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1.
BMC Plant Biol ; 21(1): 411, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496770

RESUMO

BACKGROUND: The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by flooding, wounding, drought, and pathogen attack as well as during abscission and fruit ripening. In kiwifruit (Actinidia spp.), fruit ripening is characterized by two distinct phases: an early phase of system-1 ethylene biosynthesis characterized by absence of autocatalytic ethylene, followed by a late burst of autocatalytic (system-2) ethylene accompanied by aroma production and further ripening. Progress has been made in understanding the transcriptional regulation of kiwifruit fruit ripening but the regulation of system-1 ethylene biosynthesis remains largely unknown. The aim of this work is to better understand the transcriptional regulation of both systems of ethylene biosynthesis in contrasting kiwifruit organs: fruit and leaves. RESULTS: A detailed molecular study in kiwifruit (A. chinensis) revealed that ethylene biosynthesis was regulated differently between leaf and fruit after mechanical wounding. In fruit, wound ethylene biosynthesis was accompanied by transcriptional increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) and members of the NAC class of transcription factors (TFs). However, in kiwifruit leaves, wound-specific transcriptional increases were largely absent, despite a more rapid induction of ethylene production compared to fruit, suggesting that post-transcriptional control mechanisms in kiwifruit leaves are more important. One ACS member, AcACS1, appears to fulfil a dominant double role; controlling both fruit wound (system-1) and autocatalytic ripening (system-2) ethylene biosynthesis. In kiwifruit, transcriptional regulation of both system-1 and -2 ethylene in fruit appears to be controlled by temporal up-regulation of four NAC (NAM, ATAF1/2, CUC2) TFs (AcNAC1-4) that induce AcACS1 expression by directly binding to the AcACS1 promoter as shown using gel-shift (EMSA) and by activation of the AcACS1 promoter in planta as shown by gene activation assays combined with promoter deletion analysis. CONCLUSIONS: Our results indicate that in kiwifruit the NAC TFs AcNAC2-4 regulate both system-1 and -2 ethylene biosynthesis in fruit during wounding and ripening through control of AcACS1 expression levels but not in leaves where post-transcriptional/translational regulatory mechanisms may prevail.


Assuntos
Actinidia/genética , Etilenos/biossíntese , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Actinidia/metabolismo , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Liases/genética , Liases/metabolismo , Lycopersicon esculentum/genética , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
2.
PLoS One ; 16(9): e0256863, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34492058

RESUMO

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene's mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5' nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5' nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5' nt identities and different pairing states between the 5' antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5' nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5' thermodynamic rules governing topical siRNA efficacy across plants and animals.


Assuntos
Proteínas Argonauta/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Tabaco/genética , Proteínas Argonauta/antagonistas & inibidores , Regulação da Expressão Gênica/genética , Inativação Gênica , Proteínas de Fluorescência Verde/antagonistas & inibidores , Proteínas de Fluorescência Verde/genética , Humanos , Liases/antagonistas & inibidores , Liases/genética , Ligação Proteica/genética , Tabaco/crescimento & desenvolvimento
3.
J Biotechnol ; 341: 30-42, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34500003

RESUMO

Phycobiliproteins are colored, active molecules with potential use in different industries. They are the union of proteins and bilins (Chromophores). The primary source of phycobiliproteins is algae; however, the traditional algae culture has production restrictions. The production in bacterial models can be a more efficient alternative to produce these molecules. However, the lack of knowledge in some steps of the phycobiliprotein metabolic pathway limits this alternative. Porphyridium cruentum is a single cell red alga with a high phycobiliprotein content. Its protein sequences were the basis for phycobilin production in this study. In this study, we cloned and characterized enzymes presumably involved in the chromophore production of P. cruentum. Using sequences obtained from its transcriptome, we characterized two cDNA sequences predicted to code respectively for a ferredoxin-dependent bilin reductase and a bilin lyase-isomerase. We expressed these enzymes in Escherichia coli to obtain in vivo evidence of their enzymatic activity on the substrate biliverdin IXα. Lastly, we analyzed them using thin-layer chromatography, spectrophotometry, and fluorescence spectroscopy. These experiments provided evidence of bilin modification. The expressed bilin lyase-isomerase did not show significant activity over the biliverdin molecule. On the contrary, the expressed ferredoxin-dependent bilin reductase showed activity over the biliverdin.


