RESUMO
Alpha-amylase (AMY) plays a significant role in regulating the growth, development, and postharvest quality formation in plants. Nevertheless, little is known about the genome-wide features, expression patterns, subcellular localization, and functional regulation of AMY genes (MaAMYs) in the common starchy banana (Musa acuminata). Twelve MaAMY proteins from the banana genome database were clustered into two groups and contained a conserved catalytic domain. These MaAMYs formed collinear pairs with the AMYs of maize and rice. Three tandem gene pairs were found within the MaAMYs and are indicative of putative gene duplication events. Cis-acting elements of the MaAMY promoters were found to be involved in phytohormone, development, and stress responses. Furthermore, MaAMY02, 08, 09, and 11 were actively expressed during fruit development and ripening. Specifically, MaAMY11 showed the highest expression level at the middle and later stages of banana ripening. Subcellular localization showed that MaAMY02 and 11 were predominately found in the chloroplast, whereas MaAMY08 and 09 were primarily localized in the cytoplasm. Notably, transient attenuation of MaAMY11 expression resulted in an obvious increase in the starch content of banana fruit, while a significant decrease in starch content was confirmed through the transient overexpression of MaAMY11. Together, these results reveal new insights into the structure, evolution, and expression patterns of the MaAMY family, affirming the functional role of MaAMY11 in the starch degradation of banana fruit.
Assuntos
Regulação da Expressão Gênica de Plantas , Musa , Filogenia , Proteínas de Plantas , alfa-Amilases , Musa/genética , Musa/enzimologia , Musa/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Regiões Promotoras Genéticas , Amido/metabolismo , Oryza/genética , Oryza/enzimologia , Oryza/crescimento & desenvolvimentoRESUMO
Biosensors can be developed using different immobilization methods. Interest in immobilization methods have increased because biosensors have been important for science. Polyphenol oxidase (PPO) was used generally in biosensor applications. For this purpose, Polyphenol oxidase from banana was purified and covalently immobilized on chitosan-gelatin bio-composite. The properties of immobilized enzyme were investigated and compared to free enzyme. Various parameters were studied such as pH, temperature and storage stability on immobilized and free enzyme. Kinetic parameters were also evaluated by different substrates on immobilized and free enzyme. Catechol was determined the best substrate for immobilized enzyme with optimum condition. In vitro effects of metal ions were studied on immobilized enzyme. Concentration range of metal ions is 1.0-10.0 x10-6 mol/L. The activity of immobilized PPO was increased by Fe+2 and Ag+1 ion. Co+3 and Cu+1 had very strong inhibitory effects with IC50 values of 19.69x10-3 mol/L and 23.49 x10-3 mol/L, respectively. Inhibition constants (Ki) and inhibition types of metal ions were determined with immobilized enzyme. Zn+2 and Cr+3 ions were showed competitive inhibition and Pb+2 ions were determined non-competitive inhibition with immobilized enzyme. Mixed type inhibition was obtained with Co+3 ion using catechol as substrate with 3.33x10-5 mol/L Ki value on immobilized PPO. Immobilized PPO can be evaluated for biosensor for the purpose of measurements of metal ions.
