Search

This chapter outlines the principles upon which strategies for fungal and bacterial resistance may be based, to evaluate the types of genetic manipulations which may lead to increased resistance. Some fungal pathogens have acquired virulence by being able to detoxify the phytoalexins the host produces as a part of its defensive arsenal. A basis of information exists for engineering modified phytoalexin structures which may be resistant to detoxification, or for transferring a phytoalexin biosynthetic pathway from one plant to another which lacks that particular pathway. Transgenic plants expressing either novel proteins from foreign organisms or overexpressing a part of their own defensive arsenal have been engineered, tested in both laboratory and field situations, and evaluated for disease resistance. Pathogenesis-related proteins are low-molecular weight proteins which accumulate to significant levels in infected plant tissues. Plants, and indeed other organisms, may contain antimicrobial proteins not necessarily associated with induced defense responses, which are potential subjects for engineered protection strategies.

Glandular trichomes are a major site of plant natural product synthesis and accumulation for protection against insect predation. However, to date few studies have attempted to obtain a global view of trichome gene expression. Two contrasting approaches have been adopted to investigate genes expressed in glandular trichomes from alfalfa (Medicago sativa L.). In the first approach, 5,674 clones from an alfalfa glandular trichome cDNA library were sequenced. The most highly abundant expressed sequence tag (EST) corresponded to a lipid transfer protein. The presence of ESTs corresponding to enzymes for all steps in the biosynthesis of flavonoids suggests that these are important metabolites in alfalfa trichome biology, as confirmed by histochemistry and metabolite profiling. No ESTs corresponded to enzymes of cyclized terpenoid biosynthesis. In a second approach, microarray analysis was used to compare levels of alfalfa transcripts corresponding to 16,086 Medicago truncatula A17 genes in stems with and without trichomes. This revealed over 1,000 genes with strong preferential expression in the trichome fraction of the stem, 70% of which are of unknown function. These define a class of genes that are not trichome-specific, since M. truncatula A17 does not itself have glandular trichomes, but has potential importance for trichome function within the stem.

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.

Medicarpin and maackiain are antifungal pterocarpan phytoalexins produced by many legumes, and are thought to be important components of the defense response of these legumes to certain fungal pathogens. The Mak1 gene from the fungal pathogen Nectria haematococca encodes an FAD-dependent mono-oxygenase, known to specifically hydroxylate the phytoalexins medicarpin and maackiain, converting them to less fungitoxic derivatives. Two binary vector constructs were made containing the coding regions from two fungal clones, a Mak1 cDNA (intronless) and a genomic (including three fungal introns) clone, regulated by an enhanced cauliflower mosaic virus 35S promoter. The constructs were introduced into tobacco to check for expression of active fungal enzyme in plant cells and for splicing of fungal introns. Leaves of tobacco plants transformed with the Mak1 cDNA construct readily metabolized infiltrated medicarpin to 1a-hydroxymedicarpin, indicating high levels of active enzyme. RT-PCR analysis of tobacco plants transformed with the Mak1 genomic construct indicated no processing of Mak1 introns, and no Mak1 activity was detected in these plants. When using plants containing the Mak1 cDNA construct, immunolocalization with a Mak1-specific antibody together with cellular fractionation indicated that Mak1 protein accumulated in the plant cytoplasm, associated with endoplasmic reticulum membranes; medicarpin biosynthetic enzymes have been localized to the same subcellular region. The Mak1 cDNA construct is therefore suitable for use in studies to selectively eliminate medicarpin accumulation to assess the relative importance of medicarpin in the antifungal defense mechanisms of alfalfa and other legumes.

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume Medicago truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated Medicago functional genomics program at the Noble Foundation (http://www.noble .org), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

An enantiomeric assay for the flavonoids vestitone and medicarpin from transgenic plant extracts was developed using capillary electrophoresis. It was found that no single cyclodextrin proved capable of resolving the enantiomers of both medicarpin and vestitone. Instead, hydroxypropyl-beta-cyclodextrin provided the best selectivity for the vestitones while hydroxypropyl-gamma-cyclodextrin was best for the medicarpins. The addition of organic modifiers improved the resolution of both enantiomers. Acetonitrile proved best for the vestitones and only methanol improved the resolution of the medicarpins. An optimization study of mixed hydroxypropyl-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin containing electrolytes revealed that the separation of the medicarpin enantiomers was intolerant to the presence of hydroxypropyl-beta-cyclodextrin. Our optimized running electrolyte was composed of 2 mM hydroxypropyl-beta-cyclodextrin, 20 mM hydroxypropyl-gamma-cyclodextrin, and 25 mM borate at pH 10.0 with 10% v/v methanol. This system provided a resolution of 1.47 and 1.80 for the medicarpin and vestitone enantiomers, respectively. This analysis was completed in 12 min. This separation provided a rapid screen to determine the enantiomeric purity of key flavonoids biosynthesized by transgenic legumes.

