Isoflavone Reductase Research Articles

Studies on the late steps of (+) pisatin biosynthesis: Evidence for (−) enantiomeric intermediates

Pisatin, a 6a-hydroxyl-pterocarpan phytoalexin from pea ( Pisum sativum L.), is relatively unique among naturally occurring pterocarpans by virtue of the (+) stereochemistry of its 6a–11a C–C bond. However, pisatin synthesizing pea tissue has an isoflavone reductase, first identified in alfalfa, which acts on the (−) antipode. In order to establish the natural biosynthetic pathway to (+) pisatin, and to evaluate the possible involvement of intermediates with a (−) chirality in its biosynthesis, we administered chiral, tritium-labeled, isoflavanones and pterocarpans to pisatin-synthesizing pea cotyledons and compared the efficiency of their incorporation. Pea incorporated the isoflavanone, (−) sophorol, more efficiently than either its (+) antipode, or the pterocarpans (+) or (−) maackiain. (−) Sophorol was also metabolized by protein extracts from pisatin-synthesizing pea seedlings in a NADPH-dependent manner. Three products were produced. One was the isoflavene (7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene), and another had properties consistent with the isoflavanol (7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol), the expected product for an isoflavanone reductase. A cDNA encoding sophorol reductase was also isolated from a cDNA library made from pisatin-synthesizing pea. The cloned recombinant sophorol reductase preferred (−) sophorol over (+) sophorol as a substrate and produced 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol. Although no other intermediates in (+) pisatin biosynthesis were identified, the results lend additional support to the involvement of intermediates of (−) chirality in (+) pisatin synthesis.

EFFECTS OF ARBUSCULAR MYCORRHIZAL FUNGI ON PLANT SECONDARY METABOLISM

Mycorrhizal fungi form the most important mutualistic symbioses on earth with plants. The most prevalent type of mycorrhizal fungi are the arbuscular mycorrhizal (AM) fungi. Much research has shown that the development of AM fungi is correlated with plant secondary metabolism. AM fungi can directly or indirectly affect plant secondary metabolic processes. Secondary metabolites are classified into 3 groups, terpenoids, phenolics and alkaloids. In this paper, we summarize the effects of AM fungi on the 3 groups of secondary metabolites. The relationship between terpenoids and AM fungi have been well studied, and some research has explored interactive mechanisms at the molecular level. Blumenin was first isolated and identified from mycorrhizal cereals, and its biosynthesis has been proven via the Glyceraldehyde 3_phosphate/ pyruvate pathway (MEP) by an isotopic labeling method. Since then, the accumulation of blumenin induced by AM fungi and differences in blumenin levels among different kinds of AM fungi have been observed. Studies on 1_deoxy_D_xylulose_5_phosphate synthase (DXS) and 1_deoxy_D_xylulose_5_phosphate reductoisomerase (DXR), two key enzymes in the biosynthesis of carotenoid metabolism via the MEP pathway, have found to increase the transcription of DXS and DXR in plants with AM fungi. Moreover it was temporarily and spatially correlated with the accumulation of apocarotenoids. Subsequently, two genes were identified: TC78589 encoding DXS2 which is highly expressed in roots inoculated with AM fungi, and TC77051 encoding mevalonate disphosphate decarboxylase, which is catalysed in the synthesis of terpenoids in the mevalonate pathway. Although both genes separately encode enzymes in different pathways, an enhancement of carotenoid biosynthesis has been observed. The interaction between phenolic compounds (such as phytoalexin, wall_bound phenol, flavonoids, isoflavonoids and their derivatives) and AM fungi also has been investigated intensively. It has been shown that some flavonoids stimulated the spore germination and hyphal growth of AM fungi, and the contents of flavonoids increased before the infection of AM fungi. Therefore some investigators hypothesized that flavonoids were a signal compound during the formation of AM fungi. Afterward, increased levels of flavonoids were found after the formation of AM fungi which was related to specific species of AM fungi. In addition, some experiments have indicated that the activity of peroxidase (POD), phenylalanine ammonia_lyase (PAL) and polyphenol oxidase (PPO) were significantly enhanced in AM plants. In phenylpropamoid metabolism, there are two different signaling pathways in the accumulation of secondary metabolites induced by the mycorrhizal fungus: one is through the induction of PAL and chalcone synthase (CHS), and the other is through the suppression of isoflavone reductase (IFR).Although little research seldom has examined the relationship between alkaloids and AM fungi, a recent study has shown that the formation of AM is beneficial to the accumulation of alkaloids. This study also showed the species specificity in AM affected biosynthesis of alkaloids.

