Eucalyptus urograndis stem proteome is responsive to short-term cold stress

Eucalyptus urograndis is a hybrid eucalyptus of major economic importance to the Brazilian pulp and paper industry. Although widely used in forest nurseries around the country, little is known about the biochemical changes imposed by environmental stress in this species. In this study, we evaluated the changes in the stem proteome after short-term stimulation by exposure to low temperature. Using two-dimensional gel electrophoresis coupled to high-resolution mass spectrometry-based protein identification, 12 proteins were found to be differentially regulated and successfully identified after stringent database searches against a protein database from a closely related species (Eucalyptus grandis). The identification of these proteins indicated that the E. urograndis stem proteome responded quickly to low temperature, mostly by down-regulating specific proteins involved in energy metabolism, protein synthesis and signaling. The results of this study represent the first step in understanding the molecular and biochemical responses of E. urograndis to thermal stress382191198FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2011/11650-0; 2011/51949-5; 2013/06370-4; 2013/06352-6; 2011/23582-

Proteomics of Araucaria angustifolia seed development

O presente trabalho teve como objetivo caracterizar o desenvolvimento da semente de Araucaria angustifolia através da proteômica comparativa, buscando compreender as alterações fisiológicas e metabólicas que ocorrem durante esse processo. Inicialmente, foram avaliados três diferentes metodologias de extração de proteínas. A metodologia composta por solução de extração contendo 7 M de uréia, 2 M de tiouréia, 1% de ditiotreitol, 2% de Triton-100, 1 mM de fluoreto de fenilmetilsulfonil e 5 µM de pepstatina, seguido de precipitação em 20% de ácido tricloroacético apresentou géis de maior resolução e reprodutibilidade, tendo sido escolhida como metodologia de extração protéica para o estudo das alterações no proteoma da semente de A. angustifolia. Uma dificuldade associada ao estudo do proteoma de espécies não sequenciadas é a baixa representatividade nos bancos de dados protéicos, resultando em identificações baseadas em homologia. Estratégias proteômicas baseadas em fracionamento em gel resultam em grandes contaminações por fragmentos de queratina. Sendo assim, foi desenvolvido um programa de remoção de espectros de baixa qualidade para utilização em proteômica baseada em homologia. As análises mostraram que o programa reduz o tempo de busca, melhora a qualidade dos alinhamentos e não resulta em perda de identificações positivas. Finalmente, utilizando as metodologias descritas, foram estudadas as alterações no proteoma durante o desenvolvimento da semente de A. angustifolia. Noventa e seis proteínas foram identificadas e agrupadas de acordo com sua função biológica e padrão de detecção. Os resultados obtidos permitiram o estabelecimento de marcadores protéicos no início e final do desenvolvimento embrionário. A análise das proteínas abundantes no início da embriogênese indica um maior controle no metabolismo oxidativo em relação aos estádios finais. Contrariamente, o final da embriogênese é caracterizado por um alto metabolismo de assimilação de carbono e acúmulo de proteínas de reserva. As implicações dos resultados obtidos no controle e melhoramento de sistemas de embriogênese somática na espécie também foram discutidas.The aim of the present work was to characterize the seed development of Araucaria angustifolia through proteomics in order to understand the physiological and biochemical changes during this process. For that, initially, three different protein extraction methods were evaluated. The extraction based on protein solubilization in 7 M urea, 2 M thiourea, 1% dithiothreitol, 2% Triton-100, 1 mM phenylmethylsulphonyl fluoride, 5 µM pepstatin, followed by 20% trichloroacetic acid precipitation showed the highest gel resolution and reprodutivity and, thus, was chosen to be used in the analysis of the proteome of A. angustifolia seeds. One aspect that hampers the proteome study of unsequenced species is the low protein representativity in databases. So, protein identification is usually carried out through homology. Strategies based on 2-DE result in high keratin contamination. In the present work a spectra filtering software was developed and evaluated for use in homology driven proteomics. The software reduced the time of search, improved alignment quality and did not result in lost of positive identifications. Finally, using the described strategies, the changes in the proteome of A. angustifolia seeds were studied. Ninety six proteins were identified and classified according to their biological functions and expression profiles during seed development. The identified proteins may be used as protein markers of early and late embryogenesis. Proteins involved in the control of oxidative metabolism were highly expressed during the early stages of seed development; while, carbon metabolism and storage proteins were highly expressed in late stages. Considerations on the improvement and control of somatic embryogenesis through medium manipulation and protein markers screening using data generated are also discussed

Large-Scale Proteome Comparative Analysis of Developing Rhizomes of the Ancient Vascular Plant Equisetum Hyemale

