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1.
BMC Plant Biol ; 21(1): 376, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399701

RESUMO

BACKGROUND: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. RESULTS: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. CONCLUSION: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.


Assuntos
Genoma de Planta , Manihot/enzimologia , Manihot/genética , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Cloroplastos/enzimologia , Mapeamento Cromossômico , Cromossomos de Plantas , Sequência Conservada , Citoplasma/enzimologia , Éxons , Flores/enzimologia , Íntrons , Família Multigênica , Oxigênio/metabolismo , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Regiões Promotoras Genéticas , Estresse Fisiológico/genética , Transcriptoma
2.
J Plant Physiol ; 265: 153495, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34411985

RESUMO

Nicotinamide adenine dinucleotides (NAD(H)) and NAD phosphates (NADP(H)) are electron carriers involved in redox reactions and metabolic processes in all organisms. NAD kinase (NADK) is the only enzyme that phosphorylates NAD+ into NADP+, using ATP as a phosphate donor. In NADP-dependent malic enzyme (NADP-ME)-type C4 photosynthesis, NADP(H) are required for dehydrogenation by NADP-dependent malate dehydrogenase (NADP-MDH) in mesophyll cells, and decarboxylation by NADP-ME in bundle sheath cells. In this study, we identified five NADK genes (FbNADK1a, 1b, 2a, 2b, and 3) from the C4 model species Flaveria bidentis. RNA-Seq database analysis revealed higher transcript abundance in one of the chloroplast-type NADK2 genes of C4F. bidentis (FbNADK2a). Comparative analysis of NADK activity in leaves of C3, C3-C4, and C4Flaveria showed that C4Flaveria (F. bidentis and F. trinervia) had higher NADK activity than the other photosynthetic-types of Flaveria. Taken together, our results suggest that chloroplastic NAD kinase appeared to increase in importance as C3 plants evolved into C4 plants in the genus Flaveria.


Assuntos
Cloroplastos/enzimologia , Cloroplastos/genética , Flaveria/enzimologia , Flaveria/genética , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , NADP/genética
3.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072887

RESUMO

FtsH metalloproteases found in eubacteria, animals, and plants are well-known for their vital role in the maintenance and proteolysis of membrane proteins. Their location is restricted to organelles of endosymbiotic origin, the chloroplasts, and mitochondria. In the model organism Arabidopsis thaliana, there are 17 membrane-bound FtsH proteases containing an AAA+ (ATPase associated with various cellular activities) and a Zn2+ metalloprotease domain. However, in five of those, the zinc-binding motif HEXXH is either mutated (FtsHi1, 2, 4, 5) or completely missing (FtsHi3), rendering these enzymes presumably inactive in proteolysis. Still, homozygous null mutants of the pseudo-proteases FtsHi1, 2, 4, 5 are embryo-lethal. Homozygous ftshi3 or a weak point mutant in FTSHi1 are affected in overall plant growth and development. This review will focus on the findings concerning the FtsHi pseudo-proteases and their involvement in protein import, leading to consequences in embryogenesis, seed growth, chloroplast, and leaf development and oxidative stress management.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/genética , Metaloendopeptidases/genética , Tilacoides/genética , Arabidopsis/enzimologia , Cloroplastos/enzimologia , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Transporte Proteico/genética , Proteólise , Tilacoides/enzimologia
4.
Nucleic Acids Res ; 49(12): 6771-6787, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34133716

