Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes

A collection of 8,000 Arabidopsis thaliana plants carrying 48,000 insertions of the maize transposable element En-1 has been generated. This population was used for reverse genetic analyses to identify insertions in individual gene loci. By using a PCR-based screening protocol, insertions were found in 55 genes. En-1 showed no preference for transcribed or untranscribed regions nor for a particular orientation relative to the gene of interest. In several cases, En-1 was inserted within a few kilobases upstream or downstream of the gene. En-1 was mobilized from such positions into the respective gene to cause gene disruption. Knock-out alleles of genes involved in flavonoid biosynthesis were generated. One mutant line contained an En-1 insertion in the flavonol synthase gene (FLS) and showed drastically reduced levels of kaempferol. Allelism tests with other lines containing En-1 insertions in the flavanone 3-hydroxylase gene (F3H) demonstrated that TRANSPARENT TESTA 6 (TT6) encodes flavanone 3-hydroxylase. The f3h and fls null mutants complete the set of A. thaliana lines defective in early steps of the flavonoid pathway. These experiments demonstrate the efficiency of the screening method and gene disruption strategy used for assigning functions to genes defined only by sequence.

Piperonylic Acid, a Selective, Mechanism-Based Inactivator of the trans-Cinnamate 4-Hydroxylase: A New Tool to Control the Flux of Metabolites in the Phenylpropanoid Pathway1

Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (KI = 17 μm, kinact = 0.064 min−1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.

Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis

The phenylpropanoid pathway provides precursors for the biosynthesis of soluble secondary metabolites and lignin in plants. Ferulate-5-hydroxylase (F5H) catalyzes an irreversible hydroxylation step in this pathway that diverts ferulic acid away from guaiacyl lignin biosynthesis and toward sinapic acid and syringyl lignin. This fact led us to postulate that F5H was a potential regulatory step in the determination of lignin monomer composition. To test this hypothesis, we have used Arabidopsis to examine the impact of F5H overexpression. Arabidopsis is a useful model system in which to study lignification because in wild-type plants, guaiacyl and syringyl lignins are deposited in a tissue-specific fashion, while the F5H-deficient fah1 mutant accumulates only guaiacyl lignin. Here we show that ectopic overexpression of F5H in Arabidopsis abolishes tissue-specific lignin monomer accumulation. Surprisingly, overexpression of F5H under the control of the lignification-associated cinnamate-4-hydroxylase promoter, but not the commonly employed cauliflower mosaic virus 35S promoter, generates a lignin that is almost entirely comprised of syringylpropane units. These experiments demonstrate that modification of F5H expression may enable engineering of lignin monomer composition in agronomically important plant species.

Regulation of Ferulate-5-Hydroxylase Expression in Arabidopsis in the Context of Sinapate Ester Biosynthesis1

Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapoylmalate, and sinapoylcholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here we show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated that F5H expression in leaves is dependent on sequences 3′ of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required for F5H transcript or sinapoylcholine accumulation in embryos.

Regulation and Functional Expression of Cinnamate 4-Hydroxylase from Parsley

A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit.

Intermediates of Salicylic Acid Biosynthesis in Tobacco1

Salicylic acid (SA) is an important component of systemic-acquired resistance in plants. It is synthesized from benzoic acid (BA) as part of the phenylpropanoid pathway. Benzaldehyde (BD), a potential intermediate of this pathway, was found in healthy and tobacco mosaic virus (TMV)-inoculated tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaf tissue at 100 ng/g fresh weight concentrations as measured by gas chromatography-mass spectrometry. BD was also emitted as a volatile organic compound from tobacco tissues. Application of gaseous BD to plants enclosed in jars caused a 13-fold increase in SA concentration, induced the accumulation of the pathogenesis-related transcript PR-1, and increased the resistance of tobacco to TMV inoculation. [13C6]BD and [2H5]benzyl alcohol were converted to BA and SA. Labeling experiments using [13C1]Phe in temperature-shifted plants inoculated with the TMV showed high enrichment of cinnamic acids (72%), BA (34%), and SA (55%). The endogenous BD, however, contained nondetectable enrichment, suggesting that BD was not the intermediate between cinnamic acid and BA. These results show that BD and benzyl alcohol promote SA accumulation and expression of defense responses in tobacco, and provide insight into the early steps of SA biosynthesis.

