Intracellular location of NADP+-linked malic enzyme in C3 plants

Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the mRNA for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for providing us with antisera to CHS and PAL, respectively. This work was supported by a grant from the Deutsche Forschungsgemeinschaft and scholarships from the Friedrich-Ebert-Stiftung (J. G.), the Landesgraduierten-förderungsgesetz Baden-Württemberg (J.-P. S) and the Gerhard-Rösch-Stiftung (D. S.). We thank R. Hofmann for her excellent technical assistance.     

Phytoalexin synthesis in soybean cells: elicitor induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs and correlation with phytoalexin accumulation

A glucan elicitor from cell walls of the fungus Phytophthora megasperma f. sp. glycinea, a pathogen of soybean (Glycine max), induced large and rapid increases in the activities of enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, and of the flavonoid pathway, acetyl-CoA carboxylase and chalcone synthase, in suspension-cultured soybean cells. The changes in phenylalanine ammonia-lyase and chalcone synthase activities were correlated with corresponding changes in the mRNA activities encoding these enzymes, as determined by enzyme synthesis in vitro in a mRNA-dependent reticulocyte lysate. The time courses of the elicitor-induced changes in mRNA activities for both enzymes were very similar with respect to each other. Following the onset of induction, the two mRNA activities increased significantly at 3 h, reached highest levels at 5 to 7 h, and subsequently returned to low values at 10 h. Similar degrees of induction of mRNA activities and of the catalytic activities of phenylalanine ammonia-lyase and chalcone synthase were observed in response to three diverse microbial compounds, the glucan elicitor from P. megasperma, xanthan, an extracellular polysaccharide from Xanthomonas campestris, and endopolygalacturonase from Aspergillus niger. However, whereas the glucan elicitor induced the accumulation of large amounts of the phytoalexin, glyceollin, in soybean cells, endopolygalacturonase induced only low, albeit significant, amounts; xanthan did not enhance glyceollin accumulation under the conditions of this study. This result might imply that enzymes other than phenylalanine ammonia-lyase or chalcone synthase exert an important regulatory function in phytoalexin synthesis in soybean cells.     

Stress responses in alfalfa (Medicago sativa L.) III. Induction of medicarpin and cytochrome P450 enzyme activities in elicitor-treated cell suspension cultures and protoplasts

Summary Cell suspension cultures of alfalfa (Medicago sativa L.) accumulated phenolic secondary metabolites in a pattern similar to that seen in alfalfa roots. Upon treatment with a crude elicitor preparation from the bean pathogen Colletotrichum lindemuthianum, the pterocarpan phytoalexin medicarpin accumulated in cells and culture medium. The extractable activities of six enzymes involved in medicarpin biosynthesis (including three cytochrome P450 activities) were induced by treatment with elicitor, and their induction kinetics correlated with the rate of medicarpin accumulation. However, protoplasts prepared from these cultures accumulated neither medicarpin nor other secondary products after treatment with elicitor. The cytochrome P450 activities were induced during the preparation of the protoplasts, but could be further induced by treatment with fungal elicitor. The results are discussed in relation to the use of alfalfa protoplasts as a system for functional analysis of cloned defense genes.Abbreviations AUFS absorption unit full scale - CHI chalcone isomerase (EC 5.5.1.6) - CHS chalcone synthase (EC 2.3.1.74) - C40H cinnamic acid 4-hydroxylase (EC 1.14.13.11) - CLE elicitor from Colletotrichum lindemuthianum - IFOH isoflavone 2-hydroxylase - IFS isoflavone synthase - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Induction of chalcone isomerase in elicitor-treated bean cells. Comparison of rates of synthesis and appearance of immunodetectable enzyme

Chalcone isomerase, an enzyme involved in the formation of flavonoid-derived compounds in plants, has been purified nearly 600-fold from cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.). Chromatofocussing yielded a single form of the enzyme of apparent pI 5.0. This preparation was used to raise rabbit anti-(chalcone isomerase) serum. Changes in the rate of synthesis of chalcone isomerase have been investigated by indirect immunoprecipitation of enzyme labelled in vivo with [35S]methionine in elicitor-treated cultures of P. vulgaris. Elicitor, heat-released from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes increased synthesis of the isomerase, with maximum synthetic rate occurring 11-12 h after exposure to elicitor. Immune blotting studies indicate that the elicitor-mediated increase in extractable activity of the isomerase is associated with increased appearance of immunodetactable isomerase protein of Mr 27 000. However, the maximum level of immunodetectable isomerase was attained approximately 6 h earlier than maximum extractable activity. Furthermore, a 2.8-fold increase in enzyme activity above basal levels at 12 h after elicitor-treatment was associated with a corresponding 5.8-fold increase in immunodetectable enzyme. It is concluded that elicitor induces the synthesis of both active and inactive chalcone isomerase of Mr 27 000, and that some activation of inactive enzyme occurs during the elicitor-mediated increase in isomerase activity. The presence of a pool of inactive chalcone isomerase in bean cell cultures has recently been suggested on the basis of density labelling experiments utilising 2H from 2H2O [Dixon et al. (1983) Planta (Berl.) 159, 561-569].     