Assuntos
Cianobactérias , Liases , Porphyridium , Rodófitas , Ficobilinas , Porphyridium/genética , Rodófitas/genética
4.
Zhonghua Nei Ke Za Zhi ; 60(9): 827-830, 2021 Sep 01.
Artigo em Chinês | MEDLINE | ID: mdl-34445820

RESUMO

The 17α-hydroxylase/17, 20-lyase deficiency (17-OHD) is a rare disease. The clinical characteristics and gene mutation of 2 late-diagnosed 17-OHD patients with testicular tumor admitted to our hospital from March 2018 to February 2019 were analyzed retrospectively. The two 17-OHD patients were female (46, XY). Laparoscopic abdominal exploration found undeveloped testicles in grey-yellow or grey-red in the groin and iliac fossa. The testicles were removed and showed malignancy in pathology study. Sequencing of the CYP17A1 gene identified c.1247G>A/c.1427T>C and c.985_987delTACinsAA/c.1306G>A complex heterozygous mutations. Taking together, the possibility of 17-OHD should be considered in patients with hypertension, hypokalemia, adrenal adenomatoid hyperplasia together with 46, XY gonadal dysplasia, so as to make early diagnosis and treatment, and avoid dysplastic testicular turning to malignancy.


Assuntos
Hiperplasia Suprarrenal Congênita , Liases , Neoplasias Testiculares , Hiperplasia Suprarrenal Congênita/genética , Feminino , Humanos , Masculino , Oxigenases de Função Mista , Mutação , Estudos Retrospectivos , Esteroide 17-alfa-Hidroxilase/genética , Neoplasias Testiculares/genética
5.
Gene ; 805: 145910, 2021 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-34419567

RESUMO

Ethylene is an important regulatory phytohormone for sex differentiation and flower development. As the rate-limiting enzyme encoding genes in ethylene biosynthesis, ACS gene family has been well studied in cucumber; however, little is known in other cucurbit crops, such as melon and watermelon, which show diverse sex types in the field. Here, we identified and characterized eight ACS genes each in the genomes of melon and watermelon. According to the conserved serine residues at C-terminal, all the ACS genes could be characterized into three groups, which were supported by the exon-intron organizations and conserved motif distributions. ACS genes displayed diverse tissue-specific expression patterns among four melon and three watermelon sex types. Furthermore, a comparative expression analysis in the shoot apex identified orthologous pairs with potential functions in sex determination, e.g., ACS1s and ACS6s. All ACS orthologs in melon and watermelon exhibited similar expression patterns in monoecious and gynoecious genotypes, except for ACS11s and ACS12s. As expected, the majority of ACS genes were responsive to exogenous ethephon; however, some orthologs exhibited opposite expression patterns, such as ACS1s, ACS9s, and ACS10s. Collectively, our findings provide valuable ACS candidates related to flower development in various sex types of melon and watermelon.


Assuntos
Cucurbitaceae/genética , Etilenos/metabolismo , Liases/metabolismo , Diferenciação Sexual/genética , Citrullus/genética , Citrullus/metabolismo , Cucumis sativus/genética , Cucumis sativus/metabolismo , Cucurbitaceae/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Genótipo , Liases/genética , Filogenia , Proteínas de Plantas/genética , Diferenciação Sexual/fisiologia
6.
Sci Rep ; 11(1): 16186, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376731

RESUMO

Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.


Assuntos
Sistemas CRISPR-Cas , Cryptomeria/genética , Edição de Genes , Liases/antagonistas & inibidores , Mutagênese , Mutação , Plantas Geneticamente Modificadas/genética , Cryptomeria/crescimento & desenvolvimento , Vetores Genéticos , Genoma de Planta , Japão , Liases/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
7.
Nat Commun ; 12(1): 3487, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108468

RESUMO

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.


Assuntos
Alquil e Aril Transferases/química , Diterpenos/metabolismo , Proteínas Fúngicas/química , Alquil e Aril Transferases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Ciclização , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeos/biossíntese , Liases/química , Liases/metabolismo , Enzimas Multifuncionais , Fosfatos de Poli-Isoprenil/metabolismo , Conformação Proteica
8.
Elife ; 102021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33973521

RESUMO

Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control.


