Molecular characterization of an Arabidopsis gene encoding hydroperoxide lyase, a cytochrome P-450 that is wound inducible.

Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules. We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum). The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase. The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity. Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower. Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E. coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited. Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate.

C6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes.

Six-Carbon (C6-) volatiles, including the aldehydes trans-2-hexenal, hexanal and cis-3-hexenal, as well as their corresponding alcohols, are produced from damaged or wounded plant tissue as a product of the enzymatic activity of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway. Aerial treatment of Arabidopsis seedlings with 10 microM concentrations of trans-2-hexenal induces several genes known to be involved in the plant's defense response, including phenylpropanoid-related genes as well as genes of the LOX pathway. Genes encoding the pathogenesis-related proteins PR-1 or PR-2, however, were not induced. Trans-2-hexenal induction thus closely mimics the group of genes induced by methyl jasmonate (MeJA), also a LOX-derived volatile. However, unlike MeJA, trans-2-hexenal did not induce hydroxymethylglutaryl-coenzyme A reductase (HMGR) or thionin2-1. The inductive effect seemed to be limited to C6-related volatiles, as C8-, C9- and other related volatiles did not induce LOX mRNA levels. As has been demonstrated for MeJA, trans-2-hexenal quantitatively reduced wild-type seed germination. Trans-2-hexenal also reduced the germination frequency of the MeJA resistant Arabidopsis mutant, jar1-1, supporting the notion that trans-2-hexenal and MeJA are recognized via different mechanisms. In addition, trans-2-hexenal had a moderate inhibitory effect on root length relative to similar concentrations of MeJA and was approximately 10-fold less effective than MeJA at inducing anthocyanin accumulation in Arabidopsis seedlings. These results suggest that C6-volatiles of the LOX pathway act as a wound signal in plants, but result in a moderate plant response relative to MeJA at both the physiological and molecular level.

Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants.

Nitrite reductase (NiR) is the second enzyme in the nitrate assimilatory pathway reducing nitrite to ammonium. The expression of the NiR gene is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between -330 to - 200, was shown to be necessary for nitrate induction of beta-glucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements involved in nitrate regulation of NiR gene expression, transgenic tobacco plants were generated with 5' deletions in the -330 to -200 region of the spinach NiR gene promoter fused to the GUS gene. Plants with the NiR promoter deleted to -230 showed a considerable increase in GUS activity in the presence of nitrate, indicating that the 30 bp region between -230 to -200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the -300 to -130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the -230 to -181 region revealed factor binding to two adjacent GATA elements separated by 24 bp. This arrangement of GATA elements is analogous to cis-regulatory sequences found in the promoters of nitrate-inducible genes of Neurospora crassa, regulated by the NIT2 Zn-finger protein. The -240 to -110 fragment of the NiR promoter, which contains two NIT2 consensus core elements, bound in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. The data presented here show that nitrate-inducible expression of the NiR gene is mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants.

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.

Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis.

Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanoid synthesis. The role of PAL in pathway regulation was investigated by measurement of product accumulation as a function of enzyme activity in a collection of near-isogenic transgenic tobacco plants exhibiting a range of PAL levels from wild type to 0.2% of wild type. In leaf tissue, PAL level is the dominant factor regulating accumulation of the major product chlorogenic acid and overall flux into the pathway. In stems, PAL at wild-type levels contributes, together with downstream steps, in the regulation of lignin deposition and becomes the dominant, rate-determining step at levels 3- to 4-fold below wild type. The metabolic impact of elevated PAL levels was investigated in transgenic leaf callus that overexpressed PAL. Accumulation of the flavonoid rutin, the major product in wild-type callus, was not increased, but several other products accumulated to similarly high levels. These data indicate that PAL is a key step in the regulation of overall flux into the pathway and, hence, accumulation of major phenylpropanoid products, with the regulatory architecture of the pathway poised so that downstream steps control partitioning into different branch pathways.

Increased disease susceptibility of transgenic tobacco plants with suppressed levels of preformed phenylpropanoid products.

It has been proposed that natural products synthesized by plants contribute to their resistance to pests and pathogens. We show here that transgenic tobacco plants with suppressed levels of the phenylpropanoid biosynthetic enzyme phenylalanine ammonia-lyase (L-phenylalanine ammonia-lyase, EC 4.3.1.5) and correspondingly low levels of chlorogenic acid, the major soluble leaf phenylpropanoid product, exhibit more rapid and extensive lesion development than wild-type plants after infection by the virulent fungal pathogen Cercospora nicotianae. These observations provide direct evidence that phenylpropanoid products contribute to disease limitation. No induction of transcripts encoding phenylalanine ammonia-lyase or the lignin branch pathway enzyme caffeic acid O-methyltransferase was observed during the infection and there was no perturbation in the pattern of soluble phenylpropanoids. Hence, increased disease susceptibility does not involve inhibition of a pathogen-induced response but likely reflects inhibition of the developmental accumulation of chlorogenic acid. Demonstration of the contribution of such preformed protectants to plant health identifies attractive targets for manipulation by breeding or gene transfer to reduce the quantitative impact of disease.

Active Translation of the D-1 Protein of Photosystem II in Senescing Leaves.

Primary leaves of bean (Phaseolus vulgaris L.) show pronounced symptoms of senescence within 21 days of planting. Total RNA levels decline by approximately fourfold, and pulse-labeling studies with [(35)S]methionine have indicated that synthesis of all thylakoid proteins except the D-1 protein of photosystem II is sharply curtailed. Measurements of transcript levels for D-1, which is encoded by psbA, and for two other thylakoid proteins, the 68- to 70-kilodalton protein of photosystem I encoded by psaA/B and the beta subunit of ATPase encoded by atpB/E, have indicated that the continued strong synthesis of D-1 relative to other proteins in senescing thylakoids does not reflect differential changes in message abundance. Specifically, although transcript quantity for each of these proteins decreases by approximately fourfold with advancing senescence, only the 68- to 70-kilodalton protein of photosystem I and the beta-subunit of ATPase show decreased synthesis. As well, the proportion of total psbA transcript associated with polysomes is higher in senescent leaves than in young leaves, whereas for each of psaA/B and atpB/E, there is less message in polysome formation in the senescent leaves than in young leaves. Thus, the continued strong synthesis of D-1 in senescent leaves appears to reflect preferential sequestering of ribosomes by psbA transcript from a dwindling ribosome pool. This ability to preferentially sequester ribosomes may be related to the unusually high turnover rate that characterizes D-1.