Flavone synthase II (CYP93B16) from soybean (Glycine max L.).

Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally different enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating 7,4'-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA (CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apigenin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNSII was stereoselective, favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid synthase activity.

Identification of a 4-coumarate:CoA ligase gene family in the moss, Physcomitrella patens.

Since the early evolution of land plants from primitive green algae, phenylpropanoid compounds have played an important role. In the biosynthesis of phenylpropanoids, 4-coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role at the divergence point from general phenylpropanoid metabolism to several major branch pathways. Although higher plant 4CLs have been extensively studied, little information is available on the enzymes from bryophytes. In Physcomitrella patens, we have identified a 4CL gene family consisting of four members, taking advantage of the available EST sequences and a draft sequence of the P. patens genome. The encoded proteins of three of the genes display similar substrate utilization profiles with highest catalytic efficiency towards 4-coumarate. Interestingly, the efficiency with cinnamate as substrate is in the same range as with caffeate and ferulate. The deduced proteins of the four genes share sequence identities between 78% and 86%. The intron/exon structures are pair wise similar. Pp4CL2 and Pp4CL3 each consists of four exons and three introns, whereas Pp4CL1 and Pp4CL4 are characterized each by five exons and four introns. Pp4CL1, Pp4CL2 and Pp4CL3 are expressed in both gametophore and protonema tissue of P. patens, unlike Pp4CL4 whose expression could not be demonstrated under the conditions employed. Phylogenetic analysis suggests an early evolutionary divergence of Pp4CL gene family members. Using Streptomyces coelicolor cinnamate:CoA ligase (ScCCL) as an outgroup, the P. patens 4CLs are clearly separated from the spermatophyte proteins, but are intercalated between the angiosperm 4CL class I and class II. A comparison of three P. patens subspecies from diverse geographical locations shows high sequence identities for the four 4CL isoforms.

Identification of a multigene family encoding putative beta-glucan-binding proteins in Medicago truncatula.

Branched 1,6-1,3-beta-glucans from Phytophthora sojae cell walls represent pathogen-associated molecular patterns (PAMPs) that have been shown to mediate the activation of plant defence reactions in many legumes. In soybean, a receptor protein complex containing a high affinity beta-glucan-binding protein (GBP) was identified and investigated in detail. In the model legume Medicago truncatula, used for functional genomic studies of various plant-microbe interactions, a high-affinity beta-glucan-binding site was characterized biochemically. However, to date, none of the genes encoding GBPs from M. truncatula have been described. Here, we report the identification of four full-length clones encoding putative beta-glucan-binding proteins from M. truncatula, MtGBP1, 2, 3, and 4, composing a multigene family encoding GBP-related proteins in this plant. Differences in expression patterns as well as in regulation on treatment with two different biotic elicitors are demonstrated for the members of the GBP family and for a selection of defence-related genes.

Activation of members of a MAPK module in beta-glucan elicitor-mediated non-host resistance of soybean.

Plants recognize microbial pathogens by discriminating pathogen-associated molecular patterns from self-structures. We study the non-host disease resistance of soybean (Glycine max L.) to the oomycete, Phytophthora sojae. Soybean senses a specific molecular pattern consisting of a branched heptaglucoside that is present in the oomycetal cell walls. Recognition of this elicitor may be achieved through a beta-glucan-binding protein, which forms part of a proposed receptor complex. Subsequently, soybean mounts a complex defense response, which includes the increase of the cytosolic calcium concentration, the production of reactive oxygen species, and the activation of genes responsible for the synthesis of phytoalexins. We now report the identification of two mitogen-activated protein kinases (MAPKs) and one MAPK kinase (MAPKK) that may function as signaling elements in triggering the resistance response. The use of specific antisera enabled the identification of GmMPKs 3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. Elicitor specificity of MAPK activation as well as the sensitivity against inhibitors suggested these kinases as part of the beta-glucan signal transduction pathway. An upstream GmMKK1 was identified based on sequence similarity to other plant MAPKKs and its interaction with the MAPKs was analyzed. Recombinant GmMKK1 interacted predominantly with GmMPK6, with concomitant phosphorylation of the MAPK protein. Moreover, a preferential physical interaction between GmMKK1 and GmMPK6 was demonstrated in yeast. These results suggest a role of a MAPK cascade in mediating beta-glucan signal transduction in soybean, similar to other triggers that activate MAPKs during innate immune responses in plants.

Spatial and temporal expression patterns of Avr1b-1 and defense-related genes in soybean plants upon infection with Phytophthora sojae.