Assuntos
Bactérias/enzimologia , Citocromos c/metabolismo , Liases/metabolismo , Mitocôndrias/metabolismo , Domínio Catalítico , Escherichia coli/metabolismo , Heme/metabolismo , Humanos , Técnicas In Vitro , Liases/química , Peptídeos/química , Especificidade por Substrato
9.
J Bacteriol ; 203(15): e0016421, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34001558

RESUMO

A previously identified transcriptional regulator in Campylobacter jejuni, termed HeuR, was found to positively regulate heme utilization. Additionally, transcriptomic work demonstrated that the putative operons CJJ81176_1390 to CJJ81176_1394 (CJJ81176_1390-1394) and CJJ81176_1214-1217 were upregulated in a HeuR mutant, suggesting that HeuR negatively regulates expression of these genes. Because genes within these clusters include a cystathionine ß-lyase (metC) and a methionine synthase (metE), it appeared HeuR negatively regulates C. jejuni methionine biosynthesis. To address this, we confirmed mutation of HeuR reproducibly results in metC overexpression under nutrient-replete conditions but did not affect expression of metE, while metC expression in the wild type increased to heuR mutant levels during iron limitation. We subsequently determined that both gene clusters are operonic and demonstrated the direct interaction of HeuR with the predicted promoter regions of these operons. Using DNase footprinting assays, we were able to show that HeuR specifically binds within the predicted -35 region of the CJJ81176_1390-1394 operon. As predicted based on transcriptional results, the HeuR mutant was able to grow and remain viable in a defined medium with and without methionine, but we identified significant impacts on growth and viability in metC and metE mutants. Additionally, we observed decreased adherence, invasion, and persistence of metC and metE mutants when incubated with human colonocytes, while the heuR mutant exhibited increased invasion. Taken together, these results suggest that HeuR regulates methionine biosynthesis in an iron-responsive manner and that the ability to produce methionine is an important factor for adhering to and invading the gastrointestinal tract of a susceptible host. IMPORTANCE As the leading cause of bacterium-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health. Investigating colonization factors that allow C. jejuni to successfully infect a host furthers our understanding of genes and regulatory elements necessary for virulence. In this study, we have begun to characterize the role of the transcriptional regulatory protein, HeuR, on methionine biosynthesis in C. jejuni. When the ability to synthesize methionine is impaired, detrimental impacts on growth and viability are observed during growth in limited media lacking methionine and/or iron. Additionally, mutations in the methionine biosynthetic pathway result in decreased adhesion, invasion, and intracellular survival of C. jejuni when incubated with human colonocytes, indicating the importance of regulating methionine biosynthesis.


Assuntos
Proteínas de Bactérias/genética , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/enzimologia , Colo/microbiologia , Regulação Bacteriana da Expressão Gênica , Liases/genética , Metionina/biossíntese , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/genética , Células HCT116 , Humanos , Liases/metabolismo , Família Multigênica , Óperon
10.
mBio ; 12(3)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947751

RESUMO

In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes c and c 1 is catalyzed by either multisubunit cytochrome c maturation system I or holocytochrome c synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of de novo protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids Trypanosoma and Leishmania parasites, attach heme to mitochondrial c-type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome c maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome c synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome c KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome c maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists.IMPORTANCE Cytochromes c are essential proteins for respiratory and photosynthetic electron transfer. They are posttranslationally modified by covalent attachment of a heme cofactor. Kinetoplastids include important tropical disease-causing parasites; many aspects of their biology differ from other organisms, including their mammalian or plant hosts. Uniquely, kinetoplastids produce cytochromes c with a type of heme attachment not seen elsewhere in nature and were the only cytochrome c-bearing taxa without evidence of protein machinery to attach heme to the apocytochrome. Using bioinformatics, biochemistry, and molecular genetics, we report how kinetoplastids make their cytochromes c Unexpectedly, they use a highly diverged version of an enzyme used for heme-protein attachment in many eukaryotes. Mutations in the human enzyme lead to genetic disease. Identification of kinetoplastid cytochrome c synthetase, thus, solves an evolutionary unknown, provides a possible target for antiparasite drug development, and an unanticipated resource for studying the mechanistic basis of a human genetic disease.


