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.

Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae.

BACKGROUND: The polyketide lactone, tylactone, is produced in Streptomyces fradiae by the TylG complex of five multifunctional proteins. As with other type I polyketide synthases, the enzyme catalysing the final elongation step (TylGV) possesses an integral thioesterase domain that is believed to be responsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide producers, S. fradiae also possesses an additional thioesterase gene (orf5) located within the cluster of antibiotic biosynthetic genes. The function of the Orf5 protein is addressed here. RESULTS: Disruption of orf5 reduced antibiotic accumulation in S. fradiae by at least 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression of orf6, which encodes a mycarose biosynthetic enzyme. High levels of desmycosin production were restored in the disrupted strain by complementation with intact orf5, or with the corresponding thioesterase gene, nbmB, from S. narbonensis, but not with DNA encoding the integral thioesterase domain of TylGV. CONCLUSIONS: Polyketide metabolism in S. fradiae is strongly dependent on the thioesterase activity encoded by orf5 (tylO). It is proposed that the TylG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TylO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae.

BACKGROUND: The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. RESULTS: DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which (tylP) encodes a gamma-butyrolactone signal receptor for which tylQ is a probable target. Two other genes (tylS and tylT) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR, has been shown by mutational analysis to control various aspects of tylosin production. CONCLUSIONS: The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin.

The tylE-J region of the tylosin-biosynthetic gene cluster of Streptomyces fradiae contains six open reading frames. The products of tylJ and tylD are nucleoside diphospho (NDP)-deoxyhexose 3-epimerase and NDP-deoxyhexose 4-ketoreductase, respectively, involved in the synthesis of NDP-6-deoxyallose from NDP-4-keto, 6-deoxyglucose. After incorporation of deoxyallose at C23-OH of the polyketide lactone, tylosin biosynthesis is completed by the products of tylE and tylF, which convert the deoxyallosyl moiety to mycinose via bis-O-methylation at 2-OH and 3-OH, respectively. Hydroxylation of the polyketide lactone at C23 is catalysed by the cytochrome P450 enzyme, TylHl. The product of tylHll is a ferredoxin of unknown specificity that could conceivably act together with TylHl.

Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements.

The tomato lat52 gene encodes an essential cysteine-rich protein preferentially transcribed in the vegetative cell during pollen maturation. Detailed analyses of the identity, organization and role of cis-regulatory elements in controlling the precise developmental and tissue-specific expression of lat52 during pollen development were performed. Analysis of a series of 5' promoter deletion mutants stably introduced into tobacco demonstrated differential developmental activation of deletion mutants during pollen development. All major cis-regulatory elements required for pollen-specific transcription were located within the upstream region -492 to -52. This region was shown to comprise three independent activator domains A, B and C, each sufficient to activate the minimal CaMV 35S promoter in a pollen-specific manner. 5' deletion and gain of function approaches were used to show that domain A and the previously defined motif PBII (sub-domain B1) were largely redundant in the presence of downstream sequences in mature pollen. Within domain B two novel pollen-specific sub-domains B2 and B3 were identified. Within domain C, the activity of the PBI motif (sub-domain C1) was shown to be strictly dependent upon a downstream 20 bp pollen-specific activator unit -72 to -52 (sub-domain C2), containing two novel co-dependent regulatory elements AGAAA and TCCACCATA. These results demonstrate that transcriptional activation of lat52 is controlled by a complex of pollen-specific cis-regulatory elements which cooperate to achieve maximum levels of gene expression throughout pollen maturation. Alternative models of the interaction of identified cis-regulatory elements with putative trans-acting factors within the lat52 promoter and their developmental utilization are presented.

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.

In vivo characterisation of a translational enhancer upstream from the coat protein open reading frame of potato virus S.

The 101 nucleotide region upstream from the ATG of the potato virus S (PVS) coat protein gene was isolated and the effect of this region on the translation of a downstream open reading frame analysed in vivo. Translation was monitored using the reporter genes B-glucuronidase (GUS) and luciferase (LUC). Translational enhancement was assayed transiently using DNA microprojectile bombardment into both leaf and pollen tissue and also by polyethylene glycol mediated transfection of tobacco protoplasts. In both cases the presence of this region resulted in a 2-3 fold increase in translation when compared to reporter expression with synthetic leader and authentic plant leader constructs. Tobacco plants stability transformed with this PVS 101 nucleotide region and downstream GUS gene gave 4 times the level of translation over synthetic leader GUS control plants.

Maturation-specific translational enhancement mediated by the 5'-UTR of a late pollen transcript.

The tomato lat52 gene encodes an abundant protein specifically expressed in the vegetative cell of the pollen grain during pollen maturation which is essential for normal pollen tube growth. Multiple upstream cis-regulatory elements controlling the level and specificity of lat52 transcription have previously been identified. This research investigated the role of the 5'-untranslated region (5'-UTR) in controlling lat52 expression. In transient expression assays, gene fusion constructs containing the lat52 5'-UTR were expressed in pollen at levels 13- to 60-fold above those in which synthetic polylinker sequences replaced the lat52 5'-UTR. This enhancement was shown to be independent of both the promoter sequences, the linked reporter gene and the 3'-UTR. Analysis of RNA and protein levels in transgenic plants containing such gene fusions demonstrated that the lat52 5'-UTR conferred a dramatically increased translational yield to heterologous transcripts in a pollen-specific and strictly developmentally regulated manner during the final stages of pollen maturation. These results represent a novel example of translational enhancement in plants in that translational yield is regulated developmentally in a cell-specific manner via sequences located within the 5'-UTR.