Assuntos
Técnicas Biossensoriais , Catecol Oxidase/metabolismo , Enzimas Imobilizadas/metabolismo , Metais/metabolismo , Musa/enzimologia , Proteínas de Plantas/metabolismo , Biocatálise/efeitos dos fármacos , Catecol Oxidase/química , Catecóis/metabolismo , Quitosana/química , Enzimas Imobilizadas/química , Gelatina/química , Concentração de Íons de Hidrogênio , Íons/metabolismo , Íons/farmacologia , Cinética , Metais/farmacologia , Proteínas de Plantas/química , Especificidade por Substrato , TemperaturaRESUMO
Banana (Musa spp.), one of the most important fruits worldwide, is generally cold sensitive. In this study, by using the cold-sensitive banana variety Tianbaojiao (Musa acuminate) as the study material, we investigated the effects of Piriformospora indica on banana cold resistance. Seedlings with and without fungus colonization were subjected to 4 °C cold treatment. The changes in plant phenotypes, some physiological and biochemical parameters, chlorophyll fluorescence parameters, and the expression of eight cold-responsive genes in banana leaves before and after cold treatment were measured. Results demonstrated that P. indica colonization reduced the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) but increased the activities of superoxide dismutase (SOD) and catalase (CAT) and the contents of soluble sugar (SS) and proline. Noteworthily, the CAT activity and SS content in the leaves of P. indica-colonized banana were significant (p < 0.05). After 24 h cold treatment, the decline in maximum photochemistry efficiency of photosystem II (Fv/Fm), photochemical quenching coefficient (qP), efficient quantum yield [Y(II)], and photosynthetic electron transport rate (ETR) in the leaves of P. indica-colonized banana was found to be lower than in the non-inoculated controls (p < 0.05). Moreover, although the difference was not significant, P. indica colonization increased the photochemical conversion efficiency and electron transport rate and alleviated the damage to the photosynthetic reaction center of banana leaves under cold treatment to some extent. Additionally, the expression of the most cold-responsive genes in banana leaves was significantly induced by P. indica during cold stress (p < 0.05). It was concluded that P. indica confers banana with enhanced cold resistance by stimulating antioxidant capacity, SS accumulation, and the expression of cold-responsive genes in leaves. The results obtained from this study are helpful for understanding the P. indica-induced cold resistance in banana.
Assuntos
Basidiomycota/fisiologia , Temperatura Baixa , Resistência à Doença , Endófitos/fisiologia , Musa/enzimologia , Basidiomycota/crescimento & desenvolvimento , Catalase/metabolismo , Clorofila/metabolismo , Contagem de Colônia Microbiana , Eletrólitos/metabolismo , Fluorescência , Regulação da Expressão Gênica de Plantas , Musa/genética , Musa/microbiologia , Peroxidase/metabolismo , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Superóxido Dismutase/metabolismoRESUMO
Carotenoids are a class of bioactive compounds that exhibit health-promoting properties for humans, but their regulation in bananas during fruit ripening remains largely unclear. Here, we found that the total carotenoid content continued to be elevated along the course of banana ripening and peaked at the ripening stage followed by a decrease, which is presumably caused by the transcript abundances of carotenoid biosynthetic genes MaLCYB1.1 and MaLCYB1.2. Moreover, a ripening-inducible transcription factor MaSPL16 was characterized, which was a nuclear protein with transactivation activity. Transient transformation of MaSPL16 in banana fruits led to enhanced transcript levels of MaLCYB1.1 and MaLCYB1.2 and hence the total carotenoid accumulation. Importantly, MaSPL16 stimulated the transcription of MaLCYB1.1 and MaLCYB1.2 through directly binding to their promoters. Collectively, our findings indicate that MaSPL16 behaves as an activator to modulate banana carotenoid biosynthesis, which may provide a new target for molecular improvement of the nutritional and bioactive qualities of agricultural crops that accumulate carotenoids.
Assuntos
Carotenoides/metabolismo , Frutas/crescimento & desenvolvimento , Liases Intramoleculares/genética , Musa/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/metabolismo , Frutas/enzimologia , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Liases Intramoleculares/metabolismo , Musa/enzimologia , Musa/genética , Musa/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genéticaRESUMO
KEY MESSAGE: Banana MaBZR1/2 interact with MaMPK14 to enhance the transcriptional inhibition of cell wall modifying genes including MaEXP2, MaPL2 and MaXET5. Fruit ripening and softening, the major attributes to perishability in fleshy fruits, are modulated by various plant hormones and gene expression. Banana MaBZR1/2, the central transcription factors of brassinosteroid (BR) signaling, mediate fruit ripening through regulation of ethylene biosynthesis, but their possible roles in fruit softening as well as the underlying mechanisms remain to be determined. In this work, we found that MaBZR1/2 directly bound to and repressed the promoters of several cell wall modifying genes such as MaEXP2, MaPL2 and MaXET5, whose transcripts were elevated concomitant with fruit ripening. Moreover, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that MaBZR1/2 physically interacted with a mitogen-activated protein kinase MaMPK14, and this interaction strengthened the MaBZR1/2-mediated transcriptional inhibitory abilities. Collectively, our study provides insight into the mechanism of MaBZR1/2 contributing to fruit ripening and softening, which may have potential for banana molecular improvement.
