Metabolism of trans-Aconitic Acid in Maize : II. Regulatory Properties of Two Compartmented Forms of Citrate Dehydrase

Kinetics of two molecular forms of K-dependent citrate dehydrase in maize (Zea mays L.) are reported. The isozymes, designated CD I and CD II, were found to be compartmented in mitochondria and cytosol, respectively.

CD I exhibited hyperbolic kinetics with respect to both citrate and potassium with K_m 2.3 and 12 millimolar, respectively. Maximum velocity was 0.38 micromole of trans-aconitic acid per minute per milligram protein. The pH optimum was 7.2. trans-aconitic synthesis by CD I is regulated by both citrate concentration and pH.

CD II exhibited hyperbolic kinetics with respect to citrate (K_m 0.6 millimolar) but sigmoidal kinetics with respect to potassium. trans-aconitic acid synthesis by CD II is regulated by potassium. This may account for the positive correlation between leaf potassium and trans-aconitic acid in certain grasses (Clark 1968 Crop Sci 8: 165).

     

基于rDNA ITS1和ITS2序列的褐飞虱、白背飞虱和灰飞虱的分子鉴定

本研究测定了褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera和灰飞虱Laodelphax striatellus的rDNA ITS1和ITS2的序列, 以探讨这3种稻飞虱的分子鉴定方法。3种飞虱的ITS1和ITS2侧翼区（18S, 5.8S和28S）序列相对稳定, 但ITS1和ITS2序列在3种飞虱中变异较大。 ITS1在所分析的438个位点中可变位点达294个, ITS2在分析的403个位点中可变位点为177个。根据3种飞虱rDNA的ITS1和ITS2序列设计了特异性引物, 应用特异性引物对样品进行了PCR扩增, 分析发现3种飞虱ITS1区的特异性引物扩增效果不理想, 而ITS2区的特异性引物可以稳定地扩增出明显的目的DNA条带. 因此, 采用ITS2区的特异性引物可以对3种飞虱进行快速的分子鉴定。     

Studies on chlorogenic acid biosynthesis in sweet potato root tissue using trans-cinnamic acid-2-14C and quinic acid-G-3H

When either trans-cinnamic acid-2-¹⁴C or quinic acid-G-³H wasadministered to sweet potato root discs, each compound was incorporatedinto chlorogenic acid. Hydrolysis analysis revealed that trans-cinnamicacid-2-¹⁴C and quinic acid-G-³H were selectively incorporatedinto the aromatic and non-aromatic moieties of chlorogenic acid,respectively. Quinic acid-G-³H was considered a more efficient precursor thantrans-cinnamic acid-2-¹⁴C, based on data of dilution values,incorporation percents and pool sizes in the tissue. No conjugatesof trans-cinnamic acid and quinic acid were detected in discsadministered trans-cinnamic acid-2-¹⁴C or quinic acid-G-³H.From these experimental results, a possible biosynthetic pathwayfor chlorogenic acid has been proposed. ¹ This paper constitutes Part 98 of the Phytopathological Chemistryof Sweet Potato with Black Rot or Injury. (Received November 2, 1971; )     

Isolation of 1-O-trans-p-Coumaroyl-r{beta}-D-glucopyranose from Sweet Potato Roots and Examination of Its Role in Chlorogenic Acid Biosynthesis

1-O-trans-p-Coumaroyl-rß-D-glucopyranose (p-coumaroyl-D-glucose)was isolated from slices of sweet potato root which had beenincubated with trans-cinnamic acid. Pre-loaded trans-cinnamicacid efficiently trapped the radioactivity from L-[U-¹⁴C]-phenylalanineand reduced its incorporation into chlorogenic acid by 75% ofcontrol values in disks of sweet potato root. In the root diskssupplied with trans-[3-¹⁴C]-cinnamic acid, the radioactivitywas transferred first to trans-cinnamoyl- D-glucose, then top-coumaroyl-D-glucose, and subsequently to chlorogenic acidand isochlorogenic acid. These results support our earlier propositionthat p-coumaroyl-D-glucose is involved in the biosynthesis ofchlorogenic acid as an intermediate adjacent in the pathwayto trans-cinnamoyl-D-glucose in sweet potato roots. (Received April 11, 1988; Accepted August 9, 1988)     