The relationship between salicylic acid (SA) accumulation, PR-1 gene expression, and the degree of systemic acquired resistance (SAR) established in Arabidopsis plants has been determined by comparing the response of wild-type ecotype Columbia (Col-0) and two hypersensitive response (HR)-defective mutants ( rps2-201 and rps2-101C ) during the SAR response induced by avirulent Pseudomonas syringae pv. tomato ( Pst). Unlike wild-type Col-0 plants, plants harboring either mutant allele were unable to manifest a SAR response. During the initiation/immunization stage of SAR, PR-1 gene expression was delayed in interactions which did not lead to SAR (avirulent inoculation of rps2-201 and rps2-101C, or inoculation with virulent bacteria in wild-type Col-0) compared to those interactions that did lead to SAR (avirulent inoculation of wild-type Col-0). PR-1 expression was reduced in the rps mutants compared to wild-type plants during the establishment and manifestation stage and therefore correlated with the ability to elicit a SAR response. SA accumulated to similar levels during the establishment and manifestation stages for Col-0 and the rps2 mutant alleles. Therefore the ability to accumulate SA was not predictive of the ability to elicit SAR, and SA accumulation alone is not sufficient to elicit the SAR response. Moreover, SA accumulated to similar levels during both compatible (virulent infections) and incompatible interactions (avirulent infections). However, SA accumulation was reduced in the initiation/immunization stage of SAR in rps2-101C compared to wild-type, but not in rps2-201 , suggesting that this reduction was related to an HR-specific defect mediated by the rps2-101C mutant allele.

Flavonoids are involved in a number of critical events in the interaction between nitrogen-fixing bacteria and legumes. To get a better understanding of the importance of flavonoids in the earliest stages of the alfalfa-Rhizobium meliloti symbiosis, we followed the expression of two chal-cone synthase (CHS) gene family members as well as of chalcone isomerase (CHI) and isoflavone reductase (IFR) genes. CHS transcripts increased 2 to 4 dpi (days post-inoculation) with wild-type rhizobia, but not after inoculation with the heterologous R. leguminosarum bv. trifolii or with an exopolysaccharide (exo) mutant of R. meliloti. CHS transcripts were detected in the root hairs and epidermal cells of the root hair zone, and infrequently in nodule pri-mordia. Insignificant CHI and IFR mRNA accumulation over control levels was observed in response to rhizobial inoculation. The slight increase in CHS transcript accumulation following wild-type R. meliloti inoculation was correlated with an observed increase in root flavonoid content as well as a change in the nod gene-inducing activity of the root exudate. The nod gene-inducing flavonoids exuded from wild-type rhizobia-inoculated roots were identified as 4′, 7-dihydroxyflavone and 4, 4′ dihydroxy-2′-methoxychalcone. Although there was a slight increase over the uninoculated controls in the level of medicarpin-3-O-glucoside 6″-O-malonate (MGM) in extracts of roots inoculated with rhizobia, IFR transcript accumulation was not significantly elevated over that of the controls. Moreover, no medicarpin aglycone was detected in the inoculated roots. Thus, although inoculation with wild-type rhizobia triggers some of the genes induced during an interaction between a host and a pathogen, the expression of these genes in the Rhizobium-legume interaction is at a very low level, suggesting that rhizobia have evolved a mechanism(s) to avoid triggering the host's defense responses.

Transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the enzyme L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) were grown from seeds of a primary transformant containing the bean PAL2 gene, which had shown homology-dependent silencing of the endogenous tobacco PAL genes. Analysis of endogenous and transgene-encoded PAL transcripts and protein in the primary transformant (T0) and first-generation (T1) overexpressor plants indicated that the transgene-encoded PAL is the cause of the greater than wild-type levels of PAL activity (up to 5- and 2-fold greater in leaf and stem tissue, respectively) in the T1 plants. Leaves of PAL-overexpressing plants contained increased levels of the hydroxycinnamic acid ester chlorogenic acid but not of the flavonoid rutin, indicating that PAL is the key control point for flux into chlorogenic acid. In addition, levels of the glucoside of 4-coumaric acid increased in the overexpressing plants, suggesting that the 4-coumarate:coenzyme A ligase or coumarate hydroxylase reactions might have become limiting. These results help to define the regulatory architecture of the phenylpropanoid pathway and indicate the possibility of engineering-selective changes in this complex metabolic pathway by overexpression of a single early pathway gene.

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.