RESUMO

Proper repair of damaged DNA is crucial for genetic integrity and organismal survival. As semi-autonomous organelles, plastids have their own genomes whose integrity must be preserved. Several factors have been shown to participate in plastid DNA damage repair; however, the underlying mechanism remains unclear. Here, we elucidate a mechanism of homologous recombination (HR) repair in chloroplasts that involves R-loops. We find that the recombinase RecA1 forms filaments in chloroplasts during HR repair, but aggregates as puncta when RNA:DNA hybrids accumulate. ssDNA-binding proteins WHY1/3 and chloroplast RNase H1 AtRNH1C are recruited to the same genomic sites to promote HR repair. Depletion of AtRNH1C or WHY1/3 significantly suppresses the binding of RNA polymerase to the damaged DNA, thus reducing HR repair and modulating microhomology-mediated double-strand break repair. Furthermore, we show that DNA polymerase IB works with AtRNH1C genetically to complete the DNA damage repair process. This study reveals the positive role of R-loops in facilitating the activities of WHY1/3 and RecA1, which in turn secures HR repair and organellar development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/genética , Proteínas de Ligação a DNA/metabolismo , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cloroplastos/enzimologia , Cloroplastos/metabolismo , Dano ao DNA , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Instabilidade Genômica , Mutação , RNA de Plantas/metabolismo
5.
J Biol Chem ; 296: 100217, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839679

RESUMO

Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.


Assuntos
Ferredoxinas/química , Heme Oxigenase-1/química , Heme/química , Ferro/química , Proteínas de Plantas/química , Soja/química , Sequência de Aminoácidos , Biliverdina/química , Biliverdina/metabolismo , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Domínio Catalítico , Cloroplastos/química , Cloroplastos/enzimologia , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heme/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Ligação de Hidrogênio , Ferro/metabolismo , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , 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 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Soja/enzimologia , Soja/genética , Tilacoides/química , Tilacoides/enzimologia
6.
FEBS Lett ; 595(11): 1525-1541, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33792910

RESUMO

In the N-degron pathway of protein degradation of Escherichia coli, the N-recognin ClpS identifies substrates bearing N-terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N-degron pathway. The residue in second position influences recognition (especially in N-terminal ends starting with leucine). N-terminal acetylation abrogates recognition. ClpF, a ClpS1-interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate-binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N-degron pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas de Transporte/química , Cloroplastos/enzimologia , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloroplastos/genética , Clonagem Molecular , Sequência Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Leucina/química , Leucina/metabolismo , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo , Tirosina/química , Tirosina/metabolismo
7.
Plant Cell Physiol ; 62(3): 401-410, 2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-33416847

RESUMO

Various proteins in plant chloroplasts are subject to thiol-based redox regulation, allowing light-responsive control of chloroplast functions. Most redox-regulated proteins are known to be reductively activated in the light in a thioredoxin (Trx)-dependent manner, but its regulatory network remains incompletely understood. Using a biochemical procedure, we here show that a specific form of phosphofructokinase (PFK) is a novel redox-regulated protein whose activity is suppressed upon reduction. PFK is a key enzyme in the glycolytic pathway. In Arabidopsis thaliana, PFK5 is targeted to chloroplasts and uniquely contains an insertion sequence harboring two Cys residues (Cys152 and Cys157) in the N-terminal region. Redox shift assays using a thiol-modifying reagent indicated that PFK5 is efficiently reduced by a specific type of Trx, namely, Trx-f. PFK5 enzyme activity was lowered with the Trx-f-dependent reduction. PFK5 redox regulation was bidirectional; PFK5 was also oxidized and activated by the recently identified Trx-like2/2-Cys peroxiredoxin pathway. Mass spectrometry-based peptide mapping analysis revealed that Cys152 and Cys157 are critical for the intramolecular disulfide bond formation in PFK5. The involvement of Cys152 and Cys157 in PFK5 redox regulation was further supported by a site-directed mutagenesis study. PFK5 catalyzes the reverse reaction of fructose 1,6-bisphosphatase (FBPase), which is reduced and activated specifically by Trx-f. Our data suggest that PFK5 redox regulation, together with that of FBPase, constitutes a checkpoint for switching light/dark metabolism in chloroplasts.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Oxirredução , Fosfofrutoquinases/metabolismo , Nucleotídeos de Adenina/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Cloroplastos/enzimologia , Cisteína/metabolismo , Redes e Vias Metabólicas , Peroxirredoxinas/metabolismo , Fosfofrutoquinases/genética
8.
Photosynth Res ; 147(2): 211-227, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33393063