The importance of linoleic acid and linolenic acid as precursors of hexanal and trans-2-hexenal in black tea

During tea fermentation, linoleic acid in the neutral fat fraction,and linolenic acid in both the neutral fat and phospholipidfractions from leaves decreased. The addition of linoleic orlinolenic acid to leaf macerates during fermentation resultedin an increase in hexanal or trans-2-hexenal in the volatilefraction. Tracer experiments showed the direct conversion oflinoleic-U-¹⁴C and linolenic-U-¹⁴C acids to labeled hexanaland trans-2-hexenal, respectively, which were identified as2,4-DNPH derivatives. Further conversion of hexanal and trans-2-hexenal into hexanoicand trans-2-hexenoic acids during tea fermentation was suggestedby the increases in these compounds after the addition of hexanaland trans-2-hexenal to leaf macerates. (Received December 21, 1971; )     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

黄绿绿僵菌对两种稻田蜘蛛捕杀褐飞虱作用的影响

在室内研究了喷施黄绿绿僵菌Metarhizium anisopliae var. acridum Driver et Milner孢子悬液对稻田蜘蛛捕杀褐飞虱作用的影响。结果表明,黄绿绿僵菌不感染拟水狼蛛Pirata subpiraticus Bsenberg et Strand和食虫沟瘤蛛Ummeliata inse cticeps Bsenberg et Strand,只对蜘蛛喷施菌液不影响它们的捕杀褐飞虱的能力。而对褐飞虱喷施黄绿绿僵菌液后,褐飞虱活力明显下降,导致蜘蛛对其捕杀效果显著提高。在喷施黄绿绿僵菌106 、107、108/mL孢子浓度后,拟水狼蛛的平均捕杀量分别为10.5头/d、11.1头/d和11.4头/d,食虫沟瘤蛛的平均捕杀量分别为3.8头/d、4.3头/d和4.7头/d,均显著大于对照组。对蜘蛛和褐飞虱同时喷施黄绿绿僵菌不影响前者的捕杀力。这些结果提示,在稻田施用黄绿绿僵菌防治稻飞虱对蜘蛛天敌没有不良影响。     

褐飞虱求偶鸣声中有效声段及其特征

对褐飞虱Nilaparvata lugens (Stal) 雌、雄虫播放求偶鸣声信号的不同声段及其组合,结果表明在它们求偶识别过程中,由多个规则连续的声脉冲所组成的声段是有效的。播放不同声脉冲重复频率和主振频率组合的模拟信号,表明褐飞虱雌、雄虫求偶信号的声脉冲重复频率是它们识别异性的敏感声学特征参数,其敏感范围较窄,分别为70～90 Hz和22 Hz左右;而对主振频率的敏感范围较宽,分别为200 ～1 700 Hz和100～300 Hz。     

Small brown planthopper resistance loci in wild rice (Oryza officinalis)

Host-plant resistance is the most practical and economical approach to control the rice planthoppers. However, up to date, few rice germplasm accessions that are resistant to the all three kinds of planthoppers (1) brown planthopper (BPH; Nilaparvata lugens Stål), (2) the small brown planthopper (SBPH; Laodelphax striatellus Fallen), and (3) the whitebacked planthopper (WBPH, Sogatella furcifera Horvath) have been identified; consequently, the genetic basis for host-plant broad spectrum resistance to rice planthoppers in a single variety has been seldom studied. Here, one wild species, Oryza officinalis (Acc. HY018, 2n = 24, CC), was detected showing resistance to the all three kinds of planthoppers. Because resistance to WBPH and BPH in O. officinalis has previously been reported, the study mainly focused on its SBPH resistance. The SBPH resistance gene(s) was (were) introduced into cultivated rice via asymmetric somatic hybridization. Three QTLs for SBPH resistance detected by the SSST method were mapped and confirmed on chromosomes 3, 7, and 12, respectively. The allelic/non-allelic relationship and relative map positions of the three kinds of planthopper resistance genes in O. officinalis show that the SBPH, WBPH, and BPH resistance genes in O. officinalis were governed by multiple genes, but not by any major gene. The data on the genetics of host-plant broad spectrum resistance to planthoppers in a single accession suggested that the most ideally practical and economical approach for rice breeders is to screen the sources of broad spectrum resistance to planthoppers, but not to employ broad spectrum resistance gene for the management of planthoppers. Pyramiding these genes in a variety can be an effective way for the management of planthoppers.     