In plants, several important classes of terpenoid compounds are synthesized via the mevalonate pathway. In addition to essential constitutive metabolites, potato (Solanum tuberosum L.) tubers synthesize antifungal sesquiterpenoid phytoalexins in response to fungal infection or arachidonic acid elicitation, and toxic steroid glycoalkaloids in response to wounding. The activity of the early pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has previously been shown to increase rapidly and then decrease in response to these stimuli. During an investigation of the possible post-translational control of this enzyme, it was found that the inclusion of the cysteine protease inhibitors leupeptin and E-64 { N-[N-( l -3-trans- carboxyran-2-carbonyl)- l -leucyl]agmatine } in the enzyme extraction buffer increased nine-fold the total HMGR activity recovered in the microsomal fraction and greatly increased the ratio of microsomal to soluble activity. Incubation of microsomal HMGR preparations with soluble protein extracts, Mg2+ and ATP caused an apparent inhibition of HMGR, consistent with published reports of post-translational inactivation of HMGR by phosphorylation. The apparent inhibition was completely reversed, however, by 5 mM mevalonate and was found to be an artefact caused by the presence of mevalonate kinase, the next enzyme in the pathway, in the soluble fraction. HPLC assays for mevalonate kinase and mevalonate phosphate kinase were developed and used to measure the activities of these enzymes following wounding and elicitation. While HMGR levels increased 30-fold following arachidonic acid treatment and 15-fold following wounding, mevalonate kinase and mevalonate phosphate kinase only increased two- to four-fold following these treatments, and the levels in arachidonic acid treated tubers were only 20–40% higher than in wounded tubers. While HMGR levels are extremely low in untreated tissues, the activities of the two kinases are ralatively high, suggesting that they do not serve as control points for the synthesis of terpenoids.

A method for the identification of flavonoid glycosides utilizing continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) is presented. Minimum detectable quantities (MDQs) were determined for three model flavonoid glycosides (rutin, naringin and esculin) by both positive ion direct insertion probe (DIP)-LSIMS (1.6 nmol, 1.7 nmol and 730 pmol, respectively) and positive ion CF-LSIMS (330 pmol, 340 pmol and 290 pmol, respectively). Optimization of CF-LSIMS instrumental parameters was performed using the model compound rutin. Parameters optimized included mobile phase composition, glycerol concentration, mobile phase flow rate, ion source temperature, acceleration lens potential (amplitude and polarity) and Cs+ primary ion energy. Final instrumental optimization yielded an MDQ of 1.0 ng (1.6 pmol) for rutin by flow-injection CF-LSIMS. The optimization parameters were utilized in the identification of flavonoid glucosides in alfalfa (Medicago sativa L) and chickpea (Cicer arietinum) extracts by high-performance liquid chromatogrphy/CF-LSIMS. The results support the controversial identification of a major extract component as formononetin-7-O-glucoside-6"-malonate as opposed to afrormosin-7-O-glucoside-6"-malonate.

The pterocarpan phytoalexins of the Leguminosae are synthesized from L-phenyl-alanine via a minimum of 11 enzymatic steps involving the central phenylpropanoid pathway, three reactions of flavonoid biosynthesis, and the isoflavonoid branch pathway. The extractable activities of all these enyzmes, and of enzymes supplying precursors from primary metabolism, increase in response to fungal infection or exposure of plant cells to elicitor macromolecules isolated from the cell walls of yeast or plant pathogenic fungi. The involvement of reductases and cytochrome P450 hydroxylases places a high demand for NADPH on elicited cells. The NADPH is most likely supplied by activation of the pentose phosphate pathway. Genes or cDNAs encoding 7 of the enzymes involved in the synthesis of the phytoalexin medicarpin have been cloned from alfalfa and/or other species. Induction of enzyme activity results from transcriptional activation of the corresponding genes, leading to increased steady state levels of translatable mRNAs. This transcriptional activation is programmed through the interaction of sets of elicitor/infection-modulated transcription factors with their cognate cis elements in the promoters of the phytoalexin biosynthetic genes. Gene activation occurs through generation of intracellular signals which lead to modulation of transcription factor activity, through either increased synthesis of the factor(s), activation via reversible post-translational modification (e.g. phosphorylation/dephos-phorylation), translocation of factors from cytoplasm to nucleus, or combinations of these. Coordinated induction of the enzymes of phytoalexin synthesis may involve multiple signals and factors for transcriptional activation, as well as feedback and feed-forward fine controls at both transcriptional and post-transcriptional levels. In beneficial mycorrhizal interactions, induction of early pathway genes is uncoupled from that of later, phytoalexin-specific genes.

Lignin is a major structural polymer of secondarily thickended plant vascular tissue and fibres, imparting mechanical strength to stems and trunks and hydrophobicity to conducting vessels. Constitutive expression of a lucerne caffeic acid 3-O-methyltransferase antisense RNA in transgenic tobacco leads to a significant reduction in lignin content, particularly in the younger parts of the stems, without apparent alterations in lignin monomer composition. These observations open up the possibility of genetically manipulating plants with reduced lignin for improved processing and biomass digestibility.