Rapid induction by fungal elicitor of the synthesis of cinnamyl-alcohol dehydrogenase, a specific enzyme of lignin synthesis

A fivefold increase in the extractable activity of cinnamyl-alcohol dehydrogenase, an enzyme of phenylpropanoid metabolism specific for lignin synthesis, was observed within 10 h of treatment of cell-suspension cultures of bean (Phaseolus vulgaris L.) with a high-molecular-mass elicitor preparation heat-released from mycelial cell walls of the bean pathogen Colletotrichum lindemuthianum. Elicitor caused a rapid, marked but transient increase in the synthesis of cinnamyl-alcohol dehydrogenase with maximum rates 2-3 h after elicitation, concomitant with the phase of rapid increase in enzyme activity. There is a close correspondence between increased polysomal mRNA activity encoding cinnamyl-alcohol dehydrogenase, as measured by incorporation of [35S]methionine into immunoprecipitable enzyme subunits in vitro, and the stimulation of enzyme synthesis in vivo in response to elicitor. This marked increase in polysomal mRNA activity represents an increase as a proportion of total cellular mRNA activity, indicating that elicitor does not stimulate synthesis of this enzyme by selective recruitment from the total pool of cellular mRNA. Elicitor stimulation of cinnamyl-alcohol dehydrogenase activity and enzyme synthesis is more rapid than previously observed for other proteins involved inducible defense mechanisms, such as enzymes of phytoalexin biosynthesis or the apoproteins of cell-wall hydroxyproline-rich glycoproteins.     

Phytoalexin Induction in French Bean : Intercellular Transmission of Elicitation in Cell Suspension Cultures and Hypocotyl Sections of Phaseolus vulgaris

Treatment of hypocotyl sections or cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.) with an abiotic elicitor (denatured ribonuclease A) resulted in increased extractable activity of the enzyme l-phenylalanine ammonia-lyase. This induction could be transmitted from treated cells through a dialysis membrane to cells which were not in direct contact with the elicitor. In hypocotyl sections, induction of isoflavonoid phytoalexin accumulation was also transmitted across a dialysis membrane, although levels of insoluble, lignin-like phenolic material remained unchanged in elicitor-treated and control sections. In bean cell suspension cultures, the induction of phenylalanine ammonia-lyase in cells separated from ribonuclease-treated cells by a dialysis membrane was also accompanied by increases in the activities of chalcone synthase and chalcone isomerase, two enzymes previously implicated in the phytoalexin defense response. Such intercellular transmission of elicitation did not occur in experiments with cells treated with a biotic elicitor preparation heat-released from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The results confirm and extend previous suggestions that a low molecular weight, diffusible factor of host plant origin is involved (in French bean) in the intercellular transmission of the elicitation response to abiotic elicitors.     

灯盏花 chi 的克隆及其生物信息学分析

查尔酮异构酶（CHI）是调控黄酮生物合成的关键酶,分离和克隆这一酶的功能基因,对利用转基因技术进行灯盏花黄酮生物合成的调控具有重要意义。本研究采用RT-PCR和RACE技术,获得了chi cDNA全序列,GenBank登录号为GU208823.1,序列全长996 bp,开放阅读框为594 bp,编码197个氨基酸,3-Race有一个多聚腺苷酸加尾信号。应用软件预测该基因编码蛋白分子量约为21.6 kD,理论等电点为4.78。该基因编码的蛋白无跨膜结构域,其二级结构的主要构件为α-螺旋和随机卷曲。对其三级结构进行了建模,表明其结构与苜蓿chi的三级结构相似。同时根据灯盏花chi N端序列变化的特征,提出了灯盏乙素的合成可能与chi在细胞亚结构的定位及其与合成代谢相关酶形成复合酶的特异性有关。研究为利用基因工程定向改变灯盏花黄酮代谢产物奠定了基础。     

大豆异黄酮生物合成关键酶及其代谢工程研究进展

异黄酮是一类具有C-6/C-3/C-6骨架的二次代谢产物,具有抗氧化和抗肿瘤活性。异黄酮与黄酮类物质具有相似的苯丙烷生物合成途径。天然的绝大部分异黄酮分布在豆科植物中,目前在大豆中已经发现了超过12个异黄酮(苷)。大豆异黄酮的生物合成主要涉及三个关键的酶查尔酮合酶(CHS)、查尔酮异构酶(CHI)和异黄酮合酶(IFS)。总结了大豆异黄酮的提取分离方法和生物合成途径,着重综述了CHI、CHS、IFS生物学特征和功能及异黄酮的代谢工程研究。     

灯盏花chi的克隆及其生物信息学分析

查尔酮异构酶(CHI)是调控黄酮生物合成的关键酶,分离和克隆这一酶的功能基因,对利用转基因技术进行灯盏花黄酮生物合成的调控具有重要意义。本研究采用RT-PCR和RACE技术,获得了chi cDNA全序列,GenBank登录号为GU208823.1,序列全长996 bp,开放阅读框为594 bp,编码197个氨基酸,3-Race有一个多聚腺苷酸加尾信号。应用软件预测该基因编码蛋白分子量约为21.6 kD,理论等电点为4.78。该基因编码的蛋白无跨膜结构域,其二级结构的主要构件为α-螺旋和随机卷曲。对其三级结构进行了建模,表明其结构与苜蓿chi的三级结构相似。同时根据灯盏花chi N端序列变化的特征,提出了灯盏乙素的合成可能与chi在细胞亚结构的定位及其与合成代谢相关酶形成复合酶的特异性有关。研究为利用基因工程定向改变灯盏花黄酮代谢产物奠定了基础。