Evolution of xyloglucan-related genes in green plants

<p>Abstract</p> <p>Background</p> <p>The cell shape and morphology of plant tissues are intimately related to structural modifications in the primary cell wall that are associated with key processes in the regulation of cell growth and differentiation. The primary cell wall is composed mainly of cellulose immersed in a matrix of hemicellulose, pectin, lignin and some structural proteins. Xyloglucan is a hemicellulose polysaccharide present in the cell walls of all land plants (Embryophyta) and is the main hemicellulose in non-graminaceous angiosperms.</p> <p>Results</p> <p>In this work, we used a comparative genomic approach to obtain new insights into the evolution of the xyloglucan-related enzymatic machinery in green plants. Detailed phylogenetic analyses were done for enzymes involved in xyloglucan synthesis (xyloglucan transglycosylase/hydrolase, α-xylosidase, β-galactosidase, β-glucosidase and α-fucosidase) and mobilization/degradation (β-(1→4)-glucan synthase, α-fucosyltransferases, β-galactosyltransferases and α-xylosyl transferase) based on 12 fully sequenced genomes and expressed sequence tags from 29 species of green plants. Evidence from Chlorophyta and Streptophyta green algae indicated that part of the Embryophyta xyloglucan-related machinery evolved in an aquatic environment, before land colonization. Streptophyte algae have at least three enzymes of the xyloglucan machinery: xyloglucan transglycosylase/hydrolase, β-(1→4)-glucan synthase from the celullose synthase-like C family and α-xylosidase that is also present in chlorophytes. Interestingly, gymnosperm sequences orthologs to xyloglucan transglycosylase/hydrolases with exclusively hydrolytic activity were also detected, suggesting that such activity must have emerged within the last common ancestor of spermatophytes. There was a positive correlation between the numbers of founder genes within each gene family and the complexity of the plant cell wall.</p> <p>Conclusions</p> <p>Our data support the idea that a primordial xyloglucan-like polymer emerged in streptophyte algae as a pre-adaptation that allowed plants to subsequently colonize terrestrial habitats. Our results also provide additional evidence that charophycean algae and land plants are sister groups.</p

Extensive Natural Epigenetic Variation At A De Novo Originated Gene.

Epigenetic variation, such as heritable changes of DNA methylation, can affect gene expression and thus phenotypes, but examples of natural epimutations are few and little is known about their stability and frequency in nature. Here, we report that the gene Qua-Quine Starch (QQS) of Arabidopsis thaliana, which is involved in starch metabolism and that originated de novo recently, is subject to frequent epigenetic variation in nature. Specifically, we show that expression of this gene varies considerably among natural accessions as well as within populations directly sampled from the wild, and we demonstrate that this variation correlates negatively with the DNA methylation level of repeated sequences located within the 5'end of the gene. Furthermore, we provide extensive evidence that DNA methylation and expression variants can be inherited for several generations and are not linked to DNA sequence changes. Taken together, these observations provide a first indication that de novo originated genes might be particularly prone to epigenetic variation in their initial stages of formation.9e100343

CALANGO: a phylogeny-aware comparative genomics tool for discovering quantitative genotype-phenotype associations across species

Living species vary significantly in phenotype and genomic content. Sophisticated statistical methods linking genes with phenotypes within a species have led to breakthroughs in complex genetic diseases and genetic breeding. Despite the abundance of genomic and phenotypic data available for thousands of species, finding genotype-phenotype associations across species is challenging due to the non-independence of species data resulting from common ancestry. To address this, we present CALANGO (comparative analysis with annotation-based genomic components), a phylogeny-aware comparative genomics tool to find homologous regions and biological roles associated with quantitative phenotypes across species. In two case studies, CALANGO identified both known and previously unidentified genotype-phenotype associations. The first study revealed unknown aspects of the ecological interaction between Escherichia coli, its integrated bacteriophages, and the pathogenicity phenotype. The second identified an association between maximum height in angiosperms and the expansion of a reproductive mechanism that prevents inbreeding and increases genetic diversity, with implications for conservation biology and agriculture

A transcriptomic analysis of gene expression in the venom gland of the snake Bothrops alternatus (urutu)

<p>Abstract</p> <p>Background</p> <p>The genus <it>Bothrops </it>is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of <it>Bothrops alternatus</it>, a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay.</p> <p>Results</p> <p>A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A₂(5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A₂were essentially acidic; no basic PLA₂were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed.</p> <p>Conclusions</p> <p><it>Bothrops alternatus </it>venom gland contains the major toxin classes described for other <it>Bothrops </it>venoms based on trancriptomic and proteomic studies. The predominance of type PIII metalloproteinases agrees with the well-known hemorrhagic activity of this venom, whereas the lower content of serine proteases and C-type lectins could contribute to less marked coagulopathy following envenoming by this species. The lack of basic PLA₂agrees with the lower myotoxicity of this venom compared to other <it>Bothrops </it>species with these toxins. Together, these results contribute to our understanding of the physiopathology of envenoming by this species.</p

The Evolutionary History Of Calreticulin And Calnexin Genes In Green Plants.

