Alpine-ice record of bismuth pollution implies a major role of military use during World War II.

Military conflicts result in local environmental damage, but documenting regional and larger scale impacts such as heavy metal pollution has proven elusive. Anthropogenic emissions of bismuth (Bi) include coal burning and various commodity productions but no emission estimates over the past century exist. Here we used Bi measurements in ice cores from the French Alps to show evidence of regional-scale Bi pollution concurrent with the Spanish Civil War and World War II. Tracers of the main sources of Bi emissions measured in the same ice-coal-burning, steel- and aluminum-industry, alloy and other metal processing-indicate a major, previously undocumented additional emissions source that we attribute to military activities between 1935 and 1945 Common Era (CE) in western Europe. These include the use of bismuth for low-melting point alloys for shells, thin-walled aluminum alloy aircraft oil, and munitions.

Homogeneous sulfur isotope signature in East Antarctica and implication for sulfur source shifts through the last glacial-interglacial cycle.

Sulfate aerosol (SO42-) preserved in Antarctic ice cores is discussed in the light of interactions between marine biological activity and climate since it is mainly sourced from biogenic emissions from the surface ocean and scatters solar radiation during traveling in the atmosphere. However, there has been a paradox between the ice core record and the marine sediment record; the former shows constant non-sea-salt (nss-) SO42- flux throughout the glacial-interglacial changes, and the latter shows a decrease in biogenic productivity during glacial periods compared to interglacial periods. Here, by ensuring the homogeneity of sulfur isotopic compositions of atmospheric nss-SO42- (δ34Snss) over East Antarctica, we established the applicability of the signature as a robust tool for distinguishing marine biogenic and nonmarine biogenic SO42-. Our findings, in conjunction with existing records of nss-SO42- flux and δ34Snss in Antarctic ice cores, provide an estimate of the relative importance of marine biogenic SO42- during the last glacial period to be 48 ± 10% of nss-SO42-, slightly lower than 59 ± 11% during the interglacial periods. Thus, our results tend to reconcile the ice core and sediment records, with both suggesting the decrease in marine productivity around Southern Ocean under the cold climate.

Alpine ice evidence of a three-fold increase in atmospheric iodine deposition since 1950 in Europe due to increasing oceanic emissions.

Iodine is an important nutrient and a significant sink of tropospheric ozone, a climate-forcing gas and air pollutant. Ozone interacts with seawater iodide, leading to volatile inorganic iodine release that likely represents the largest source of atmospheric iodine. Increasing ozone concentrations since the preindustrial period imply that iodine chemistry and its associated ozone destruction is now substantially more active. However, the lack of historical observations of ozone and iodine means that such estimates rely primarily on model calculations. Here we use seasonally resolved records from an Alpine ice core to investigate 20th century changes in atmospheric iodine. After carefully considering possible postdepositional changes in the ice core record, we conclude that iodine deposition over the Alps increased by at least a factor of 3 from 1950 to the 1990s in the summer months, with smaller increases during the winter months. We reproduce these general trends using a chemical transport model and show that they are due to increased oceanic iodine emissions, coupled to a change in iodine speciation over Europe from enhanced nitrogen oxide emissions. The model underestimates the increase in iodine deposition by a factor of 2, however, which may be due to an underestimate in the 20th century ozone increase. Our results suggest that iodine's impact on the Northern Hemisphere atmosphere accelerated over the 20th century and show a coupling between anthropogenic pollution and the availability of iodine as an essential nutrient to the terrestrial biosphere.

Feedback mechanisms between snow and atmospheric mercury: Results and observations from field campaigns on the Antarctic plateau.

