Nancy L. Paiva

Stress-Induced Phenylpropanoid Metabolism.

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).

Overexpression of L-Phenylalanine Ammonia-Lyase in Transgenic Tobacco Plants Reveals Control Points for Flux into Phenylpropanoid Biosynthesis.

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.

Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses--a review.

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.

Constitutive Accumulation of a Resveratrol-Glucoside in Transgenic Alfalfa Increases Resistance to Phoma medicaginis

Alfalfa (Medicago sativa) was transformed with a peanut (Arachis hypogaea) cDNA encoding resveratrol synthase (RS) transcriptionally regulated by an enhanced Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated a new compound, not present in wild-type or vector-transformed alfalfa, that was identified as trans-resveratrol-3-O-beta-D-glucopyranoside (RGluc) by high-pressure liquid chromatography (HPLC), UV, 1H- and 13C-nuclear magnetic resonance (NMR) analyses. RGluc concentration was highest in the youngest leaves (>15 microg per g fresh weight) and oldest stem internode segments (>10 microg per g fresh weight) while roots contained only trace amounts (<0.2 microg per g fresh weight). RS transcript levels were highest in leaves and stems, with comparatively little transcript accumulation in the roots, while an inverse pattern was observed for chalcone synthase (CHS) transcript levels. CHS directly competes with RS for the metabolic precursors p-coumaroyl CoA and malonyl CoA, and may also contribute to the developmental variations in RGluc levels by limiting the availability of substrates. Agar-plate bioassays indicated that both RGluc and resveratrol greatly inhibit hyphal growth of the alfalfa fungal pathogen Phoma medicaginis. Subsequently, RGluc-containing leaves were wound inoculated and showed a significant reduction (relative to control leaves) in the size of necrotic lesions, intensity of adjacent chlorosis, and number of fungal reproductive structures (pycnidia). Decreasing sporulation of this pathogen may greatly reduce disease spread and severity throughout the field.

The Medicago Genome Initiative: a model legume database

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.

Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis

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 (35400) 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.

Reduced lignin in transgenic plants containing a caffeic acidO-methyltransferase antisense gene

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.

Transcriptome analysis of alfalfa glandular trichomes

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.

Molecular and Biochemical Analysis of Two cDNA Clones Encoding Dihydroflavonol-4-Reductase from Medicago truncatula

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.

The elicitor-inducible alfalfa isoflavone reductase promoter confers different patterns of developmental expression in homologous and heterologous transgenic plants.

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.