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Δ3,Δ2-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the β-oxidation cycle. Three genes encoding Δ3,Δ2-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Δeci1 mutant to degrade 10 Z-heptadecenoic acid, demonstrating Δ3,Δ2-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Δ3,Δ2-enoyl CoA isomerase, indicating that evolution of cytosolic Δ3,Δ2-enoyl CoA isomerases is restricted to the plant kingdom. [ABSTRACT FROM AUTHOR]

The degradation of fatty acids in plants occurs primarily in the peroxisomes through the β-oxidation cycle. Enzymes that are involved in various aspects of β-oxidation have been identified recently and shown to act biochemically on a diversity of fatty acids and derivatives. Analysis of several mutants has revealed essential roles for β-oxidation in the breakdown of reserve triacylglycerols, seed development, seed germination and post-germinative growth before the establishment of photosynthesis. β-Oxidation has also a considerable importance during the vegetative and reproductive growth phases, and plays a role in plant responses to stress, particularly in the synthesis of jasmonic acid. [Copyright &y& Elsevier]

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high β-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the β-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or under-expressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core β-oxidation cycle. [ABSTRACT FROM AUTHOR]

Degradation of unsaturated fatty acids through the peroxisomal β-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core β-oxidation cycle. The auxiliary enzyme Δ3,5,Δ2,4-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) Δ3,5,Δ2,4-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have Δ3,5,Δ2,4-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the β-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the Δ3,5,Δ2,4-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has Δ3,5,Δ2,4-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes. [ABSTRACT FROM AUTHOR]

The clove (Syzygium aromaticum) is an important tropical spice crop in global trade. Evolving environmental pressures necessitate modern characterization and selection techniques that are currently inaccessible to clove growers owing to the scarcity of genomic and genetic information. Here, we present a 370-Mb high-quality chromosome-scale genome assembly for clove. Comparative genomic analysis between S. aromaticum and Eucalyptus grandis—both species of the Myrtaceae family—reveals good genome structure conservation and intrachromosomal rearrangements on seven of the eleven chromosomes. We report genes that belong to families involved in the biosynthesis of eugenol, the major bioactive component of clove products. On the basis of our transcriptomic and metabolomic findings, we propose a hypothetical scenario in which eugenol acetate plays a key role in high eugenol accumulation in clove leaves and buds. The clove genome is a new contribution to omics resources for the Myrtaceae family and an important tool for clove research. A newly assembled clove genome is compared with E. grandis to investigate genome evolution between the two genera of the Myrtaceae family, and putative genes involved in the biosynthesis of eugenol are identified through transcriptomics and metabolomics. [ABSTRACT FROM AUTHOR]

Background: Anatabine, although being one of four major tobacco alkaloids, is never accumulated in high quantity in any of the naturally occurring species from the Nicotiana genus. Previous studies therefore focused on transgenic approaches to synthetize anatabine, most notably by generating transgenic lines with suppressed putrescine methyltransferase (PMT) activity. This led to promising results, but the global gene expression of plants with such distinct metabolism has not been analyzed. In the current study, we describe how these plants respond to topping and the downstream effects on alkaloid biosynthesis. Results: The surge in anatabine accumulation in PMT transgenic lines after topping treatment and its effects on gene expression changes were analyzed. The results revealed increases in expression of isoflavone reductase-like (A622) and berberine bridge-like enzymes (BBLs) oxidoreductase genes, previously shown to be crucial for the final steps of nicotine biosynthesis. We also observed significantly higher methylputrescine oxidase (MPO) expression in all plants subjected to topping treatment. In order to investigate if MPO suppression would have the same effects as that of PMT, we generated transgenic plants. These plants with suppressed MPO expression showed an almost complete drop in leaf nicotine content, whereas leaf anatabine was observed to increase by a factor of ~ 1.6X. Conclusion: Our results are the first concrete evidence that suppression of MPO leads to decreased nicotine in favor of anatabine in tobacco roots and that this anatabine is successfully transported to tobacco leaves. Alkaloid transport in plants remains to be investigated to higher detail due to high variation of its efficiency among Nicotiana species and varieties of tobacco. Our research adds important step to better understand pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine. [ABSTRACT FROM AUTHOR]