The Antarctic Plateau snowpack is an important environment for the mercury geochemical cycle. We have extensively characterized and compared the changes in surface snow and atmospheric mercury concentrations that occur at Dome C. Three summer sampling campaigns were conducted between 2013 and 2016. The three campaigns had different meteorological conditions that significantly affected mercury deposition processes and its abundance in surface snow. In the absence of snow deposition events, the surface mercury concentration remained stable with narrow oscillations, while an increase in precipitation results in a higher mercury variability. The Hg concentrations detected confirm that snowfall can act as a mercury atmospheric scavenger. A high temporal resolution sampling experiment showed that surface concentration changes are connected with the diurnal solar radiation cycle. Mercury in surface snow is highly dynamic and it could decrease by up to 90% within 4/6 h. A negative relationship between surface snow mercury and atmospheric concentrations has been detected suggesting a mutual dynamic exchange between these two environments. Mercury concentrations were also compared with the Br concentrations in surface and deeper snow, results suggest that Br could have an active role in Hg deposition, particularly when air masses are from coastal areas. This research presents new information on the presence of Hg in surface and deeper snow layers, improving our understanding of atmospheric Hg deposition to the snow surface and the possible role of re-emission on the atmospheric Hg concentration.

Sporopollenin biosynthetic enzymes interact and constitute a metabolon localized to the endoplasmic reticulum of tapetum cells.

The sporopollenin polymer is the major constituent of exine, the outer pollen wall. Recently fatty acid derivatives have been shown to be the precursors of sporopollenin building units. ACYL-COA SYNTHETASE, POLYKETIDE SYNTHASE A (PKSA) and PKSB, TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 have been demonstrated to be involved in sporopollenin biosynthesis in Arabidopsis (Arabidopsis thaliana). Here all these sporopollenin biosynthetic enzymes but TKPR2 have been immunolocalized to endoplasmic reticulum of anther tapetal cells. Pull-down experiments demonstrated that tagged recombinant proteins interacted to form complexes whose constituents were characterized by immunoblotting. In vivo protein interactions were evidenced by yeast (Saccharomyces cerevisiae) two-hybrid analysis and by fluorescence lifetime imaging microscopy/Förster resonance energy transfer studies in transgenic Nicotiana benthamiana, which were used to test the possibility that the enzymes interact to form a biosynthetic metabolon. Various pairs of proteins fused to two distinct fluorochromes were coexpressed in N. benthamiana leaf tissues and fluorescence lifetime imaging microscopy/Förster resonance energy transfer measurements demonstrated that proteins interacted pairwise in planta. Taken together, these results suggest the existence of a sporopollenin metabolon.

The interplay of lipid acyl hydrolases in inducible plant defense.

Lipid acyl hydrolases (LAH) have received recently increased attention in the context of plant defense. Multiple structurally unrelated gene families have been annotated in Arabidopsis as encoding potential lipid deacylating enzymes with numerous members being transcriptionally activated upon biotic stress. Confirming in silico predictions, experimental data have illustrated the wide subcellular distribution of LAHs indicating they likely interact with distinct membrane systems to initiate specific cellular responses. While recombinant LAHs are active in vitro on a small set of polar lipids, precise knowledge of in vivo substrates and hydrolysis products is generally lacking. Functional analysis of a few LAHs has revealed their roles in initiating oxylipin biosynthesis, cell death execution, signalling or direct antimicrobial activity. The picture emerging is that pathogenic challenge triggers a complex network of lipid hydrolysis events across the cellular compartments resulting in changes in membrane structures and release of signal precursors involved in the building-up of an adequate immune response.

LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana.

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.

Analysis of TETRAKETIDE α-PYRONE REDUCTASE function in Arabidopsis thaliana reveals a previously unknown, but conserved, biochemical pathway in sporopollenin monomer biosynthesis.

The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors.

The Arabidopsis patatin-like protein 2 (PLP2) plays an essential role in cell death execution and differentially affects biosynthesis of oxylipins and resistance to pathogens.