Identification and Characterization of Cor33p, a Novel Protein Implicated in Tolerance towards Oxidative Stress inCandida albicans

We applied two-dimensional gel electrophoresis to identify downstream effectors of CPH1 and EFG1 under hypha-inducing conditions in Candida albicans. Among the proteins that were expressed in wild-type cells but were strongly downregulated in a cph1Delta/efg1Delta double mutant in alpha-minimal essential medium at 37 degrees C, we could identify not-yet-characterized proteins, including Cor33-1p and Cor33-2p. The two proteins are almost identical (97% identity) and represent products of allelic isoforms of the same gene. Cor33p is highly similar to Cip1p from Candida sp. but lacks any significant homology to proteins from Saccharomyces cerevisiae. Strikingly, both proteins share homology with phenylcoumaran benzylic ether reductases and isoflavone reductases from plants. For other hypha-inducing media, like yeast-peptone-dextrose (YPD) plus serum at 37 degrees C, we could not detect any transcription for COR33 in wild-type cells, indicating that Cor33p is not hypha specific. In contrast, we found a strong induction for COR33 when cells were treated with 5 mM hydrogen peroxide. However, under oxidative conditions, transcription of COR33 was not dependent on EFG1, indicating that other regulatory factors are involved. In fact, upregulation depends on CAP1 at least, as transcript levels were clearly reduced in a Deltacap1 mutant strain under oxidative conditions. Unlike in wild-type cells, transcription of COR33 in a tsa1Delta mutant can be induced by treatment with 0.1 mM hydrogen peroxide. This suggests a functional link between COR33 and thiol-specific antioxidant-like proteins that are important in the oxidative-stress response in yeasts. Concordantly, cor33Delta deletion mutants show retarded growth on YPD plates supplemented with hydrogen peroxide, indicating that COR33 in general is implicated in conferring tolerance toward oxidative stress on Candida albicans.

Proteins Related to the Biocontrol of Pythium Damping-off in Maize with Trichoderma harzianum Rifai

Abstract: Induced resistance has been evidenced as one of mechanisms of Trichoderma to control plant diseases, however, no study showed the change of host proteomics in Trichoderma-induced resistance of maize against damping-off caused by Pythium ultimum Trow. The mechanism of Trichoderma harzianum Rifai for controlling maize seedling disease caused by Pythium ultimum Trow was investigated firstly by proteome technique and the result suggested that T. harzianum strain T22 was not only able to promote seedling growth but also protein accumulation. One-dimensional electrophoresis assay showed that more bands appeared on the gel with T22 or T22 combined with P. ultimum (T22 +P. ultimum) treatment than with other treatments. Enzyme assay showed that two chitinases of the root sample were more activated in the treatments with T22 than in the other treatments without T22. Proteins in the seedling roots from the various treatments were separated through protein extraction and 2-D electrophoresis technique. In the seedlings produced from the T22-treated seeds, there were 104 up-regulated proteins and 164 down-regulated proteins relative to the control, and 97 and 150, respectively, after treatment with T22 +P. ultimum; however, with P. ultimum alone the values were much lower than with the other two treatments. The correlation coefficient values were 0.72, 0.51 and 0.49 for the comparison of protein spot distribution on gel among control with T22, P. ultimum and T22 +P. ultimum, respectively. So it seemed that P. ultimum infection was more effective than T22 in interfering with the host proteome profile. Furthermore, analysis with MALDI-TOF-MAS showed that some important proteins associated with defensive reactions were identified in T22 or T22 +P. ultimum treatments, including endochitinase, pathogenesis-related protein PRMS (pathogenesis-related maize seed), GTP-binding protein, isoflavone reductase and other proteins related to respiration. All those proteins are probably part of the network of resistance or development-related proteins. Interestingly, P. ultimum treatment resulted in elimination of pathogenesis-related protein PRMS on gel, and therefore damping-off could be in part attributed to inhibition of the expression of this protein by P. ultimum infection. Some unknown proteins are also related to the defensive reaction of the host. (Managing editor: Li-Hui ZHAO)

Identification of a Vinyl Reductase Gene for Chlorophyll Synthesis in Arabidopsis thaliana and Implications for the Evolution of Prochlorococcus Species

Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet. In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis. Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR. DVR is encoded by a single copy gene in the A. thaliana genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of A. thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b. Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls. A DVR homolog was not found in the genome of P. marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus.