RESUMO

C4-like plants represent the penultimate stage of evolution from C3 to C4 plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C4 plant, its leaf anatomy and gas exchange traits suggest that it may be a C4-like plant. Here, we reexamined the leaf structure and biochemical and physiological traits of photosynthesis in this grass. The large vascular bundles were surrounded by two layers of bundle sheath (BS): a colorless outer BS and a chloroplast-rich inner BS. Small vascular bundles, which generally had a single BS layer with various vascular structures, also occurred throughout the mesophyll together with BS cells not associated with vascular tissue. The mesophyll cells did not show a radial arrangement typical of Kranz anatomy. These features suggest that the leaf anatomy of C. prionitis is on the evolutionary pathway to a complete C4 Kranz type. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, Pi dikinase occurred in the mesophyll and outer BS. Glycine decarboxylase was confined to the inner BS. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in the mesophyll and both BSs. C. prionitis had biochemical traits of NADP-malic enzyme type, whereas its gas exchange traits were close to those of C4-like intermediate plants rather than C4 plants. A gas exchange study with a PEPC inhibitor suggested that Rubisco in the mesophyll could fix atmospheric CO2. These data demonstrate that C. prionitis is not a true C4 plant but should be considered as a C4-like plant.


Assuntos
Dióxido de Carbono/metabolismo , Fotossíntese , Poaceae/fisiologia , Cloroplastos/enzimologia , Cloroplastos/fisiologia , Cloroplastos/ultraestrutura , Glicina Desidrogenase (Descarboxilante)/metabolismo , Malato Desidrogenase/metabolismo , Células do Mesofilo/enzimologia , Células do Mesofilo/fisiologia , Células do Mesofilo/ultraestrutura , Fenótipo , Fosfoenolpiruvato Carboxilase/antagonistas & inibidores , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Folhas de Planta/ultraestrutura , Proteínas de Plantas/metabolismo , Poaceae/enzimologia , Poaceae/ultraestrutura , Ribulose-Bifosfato Carboxilase/metabolismo
9.
Plant Cell Physiol ; 62(1): 156-165, 2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33289530

RESUMO

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) limits the regeneration of ribulose 1,5-bisphosphate (RuBP) in the Calvin-Benson cycle. However, it does not always limit the rate of CO2 assimilation. In the present study, the effects of overproduction of GAPDH on the rate of CO2 assimilation under elevated [CO2] conditions, where the capacity for RuBP regeneration limits photosynthesis, were examined in transgenic rice (Oryza sativa). GAPDH activity was increased to 3.2- and 4.5-fold of the wild-type levels by co-overexpression of the GAPDH genes, GAPA and GAPB, respectively. In the transgenic rice plants, the rate of CO2 assimilation under elevated [CO2] conditions increased by approximately 10%, whereas that under normal and low [CO2] conditions was not affected. These results indicate that overproduction of GAPDH is effective in improving photosynthesis under elevated [CO2] conditions, although its magnitude is relatively small. By contrast, biomass production of the transgenic rice plants was not greater than that of wild-type plants under elevated [CO2] conditions, although starch content tended to increase marginally.


Assuntos
Cloroplastos/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Oryza/metabolismo , Fotossíntese , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Citocromos f/metabolismo , Regulação da Expressão Gênica de Plantas , Gliceraldeído-3-Fosfato Desidrogenases/fisiologia , Oryza/enzimologia , Oryza/fisiologia , Folhas de Planta/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo
10.
Plant J ; 105(4): 942-956, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33190327