We previously reported that patatin-like protein 2 (PLP2), a pathogen-induced patatin-like lipid acyl hydrolase, promotes cell death and negatively affects Arabidopsis resistance to the fungus Botrytis cinerea and to the bacteria Pseudomonas syringae. We show here that, on the contrary, PLP2 contributes to resistance to Cucumber mosaic virus, an obligate parasite inducing the hypersensitive response. These contrasted impacts on different pathosystems were also reflected by differential effects on defense gene induction. To examine a possible link between PLP2 lipolytic activity and oxylipin metabolism, gene expression profiling was performed and identified B. cinerea among these pathogens as the strongest inducer of most oxylipin biosynthetic genes. Quantitative oxylipin profiling in wild-type and PLP2-modified, Botrytis-challenged plants established the massive accumulation of oxidized fatty acid derivatives in infected leaves. Several compounds previously described as modulating plant tissue damage and issued from the alpha-dioxygenase pathway were found to accumulate in a PLP2-dependent manner. Finally, the contribution of PLP2 to genetically controlled cell death was evaluated using PLP2-silenced or -overexpressing plants crossed with the lesion mimic mutant vascular-associated death 1 (vad1). Phenotypic analysis of double-mutant progeny showed that PLP2 expression strongly promotes necrotic symptoms in vad1 leaves. Collectively, our data indicate that PLP2 is an integral component of the plant cell death execution machinery, possibly providing fatty acid precursors for the biosynthesis of specific oxylipins and differentially affecting resistance to pathogens with distinct lifestyles.

A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines.

BAHD acyltransferases catalyze the acylation of many plant secondary metabolites. We characterized the function of At2g19070, a member of the BAHD gene family of Arabidopsis thaliana. The acyltransferase gene was shown to be specifically expressed in anther tapetum cells in the early stages of flower development. The impact of gene repression was studied in RNAi plants and in a knockout (KO) mutant line. Immunoblotting with a specific antiserum raised against the recombinant protein was used to evaluate the accumulation of At2g19070 gene product in flowers of various Arabidopsis genotypes including the KO and RNAi lines, the male sterile mutant ms1 and transformants overexpressing the acyltransferase gene. Metabolic profiling of flower bud tissues from these genetic backgrounds demonstrated a positive correlation between the accumulation of acyltransferase protein and the quantities of metabolites that were putatively identified by tandem mass spectrometry as N(1),N(5),N(10)-trihydroxyferuloyl spermidine and N(1),N(5)-dihydroxyferuloyl-N(10)-sinapoyl spermidine. These products, deposited in pollen coat, can be readily extracted by pollen wash and were shown to be responsible for pollen autofluorescence. The activity of the recombinant enzyme produced in bacteria was assayed with various hydroxycinnamoyl-CoA esters and polyamines as donor and acceptor substrates, respectively. Feruloyl-CoA and spermidine proved the best substrates, and the enzyme has therefore been named spermidine hydroxycinnamoyl transferase (SHT). A methyltransferase gene (At1g67990) which co-regulated with SHT during flower development, was shown to be involved in the O-methylation of spermidine conjugates by analyzing the consequences of its repression in RNAi plants and by characterizing the methylation activity of the recombinant enzyme.

Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth.

In Arabidopsis thaliana, silencing of hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), a lignin biosynthetic gene, results in a strong reduction of plant growth. We show that, in HCT-silenced plants, lignin synthesis repression leads to the redirection of the metabolic flux into flavonoids through chalcone synthase activity. Several flavonol glycosides and acylated anthocyanin were shown to accumulate in higher amounts in silenced plants. By contrast, sinapoylmalate levels were barely affected, suggesting that the synthesis of that phenylpropanoid compound might be HCT-independent. The growth phenotype of HCT-silenced plants was shown to be controlled by light and to depend on chalcone synthase expression. Histochemical analysis of silenced stem tissues demonstrated altered tracheary elements. The level of plant growth reduction of HCT-deficient plants was correlated with the inhibition of auxin transport. Suppression of flavonoid accumulation by chalcone synthase repression in HCT-deficient plants restored normal auxin transport and wild-type plant growth. By contrast, the lignin structure of the plants simultaneously repressed for HCT and chalcone synthase remained as severely altered as in HCT-silenced plants, with a large predominance of nonmethoxylated H units. These data demonstrate that the reduced size phenotype of HCT-silenced plants is not due to the alteration of lignin synthesis but to flavonoid accumulation.

Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth.

The Agrobacterium T-DNA oncogene 6b induces tumors and modifies the growth of transgenic plants by an unknown mechanism. We have investigated changes in roots of tobacco seedlings that express a dexamethasone-inducible T-6b (dex-T-6b) gene. On induction medium with sucrose, intact or isolated dex-T-6b roots accumulated sucrose, glucose, and fructose and changed their growth, contrary to noninduced roots. Root fragments bridging agar blocks with or without sucrose accumulated sugars at the site of sucrose uptake, resulting in local growth. Induced root fragments showed enhanced uptake of 14C-labeled sucrose, glucose, and fructose. When seedlings were placed on sucrose-free induction medium, sugar levels strongly decreased in roots and increased in cotyledons. Collectively, these results demonstrate that 6b stimulates sugar uptake and retention with drastic effects on growth. Apart from sugars, phenolic compounds also have been found to accumulate in 6b tissues and have been proposed earlier to play a role in 6b-induced growth. Induced dex-T-6b roots accumulated high levels of 5-caffeoylquinic acid (or chlorogenic acid [CGA]), but only under conditions where endogenous sugars increased. Inhibition of phenylalanine ammonia-lyase with the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP) abolished CGA accumulation without modifying sugar accumulation or affecting the 6b phenotype. We conclude that the absorption, retention, and abnormal accumulation of sugars are essential factors in 6b-induced growth changes, whereas phenylpropanoids only marginally contribute to the 6b seedling phenotype.

A pathogen-inducible patatin-like lipid acyl hydrolase facilitates fungal and bacterial host colonization in Arabidopsis.

Genes and proteins related to patatin, the major storage protein of potato tubers, have been identified in many plant species and shown to be induced by a variety of environmental stresses. The Arabidopsis patatin-like gene family (PLPs) comprises nine members, two of which (PLP2 and PLP7) are strongly induced in leaves challenged with fungal and bacterial pathogens. Here we show that accumulation of PLP2 protein in response to Botrytis cinerea or Pseudomonas syringae pv. tomato (avrRpt2) is dependent on jasmonic acid and ethylene signaling, but is not dependent on salicylic acid. Expression of a PLP2-green fluorescent protein (GFP) fusion protein and analysis of recombinant PLP2 indicates that PLP2 encodes a cytoplasmic lipid acyl hydrolase with wide substrate specificity. Transgenic plants with altered levels of PLP2 protein were generated and assayed for pathogen resistance. Plants silenced for PLP2 expression displayed enhanced resistance to B. cinerea, whereas plants overexpressing PLP2 were much more sensitive to this necrotrophic fungus. We also established a positive correlation between the level of PLP2 expression in transgenic plants and cell death or damage in response to paraquat treatment or infection by avirulent P. syringae. Interestingly, repression of PLP2 expression increased resistance to avirulent bacteria, while PLP2-overexpressing plants multiplied avirulent bacteria close to the titers reached by virulent bacteria. Collectively, the data indicate that PLP2-encoded lipolytic activity can be exploited by pathogens with different lifestyles to facilitate host colonization. In particular PLP2 potentiates plant cell death inflicted by Botrytis and reduces the efficiency of the hypersensitive response in restricting the multiplication of avirulent bacteria. Both effects are possibly mediated by providing fatty acid precursors of bioactive oxylipins.

Metabolic reprogramming in plant innate immunity: the contributions of phenylpropanoid and oxylipin pathways.

