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1.
Nat Commun ; 13(1): 348, 2022 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-35039506

RESUMO

2-(2-Phenylethyl)chromones (PECs) are the principal constituents contributing to the distinctive fragrance of agarwood. How PECs are biosynthesized is currently unknown. In this work, we describe a diarylpentanoid-producing polyketide synthase (PECPS) identified from Aquilaria sinensis. Through biotransformation experiments using fluorine-labeled substrate, transient expression of PECPS in Nicotiana benthamiana, and knockdown of PECPS expression in A. sinensis calli, we demonstrate that the C6-C5-C6 scaffold of diarylpentanoid is the common precursor of PECs, and PECPS plays a crucial role in PECs biosynthesis. Crystal structure (1.98 Å) analyses and site-directed mutagenesis reveal that, due to its small active site cavity (247 Å3), PECPS employs a one-pot formation mechanism including a "diketide-CoA intermediate-released" step for the formation of the C6-C5-C6 scaffold. The identification of PECPS, the pivotal enzyme of PECs biosynthesis, provides insight into not only the feasibility of overproduction of pharmaceutically important PECs using metabolic engineering approaches, but also further exploration of how agarwood is formed.


Assuntos
Vias Biossintéticas , Flavonoides/metabolismo , Policetídeo Sintases/metabolismo , Thymelaeaceae/enzimologia , Madeira/enzimologia , Biocatálise , Biotransformação , Clonagem Molecular , Flavonoides/química , Modelos Moleculares , Mutação/genética , Policetídeo Sintases/genética , Tabaco/enzimologia
2.
Biomed Res Int ; 2022: 1382604, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35047628

RESUMO

Anthocyanins are important pigments for flower color, determining the ornamental and economic values of horticultural plants. As a key enzyme in the biosynthesis of anthocyanidins, dihydroflavonol 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to generate the precursors for anthocyanidins (i.e., leucoanthocyanidins) and anthocyanins. To investigate the functions of DFRs in plants, we cloned the GlaDFR1 and GlaDFR2 genes from the petals of Gentiana lutea var. aurantiaca and transformed both genes into Nicotiana tabacum by Agrobacterium-mediated leaf disc method. We further investigated the molecular and phenotypic characteristics of T1 generation transgenic tobacco plants selected based on the hygromycin resistance and verified by both PCR and semiquantitative real-time PCR analyses. The phenotypic segregation was observed in the flower color of the transgenic tobacco plants, showing petals darker than those in the wild-type (WT) plants. Results of high-performance liquid chromatography (HPLC) analysis showed that the contents of gentiocyanin derivatives were decreased in the petals of transgenic plants in comparison to those of WT plants. Ours results revealed the molecular functions of GlaDFR1 and GlaDFR2 in the formation of coloration, providing solid theoretical foundation and candidate genes for further genetic improvement in flower color of plants.


Assuntos
Oxirredutases do Álcool , Flores , Gentiana , Pigmentação/fisiologia , Proteínas de Plantas , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Clonagem Molecular , Flores/enzimologia , Flores/genética , Gentiana/enzimologia , Gentiana/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Tabaco/enzimologia , Tabaco/genética
3.
Plant Physiol ; 187(1): 103-115, 2021 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-34618129

RESUMO

Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.


Assuntos
Aminoácidos/metabolismo , Dioxigenases/metabolismo , Proteínas de Plantas/metabolismo , Tabaco/enzimologia , Oxirredução
4.
Biochem Soc Trans ; 49(5): 2007-2019, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34623388

