RESUMO
In recent years, many attempts have been made to find new plant proteases to make artisan cheeses. The global increase in cheese consumption, together with a lower supply and increasing cost of calf rennet, religious factors (Islam and Judaism) and food choices (vegetarianism) have led to the search for suitable rennet substitutes for milk clotting. This study describes the milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits (SoAP) on individual caseins to explore its potential use as an alternative to animal rennet. The milk-clotting index obtained for SoAP was 8.4 times lower than that obtained for chymosin. SoAP showed a higher degree of hydrolysis on α-casein than on the other fractions under the proposed conditions. RP-HPLC, mass spectrometry analyses and sequencing of the hydrolysates allowed identifying five peptides from α-casein, one peptide from ß-casein, and three peptides from k-casein. In silico analysis showed that the peptides identified may display a wide variety of potential biological activities. These results demonstrate the possibility of using SoAP for the manufacture of new types or artisan cheeses, with the simultaneous added value of the potential health-promoting benefits of the bioactive peptides generated during the hydrolysis.
Assuntos
Ácido Aspártico Proteases/química , Caseínas/química , Frutas/enzimologia , Leite/química , Solanaceae/enzimologia , Animais , Ácido Aspártico Proteases/isolamento & purificação , Queijo/análise , Fenômenos Químicos , Ativação Enzimática , Frutas/química , Hidrólise , Cinética , Extratos Vegetais , Solanaceae/química , Relação Estrutura-AtividadeRESUMO
Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside.
Assuntos
Oxirredutases do Álcool/biossíntese , Flores/genética , Regulação da Expressão Gênica de Plantas , Petunia/genética , Proteínas de Plantas/biossíntese , Plantas Geneticamente Modificadas , Oxirredutases do Álcool/genética , Antocianinas/biossíntese , Cor , Flavonoides/metabolismo , Flores/anatomia & histologia , Flores/enzimologia , Petunia/anatomia & histologia , Petunia/enzimologia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Rosa/química , Rosa/enzimologia , Solanaceae/química , Solanaceae/enzimologia , TransgenesRESUMO
PREMISE OF THE STUDY: White forms of typically pigmented flowers are one of the most common polymorphisms in flowering plants. Although the range of genetic changes that give rise to white phenotypes is well known from model systems, few studies have identified causative mutations in natural populations. METHODS: Here we combine genetic studies, in vitro enzyme assays, and biochemical analyses to identify the mechanism underlying the loss of anthocyanin pigment production in the naturally occurring white-flowered morph of Iochroma calycinum (Solanaceae). KEY RESULTS: Comparison of anthocyanin gene sequences revealed a putative loss-of-function mutation, an 11 amino-acid deletion in dihydroflavonol 4-reductase (DFR), in the white morph. Functional assays of Dfr alleles from blue and white morphs demonstrated that this deletion results in a loss of enzymatic activity, indicating that the deletion could be solely responsible for the lack of pigment production. Consistent with this hypothesis, quantitative PCR showed no significant differences in expression of anthocyanin genes between the morphs. Also, thin layer chromatography confirmed that the white morph continues to accumulate compounds upstream of the DFR enzyme. CONCLUSIONS: Collectively, these experiments indicate that the structural mutation at Dfr underlies the rare white flower morph of I. calycinum. This study is one of only a few examples where a flower color polymorphism is due to a loss-of-function mutation in the coding region of an anthocyanin enzyme. The rarity of such mutations in nature suggests that negative consequences prevent fixation across populations.
Assuntos
Oxirredutases do Álcool/genética , Antocianinas/genética , Flores/metabolismo , Mutação , Pigmentação/genética , Polimorfismo Genético , Solanaceae/genética , Oxirredutases do Álcool/metabolismo , Alelos , Sequência de Aminoácidos , Antocianinas/biossíntese , Flores/enzimologia , Genes de Plantas , Fenótipo , Pigmentos Biológicos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Solanaceae/enzimologia , Solanaceae/metabolismo , América do Sul , Especificidade da EspécieRESUMO
BACKGROUND: The most frequent case of horizontal transfer in plants involves a group I intron in the mitochondrial gene cox1, which has been acquired via some 80 separate plant-to-plant transfer events among 833 diverse angiosperms examined. This homing intron encodes an endonuclease thought to promote the intron's promiscuous behavior. A promising experimental approach to study endonuclease activity and intron transmission involves somatic cell hybridization, which in plants leads to mitochondrial fusion and genome recombination. However, the cox1 intron has not yet been found in the ideal group for plant somatic genetics - the Solanaceae. We therefore undertook an extensive survey of this family to find members with the intron and to learn more about the evolutionary history of this exceptionally mobile genetic element. RESULTS: Although 409 of the 426 species of Solanaceae examined lack the cox1 intron, it is uniformly present in three phylogenetically disjunct clades. Despite strong overall incongruence of cox1 intron phylogeny with angiosperm phylogeny, two of these clades possess nearly identical intron sequences and are monophyletic in intron phylogeny. These two clades, and possibly the third also, contain a co-conversion tract (CCT) downstream of the intron that is extended relative to all previously recognized CCTs in angiosperm cox1. Re-examination of all published cox1 genes uncovered additional cases of extended co-conversion and identified a rare case of putative intron loss, accompanied by full retention of the CCT. CONCLUSIONS: We infer that the cox1 intron was separately and recently acquired by at least three different lineages of Solanaceae. The striking identity of the intron and CCT from two of these lineages suggests that one of these three intron captures may have occurred by a within-family transfer event. This is consistent with previous evidence that horizontal transfer in plants is biased towards phylogenetically local events. The discovery of extended co-conversion suggests that other cox1 conversions may be longer than realized but obscured by the exceptional conservation of plant mitochondrial sequences. Our findings provide further support for the rampant-transfer model of cox1 intron evolution and recommend the Solanaceae as a model system for the experimental analysis of cox1 intron transfer in plants.