Calreticulin and calnexin are Ca(2+)-binding chaperones localized in the endoplasmic reticulum of eukaryotes acting in glycoprotein folding quality control and Ca(2+) homeostasis. The evolutionary histories of calreticulin and calnexin gene families were inferred by comprehensive phylogenetic analyses using 18 completed genomes and ESTs covering the major green plants groups, from green algae to angiosperms. Calreticulin and calnexin possibly share a common origin, and both proteins are present along all green plants lineages. The calreticulin founder gene within green plants duplicated in early tracheophytes leading to two possible groups of orthologs with specialized functions, followed by lineage-specific gene duplications in spermatophytes. Calnexin founder gene in land plants was inherited from basal green algae during evolution in a very conservative copy number. A comprehensive classification in possible groups of orthologs and a catalog of calreticulin and calnexin genes from green plants are provided.139255-

Phylogenetic analysis of Xyloglucan's hydrolytic enzymes in the Viridiplantae kingdom and construction of cDNA libraries from Jatoba (Hymenaea courbaril)

Orientador: Michel Georges Albert VincentzDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Introdução: Os xiloglucanos são os polímeros de açúcar mais abundantes na hemicelulose da maioria das espécies de plantas terrestres, em especial nas eudicotiledôneas. Possuem papel estrutural na parede celular vegetal, interagindo com os filamentos de celulose, e podem ser utilizados como reserva em sementes de várias espécies de eudicotiledôneas, como o Jatobá (Hymenaea courbaril), onde correspondem a quase 50% do peso seco da semente. Este polímero é formado por uma cadeia central de ß-glucano com ramificações que contêm xilose, galactose e fucose. O mecanismo de degradação deste polímero é realizado por cinco hidrolases: XTH, ß-Galactosidase, ß-Glucosidase,? a-Xilosidase e? a-Fucosidase. Estas enzimas são codificadas por genes que constituem famílias multigênicas nos genomas de plantas, e sua atividade na degradação seletiva de xiloglucano têm papel central na regulação do crescimento e morfogênese da célula vegetal. O Jatobá (Leguminosae) é uma árvore tropical, nativa do Brasil, que vem sendo utilizada como modelo vegetal para estudos de impacto ambiental por efeito estufa e estresses abióticos oriundos do aquecimento global. Foi observado que mudas de Jatobá, crescidas numa atmosfera com 720 PPM de CO2 (dobro da concentração atual), apresentam até 50% de aumento de biomassa aos 100 dias. O entendimento das respostas transcripcionais desta planta, em resposta a estes estresses, pode levar a conclusões à cerca de como as florestas tropicais responderão ao aumento inexorável na concentração de CO2, num quadro de aquecimento global. Resultados: Construímos bibliotecas de cDNA de folhas, caule, cotilédones e raízes de plântulas de 45 dias de Jatobá. Um seqüenciamento amostral dos ESTs levou à obtenção de 103 seqüências, parciais ou completas, de proteínas de Jatobá. São os primeiros dados de ESTs numa árvore tropical brasileira. Análises filogenéticas das enzimas que constituem o mecanismo de degradação de xiloglucano foram conduzidas ao longo de 13 genomas completos e 27 bancos de ESTs de espécies dos mais diversos grupos no reino Viridiplantae. Isso nos permitiu organizar a diversidade destas cinco famílias multigênicas em possíveis grupos de ortólogos (PoGOs). As XTHs foram divididas em seis grupos de genes homólogos e 19 PoGOs. As ß-Galactosidases foram divididas em dois grupos de genes homólogos e 10 PoGOs. As ß-Glucosidases foram divididas em dois grupos de genes homólogos e dois PoGOs. As? a-Xilosidase foram divididas em três PoGOs e as a-Fucosidase em dois PoGOs não relacionados evolutivamente. Conclusões e Perspectivas: As 103 seqüências peptídicas obtidas de Jatobá foram anotadas por comparação e serão disponibilizadas nos bancos de dados internacionais. A perspectiva de seqüenciar mais clones poderá levar à montagem do transcriptoma do Jatobá, algo inédito para uma árvore tropical. Concluímos, com as análises filogenéticas, que as XTHs, que formam um grupo monofilético de genes em Streptophyta, surgiram antes da conquista do ambiente terrestre. Estes genes foram progressivamente amplificados ao longo da evolução das plantas terrestres, o que sugere um ganho progressivo de complexidade, que teve seu auge nas Angiospermas. Apresentamos evidências que podem unir evolutivamente as XTHs exclusivas de plantas a enzimas transglicosiladoras de cadeias de ß -glucano em fungos, o que sugere uma origem comum do processo de transglicosilação de cadeias de ß-glucano como mecanismo de controle do crescimento e formato celular em eucariotos com parede celular. As ß-Galactosidases formam um grupo monofilético em Embryophytas com nove PoGOs, no entanto sua grande diversificação (seis PoGOs) ocorreu apenas em