RESUMO

Rising human population, along with the reduction in arable land and the impacts of global change, sets out the need for continuously improving agricultural resource use efficiency and crop yield (CY). Bioengineering approaches for photosynthesis optimization have largely demonstrated the potential for enhancing CY. This review is focused on the improvement of Rubisco functioning, which catalyzes the rate-limiting step of CO2 fixation required for plant growth, but also catalyzes the ribulose-bisphosphate oxygenation initiating the carbon and energy wasteful photorespiration pathway. Rubisco carboxylation capacity can be enhanced by engineering the Rubisco large and/or small subunit genes to improve its catalytic traits, or by engineering the mechanisms that provide enhanced Rubisco expression, activation and/or elevated [CO2] around the active sites to favor carboxylation over oxygenation. Recent advances have been made in the expression, assembly and activation of foreign (either natural or mutant) faster and/or more CO2-specific Rubisco versions. Some components of CO2 concentrating mechanisms (CCMs) from bacteria, algae and C4 plants has been successfully expressed in tobacco and rice. Still, none of the transformed plant lines expressing foreign Rubisco versions and/or simplified CCM components were able to grow faster than wild type plants under present atmospheric [CO2] and optimum conditions. However, the results obtained up to date suggest that it might be achievable in the near future. In addition, photosynthetic and yield improvements have already been observed when manipulating Rubisco quantity and activation degree in crops. Therefore, engineering Rubisco carboxylation capacity continues being a promising target for the improvement in photosynthesis and yield.


Assuntos
Bioengenharia/métodos , Produção Agrícola/métodos , Fotossíntese/genética , Engenharia de Proteínas/métodos , Ribulose-Bifosfato Carboxilase/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Catálise , Cloroplastos/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Ativação Enzimática/genética , Oryza/enzimologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Ribulose-Bifosfato Carboxilase/genética , Tabaco/enzimologia , Tabaco/genética , Tabaco/crescimento & desenvolvimento
5.
BMC Plant Biol ; 21(1): 501, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34717531

RESUMO

BACKGROUND: GDSL esterases/lipases are a large protein subfamily defined by the distinct GDSL motif, and play important roles in plant development and stress responses. However, few studies have reported on the role of GDSLs in the growth and development of axillary buds. This work aims to identify the GDSL family members in tobacco and explore whether the NtGDSL gene contributes to development of the axillary bud in tobacco. RESULTS: One hundred fifty-nine GDSL esterase/lipase genes from cultivated tobacco (Nicotiana tabacum) were identified, and the dynamic changes in the expression levels of 93 of these genes in response to topping, as assessed using transcriptome data of topping-induced axillary shoots, were analysed. In total, 13 GDSL esterase/lipase genes responded with changes in expression level. To identify genes and promoters that drive the tissue-specific expression in tobacco apical and axillary buds, the expression patterns of these 13 genes were verified using qRT-PCR. GUS activity and a lethal gene expression pattern driven by the NtGDSL127 promoter in transgenic tobacco demonstrated that NtGDSL127 is specifically expressed in apical buds, axillary buds, and flowers. Three separate deletions in the NtGDSL127 promoter demonstrated that a minimum upstream segment of 235 bp from the translation start site can drive the tissue-specific expression in the apical meristem. Additionally, NtGDSL127 responded to phytohormones, providing strategies for improving tobacco breeding and growth. CONCLUSION: We propose that in tobacco, the NtGDSL127 promoter directs expression specifically in the apical meristem and that expression is closely correlated with axillary bud development.


Assuntos
Esterases/genética , Lipase/genética , Meristema/crescimento & desenvolvimento , Meristema/genética , Tabaco/enzimologia , Tabaco/crescimento & desenvolvimento , Tabaco/genética , Produtos Agrícolas/enzimologia , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Esterases/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Estudo de Associação Genômica Ampla , Lipase/metabolismo , Filogenia , Transcriptoma
6.
Proc Natl Acad Sci U S A ; 118(33)2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34380739

RESUMO

The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, ß-CA1 and ß-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δß-ca1 and Δß-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δß-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δß-ca1ca5 plants normalized at 9,000 ppm CO2 Leaves of Δß-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δß-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δß-ca1ca5 seedling germination and development is negatively affected at ambient CO2 Transgenes expressing full-length ß-CA1 and ß-CA5 proteins complemented the Δß-ca1ca5 mutation but inactivated (ΔZn-ßCA1) and cytoplasm-localized (Δ62-ßCA1) forms of ß-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-ßCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δß-ca1 and Δß-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.