Assuntos
Ciclo-Oxigenase 1/genética , Evolução Molecular , Transferência Genética Horizontal/genética , Íntrons/genética , Filogenia , Solanaceae/enzimologia , Sequência de Bases , Biologia Computacional , Primers do DNA/genética , Funções Verossimilhança , Modelos Genéticos , Alinhamento de Sequência , Solanaceae/genéticaRESUMO
Self-incompatibility in the Solanaceae is mediated by S-RNase alleles expressed in the style, which confer specificity for pollen recognition. Nicotiana alata has been successfully used as an experimental model to elucidate cellular and molecular aspects of S-RNase-based self-incompatibility in Solanaceae. However, S-RNase alleles of this species have not been surveyed from natural populations and consequently the S-haplotype diversity is poorly known. Here the molecular and functional characterization of seven S-RNase candidate sequences, identified from a natural population of N. alata, are reported. Six of these candidates, S ( 5 ), S ( 27 ), S ( 70 ), S ( 75 ), S ( 107 ), and S ( 210 ), showed plant-specific amplification in the natural population and style-specific expression, which increased gradually during bud maturation, consistent with the reported S-RNase expression. In contrast, the S ( 63 ) ribonuclease was present in all plants examined and was ubiquitously expressed in different organs and bud developmental stages. Genetic segregation analysis demonstrated that S ( 27 ), S ( 70 ), S ( 75 ), S ( 107 ), and S ( 210 ) alleles were fully functional novel S-RNases, while S ( 5 ) and S ( 63 ) resulted to be non-S-RNases, although with a clearly distinct pattern of expression. These results reveal the importance of performing functional analysis in studies of S-RNase allelic diversity. Comparative phylogenetic analysis of six species of Solanaceae showed that N. alata S-RNases were included in eight transgeneric S-lineages. Phylogenetic pattern obtained from the inclusion of the novel S-RNase alleles confirms that N. alata represents a broad sample of the allelic variation at the S-locus of the Solanaceae.
Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Nicotiana/enzimologia , Nicotiana/genética , Ribonucleases/genética , Autofertilização , Alelos , Regulação da Expressão Gênica no Desenvolvimento/genética , Variação Genética/genética , Haplótipos/genética , Estruturas Vegetais/enzimologia , Estruturas Vegetais/genética , Estruturas Vegetais/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , RNA de Plantas/genética , RNA de Plantas/metabolismo , Ribonucleases/metabolismo , Solanaceae/enzimologia , Solanaceae/genética , Nicotiana/crescimento & desenvolvimentoRESUMO
A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.
Assuntos
Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Frutas/enzimologia , Frutas/crescimento & desenvolvimento , Solanaceae/enzimologia , Solanaceae/crescimento & desenvolvimento , Sequência de Aminoácidos , Domínio Catalítico , Resinas de Troca de Cátion , Cisteína Endopeptidases/química , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Extratos Vegetais/metabolismoRESUMO
A biosensor based on stearic acid-graphite powder modified with sweet potato (Ipomoea batatas (L.) Lam.) tissue as peroxidase source was constructed and applied in organic solvents. Several parameters were studied to evaluate the performance of this biosensor such as stearic acid-graphite powder and tissue composition, type and concentration of supporting electrolyte, organic solvents, water/organic solvent ratio (% v/v) and hydrogen peroxide concentration. After selection of the best conditions, the biosensor was applied for the determination of hydroquinone in cosmetic creams in methanol. At the peroxidase electrode hydroquinone is oxidized in the presence of hydrogen peroxide and the radical formed was reduced back electrochemically at -180 mV vs Ag/AgCl (3.0 mol L(-1) KCl). The reduction current obtained was proportional to the concentration of hydroquinone from 6.2 x 10(-5) to 1.5 x 10(-3) mol L(-1) (r = 0.9990) with a detection limit of 8.5 x 10(-6) mol L(-1). The recovery of hydroquinone from two samples ranged from 98.8 to 104.1% and an RSD lower than 1.0% for a solution containing 7.3 x 10(-4) mol L(-1) hydroquinone and 1.0 x 10(-3) mol L(-1) hydrogen peroxide in 0.10 mol L(-1) tetrabutylammonium bromide methanol-phosphate buffer solution (95:5% v/v) (n = 10) was obtained.
Assuntos
Técnicas Biossensoriais/métodos , Solanaceae , Solventes/farmacologia , Antioxidantes/análise , Cosméticos/química , Cosméticos/normas , Cosméticos/toxicidade , Grafite , Hidroquinonas/análise , Compostos Orgânicos/farmacologia , Peroxidase , Protetores contra Radiação/análise , Reprodutibilidade dos Testes , Solanaceae/enzimologia , Ácidos EsteáricosRESUMO
Identification of pistil-expressed genes is an important step in understanding pistil development and function in plant reproduction. A tobacco stigma/style cDNA library was differentially screened and several cDNA clones were isolated. One of these tobacco genes, designated tobP1, is characterized here. TobP1 encodes a protein highly homologous to plant polyphenol oxidases. Northern blot analysis of total RNA extracted from different organs and probed with tobP1 cDNA identified a single transcript that is exclusively present in flower organs (petals, stamens, and predominantly in pistils). The tobP1 gene is co-ordinately regulated during development in pistils and stamens, and is not induced in mature leaves even under stress conditions. TobP1 belongs to a multigene family, as reported for PPO in other plant species.