LEAPdb: a database for the late embryogenesis abundant proteins

<p>Abstract</p> <p>Background</p> <p>Late Embryogenesis Abundant Proteins database (LEAPdb) contains resource regarding LEAP from plants and other organisms. Although LEAP are grouped into several families, there is no general consensus on their definition and on their classification. They are associated with abiotic stress tolerance, but their actual function at the molecular level is still enigmatic. The scarcity of 3-D structures for LEAP remains a handicap for their structure-function relationships analysis. Finally, the growing body of published data about LEAP represents a great amount of information that needs to be compiled, organized and classified.</p> <p>Results</p> <p>LEAPdb gathers data about 8 LEAP sub-families defined by the PFAM, the Conserved Domain and the InterPro databases. Among its functionalities, LEAPdb provides a browse interface for retrieving information on the whole database. A search interface using various criteria such as sophisticated text expression, amino acids motifs and other useful parameters allows the retrieving of refined subset of entries. LEAPdb also offers sequence similarity search. Information is displayed in re-ordering tables facilitating the analysis of data. LEAP sequences can be downloaded in three formats. Finally, the user can submit his sequence(s). LEAPdb has been conceived as a user-friendly web-based database with multiple functions to search and describe the different LEAP families. It will likely be helpful for computational analyses of their structure - function relationships.</p> <p>Conclusions</p> <p>LEAPdb contains 769 non-redundant and curated entries, from 196 organisms. All LEAP sequences are full-length. LEAPdb is publicly available at <url>http://forge.info.univ-angers.fr/~gh/Leadb/index.php</url>.</p

Efficient protocol for isolation of functional RNA from different grape tissue rich in polyphenols and polysaccharides for gene expression studies

Extraction of RNA from plant tissue containing high levels of polyphenols and polysaccharides is tedious and difficult in grapes. Although several protocols have been published for plant RNA isolation, most have failed to yield high quality RNA in sufficient quantity from mature and diseased grape tissue. We describe a protocol for isolating intact and high quality RNA from various grape tissues as evident by high A260/A280 absorbance ratio (1.8 to 1.9) and electrophoretic profile on denaturing formaldehyde agarose gel. On an average, 205 \ub5g RNA per g of fresh tissue were obtained using this modified protocol. RNA quality was further assessed through RT-PCR, differential display RT-PCR and subtractive hybridization, and found to be suitable for molecular studies

A novel bacterial Water Hypersensitivity-like protein shows in vivo protection against cold and freeze damage

Metagenomic library screening, by functional or sequence analysis, has become an established method for the identification of novel genes and gene products, including genetic elements implicated in microbial stress response and adaptation. We have identified, using a sequence based approach, a fosmid clone from an Antarctic desert soil metagenome library containing a novel gene which codes for a protein homologous to a Water Hypersensitive domain (WHy). The WHy domain is typically found as a component of specific LEA (Late Embryogenesis Abundant) proteins, particularly the LEA-14 (LEA-8) variants, which occur widely in plants, nematodes, bacteria and archaea and which are typically induced by exposure to stress conditions. The novel Why-like protein, (165 amino acid, 18.6 kDa) exhibits a largely invariant NPN motif at the N-terminus and has high sequence identity to genes identified in Pseudomonas genomes. Expression of this protein in E. coli significantly protected the recombinant host against cold and freeze stress.South African National Antarctic Program of the National Research Foundation.http://femsle.oxfordjournals.org2016-08-31hb201

Cell envelope constituents of Pseudomonas putida contributing to growth and survival in low-water-content habitats