Assuntos
Anidrases Carbônicas/metabolismo , Cloroplastos/enzimologia , Tabaco/enzimologia , Sistemas CRISPR-Cas , Cloroplastos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica de Plantas/fisiologia , Mutação , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Tabaco/genética
7.
Org Biomol Chem ; 19(30): 6650-6656, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34264250

RESUMO

The exquisite chemodiversity of terpenoids is the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue (Cys440 in Nicotiana tabacum 5-epi-aristolochene synthase) proximal to an ion-binding motif common to all TPSs and named the preNSE/DTE residue, which determines the product specificity of sesquiterpene synthases from different plant species. In sesquiterpene synthases catalyzing 1,10-cyclization (1,10-cyclases) of farnesyl diphosphate, mutation of the residue in both specific and promiscuous 1,10-cyclases from different lineages leads to the accumulation of monocyclic germacrene A-11-ol, which is "short-circuited" from complex cyclization cascades, suggesting a key role of this residue in generating the first common intermediate of 1,10-cyclization. Altering this residue in a specific 1,11-cyclase results in alternative 1,10-cyclization products. Moreover, the preNSE/DTE residue can be harnessed to engineer highly specific sesquiterpene synthases for an improved proportion of high-value terpenoids, such as patchoulol, a main constituent of several traditional Chinese medicines that could treat SARS-CoV-2.


Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Biocatálise , Alquil e Aril Transferases/genética , Domínio Catalítico , Ciclização , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Tabaco/enzimologia
8.
Sci Rep ; 11(1): 13435, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183701

RESUMO

Stenotrophomonas maltophilia has plant growth-promoting potential, and interaction with Arachis hypogaea changes host-plant physiology, biochemistry, and metabolomics, which provides tolerance under the N2 starvation conditions. About 226 suppression subtractive hybridization clones were obtained from plant-microbe interaction, of which, about 62% of gene sequences were uncharacterized, whereas 23% of sequences were involved in photosynthesis. An uncharacterized SSH clone, SM409 (full-length sequence showed resemblance with Cytb6), showed about 4-fold upregulation during the interaction was transformed to tobacco for functional validation. Overexpression of the AhCytb6 gene enhanced the seed germination efficiency and plant growth under N2 deficit and salt stress conditions compared to wild-type and vector control plants. Results confirmed that transgenic lines maintained high photosynthesis and protected plants from reactive oxygen species buildup during stress conditions. Microarray-based whole-transcript expression of host plants showed that out of 272,410 genes, 8704 and 24,409 genes were significantly (p < 0.05) differentially expressed (> 2 up or down-regulated) under N2 starvation and salt stress conditions, respectively. The differentially expressed genes belonged to different regulatory pathways. Overall, results suggested that overexpression of AhCytb6 regulates the expression of various genes to enhance plant growth under N2 deficit and abiotic stress conditions by modulating plant physiology.


Assuntos
Arachis/genética , Citocromos b6/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fixação de Nitrogênio/genética , Nitrogênio/deficiência , Proteínas de Plantas/genética , Estresse Salino/genética , Stenotrophomonas maltophilia/fisiologia , Simbiose/genética , Tabaco/genética , Arachis/enzimologia , Biomassa , Mudança Climática , Simulação por Computador , Citocromos b6/fisiologia , Modelos Genéticos , Nitrogênio/metabolismo , Fotossíntese , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Tabaco/enzimologia , Tabaco/crescimento & desenvolvimento , Tabaco/microbiologia , Regulação para Cima
9.
Plant Signal Behav ; 16(10): 1938442, 2021 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-34120568

RESUMO

Fructose 1,6-bisphosphate aldolase (FBA) as a key enzyme play crucial roles in glycolysis, gluconeogenesis and Calvin cycle processes in plants. However, limited information is known regarding FBA genes in Nicotiana tabacum. In this study, 16 FBAs were identified and characterized in Nicotiana tabacum. Phylogenetic analysis revealed that these genes can be categorized as type I (NtFBA1-10 located in chloroplast) and type II (NtFBA11-16 located in cytoplasm) subfamilies. According to the conserved motifs and gene structure analysis, NtFBA protein sequences had the highly homologous to FBAs in other species. Most members of the NtFBA gene family responded positively to NaHCO3 stress, especially the expression of NtFBA13/14 increased by 642%. In addition, the expression results of NtFBAs under five abiotic stress (light, NaCl, NaHCO3, drought, and cold) conditions were showed that NtFBA13/14 were highly up-regulated. qRT-PCR results showed that most of the NtFBAs expressed higher in leaves. NtFBA7/8 and NtFBA13/14 have important significance in photosynthesis and abiotic stress, respectively. This study provides a basis foundation for further elucidating the function of NtFBAs and the N. tabacum mechanism of resistance under abiotic stress.