Phytophthora nicotianae and Ralstonia solanacearum are two of the most important pathogens affecting tobacco worldwide. Greater insight regarding genetic systems controlling resistance to these two soilborne pathogens, as well as identification of DNA markers associated with genomic regions controlling this resistance, could aid in variety development. An evaluation of 50 historical tobacco lines revealed a high positive correlation between resistances to the two pathogens, preliminarily suggesting that some genomic regions may confer resistance to both pathogens. A quantitative trait loci (QTL) mapping experiment designed to investigate the genetic control of soilborne disease resistance of highly resistant 'K346' tobacco identified four QTL significantly associated with resistance to P. nicotianae (explaining 60.0% of the observed phenotypic variation) and three QTL to be associated with R. solanacearum resistance (explaining 50.3% of the observed variation). The two QTL with the largest effect on Phytophthora resistance were also found to be the QTL with the greatest effects on resistance to Ralstonia. This finding partially explains previously observed associations between resistances to these two pathogens among U.S. current cultivars and within breeding populations. Further study is needed to determine whether these relationships are due to the same genes (i.e., pleiotropy) or favorable coupling-phase linkages that have been established over time. [ABSTRACT FROM AUTHOR]

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal β-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward β-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through β-oxidation than the expression profile of genes involved in lipid metabolism. [ABSTRACT FROM AUTHOR]

Pyridine alkaloids produced in tobacco can react with nitrosating agents such as nitrite to form tobacco-specific nitrosamines (TSNA), which are among the most notable toxicants present in tobacco smoke. The market type known as burley tobacco is particularly susceptible to TSNA formation because its corresponding cultivars exhibit a nitrogen-use-deficiency phenotype which results in high accumulation of nitrate, which, in turn, is converted to nitrite by leaf surface microbes. We have previously shown that expression of a constitutively activated nitrate reductase (NR) enzyme dramatically decreases leaf nitrate levels in burley tobacco, resulting in substantial TSNA reductions without altering the alkaloid profile. Here, we show that plants expressing a constitutively active NR construct, designated 35S:S523D-NR, display an early-flowering phenotype that is also associated with a substantial reduction in plant biomass. We hypothesized that crossing 35S:S523D-NR tobaccos with burley cultivars that flower later than normal would help mitigate the undesirable early-flowering/reduced-biomass traits while maintaining the desirable low-nitrate/TSNA phenotype. To test this, 35S:S523D-NR plants were crossed with two late-flowering cultivars, NC 775 and NC 645WZ. In both cases, the plant biomass at harvest was restored to levels similar to those in the original cultivar used for transformation while the low-nitrate/TSNA trait was maintained. Interestingly, the mechanism by which yield was restored differed markedly between the two crosses. Biomass restoration in F1 hybrids using NC 645WZ as a parent was associated with delayed flowering, as originally hypothesized. Unexpectedly, however, crosses with NC 775 displayed enhanced biomass despite maintaining the early-flowering trait of the 35S:S523D-NR parent. [ABSTRACT FROM AUTHOR]

In tobacco, the heavy metal P1B-ATPases HMA4.1 and HMA4.2 function in root-to-shoot zinc and cadmium transport. We present greenhouse and field data that dissect the possibilities to impact the two homeologous genes in order to define the best strategy for leaf cadmium reduction. In a first step, both genes were silenced using an RNAi approach leading to >90% reduction of leaf cadmium content. To modulate HMA4 function more precisely, mutant HMA4.1 and HMA4.2 alleles of a Targeting Induced Local Lesions IN Genomes (TILLING) population were combined. As observed with RNAi plants, knockout of both homeologs decreased cadmium root-to-shoot transfer by >90%. Analysis of plants with segregating null and wild-type alleles of both homeologs showed that one functional HMA4 allele is sufficient to maintain wild-type cadmium levels. Plant development was affected in HMA4 RNAi and double knockout plants that included retarded growth, necrotic lesions, altered leaf morphology and increased water content. The combination of complete functional loss (nonsense mutation) in one homeologous HMA4 gene and the functional reduction in the other HMA4 gene (missense mutation) is proposed as strategy to limit cadmium leaf accumulation without developmental effects. [ABSTRACT FROM AUTHOR]

