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1.
BMC Genomics ; 25(1): 776, 2024 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-39123103

RESUMEN

BACKGROUND: Transcription factors (TFs) of plant-specific SHORT INTERNODES (SHI) family play a significant role in regulating development and metabolism in plants. In Artemisia annua, various TFs from different families have been discovered to regulate the accumulation of artemisinin. However, specific members of the SHI family in A. annua (AaSHIs) have not been identified to regulate the biosynthesis of artemisinin. RESULTS: We found five AaSHI genes (AaSHI1 to AaSHI5) in the A. annua genome. The expression levels of AaSHI1, AaSHI2, AaSHI3 and AaSHI4 genes were higher in trichomes and young leaves, also induced by light and decreased when the plants were subjected to dark treatment. The expression pattern of these four AaSHI genes was consistent with the expression pattern of four structural genes of artemisinin biosynthesis and their specific regulatory factors. Dual-luciferase reporter assays, yeast one-hybrid assays, and transient transformation in A. annua provided the evidence that AaSHI1 could directly bind to the promoters of structural genes AaADS and AaCYP71AV1, and positively regulate their expressions. This study has presented candidate genes, with AaSHI1 in particular, that can be considered for the metabolic engineering of artemisinin biosynthesis in A. annua. CONCLUSIONS: Overall, a genome-wide analysis of the AaSHI TF family of A. annua was conducted. Five AaSHIs were identified in A. annua genome. Among the identified AaSHIs, AaSHI1 was found to be localized to the nucleus and activate the expression of structural genes of artemisinin biosynthesis including AaADS and AaCYP71AV1. These results indicated that AaSHI1 had positive roles in modulating artemisinin biosynthesis, providing candidate genes for obtaining high-quality new A. annua germplasms.


Asunto(s)
Artemisia annua , Artemisininas , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Factores de Transcripción , Artemisia annua/genética , Artemisia annua/metabolismo , Artemisininas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Filogenia
2.
Commun Biol ; 7(1): 102, 2024 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-38267515

RESUMEN

Serine metabolism is involved in various biological processes. Here we investigate primary functions of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH encoding 3-phosphoglycerate dehydrogenase in this pathway. Growth phenotypes indicate that serine from the phosphorylated pathway in the dark is crucial for thallus growth. Sperm development requires serine from the phosphorylated pathway, while egg formation does not. Functional MpPGDH in the maternal genome is necessary for embryo and sporophyte development. Under high CO2 where the glycolate pathway of serine biosynthesis is inhibited, suppressed thallus growth of the mutants is not fully recovered by exogenously-supplemented serine, suggesting the importance of serine homeostasis involving the phosphorylated and glycolate pathways. Metabolomic phenotypes indicate that the phosphorylated pathway mainly influences the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lipid metabolism. These results indicate the importance of the phosphorylated pathway of serine biosynthesis in the dark, in the development of sperm, embryo, and sporophyte, and metabolism in M. polymorpha.


Asunto(s)
Marchantia , Serina , Marchantia/genética , Semillas , Espermatozoides , Glicolatos
3.
J Exp Bot ; 75(6): 1741-1753, 2024 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-37647764

RESUMEN

Tobacco (Nicotiana tabacum L.) is a widely cultivated crop of the genus Nicotiana. Due to the highly addictive nature of tobacco products, tobacco smoking remains the leading cause of preventable death and disease. There is therefore a critical need to develop tobacco varieties with reduced or non-addictive nicotine levels. Nicotine and related pyridine alkaloids biosynthesized in the roots of tobacco plants are transported to the leaves, where they are stored in vacuoles as a defense against predators. Jasmonate, a defense-related plant hormone, plays a crucial signaling role in activating transcriptional regulators that coordinate the expression of downstream metabolic and transport genes involved in nicotine production. In recent years, substantial progress has been made in molecular and genomics research, revealing many metabolic and regulatory genes involved in nicotine biosynthesis. These advances have enabled us to develop tobacco plants with low or ultra-low nicotine levels through various methodologies, such as mutational breeding, genetic engineering, and genome editing. We review the recent progress on genetic manipulation of nicotine production in tobacco, which serves as an excellent example of plant metabolic engineering with profound social implications.