Although the addition of shikimic acid to the medium had no effect on the level of production of rapamycin byStreptomyces hygroscopicus,14C-shikimic acid was incorporated into rapamycin to a very high degree.13C-Shikimic acid was successfully prepared from 1-[13C]-glucose using a mutant ofKlebsiella pneumoniae, and used to label rapamycin. It was found that13C-shikimic acid was incorporated into the cyclohexane moiety of rapamycin, thereby establishing the shikimic acid pathway origin of the seven-carbon starter unit.

Studies were carried out to determine the precursor of the nitrogen-containing ring of rapamycin. The addition of l -lysine and d,l -pipecolate to the medium of Streptomyces hygroscopicus did not affect the specific production of rapamycin. In a basal medium containing no l -lysine except for that in the yeast extract component, l - 14 C-lysine and d,l - 3 H-pipecolate were incorporated to a high degree into rapamycin, d,l - 3 H-pipecolate being incorporated at a higher efficiency. Unlabeled pipecolate reduced the incorporation of radioactive lysine much more than unlabeled lysine decreased the incorporation of radioactive pipecolate. These results indicate that pipecolate is the more direct precursor of rapamycin, lysine being converted to pipecolate and free pipecolate being incorporated directly into rapamycin. Rapamycin samples enriched with 14 C-lysine and 3 H-pipecolate were hydrolyzed and analyzed to provide evidence of specific incorporation of these precursors into the pipecolate moiety .

Transgenic alfalfa (Medicago sativa L.), which accumulated resveratrol-glucoside (RG), was incorporated into diets and fed to female, 6-wk-old CF-1 mice for 5 wk. Mice fed diets containing transgenic alfalfa with supplemented alpha -galactosidase had significantly fewer azoxymethane (AOM)-induced aberrant crypt foci (ACF) in their colon relative to mice fed the transgenic alfalfa diets without added alpha -galactosidase (P = 0.02). Resveratrol-aglycone (Rag) was detected in the colon of 100% of mice fed transgenic alfalfa diets with supplemented alpha -galactosidase and in 60% of mice fed transgenic alfalfa without alpha -galactosidase (P0.05). Colonic concentrations of Rag (0.5 nmol/g tissue) in mice fed transgenic alfalfa with alpha -galactosidase (0.22 +/- 0.18 nmol/g tissue) tended to be higher than in animals fed diets without alpha -galactosidase (0.1 +/- 0.08 nmol/g tissue; P = 0.09). The use of N-(Bn-butyl)-deoxygalactonojirimycin, an inhibitor of lactase-phlorizin hydrolase (LPH), in transport studies with everted jejunal sacs from CF-1 mice (N = 8) suggested that LPH is involved in the intestinal deglycosylation of RG. Our collective findings suggest that RG from transgenic alfalfa is metabolized and absorbed in the upper intestine and does not reach the colon in sufficient amounts to inhibit ACF.

The alfalfa ( Medicago sativa L.) phytoalexin medicarpin and four biosynthetic intermediates immediately preceding medicarpin in the phytoalexin pathway were tested for antimicrobial activity against eight phytopathogenic fungi. Medicarpin, vestitone, 2′-hydroxyformononetin (2′-OHF), formononetin and daidzein were tested at concentrations from 0·1 m m to 0·5 m m in agar plate assays measuring inhibition of linear fungal mycelial growth. Medicarpin at 0·1 m m caused little or no inhibition of the growth of the four known alfalfa pathogens. Unlike the other alfalfa pathogens, Phytophthora megasperma f.sp. medicaginis ( Pmm ) showed a very significant sensitivity to 0·1 m m vestitone and 2′-OHF. Pmm was strongly inhibited by medicarpin, vestitone, and 2′-OHF at 0·5 m m . Phoma medicaginis was strongly inhibited by 0·5 m m medicarpin and vestitone, whereas 2′-OHF was not a significant inhibitor. Three strains of Nectria haematococca , varying in their phytoalexin detoxification capabilities, were inhibited to different extents by medicarpin and its precursors. The only growth inhibition observed at 0·1 m m was of strain 156-2-1 by medicarpin. At 0·5 m m , 2′-OHF and vestitone caused weak to moderate inhibition of the growth of all three strains; in contrast, 0·5 m m medicarpin greatly inhibited the growth of strain 156-2-1 compared to the other two strains. These results are discussed in relation to a potential strategy for testing the role of phytoalexins in disease resistance based upon transgenic plants blocked in the formation of vestitone from 2′-OHF.

Labelling experiments with 13C-labelled precursors demonstrated that the majority of the macrolide ring of rapamycin is formed from six acetate and seven propionate units. A two-carbon unit in the ring was not labelled by acetate, propionate, glycine, or methionine. Methionine and glycine did, however, label the three methoxy groups of rapamycin to a high degree. Results from the incorporation experiments demonstrated that there were errors in the published 13C-nmr spectral assignments for rapamycin; these have now been corrected.