Assuntos
Parede Celular/metabolismo , Frutas/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Musa/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Brassinosteroides/metabolismo , Proteínas de Ligação a DNA/metabolismo , Etilenos/metabolismo , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Musa/enzimologia , Musa/genética , Musa/metabolismo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
Protein cleaving enzymes, called proteases are one of the most commercially used enzymes possessing extensive applications in various industries. The present study was focused on screening, extraction, purification and characterization of protease from banana peel. Twelve different varieties of banana peels were screened for the protease activity. The maximum activity of the isolated enzyme was 230.4 CDU/mg from Nendran banana. The crude enzyme was purified up to 8.1 fold with the yield of 61.34%. The molecular weight of the purified fraction was found to be 40â¯kDa. The optimum conditions for maximum protease activity were achieved at 30⯰C and pHâ¯7. The Lineweaver-Burk plot exhibited the kinetic parameters of the enzyme such as Vmax and Km as 588.4 CDU/mg and 15.2â¯mg/ml respectively. The influence of different inhibitors, metal ions, organic solvents, detergents, oxidising agents and salinity were examined. The collagenolytic activity was tested with purified type I collagen as a substrate. The enzyme was tested for cell cytotoxicity, detection of apoptosis using fluorescent dyes and antitumor activity. The results demonstrated that protease from banana peel was of high significance and potential for usage in therapeutic for breaking down collagen/peptide bonds.
Assuntos
Metaloproteases/química , Musa/enzimologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fenômenos Químicos , Ativação Enzimática , Estabilidade Enzimática , Hidrólise , Íons , Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Metaloproteases/farmacologia , Metais , Camundongos , Peso Molecular , Solventes , Análise Espectral , Especificidade por SubstratoRESUMO
Polyphenol oxidase (Tyrosinase, PPO) has received considerable attention, since it is the key enzyme in melanin biosynthesis. In this study, we investigated prepared novel carbazole-containing pyridopyrimidine-substituted with urea and thiourea derivatives and their PPO activities on the diphenolase activity of banana tyrosinase. The structures of the compounds synthesized were confirmed by 1 H NMR, 13 C NMR, FTIR and elemental analysis. PPO enzyme was purified from banana on an affinity gel comprised of Sepharose 4B-L-tyrosine-p-amino benzoic acid. For evaluating the enzyme activity, the synthesised compounds were subjected to tyrosinase inhibition assay using catechol as substrate. While some of the compounds (6, 7, 8f, 8h, 8i, 8j) showed enzyme inhibitor effect, some of them (8a, 8b, 8c, 8d, 8e, 8g, 8k) activated the PPO enzyme activity. Gaussian software was used for the molecular calculations to explain the results for the prepared compounds.
Assuntos
Carbazóis/química , Catecol Oxidase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Musa/enzimologia , Proteínas de Plantas/antagonistas & inibidores , Tioureia/química , Ureia/química , Inibidores Enzimáticos/química , Modelos Teóricos , Estrutura Molecular , Piridinas/química , Pirimidinas/químicaRESUMO
An ascorbate peroxidase from a new source Musa paradisiaca leaf juice has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and anion exchange chromatography on diethyl amino ethyl [DEAE] cellulose column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis [SDS-PAGE] analysis of the purified enzyme has shown a single protein band of molecular mass 208.9â¯kDa which has been confirmed by native-PAGE and intact mass analysis by mass spectrometry. The Km and kcat values of the enzyme using ascorbate and H2O2 as the variable substrates were 0.13â¯mâ¯molâ¯L-1, 40.42â¯s-1 and 0.23â¯mâ¯molâ¯L-1, 27.24â¯s-1, respectively. The pH and temperature optima of the enzyme were 7.0 and 298â¯K, respectively. The enzyme transformed approximately 97% methyl phenyl sulfide to its sulfoxide. The product was racemic mixture.