水稻褐飞虱内生共生细菌Arsenophonus的鉴定和系统分析

利用16S rDNA通用引物扩增了水稻褐飞虱Nilaparvata lugens（Stål）体内共生细菌的序列,经克隆、测序和NCBI数据库比对,发现褐飞虱体内存在杀雄菌属Arsenophonus类共生细菌,系统发育上与粉虱科和木虱科体内的Arsenophonus属亲源关系较近。在褐飞虱体内该共生细菌具有两种长度不同的16S rDNA序列,分别为1 504 bp和547 bp,其中后者为前者中间缺失了957 bp,其余序列相同。通过重新设计两对引物进行扩增,进一步确认不同褐飞虱地理种群及寄主种群均存在两种片段。Arsenophonus特异的 23S rDNA引物的扩增结果表明,Arsenophonus存在于所有检测的褐飞虱种群中,但不存在于水稻寄主中。荧光定量PCR检测发现3个褐飞虱室内寄主种群Arsenophonus属共生细菌含量不同,其中TN1种群明显高于Mudgo种群和ASD7种群。此为水稻褐飞虱体内存在Arsenophonus属共生细菌的首次报道。     

Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Trypanosoma cruzi, the agent of Chagas disease, expresses onits surface a trans-sialidase that catalyzes preferentiallythe transference of -2,3-linked sialic acid to acceptors containingterminal β-galactosyl residues, instead of the typicalhydrolysis reaction, found in most sialidases. The trans-sialidaseis responsible for the acquisition of the host sialic acid bythis protozoan parasite, which does not synthesize sialic acids.Here, we have studied some kinetic properties of a recombinanttrans-sialidase expressed in Escherichia coli We found thatit has sequential-type kinetics for the transferase reaction,as shown for the parasite-derived enzyme. The rates of sialicacid transfer to water (hydrolysis), and to β-galactosylresidues have a unique behavior with respect to the reactiontemperature. While the hydrolysis rate of sialyUactose increasescontinuously up to 35°C, the temperature for the maximalrate of trans-glycosylation depends on the acceptor concentration.At low acceptor concentrations the rate of trans-glycosylationis maximal at 13°C and independent of the amount of sialicacid donors. With increasing acceptor concentrations, maximalrates of trans-glycosylation are shifted to higher temperatures.This finding is explained by an 8-fold increase in the K_m forthe acceptor from 13°C to 33°C. Differences in hydrolysisand transfer rates were also obtained by using 4-methyl-umbelliferyl-N-acetyl-neuraminicacid. However, its hydrolysis rate is much higher than the rateof transference to lactose, suggesting that a long-lived enzyme-sialosylintermediate is not formed. In addition, lactose does not increasethe rate of methyl-umbelliferone release at any temperature,indicating that the rate limiting step is the aglycon release.Based on these results we propose that trans-glycosylation inT.cruzi sialidase is favored by the existence of a binding sitefor β-galactosyl residues, which accepts the new glycosidicbond as sialic acid is released from the donor. With increasingtemperature the affinity for the acceptor decreases, resultingin a concomitant increase in the rate of transfer to water,which, in turn, can be suppressed by increasing the acceptorconcentration. Trypanosoma cruzi sialidase kinetics reaction mechanism temperature     

The Role of Farnesol as a Regulator of Stomatal Opening in Sorghum

Fine, very dilute aqueous emulsions of all-trans farnesol appliedto intact leaves of Sorghum bicolor caused appreciable inhibitionof stomatal opening which persisted for 2 d, after which timethe stomata regained their capacity to open. The inhibitoryeffect of farnesol was not overcome by flushing the leaves withCO₂-free air, indicating that it was not the result of an accumulationof CO₂. This conclusion was supported by measurements of CO₂compensation, which increased only slightly after farnesol treatment. All-trans farnesol has previously been reported to be formedin water-stressed plants of Sorghum. The data presented heresuggest that it could be acting as an endogenous antitranspirant,in a comparable role to that already established for abscisicacid in several species. It would appear, however, to have aless prolonged inhibitory effect than abscisic acid, and itcould be responsible for the rapid responses of Sorghum stomatato water stress and their quick recovery after the plant hasregained turgor, a characteristic which distinguishes Sorghumfrom many other genera so far investigated.     

Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

稻虱缨小蜂对褐飞虱和白背飞虱卵的识别机制

研究了室内条件下信息化合物及稻飞虱卵的形态特征在稻虱缨小蜂识别2种卵寄主褐飞虱和白背飞虱中的作用。结果表明,褐飞虱和白背飞虱雌成虫诱导的水稻挥发物对稻虱缨小蜂的引诱作用无显著差异,各自的引诱比例(头数)分别为57.50%(23头)和42.50%(17头)。稻虱缨小蜂对2种飞虱4组材料,完整卵、磨碎卵、带卵叶鞘和若虫为害叶鞘的行为反应,除了在褐飞虱完整卵上的搜索时间极显著地长于在白背飞虱完整卵上的以外,其余的均不存在差异。挥发物捕集结果表明,褐飞虱和白背飞虱雌成虫诱导的水稻挥发物组成相非常一致。上述结果表明水稻挥发物、稻飞虱利它素及飞虱卵的形态特征在稻虱缨小蜂识别褐飞虱和白背飞虱卵中的作用不明显。文中就稻虱缨小蜂识别2种寄主的机制进行了讨论。     

家蚕保存系统红色卵的遗传分析

用遗传背景清楚的家蚕Bombyx mori红卵（re）、白卵(w-2、pe)、第4褐卵（b-4）的标志基因系统和正常型黑卵系统与我国家蚕基因库保存的20个红色卵系统杂交,进行顺反测验,分析了它们的卵色支配基因及遗传规律。结果发现：①在03-310系统中存在家蚕卵色新突变pink egg,与红卵re 等位,基因符号为re^p,表型特征为：卵淡红色,成虫蛾眼也为淡红色;②6个系统为红卵（re）的纯合系统,还有５个系统除具有re／re基因型外,还具有支配白色卵或浅红色或橙红色卵的突变基因;③２个系统为第4褐卵（b4）的纯合系统; ④6个系统的红褐色卵为母性影响遗传;⑤发现家蚕卵色基因b-4和r-e的互补关系,b-4/b-4 re/re基因型表现为新的卵色——橙黄色。     

Distribution of Diacylglycerylhydroxymethyltrimethyl-{beta}-alanine (DGTA) and Phosphatidylcholine in Brown Algae

Lipids were analyzed in thirteen species of brown algae collectedat the seashore near Tokyo, Japan. Diacylglycerylhydroxymethyltrimethyl-ß-alanine(DGTA), a recently identified betaine lipid, was found as amajor lipid component in eight species of brown algae examined,namely, Ishige okamurai, Dictyota dichotoma, Pachydictyon coriaceum,Padina arborescens, Hizikia fusiformis, Sargassum horneri, S.ringgoldianum and S. thunbergii. However, phosphatidylcholine(PC) was not detected in any of these algae except I. okamurai.By contrast, PC was found as a major lipid component in fiveother species, namely, Colpomenia sinuosa, Endarachne binghamiae,Scytosiphon lomentarius, Eisenia bicyclis, Undaria pinnatifida.These algae in turn did not contain detectable amounts of DGTA.The fatty acid composition of four selected species, S. lomentarius,U. pinnatifida, D. dichotoma and H. fusiformis, was also studied.The fatty acid components of DGTA in D. dichotoma and H. fusiformiswere similar to those of PC in U. pinnatifida, the major componentsbeing 16:0, 18:2 and 20:46 (also 16:1 in D. dichotoma). (Received December 14, 1990; Accepted April 10, 1991)     

Studies on the Metabolism of Gallic Acid by Microorganisms

The intermediary metabolism of gallic acid by Aspergillus niger under the influence of some added inhibitors has been studied. The decomposition of gallic acid by lyophilized cells under fluoroacetate inhibition allowed cis-aconitic acid, α-ketoglutaric acid and citric acid to accumulate. A mechanism of gallic acid decomposition via cis-aconitic acid has been inferred.     