Assuntos
Citocromos c/genética , Citocromos c/fisiologia , Eucariotos/fisiologia , Biologia Computacional , Leishmania mexicana/genética , Leishmania mexicana/fisiologia , Liases/química , Liases/genética , Liases/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiologia
11.
Nat Chem Biol ; 17(7): 800-805, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33958791

RESUMO

The covalent attachment of one or multiple heme cofactors to cytochrome c protein chains enables cytochrome c proteins to be used in electron transfer and redox catalysis in extracytoplasmic environments. A dedicated heme maturation machinery, whose core component is a heme lyase, scans nascent peptides after Sec-dependent translocation for CXnCH-binding motifs. Here we report the three-dimensional (3D) structure of the heme lyase CcmF, a 643-amino acid integral membrane protein, from Thermus thermophilus. CcmF contains a heme b cofactor at the bottom of a large cavity that opens toward the extracellular side to receive heme groups from the heme chaperone CcmE for cytochrome maturation. A surface groove on CcmF may guide the extended apoprotein to heme attachment at or near a loop containing the functionally essential WXWD motif, which is situated above the putative cofactor binding pocket. The structure suggests heme delivery from within the membrane, redefining the role of the chaperone CcmE.


Assuntos
Membrana Celular/metabolismo , Liases/metabolismo , Membrana Celular/química , Liases/química , Thermus thermophilus/enzimologia
12.
Biochemistry ; 60(20): 1609-1618, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33949189

RESUMO

d-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of d-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D2O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.


Assuntos
Amônia-Liases/metabolismo , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Fosfato de Piridoxal/metabolismo , Aminoácidos/metabolismo , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Glucosamina/metabolismo , Glucose-6-Fosfato/metabolismo , Cinética , Liases/metabolismo , Modelos Moleculares , Fosfatos , Bases de Schiff , Especificidade por Substrato , Transaminases/metabolismo
13.
Biomolecules ; 11(4)2021 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-33920493

RESUMO

Listeria monocytogenes, the bacterial foodborne pathogen responsible for the severe disease listeriosis, frequently exhibits heavy metal resistance. Concurrent resistance to cadmium and arsenic in L. monocytogenes is strongly associated with the 35-kb chromosomal island LGI2. LGI2 has been encountered repeatedly among L. monocytogenes serotype 4b hypervirulent clones but, surprisingly, not among non-pathogenic Listeria spp. Here we describe a novel LGI2 variant, LGI2-3, in two L. welshimeri strains from an urban aquatic environment. Whole genome sequence analysis revealed that the genomes were closely related except for one prophage region and confirmed a chromosomally integrated LGI2-3. It harbored a cystathionine beta-lyase gene previously only encountered in LGI2-1 of L. monocytogenes clonal complex 1 but was otherwise most closely related to LGI2. LGI2-3 harbored a novel cadAC cassette (cadA7C7) that, like LGI2's cadA4C4, was associated with lower-level tolerance to cadmium (MIC 50 µg/mL) than other cadAC cassettes (MIC ≥ 140 µg/mL). CadA sequence analysis identified two amino acids that may be important for mediating different levels of cadmium tolerance. Our findings clearly demonstrated the potential for LGI2-like islands to be harbored by non-pathogenic Listeria spp. and generate intriguing hypotheses on the genetic diversity mediated by this island and its transfer among Listeria spp.


Assuntos
Arsênio/toxicidade , Cádmio/toxicidade , Farmacorresistência Bacteriana , Ilhas Genômicas , Listeria/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Listeria/efeitos dos fármacos , Liases/genética , Liases/metabolismo
14.
Plant Cell ; 33(2): 306-321, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793793

RESUMO

Unisexual flowers provide a useful system for studying plant sex determination. In cucumber (Cucumis sativus L.), three major Mendelian loci control unisexual flower development, Female (F), androecious [a; 1-aminocyclopropane-1-carboxylate {ACC} synthase 11, acs11], and Monoecious (M; ACS2), referred to here as the Female, Androecious, Monoecious (FAM) model, in combination with two genes, gynoecious (g, the WIP family C2H2 zinc finger transcription factor gene WIP1) and the ethylene biosynthetic gene ACC oxidase 2 (ACO2). The F locus, conferring gynoecy and the potential for increasing fruit yield, is defined by a 30.2-kb tandem duplication containing three genes. However, the gene that determines the Female phenotype, and its mechanism, remains unknown. Here, we created a set of mutants and revealed that ACS1G is responsible for gynoecy conferred by the F locus. The duplication resulted in ACS1G acquiring a new promoter and expression pattern; in plants carrying the F locus duplication, ACS1G is expressed early in floral bud development, where it functions with ACO2 to generate an ethylene burst. The resulting ethylene represses WIP1 and activates ACS2 to initiate gynoecy. This early ACS1G expression bypasses the need for ACS11 to produce ethylene, thereby establishing a dominant pathway for female floral development. Based on these findings, we propose a model for how these ethylene biosynthesis genes cooperate to control unisexual flower development in cucumber.