Assuntos
Ascorbato Peroxidases/metabolismo , Musa/enzimologia , Safrol/análogos & derivados , Sulfetos/metabolismo , Sequência de Aminoácidos , Ascorbato Peroxidases/química , Biotransformação , Concentração de Íons de Hidrogênio , Folhas de Planta/enzimologia , Safrol/metabolismo , TemperaturaRESUMO
The U-box gene family is a family of genes which encode U-box domain-containing proteins. However, little is known about U-box genes in banana (Musa acuminata). In this study, 91 U-box genes were identified in banana based on its genome sequence. The banana U-box genes were distributed across all 12 chromosomes at different densities. Phylogenetic analysis of U-box genes from banana, Arabidopsis, and rice suggested that they can be clustered into seven subgroups (Iâ»VII), and most U-box genes had a closer relationship between banana and rice relative to Arabidopsis. Typical U-box domains were found in all identified MaU-box genes through the analysis of conserved motifs. Four conserved domains were found in major banana U-box proteins. The MaU-box gene family had the highest expression in the roots at the initial fruit developmental stage. The MaU-box genes exhibited stronger response to drought than to salt and low temperatures. To the best of our knowledge, this report is the first to perform genome-wide identification and analysis of the U-box gene family in banana, and the results should provide valuable information for better understanding of the function of U-box in banana.
Assuntos
Genoma de Planta , Família Multigênica , Musa/enzimologia , Musa/genética , Proteínas de Plantas/genética , Ubiquitina-Proteína Ligases/genética , Arabidopsis/genética , Cromossomos de Plantas/genética , Sequência Conservada/genética , Frutas/genética , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Motivos de Nucleotídeos/genética , Oryza/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Domínios Proteicos , Estresse Fisiológico/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Plant casein kinase II (CKII) plays an essential role in regulating plant growth and development, and responses to biotic and abiotic stresses. Here, we report the identification and characterization of the CKII family genes in Musa spp. cv. 'Tianbaojiao' (AAA group) and the wild banana (Musa itinerans). The 13 cDNA sequences of the CKII family members were identified both in 'Tianbaojiao' and wild banana, respectively. The differences between CKII α and CKII ß members are corroborated through the subcellular localizations, phosphorylation sites and gene structures. The cloning of CKII ß-like-2 gDNA sequences in wild banana and 'Tianbaojiao' and the analysis of gene structures showed MiCKIIß-like-2b and MaCKIIß-like-2 are likely alternatively spliced transcripts, which were derived from the alternative splicing events that involved exon deletion. The qPCR validation showed differential expression CKII family members in response to cold stress and also in all tested tissues (leaf, pseudostem and root) of wild banana. In particular, the normal transcript MiCKIIß-like-2a was highly expressed in response to cold stress in wild banana; oppositely, the alternatively spliced transcript MiCKIIß-like-2b was quite lowly expressed. The complex origin and long-term evolution of Musa lineage might explain the alternative splicing events of CKII ß-like-2.
