RESUMO
Barley malting depends on hydrolytic enzymes that degrade storage macromolecules. Identifying barley cultivars with proteolytic activity that guarantees appropriate foaming, flavor, and aroma in the beer is of great importance. In this work, the proteolytic activity and profiles of brewing malt from Mexican barley cultivars were analyzed. Data showed that Cys- (at 50°C) and Ser-proteases (at 70°C) are the major contributors to proteolytic activity during mashing. Essential amino acids, necessary for fermentation and production of good flavor and aroma in beer, were detected at the end of mashing. According to our results, Mexican cultivar HV2005-19 exhibits similar proteolytic activities as those from cultivar Metcalfe, which is one of the most utilized for the brewing industry. Moreover, we propose Cys- and Ser-proteases as biochemical markers during mashing at 50 and 70°C, respectively, to select barley cultivars for beer production. PRACTICAL APPLICATIONS: Proteolytic activity, which depends on activation and de novo synthesis of proteases in the aleurone layer of barley seeds, is crucial in beer production. Identifying new barley varieties that have optimal proteolytic activities is of great interest for genetic improvement programs. In this study, we propose the variety HV2005-19 as a genotype with Cys- and Ser-proteases activity similar to that from Metcalfe, which is a top variety in the brewing industry.
Assuntos
Hordeum , Cerveja/análise , Fermentação , Hordeum/química , Hordeum/genética , Peptídeo Hidrolases/genética , Sementes/químicaRESUMO
The marine-facultative Aspergillus sp. MEXU 27854, isolated from the Caleta Bay in Acapulco, Guerrero, Mexico, has provided an interesting diversity of secondary metabolites, including a series of rare dioxomorpholines, peptides, and butyrolactones. Here, we report on the genomic data, which consists of 11 contigs (N50~3.95 Mb) with a ~30.75 Mb total length of assembly. Genome annotation resulted in the prediction of 10,822 putative genes. Functional annotation was accomplished by BLAST searching protein sequences with different public databases. Of the predicted genes, 75% were assigned gene ontology terms. From the 67 BGCs identified, ~60% belong to the NRPS and NRPS-like classes. Putative BGCs for the dioxomorpholines and other metabolites were predicted by extensive genome mining. In addition, metabolomic molecular networking analysis allowed the annotation of all isolated compounds and revealed the biosynthetic potential of this fungus. This work represents the first report of whole-genome sequencing and annotation from a marine-facultative fungal strain isolated from Mexico.
Assuntos
Aspergillus/metabolismo , Metabolômica , Morfolinas/metabolismo , Peptídeos Cíclicos/metabolismo , Aspergillus/genética , Aspergillus/isolamento & purificação , México , Estrutura Molecular , Morfolinas/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genéticaRESUMO
Thioredoxins are regulatory proteins that reduce disulfide bonds on target proteins. NaTrxh, which belongs to the plant thioredoxin family h subgroup 2, interacts and reduces the S-RNase enhancing its ribonuclease activity seven-fold, resulting an essential protein for pollen rejection inNicotiana.Here, the crystal structure of NaTrxh at 1.7 Å by X-ray diffraction is reported. NaTrxh conserves the typical fold observed in other thioredoxins from prokaryotes and eukaryotes, but it contains extensions towards both N- and C-termini.The NaTrxh N-terminal extension participates in the reduction of S-RNase, and in the structure reported here, this is orientated towards the reactive site. The interaction between SF11-RNase and the NaTrxh N-terminal was simulated and the short-lived complex observed lasted for a tenth of ns. Moreover, we identified certain amino acids as SF11-RNase-E155 and NaTrxh-M104 as good candidates to contribute to the stability of the complex. Furthermore, we simulated the reduction of the C153-C186 SF11-RNase disulfide bond and observed subtle changes that affect the entire core, which might explain the increase in the ribonuclease activity of S-RNase when it is reduced by NaTrxh.