The Avr1b locus is required for avirulence of the oomycete pathogen Phytophthora sojae on soybeans carrying resistance gene Rps1b. One of the Avr genes of the locus (Avr1b-1) was shown to encode an elicitor. We have analyzed the spatial and temporal expression patterns of Avr1b-1 in comparison to defense-related genes induced in soybean. Avr1b-1 expression was detectable mainly in close proximity to the site of infection, in wound-inoculated hypocotyls as well as in roots infected with zoospores. Usually, in compatible interactions, higher expression levels of Avr1b-1 were observed in roots when compared with incompatible P. sojae-soybean interactions, whereas neither the timing nor the amount of transcript accumulation of defense-related genes showed cultivar-specific differences. In contrast, the PsojNIP gene encoding a proposed virulence factor was expressed only during the necrotrophic phase in the compatible interaction.

Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases.

The beta-glucan-binding protein (GBP) of soybean (Glycine max L.) has been shown to contain two different activities. As part of the plasma membrane-localized pathogen receptor complex, it binds a microbial cell wall elicitor, triggering the activation of defence responses. Additionally, the GBP is able to hydrolyze beta-1,3-glucans, as present in the cell walls of potential pathogens. The substrate specificity, the mode of action, and the stereochemistry of the catalysis have been elucidated. This defines for the first time the inverting mode of the catalytic mechanism of glycoside hydrolases belonging to family 81.

Cation fluxes cause plasma membrane depolarization involved in beta-glucan elicitor-signaling in soybean roots.

Inducible and specific ion fluxes on plasma membranes represent very early events during elicitation of plant cells. The hierarchy of such ion fluxes involved is still unknown. The effect of Phytophthora sojae-derived beta-glucan elicitors on the plasma membrane potential as well as on surface K+, Ca2+, and H+ fluxes has been investigated on soybean roots using ion-selective microelectrodes. Beta-Glucans with different degrees of polymerization transiently depolarized the plasma membrane. The elicitor concentration necessary for half-maximal depolarization closely resembled the corresponding binding affinities of soybean root membranes toward the respective beta-glucans. Upon repeated elicitor treatment, the root cells responded partially refractory, suggesting a complex responsiveness of the system. Within the root hair space, characteristic decreasing K(+)- and Ca(2+)-free concentrations were induced by the elicitors, probably causing depolarization through the influx of positive charges. Whereas K+ fluxes were inverted after passing the K+ equilibrium (Nernst-) potential, Ca2+ influx continued. No anion fluxes sufficient to account for charge compensation were observed under the same experimental conditions. K+ and Ca2+ fluxes as well as depolarization were inhibited by 100 microM or less of the Ca2+ antagonist La3+. Contrasting other systems, in soybean the main cause for elicitor-induced plasma membrane depolarization is the activation of cation instead of anion fluxes.

Coronalon: a powerful tool in plant stress physiology.

Coronalon, a synthetic 6-ethyl indanoyl isoleucine conjugate, has been designed as a highly active mimic of octadecanoid phytohormones that are involved in insect and disease resistance. The spectrum of biological activities that is affected by coronalon was investigated in nine different plant systems specifically responding to jasmonates and/or 12-oxo-phytodienoic acid. In all bioassays analyzed, coronalon demonstrated a general strong activity at low micromolar concentrations. The results obtained showed the induction of (i) defense-related secondary metabolite accumulation in both cell cultures and plant tissues, (ii) specific abiotic and biotic stress-related gene expression, and (iii) root growth retardation. The general activity of coronalon in the induction of plant stress responses together with its simple and efficient synthesis suggests that this compound might serve as a valuable tool in the examination of various aspects in plant stress physiology. Moreover, coronalon might become employed in agriculture to elicit plant resistance against various aggressors.

An ancient enzyme domain hidden in the putative beta-glucan elicitor receptor of soybean may play an active part in the perception of pathogen-associated molecular patterns during broad host resistance.

A successful defense against potential pathogens requires that a host organism is able to discriminate between self and nonself structures. Soybean (Glycine max L.) exploits a specific molecular pattern, a 1,6-beta-linked and 1,3-beta-branched heptaglucoside (HG), present in cell walls of the oomycetal pathogen Phytophthora sojae, as a signal compound eliciting the onset of defense reactions. The specific and high affinity HG-binding site is contained in the beta-glucan-binding protein (GBP), which in turn is part of a proposed receptor complex. The ability to perceive and respond to Phytophthora cell wall-derived beta-glucan elicitors is exclusive to plants that belong to the Fabaceae. However, we propose that the presence of the GBP is essential, but not sufficient for beta-glucan elicitor-dependent disease resistance because genes encoding GBP-related proteins can be retrieved from many plant species. Furthermore, we show that the GBP is composed of two different carbohydrateactive protein domains, one containing the beta-glucan-binding site, and the other related to glucan endoglucosidases of fungal origin. The glucan hydrolase displays most likely an endo-specific mode of action, cleaving only 1,3-beta-d-glucosidic linkages of oligoglucosides consisting of at least four moieties. Thus, the intrinsic endo-1,3-beta-glucanase activity of the GBP is perfectly suited during initial contact with Phytophthora to release oligoglucoside fragments enriched in motifs that constitute ligands for the high affinity binding site present in the same protein. The concept of innate immunity in plants receives substantial support by this highly sophisticated system using ancient enzyme modules as an active part of the recognition mechanism.