Assuntos
Caseína Quinase II/genética , Genes de Plantas , Musa/enzimologia , Musa/genética , Melhoramento Vegetal , Processamento Alternativo , Resposta ao Choque Frio/genética , Resposta ao Choque Frio/fisiologia , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Fosforilação , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Sulfoxidation of methionine in proteins by reactive oxygen species can cause conformational alteration or functional impairment, and can be reversed by methionine sulfoxide reductase (Msr). Currently, only a few potential Msr substrates have been confirmed in higher plants. Here, we investigated Msr-mediated sulfoxidation regulation of calmodulin (CaM) and its underlying biological significance in relation to banana fruit ripening and senescence. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. We verified that MaCaM1 interacts with MaMsrA7 in vitro and in vivo, and sulfoxidated MaCaM1 could be partly repaired by MaMsrA7 (MaMsrA7 reduces oxidized residues Met77 and Met110 in MaCaM1). Furthermore, we investigated two known CaM-binding proteins, catalase (MaCAT1) and MaHY5-1. MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3) in banana fruit. MaCaM1 could enhance the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1 toward MaPSY2. Mimicked sulfoxidation in MaCaM1 did not affect the physical interactions of the protein with MaHY5-1 and MaCAT1, but reduced the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1. Our data suggest that sulfoxidation modification in MaCaM1 by MaMsrA7 regulates antioxidant response and gene transcription, thereby being involved in regulation of ripening and senescence of banana fruit.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Regulação da Expressão Gênica de Plantas , Metionina Sulfóxido Redutases/metabolismo , Musa/genética , Espécies Reativas de Oxigênio/metabolismo , Calmodulina/genética , Proteínas de Ligação a Calmodulina/genética , Frutas/enzimologia , Frutas/genética , Frutas/fisiologia , Genes Reporter , Metionina Sulfóxido Redutases/genética , Musa/enzimologia , Musa/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Técnicas do Sistema de Duplo-HíbridoRESUMO
In this paper, a new and facile method for the electrochemical determination of l-tyrosine was designed. First, 3-mercaptopropyl trimethoxysilane-functionalized silica nanoparticles were added to a paper disc. Then, the banana peel tissue and the mediator potassium hexacyanoferrate were dropped onto the paper, respectively. The modified paper disc was placed on the top of the graphite screen printed electrode and electrochemical characterization of this biosensor was studied by cyclic voltammetry and electrochemical impedance spectroscopy methods. The effective parameters like pH, banana peel tissue percentage, and the amount of mediator loading were optimized. l-tyrosine measurements were done by differential pulse voltammetry with a little sample (3⯵L) for analysis. The biosensor showed a linear response for l-tyrosine in the wide concentration range of 0.05-600⯵M and a low detection limit about 0.02⯵M because of the co-catalytic effect of enzyme and nanoparticles. The stability of the biosensor and its selectivity were evaluated. This biosensor was applied for the voltammetric determination of l-tyrosine in the blood plasma sample. The results of the practical application study were comparable with the standard method (HPLC). In conclusion, a simple, inexpensive, rapid, sensitive and selective technique was successfully applied to the l-tyrosine analysis of the little samples.
Assuntos
Biocatálise , Técnicas Biossensoriais/métodos , Monofenol Mono-Oxigenase/metabolismo , Musa/enzimologia , Nanopartículas/química , Papel , Dióxido de Silício/química , Tirosina/análise , Eletroquímica , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Limite de Detecção , Monofenol Mono-Oxigenase/química , Tirosina/químicaRESUMO
Diagnostic reagents based on food allergen extracts often lack sufficient sensitivity. The introduction of well characterized food allergens in molecular allergy diagnosis has been recognized as valid approach to circumvent unstandardized allergen extracts. Banana fruit (Musa acuminata) is a well-established allergen source which besides six characterized allergens, contains unidentified IgE reactive proteins whose clinical relevance remains undefined. By employment of a combinatorial peptide ligand library (CPLL) methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis, a novel allergen from banana fruit was detected in banana as catalase. A recombinant homologue of natural catalase was produced, isolated and biochemically characterized. The recombinant protein showed IgE reactivity in 7 out of 13 tested patients with suspected allergy to banana in immunoblot. Novel banana fruit allergens should be added as components to allergen-microarrays for the diagnosis and the monitoring of banana allergy. SIGNIFICANCE: By employment of CPLL methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis catalase from banana fruit is identified as a novel allergen, with proposed designation as Mus a 7. IgE reactive recombinant Mus a 7 was produced and should be included in a component-resolved allergy diagnosis.