Horsetail ( Equisetum hyemale ) is a widespread vascular plant species, whose reproduction is mainly dependent on the growth and development of the rhizomes. Due to its key evolutionary position, the identification of factors that could be involved in the existence of the rhizomatous trait may contribute to a better understanding of the role of this underground organ for the successful propagation of this and other plant species. In the present work, we characterized the proteome of E. hyemale rhizomes using a GeLC-MS spectral-counting proteomics strategy. A total of 1,911 and 1,860 non-redundant proteins were identified in the rhizomes apical tip and elongation zone, respectively. Rhizome-characteristic proteins were determined by comparisons of the developing rhizome tissues to developing roots. A total of 87 proteins were found to be up-regulated in both horsetail rhizome tissues in relation to developing roots. Hierarchical clustering indicated a vast dynamic range in the regulation of the 87 characteristic proteins and revealed, based on the regulation profile, the existence of nine major protein groups. Gene ontology analyses suggested an over-representation of the terms involved in macromolecular and protein biosynthetic processes, gene expression, and nucleotide and protein binding functions. Spatial difference analysis between the rhizome apical tip and the elongation zone revealed that only eight proteins were up-regulated in the apical tip including RNA-binding proteins and an acyl carrier protein, as well as a KH domain protein and a T-complex subunit; while only seven proteins were up-regulated in the elongation zone including phosphomannomutase, galactomannan galactosyltransferase, endoglucanase 10 and 25, and mannose-1-phosphate guanyltransferase subunits alpha and beta. This is the first large-scale characterization of the proteome of a plant rhizome. Implications of the findings were discussed in relation to other underground organs and related species

Structure, organization, and expression of the alpha prolamin multigenic family bring new insights into the evolutionary relationships among grasses

Prolamins are the major seed storage proteins of grasses. In maize and related species, prolamins are classified into alpha-, beta-, gamma-, and delta-subclasses by their solubility properties. alpha-prolamins are encoded by multigene families and have a secondary structure that consists of tandem alpha-helix repeats. Maize has two alpha-prolamin subclasses, namely the 19 and 22 kDa subclasses that contain nine and 10 alpha-helix repeats, respectively. Here, we present an evolutionary study based on the structure, organization, and expression of alpha-prolamins in maize, sugarcane, sorghum, and coix. True 22 kDa subclasses containing 10 repeats are conserved in all four species, but true 19 kDa subclasses containing nine repeats are found only in maize and sugarcane. We discovered a 19 kD alpha-like a-coixin that, as in sorghum, is encoded by few genes. These data suggest that a 19 kDa progenitor present in the ancestor common to maize, coix, sorghum, and sugarcane was preserved at low copy number in coix and sorghum, while amplified into multigene family architecture in maize and sugarcane. The expression profiling of alpha-prolamins, verified by two-dimensional gels, showed highly conserved multispot composition for the 19 kDa alpha-prolamins in maize and sugarcane. Coix and sorghum did not present true 19 kDa alpha-prolamin spots. Our data show remarkable similarity between maize and sugarcane 19 kDa alpha-prolamins regarding both gene structure and expression. Since the multigene architecture of 19 kDa alpha-canein appeared after sugarcane diverged from sorghum, our data suggest that maize and sugarcane might have acquired the multigene family encoding these storage proteins from a common ancestor.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Phylogenomics Resolves the Phylogeny of Theaceae by Using Low-Copy and Multi-Copy Nuclear Gene Makers and Uncovers a Fast Radiation Event Contributing to Tea Plants Diversity

Tea is one of the three most popular nonalcoholic beverages globally and has extremely high economic and cultural value. Currently, the classification, taxonomy, and evolutionary history of the tea family are largely elusive, including phylogeny, divergence, speciation, and diversity. For understanding the evolutionary history and dynamics of species diversity in Theaceae, a robust phylogenetic framework based on 1785 low-copy and 79,103 multi-copy nuclear genes from 91 tea plant genomes and transcriptome datasets had been reconstructed. Our results maximumly supported that the tribes Stewartieae and Gordonieae are successive sister groups to the tribe Theeae from both coalescent and super matrix ML tree analyses. Moreover, in the most evolved tribe, Theeae, the monophyletic genera Pyrenaria, Apterosperma, and Polyspora are the successive sister groups of Camellia. We also yield a well-resolved relationship of Camellia, which contains the vast majority of Theaceae species richness. Molecular dating suggests that Theaceae originated in the late L-Cretaceous, with subsequent early radiation under the Early Eocene Climatic Optimal (EECO) for the three tribes. A diversification rate shift was detected in the common ancestors of Camellia with subsequent acceleration in speciation rate under the climate optimum in the early Miocene. These results provide a phylogenetic framework and new insights into factors that likely have contributed to the survival of Theaceae, especially a successful radiation event of genus Camellia members to subtropic/tropic regions. These novel findings will facilitate the efficient conservation and utilization of germplasm resources for breeding cultivated tea and oil-tea. Collectively, these results provide a foundation for further morphological and functional evolutionary analyses across Theaceae