The role of octadecanoids and functional mimics in soybean defense responses.

Oxylipins of the jasmonate pathway and synthetic functional analogs have been analyzed for their elicitor-like activities in an assay based on the induced accumulation of glyceollins, the phytoalexins of soybean (Glycine max L.), in cell suspension cultures of this plant. Jasmonic acid (JA) and its methyl ester showed weak phytoalexin-inducing activity when compared to an early jasmonate biosynthetic precursor, 12-oxo-phytodienoic acid (OPDA), as well as to the bacterial phytotoxin coronatine and certain 6-substituted indanoyl-L-isoleucine methyl esters, which all were highly active. Interestingly, different octadecanoids and indanoyl conjugates induced the accumulation of transcripts of various defense-related genes to different degrees, indicating distinct induction competencies. Therefore, these signaling compounds and mimics were further analyzed for their effects on signal transduction elements, such as the transient enhancement of the cytosolic Ca2+ concentration and MAP kinase activation, which are known to be initiated by a soybean pathogen-derived beta-glucan elicitor. In contrast to the beta-glucan elicitor, none of the other compounds tested triggered these early signaling elements. Moreover, endogenous levels of OPDA and JA in soybean cells were shown to be unaffected after treatment with beta-glucans. Thus, OPDA and JA, which are functionally mimicked by coronatine and a variety of 6-substituted derivatives of indanoyl-L-isoleucine methyl ester, represent highly efficient signaling compounds of a lipid-based pathway not deployed in the beta-glucan elicitor-initiated signal transduction.

Deletion of a single amino acid residue from different 4-coumarate:CoA ligases from soybean results in the generation of new substrate specificities.

Plant 4-coumarate:coenzyme A ligases, acyl-CoA ligases, peptide synthetases, and firefly luciferases are grouped in one family of AMP-binding proteins. These enzymes do not only use a common reaction mechanism for the activation of carboxylate substrates but are also very likely marked by a similar functional architecture. In soybean, four 4-coumarate:CoA ligases have been described that display different substrate utilization profiles. One of these (Gm4CL1) represented an isoform that was able to convert highly ring-substituted cinnamic acids. Using computer-based predictions of the conformation of Gm4CL1, a peptide motif was identified and experimentally verified to exert a critical influence on the selectivity toward differently ring-substituted cinnamate substrates. Furthermore, one unique amino acid residue present in the other isoenzymes of soybean was shown to be responsible for the incapability to accommodate highly substituted substrates. The deletion of this residue conferred the ability to activate sinapate and, in one case, also 3,4-dimethoxy cinnamate and was accompanied by a significantly better affinity for ferulate. The engineering of the substrate specificity of the critical enzymes that activate the common precursors of a variety of phenylpropanoid-derived secondary metabolites may offer a convenient tool for the generation of transgenic plants with desirably modified metabolite profiles.

Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family.

4-Coumarate:CoA ligase (4CL) is involved in the formation of coenzyme A thioesters of hydroxycinnamic acids that are central substrates for subsequent condensation, reduction, and transfer reactions in the biosynthesis of plant phenylpropanoids. Previous studies of 4CL appear to suggest that many isoenzymes are functionally equivalent in supplying substrates to various subsequent branches of phenylpropanoid biosyntheses. In contrast, divergent members of a 4CL gene family were identified in soybean (Glycine max L.). We isolated three structurally and functionally distinct 4CL cDNAs encoding 4CL1, 4CL2, and 4CL3 and the gene Gm4CL3. A fourth cDNA encoding 4CL4 had high similarity with 4CL3. The recombinant proteins expressed in Escherichia coli possessed highly divergent catalytic efficiency with various hydroxycinnamic acids. Remarkably, one isoenzyme (4CL1) was able to convert sinapate; thus the first cDNA encoding a 4CL that accepts highly substituted cinnamic acids is available for further studies on branches of phenylpropanoid metabolism that probably lead to the precursors of lignin. Surprisingly, the activity levels of the four isoenzymes and steady-state levels of their transcripts were differently affected after elicitor treatment of soybean cell cultures with a beta-glucan elicitor of Phytophthora sojae, revealing the down-regulation of 4CL1 vs. up-regulation of 4CL3/4. A similar regulation of the transcript levels of the different 4CL isoforms was observed in soybean seedlings after infection with Phytophthora sojae zoospores. Thus, partitioning of cinnamic acid building units between phenylpropanoid branch pathways in soybean could be regulated at the level of catalytic specificity and the level of expression of the 4CL isoenzymes.