Asunto(s)
Alcaloides , Nicotiana , Nicotiana/genética , Nicotina , Regulación de la Expresión Génica de las Plantas , Fitomejoramiento , Proteínas de Plantas/metabolismo
4.
PNAS Nexus ; 2(10): pgad326, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37920550

RESUMEN

Plants produce specialized metabolites with defensive properties that are often synthesized through the coordinated regulation of metabolic genes by transcription factors in various biological contexts. In this study, we investigated the regulatory function of the transcription factor PhERF1 from petunia (Petunia hybrida), which belongs to a small group of ETHYLENE RESPONSE FACTOR (ERF) family members that regulate the biosynthesis of bioactive alkaloids and terpenoids in various plant lineages. We examined the effects of transiently overexpressing PhERF1 in petunia leaves on the transcriptome and metabolome, demonstrating the production of a class of specialized steroids, petuniolides, and petuniasterones in these leaves. We also observed the activation of many metabolic genes, including those involved in sterol biosynthesis, as well as clustered genes that encode new metabolic enzymes, such as cytochrome P450 oxidoreductases, 2-oxoglutarate-dependent dioxygenases, and BAHD acyltransferases. Furthermore, we determined that PhERF1 transcriptionally induces downstream metabolic genes by recognizing specific cis-regulatory elements in their promoters. This study highlights the potential of evolutionarily conserved transcriptional regulators to induce the production of specialized products through transcriptional reprogramming.

5.
Plant Biotechnol (Tokyo) ; 40(1): 71-76, 2023 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-38213915

RESUMEN

A group of anti-nutritional specialized metabolites called steroidal glycoalkaloids (SGAs) are produced in Solanum species such as tomato, potato, and eggplant. The transcription factor JASMONATE-RESPONSIVE ETHYLENE RESPONSE FACTOR 4 (JRE4) regulates many SGA biosynthesis genes in tomato and potato. Here we report that the expression of a cluster of genes encoding nitrate transporter 1/peptide transporter family (NPF) members is downregulated in the jre4-1 loss-of-function tomato mutant, which has a low-SGA phenotype compared to the wild type. NPFs are a large family of plant membrane transporters that transport a wide range of substrates, including specialized metabolites. We found that the JRE4-regulated NPF genes are induced by the defense-related phytohormone jasmonate. Conversely, jasmonate-mediated induction of gene expression was attenuated by ethylene treatment of the leaves. The co-regulation of the NPF genes with SGA biosynthesis genes by JRE4 suggests that NPF transporters are involved in the SGA pathway.

6.
Plants (Basel) ; 11(23)2022 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-36501375

RESUMEN

Steroidal glycoalkaloids (SGAs) are a class of cholesterol-derived anti-nutritional defense compound that are produced in species of the genus Solanum, such as tomato (S. lycopersicum), potato (S. tuberosum), and eggplant (S. melongena). However, the regulation of defense-related metabolites in eggplant remains underexplored. In tomato and potato, the JASMONATE-RESPONSIVE ETHYLENE RESPONSE FACTOR 4 (JRE4) transcription factor positively regulates a large number of genes involved in SGA biosynthesis. Here, we report that the overexpression of eggplant JRE4 (SmJRE4) induces numerous metabolic genes involved in SGA biosynthesis in leaves. We demonstrate the jasmonate-dependent induction of SmJRE4 and its downstream metabolic genes and show that ethylene treatment attenuates this induction. Our findings thus provide molecular insights into SGA biosynthesis and its regulation in this major crop.

7.
Plant J ; 111(6): 1768-1779, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35883194

RESUMEN

In tobacco, the homologous ETHYLENE RESPONSE FACTOR (ERF) transcription factors ERF199 and ERF189 coordinate the transcription of multiple metabolic genes involved in nicotine biosynthesis. Natural alleles at the NIC1 and NIC2 loci greatly affect alkaloid accumulation and overlap with ERF199 and ERF189 in the tobacco genome, respectively. In this study, we identified several low-nicotine tobacco varieties lacking ERF199 or ERF189 from a tobacco germplasm collection. We characterized the sequence of these new nic1 and nic2 alleles, as well as the previously defined alleles nic1-1 and nic2-1. Moreover, we examined the influence of different nic alleles on alkaloid contents and expression levels of genes related to nicotine biosynthesis. We also demonstrated that the deletion of a distal genomic region attenuates ERF199 expression, resulting in a moderately negative effect on the alkaloid phenotype. Our study provides new insights into the regulation of nicotine biosynthesis and novel genetic resources to breed low-nicotine tobacco.