Angiospermas. As ß -Glucosidases formam um grupo monofilético em Embryophytas, seqüências similares em bactérias fotossintetizantes podem sugerir uma origem no ancestral dos cloroplastos. As a -Xilosidase, que são monofiléticas nas Spermatophytas, derivaram das ?a-Glucosidases que se encontram dispersas entre todos os eucariotos, é um caso de neofuncionalização. Duas linhagens distintas evolutivamente de a-Fucosidases foram encontradas, uma delas é monofilética em Embryophytas e a outra pertence a uma grande família multigênica (GDSL-motif) da qual pouco se sabe. Mostramos que o mecanismo completo (cinco hidrolases) de degradação de xiloglucano existia no ancestral comum das Spermatophytas (plantas com semente). Como perspectivas este trabalho permite a racionalização de estudos funcionais destas hidrolases o que, em longo prazo, pode contribuir com processos biotecnológicos que passem pela modificação seletiva da parede celular vegetal.Abstract: Introduction: Xyloglucans are the main hemicelulose in most of land plants, especially in eudicots. It is a structural compound of plant cell-wall that interacts with cellulose and can be used as seed's energy storage of many species, like Jatoba (Hymenaea courbaril). Xyloglucan structure is composed of a ß-glucan backbone that it branched with xylose, galactose and fucose. Its degradation machinery is composed by five glycosil hydrolases: XTH, ß-Galactosidase, ß-Glucosidase,?a-Xylosidase and? a- Fucosidase. These enzymes are codified by multigenic families in plant's genomes and it plays a central role in key processes like growth and morphogenesis of plant cells. Jatoba (Leguminosae) is a tropical tree, native of Brazil. It's been used as a model tree in researches of plant's responses to stresses caused by global warming and high atmospheric CO2 concentration. It was observed a 50% increase in biomass of a 100 days Jatoba seedling when grown in a 720 PPM of CO2 atmosphere (two times bigger than today's atmospheric concentration). Understand the transcriptional responses to these stresses can lead to conclusions about how tropical forests will respond to high concentrations of CO2 and global warming. Results: We made cDNA libraries of leaves, stem, cotyledons and roots of 45 days seedlings of Jatoba. A preliminary sequencing of these libraries reveled 103 predict protein sequences (most partial sequences). Phylogenetic analyses of xyloglucan hydrolytic enzymes were conducted using 13 completed genomes and 27 ESTs assemblies, from a wild range of taxonomic groups in the Viridiplantae kingdom. It allowed us to divide XTH's diversity of genes into six homology groups and 19 possible groups of orthologues (PoGOs). ß-Galactosidases were divided into two groups of homologues and 10 PoGOs. ß -Glucosidases were divided into two groups of homologues and two PoGOs. a-Xylosidase were divided into three PoGOs and a-Fucosidase into two PoGOs evolutionarily unrelated. Conclusions and Perspectives: The 103 protein sequences of Jatoba were annotated by comparison to known proteins and will be deposited in international sequences assemblies. As a perspective, the sequencing of Jatoba ESTs will lead to the assembly of its transcriptome, something never done before in a tropical tree. We concluded that XTHs are monophyletic group o genes in Streptophyta, what means they emerged before lands conquest by plants. These genes were progressively amplified in land plants evolution, especially in Angiosperms, what suggests a progressive gain in complexity. We showed evidences of a possible evolutionary relation between plant's XTHs and fungus hydrolases/transglycosylases enzymes. It suggests a eukaryotic ancestral mechanism to control cell expansion and shape based in ß -glucan transglycosylation and its interaction to cellulose (in plants) or chitin (in fungus). The ß -Galactosidases are a monophyletic group in Embryophytas that were divided into nine PoGOs, six PoGOs only appeared in Angiosperms. The ß -Glucosidases belongs to a monophyletic group in Embryophytas that has sequence similarity to bacterial proteins, especially ones from photosynthetic bacteria species. The a-Xylosidases are a PoGO in Spermatophyta that probably emerged from a-Glucosidases presents in all eukaryotes. It's probably a neofunctionalization process. Two evolutionary distinct lineages of a-Fucosidases where found, one monophyletic in Embryophytas and another that belongs to the poorly understood multigenic family "GDSL-motif proteins". We showed that the complete machinery (all the five hydrolases) of Xyloglucan degradation already exists in Spermatophytas common ancestor. As a perspective, we expect to rationalize the functional characterization works among these multigenic families and to contribute in biotechnology processes that pass through cell-wall modification and selective control.MestradoGenetica Vegetal e MelhoramentoMestre em Genética e Biologia Molecula