RESUMO

Lesion-mimic mutants (LMMs) provide a valuable tool to reveal the molecular mechanisms determining programmed cell death (PCD) in plants. Despite intensive research, the mechanisms behind PCD and the formation of lesions in various LMMs still remain to be elucidated. Here, we identified a rice (Oryza sativa) LMM, early lesion leaf 1 (ell1), cloned the causal gene by map-based cloning, and verified this by complementation. ELL1 encodes a cytochrome P450 monooxygenase, and the ELL1 protein was located in the endoplasmic reticulum. The ell1 mutant exhibited decreased chlorophyll contents, serious chloroplast degradation, upregulated expression of chloroplast degradation-related genes, and attenuated photosynthetic protein activity, indicating that ELL1 is involved in chloroplast development. RNA sequencing analysis showed that genes related to oxygen binding were differentially expressed in ell1 and wild-type plants; histochemistry and paraffin sectioning results indicated that hydrogen peroxide (H2 O2 ) and callose accumulated in the ell1 leaves, and the cell structure around the lesions was severely damaged, which indicated that reactive oxygen species (ROS) accumulated and cell death occurred in the mutant. TUNEL staining and comet experiments revealed that severe DNA degradation and abnormal PCD occurred in the ell1 mutants, which implied that excessive ROS accumulation may induce DNA damage and ROS-mediated cell death in the mutant. Additionally, lesion initiation in the ell1 mutant was light dependent and temperature sensitive. Our findings revealed that ELL1 affects chloroplast development or function, and that loss of ELL1 function induces ROS accumulation and lesion formation in rice.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Morte Celular , Cloroplastos/enzimologia , Cloroplastos/metabolismo , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/fisiologia , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Oryza/enzimologia , Oryza/genética , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia
11.
Plant Physiol Biochem ; 156: 407-419, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33010551

RESUMO

Carotenoids are a group of natural tetraterpenoid pigments with essential roles in a variety of physiological processes of plants. Although carotenoid biosynthesis has been well characterized, the genetic basis of the pathway, especially in crop plants, is largely unknown. In this study, we characterized a new albino maize mutant called albino1 (alb1), which was obtained from a Mutator mutagenized population. The alb1 mutant showed defective chloroplast development and declined photosynthetic pigments, leading to a seedling-lethal phenotype. Genetic and molecular analyses indicated that ALB1 encoded a putative ζ-carotene desaturase (ZDS) involved in carotenoid biosynthesis. Measurement of carotenoids revealed that several major carotenoid compounds downstream of the ZDS were significantly reduced in alb1 mutant, indicating that ALB1 is a functional ZDS. Further transcriptome analysis revealed that several groups of nuclear genes involved in photosynthesis, such as light-harvesting complex, pigment metabolism, and chloroplast function, were significantly down-regulated in alb1 compared with wide type. Interestingly, expression of some maize plastid-localized nuclear genes, including POR, CAO, Lhcb, and RbcS, was substantially reduced in alb1 plants. Furthermore, treatment of the inhibitor fluridone significantly rescued gene transcripts of these nucleus-encoded genes in alb1 mutant, which supported the retrograde signaling of ζ-carotene/phytofluene derived molecules. These results suggested that ALB1/ZDS might function as a regulator to coordinate nuclear photosynthetic gene expression in plastid-to-nucleus retrograde signaling during development of maize plants. Together, these results have demonstrated that ALB1/ZDS is essential for carotenoids biosynthesis and plays crucial roles in chloroplast biogenesis and development in maize.


Assuntos
Cloroplastos/enzimologia , Oxirredutases/fisiologia , Proteínas de Plantas/fisiologia , Zea mays/enzimologia , Carotenoides/metabolismo , Cloroplastos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oxirredutases/genética , Proteínas de Plantas/genética , Zea mays/genética
12.
Int J Mol Sci ; 21(19)2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32977586