The characterization of defense responses to fungal infection in alfalfa

The enzyme activity and transcript level of three enzymes involved in flavonoid biosynthesis, phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CA4H), and isoflavone reductase (IFR), were monitored in alfalfa seedlings (Medicago sativa) that had been challenged with the fungal pathogen Colletotrichum trifoliii. In many legumes, a pathogen–host infection leads to an induced synthesis of fungitoxic phytoalexins produced via these key enzymes. For example, when alfalfa is exposed to an avirulent fungal type, phytoalexins are produced by the plant providing protection from additional exposure to a virulent fungal race. This defensive plant protection response was accompanied by increases in transcript levels of PAL, CA4H and IFR gene expression, by increases in PAL enzyme activity, and by production of the end product phytoalexins, medicarpin and sativan. The expression of these defense genes and PAL enzyme activity were significantly greater in seedlings responding to combined inoculations of avirulent and virulent fungi, compared to plants inoculated with the avirulent fungus alone. Inoculation with the virulent race alone elevated the gene transcripts compared with control plants but these levels were less than found in plants inoculated with the avirulent race or those inoculated with both. Production of gene transcripts reached its peak in treated plants after 49 h. The phytoalexins medicarpin and sativan increased rapidly and reached maximal levels 97 h after inoculation with the avirulent fungal race. The plants challenged with combined inoculations of the avirulent and the virulent fungi showed significantly greater accumulation of medicarpin than in any other treatment. These results suggest that the increased medicarpin accumulation produced in induced resistant tissues, following challenge inoculation with virulent race, is attributable to increased expression of genes of flavonoid biosynthesis.

Introduction of plant and fungal genes into pea (Pisum sativum L.) hairy roots reduces their ability to produce pisatin and affects their response to a fungal pathogen.

Pisatin is an isoflavonoid phytoalexin synthesized by pea (Pisum sativum L.). Previous studies have identified two enzymes apparently involved in the synthesis of this phytoalexin, isoflavone reductase (IFR), which catalyzes an intermediate step in pisatin biosynthesis, and (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM), an enzyme catalyzing the terminal step. To further evaluate the involvement of these enzymes in pisatin biosynthesis, sense- and antisense-oriented cDNAs of Ifr and Hmm fused to the 35s CaMV promoter, and Agrobacterium rhizogenes, were used to produce transgenic pea hairy root cultures. PDA, a gene encoding pisatin demethylating activity (pda) in the pea-pathogenic fungus Nectria haematococca, also was used in an attempt to reduce pisatin levels. Although hairy root tissue with either sense or antisense Ifr cDNA produced less pisatin, the greatest reduction occurred with sense or antisense Hmm cDNA. The reduced pisatin production in these lines was associated with reduced amounts of Hmm transcripts, HMM protein, and HMM enzyme activity. Hairy roots containing the PDA gene also produced less pisatin. To evaluate the role of pisatin in disease resistance, the virulence of N. haematococca on the transgenic roots that produced the lowest levels of pisatin was tested. Hairy roots expressing antisense Hmm were more susceptible than the control hairy roots to isolates of N. haematococca that are either virulent or nonvirulent on wild-type pea plants. This appears to be the first case of producing transgenic plant tissue with a reduced ability to produce a phytoalexin and demonstrating that such tissue is less resistant to fungal infection: these results support the hypothesis that phytoalexin production is a disease resistance mechanism.

