Identification and Metabolism of 1-(Malonylamino)cyclopropane-1-carboxylic Acid in Germinating Peanut Seeds

Peanut seeds (Arachis hypogea L. Yue-you 551) contain 50 to 100 nanomoles per gram conjugated 1-aminocyclopropanecarboxylic acid (ACC). Based on paper chromatography, paper electrophoresis, and gas chromatography-mass spectrometry, it was verified that the major ACC conjugate was N-malonyl-ACC (MACC). Germinating peanut seeds converted [2-¹⁴C]ACC to ethylene 70 times more efficiently than N-malonyl-[2-¹⁴C]ACC; when ACC was administered, most of it was metabolized to MACC. Germinating peanut seeds produced ethylene and converted l-[3,4-¹⁴C]methionine to ethylene; this ethylene biosynthesis was inhibited by aminoethoxyvinylglycine. These data indicate that MACC occurs in peanut seeds but does not serve as the source of ethylene during germination; ethylene is, however, synthesized from methionine via ACC.     

The Conversion of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid to 1-Aminocyclopropane-1-Carboxylic Acid in Plant Tissues

Since 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the major conjugate of 1-aminocyclopropane-1-carboxylic acid (ACC) in plant tissues, is a poor ethylene producer, it is generally thought that MACC is a biologically inactive end product of ACC. In the present study we have shown that the capability of watercress (Nasturtium officinale R. Br) stem sections and tobacco (Nicotiana tabacum L.) leaf discs to convert exogenously applied MACC to ACC increased with increasing MACC concentrations (0.2-5 millimolar) and duration (4-48 hours) of the treatment. The MACC-induced ethylene production was inhibited by CoCl₂ but not by aminoethoxyvinylglycin, suggesting that the ACC formed is derived from the MACC applied, and not from the methionine pathway. This was further confirmed by the observation that radioactive MACC released radioactive ACC and ethylene. A cell-free extract, which catalyzes the conversion of MACC to ACC, was prepared from watercress stems which were preincubated with 1 millimolar MACC for 24 hours. Neither fresh tissues nor aged tissues incubated without external MACC exhibited enzymic activity, confirming the view that the enzyme is induced by MACC. The enzyme had a K_m of 0.45 millimolar for MACC and showed maximal activity at pH 8.0 in the presence of 1 millimolar MnSO₄. The present study indicates that high MACC levels in the plant tissue can induce to some extent the capability to convert MACC to ACC.     

Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K_m of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30°C. Several effectors known to inhibit ATPase (N,N′-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H⁺ electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN⁻ and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH—, OH—, and NH₂— groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.     

Isolation and Identification of ( + )-Hexylitaconic Acid as a Plant Growth Regulator

( + )-Hexylitaconic acid was isolated as a root-growth stimulating substance from a culture filtrate of Aspergillus niger K-88.     

Studies on the Translocation and Accumulation of 14C-Labelled N-Dimethylaminosuccinmic Acid (B) in Peanut Plant

The 14C-B9 was used by smearing the surface of the leaf of peanut plant (Arachis hypogeae L.) during the flowering stage and pegging stage. The results obtained with the measurement of radioactivity were as follows: The incorporation of B9 into peanut plant was very fast, and the radioactive isotope was so much accumulated in the leaf of the main stem one hour after treatment. Foul hours after treatment, B9 would also be occurred so much in the flower. After 3 days, the radioactive isotope was accumulated in the organs of peanut plant up to the maximum amount. In the pegging stage the translocation of B9 into the organs of peanut plant was faster than that in the flowering stape having the maximum amount of accumulation in the first day. The rate of out flow of B9 from the smearing leaf was high. It was shown that the radioactivity was mainly concentrated in the young tissues of stem and leaf, and in the flower and small pod. By using microautoradiography, the radioactivity was translocated through the vascular bundle in the petiole and stem of the peanut plant. Later, it was chiefly distributed in the cortex of the stem and the palisade tissues of the leaf. In the flower, the 14C-B9 was firstly found in the vascular bundle of the filament and the petal. After 3 days, the radioactive isotope was transported into the pollen grain and concentrated in the inner wall of the pollen sac. The chromatogram of the radioactive matter extracted from the peanut plant was showed that the compound of B9 was biochemically stable and degradated not easily in the peanut plant.     