Reduction of Light-Induced Anthocyanin Accumulation in Inoculated Sorghum Mesocotyls1 : Implications for a Compensatory Role in the Defense Response

Sorghum (Sorghum bicolor L. Moench) accumulates the anthocyanin cyanidin 3-dimalonyl glucoside in etiolated mesocotyls in response to light. Inoculation with the nonpathogenic fungus Cochliobolus heterostrophus drastically reduced the light-induced accumulation of anthocyanin by repressing the transcription of the anthocyanin biosynthesis genes encoding flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. In contrast to these repression effects, fungal inoculation resulted in the synthesis of the four known 3-deoxyanthocyanidin phytoalexins and a corresponding activation of genes encoding the key branch-point enzymes in the phenylpropanoid pathway, phenylalanine ammonia-lyase and chalcone synthase. In addition, a gene encoding the pathogenesis-related protein PR-10 was strongly induced in response to inoculation. The accumulation of phytoalexins leveled off by 48 h after inoculation and was accompanied by a more rapid increase in the rate of anthocyanin accumulation. The results suggest that the plant represses less essential metabolic activities such as anthocyanin synthesis as a means of compensating for the immediate biochemical and physiological needs for the defense response.

4-Coumarate:Coenzyme A Ligase in Hybrid Poplar1 : Properties of Native Enzymes, cDNA Cloning, and Analysis of Recombinant Enzymes

The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa × Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.

Two Isoforms of NADPH:Cytochrome P450 Reductase in Arabidopsis thaliana : Gene Structure, Heterologous Expression in Insect Cells, and Differential Regulation

We have investigated two NADPH-cytochrome (Cyt) P450 reductase isoforms encoded by separate genes (AR1 and AR2) in Arabidopsis thaliana. We isolated AR1 and AR2 cDNAs using a mung bean (Phaseolus aureus L.) NADPH-Cyt P450 reductase cDNA as a probe. The recombinant AR1 and AR2 proteins produced using a baculovirus expression system showed similar Km values for Cyt c and NADPH, respectively. In the reconstitution system with a recombinant cinnamate 4-hydroxylase (CYP73A5), the recombinant AR1 and AR2 proteins gave the same level of cinnamate 4-hydroxylase activity (about 70 nmol min−1 nmol−1 P450). The AR2 gene expression was transiently induced by 4- and 3-fold within 1 h of wounding and light treatments, respectively, and the induction time course preceded those of CYP73A5 and a phenylalanine ammonia-lyase (PAL1) gene. On the contrary, the AR1 expression level did not change during the treatments. Analysis of the AR1 and AR2 gene structure revealed that only the AR2 promoter contained three putative sequence motifs (boxes P, A, and L), which are involved in the coordinated expression of CYP73A5 and other phenylpropanoid pathway genes. These results suggest the possibility that AR2 transcription may be functionally linked to the induced levels of phenylpropanoid pathway enzymes.

Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.

Biosynthesis and metabolism of salicylic acid.

Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-beta-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance.

Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells.

The accumulation of phenylalanine-derived phenolic compounds is a well-known element of a plant's defense in response to pathogen attack. Phenylalanine, as well as the other two aromatic amino acids, tyrosine and tryptophan, is synthesized by way of the shikimate pathway. The first seven steps of the shikimate pathway (the prechorismate pathway) are common for the biosynthesis of all three aromatic amino acids. We have studied transcript levels of six genes--i.e., two 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes, one shikimate kinase gene, one 5-enolpyruvylshikimate 3-phosphate synthase gene, and two chorismate synthase genes--corresponding to four steps of the prechorismate pathway, in cultured tomato cells exposed to fungal elicitors. The abundance of transcripts specific for some of these genes increased 10- to 20-fold within 6 h after elicitor treatment, as did the abundance of phenylalanine ammonialyase-specific transcripts and the synthesis of ethylene. Interestingly, transcript accumulation occurred more rapidly for shikimate kinase than for the enzymes preceding or following it in the prechorismate pathway. Neither the inhibition of ethylene biosynthesis by aminoethoxyvinylglycine nor inhibition of phenylalanine ammonia-lyase (EC 4.3.1.5) activity by 2-aminoindan-2-phosphonic acid affected the time course or extent of transcript accumulation. Thus, the increased demand for phenylalanine in the phenylpropanoid pathway required after elicitor treatment appears to be met by increased de novo synthesis of its biosynthetic enzymes.