Assuntos
Western Blotting/métodos , Catalase/isolamento & purificação , Hipersensibilidade Alimentar/etiologia , Musa/imunologia , Proteômica/métodos , Alérgenos/análise , Catalase/análise , Hipersensibilidade Alimentar/diagnóstico , Frutas/enzimologia , Frutas/imunologia , Humanos , Imunoglobulina E/imunologia , Musa/enzimologia , Proteínas de Plantas/análise , Proteínas de Plantas/imunologiaRESUMO
The aim of this study was to develop a cantilever nanobiosensor for atrazine detection in liquid medium by immobilising the biological recognition element (tyrosinase vegetal extract) on its surface with self-assembled monolayers using gold, 16-mercaptohexadecanoic acid, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/n-hydroxysuccinimide. Cantilever nanobiosensors presented a surface compression tension increase when atrazine concentrations were increased, with a limit of detection and limit of quantification of 7.754 ppb (parts per billion) and 22.792 ppb, respectively. From the voltage results obtained, the evaluation of atrazine contamination in river and drinking water were very close to those of the reference sample and ultrapure water, demonstrating the ability of the cantilever nanobiosensor to distinguish different water samples and different concentrations of atrazine. Cantilever nanosensor surface functionalization was characterised by combining polarisation modulation infrared reflection-absorption spectroscopy and atomic force microscopy and indicating film thickness in nanometric scale (80.2 ± 0.4 nm). Thus, the cantilever nanobiosensor developed for this study using low cost tyrosinase vegetal extract was adequate for atrazine detection, a potential tool in the environmental field.
Assuntos
Atrazina/análise , Técnicas Biossensoriais , Monofenol Mono-Oxigenase/metabolismo , Nanotecnologia , Água Potável/química , Contaminação de Alimentos/análise , Ouro/química , Herbicidas/análise , Imidas/química , Limite de Detecção , Musa/química , Musa/enzimologia , Ácidos Palmíticos/química , Extratos Vegetais/química , Propilaminas/química , Rios/química , Propriedades de SuperfícieRESUMO
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1-5) or deletion (1-4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma de Planta , Musa/enzimologia , Musa/genética , Oxirredutases/genética , Sequência de Aminoácidos , Sequência de Bases , Carotenoides/metabolismo , Clorofila/metabolismo , Especificidade de Órgãos , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Filogenia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
ADP-glucose pyrophosphorylase (AGPase) is the first rate-limiting enzyme in starch biosynthesis and plays crucial roles in multiple biological processes. Despite its importance, AGPase is poorly studied in starchy fruit crop banana (Musa acuminata L.). In this study, eight MaAGPase genes have been identified genome-wide in M. acuminata, which could be clustered into the large (APL) and small (APS) subunits. Comprehensive transcriptomic analysis revealed temporal and spatial expression variations of MaAPLs and MaAPSs and their differential responses to abiotic/biotic stresses in two banana genotypes, Fen Jiao (FJ) and BaXi Jiao (BX). MaAPS1 showed generally high expression at various developmental and ripening stages and in response to abiotic/biotic stresses in both genotypes. MaAPL-3 and -2a were specifically induced by abiotic stresses including cold, salt, and drought, as well as by fungal infection in FJ, but not in BX. The presence of hormone-related and stress-relevant cis-acting elements in the promoters of MaAGPase genes suggests that MaAGPases may play an important role in multiple biological processes. Taken together, this study provides new insights into the complex transcriptional regulation of AGPases, underlying their key roles in promoting starch biosynthesis and enhancing stress tolerance in banana.
Assuntos
Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Musa/enzimologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Família Multigênica , Musa/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Estresse FisiológicoRESUMO
The mitogen-activated protein kinase (MAPK) cascade, which is a major signal transduction pathway widely distributed in eukaryotes, has an important function in plant development and stress responses. However, less information is known regarding the MAPKKK and MAPKK gene families in the important fruit crop banana. In this study, 10 MAPKK and 77 MAPKKK genes were identified in the banana genome, and were classified into 4 and 3 subfamilies respectively based on phylogenetic analysis. Majority of MAPKKK and MAPKK genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis indicated that MAPKKK-MAPKK genes is involved in tissue development, fruit development and ripening, and response to abiotic stress of drought, cold and salt in two banana genotypes. Interaction networks and co-expression assays demonstrated that MAPK signaling cascade mediated network participates in multiple stress signaling, which was strongly activated in Fen Jiao (FJ). The findings of this study advance understanding of the intricately transcriptional control of MAPKKK-MAPKK genes and provide robust candidate genes for further genetic improvement of banana.