Plants have been genetically enhanced to produce a number of products for agricultural, industrial and pharmaceutical purposes. This technology could potentially be applied to providing chemoprevention strategies to the general population. Resveratrol (3,5,4'-trihydroxystilbene) is a compound that has been shown to have protective activity against a number of cancers and could be an ideal candidate for such an application. Alfalfa that was genetically modified to express resveratrol-synthase was used as a model in applying biotechnological approaches to cancer prevention. The transgenic alfalfa, which accumulates resveratrol as a glucoside (piceid = trans-resveratrol-3-O-Beta-D-glucopyranoside) (152 +/- 17.5 microg piceid/g dry weight), was incorporated into a standard mouse diet at 20% of the diet by weight and fed for 5 wk to 6-wk-old, female CF-1 mice (N = 17-30) that were injected with a single dose of azoxymethane (5 mg/kg body weight). While the addition of resveratrol-aglycone (20 mg/kg diet) to the basal diet reduced the number of aberrant crypt foci/mouse, the transgenic alfalfa did not inhibit the number, size, or multiplicity of aberrant crypt foci in the colon of the CF-1 mice relative to control alfalfa which does not accumulate resveratrol-glucoside. However, diets containing transgenic alfalfa with an exogenous Beta-glucosidase (860 U/kg diet) did significantly inhibit the number of aberrant crypt foci in the distal 2 cm of the colon of the mice relative to mice fed diets containing the transgenic alfalfa without the enzyme (P < 0.05; Fisher's Combination of p-values). The Beta-glucosidase alone appeared to have no effect on the inhibition of aberrant crypt foci. These results suggest that piceid in transgenic piceid-accumulating alfalfa was not bioavailable.

Dihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Two DFR cDNA clones (MtDFR1 and MtDFR2) were isolated from the model legume Medicago truncatula cv Jemalong. Both clones were functionally expressed in Escherichia coli, confirming that both encode active DFR proteins that readily reduce taxifolin (dihydroquercetin) to leucocyanidin. M. truncatula leaf anthocyanins were shown to be cyanidin-glucoside derivatives, and the seed coat proanthocyanidins are known catechin and epicatechin derivatives, all biosynthesized from leucocyanidin. Despite high amino acid similarity (79% identical), the recombinant DFR proteins exhibited differing pH and temperature profiles and differing relative substrate preferences. Although no pelargonidin derivatives were identified in M. truncatula, MtDFR1 readily reduced dihydrokaempferol, consistent with the presence of an asparagine residue at a location known to determine substrate specificity in other DFRs, whereas MtDFR2 contained an aspartate residue at the same site and was only marginally active on dihydrokaempferol. Both recombinant DFR proteins very efficiently reduced 5-deoxydihydroflavonol substrates fustin and dihydrorobinetin, substances not previously reported as constituents of M. truncatula. Transcript accumulation for both genes was highest in young seeds and flowers, consistent with accumulation of condensed tannins and leucoanthocyanidins in these tissues. MtDFR1 transcript levels in developing leaves closely paralleled leaf anthocyanin accumulation. Overexpression of MtDFR1 in transgenic tobacco (Nicotiana tabacum) resulted in visible increases in anthocyanin accumulation in flowers, whereas MtDFR2 did not. The data reveal unexpected properties and differences in two DFR proteins from a single species.

Condensed tannins (CTs) are flavonoid oligomers, many of which have beneficial effects on animal and human health. The flavanol (–)-epicatechin is a component of many CTs and contributes to flavor and astringency in tea and wine. We show that the BANYULS ( BAN ) genes from Arabidopsis thaliana and Medicago truncatula encode anthocyanidin reductase, which converts anthocyanidins to their corresponding 2,3- cis -flavan-3-ols. Ectopic expression of BAN in tobacco flower petals and Arabidopsis leaves results in loss of anthocyanins and accumulation of CTs.

Legumes such as alfalfa (Medicago sativa L.), barrel medic (Medicago truncatula), white sweet clover (Melilotus alba) and fenugreek (Trigonella graecum), normally accumulate (-)-medicarpin and its malonated glucose conjugate as natural inhibitors of fungal pathogens. These plants also accumulate the biosynthetic precursor formononetin as well as the malonated glycoside. We were interested in developing a robust high-throughput method to quantitate the levels of these two isoflavonoids, both free and conjugated, in legume root extracts, for use in screening for mutant plants accumulating altered levels of these compounds. Capillary electrophoresis was examined as an alternative to current high-performance liquid chromatography (HPLC) methods to generate isoflavonoid profiles. The developed assay used micellar electrokinetic capillary chromatography (MEKC) to provide the required selectivity in complex root extracts. The addition of 1,2-hexanediol to the sodium dodecyl sulfate (SDS) electrolyte provided improved resolution of adjacent isoflavonoids. We examined the role of several factors including sample preparation, buffer composition, buffer pH, and organic component in the injected sample. The use of capillaries with longer path lengths were also examined to increase sensitivity. A comparison of results obtained using MEKC and HPLC showed good correlation in the relative amounts of the isoflavonoids studied.