Assuntos
Cucumis sativus/enzimologia , Cucumis sativus/genética , Flores/enzimologia , Flores/genética , Liases/genética , Sequência de Aminoácidos , Regulação da Expressão Gênica de Plantas , Loci Gênicos , Genoma de Planta , Genótipo , Glucuronidase/metabolismo , Liases/química , Fenótipo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
Mol Biol (Mosk) ; 55(2): 269-276, 2021.
Artigo em Russo | MEDLINE | ID: mdl-33871440

RESUMO

One of the most common DNA lesions is the appearance of apurinic/apyrimidinic (AP-) sites. The main repair pathway for AP sites is initiated by apurinic/apyrimidinic endonuclease 1 (APE1). Upon hydrolysis of the phosphodiester bond by this enzyme, a one nucleotide gap flanked by 3'-hydroxyl and 5'-deoxyribose phosphate groups on the 5'-side of the AP site is formed. After hydrolysis of the AP site, APE1 remains associated with the product for some time. In the present work, the ability of APE1 to form a product of covalent attachment of APE1 to DNA containing a gap with a 5'-deoxyribose phosphate residue was demonstrated. In addition, it was found that while in a complex with the product of hydrolysis of the AP site, APE1 exhibits 5'-deoxyribose phosphate lyase activity, cleaving off the 5'-deoxyribose phosphate residue. The presence of lyase activity in APE1 may be important for the repair of AP sites if there is a deficiency of, or mutations in DNA polymerase ß, the main enzyme that removes the 5'-deoxyribose phosphate group.


Assuntos
Liases , Reparo do DNA , Endonucleases , Fósforo-Oxigênio Liases
16.
Plant Sci ; 307: 110902, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33902860

RESUMO

During leaf senescence, the degradation of photosystems and photosynthetic pigments proceeds in a coordinated manner, which would minimize the potential photodamage to cells. Both photosystem I and II are composed of core complexes and peripheral antenna complexes, with the former binding chlorophyll a and the latter binding chlorophyll a and b. Although the degradation of peripheral antenna complexes is initiated by chlorophyll degradation, it remains unclear whether the degradation of core complexes and chlorophyll is coordinated. In this study, we examined the degradation of peripheral antenna and core complexes in the Arabidopsis sgr1/sgr2/sgrl triple mutant, lacking all the isoforms of chlorophyll a:Mg2+ dechelatase. In this mutant, the degradation of peripheral antenna complexes and photosystem I core complexes was substantially retarded, but the core complexes of photosystem II were rapidly degraded during leaf senescence. On the contrary, the photosynthetic activity declined at a similar rate as in the wild type plants. These results suggest that the degradation of photosystem II core complexes is regulated independently of the major chlorophyll degradation pathway mediated by the dechelatase. The study should contribute to the understanding of the complex molecular mechanisms underlying the degradation of photosystems, which is an essential step during leaf senescence.


Assuntos
Envelhecimento/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Clorofila/metabolismo , Liases/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Envelhecimento/genética , Clorofila/genética , Variação Genética , Mutação , Complexo de Proteína do Fotossistema II/genética
17.
J Biol Chem ; 296: 100571, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33753170

RESUMO

It has been recognized for >50 years that cytochrome b5 (b5) stimulates some cytochrome P450 (P450)-catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b5 function. One of the issues in studying b5-P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b5 that can be used in titrations. The labeled b5 bound to WT P450 17A1 with a Kd of 2.5 nM and rapid kinetics, on the order of 1 s-1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b5 binding. Kd values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b5 fluorescence at higher concentrations. The addition of NADPH-P450 reductase (POR) to an Alexa 488-T70C-b5:P450 17A1 complex resulted in a concentration-dependent partial restoration of b5 fluorescence, indicative of a ternary P450:b5:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.