Assuntos
Perfilação da Expressão Gênica , MAP Quinase Quinase Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Musa/enzimologia , Musa/crescimento & desenvolvimento , Redes Reguladoras de Genes , Genoma de Planta , MAP Quinase Quinase Quinases/biossíntese , Quinases de Proteína Quinase Ativadas por Mitógeno/biossíntese , Musa/genética , Filogenia , Análise de Sequência de DNA , Homologia de Sequência , Transdução de Sinais , Estresse FisiológicoRESUMO
We developed a label-free potentiometric biosensor using tyrosinase extracted from Musa acuminata and immobilized by covalent bond on a surface of a solid-contact transducer. The transducer was manufactured containing two layers. The first layer contained a blend of poly(vinyl) chloride carboxylated (PVC-COOH), graphite and potassium permanganate. On this layer, we deposited a second layer containing just a mixture of poly(vinyl chloride) carboxylated and graphite. On the last layer of the transducer, we immobilized the tyrosinase enzyme by reaction with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The solid-contact potentiometric biosensor presented at low detection limit of 7.3×10-7M and a linear range to catechol concentration between 9.3×10-7M and 8.3×10-2M. This biosensor was applied to determine the amount of total phenols in different samples of honey and propolis. The results agreed with the Folin-Ciocalteu method.
Assuntos
Técnicas Biossensoriais/métodos , Mel/análise , Fenóis/análise , Própole/química , Cinética , Fenômenos Mecânicos , Monofenol Mono-Oxigenase/metabolismo , Musa/enzimologia , PotenciometriaRESUMO
BACKGROUND: The impact of high-pressure processing (HPP) on the polyphenol (PP) content and antioxidant activity (AOX) of plantain pulp was evaluated. Pressures of 400, 500 and 600 MPa were applied to plantain pulp for 90 and 180 s at room temperature (25 °C). Polyphenoloxidase activity, extractable (EPP) and non-extractable PP (NEPP) contents, flavonoid content and AOX (FRAP, ABTSâ¢+ ) were evaluated. In addition, PP identification was performed using high-performance liquid chromatography. RESULTS: Polyphenoloxidase activity was inhibited after HPP under all of the conditions studied. Increases of 110.80% and 137.40% in EPP content under conditions of 500 MPa/180 s and 600 MPa/90 s were observed with a simultaneous improvement in the AOX with increments of up to 128.71%. The treatment under conditions of 500 MPa/90 s had the highest total PP content, including the highest content of flavonoids (0.22 g ellagic acid equivalents kg-1 dry weight) and the proportion of NEPP that contained hydrolysable PPs (91.12 g gallic acid equivalents kg-1 dry weight with high AOX. The identified PPs included catechin, quercetin, gallic and hydroxybenzoic acids. CONCLUSION: HPP performed at a room temperature can be used for improving the total content of PP compounds in plantain pulp under specific pressure and time conditions. © 2016 Society of Chemical Industry.
Assuntos
Antioxidantes/química , Manipulação de Alimentos/métodos , Frutas/química , Musa/química , Polifenóis/química , Catecol Oxidase/análise , Catecol Oxidase/metabolismo , Cromatografia Líquida de Alta Pressão , Manipulação de Alimentos/instrumentação , Frutas/enzimologia , Pressão Hidrostática , Musa/enzimologia , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , TemperaturaRESUMO
Effect of exogenous nitric oxide (NO) on polyamines (PAs) catabolism, γ-aminobutyric acid (GABA) shunt, proline accumulation and chilling injury of banana fruit under cold storage was investigated. Banana fruit treated with NO sustained lower chilling injury index than the control. Notably elevated nitric oxide synthetase activity and endogenous NO level were observed in NO-treated banana fruit. PAs contents in treated fruit were significantly higher than control fruit, due to the elevated activities of arginine decarboxylase and ornithine decarboxylase. NO treatment increased the activities of diamine oxidase, polyamine oxidase and glutamate decarboxylase, while reduced GABA transaminase activity to lower levels compared with control fruit, which resulted the accumulation of GABA. Besides, NO treatment upregulated proline content and significantly enhanced the ornithine aminotransferase activity. These results indicated that the chilling tolerance induced by NO treatment might be ascribed to the enhanced catabolism of PAs, GABA and proline.