A new latex allergen classified into the plant defense-related reductase family

Rationale As sequence information on genes and proteins is accumulated in databases, it becomes easy to identify a large number of proteins in a short time with proteomics approaches. Application of such techniques to allergenic proteins is referred to allergenomics. In this study, we tried to find unidentified latex allergens with an allergenomics method. Methods Proteins extracted from non-ammoniated rubber latex was two-dimensionally separated according to their isoelectric points (pI) and molecular weights then transferred onto a membrane. IgE-recognizing proteins were visualized by immunoblotting using a pooled serum from latex-allergic people. Spots of the allergenic proteins were cut out form the gel and the allergens were site-specifically in-gel digested by trypsin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) spectra of the resultant fragments and MS/MS spectra of some prominent peptide-ions were recorded on a TOF-TOF instrument. From these mass-spectrometric data, the allergens were assigned by database searches. Results A few new latex allergens as well as several previously reported allergens such as patatin-like proteins (Hev b 7), enolase (Hev b 9), and havamine were identified with this approach. One of the novel latex allergens (32 kD pI 5.2) was sequentially homologous to plant defense-related reductases like isoflavone reductase and phenylcoumaran benzylic ether reductase. Conclusions We first identified a defense-related reductase as a latex allergen. Several allergens belonging to the plant defense-related reductase family (Bet v 6-related allergens) have already been found from fruits, vegetables, and pollen. Considering the sequence similarities among them, we can predict the cross-reactivity of the defense-related reductase found from natural rubber.

Crystal Structures of Pinoresinol-Lariciresinol and Phenylcoumaran Benzylic Ether Reductases and Their Relationship to Isoflavone Reductases

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells.

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.

Proanthocyanidin Biosynthesis in Plants: PURIFICATION OF LEGUME LEUCOANTHOCYANIDIN REDUCTASE AND MOLECULAR CLONING OF ITS cDNA

Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and thus is the first committed step in PA biosynthesis. The enzyme was purified to near homogeneity from PA-rich leaves of the legume Desmodium uncinatum (Jacq.) DC, partially sequenced and the corresponding cDNA cloned. The identity of the enzyme was confirmed by expressing active recombinant LAR in Escherichia coli and in tobacco and white clover. The enzyme is a monomer of 43 kDa (382 amino acids) and is most active synthesizing catechin (specific activity of approximately 10 micromol min+1 mg of protein+1) but also synthesizes afzelechin and gallocatechin. LAR is most closely related to the isoflavone reductase group of plant enzymes that are part of the Reductase-Epimerase-Dehydrogenase (RED) family of proteins. Unlike all other plant isoflavone reductase homologues that are about 320 amino acids long, LAR has an additional 65-amino acid C-terminal extension whose function is not known. Curiously, although Arabidopsis makes PA, there is no obvious LAR orthologue in the Arabidopsis genome. This may be because Arabidopsis seems to produce only an epicatechin, rather than a dual catechin/epicatechin-based PA similar to many other plants.

Immunolocalization of vestitone reductase and isoflavone reductase, two enzymes involved in the biosynthesis of the phytoalexin medicarpin

Vestitone reductase (VR; E.C. 1.1.1._) and isoflavone reductase (IFR; E.C. 1.3.1.45) are phytoalexin-specific enzymes involved in the biosynthesis of medicarpin in alfalfa ( Medicago sativa L.). Immunoblot analysis of total soluble protein extracts indicated that these proteins constitutively accumulate in roots and nodules of alfalfa plants, and in alfalfa cell cultures. VR and IFR proteins are not present in healthy leaves but are rapidly synthesized upon fungal infection, and levels in cell cultures increase following elicitation. Immunolocalization studies in alfalfa organs at the cellular level indicated that VR accumulates in a narrow zone of leaf cells surrounding fungal lesions, in all cell types of mature and young roots, and in nodule meristem and lateral nodular tissues. Subcellular localization studies using immunogold labelling clearly indicated that VR is a cytosolic enzyme in alfalfa, with the majority of VR appearing free in cytosol. Association of VR with preserved endoplasmic reticulum (ER) membrane fragments was observed in several sections, but sucrose density gradient fractionation did not confirm the ER association. Although VR exhibits maximum activity in vitro in relatively acidic buffers (pH 6.0, approaching the pH of the vacuole), and the final product of the pathway (medicarpin-3-O-glucoside-6″-O-malonate) accumulates in the vacuole, no immunogold signal was observed in the vacuole. Subcellular analysis of CaMV35S∷IFR transgenic tobacco indicated that IFR is cytosolic as well. These results clearly indicate that the late steps of pterocarpan biosynthesis occur in the plant cytosol.

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family.

Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.