Publisher Summary Genetic engineering of resveratrol accumulation in crop species may enhance their disease resistance and nutritional value because of the antifungal and medicinal properties of resveratrol. In multiple species, the expression of a resveratrol synthase gene has allowed the accumulation of resveratrol and/or resveratrol glucoside. Despite the use of similar stilbene synthase binary expression constructs, different product profiles and plant phenotypes have been reported. These may be due to differences in the endogenous glucosyl transferases, glucosidases, and/or differences in promoter strengths in different species. While it is clear that several species transformed to date have produced resveratrol, the possible derivatives that accumulate (free or glycosylated) may vary from species to species. Factors affecting the ratio of resveratrol aglycone to glucoside are being investigated because glycosylation may affect the phytotoxicity, bioavailability, and stability of resveratrol.

Plants accumulate a diverse array of natural products, which can serve either to defend the plant against various microbes in its environment or to attract various microbes, both beneficial and pathogenic. Plants must also attract pollinators, repel or poison herbivores, compete with other plant species, and protect themselves from environmental dangers such as high light intensities. Some compounds have been implicated in playing a role in multiple interactions. Although the structures vary immensely in size and complexity, most are derived from a limited number of core biosynthetic pathways. This review briefly summarizes the biosynthetic origins of phenylpropanoid (including simple phenolics, flavonoids, anthocyanins and isoflavonoids), polyacetate, terpenoid, and alkaloid classes of metabolites. Compounds reported to be important in plant-microbe, plant-animal, and plant-plant interactions will be given as examples of each of these classes. Other aspects of biosynthesis also will be discussed, including the timing or location of biosynthesis, the potential for genetic manipulation of these pathways, and various questions regarding the biosynthesis of these compounds.

Isoflavonoids/flavonoids have been found to be involved in a number of critical plant activities, such as plant defense, pollen development, pigmentation, and the Rhizobium-legume symbiosis. The biosynthetic pathways for isoflavonoids/flavonoids are well established, and the genes for the majority of enzymes involved in these pathways have been isolated and cloned. For alfalfa (Medicago sativa L.), genes encoding phenylalanine ammonia lyase (PAL) (Gowri et al., 1991), chalcone synthase (CHS) (Esnault et al., 1993; Junghans et al., 1993; McKhann and Hirsch, 1994a), chalcone reductase (CHR) (Ballance and Dixon, 1995; Sallaud et al., 1995), chalcone isomerase (CHI) (McKhann and Hirsch, 1994a), chalcone 4′-O-methyltransferase (Maxwell et al., 1993), isoflavone reductase (IFR) (Paiva et al., 1991), and vestitone reductase (Guo and Paiva, 1995) have been cloned and sequenced. Earlier, we utilized two CHS cDNAs and CHI and IFR clones to study the temporal and spatial expression patterns of these genes during the establishment of the Rhizobium meliloti-alfalfa symbiosis (McKhann et al., 1997). Chalcone synthase is a key enzyme in the phenypropanoid biosynthetic pathway, catalyzing the condensation of malonyl-CoA with 4-coumaroyl CoA to yield naringenin chalcone, whereas chalcone isomerase catalyzes the isomerization of chalcones to their corresponding (-)- rlavanones. Isoflavone reductase catalyzes the penultimate step in the biosynthesis of medicarpin, an isoflavonoid phytoalexin.

In leguminous plants such as the forage legume alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms (flavonoid inducers of the Rhizobium symbiosis), and in defense against pathogens (isoflavonoid phytoalexins). In addition, the phenylpropane polymer lignin is a major structural component of secondary vascular tissue and fibers in higher plants. the recent isolation of genes encoding key enzymes of the various phenylpropanoid branch pathways opens up the possibility of engineering important crop plants such as alfalfa for: (a) improved forage digestibility, by modification of lignin composition and/or content; (b) increased or broader-spectrum disease resistance, by introducing novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways; and (c) enhanced nodulation efficiency, by engineering over-production of flavonoid nod gene inducers. The basic biochemistry and molecular biology underlying these strategies is briefly reviewed, and recent progress with transgenic plants summarized. The potential importance of metabolic compartmentation for attempts to engineer phenylpropanoid biosynthetic pathways is also discussed. Over-expression of an alfalfa glucanase-encoding gene confers significant protection against Phytophthora in alfalfa, possibly via indirect effects on phenylpropanoid metabolism.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the beta-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.