Assuntos
Evolução Molecular , Frutose-Bifosfato Aldolase/genética , Genes de Plantas , Luz , Tabaco/enzimologia , Tabaco/genética , Frutose-Bifosfato Aldolase/metabolismo , Perfilação da Expressão Gênica , Família Multigênica , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Estresse Fisiológico/genética , Estresse Fisiológico/efeitos da radiação , Tabaco/efeitos da radiação
10.
Virology ; 559: 15-29, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33799077

RESUMO

Positive-strand RNA viruses build viral replication organelles (VROs) with the help of co-opted host factors. The energy requirement of intensive viral replication processes is less understood. Previous studies on tomato bushy stunt virus (TBSV) showed that tombusviruses hijack two ATP-producing glycolytic enzymes to produce ATP locally within VROs. In this work, we performed a cDNA library screen with Arabidopsis thaliana proteins and the TBSV p33 replication protein. The p33 - plant interactome contained highly conserved glycolytic proteins. We find that the glycolytic Hxk2 hexokinase, Eno2 phosphopyruvate hydratase and Fba1 fructose 1,6-bisphosphate aldolase are critical for TBSV replication in yeast or in a cell-free replicase reconstitution assay. The recruitment of Fba1 is important for the local production of ATP within VROs. Altogether, our data support the model that TBSV recruits and compartmentalizes possibly most members of the glycolytic pathway. This might allow TBSV to avoid competition with the host for ATP.


Assuntos
Trifosfato de Adenosina/metabolismo , Glicólise , Tabaco/enzimologia , Tombusvirus/fisiologia , Replicação Viral/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Interações entre Hospedeiro e Microrganismos , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/genética , Tombusvirus/metabolismo
11.
Plant J ; 106(5): 1468-1483, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33768632

RESUMO

Suberin is a complex hydrophobic polymer of aliphatic and phenolic compounds which controls the movement of gases, water, and solutes and protects plants from environmental stresses and pathogenic infection. The synthesis and regulatory pathways of suberin remain unknown in Brachypodium distachyon. Here we describe the identification of a B. distachyon gene, BdFAR4, encoding a fatty acyl-coenzyme A reductase (FAR) by a reverse genetic approach, and investigate the molecular relevance of BdFAR4 in the root suberin synthesis of B. distachyon. BdFAR4 is specifically expressed throughout root development. Heterologous expression of BdFAR4 in yeast (Saccharomyces cerevisiae) afforded the production of C20:0 and C22:0 fatty alcohols. The loss-of-function knockout of BdFAR4 by CRISPR/Cas9-mediated gene editing significantly reduced the content of C20:0 and C22:0 fatty alcohols associated with root suberin. In contrast, overexpression of BdFAR4 in B. distachyon and tomato (Solanum lycopersicum) resulted in the accumulation of root suberin-associated C20:0 and C22:0 fatty alcohols, suggesting that BdFAR4 preferentially accepts C20:0 and C22:0 fatty acyl-CoAs as substrates. The BdFAR4 protein was localized to the endoplasmic reticulum in Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf epidermal cells. BdFAR4 transcript levels can be increased by abiotic stresses and abscisic acid treatment. Furthermore, yeast one-hybrid, dual-luciferase activity, and electrophoretic mobility shift assays indicated that the R2R3-MYB transcription factor BdMYB41 directly binds to the promoter of BdFAR4. Taken together, these results imply that BdFAR4 is essential for the production of root suberin-associated fatty alcohols, especially under stress conditions, and that its activity is transcriptionally regulated by the BdMYB41 transcription factor.