Protein identification pipeline for the homology-driven proteomics

Homology-driven proteomics is a major tool to characterize proteomes of organisms with unsequenced genomes. This paper addresses practical aspects of automated homology-driven protein identifications by LC-MS/MS on a hybrid LTQ orbitrap mass spectrometer. All essential software elements supporting the presented pipeline are either hosted at the publicly accessible web server, or are available for free download. (C) 2008 Elsevier B.V. All rights reserved.U.S. National Institutes of Health (NIH)NIH NIGMS[1R01GM070986-01A1

Controlled Atmosphere Storage and Sorbitol Dipping Minimize Chilling Injuries in ‘Palmer’ Mangoes

Our previous studies have shown that ‘Palmer’ mangoes immersed in solutions containing 2.5% sorbitol and stored under a controlled atmosphere (CA) at 8 °C for 30 days had fewer symptoms of a chilling injury. However, there is no information regarding the effectiveness of sorbitol treatment in other atmospheres and/or in combination with lower temperatures. Thus, the objective of this study was to assess the impact of dipping ‘Palmer’ mangoes in 0.1% and 2.5% (w/v) sorbitol solutions and storing the fruit under a CA without atmosphere modification (21 kPa O2 + 0.03 kPa CO2) at 8 °C/95% relative humidity (RH) or with 5 kPa O2 + 5 kPa CO2 at 4 °C/95% RH for 28 days. The fruits were evaluated periodically for chilling injuries, quality, and oxidative metabolism. A chilling injury (CI) was correlated with increased fresh weight loss (FWL) and changes in the color of the epicarp (Lpeel, h°peel, and Cpeel) and mesocarp (L*pulp). Lipid peroxidation (LPpulp and LPpeel) and the hydrogen peroxide content (H2O2peel and H2O2pulp) were associated with the development of a CI, particularly after being transferred to ambient. The treatment with 2.5% sorbitol was more effective in minimizing the chilling injury symptoms and did not compromise the fruit quality, especially when it was stored at 4 °C in association with a CA containing 5 kPa O2 + 5 kPa CO2. This treatment reduced lipid peroxidation and increased the activities of the superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzymes in the epicarp and mesocarp, providing greater cold tolerance. The use of 2.5% sorbitol has been identified as the most efficacious approach for mitigating the adverse impacts of chilling injuries, preserving the fruit quality, and enhancing oxidative metabolism, even at lower temperatures. Thus, this treatment represents a viable alternative for managing chilling injuries in mangoes

Análise proteômica comparativa durante a maturação de culturas embriogênicas de Araucaria angustifolia

A evolução morfogenética dos embriões, durante a embriogênese em plantas, é acompanhada por uma intensa síntese de proteínas LEAs (proteínas da embriogênese tardia) e proteínas de reserva que possuem papel fundamental durante a fase de maturação. O objetivo deste trabalho foi avaliar o efeito do ABA e dos agentes osmóticos, polietilenoglicol (PEG) e maltose, na expressão diferencial de proteínas durante a maturação de culturas embriogênicas de A. angustifolia. Culturas embriogênicas de A. angustifolia foram submetidas a cinco tratamentos de maturação: 1) Controle, meio de cultura BM; 2) ABA, meio BM suplementado com ABA (200 µM); 3) PEG, meio BM suplementado com PEG (9%); 4) Maltose, meio BM suplementado com maltose (9%) e; 5) APM, meio BM suplementado com ABA (200 µM), PEG (9%) e maltose (9%). Após 60 dias de cultivo as proteínas totais foram extraídas com tampão uréia:tiouréia (7M:2M), seguido de precipitação em 10% ácido tricloroacético (TCA)/acetona. Alíquotas de 120 µg de proteínas foram isofocalizadas em tiras de gel IPG (gradiente imobilizado de pH) de 11 cm e faixa de pH 4-7, sendo posteriormente submetidas à separação eletroforética em gel de poliacrilamida SDSPAGE 12%. As proteínas foram visualizadas pela coloração com prata e os géis analisados através do programa Image Master 2-D Platinum®. O tratamento PEG resultou na expressão de maior número de proteínas (420 polipeptídeos), enquanto a adição de ABA apresentou 164 polipeptídeos. Os demais tratamentos: Controle, Maltose e APM apresentaram, respectivamente, 347, 322 e 273 polipeptídeos. Todos os tratamentos apresentaram predominância de proteínas com peso molecular entre 30 kDa e 60 kDa. O tratamento suplementado com Maltose apresentou maior porcentagem de polipeptídeos de alto peso molecular (31,4%) quando comparado com os demais tratamentos. A identificação das proteínas expressas diferencialmente pode contribuir para a compreensão do desenvolvimento embrionário, além de auxiliar na otimização dos protocolos de maturação das culturas embriogênicas em A. angustifolia. (FAPESP, CNPq