Vestitone reductase and 7,2′-dihydroxy-4′-methoxy-isoflavanol (DMI) dehydratase are the two final enzymes in medicarpin biosynthesis in alfalfa (Medicago sativa). Although two independent enzymes, vestitone reductase and DMI dehydratase can be loosely associated in low ionic strength buffers, presumably by a weak protein—protein interaction. The activities of vestitone reductase and DMI dehydratase increased approximately 3-fold 6 hours after elicitor treatment in alfalfa suspension cell culture. The activities remained at maximal levels for 40 hours, correlating with a steady increase in the medicarpin content of the cells. Medicarpin produced in vitro from vestitone by the action of vestitone reductase and DMI dehydratase was found to be (−)-medicarpin (6aR, 11aR-medicarpin), possessing the same stereochemistry as medicarpin produced in vivo.

Isoflavone reductase (IFR) reduces achiral isoflavones to chiral isoflavanones during the biosynthesis of chiral pterocarpan phytoalexins. A cDNA clone for IFR from pea ( Pisum sativum ) was isolated using the polymerase chain reaction and expressed in Escherichia coli . Analysis of circular dichroism (CD) spectra of the reduction product sophorol obtained using the recombinant enzyme indicated that the isoflavanone possessed the 3 R stereochemistry, in contrast to previous reports indicating a 3 S -isoflavanone as the product of the pea IFR. Analysis of CD spectra of sophorol produced using enzyme extracts of CuCl 2 -treated pea seedlings confirmed the 3 R stereochemistry. Thus, the stereochemistry of the isoflavanone intermediate in (+)-pisatin biosynthesis in pea is the same as that in (−)-medicarpin biosynthesis in alfalfa, although the final pterocarpans have the opposite stereochemistry. At the amino acid level the pea IFR cDNA was 91.8 and 85.2% identical to the IFRs from alfalfa and chickpea, respectively. IFR appears to be encoded by a single gene in pea. Its transcripts are highly induced in CuCl 2 -treated seedlings, consistent with the appearance of IFR enzyme activity and pisatin accumulation.

Increasing knowledge of the biochemistry of plant secondary product synthesis, and the cloning of biosynthetic pathway genes, has opened up the possibility of engineering novel pathways or reducing unwanted metabolites by genetic engineering strategies. Such approaches should lead to significant improvements in agronomic performance and post-harvest processing. At present, the necessary knowledge base is most advanced in the area of phenylpropanoid derivatives and certain alkaloids. Further advances in our understanding of terpenoid biochemistry, and our ability to genetically transform cereals and large seeded legumes, are required to underpin fuller exploitation of genetic manipulation of secondary metabolism for plant improvement.

In legumes, isoflavonoid derivatives function as antimicrobial phytoalexins, whereas phytoalexins of solanaceous species are of terpenoid origin. The phenylpropanoid and isoprenoid pathways leading to these phytoalexins are involved in the synthesis of a wide range of secondary metabolites with important functions in plant growth, development and responses to the environment. Elicitation of phytoalexin biosynthesis involves transcriptional activation of the genes encoding enzymes of general phenylpropanoid/terpenoid biosynthesis, and of the genes for the specific branch pathways leading to antimicrobial compounds. In order to understand the molecular controls determining the developmental and environmental regulation of the general and specific enzymes of phytoalexin synthesis, we are studying the promoter regions of three elicitor inducible genes, chalcone synthase (chs, isoflavonoid pathway, general), isoflavone reductase (ifr,isoflavonoid pathway, specific) and 3-hydroxy-3-methylglutaryl CoA reductase (hmgr,terpenoid pathway, general). We describe cis-elements and trans-factors involved in the expression of these genes in relation to their tissue specific expression and response to biotic stress. Two elements, the G-box and H-box, located within 50 bp of the TATA box, are important for regulation of expression of chs and probably hmgr, but are not present in the alfalfa ifr promoter.

Alfalfa (Medicago sativa L.) is probably the world’s most important forage crop. As a nitrogen-fixing legume, it is of value as a source of reduced nitrogen, and is becoming increasingly popular as a soil-conserving perennial in low-input sustainable agricultural systems. It is a cross-pollinated autotetraploid which, being highly heterozygous, exhibits substantial genetic diversity between individual plants of a particular cultivar. The complexity of alfalfa genetics is a hindrance to any form of molecular investigation which involves genetic crosses; however, alfalfa is amenable to stable genetic transformation with Agro-bacterium tumefaciens or A. rhizogenes, 1-6 and is readily, if slowly, regenerable from culture via somatic embryogenesis.7-9 It is therefore a suitable target for improvement by recombinant DNA technology.