RESUMO

Floral scent is a key ornamental trait that determines the quality and commercial value of orchids. Geraniol, an important volatile monoterpene in orchids that attracts pollinators, is also involved in responses to stresses but the geraniol synthase (GES) responsible for its synthesis in the medicinal orchid Dendrobium officinale has not yet been identified. In this study, three potential geraniol synthases were mined from the D. officinale genome. DoGES1, which was localized in chloroplasts, was characterized as a geraniol synthase. DoGES1 was highly expressed in flowers, especially in petals. DoGES1 transcript levels were high in the budding stage of D. officinale flowers at 11:00 a.m. DoGES1 catalyzed geraniol in vitro, and transient expression of DoGES1 in Nicotiana benthamiana leaves resulted in the accumulation of geraniol in vivo. These findings on DoGES1 advance our understanding of geraniol biosynthesis in orchids, and lay the basis for genetic modification of floral scent in D. officinale or in other ornamental orchids.


Assuntos
Proteínas de Cloroplastos , Cloroplastos , Dendrobium , Flores , Odorantes , Monoéster Fosfórico Hidrolases , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/enzimologia , Cloroplastos/genética , Dendrobium/enzimologia , Dendrobium/genética , Flores/enzimologia , Flores/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Tabaco/enzimologia , Tabaco/genética
13.
Nat Commun ; 11(1): 4509, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908151

RESUMO

Glycolysis is one of the primordial pathways of metabolism, playing a pivotal role in energy metabolism and biosynthesis. Glycolytic enzymes are known to form transient multi-enzyme assemblies. Here we examine the wider protein-protein interactions of plant glycolytic enzymes and reveal a moonlighting role for specific glycolytic enzymes in mediating the co-localization of mitochondria and chloroplasts. Knockout mutation of phosphoglycerate mutase or enolase resulted in a significantly reduced association of the two organelles. We provide evidence that phosphoglycerate mutase and enolase form a substrate-channelling metabolon which is part of a larger complex of proteins including pyruvate kinase. These results alongside a range of genetic complementation experiments are discussed in the context of our current understanding of chloroplast-mitochondrial interactions within photosynthetic eukaryotes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Cloroplastos/enzimologia , Glicólise/fisiologia , Mitocôndrias/enzimologia , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Metabolismo Energético/fisiologia , Mutação , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/fisiologia , Piruvato Quinase/genética , Piruvato Quinase/metabolismo
14.
J Agric Food Chem ; 68(37): 10109-10117, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32829629

RESUMO

Linalool is abundant in tea leaves and contributes greatly to tea aroma. The two isomers of linalool, (R)-linalool and (S)-linalool, exist in tea leaves. Our study found that (R)-linalool was the minor isomer in nine of Camellia sinensis var. sinensis cultivars. The (R)-linalool synthase of tea plant CsRLIS was identified subsequently. It is a chloroplast-located protein and specifically catalyzes the formation of (R)-linalool in vitro and in vivo. CsRLIS was observed to be a stress-responsive gene and caused the accumulation of internal (R)-linalool during oolong tea manufacture, mechanical wounding, and insect attack. Further study demonstrated that the catalytic efficiency of CsRLIS was much lower than that of (S)-linalool synthase CsSLIS, which might explain the lower (R)-linalool proportion in C. sinensis var. sinensis cultivars. The relative expression levels of CsRLIS and CsSLIS may also affect the (R)-linalool proportions among C. sinensis var. sinensis cultivars. This information will help us understand differential distributions of chiral aroma compounds in tea.


Assuntos
Monoterpenos Acíclicos/química , Camellia sinensis/enzimologia , Hidroliases/metabolismo , Proteínas de Plantas/metabolismo , Monoterpenos Acíclicos/metabolismo , Biocatálise , Camellia sinensis/química , Camellia sinensis/genética , Camellia sinensis/metabolismo , Cloroplastos/enzimologia , Cloroplastos/genética , Cloroplastos/metabolismo , Hidroliases/química , Hidroliases/genética , Odorantes/análise , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estereoisomerismo , Chá/química
15.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32630078

RESUMO

Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA's ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a KD of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein-ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket-a surface cavity around Asn35-would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP+ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data.