Vacuolar Release of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid, the Conjugated Form of the Ethylene Precursor

The mechanisms underlying the vacuolar retention or release of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, has been studied in grape (Vitis vinifera) cells grown in vitro using the technique of compartmental analysis of radioisotope elution. Following its accumulation in the vacuole, M[2,3-¹⁴C]ACC could be released from cells when the vacuolar pH was artificially lowered by external buffers from its initial value of 6.2 to below the critical pH of 5.5. Successive release and retention of vacuolar MACC could be achieved by switching the vacuolar pH from values lower and higher than 5.5. The rate constant of efflux was highly correlated with the vacuolar pH. In plant tissues having low vacuolar pH under natural conditions, e.g. apple fruits (pH 4.2) and mung bean hypocotyls (pH 5.3), an efflux of M[2,3-¹⁴C]ACC also occurred. Its rate constant closely corresponded to the theorical values derived from the correlation established for grape cells. Evidence is presented that the efflux proceeded by passive lipophilic membrane diffusion only when MACC was in the protonated form. In contrast to other organic anions like malic acid, the mono and diionic species could not permeate the tonoplast, thus indicating the strict dependence of MACC retention upon the ionic status of the molecule and the absence of carrier-mediated efflux.     

Intracellular Sites of Synthesis and Storage of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Acer pseudoplatanus Cells

Vacuoles were isolated from Acer pseudoplatanus cells that were incubated with [¹⁴C]1-aminocyclopropane-1-carboxylic acid (ACC). The kinetics of [¹⁴C]1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) formation are consistent with the interpretation that MACC is synthesized in the cytosol, transported through the tonoplast, and accumulated in the vacuole. Twenty hours after chasing the labeled ACC with unlabeled ACC and adding 1 millimolar unlabeled MACC, all the [¹⁴C]MACC synthesized is located in the vacuole. Whole cells preloaded with [¹⁴C]MACC and then submitted to a continuous washing out, readily release their cytosolic MACC until complete exhaustion. The half-time of MACC efflux from the cytosol, calculated by the technique of compartmental analysis, is about 22 minutes. In contrast, vacuolar MACC remains sequestered within the vacuole. The transport of labeled MACC into the vacuole is stimulated by the presence of unlabeled MACC in the suspension medium, probably as a result of a reduced efflux of the labeled MACC from the cytosol into the suspending medium.     

花生慢生根瘤菌的分离与鉴定

目的:从野生花生的新鲜根瘤中分离纯化花生慢生根瘤菌。方法:将野生型花生的新鲜根瘤灭菌后捣碎制成悬浮液,通过稀释涂布、划线和贴片三种方法,在YMA结晶紫和YMA刚果红选择培养基上逐步分离纯化得到单菌落。结果:三种方法均分离得到表面性状一致的单菌落,其中用稀释53~54倍的悬浮液划线分离的效果最好。所得菌株经理化性质和回接试验鉴定为慢生根瘤菌,从而为接着的花生根瘤菌及其结瘤基因的研究提供了基础。     

Isolation of Ciliatine (2-Aminoethylphosphonic Acid) from the Bile of the Bovine

To elucidate the formation mechanism of N,N′-dialkylpyrazine cation radical during browning reaction of sugars with amino compounds, main products in an early stage of the reaction were determined quantitatively by TLC and GLC. It was shown that the Schiff base, two-carbon fragmental product of sugar, the free radical and deoxyosone were successively produced prior to the browning. Polarographical measurements indicated that the radical formation was induced by the production of some reducing substances in the reaction mixture. These results suggest that the free radical was formed by the reduction of N,N′-dialkylpyrazinium; a compound, which demonstrated to have a strong activity to browning, might be formed by condensation of two-carbon enaminol followed by oxidation.     

间接竞争ELISA测定花生植株内源水杨酸含量

以SA-N=CH-（CH2）3-CH=N-0VA为包被抗原,利用SA-NH-CH2-NH-BsA为免疫原产生的抗体,来建立能定量测定花生组织中游离水杨酸（sA）的间接竞争ELISAS-作程序,并明确该方法的工作条件以及基本参数。结果表明：该方法对sA标准品的最低检测量为10ng-mL-1,线性检测范围为10ng·mL～10·mL-1。在该范围内,标准曲线的批内和批间变异系数分别为1．90％和6．81％;花生叶片和根组织中添sr,SA的平均回收率分别为93．9％和91．0％。     

Evidence for 1-(Malonylamino)cyclopropane-1-Carboxylic Acid Being the Major Conjugate of Aminocyclopropane-1-Carboxylic Acid in Tomato Fruit

Tomato (Lycopersicon esculentum Miller) fruit discs fed with [2,3-¹⁴C]1-aminocyclopropane-1-carboxylic acid (ACC) formed 1-malonyl-ACC (MACC) as the major conjugate of ACC in fruit throughout all ripening stages, from immature-green through the red-ripe stage. Another conjugate of ACC, γ-glutamyl-ACC (GACC), was formed only in mature-green fruit in an amount about 10% of that of MACC; conjugation of ACC into GACC was not detected in fruits at other ripening stages. No GACC formation was observed from etiolated mung bean (Vigna radiata [L.] Wilczek) hypocotyls, etiolated common vetch (Vicia sativum L.) epicotyls, or pea (Pisum sativum L.) root tips, etiolated epicotyls, and green stem tissue, where active conversion of ACC into MACC was observed. GACC was, however, formed in vitro in extracts from fruit of all ripening stages. GACC formation in an extract from red fruit at pH 7.15 was only about 3% of that at pH 8.0, the pH at which most assays were run. Our present in vivo data support the previous contention that MACC is the major conjugate of ACC in plant tissues, whereas GACC is a minor, if any, conjugate of ACC. Thus, our data do not support the proposal that GACC formation could be more important than MACC formation in tomato fruit.     