Summary - The feasibility of utilizing genes from DNA banks is examined. A summary of the efforts to clone genes of phenylpropanoid metabolism is presented. These genes have been cloned from five major phenylpropanoid pathways: central phenylpropanoid, lignin branch, flavonoid branch, isoflavonoid branch and the stilbene branch pathway. Genes have been cloned from the following alkaloid classes: monoterpene indole, indole and isoquinoline, isoquinoline, and tropane. Two other gene classes are reviewed: genes that encode bioactive proteins and regulatory proteins. The major limitation for utilization of DNA from banks does not appear to be technology, but the lack of detailed basic knowledge concerning the biochemistry, bioactivity, and pharmacology of plant chemicals.

The major phytoalexin in alfalfa is the isoflavonoid (-)-medicarpin (or 6aR, 11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in medicarpin biosynthesis, is responsible for introducing one of two chiral centers in (-)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1) which, when expressed in Escherichia coli, converts 2'-hydroxyformononetin stereospecifically to (3R)-vestitone, as would be predicted for IFR from alfalfa. The calculated molecular weight of the polypeptide (35,400) derived from the 954 bp open reading frame compares favorably to estimated Mrs determined for IFR proteins purified from other legumes. The transcript (1.4 kb) is highly induced in elicited alfalfa cell cultures. The kinetics of induction are consistent with the appearance of IFR activity, the accumulation of medicarpin, and the observed induction of other enzymes in the pathway. Low levels of IFR transcripts were found in healthy plant parts (roots and nodules) which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded by a single gene in alfalfa. The cloning of IFR opens up the possibility of genetic manipulation of phytoalexin biosynthesis in alfalfa by altering isoflavonoid stereochemistry.

An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa. A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5'-untranslated leader and 128 bp 3'-non-coding region. The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide of Mr 78,865. A second PAL cDNA species was isolated, whose 3'-untranslated region was 86% identical to that of APAL1. Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa. PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast. PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings. These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation.

Phenylpropanoid compounds encompass a wide range of structural classes and biological functions. Limiting discussion to stress-induced phenylpropanoids eliminates few of the structural classes, because many compounds thst are constitutive in one plant species or tissue can be induced by various stresses in another species or in another tissue of the same plant (Beggs et al., 1987; Christie et al., 1994).

In legumes, the synthesis of infection- and elicitor-inducible antimicrobial phytoalexins occurs via the isoflavonoid branch of the phenylpropanoid pathway. To study transcriptional regulation of isoflavonoid pathway-specific genes, we have isolated the gene encoding isoflavone reductase (IFR), which is the enzyme that catalyzes the penultimate step in the synthesis of the phytoalexin medicarpin in alfalfa. Chimeric gene fusions were constructed between 765- and 436-bp promoter fragments of the IFR gene and the beta-glucuronidase reporter gene and transferred to alfalfa and tobacco by Agrobacterium-mediated transformation. Both promoter fragments conferred elicitor-mediated expression in cell suspension cultures derived from transgenic plants of both species and fungal infection-mediated expression in leaves of transgenic alfalfa. Developmental expression directed by both promoter fragments in transgenic alfalfa was observed only in the root meristem, cortex, and nodules, which is consistent with the accumulation of endogenous IFR transcripts. However, in transgenic tobacco, expression from the 765-bp promoter was observed in vegetative tissues (root meristem and cortex, inner vascular tissue of stems and petioles, leaf tips, and stem peripheries adjacent to petioles) and in reproductive tissues (stigma, placenta, base of the ovary, receptacle, seed, tapetal layer, and pollen grains), whereas the 436-bp promoter was expressed only in fruits, seed, and pollen. These data indicate that infection/elicitor inducibility of the IFR promoter in both species and developmental expression in alfalfa are determined by sequences downstream of position -436, whereas sequences between -436 and -765 confer a complex pattern of strong ectopic developmental expression in the heterologous species that lacks the isoflavonoid pathway.

Summary: Glutamine synthetase, glutamate synthase and alanine dehydrogenase activities were detected in crude extracts of Streptomyces clavuligerus. Glutamate dehydrogenase and alanine: 2-oxoglutarate aminotransferase could not be found. Glutamate synthase levels were independent of the nitrogen source in the culture medium. Glutamine synthetase activity was repressed in the presence of ammonium, and a rapid inactivation of the enzyme was seen after ammonium shock. Alanine dehydrogenase was induced by alanine or ammonium concentrations higher than 20 mm. Glutamate and, in lower amounts, glutamine, alanine, βgM-aminobutyrate and aspartate were the main constituents of the intracellular pool of free amino acids. Only the pool concentrations of glutamine and alanine were markedly influenced by the nitrogen source used for growth. Mutants devoid of glutamine synthetase, glutamate synthase, or alanine dehydrogenase were isolated. The pattern of utilization of nitrogen sources by the three classes of mutants indicated that the glutamine synthetase–glutamate synthase pathway is the only means of assimilation of ammonium in S. clavuligerus. Alanine dehydrogenase is not directly involved in that process, but a possible related function is discussed.