Immunolocalization of vestitone reductase and isoflavone reductase, two enzymes involved in the biosynthesis of the phytoalexin medicarpin

Immunolocalization of vestitone reductase and isoflavone reductase, two enzymes involved in the biosynthesis of the phytoalexin medicarpin

Biotransformation of an exogenously supplied isoflavonoid by transgenic tobacco cells expressing alfalfa isoflavone reductase

Isoflavone reductase (IFR) from alfalfa catalyzes the NADPH-dependent reduction of 2′-hydroxy isoflavones to 2′-hydroxyisoflavanones such as vestitone, thereby performing a key step in isoflavonoid phytoalexin biosynthesis. Tobacco (Nicotiana tabacum L. cv. Xanthi) was transformed with an alfalfa cDNA encoding IFR regulated by an enhanced cauliflower mosaic virus 35S promoter and used to generate cell suspension cultures. Transformed cells expressed high levels of IFR activity and could take up 2′-hydroxyformononetin (2′OHF; IFR substrate) and convert it to vestitone. Both 2′OHF and vestitone were each converted to two sugar conjugates. The structure of the most abundant vestitone conjugate was determined using NMR spectroscopic analysis to be a novel vestitone 2′-O-glucoside. Liquid chromatography-mass spectroscopy (LC-MS) indicated that the molecular weights of the three less abundant conjugates were consistent with hexose conjugates of vestitone and 2′OHF. The IFR reduction and conjugation reactions were complete within 4 h, with the net percentage conversion of 2′OHF to vestitone ranging as high as 60%.

Expression patterns of two tobacco isoflavone reductase-like genes and their possible roles in secondary metabolism in tobacco.

Plants contain a large number of proteins homologous to isoflavone reductase, an NADPH-dependent reductase involved in the biosynthesis of isoflavonoid phytoalexins in legumes. Although some are bonafide isoflavone reductases, others may catalyze distinct reductase reactions. Two tobacco genes, TP7 and A622, encoding isoflavone reductase-like proteins, had been previously identified from their unique expression patterns, but their functions were not known. We show here that TP7 is a tobacco phenylcoumaran benzylic ether reductase involved in lignan biosynthesis, but that A622 is not. To gain insight into the possible function of A622, we analyzed in detail the expression patterns of the A622 gene by RNA and protein blots, immunohistochemistry, and its promoter expression in transgenic Nicotiana sylvestris roots. The A622 expression patterns were qualitatively similar to those of putrescine N-methyltransferase, the first enzyme in nicotine biosynthesis, suggesting that A622 may function in the metabolism of nicotine or related alkaloids.

In silico differential display of defense-related expressed sequence tags from sugarcane tissues infected with diazotrophic endophytes

The expression patterns of 277 sugarcane expressed sequence tags (EST)-contigs encoding putative defense-related (DR) proteins were evaluated using the Sugarcane EST database. The DR proteins evaluated included chitinases, beta-1,3-glucanases, phenylalanine ammonia-lyases, chalcone synthases, chalcone isomerases, isoflavone reductases, hydroxyproline-rich glycoproteins, proline-rich glycoproteins, peroxidases, catalases, superoxide dismutases, WRKY-like transcription factors and proteins involved in cell death control. Putative sugarcane WRKY proteins were compared and their phylogenetic relationships determined. A hierarchical clustering approach was used to identify DR ESTs with similar expression profiles in representative cDNA libraries. To identify DR ESTs differentially expressed in sugarcane tissues infected with Gluconacetobacter diazotrophicus or Herbaspirillum rubrisubalbicans, 179 putative DR EST-contigs expressed in non-infected tissues (leaves and roots) and/or infected tissues were selected and arrayed by similarity of their expression profiles. Changes in the expression levels of 124 putative DR EST-contigs, expressed in non-infected tissues, were evaluated in infected tissues. Approximately 42% of these EST-contigs showed no expression in infected tissues, whereas 15% and 3% showed more than 2-fold suppression in tissues infected with G. diazotrophicus or H. rubrisubalbicans, respectively. Approximately 14 and 8% of the DR EST-contigs evaluated showed more than 2-fold induction in tissues infected with G. diazotrophicus or H. rubrisubalbicans, respectively. The differential expression of clusters of DR genes may be important in the establishment of a compatible interaction between sugarcane and diazotrophic endophytes. It is suggested that the hierarchical clustering approach can be used on a genome-wide scale to identify genes likely involved in controlling plant-microorganism interactions.