Assuntos
Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Ribonucleases/metabolismo , Sítios de Ligação/fisiologia , Eucariotos/metabolismo , Células Procarióticas/metabolismo , Transporte Proteico/fisiologiaRESUMO
KEY MESSAGE: PCD role in unisexual flowers. The developmental processes underlying the transition from hermaphroditism to unisexuality are key to understanding variation and evolution of floral structure and function. A detailed examination of the cytological and histological patterns involved in pollen and ovule development of staminate and pistillate flowers in the dioecious Opuntia robusta was undertaken, and the potential involvement of programmed cell death in the abortion of the sex whorls was explored. Flowers initiated development as hermaphrodites and became functionally unisexual by anthesis. Female individuals have pistillate flowers with a conspicuous stigma, functional ovary, collapsed stamens and no pollen grains. Male individuals have staminate flowers, with large yellow anthers, abundant pollen grains, underdeveloped stigma, style and an ovary that rarely produced ovules. In pistillate flowers, anther abortion resulted from the premature degradation of the tapetum by PCD, followed by irregular deposition of callose wall around the microsporocytes, and finally by microspore degradation. In staminate flowers, the stigma could support pollen germination; however, the ovaries were reduced, with evidence of placental arrest and ovule abortion through PCD, when ovules were present. We demonstrate that PCD is recruited in both pistillate and staminate flower development; however, it occurs at different times of floral development. This study contributes to the understanding of the nature of the O. robusta breeding system and identifies developmental landmarks that contribute to sexual determination in Cactaceae.
Assuntos
Apoptose , Opuntia/crescimento & desenvolvimento , Infertilidade das Plantas , Flores/crescimento & desenvolvimento , Flores/fisiologia , Opuntia/fisiologia , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/fisiologia , Melhoramento Vegetal , Pólen/crescimento & desenvolvimento , Pólen/fisiologia , Polinização , ReproduçãoRESUMO
Barley malting quality depends on seed characteristics achieved during grain development and germination. One important parameter is protein accumulation in the mature seed, which may vary between cultivars. Here we conducted a protein pattern analysis in the range of pI 4-7 of mature grains from five Mexican barley cultivars, commonly used for malt and beer production. Reproducibly distinct protein spots, separated by 2D SDS PAGE, were identified by mass spectrometry and considered as potential markers for cultivars with distinct seed protein accumulation. The expression patterns of glutamate decarboxylase (GAD) and protein disulfide isomerase (PDI1-1) were followed at transcript level during grain development for three independent growth cycles to establish whether differences between cultivars were reproducible. Quantitative determination of PDI1-1 protein levels by ELISA confirmed a reproducibly, distinctive accumulation and post-translational modifications between cultivars, which were independent of plant growth regimes. According to its impact on differential storage protein accumulation, we propose the PDI1-1 protein as potential biomarker for Mexican malting barley cultivars.
Assuntos
Regulação da Expressão Gênica de Plantas , Hordeum/enzimologia , Hordeum/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Glicosilação , Hordeum/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/crescimento & desenvolvimentoRESUMO
In Solanaceae, the S-specific interaction between the pistil S-RNase and the pollen S-Locus F-box protein controls self-incompatibility (SI). Although this interaction defines the specificity of the pollen rejection response, the identification of three pistil essential modifier genes unlinked to the S-locus (HT-B, 120K, and NaStEP) unveils a higher degree of complexity in the pollen rejection pathway. We showed previously that NaStEP, a stigma protein with homology with Kunitz-type protease inhibitors, is essential to SI in Nicotiana spp. During pollination, NaStEP is taken up by pollen tubes, where potential interactions with pollen tube proteins might underlie its function. Here, we identified NaSIPP, a mitochondrial protein with phosphate transporter activity, as a novel NaStEP-interacting protein. Coexpression of NaStEP and NaSIPP in pollen tubes showed interaction in the mitochondria, although when expressed alone, NaStEP remains mostly cytosolic, implicating NaSIPP-mediated translocation of NaStEP into the organelle. The NaSIPP transcript is detected specifically in mature pollen of Nicotiana spp.; however, in self-compatible plants, this gene has accumulated mutations, so its coding region is unlikely to produce a functional protein. RNA interference suppression of NaSIPP in Nicotiana spp. pollen grains disrupts the SI by preventing pollen tube inhibition. Taken together, our results are consistent with a model whereby the NaStEP and NaSIPP interaction, in incompatible pollen tubes, might destabilize the mitochondria and contribute to arrest pollen tube growth.