The ability of bacteria to respond to the ever changing environmental conditions they encounter requires the utilization of a broad range of colonization and survival strategies. One of the most important and probably least understood factors determining bacterial activity in terrestrial habitats is water availability. The goal of this dissertation research was to identify traits that contribute to maintaining a functional cell envelope when cells are dehydrated, which will help us to assess the scope of physiological changes necessary for growth in low-water-content habitats. The primary objective was to identify genes that are specifically regulated by dehydration (matric stress), and not by a thermodynamically-equivalent solute (osmotic) stress. We used transposon mutagenesis combined with a screen to identify genes in the soil and rhizosphere colonizing Pseudomonas putida strain mt-2. Although some of the w&barbelow;ater d&barbelow;eprivation-c&barbelow;ontrolled (wdc) genes were regulated by growth phase, temperature, or toluene exposure, most were specifically induced by matric stress, indicating that bacteria respond differently to a matric stress than to a solute stress. The knowledge of the function of these matric stress-regulated wdc genes allowed us to develop a model of tolerance mechanisms necessary for growth and survival in water-limited environments. Moreover, we found that most wdc loci contributed to survival in low-water-content habitats. One of the identified wdc genes encodes a putative periplasmic lysophospholipase, indicating that dehydration stress disrupts membrane integrity. We determined that lysophospholipids (LPL) accumulated in the wild type when grown under matric stress conditions, but not when grown in the absence of stress or under solute stress conditions. In contrast, the putative lysophospholipase-deficient mutant accumulated LPL even in the absence of a matric stress, indicating that this periplasmic lysophospholipase removes LPL. Exogenously supplied LPL was toxic to P. putida and the mutant was more sensitive than the wild type to LPL. Finally, we observed that biofilm cells are more desiccation-tolerant than planktonic cells and that matric stress is more stressful to cells than solute stress

Origen y función de las espermidina aminopropil transferasas en Arabidopsis thaliana

"Origen y función de las espermidina aminopropil transferasas en Arabidopsis thaliana " Las poliaminas son pequeñas moléculas con carga positiva a pH fisiológico. Las más abundantes y más ampliamente distribuidas entre todos los seres vivos son la putrescina y la espermidina con dos y tres grupos amino, respectivamente. La espermidina se forma a partir de la putrescina por adición de un grupo aminopropilo. La espermina, con cuatro grupos amino y presente sólo en eucariotas, se forma a partir de la espermidina por adición de un segundo grupo aminopropilo. Las poliaminas han sido relacionadas con procesos fundamentales para la vida, como son la división, el crecimiento, la diferenciación y la muerte celular, habiéndose demostrado en todos los organismos en los que se han conseguido mutantes deficientes en su síntesis que las poliaminas son esenciales. En plantas se han encontrado múltiples correlaciones entre la variación en la concentración de las poliaminas y procesos tales como la germinación, la embriogénesis, la formación de raíces, la iniciación floral o el desarrollo de flores y frutos. Al inicio de esta tesis se publicó la identificación de la primera putativa espermina sintasa en plantas (ACL5), cuya pérdida de función da lugar a un defecto en la elongación del tallo y alteración del patrón normal de los haces vasculares; sin embargo, nuestro análisis del mutante acl5 nos ha revelado que no había perdido la capacidad de sintetizar espermina. Nuestro rastreo del genoma de Arabidopsis thaliana nos permitió identificar y caracterizar el gen SPM, otra putativa espermina sintasa regulada por ácido abscísico. Los mutantes nulos para este gen no muestran diferencias fenotípicas respecto del silvestre pero el doble mutante spm/acl5 nos ha permitido confirmar que no hay más genes responsables de la síntesis de espermina. No obstante, la sobreexpresión de SPM en el mutante acl5 no ha sido capaz de aliviar su fenotipo.Gómez Minguet, E. (2008). Origen y función de las espermidina aminopropil transferasas en Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/831

Unfolding plant desiccation tolerance : evolution, structure, and function of LEA proteins

When plants colonized land they developed a wide range of adaptations to cope with life in a drier environment. One key adaptation was desiccation tolerance (DT) which is the ability to survive the removal of almost all cellular water without irreparable damage. DT is recurrent in orthodox seeds and in the vegetative body of species commonly known as ‘resurrection plants’. In this thesis a multilevel approach, combining genomics, transcriptomics, gene family evolution, protein structural and functional analysis, and seed physiology was employed in order to tackle curiosity-driven fundamental questions about the major mechanisms governing DT. Several mechanisms were found to be important for DT, including the coordinated activation of cell protection through Late Embryogenesis Abundant (LEA) proteins, which were shown to be common amongst resurrection plants and orthodox seeds. These findings aid to the comprehension of the complexity of DT in plants, and may provide transferrable knowledge to design more water-stress tolerant crops.</p

The Role of OsWRKY71 and Its Interacting Proteins in Seed Germination and Early Growth of Cereal Grains