灰飞虱体内一种酵母类共生菌的分子鉴定

为明确稻飞虱体内酵母类共生菌（yeast-like symbiont,YLS）的种类,采用超速离心的方法分离纯化灰飞虱Laodelphax striatellus（Fallén）体内YLS,用真菌的通用引物对其18S rDNA、5.8S-ITS rDNA全长序列进行扩增。结果得到一条分子量约为2 340 bp的序列。序列同源性分析表明,该菌与Noda等所报道的类酵母菌的18S rDNA序列差异较大（同源性只有89.6％）,而与季也蒙毕赤酵母Pichia guilliermondii有99.8％的同源性。原位杂交（ISH）和巢氏PCR均证明该菌存在于灰飞虱脂肪体和卵内,但数量较少。因此,灰飞虱体内YLS除了Noda等报道的类酵母菌外,尚存在另外一种季也蒙毕赤酵母菌。     

Enzyme system catalyzing formation of cis-3-hexenal and n-hexanal from linolenic and linoleic acids in Japanese silver (Farfugium iaponicum Kitamura) leaves

Leaf alcohol (cis-3-hexenol) and leaf aldehyde (trans-2-hexenal)are responsible for the green odor in leaves and fruits. cis-3-Hexenal,a precursor of cis-3-hexenol and trans-2-hexenal, was producedfrom linolenic acid by a homogenate of Farfugium japonicum (Japanesesilver) leaves. n-Hexanal was produced from linoleic acid bya homogenate of the leaves. The enzyme system catalyzing formationof C₆-aldehydes from linolenic and linoleic acids was localizedin chloroplast lamellae, and required oxygen for reaction. C₁₈-unsaturatedfatty acids such as linolenic acid, linoleic acid and -linolenicacid, which have carboxyl groups and cis-1, cis-4-pentadienesystems including a double bond at C-12, acted as substrates,and C₆-aldehydes (cis-3-hexenal or n-hexanal), but not C₉-aldehydes,were produced from them. The properties of the enzyme systemin chloroplasts were as follows: optimal pH 7.0; stable at pH5 to 7; thermolabile and no activity at 50?C. These propertieswere very similar to those of tea chloroplasts. The enzyme systemcould be solubilized from chloroplasts by 2% Triton X-100, butwas very unstable in solubilized form. (Received July 9, 1976; )     

Thermodynamics of Malate Transport across the Tonoplast of Leaf Cells of CAM Plants

The electrochemical potential difference for each dissociationstate of malic acid across the tonoplast of leaf cells was examinedin two CAM plants, Graptopetalum paraguayense and Kalanchoëdaigremontiana. The concentration of malic acid in each dissociationstate was estimated from an analysis of pH and concentrationsof ionic species that included calcium, malate and isocitrate.The vacuoles contained 30–40 mM isocitrate and 50–70mM calcium in G. paraguayense, and 20–30 mM isocitrateand 70–100 mM calcium in K. daigremontiana. For the calculationof the pattern of dissociation of malic acid, the formationof chelates of calcium with malate and isocitrate, which havedifferent stability constants depending on the dissociationof the acids, were also taken into consideration. The vacuolarconcentrations of the divalently dissociated form of malic acid(mal^2– were 4–7 mM and 1-3 mM in G. paraguayenseand in K. daigremontiana, respectively. To obtain informationabout the cytoplasmic concentration of malate, the apparentinhibition constant for malate of phosphoenolpyruvate carboxylasewas measured. It was about 330 µM in the dark period and60 µM in the light period. Considering an inside-positivemembrane potential, we conclude that mal^2– can be takenup passively into the vacuole during the dark period and canbe released passively from the vacuole during the light period.Two types of channel (the "SV-type" channel and a novel "MU-type"channel) which we found recently in G. paraguayense [Iwasakiet al. (1992) Plant Physiol. 98: 1494] are probably involvedin the uptake and the release of malate in the diurnal CAM rhythm.The existence of a large pH-buffering capacity due to isocitricacid in the vacuole allows the accumulation of a large amountof malic acid during the diurnal CAM rhythm. (Received February 12, 1992; Accepted July 10, 1992)