Assuntos
Proteínas Mitocondriais/metabolismo , Nicotiana/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Plantas/metabolismo , Autoincompatibilidade em Angiospermas , Regulação da Expressão Gênica de Plantas , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Mutação/genética , Proteínas de Transporte de Fosfato/química , Células Vegetais/metabolismo , Proteínas de Plantas/química , Tubo Polínico/metabolismo , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Frações Subcelulares/metabolismo , Nicotiana/genéticaRESUMO
BACKGROUND AND AIMS: The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described. METHODS: The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD. KEY RESULTS: PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption. CONCLUSIONS: Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.
Assuntos
Apoptose/fisiologia , Flores/fisiologia , Opuntia/fisiologia , Infertilidade das Plantas/fisiologia , Sobrevivência Celular , Fragmentação do DNA , Flores/crescimento & desenvolvimento , Flores/ultraestrutura , Meiose , México , Microscopia Eletrônica de Varredura , Opuntia/crescimento & desenvolvimento , Opuntia/ultraestrutura , ReproduçãoRESUMO
In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.
Assuntos
Inibidores Enzimáticos/metabolismo , Nicotiana/metabolismo , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Tubo Polínico/metabolismo , Autoincompatibilidade em Angiospermas , Sequência de Aminoácidos , Ativação Enzimática , Genes de Plantas , Dados de Sequência Molecular , Peptídeos/genética , Extratos Vegetais/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tubo Polínico/genética , Polinização , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteólise , Interferência de RNA , Subtilisina/antagonistas & inibidores , Nicotiana/genéticaRESUMO
In Opuntia stenopetala, flowers initiate as hermaphrodite; however, at maturity, only the stamens in male flowers and the gynoecium in female flowers become functional. At early developmental stages, growth and morphogenesis of the gynoecium in male flowers cease, forming a short style lacking stigmatic tissue at maturity. Here, an analysis of the masculinization process of this species and its relationship with auxin metabolism during gynoecium morphogenesis is presented. Histological analysis and scanning electron microscopy were performed; auxin levels were immunoanalyzed and exogenous auxin was applied to developing gynoecia. Male flower style-tissue patterning revealed morphological defects in the vascular bundles, stylar canal, and transmitting tissue. These features are similar to those observed in Arabidopsis thaliana mutant plants affected in auxin transport, metabolism, or signaling. Notably, when comparing auxin levels between male and female gynoecia from O. stenopetala at an early developmental stage, we found that they were particularly low in the male gynoecium. Consequently, exogenous auxin application on male gynoecia partially restored the defects of gynoecium development. We therefore hypothesize that, the arrest in male flower gynoecia patterning could be related to altered auxin homeostasis; alternatively, the addition of auxin could compensate for the lack of another unknown factor affecting male flower gynoecium development.
Assuntos
Flores/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Morfogênese/fisiologia , Opuntia/crescimento & desenvolvimento , Opuntia/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Flores/ultraestrutura , Organismos Hermafroditas/citologia , Organismos Hermafroditas/crescimento & desenvolvimento , Caracteres Sexuais , Processos de Determinação SexualRESUMO
Aspergillus nidulans produces StcI esterase, which is involved in the biosynthesis of sterigmatocystin, a precursor of aflatoxins. Previous reports of this esterase in A. nidulans suggest that it is composed of 286 amino acid residues with a theoretical molecular mass of 31 kDa. Various conditions were evaluated to determine the optimal expression conditions for StcI; the highest level was observed when A. nidulans was cultured in solid oat media. Various esterases were expressed differentially according to the culture media used. However, specific antibodies designed to detect StcI reacted with a protein with an unexpected molecular mass of 35 kDa in cell extracts from all expression conditions. Analysis of the gene sequence and already reported expressed sequence tags indicated the presence of an additional 29-amino-acid N-terminal region of StcI, which is not a signal peptide and which has not been previously reported. We also detected the presence of this additional N-terminal region using reverse-transcriptase polymerase chain reaction. The complete protein (NStcI) was cloned and successfully expressed in Pichia pastoris.