The Isolation of DNA from Plant Materials

Total DNA was isolated from seedling and tissues of many plants. The nuclei and chloroplasts were prepared from plant tissues, and then the nuclear DNA and chloroplast DNA were isolated from them. According to the chemical analysis and the physical properties determined by ultraviolet absorbance, hyperchromicities, ultracentrifugation, gel electrophoresis and the electro-microscopical observations it is suggested that DNA obtained possessed a considerable purity and to a certain extent retained the natural status of the large molecule.     

三种花生幼胚蛋白质提取方法比较研究

植物组织(或细胞)的蛋白质提取效率与效果直接影响蛋白质双向凝胶电泳等实验的结果。为探索建立适用于花生幼胚蛋白质(双向凝胶电泳用)提取的最佳条件,尝试了磷酸缓冲液直接提取法、改良的荔枝胚胎蛋白提取法和Trizol(附加)提取法等3种提取方法,根据蛋白提取得率、试剂成本、双向电泳图谱的质量(蛋白质斑点的丰度、分布特点)进行初步评价。结果表明,磷酸缓冲液直接提取法简单但总体效果较差,改良的荔枝胚胎蛋白提取法综合评价最好,与双向凝胶电泳条件更兼容。     

Isolation of Fulvic Acid from Cercospora beticola

Phosphatidylethanolamine (PE) is generally more oxidizable than phosphatidylcholine (PC). To determine the difference in reactivities to oxidation between PE and PC, it is necessary for their fatty acid moieties to be uniform. Experimental results of the ferrous ion-catalyzed oxidation of dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylethanolamine, and dilinoleoyldiglyceride revealed that the rate of oxidation depends on the type of base. Ferrous ion possessed a high catalytic activity in hydroperoxide formation at pH 5.8. Iron ions might initiate the oxidation of phospholipids by forming free radicals. Phosphoethanolamine was capable of trapping ferrous ion and preventing it from being autoxidized to ferric ion. Trapping of ferrous ion might be responsible for the significant oxidizability of PE at pH 5.8 ~ 7.0. In the ferrous ion-ascorbic acid (AsA) catalyzed oxidation system, PC oxidation was remarkably enhanced at pH 7.0. In this case, no reduction of ferric ion occurred, but AsA had a prooxidant effect of accelerating the formation of free radicals.     

5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles

Cells release ATP in response to physiologic stimuli. Extracellular ATP regulates a broad range of important cellular functions by activation of the purinergic receptors in the plasma membrane. The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. FM1-43 fluorescence was used to measure exocytosis and bioluminescence to measure ATP release in HTC rat hepatoma and Mz-Cha-1 human cholangiocarcinoma cells. Exposure to a Cl⁻ channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50–300 μm) stimulated a 5–100-fold increase in extracellular ATP levels within minutes of the exposure. This rapid response was not a result of changes in cell viability or Cl⁻ channel activity. NPPB also potently stimulated ATP release in HEK293 cells and HEK293 cells expressing a rat P2X7 receptor indicating that P2X7 receptors are not involved in stimulation of ATP release by NPPB. In all cells studied, NPPB rapidly stimulated vesicular exocytosis that persisted many minutes after the exposure. The kinetics of NPPB-evoked exocytosis and ATP release were similar. Furthermore, the magnitudes of NPPB-evoked exocytosis and ATP release were correlated (correlation coefficient 0.77), indicating that NPPB may stimulate exocytosis of a pool of ATP-enriched vesicles. These findings provide further support for the concept that vesicular exocytosis plays an important role in cellular ATP release and suggest that NPPB can be used as a biochemical tool to specifically stimulate ATP release through exocytic mechanisms.     

Isolation and Identification of Fatty Acid Component from Seed Oil of Microula sikkimensis (Clarke) Hemsl

The seed oil content of Microula sikkimensis (Clarke) Hemsl. is up to 45% There is 8.1% of γ-linolenic acid which has the pharmacological action in the fatty acids composition, It has showed that this oil has a stronger effect on reducing triglyceride in serum. Fifteen different kinds of fatty acids were analysed. The unsaturated C20, C22, C24 acid, C18 triene-acid and tetraene-acid of the seed oil were separated on AgNO3-silica gel column and HPLC. and were identified by Periodata-Permanganate Oxidation, GLC, IR, UV, and MS. They are cis-11-eicosenoic, cis-13-docosenoic, cis-15-tetracosenoic, cis-6,9,12-octadecatrienoic and cis-6,9,12,15- octadecatetraenoic acids.