light chain 3 (LC3) is a homologue of yeast Atg8, an essential component of autophagy. Following synthesis, the C-terminus of rat LC3 is cleaved by a cysteine protease-Atg4, to produce LC3-I, which is located in cytosolic fraction. LC3-I can be converted to LC3-II through the processing by Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme). LC3-II is modified by phosphatidylethanolamine on C-terminus and binds tightly to autophagosomal membrane. Here we reported the cloning of two novel variants of rat LC3, named LC3A and LC3B, respectively, and LC3B is an alternative splicing variant of LC3. LC3A, LC3B, and LC3 showed different expression patterns in rat tissues, suggesting a functional divergence among these proteins. When LC3A and LC3B were overexpressed, both exhibited two forms (18 and 16 kDa, representing types of I and II, separately), which might be due to post-translational modification including the characteristic C-terminal cleavage at these two proteins as similar to that found in rat LC3 and yeast Atg8. Subcellular localization demonstrated that both LC3A and LC3B are colocalized with LC3 and associated with the autophagic membranes. Mutation analysis further revealed that the conserved Gly120 residues of LC3A and LC3B are essential for their characteristic C-terminal cleavage and localization to autophagic membranes. Present data suggested that LC3A and LC3B could also be used as two novel autophagosomal markers.