Asunto(s)
Nicotiana , Nicotina , Ciclopentanos/metabolismo , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Genes Reguladores , Nicotina/genética , Nicotina/metabolismo , Oxilipinas/metabolismo , Fenotipo , Fitomejoramiento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
8.
Plant Biotechnol (Tokyo) ; 39(4): 421-425, 2022 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-37283616

RESUMEN

RING membrane-anchor (RMA) E3 ubiquitin ligases are involved in endoplasmic reticulum (ER)-associated protein degradation, which mediates the regulated destruction of ER-resident enzymes in various organisms. We determined that the transcription factor JASMONATE-RESPONSIVE ETHYLENE RESPONSE FACTOR 4 (JRE4) co-regulates the expression of the RMA-type ligase gene SlRMA1, but not its homolog SlRMA2, with steroidal glycoalkaloid biosynthesis genes in tomato, perhaps to prevent the overaccumulation of these metabolites.

9.
Plant Mol Biol ; 109(4-5): 401-411, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34114167

RESUMEN

KEY MESSAGE: A number of mutational changes in transcriptional regulators of defense metabolism have occurred during plant domestication and improvement. Plant domestication and improvement entail genetic changes that underlie divergence in development and metabolism, providing a tremendous model of biological evolution. Plant metabolism produces numerous specialized alkaloids, terpenoids, phenolics, and cyanogenic glucosides with indispensable roles in defense against herbivory and microbial infection. Many compounds toxic or deterrent to predators have been eliminated through domestication and breeding. Series of genes involved in defense metabolism are coordinately regulated by transcription factors that specifically recognize cis-regulatory elements in promoter regions of downstream target genes. Recent developments in DNA sequencing technologies and genomic approaches have facilitated studies of the metabolic and genetic changes in chemical defense that have occurred via human-mediated selection, many of which result from mutations in transcriptional regulators of defense metabolism. In this article, we review such examples in almond (Prunus dulcis), cucumber (Cucumis sativus), pepper (Capsicum spp.), potato (Solanum tuberosum), quinoa (Chenopodium quinoa), sorghum (Sorghum bicolor), and related species and discuss insights into the evolution and regulation of metabolic pathways for specialized defense compounds.


Asunto(s)
Cucumis sativus , Solanum tuberosum , Sorghum , Cucumis sativus/genética , Domesticación , Regulación de la Expresión Génica de las Plantas , Fitomejoramiento , Solanum tuberosum/genética , Sorghum/genética , Factores de Transcripción/genética
10.
Biosci Biotechnol Biochem ; 85(12): 2404-2409, 2021 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-34562094

RESUMEN

Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A noncanonical type III polyketide synthase, pyrrolidine ketide synthase (PYKS) catalyzes a two-step decarboxylative reaction, which involves imine-ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.


Asunto(s)
Atropa belladonna
11.
Plant Physiol ; 186(1): 270-284, 2021 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-33619554

RESUMEN

Saponins are the group of plant specialized metabolites which are widely distributed in angiosperm plants and have various biological activities. The present study focused on α-tomatine, a major saponin present in tissues of tomato (Solanum lycopersicum) plants. α-Tomatine is responsible for defense against plant pathogens and herbivores, but its biological function in the rhizosphere remains unknown. Secretion of tomatine was higher at the early growth than the green-fruit stage in hydroponically grown plants, and the concentration of tomatine in the rhizosphere of field-grown plants was higher than that of the bulk soil at all growth stages. The effects of tomatine and its aglycone tomatidine on the bacterial communities in the soil were evaluated in vitro, revealing that both compounds influenced the microbiome in a concentration-dependent manner. Numerous bacterial families were influenced in tomatine/tomatidine-treated soil as well as in the tomato rhizosphere. Sphingomonadaceae species, which are commonly observed and enriched in tomato rhizospheres in the fields, were also enriched in tomatine- and tomatidine-treated soils. Moreover, a jasmonate-responsive ETHYLENE RESPONSE FACTOR 4 mutant associated with low tomatine production caused the root-associated bacterial communities to change with a reduced abundance of Sphingomonadaceae. Taken together, our results highlight the role of tomatine in shaping the bacterial communities of the rhizosphere and suggest additional functions of tomatine in belowground biological communication.