Biosynthesis of secondary metabolites in sugarcane

A set of genes related to secondary metabolism was extracted from the sugarcane expressed sequence tag (SUCEST) database and was used to investigate both the gene expression pattern of key enzymes regulating the main biosynthetic secondary metabolism pathways and the major classes of metabolites involved in the response of sugarcane to environmental and developmental cues. The SUCEST database was constructed with tissues in different physiological conditions which had been collected under varied situation of environmental stress. This database allows researchers to identify and characterize the expressed genes of a wide range of putative enzymes able to catalyze steps in the phenylpropanoid, isoprenoid and other pathways of the special metabolic mechanisms involved in the response of sugarcane to environmental changes. Our results show that sugarcane cDNAs encoded putative ultra-violet induced sesquiterpene cyclases (SC); chalcone synthase (CHS), the first enzyme in the pathway branch for flavonoid biosynthesis; isoflavone synthase (IFS), involved in plant defense and root nodulation; isoflavone reductase (IFR), a key enzyme in phenylpropanoid phytoalexin biosynthesis; and caffeic acid-O-methyltransferase, a key enzyme in the biosynthesis of lignin cell wall precursors. High levels of CHS transcripts from plantlets infected with Herbaspirillum rubri or Gluconacetobacter diazotroficans suggests that agents of biotic stress can elicit flavonoid biosynthesis in sugarcane. From this data we have predicted the profile of isoprenoid and phenylpropanoid metabolism in sugarcane and pointed the branches of secondary metabolism activated during tissue-specific stages of development and the adaptive response of sugarcane to agents of biotic and abiotic stress, although our assignment of enzyme function should be confirmed by careful biochemical and genetic supporting evidence.

Cross-reactivity of IgE antibodies to allergens.

The cross-reactivity of IgE antibodies is of interest for various reasons, three of which are discussed. Firstly, from the clinical view, it is important to know the patterns of cross-reactivity, because they often (but not always) reflect the pattern of clinical sensitivities. We discuss the cross-reactivities associated with sensitization to pollen and vegetable foods: PR-10 (Bet v 1-related), profilin, the cross-reactive carbohydrate determinant (CCD), the recently described isoflavone reductase, and the (still elusive) mugwort allergen that is associated with celery anaphylaxis; cross-reactivities between allergens from invertebrates, particularly tropomyosin, paramyosin, and glutathione S-transferase (GST); and latex-associated cross-reactivities. Clustering cross-reactive allergens may simplify diagnostic procedures and therapeutic regimens. Secondly, IgE cross-reactivity is of interest for its immunologic basis, particularly in relation to the regulation of allergic sensitization: are IgE antibodies to allergens more often cross-reactive than IgG antibodies to "normal" antigens? If so, why? For this discussion, it is relevant to compare not only the structural relation between the two allergens in question, but also the relatedness to the human equivalent (if any) and how the latter influences the immune repertoire. Thirdly, prediction of IgE cross-reactivity is of interest in relation to allergic reactivity to novel foods. Cross-reactivity is a property defined by individual antibodies to individual allergens. Quantitative information (including relative affinity) is required on cross-reactivity in the allergic population and with specific allergens (rather than with whole extracts). Such information is still scarce, but with the increasing availability of purified (usually recombinant) allergens, such quantitative information will soon start to accumulate. It is expected that similarity in short stretches of the linear amino-acid sequence is unlikely to result in relevant cross-reactivity between two proteins unless there is similarity in the protein fold.

The defense response elicited by the pathogen Rhizoctonia solani is suppressed by colonization of the AM-fungus Glomus intraradices

Defense responses of alfalfa roots to the pathogenic fungus Rhizoctonia solani were reduced significantly in roots simultaneously infected with the vesicular arbuscular mycorrhizal (AM) fungus Glomus intraradices. R. solani induced five- to tenfold increases in the steady-state levels of chalcone isomerase and isoflavone reductase mRNAs a doubling of root peroxidase activity and a marked autofluorescence in the infected tissue. These changes were inhibited by the presence of G. intraradices. Interestingly, germination of G. intraradices spores and hyphal elongation were sensitive to low concentrations (2 μM) of medicarpin-3- O-glucoside, an isoflavonoid phytoalexin that accumulated both in roots colonized by the pathogenic fungus as well as in AM-treated roots receiving high P, where no colonization by the beneficial fungus occurred. These data support the hypothesis that during early stages of colonization by G. intraradices, suppression of defense-related properties is associated with the successful establishment of AM symbiosis.