Assuntos
Aldeído Oxirredutases/metabolismo , Brachypodium/genética , Álcoois Graxos/metabolismo , Regulação da Expressão Gênica de Plantas , Lipídeos/biossíntese , Aldeído Oxirredutases/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/fisiologia , Brachypodium/enzimologia , Brachypodium/fisiologia , Edição de Genes , Técnicas de Inativação de Genes , Mutação com Perda de Função , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Poliésteres/metabolismo , Estresse Fisiológico , Tabaco/enzimologia , Tabaco/genética , Tabaco/fisiologia
12.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670111

RESUMO

The gene coding for the telomerase reverse transcriptase (TERT) is essential for the maintenance of telomeres. Previously we described the presence of three TERT paralogs in the allotetraploid plant Nicotiana tabacum, while a single TERT copy was identified in the paleopolyploid model plant Arabidopsis thaliana. Here we examine the presence, origin and functional status of TERT variants in allotetraploid Nicotiana species of diverse evolutionary ages and their parental genome donors, as well as in other diploid and polyploid plant species. A combination of experimental and in silico bottom-up analyses of TERT gene copies in Nicotiana polyploids revealed various patterns of retention or loss of parental TERT variants and divergence in their functions. RT-qPCR results confirmed the expression of all the identified TERT variants. In representative plant and green algal genomes, our synteny analyses show that their TERT genes were located in a conserved locus that became advantageous after the divergence of eudicots, and the gene was later translocated in several plant groups. In various diploid and polyploid species, translocation of TERT became fixed in target loci that show ancient synapomorphy.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Dosagem de Genes , Poliploidia , Telomerase , Tabaco , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Telomerase/genética , Telomerase/metabolismo , Tabaco/enzimologia , Tabaco/genética
13.
Plant Sci ; 305: 110829, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33691963

RESUMO

Serine hydroxymethyltransferase 1 (SHMT1) is a key enzyme in the photorespiration pathway in higher plants. Our previous study showed that AtSHMT1 controls the assimilation of HCHO to sugars in Arabidopsis. The expression of SHMT1 was induced in Arabidopsis but was inhibited in tobacco under HCHO stress. To investigate whether the function of AtSHMT1 in the HCHO assimilation could be exerted in tobacco, AtSHMT1 was overexpressed alone (S5) or co-overexpressed (SF6) with Arabidopsis formate dehydrogenase (AtFDH) in leaves using a light-inducible promoter in this study. 13C NMR analyses showed that the 13C-metabolic flux from H13CHO was introduced to sugar synthesis in SF6 leaves but not in S5 leaves. The increase in the production of metabolites via the original pathways was particularly greater in SF6 leaves than in S5 leaves, suggesting that co-overexpression of AtSHMT1 and AtFDH is more effective than overexpression of AtSHMT1 alone in the enhancement of HCHO metabolism in tobacco leaves. Consequently, the increase in HCHO uptake and resistance was greater in SF6 leaves than in S5 leaves. The mechanism underlying the role of overexpressed AtSHMT1 and AtFDH was discussed based on changes in photosynthetic parameters, chlorophyll content, antioxidant enzyme activity and the oxidative level in leaves.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Formaldeído/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Açúcares/metabolismo , Tabaco/enzimologia , Tabaco/genética , Transporte Biológico , Vias Biossintéticas/genética , Clorofila/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fotossíntese/genética , Fotossíntese/fisiologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo
14.
J Nat Prod ; 84(3): 694-706, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33687206

RESUMO

Chlorogenic acid (CGA) and guaiacyl/syringyl (G/S) lignin formation involves hydroxycinnamoyl ester intermediacy, the latter formed via hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) activities. HQT and HCT RNAi silencing of a commercial tobacco (Nicotiana tabacum) K326 line was examined herein. NtHQT gene silencing gave relatively normal plant phenotypes, with CGA levels reduced (down to 1% of wild type) with no effects on lignin. RNAi NtHCT silencing had markedly adverse phenotypes (e.g., stunted, multiple stems, delayed flowering, with senescence delayed by several months). Lignin contents were partially lowered, with a small increase in cleavable p-hydroxyphenyl (H) monomers; those plants had no detectable CGA level differences relative to wild type. In vitro NtHCT kinetic parameters revealed preferential p-coumaroyl CoA and shikimate esterification, as compared to other structurally related potential acyl group donors and acceptors. In the presence of coenzyme A, NtHCT catalyzed the reverse reaction. Site-directed mutagenesis of NtHCT (His153Ala) abolished enzymatic activity. NtHQT, by comparison, catalyzed preferential conversion of p-coumaroyl CoA and quinic acid to form p-coumaroyl quinate, the presumed CGA precursor. In sum, metabolic pathways to CGA and lignins appear to be fully independent, and previous conflicting reports of substrate versatilities and metabolic cross-talk are resolved.