Assuntos
Arabidopsis/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ácido Salicílico/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cloroplastos/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , NAD , Mutação Puntual
16.
Planta ; 252(2): 29, 2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32725285

RESUMO

MAIN CONCLUSION: CsERF2, an ethylene response factor, plays a role in leaf variegation. Leaf variegation is a main ornamental characteristic in Cymbidium sinense (C. sinense). However, the mechanisms of leaf color variegation remain largely unclear. In the present study, we analyzed the cytological and physiological features, as well as molecular analyses of leaves from wild-type (WT) and leaf variegation mutants of Cymbidium sinense 'Dharma'. Chloroplasts with typical and functional structures were discovered in WT and green sectors of the mutants leaves (MG), but not in yellow sectors of the mutant leaves (MY). The activities of key enzymes involved in chlorophyll (Chl) degradation and their substrate contents were significantly increased in MY. Genes related to Chl degradation also showed a significant up-regulation in MY. Transcriptomic analysis showed that the expression of all identified ethylene response factors (ERFs) was significantly up-regulated, and the 1-aminocyclopropane-1-carboxylic acid (ACC) content in MY was significantly higher compared with MG. QRT-PCR analysis validated that the expression levels of genes related to Chl degradation could be positively affected by ethylene (ETH) treatment. Stable overexpression of CsERF2 in Nicotiana tabacum (N. tabacum) led to a decrease in Chl content and abnormal chloroplast. Transcriptomic analysis and qRT-PCR results showed that the KEGG pathway related to chloroplast development and function showed significant change in transgenic N. tabacum. Therefore, the leaf color formation of C. sinense was greatly affected by chloroplast development and Chl metabolism. CsERF2 played an important role in leaf variegation of C. sinense.


Assuntos
Orchidaceae/fisiologia , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Clorofila/metabolismo , Cloroplastos/enzimologia , Cloroplastos/ultraestrutura , Regulação da Expressão Gênica de Plantas , Mutação/genética , Orchidaceae/enzimologia , Orchidaceae/genética , Fenótipo , Fotossíntese/genética , Pigmentação/genética , Folhas de Planta/enzimologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Tabaco/genética , Regulação para Cima
17.
Planta ; 252(1): 13, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32621079

RESUMO

MAIN CONCLUSION: NtALS1 is specifically expressed in glandular trichomes, and can improve the content of acylsugars in tobacco. ABTRACT: The glandular trichomes of many species in the Solanaceae family play an important role in plant defense. These epidermal outgrowths exhibit specialized secondary metabolism, including the production of structurally diverse acylsugars that function in defense against insects and have substantial developmental potential for commercial uses. However, our current understanding of genes involved in acyl chain biosynthesis of acylsugars remains poor in tobacco. In this study, we identified three acetolactate synthase (ALS) genes in tobacco through homology-based gene prediction using Arabidopsis ALS. Quantitative real-time PCR (qRT-PCR) and tissue distribution analyses suggested that NtALS1 was highly expressed in the tips of glandular trichomes. Subcellular localization analysis showed that the NtALS1 localized to the chloroplast. Moreover, in the wild-type K326 variety background, we generated two ntals1 loss-of-function mutants using the CRISPR-Cas9 system. Acylsugars contents in the two ntals1 mutants were significantly lower than those in the wild type. Through phylogenetic tree analysis, we also identified NtALS1 orthologs that may be involved in acylsugar biosynthesis in other Solanaceae species. Taken together, these findings indicate a functional role for NtALS1 in acylsugar biosynthesis in tobacco.