During seed germination and early seedling growth, complex molecular and physiological events occur in rice (Oryza sativa) and other cereal grains. As the seed transitions to vegetative tissue, it responds to both favorable and unfavorable environmental conditions and is vulnerable to attack by predation and disease. Although seeds are relatively small and tender in size, extensive and sophisticated molecular networks enables the immobile seed to grow, survive and adapt in its environment. One of the networks I am interested in is in the crosstalk between the gibberellin (GA) and abscisic acid (ABA) signaling pathways. These pathways are interesting because they are largely antagonistic. GA is a hormone that generally promotes germination and growth- related processes while ABA, also a hormone, promotes seed dormancy and represses growth. Although a great deal of research has been dedicated towards understanding these two pathways, the actual mechanism of crosstalk during seed germination is less understood. Any deficiencies in GA and ABA regulation and response may result in altered interpretation of environmental signals and aberrations in seed development and germination, leading to lower grain yields. My research is dedicated towards deciphering the specific role of Oryza sativa WRKY71 (OsWRKY71; amino acid W-R-K-Y) and harpin-induced1-like (HIL) members in the crosstalk between GA and ABA in rice, with the goal that this research will be used to improve cereal grain yield in areas of the world with limited plant productivity. The crosstalk between GA and ABA directs the synthesis of α-amylase, which is an enzyme that breaks down starch in seeds to provide energy for germination. OsWRKY71 was shown to be a transcriptional regulator of α-amylase and was regulated by both GA and ABA in barley. In this study, I have provided a model of the regulation of OsWRKY71 in seed germination in rice. Although it was previously determined that OsWRKY71 negatively regulated α-amylase, I show that it positively regulated not only germination but also root growth. To support this, I performed seed germination and root elongation assays using knockout mutants of OsWRKY71. Mutant analysis determined that germination in oswrky71 was delayed for approximately 1 day and was able to recover from the delay. Additionally, after 4 days, oswrky71 seedling roots were nearly 2 cm shorter than wildtype (wt), suggesting that OsWRKY71 may regulate other aspects of plant development. This is further supported by analysis of β-Glucuronidase (GUS) reporter expression of OsWRKY71p-GUS, which indicated that OsWRKY71 was localized to the third node of rice culms. Thus, the function of OsWRKY71 appears to be more complex and versatile than predicted. To further understand the mechanism of OsWRKY71 regulation in rice seed germination, I investigated the role of one of its interacting partners, Oryza sativa harpin-induced1-like 58 (OsHIL58). Using rice aleurone RNA-sequencing data, I found that OsHIL58 was induced upon ABA treatment. Thus, the two proteins may interact during ABA induction. I also annotated the HIL family using in silico methods and identified several other HIL members that were differentially and significantly expressed in the aleurone. One member, OsHIL16, was highly expressed and also coexpressed with an ABA receptor, regulatory component of ABA receptor 9 (RCAR9). Surprisingly, both were repressed by ABA, suggesting that they be involved in the same pathway in the aleurone aside from OsWRKY71 regulation. From this annotation, I also identified and compiled a large family of 104 unique HIL members expressed in various rice tissues. A classification system was designed based on the presence of several conserved amino acid motifs: NPN, RPP, and YQYF. Most HIL members, including OsHIL16 and -58, were Group I members with all three motifs present. These and further analyses suggest that HILs may have multiple roles in plant development, including in seed germination

Proteômica e histopatologia associadas aos mecanismos de infecção e defesa da videira (Vitis sp) ao patógeno Plasmopara viticola (Berk. &amp; M. A. Curtis) Berl. &amp; de Toni