In their environment, plants interact with a multitude of living organisms and have to cope with a large variety of aggressions of biotic or abiotic origin. To survive, plants have acquired, during evolution, complex mechanisms to detect their aggressors and defend themselves. Receptors and signaling pathways that are involved in such interactions with the environment are just beginning to be uncovered. What has been known for several decades is the extraordinary variety of chemical compounds the plants are capable to synthesize, and many of these products are implicated in defense responses. The number of natural products occurring in plants may be estimated in the range of hundreds of thousands, but only a fraction have been fully characterized. Despite the great importance of these metabolites for plant and also for human health, our knowledge about their biosynthetic pathways and functions is still fragmentary. Recent progress has been made particularly for phenylpropanoid and oxylipin metabolism, which are emphasized in this review. Both pathways are involved in plant resistance at several levels: by providing building units of physical barriers against pathogen invasion, by synthesizing an array of antibiotic compounds, and by producing signals implicated in the mounting of plant resistance.

Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis.

The hydroxyl group in the 3-position of the phenylpropanoid compounds is introduced at the level of coumarate shikimate/quinate esters, whose synthesis implicates an acyltransferase activity. Specific antibodies raised against the recombinant tobacco (Nicotiana tabacum) acyltransferase revealed the accumulation of the enzyme in stem vascular tissues of tobacco, in accordance with a putative role in lignification. For functional analysis, the acyltransferase gene was silenced in Arabidopsis thaliana and N. benthamiana by RNA-mediated posttranscriptional gene silencing. In Arabidopsis, gene silencing resulted in a dwarf phenotype and changes in lignin composition as indicated by histochemical staining. An in-depth study of silenced N. benthamiana plants by immunological, histochemical, and chemical methods revealed the impact of acyltransferase silencing on soluble phenylpropanoids and lignin content and composition. In particular, a decrease in syringyl units and an increase in p-hydroxyphenyl units were recorded. Enzyme immunolocalization by confocal microscopy showed a correlation between enzyme accumulation levels and lignin composition in vascular cells. These results demonstrate the function of the acyltransferase in phenylpropanoid biosynthesis.

Lignification and structural biomass production in tobacco with suppressed caffeic/5-hydroxy ferulic acid-O-methyl transferase activity under ambient and elevated CO concentrations.

To investigate the relationship between growth, biomass partitioning and lignification we used tobacco (Nicotiana tabacum) in which O-methyl transferase (OMT) activity, an enzyme involved in the pathway of sinapyl alcohol formation for lignin synthesis, was suppressed by antisense transformation. To modulate growth, controls and transformed tobacco plants were grown under ambient (approximately 380 p.p.m) or elevated CO(2) (700 p.p.m), respectively. Lignin concentrations and composition were determined with spectrophotometric methods (thioglycolate and acetyl bromide) and Fourier transform infrared (FTIR) spectroscopy, respectively. A comparison of the thioglycolate and acetylbromide method suggested that the thioglycolate method was sensitive to changes in the syringyl/guaiacyl (S/G)-ratio in lignin and therefore not suitable for quantification in tissues with altered lignin composition. Growth under elevated CO(2) increased leaf and stem biomass of both genotypes by 40 and 20%, respectively, compared with ambient CO(2) and had no effect on root biomass. OMT suppression did not affect lignin concentrations in the leaves but caused a shift in biomass partitioning from the structural to the non-structural fraction. Elevated CO(2) caused a shift towards production of structural compounds resulting in decreased foliar lignin concentrations and indicating that the lignin/structural mass ratio is flexible in leaves. By contrast, the lignin concentrations of stems were unaffected by elevated CO(2) or OMT suppression and increased about three-fold from the apex to the base. Antisense-OMT plants produced more stem biomass than controls but showed no changes of the relative partitioning of biomass to the different pools. This indicates that the metabolic control of carbon fluxes to the production of structural versus non-structural fractions is tighter in stems than in leaves. FTIR spectroscopy indicated a relative increase in guaiacyl- as compared with syringyl-units in antisense-OMT tobacco, which was more pronounced under elevated as compared with ambient CO(2). Since there was no evidence for decreased lignin concentrations in the antisense-OMT plants but increased biomass formation we suggest that less methylated lignins are 'cheaper' in biosynthetic carbon and energy demand and, thus, may enable plants to allocate increased resources to growth.