Asunto(s)
Microbiota/fisiología , Raíces de Plantas/metabolismo , Rizosfera , Solanum lycopersicum/metabolismo , Tomatina/metabolismo , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Solanum lycopersicum/microbiología , Raíces de Plantas/microbiología
12.
Trends Plant Sci ; 26(1): 23-32, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32883605

RESUMEN

Plants produce structurally diverse specialized metabolites, including bioactive alkaloids and terpenoids, in response to biotic and abiotic environmental stresses. The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family of transcription factors (TFs) play key roles in regulating biosynthesis of specialized metabolites. Increasing genomic and functional evidence shows that a subset of the ERF genes occurs in clusters on the chromosomes. These jasmonate-responsive ERF TF gene clusters control the biosynthesis of many important metabolites, from natural products, such as nicotine and steroidal glycoalkaloids (SGAs), to pharmaceuticals, such as artemisinin, vinblastine, and vincristine. Here, we review the function, regulation, and evolution of ERF clusters and highlight recent advances in understanding the distinct roles of clustered ERF genes and their possible application in metabolic engineering.


Asunto(s)
Familia de Multigenes , Proteínas de Plantas , Regulación de la Expresión Génica de las Plantas , Filogenia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
13.
Plant Cell Physiol ; 61(6): 1041-1053, 2020 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-32191315

RESUMEN

The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.


Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Nicotiana/genética , Nicotina/biosíntesis , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Edición Génica , Técnicas de Inactivación de Genes , Redes y Vías Metabólicas/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Factores de Transcripción/genética
14.
Plant Biotechnol (Tokyo) ; 36(1): 29-37, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31275046

RESUMEN

In Solanum lycoperisicum (tomato), a transcription factor of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family, JASMONATE-RESPONSIVE ERF 3 (JRE3), is a closest homolog of JRE4, a master transcriptional regulator of steroidal glycoalkaloid (SGA) biosynthesis. In tomato genome, JRE3 resides in a gene cluster with JRE4 and related JRE1, JRE2, and JRE5, while JRE6 exists as a singleton on a different chromosome. All of the JREs are induced by jasmonates (JAs), whereas sodium chloride (NaCl) treatment drastically increases the expression of the JREs except for JRE4 and JRE6. In this study, to get insights into the regulatory function of the JA- and NaCl-inducible JRE3, a series of genes upregulated by ß-estradiol-induced overexpression of JRE3 are identified with microarray analysis in transgenic tomato hairy roots. No gene involved in the SGA pathway has been identified through the screening, confirming the functional distinction between JRE3 and JRE4. Among the JRE3-regulated genes, we characterize the stress-induced expression of genes encoding malate synthase and tonoplast dicarboxylate transporter both involved in malate accumulation. In transient transactivation assay, we reveal that both terminal regions of JRE4, but not a central DNA-binding domain, are indispensable for the induction of a gene involved in the JRE4 regulon. Functional differentiation of the JREs is discussed.

15.
Front Plant Sci ; 10: 560, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31156658

RESUMEN

Plants produce a vast array of structurally diverse specialized metabolites with various biological activities, including medicinal alkaloids and terpenoids, from relatively simple precursors through a series of enzymatic steps. Massive metabolic flow through these pathways usually depends on the transcriptional coordination of a large set of metabolic, transport, and regulatory genes known as a regulon. The coexpression of genes involved in certain metabolic pathways in a wide range of developmental and environmental contexts has been investigated through transcriptomic analysis, which has been successfully exploited to mine the genes involved in various metabolic processes. Transcription factors are DNA-binding proteins that recognize relatively short sequences known as cis-regulatory elements residing in the promoter regions of target genes. Transcription factors have positive or negative effects on gene transcription mediated by RNA polymerase II. Evolutionarily conserved transcription factors of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) and basic helix-loop-helix (bHLH) families have been identified as jasmonate (JA)-responsive transcriptional regulators of unrelated specialized pathways in distinct plant lineages. Here, I review the current knowledge and propose a conceptual model for the evolution of metabolic pathways, termed "recruitment model of metabolic evolution." According to this model, structural genes are repeatedly recruited into regulons under the control of conserved transcription factors through the generation of cognate cis-regulatory elements in the promoters of these genes. This leads to the adjustment of catalytic activities that improve metabolic flow through newly established passages.