Analysis of a translational enhancer upstream from the coat protein open reading frame of potato virus S.

Evidence has suggested that the subgenomic RNA of the carlavirus potato virus S is an efficient message for the coat protein, even though evidence suggests it is uncapped at its 5' terminus. We have investigated the effect of the upstream region of the coat protein gene of potato virus S on the level of reporter gene expression in vitro. The region of 101 nucleotides upstream of the coat protein, designated VTE (viral translational enhancer) was found to increase levels of translation in comparison to a synthetic leader when linked to the beta-glucuronidase (GUS) reporter gene in vitro in rabbit reticulocyte and wheat germ lysate. VTE was also able to increase translation of the reporter gene luciferase (LUC) in vitro above the levels obtained for both a synthetic leader and a leader obtained from a plant gene isolated from Arabidopsis thaliana. The level of enhancement was evident with both capped and uncapped transcripts. When the VTE sequence was deleted to 20 nucleotides of the upstream region, thus removing the nucleotide block homologous among carlaviruses, the ability to enhance levels of translation was removed. In vitro translation studies indicated that the translational enhancement activity of VTE was at least partially cap independent. Translation of VTE linked to reporter genes in the presence of cap analogue was relatively unaffected whereas synthetic leader and a plant leader constructs were both more sensitive. In vitro competition analysis revealed that when short RNA transcripts representing the 101 nucleotides of VTE were added in trans to functional VTE leader LUC constructs there was a marked decrease in the level of translation when compared with a synthetic leader added in trans. These results suggest that the upstream region of the coat protein ORF of potato virus S promotes translation in a cap-independent manner that may involve the binding of proteins and/or ribosomes to the 101 nucleotides of the VTE sequence.

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.

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.

The tylosin-biosynthetic genes of Streptomyces fradiae.

The tylosin-biosynthetic (tyl) gene cluster occupies about 1% of the genome of Streptomycesfradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tyl cluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of y-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

The small cysteine-rich protein P14 of beet necrotic yellow vein virus regulates accumulation of RNA 2 in cis and coat protein in trans.

The effect of null mutations of the small cysteine-rich protein P14 encoded by RNA 2 of beet necrotic yellow vein virus has been investigated using in vitro transcripts of viral RNA to infect Chenopodium quinoa protoplasts. The P14 mutations down-regulated RNA 2 accumulation by approximately 10- to 50-fold. Accumulation of minus-strand RNA 2 was also diminished but RNA 1 accumulation was much less affected. The inhibition of RNA 2 accumulation could not be complemented in trans by providing P14 from another source (either a second molecule of RNA 2 or an RNA 3-based replicon) containing and expressing the P14 gene. The P14 null mutations dramatically inhibited accumulation of viral coat protein, which is encoded by the 5'-proximal gene on RNA 2, but this effect could be complemented in trans, indicating that it occurs by a mechanism distinct from that affecting RNA 2 accumulation. Transient expression experiments were also carried out in which a plasmid expressing P14 and plasmids expressing a reporter gene placed downstream of potential translational control sequences (the 5'-noncoding sequences of RNAs 2, 3, or 4) were introduced into C. quinoa or Nicotiana tabacum leaves by microprojectile bombardment. Coexpression of P14 produced a 3- to 4-fold stimulation of reporter gene expression levels for all the constructs. The lack of sequence specificity suggests that this phenomenon is not directly related to the RNA 2-specific stimulation of coat protein accumulation observed in a viral infection.

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.

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.

Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10.

The tobacco gene g10 is preferentially and maximally expressed in mature pollen, shows homology to pectate lyases, and is the putative homologue of the tomato gene lat56. Analysis of regulatory elements within the g10 promoter was carried out to verify the importance of putative regulatory sequence motifs. Analysis of transgenic plants showed that 1190 bp of g10 5' sequence directed preferential expression of GUS in pollen, with bimodal peaks of expression just before and during pollen mitosis I, and in mature anthers. This was confirmed by northern analysis of native g10 transcripts in isolated spores. Transient expression analysis defined the minimal g10 promoter region capable of directing expression in pollen as -86 to +217. Three upstream regions within -427 bp modulate the expression from g10. Gain-of-function analyses showed that the region from -106 to -53 could enhance pollen-specific expression of a minimal CaMV 35S promoter. These analyses further showed that sequences upstream of -86 modulate expression in pollen, but are not essential for preferential pollen expression. The function of a conserved GTGA motif shared between the tobacco g10 and tomato lat56 promoters was demonstrated in g10. Thus, further functional evidence is provided for the conservation of mechanisms for the regulation of late pollen genes across species.

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.