In the tobacco plant, nicotine N -demethylase enzymes (NND) belonging to the cytochrome P450 family catalyse the conversion of nicotine to nornicotine, the precursor of the carcinogenic tobacco-specific N -nitrosamine, N -nitrosonornicotine. To date three demethylase genes, namely CYP82E4 , CYP82E5 and CYP82E10 , have been shown to be involved in this process, while the related CYP82E2 and CYP82E3 genes are not functional. We have identified a further gene named CYP82E21 encoding a putative nicotine N -demethylase closely related to the CYP82E genes. The CYP82E21 gene was found in all Nicotiana tabacum cultivars analysed and originates from the tobacco ancestor Nicotiana tomentosiformis . We show that, in contrast to all other previously characterized NND genes, CYP82E21 is not expressed in green or senescent leaves, but in flowers, more specifically in ovaries. The nicotine N -demethylase activity of CYP82E21 was confirmed by ectopic expression of the coding sequence in a tobacco line lacking functional CYP82E4 , CYP82E5 and CYP82E10 genes, resulting in an eightfold increase of nicotine demethylation compared to the control plants. Furthermore, nornicotine formation can be reduced in ovaries by introducing a CYP82E21 -specific RNAi construct. Together, our results demonstrate that the CYP82E21 gene encodes a functional ovary-specific nicotine N -demethylase. [ABSTRACT FROM AUTHOR]

Burley tobaccos ( Nicotiana tabacum) display a nitrogen-use-deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco-specific nitrosamines ( TSNAs). Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( NNK) and N-nitrosonornicotine ( NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen-assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase ( NR) dramatically decreased free nitrate levels in the leaves. Field-grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well-documented animal carcinogens found in tobacco products. [ABSTRACT FROM AUTHOR]

Leaves of tobacco ( Nicotiana tabacum) are covered with glandular trichomes that produce sucrose esters and diterpenoids in varying quantities, depending on cultivar type. The bicyclic diterpene Z-abienol is the major labdanoid present in some oriental tobacco cultivars, where it constitutes a precursor of important flavours and aromas. We describe here the identification and characterization of two genes governing the biosynthesis of Z-abienol in N. tabacum. As for other angiosperm labdanoid diterpenes, the biosynthesis of Z-abienol proceeds in two steps. NtCPS2 encodes a class-II terpene synthase that synthesizes 8-hydroxy-copalyl diphosphate, and NtABS encodes a kaurene synthase-like (KSL) protein that uses 8-hydroxy-copalyl diphosphate to produce Z-abienol. Phylogenetic analysis indicates that NtABS belongs to a distinct clade of KSL proteins that comprises the recently identified tomato ( Solanum habrochaites) santalene and bergamotene synthase. RT-PCR results show that both genes are preferentially expressed in trichomes. Moreover, microscopy of NtCPS2 promoter-GUS fusion transgenics demonstrated a high specificity of expression to trichome glandular cells. Ectopic expression of both genes, but not of either one alone, driven by a trichome-specific promoter in transgenic Nicotiana sylvestris conferred Z-abienol formation to this species, which does not normally produce it. Furthermore, sequence analysis of over 100 tobacco cultivars revealed polymorphisms in NtCPS2 that lead to a prematurely truncated protein in cultivars lacking Z-abienol, thus establishing NtCPS2 as a major gene controlling Z-abienol biosynthesis in tobacco. These results offer new perspectives for tobacco breeding and the metabolic engineering of labdanoid diterpenes, as well as for structure-function relationship studies of terpene synthases. [ABSTRACT FROM AUTHOR]

Together in silico and genetic mining approaches have recently designated the CYP98 family of plant cytochromes P450 as the family of enzymes that catalyzes the meta-hydroxylation step in the phenylpropanoid pathway. This meta-hydroxylation is not catalyzed on the free p-coumaric acid as anticipated, but on its conjugates with shikimic, quinic, or phenyllactic acids. While all CYP98s have in common phenol meta-hydroxylase activity, p-coumaroylshikimate remains their preferred substrate. High expression of CYP98s is detected in lignifying tissues in stems, roots, and siliques. The CYP98A3 gene disruption in Arabidopsis thaliana leads to a drastic inhibition of lignin synthesis, cell growth, and plant development. The meta-hydroxylation of phenolic precursors is thus essential for higher plant development. Isolation of coding sequences belonging to the CYP98 family from basil, wheat, and extensive functional analysis of the recombinant enzymes, together with CYP98s from other plant taxa, helps shedding some light on mechanisms of P450s evolution. Most importantly, the occurrence of the meta-hydroxylation on esters of shikimic or quinic acids introduces a new biochemical regulation mechanism in the phenylpropanoid pathway. [ABSTRACT FROM AUTHOR]