16.
J Plant Res ; 132(2): 173-180, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30478481

RESUMEN

The jasmonate-responsive transcription factor ERF189 in tobacco (Nicotiana tabacum) and its ortholog JRE4 in tomato (Solanum lycopersicum) regulate a series of biosynthetic genes involved in the nicotine and steroidal glycoalkaloid pathways. In tobacco, QUINOLINATE PHOSPHORIBOSYL TRANSFERASE 2 (NtQPT2) is regulated by ERF189; however, we found that the tomato QPT gene is not regulated by JRE4. Here, we explored whether and how NtQPT2 is regulated in a heterogenous tomato host. We used a NtQPT2 promoter-driven reporter gene to examine the cell type-specific and jasmonate-induced expression of this gene in transgenic tomato hairy roots. The downregulation of the reporter in the jre4 loss-of-function tomato mutant and its transactivation by JRE4 in transient expression experiments suggested that JRE4, like its ortholog ERF189 in tobacco, activates the NtQPT2 promoter in tomato. We discuss the evolution of QPT2 in the Nicotiana lineage, which mainly occurred through mutational changes in the promoter that altered the control of the functionally conserved transcription factors.


Asunto(s)
Regulación de la Expresión Génica de las Plantas , Nicotiana/genética , Pentosiltransferasa/genética , Solanum lycopersicum/metabolismo , Factores de Transcripción/metabolismo , Evolución Molecular , Genes Reporteros , Solanum lycopersicum/genética , Nicotina/biosíntesis , Pentosiltransferasa/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Activación Transcripcional
17.
Plant J ; 94(6): 975-990, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29569783

RESUMEN

Steroidal glycoalkaloids (SGAs) are specialized anti-nutritional metabolites that accumulate in Solanum lycopersicum (tomato) and Solanum tuberosum (potato). A series of SGA biosynthetic genes is known to be upregulated in Solanaceae species by jasmonate-responsive Ethylene Response Factor transcription factors, including JRE4 (otherwise known as GAME9), but the exact regulatory significance in planta of each factor has remained unaddressed. Here, via TILLING-based screening of an EMS-mutagenized tomato population, we isolated a JRE4 loss-of-function line that carries an amino acid residue missense change in a region of the protein important for DNA binding. In this jre4 mutant, we observed downregulated expression of SGA biosynthetic genes and decreased SGA accumulation. Moreover, JRE4 overexpression stimulated SGA production. Further characterization of jre4 plants revealed their increased susceptibility to the generalist herbivore Spodoptera litura larvae. This susceptibility illustrates that herbivory resistance is dependent on JRE4-mediated defense responses, which include SGA accumulation. Ethylene treatment attenuated the jasmonate-mediated JRE4 expression induction and downstream SGA biosynthesis in tomato leaves and hairy roots. Overall, this study indicated that JRE4 functions as a primary master regulator of SGA biosynthesis, and thereby contributes toward plant defense against chewing insects.


Asunto(s)
Proteínas de Plantas/metabolismo , Alcaloides Solanáceos/metabolismo , Solanum lycopersicum/metabolismo , Factores de Transcripción/metabolismo , Animales , Regulación de la Expresión Génica de las Plantas , Herbivoria , Larva , Solanum lycopersicum/fisiología , Hojas de la Planta/metabolismo , Proteínas de Plantas/fisiología , Raíces de Plantas/metabolismo , Spodoptera , Factores de Transcripción/fisiología
18.
Methods Mol Biol ; 1694: 215-223, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29080170

RESUMEN

The intracellular localization of enzymes provides key information for understanding complex metabolic pathways. Based on enzyme localization data, the involvement of multiple organelles and the movement of metabolites between cellular compartments have been suggested for a number of pathways. Transient expression of fluorescently tagged proteins in the leaves of Nicotiana benthamiana through Agrobacterium infiltration is a simple and versatile way to examine the intracellular localization of proteins of interest. Here, this method was applied to demonstrate the peroxisomal localization of a pair of homologous copper-containing amine oxidases (CuAOs) from tobacco with distinct substrate preferences: diamine oxidase (DAO), which mediates polyamine catabolism, and N-methylputrescine oxidase (MPO), which is involved in nicotine biosynthesis. Our results demonstrate that the Agrobacterium infiltration protocol can be effectively used to study the intracellular localization of oxidases that localize to the peroxisome.