Assuntos
Ácido Clorogênico/metabolismo , Lignina/metabolismo , Interferência de RNA , Tabaco/enzimologia , Aciltransferases/genética , Estrutura Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Tabaco/genética
15.
Plant Mol Biol ; 106(1-2): 85-108, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33629224

RESUMO

KEY MESSAGE: Overexpression of StCaM2 in tobacco promotes plant growth and confers increased salinity and drought tolerance by enhancing the photosynthetic efficiency, ROS scavenging, and recovery from membrane injury. Calmodulins (CaMs) are important Ca2+ sensors that interact with effector proteins and drive a network of signal transduction pathways involved in regulating the growth and developmental pattern of plants under stress. Herein, using in silico analysis, we identified 17 CaM isoforms (StCaM) in potato. Expression profiling revealed different temporal and spatial expression patterns of these genes, which were modulated under abiotic stress. Among the identified StCaM genes, StCaM2 was found to have the largest number of abiotic stress responsive promoter elements. In addition, StCaM2 was upregulated in response to some of the selected abiotic stress in potato tissues. Overexpression of StCaM2 in transgenic tobacco plants enhanced their tolerance to salinity and drought stress. Accumulation of reactive oxygen species was remarkably decreased in transgenic lines compared to that in wild type plants. Chlorophyll a fluorescence analysis suggested better performance of photosystem II in transgenic plants under stress compared to that in wild type plants. The increase in salinity stress tolerance in StCaM2-overexpressing plants was also associated with a favorable K+/Na+ ratio. The enhanced tolerance to abiotic stresses correlated with the increase in the activities of anti-oxidative enzymes in transgenic tobacco plants. Overall, our results suggest that StCaM2 can be a novel candidate for conferring salt and drought tolerance in plants.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Secas , Proteínas de Plantas/metabolismo , Salinidade , Solanum tuberosum/metabolismo , Estresse Fisiológico , Tabaco/genética , Tabaco/fisiologia , Antioxidantes/metabolismo , Proteínas de Ligação ao Cálcio/genética , Calmodulina/genética , Calmodulina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genoma de Planta , Germinação/efeitos dos fármacos , Germinação/genética , Íons , Membranas , Fotossíntese/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Prolina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Tabaco/enzimologia , Tabaco/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Água/metabolismo
16.
Plant Physiol ; 185(2): 441-456, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33580795

RESUMO

Age-dependent changes in reactive oxygen species (ROS) levels are critical in leaf senescence. While H2O2-reducing enzymes such as catalases and cytosolic ASCORBATE PEROXIDASE1 (APX1) tightly control the oxidative load during senescence, their regulation and function are not specific to senescence. Previously, we identified the role of ASCORBATE PEROXIDASE6 (APX6) during seed maturation in Arabidopsis (Arabidopsis thaliana). Here, we show that APX6 is a bona fide senescence-associated gene. APX6 expression is specifically induced in aging leaves and in response to senescence-promoting stimuli such as abscisic acid (ABA), extended darkness, and osmotic stress. apx6 mutants showed early developmental senescence and increased sensitivity to dark stress. Reduced APX activity, increased H2O2 level, and altered redox state of the ascorbate pool in mature pre-senescing green leaves of the apx6 mutants correlated with the early onset of senescence. Using transient expression assays in Nicotiana benthamiana leaves, we unraveled the age-dependent post-transcriptional regulation of APX6. We then identified the coding sequence of APX6 as a potential target of miR398, which is a key regulator of copper redistribution. Furthermore, we showed that mutants of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), the master regulator of copper homeostasis and miR398 expression, have a higher APX6 level compared with the wild type, which further increased under copper deficiency. Our study suggests that APX6 is a modulator of ROS/redox homeostasis and signaling in aging leaves that plays an important role in developmental- and stress-induced senescence programs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ascorbato Peroxidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Ascorbato Peroxidases/genética , Cobre/deficiência , Proteínas de Ligação a DNA/genética , Escuridão , Homeostase , Peróxido de Hidrogênio/metabolismo , MicroRNAs/genética , Oxirredução , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Tabaco/enzimologia , Tabaco/genética , Tabaco/fisiologia , Fatores de Transcrição/genética
17.
Plant Sci ; 304: 110714, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33568313