Structure of the tobacco caffeic acid O-methyltransferase (COMT) II gene: identification of promoter sequences involved in gene inducibility by various stimuli.

The tobacco gene encoding caffeic acid-O-methyltransferase of class II (COMT II) was isolated, including a 1.7 kb 5'-flanking region. Sequence motifs were identified in COMT II gene promoter which are present in many genes of the phenylpropanoid pathway or in stress-inducible pathogenesis-related (PR) genes. A 1215 bp COMT II promoter fragment was transcriptionally fused to the GUS coding region and its activity pattern studied by stable expression of the fusion gene in tobacco. Transgenic lines were analysed for GUS and OMT activities upon infection, UV irradiation, wounding and treatment by various signalling compounds. The promoter proved responsive to various biotic and abiotic elicitors and to infection by avirulent and virulent pathogens. During the course of the hypersensitive reaction of tobacco to TMV two peaks were detected, an early one induced by the inoculation process and a second one at the onset of lesion formation. Parallel changes were observed between GUS activity that reflected the activity of the COMT II promoter fragment and COMT II activity that mirrored expression of the endogenous COMT II gene, indicating that COMT II pattern of expression is established at the transcriptional level. Various promoter fragments were fused to the GUS gene and revealed that gene induction by MeJA or UV and by TMV or wounding requires different sequences included in a 74 bp fragment. When the 74 bp sequence was multimerized and inserted ahead of the CaMV 35S RNA minimal promoter, one construct was shown to be capable of driving expression of the reporter gene around the TMV-infected sites in transgenic tobacco plants.

Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism.

A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.

Spatio-temporal expression of patatin-like lipid acyl hydrolases and accumulation of jasmonates in elicitor-treated tobacco leaves are not affected by endogenous levels of salicylic acid.

We have previously isolated three tobacco genes (NtPat) encoding patatin-like proteins, getting rapidly induced during the hypersensitive response (HR) to tobacco mosaic virus, in advance to jasmonate accumulation. NtPAT enzymes are lipid acyl hydrolases that display high phospholipase A2 (PLA2) activity and may mobilize fatty acid precursors of oxylipins. Here, we performed a detailed study of NtPat gene regulation under various biotic and abiotic stresses. PLA2 activity was poorly induced in response to drought, wounding, reactive oxygen intermediates, salicylic acid (SA) or methyl-jasmonate (MJ) whereas the ethylene (ET) precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), provoked a moderate induction. In contrast, PLA2 activity was strongly induced when ACC was combined with MJ, and in response to the bacterium Erwinia carotovora or to the fungus Botrytis cinerea, as well as to treatment with beta-megaspermin, a cell death-inducing protein elicitor. A simplified system based on the infiltration of beta-megaspermin into leaves was used to dissect the spatio-temporal activation of PLA2 activity with regards to the accumulation of jasmonates and to the influence of endogenous SA. NtPat-encoded PLA2 activity was rapidly induced in the infiltrated zone before the appearance of cell death and with some delay in the surrounding living cells. A massive accumulation of 12-oxo-phytodienoic and jasmonic acids occurred in the elicitor-infiltrated zone, but only low levels were detectable outside this area. A similar picture was found in SA-deficient plants, showing that in tobacco, accumulation of jasmonates is not affected by the concomitant HR-induced build-up of endogenous SA. Finally, ET-insensitive plants showed a weakened induction of PLA2 activity outside the elicitor-infiltrated tissue.

Simultaneous suppression of multiple genes by single transgenes. Down-regulation of three unrelated lignin biosynthetic genes in tobacco.

Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.