Evolution of multigenic families and genetic networks in plants

Orientadores: Michel Georges Albert Vincentz, Renato Vicentini dos SantosTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O sequenciamento de um número crescente de genomas completos tem transformado a biologia. Mais especificamente, no campo da biologia evolutiva, tem se tornado possível endereçar perguntas centrais sobre o funcionamento ultimato dos mecanismos de transformação genética, com potencial impacto em todos os campos da biologia, assim como na filosofia. Esta tese está dividida em dois aspectos importantes da evolução de genomas: o processo de duplicação e fixação de genes duplicados, que é a base do surgimento de famílias multigênicas, e a evolução de redes de regulação, que determinam as relações de causalidade nos processos celulares. Os dois aspectos se relacionam à evolução da complexidade, tanto no que tange o conteúdo gênico dos seres vivos quanto nas interações mecanistica entre os genes via seus produtos (RNAs e proteínas basicamente). No primeiro aspecto abordamos a evolução de dois mecanismos biológicos que depende da ação integrada entre proteínas de famílias distintas: o mecanismo de síntese e degradação do polissacarídeo de parede celular xiloglucano, e o ciclo das chaperonas calreticulina/calnexina envolvidas no controle de qualidade de proteínas sintetizadas no retículo endoplasmático. Nossos trabalhos mostraram que uma forma primordial de xiloglucano, mais simples, surgiu antes da conquista do meio terrestre pela linhagem das plantas, ao contrário do que se imaginava, e que o ciclo calreticulina/calnexina é produto da subfuncionalização em eucariotos basais de uma chaperona ancestral, além do surgimento de funções específicas na família da calreticulina em plantas terrestres. O interesse em evolução de famílias multigênicas nos levou a desenvolver um método (Phylexpress) para análise de ortologia em larga escala, bem como permitir a integração de dados de expressão na tentativa de entender a dinâmica evolutiva da expressão gênica em famílias multigênicas. Utilizamos nosso método para revisitar o conteúdo gênico dos ESTs públicos de cana-de-açúcar, como prova de conceito, numa análise comparativa com o proteoma predito de sorgo. Nossos resultados mostram uma cobertura em termos de ortólogos para apenas ~58% do proteoma predito de sorgo em contrates com estimativas anteriores, com métodos mais simples, que chegaram a 90% do proteoma hipotético de cana. Para abordar a dinâmica evolutiva de redes de regulação, realizamos medições, em escala genômica, das alterações nos níveis de mRNAs de plântulas de sorgo e arroz em resposta a tratamentos de curta duração (2hrs) com sinais exógenos de ABA (hormônio vegetal) e dos açúcares glicose e sacarose. Utilizamos dados públicos e experimentalmente comparáveis de Arabidopsis thaliana em resposta aos mesmos sinais para realizar comparações que revelassem respostas conservadas ou divergentes entre ortólogos. Além disso, buscamos entender a dinâmica evolutiva das respostas transcricionais num contexto de duplicação gênica em famílias multigênicas, onde há diversos genes potencialmente redundantes do ponto de vista bioquímico/estrutural. Nossa abordagem sugere que redes de regulação gênica em eucariotos complexos evoluem majoritariamente de forma neutra, pois parecem apresentar uma taxa de divergência constante, que independe da rede (disparada por cada um dos diferentes sinais) e das espécies envolvidas. Nossos dados são complementares e potencialmente confirmadores de modelos recentes de evolução não-adaptativa em redes de regulação gênica. Concluímos que a evolução da complexidade em sistemas biológicos está parcialmente ligada à diminuição da eficiência da seleção, causada majoritariamente por números populacionais efetivos restritivos presentes nas linhagens de eucariotos complexos (vertebrados e plantas terrestres)Abstract: The availability of complete sequences of a growing number of genomes is transforming biology. More specifically, in the field of evolutionary biology, it became possible to address central questions on the ultimate mechanisms underlying genetic changes. It has a broad impact on biology and philosophy as well. This thesis deals with two important aspects of genome evolution: the process of gene duplication and fixation of duplicated genes, which is the basis of the origins of multigenic families, and the evolution of genetic regulatory networks that determines the causality of the cellular processes. Both aspects are related to the evolution of complexity regarding the gene content of living forms and the mechanistic interaction between the gene products (mainly RNAs and proteins). In the first aspect we studied the evolution of two biological mechanisms depending on the integrated function of proteins from distinct families. The mechanism of synthesis and remobilization of xyloglucan, a plant cell wall polysaccharide, and the calreticulin/calnexin cycle of protein folding that takes place in the endoplasmic reticulum. Our work showed that a primordial form of xyloglucan already existed before the land conquest by plants. We propose that the calreticulin/calnexin cycle is the product of subfuncionalization of an ancestral eukaryotic chaperone, and plants evolved specific calreticulin functions due to gene duplication. Our interest in the evolution of multigenic families impelled the development of Phylexpress, a method dedicated to large-scale orthology analyses. It can integrate expression data in the context of multigenic families with the goal of understand the evolutionary dynamics of gene expression. We used Phylexpress to revisit the gene content of the publicly available sugarcane ESTs as a proof of concept. Our results showed that the ESTs sampled orthologs for just ~58% of the predict sorghum proteome, in contrast with previous estimations acconting for 90% of the hypotethical sugarcane proteome. In order to approach the evolutionary dynamics of regulatory networks, we measured global changes in gene expression of sorghum and rice plantlets in response to short-term treatments (2hrs) with exogenous ABA (plant hormone) and the sugars glucose and sucrose. We took public data from comparable experiments using Arabidopsis thaliana in order to unravel conserved and divergent responses across orthologs. Furthermore, we analyzed the evolutionary change in transcriptional responses in a context of gene duplications in multigenic families, leading to a set of potentially redundant genes in terms of biochemical/structural properties. Our approach suggests that gene regulatory networks in complex eukaryotes evolve mainly neutrally, in a constant rate that is independent of the analyzed network (triggered by each one of the signals) and the species. Our data is complementary and potentially confirmatory of recent models of nonadaptive evolution in regulatory networks. We concluded that the evolution of the complexity in biological systems is partially connected to the attenuation of the efficiency, mainly due to low effective population sizes present in the lineages that gave rise to complex eukaryotes (vertebrates and land plants)DoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecula

The Evolution of tRNA Copy Number and Repertoire in Cellular Life

tRNAs are universal decoders that bridge the gap between transcriptome and proteome. They can also be processed into small RNA fragments with regulatory functions. In this work, we show that tRNA copy number is largely controlled by genome size in all cellular organisms, in contrast to what is observed for protein-coding genes that stop expanding between ~20,000 and ~35,000 loci per haploid genome in eukaryotes, regardless of genome size. Our analyses indicate that after the bacteria/archaea split, the tRNA gene pool experienced the evolution of increased anticodon diversity in the archaeal lineage, along with a tRNA gene size increase and mature tRNA size decrease. The evolution and diversification of eukaryotes from archaeal ancestors involved further expansion of the tRNA anticodon repertoire, additional increase in tRNA gene size and decrease in mature tRNA length, along with an explosion of the tRNA gene copy number that emerged coupled with accelerated genome size expansion. Our findings support the notion that macroscopic eukaryotes with a high diversity of cell types, such as land plants and vertebrates, independently evolved a high diversity of tRNA anticodons along with high gene redundancy caused by the expansion of the tRNA copy number. The results presented here suggest that the evolution of tRNA genes played important roles in the early split between bacteria and archaea, and in eukaryogenesis and the later emergence of complex eukaryotes, with potential implications in protein translation and gene regulation through tRNA-derived RNA fragments