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Recursos Genéticos Vegetais, Florianópolis, 2014.O cultivo de espécies frutíferas no Brasil encontra na incidência de pragas e doenças suas principais limitações. Uma estratégia adequada para o controle das moléstias é o uso dos mecanismos de defesas natos das plantas. Para isto as plantas apresentam um sistema de resposta basal associado a um sistema de defesa especifica para cada tipo de patógeno, o que gera respostas sistêmicas e duradouras, como transdução de sinais e transcrição dos genes de defesa. As proteínas relacionadas a patogenene (PR) bem como os fitohormônios, importantes na sinalização, são alvos importantes de estudos relacionados à resistência contra patógenos. Dentre as doenças de grande impacto em culturas com relevância econômica está o míldio da videira (Plasmopara viticola). A videira (Vitis vinifera) além da sua importância econômica é a primeira espécie frutífera cujo genoma foi sequenciado, tornando-se assim um ótimo modelo de estudo. Análises proteômicas possibilitam avanços significativos no conhecimento dos mecanismos moleculares de resistência a patógenos e a associação de diferentes técnicas relacionadas a proteomica para estudar as diversas partes do mecanismo de resistência na planta pode auxiliar no entendimento da relação planta-patógeno. Assim, o presente estudo tem como objetivo contribuir para o esclarecimento dos mecanismos moleculares e bioquímicos associados à resistência à doença causada por Plasmopara viticola em videira (Vitis sp). Os estudos histológicos tiveram como objetivo caracterizar o desenvolvimento do P. viticola, bem como determinar diferenças estruturais nas variedades escolhidas para o estudo: uma resistente (Bordô) e outra susceptível (Cabernet Sauvignon). Nessas avaliações histológicas foram observadas diferenças estruturais e bioquímicas entre as duas variedades como presença de pêlos e mesofilo mais compactos na var. resistente. Já para o desenvolvimento do patógeno constatou-se a dificuldade dos esporos na penetração dos tubos germinativos através dos estômatos também na variedade resistente. Para as analises da expressão protéica foi utilizado uma combinação de eletroforese bidimensional (2-DE) associado ao nanoLC- MS/MS. Foram observadas diferenças protéicas quantitativas e qualitativas nos diferentes tempos após a inoculação. Foram identificadas 44 proteínas exclusivas, sendo 33 da var. resistente e 10 da var. suscetível. A ativação da resposta de defesa foi observada somente na var. Bordô com aumento constante na expressão das proteínas no decorrer dos tempos analisados, principalmente as 96 hai(horas após a infecção). As analises do proteoma das linhagens de videira contendo o locus de resistência Rpv1 e Rpv3 também estudadas nesse projeto apresentaram resultados semelhantes os da var. Bordô. Nessas analises foram identificados 41 proteínas. As proteínas foram classificadas em diferentes categorias funcionais: metabolismo energético, metabolismo de proteínas, resposta ao estresse e resposta de resistência. Proteínas relacionadas à resistência estavam presentes apenas as 96 hai. A ativação de uma reação de defesa, com um aumento da expressão das proteínas foi observada mais frequentemente em 48 hpi, o que é consistente com o estabelecimento da interação incompatível para P. viticola.Abstract: Cultivation of fruit species in Brazil has its limitations due to the incidence of pests and diseases. An adequate strategy for disease control is the use of a basal response system intrinsic to the plant. For that, plants show a basal response system associated with a specific defense system for each type of pathogen, which creates lasting and systemic responses, such as signal transduction and transcription of defense genes. Pathogenesis-related proteins (PR), as well as phytohormones (important for signaling), are important targets for studies related to resistance against pathogens. The Grapevine Powdery Mildew (Plasmopara viticola) is among the diseases of great impact in economically relevant cultures. Grapevine (Vitis vinifera), besides its economic importance, is the first fruit specie whose genome was sequenced, becoming a great model of study. Proteomic analyzes enable significant advances in knowledge of molecular mechanism on pathogen resistance and the association of different techniques to study different parts of resistance mechanisms in plants can help to understand the plant-pathogen relation. Therefore, this study aimed to contribute to the elucidation of the molecular and biochemical mechanisms associated to disease resistance caused by Plasmopara viticola in Grapevine (Vitis sp.). Histological studies aimed to characterize the development of P. viticola, as well as to determine structural differences in the chosen varieties for the study: a resistant one (Bordo) and susceptible one (Cabernet Sauvignon). Structural and biochemical differences were observed between the varieties on the histological analyzes, such as presence of leaf hair and more compact mesophyll on the resistant variety. For the pathogen development it was found a difficulty for the spores to penetrate the germ tubes through stomata for the resistant variety. The combination of bidimensional electrophoresis (2-DE) associated with nanoLC -MS/MS was used for the protein expression analyzes. Quantitative and qualitative differences were found in the protein analyzes for different hour after infection. 44 exclusive proteins were found, 34 for the resistant variety and 10 for the susceptible variety. The activation of defense response was observed only in the Bordo variety with a constant increase on protein expression over time, mainly at 96 hours after infection. Proteomic analyzes of Grapevine lines containing Rpv1 and Rpv3 resistant locus, also studied in this project, showed similar results to the Bordo variety, where 41 proteins were identified. The proteins were classified in different functional categories: energetic metabolism, protein metabolism, stress response and resistance response. Proteins related to resistance were present only at 96 hours after infection. The activation of a defense reaction with an increase of protein expression was observed most often at 48 hours after infection, which is consistent with the establishment of incompatible interaction for P. viticola