RESUMO

Plant NADH glutamate dehydrogenase (GDH) is an intriguing enzyme, since it is involved in different metabolic processes owing to its reversible (anabolic/catabolic) activity and due to the oligomeric nature of the enzyme, that gives rise to several isoforms. The complexity of GDH isoenzymes pattern and the variability of the spatial and temporal localization of the different isoforms have limited our comprehension of the physiological role of GDH in plants. Genetics, immunological, and biochemical approaches have been used until now in order to shed light on the regulatory mechanism that control GDH expression in different plant systems and environmental conditions. We describe here the validation of a simple in planta GDH activity staining procedure, providing evidence that it might be used, with different purposes, to determine GDH expression in plant organs, tissues, extracts and also heterologous systems.


Assuntos
Glutamato Desidrogenase/metabolismo , Plantas/enzimologia , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Corantes , Ensaios Enzimáticos/métodos , Regulação da Expressão Gênica de Plantas , Extratos Vegetais/metabolismo , Plantas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Tabaco/enzimologia , Tabaco/metabolismo
18.
Plant J ; 106(3): 876-887, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33576096

RESUMO

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.


Assuntos
Edição de Genes/métodos , Genes de Plantas/genética , Mutação Puntual/genética , Ribulose-Bifosfato Carboxilase/genética , Tabaco/genética , Cloroplastos/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Tabaco/enzimologia
19.
Int J Biol Macromol ; 171: 89-99, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33412202

RESUMO

In this study we describe the crystal structures of the apoform, the binary and the ternary complexes of a double bond reductase from Malus domestica L. (MdDBR) and explore a range of potential substrates. The overall fold of MdDBR is similar to that of the medium chain reductase/dehydrogenase/zinc-dependent alcohol dehydrogenase-like family. Structural comparison of MdDBR with Arabidopsis thaliana DBR (AtDBR), Nicotiana tabacum DBR (NtDBR) and Rubus idaeus DBR (RiDBR) allowed the identification of key amino acids involved in cofactor and ligands binding and shed light on how these residues may guide the orientation of the substrates. The enzyme kinetic for the substrate trans-4-phenylbuten-2-one has been analyzed, and MdDBR activity towards a variety of substrates was tested. This enzyme has been reported to be involved in the phenylpropanoid pathway where it would catalyze the NADPH-dependent reduction of the α, ß-unsaturated double bond of carbonyl metabolites. Our study provides new data towards the identification of MdDBR natural substrate and the biosynthetic pathway where it belongs. Furthermore, the originally proposed involvement in dihydrochalcone biosynthesis in apple must be questioned.


Assuntos
Apoproteínas/química , Butanonas/química , Malus/química , NADP/química , Oxirredutases/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Arabidopsis/química , Arabidopsis/enzimologia , Sítios de Ligação , Butanonas/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Malus/enzimologia , Modelos Moleculares , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rubus/química , Rubus/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , Tabaco/química , Tabaco/enzimologia
20.
Science ; 371(6526): 255-260, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33446550

RESUMO

Many plant specialized metabolites function in herbivore defense, and abrogating particular steps in their biosynthetic pathways frequently causes autotoxicity. However, the molecular mechanisms underlying their defense and autotoxicity remain unclear. Here, we show that silencing two cytochrome P450s involved in diterpene biosynthesis in the wild tobacco Nicotiana attenuata causes severe autotoxicity symptoms that result from the inhibition of sphingolipid biosynthesis by noncontrolled hydroxylated diterpene derivatives. Moreover, the diterpenes' defensive function is achieved by inhibiting herbivore sphingolipid biosynthesis through postingestive backbone hydroxylation products. Thus, by regulating metabolic modifications, tobacco plants avoid autotoxicity and gain herbivore defense. The postdigestive duet that occurs between plants and their insect herbivores can reflect the plant's solutions to the "toxic waste dump" problem of using potent chemical defenses.


Assuntos
Diterpenos/metabolismo , Glucosídeos/biossíntese , Herbivoria , Manduca/fisiologia , Esfingolipídeos/biossíntese , Tabaco/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Manduca/enzimologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Tabaco/enzimologia
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