The pleiotropic effects of zinc deficiency on ion homeostasis have already been described in several plants. Tobacco (Nicotiana tabacum) heavy metal ATPases HMA4.1 and HMA4.2 are involved in zinc and cadmium root-to-shoot translocation. In previous research, we have shown that N. tabacum HMA4 RNAi plants and HMA4 double-nonsense mutants exhibit strongly reduced zinc and cadmium levels in leaves as well as stunted growth. In this study, the ionome and transcriptome of these lines were investigated to better characterize the effect of reduced zinc levels and to understand the impaired growth phenotype. We found that, under standard greenhouse fertilization rates, these lines accumulated up to 4- to 6-fold more phosphorus, iron, manganese, and copper than their respective controls. Under field conditions, HMA4 double-mutant plants also exhibited similar accumulation phenotypes, albeit to a lower extent. In both HMA4 RNAi plants and HMA4 mutants, transcription analysis showed a local zinc-deficiency response in leaves as well as an FIT1-mediated iron-deficiency response in roots, likely contributing to iron and manganese uptake at the root level. A phosphate-starvation response involving HHO2 was also observed in HMA4 -impaired plant leaves. The high level of phosphorus observed in HMA4 -impaired plants is correlated with leaf swelling and necrosis. The upregulation of aquaporin genes is in line with cellular water influx and the observed leaf swelling phenotype. These results highlight the involvement of HMA4 in zinc homeostasis and related regulatory processes that balance the micro- and macroelements in above-ground organs. N. tabacum plants lacking HMA4 transport activity are zinc-deficient in the shoots and accumulate high levels of phosphorous, iron, manganese, and copper, accompanied by gene expression changes. [Display omitted] • Tobacco lacking HMA4 exhibits reduced Zn and increased P, Fe, Mn, and Cu content. • Accumulation of P and lack of Zn impact the growth of tobacco plants. • Nutrient accumulation was confirmed in field experiments. • Diverse transport mechanisms were upregulated in HMA4 -deficient plants. • HMA4 -deficient tobacco plants can be a model for dissecting Zn-deficiency response. [ABSTRACT FROM AUTHOR]

In this study, we determined the pyridine alkaloid content (nicotine, nornicotine, anabasine, anatabine, cotinine, and myosmine) of 58 species and 2 subspecies of the Nicotiana genus by ultra-high-performance liquid chromatography coupled with mass spectrometry. We observed clear correlation between Noctiflorae and Suaveolentes sections and their above average accumulation of anabasine in the genus. In addition, the results demonstrated the presence of not only trace amounts but quantifiable levels of myosmine, an alkaloid previously detected in only minute quantities, in the leaves and roots of 16 species. In this study, analysis of gene expression of 58 species and 2 subspecies from the Nicotiana genus by mRNA sequencing was performed for the first time. Sequencing reads were mapped against annotated genes of a Nicotiana tabacum reference genome and expression values were subsequently calculated. Hierarchical clustering of alkaloid biosynthesis pathway genes and alkaloid content composition revealed patterns clearly segregating Nicotiana sections. Correlation of gene expression with alkaloid accumulation phenotypes was evident, including low putrescine methyltransferase expression for all species in the Suaveolentes section or clear correlation of nicotine demethylase with conversion rates of nicotine to nornicotine in the majority of species. Multiple additional correlations between alkaloid accumulation and gene expression values were identified, which makes this study an important fundament toward future scientific exploration of the Nicotiana genus. Image 1 • Pyridine alkaloid content in Nicotiana genus correlates with sectional classification. • Myosmine was detected in 16 Nicotiana species. • Noctiflorae and Suaveolentes sections accumulated above average levels of anabasine. • Clustering of Nicotiana gene expression reflected sectional classification. • Correlation of gene expression with alkaloid accumulation was evident. [ABSTRACT FROM AUTHOR]

Multiple factors act simultaneously on plants to establish complex interaction networks involving nutrients, elicitors and metabolites. Metabolomics offers a better understanding of complex biological systems, but evaluating the simultaneous impact of different parameters on metabolic pathways that have many components is a challenging task. We therefore developed a novel approach that combines experimental design, untargeted metabolic profiling based on multiple chromatography systems and ionization modes, and multiblock data analysis, facilitating the systematic analysis of metabolic changes in plants caused by different factors acting at the same time. Using this method, target geraniol compounds produced in transgenic tobacco cell cultures were grouped into clusters based on their response to different factors. We hypothesized that our novel approach may provide more robust data for process optimization in plant cell cultures producing any target secondary metabolite, based on the simultaneous exploration of multiple factors rather than varying one factor each time. The suitability of our approach was verified by confirming several previously reported examples of elicitor-metabolite crosstalk. However, unravelling all factor-metabolite networks remains challenging because it requires the identification of all biochemically significant metabolites in the metabolomics dataset. [ABSTRACT FROM AUTHOR]