Assuntos
Androgênios/biossíntese , Citocromos b5/metabolismo , Liases/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Humanos , Cinética , Mutação , Ligação Proteica , Esteroide 17-alfa-Hidroxilase/genética
18.
Genes (Basel) ; 12(2)2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670341

RESUMO

Mitochondrial disorders, although heterogeneous, are traditionally described as conditions characterized by encephalomyopathy, hypotonia, and progressive postnatal organ failure. Here, we provide a systematic review of Linear Skin Defects with Multiple Congenital Anomalies (LSDMCA), a rare, unconventional mitochondrial disorder which presents as a developmental disease; its main clinical features include microphthalmia with different degrees of severity, linear skin lesions, and central nervous system malformations. The molecular basis of this disorder has been elusive for several years. Mutations were eventually identified in three X-linked genes, i.e., HCCS, COX7B, and NDUFB11, which are all endowed with defined roles in the mitochondrial respiratory chain. A peculiar feature of this condition is its inheritance pattern: X-linked dominant male-lethal. Only female or XX male individuals can be observed, implying that nullisomy for these genes is incompatible with normal embryonic development in mammals. All three genes undergo X-inactivation that, according to our hypothesis, may contribute to the extreme variable expressivity observed in this condition. We propose that mitochondrial dysfunction should be considered as an underlying cause in developmental disorders. Moreover, LSDMCA should be taken into consideration by clinicians when dealing with patients with microphthalmia with or without associated skin phenotypes.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Microftalmia/genética , Doenças Mitocondriais/genética , Anormalidades da Pele/genética , Cromossomos Humanos X/genética , Complexo I de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Liases/genética , Masculino , Microftalmia/patologia , Doenças Mitocondriais/patologia , Mutação/genética , Pele/patologia , Anormalidades da Pele/patologia
19.
J Exp Bot ; 72(12): 4535-4547, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33770168

RESUMO

Plant responses to pathogens comprise a complex process, implying a plethora of signals and reactions. Among them, endogenous production of hydrogen cyanide (HCN) has been shown to induce resistance in Arabidopsis to the hemibiotrophic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000. ß-cyanoalanine synthase (CAS-C1) is responsible for the detoxification of HCN in Arabidopsis mitochondria. Here, we show that green fluorescent protein-tagged CAS-C1 is transiently reduced in leaves infected with an avirulent strain of Pst during early interactions and increased in leaves infected with a virulent strain of Pst, supporting previous transcriptional data. Genetic crosses show that mutation in CAS-C1 in Arabidopsis resembles the action of the NADPH oxidase RbohD independently of reactive oxygen species production and that the accumulation of salicylic acid is required for HCN-stimulated resistance to Pst. Finally, we show that the cas-c1 mutation acts on the salicylic acid-dependent response to pathogens by mechanisms other than protein ubiquitination or the increase of monomerization and entry to the nucleus of NPR1, the central regulator of the salicylic acid-mediated response. Considering these results, we propose new mechanisms for modulation of the immune response by HCN.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Liases , Mutação , NADPH Oxidases/metabolismo , Doenças das Plantas/genética , Pseudomonas syringae/metabolismo , Ácido Salicílico
20.
Plant Physiol ; 185(2): 491-502, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33721891

RESUMO

The genus Cuscuta comprises stem holoparasitic plant species with wide geographic distribution. Cuscuta spp. obtain water, nutrients, proteins, and mRNA from their host plants via a parasitic organ called the haustorium. As the haustorium penetrates into the host tissue, search hyphae elongate within the host tissue and finally connect with the host's vascular system. Invasion by Cuscuta spp. evokes various reactions within the host plant's tissues. Here, we show that, when Arabidopsis (Arabidopsis thaliana) is invaded by Cuscuta campestris, ethylene biosynthesis by the host plant promotes elongation of the parasite's search hyphae. The expression of genes encoding 1-aminocylclopropane-1-carboxylic acid (ACC) synthases, ACC SYNTHASE2 (AtACS2) and ACC SYNTHASE6 (AtACS6), was activated in the stem of Arabidopsis plants upon invasion by C. campestris. When the ethylene-deficient Arabidopsis acs octuple mutant was invaded by C. campestris, cell elongation and endoreduplication of the search hyphae were significantly reduced, and the inhibition of search hyphae growth was complemented by exogenous application of ACC. In contrast, in the C. campestris-infected Arabidopsis ethylene-insensitive mutant etr1-3, no growth inhibition of search hyphae was observed, indicating that ETHYLENE RESPONSE1-mediated ethylene signaling in the host plant is not essential for parasitism by C. campestris. Overall, our results suggest that C. campestris recognizes host-produced ethylene as a stimulatory signal for successful invasion.


Assuntos
Arabidopsis/genética , Cuscuta/fisiologia , Etilenos/metabolismo , Doenças das Plantas/parasitologia , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Arabidopsis/metabolismo , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Crescimento Celular , Cuscuta/genética , Endorreduplicação , Interações Hospedeiro-Parasita , Liases/genética , Liases/metabolismo , Mutação , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
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