Assuntos
Acetolactato Sintase/genética , Açúcares/metabolismo , Tabaco/metabolismo , Tricomas/enzimologia , Acetolactato Sintase/metabolismo , Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas , Cloroplastos/enzimologia , Diploide , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Tabaco/genética , Tricomas/genética
18.
Plant Physiol ; 184(1): 110-129, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32663165

RESUMO

Chloroplast proteostasis is governed by a network of peptidases. As a part of this network, we show that Arabidopsis (Arabidopsis thaliana) chloroplast glutamyl peptidase (CGEP) is a homo-oligomeric stromal Ser-type (S9D) peptidase with both exo- and endo-peptidase activity. Arabidopsis CGEP null mutant alleles (cgep) had no visible phenotype but showed strong genetic interactions with stromal CLP protease system mutants, resulting in reduced growth. Loss of CGEP upregulated the chloroplast protein chaperone machinery and 70S ribosomal proteins, but other parts of the proteostasis network were unaffected. Both comparative proteomics and mRNA-based coexpression analyses strongly suggested that the function of CGEP is at least partly involved in starch metabolism regulation. Recombinant CGEP degraded peptides and proteins smaller than ∼25 kD. CGEP specifically cleaved substrates on the C-terminal side of Glu irrespective of neighboring residues, as shown using peptide libraries incubated with recombinant CGEP and mass spectrometry. CGEP was shown to undergo autocatalytic C-terminal cleavage at E946, removing 15 residues, both in vitro and in vivo. A conserved motif (A[S/T]GGG[N/G]PE946) immediately upstream of E946 was identified in dicotyledons, but not monocotyledons. Structural modeling suggested that C-terminal processing increases the upper substrate size limit by improving catalytic cavity access. In vivo complementation with catalytically inactive CGEP-S781R or a CGEP variant with an unprocessed C-terminus in a cgep clpr2-1 background was used to demonstrate the physiological importance of both CGEP peptidase activity and its autocatalytic processing. CGEP homologs of photosynthetic and nonphotosynthetic bacteria lack the C-terminal prosequence, suggesting it is a recent functional adaptation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/enzimologia , Peptídeo Hidrolases/metabolismo , Catálise , Regulação da Expressão Gênica de Plantas , Proteínas Ribossômicas/metabolismo , Especificidade por Substrato
19.
Sci Rep ; 10(1): 10846, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616740

RESUMO

In plants, the shikimate pathway generally occurs in plastids and leads to the biosynthesis of aromatic amino acids. Chorismate synthase (CS) catalyses the last step of the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, but the role of CS in the metabolism of higher plants has not been reported. In this study, we found that PhCS, which is encoded by a single-copy gene in petunia (Petunia hybrida), contains N-terminal plastidic transit peptides and peroxisomal targeting signals. Green fluorescent protein (GFP) fusion protein assays revealed that PhCS was localized in chloroplasts and, unexpectedly, in peroxisomes. Petunia plants with reduced PhCS activity were generated through virus-induced gene silencing and further characterized. PhCS silencing resulted in reduced CS activity, severe growth retardation, abnormal flower and leaf development and reduced levels of folate and pigments, including chlorophylls, carotenoids and anthocyanins. A widely targeted metabolomics analysis showed that most primary and secondary metabolites were significantly changed in pTRV2-PhCS-treated corollas. Overall, the results revealed a clear connection between primary and specialized metabolism related to the shikimate pathway in petunia.


Assuntos
Antocianinas/metabolismo , Cloroplastos/enzimologia , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Peroxissomos/enzimologia , Petunia/crescimento & desenvolvimento , Fósforo-Oxigênio Liases/metabolismo , Flores/metabolismo , Petunia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
20.
Plant J ; 103(3): 1140-1154, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32365245

RESUMO

Thiol-based redox-regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin-dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione-mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo-lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH ) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild-type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.


Assuntos
Cloroplastos/metabolismo , Glutationa Redutase/metabolismo , Fotossíntese , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Bryopsida/enzimologia , Bryopsida/metabolismo , Bryopsida/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Cloroplastos/enzimologia , Cloroplastos/fisiologia , Técnicas de Inativação de Genes , Glutationa/metabolismo , Glutationa Redutase/fisiologia , Oxirredução
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