Asunto(s)
Agrobacterium/metabolismo , Amina Oxidasa (conteniendo Cobre)/metabolismo , Nicotiana/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Agrobacterium/genética , Amina Oxidasa (conteniendo Cobre)/genética , Expresión Génica , Genes Reporteros , Redes y Vías Metabólicas , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Plásmidos/genética , Poliaminas/metabolismo , Nicotiana/genética
19.
Plant Signal Behav ; 12(6): e1338225, 2017 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-28613112

RESUMEN

In tobacco, the defense alkaloid nicotine is produced in roots and accumulates mainly in leaves. Signaling mediated by jasmonates (JAs) induces the formation of nicotine via a series of structural genes that constitute a regulon and are coordinated by JA-responsive transcription factors of the ethylene response factor (ERF) family. Early steps in the pyrrolidine and pyridine biosynthesis pathways likely arose through duplication of the polyamine and nicotinamide adenine dinucleotide (NAD) biosynthetic pathways, respectively, followed by recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon. Transcriptional regulation of nicotine biosynthesis by ERF and cooperatively-acting MYC2 transcription factors is implied by the frequency of cognate cis-regulatory elements for these factors in the promoter regions of the downstream structural genes. Indeed, a mutant tobacco with low nicotine content was found to have a large chromosomal deletion in a cluster of closely related ERF genes at the nicotine-controlling NICOTINE2 (NIC2) locus.


Asunto(s)
Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Modelos Biológicos , Nicotiana/genética , Nicotina/biosíntesis , Regulón/genética , Vías Biosintéticas/genética , Duplicación de Gen , Genes de Plantas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
20.
Plant Physiol ; 174(2): 999-1011, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28584068

RESUMEN

In tobacco (Nicotiana tabacum), nicotine is the predominant alkaloid. It is produced in the roots and accumulated mainly in the leaves. Jasmonates play a central signaling role in damage-induced nicotine formation. The genome sequence of tobacco provides us an almost complete inventory of structural and regulatory genes involved in nicotine pathway. Phylogenetic and expression analyses revealed a series of structural genes of the nicotine pathway, forming a regulon, under the control of jasmonate-responsive ETHYLENE RESPONSE FACTOR (ERF) transcription factors. The duplication of NAD and polyamine metabolic pathways and the subsequent recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon were suggested to be the drivers for pyridine and pyrrolidine ring formation steps early in the pathway. Transcriptional regulation by ERF and cooperatively acting MYC2 transcription factors are corroborated by the frequent occurrence of cognate cis-regulatory elements of the factors in the promoter regions of the downstream structural genes. The allotetraploid tobacco has homologous clusters of ERF genes on different chromosomes, which are possibly derived from two ancestral diploids and include either nicotine-controlling ERF189 or ERF199 A large chromosomal deletion was found within one allele of the nicotine-controlling NICOTINE2 locus, which is part of one of the ERF gene clusters, and which has been used to breed tobacco cultivars with a low-nicotine content.


Asunto(s)
Vías Biosintéticas/genética , Evolución Molecular , Genoma de Planta , Nicotiana/genética , Nicotina/biosíntesis , Secuencia de Bases , Vías Biosintéticas/efectos de los fármacos , Cromosomas de las Plantas/genética , Ciclopentanos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Sitios Genéticos , Glucuronidasa/metabolismo , Familia de Multigenes , Mutación/genética , NAD/metabolismo , Oxilipinas/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poliaminas/metabolismo , Regiones Promotoras Genéticas , Eliminación de Secuencia/genética , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Nicotiana/efectos de los fármacos
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