Genetic variability in wild and domestic populations of Inga edulis Mart. (Fabaceae) in Peruvian Amazon

Human activity in the Peruvian Amazon causes native vegetation fragmentation into smaller units resulting on the increase of agricultural systems. Understanding the level, the structure and the origin of morphologic within and among populations variation is essential for planning better management strategies aimed at sustainable use and conservation of Inga edulis Mart. species. We evaluated the genetic variability in wild and domestic population to unfold cultivation changes over the species genetic resources. We have studied 400 adult trees: 200 cultivated on arable land and 200 wild growing in untouched lowland rain forest. The individuals were randomly selected. Sampling sites were selected and defined on the basis of the geographical coordinates: longitude, latitude and altitude. Phenotypic variation was monitored using the proposed descriptor of qualitative and quantitative features (e.g., weight of hundred seeds). For each individual a voucher specimen was kept. The total genomic DNA was extracted from young leaves, conserved in silica gel, with INVITEK, Invisorb ®Spin Plant Mini Kit. Samples were then genotyped with five microsatellite (SSR) loci. One locus (Pel5) was cross-transferred, developed previously for Pithecellobium elegans. The remaining four loci (Inga03, 05, 08, 33) were previously developed for the species. Polymerase chain reaction (PCR) was made using a Biometra® T1 Thermocycler using the following profile: 95 °C for 2 min; 95°C for 15 s, 55/59 °C for 30 s, 72 °C for 30 s, 30 cycles; 72 °C for 15 min. The PCR products were fluorescently labelled. The visualization of fragments was carried out according to standard protocols on genetic analyser, ABI PRISM® 310 (Applied Biosystems), using ABI GENESCAN and GENOTYPER software. The phenotypic and genotypic results of wild versus domestic populations are under evaluation to verify if cultivation is altering the allelic variation considering that morphology is considerably changed.Czech Development Cooperation Project entitled “Sustainable use of natural resources in Peruvian Amazon” Project No. 23/MZe/B/07-10; The Academy of Science of The Czech Republic and the National Council for Science, Technology and Technological Innovation, Peru, binational project entitled “Morphological and genetic diversity of indigenous tropical trees in the Amazon – model study of Inga edulis Mart. in Peruvian Amazon”; Foundation “Nadace Nadání Josefa, Marie a Zdeňky Hlávkových”, Czech Republi

Fluorescence-Tagged Transgenic Lines Reveal Genetic Defects in Pollen Growth—Application to the Eif3 Complex

BACKGROUND: Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis. METHODOLOGY/PRINCIPAL FINDINGS: Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation. CONCLUSION/SIGNIFICANCE: eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis

Transcriptome analysis of haploid male gametophyte development in Arabidopsis

BACKGROUND: The haploid male gametophyte generation of flowering plants consists of two- or three-celled pollen grains. This functional specialization is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen ontogeny is also an attractive model in which to dissect cellular networks that control cell growth, asymmetric cell division and cellular differentiation. Our objective, and an essential step towards the detailed understanding of these processes, was to comprehensively define the male haploid transcriptome throughout development. RESULTS: We have developed staged spore isolation procedures for Arabidopsis and used Affymetrix ATH1 genome arrays to identify a total of 13,977 male gametophyte-expressed mRNAs, 9.7% of which were male-gametophyte-specific. The transition from bicellular to tricellular pollen was accompanied by a decline in the number of diverse mRNA species and an increase in the proportion of male gametophyte-specific transcripts. Expression profiles of regulatory proteins and distinct clusters of coexpressed genes were identified that could correspond to components of gametophytic regulatory networks. Moreover, integration of transcriptome and experimental data revealed the early synthesis of translation factors and their requirement to support pollen tube growth. CONCLUSIONS: The progression from proliferating microspores to terminally differentiated pollen is characterized by large-scale repression of early program genes and the activation of a unique late gene-expression program in maturing pollen. These data provide a quantum increase in knowledge concerning gametophytic transcription and lay the foundations for new genomic-led studies of the regulatory networks and cellular functions that operate to specify male gametophyte development

A Plant-Specific Transcription Factor IIB-Related Protein, pBRP2, Is Involved in Endosperm Growth Control

General transcription factor IIB (TFIIB) and TFIIB-related factor (BRF), are conserved RNA polymerase II/III (RNAPII/III) selectivity factors that are involved in polymerase recruitment and transcription initiation in eukaryotes. Recent findings have shown that plants have evolved a third type of B-factor, plant-specific TFIIB-related protein 1 (pBRP1), which seems to be involved in RNAPI transcription. Here, we extend the repertoire of B-factors in plants by reporting the characterization of a novel TFIIB-related protein, plant-specific TFIIB-related protein 2 (pBRP2), which is found to date only in the Brassicacea family. Unlike other B-factors that are ubiquitously expressed, PBRP2 expression is restricted to reproductive organs and seeds as shown by RT-PCR, immunofluorescence labelling and GUS staining experiments. Interestingly, pbrp2 loss-of-function specifically affects the development of the syncytial endosperm, with both parental contributions required for wild-type development. pBRP2, is the first B-factor to exhibit cell-specific expression and regulation in eukaryotes, and might play a role in enforcing bi-parental reproduction in angiosperms

An “Electronic Fluorescent Pictograph” Browser for Exploring and Analyzing Large-Scale Biological Data Sets

Background. The exploration of microarray data and data from other high-throughput projects for hypothesis generation has become a vital aspect of post-genomic research. For the non-bioinformatics specialist, however, many of the currently available tools provide overwhelming amounts of data that are presented in a non-intuitive way. Methodology/Principal Findings. In order to facilitate the interpretation and analysis of microarray data and data from other large-scale data sets, we have developed a tool, which we have dubbed the electronic Fluorescent Pictograph – or eFP – Browser, available a

Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil

Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction

Molecular Foundations of Reproductive Lethality in Arabidopsis thaliana

The SeedGenes database (www.seedgenes.org) contains information on more than 400 genes required for embryo development in Arabidopsis. Many of these EMBRYO-DEFECTIVE (EMB) genes encode proteins with an essential function required throughout the life cycle. This raises a fundamental question. Why does elimination of an essential gene in Arabidopsis often result in embryo lethality rather than gametophyte lethality? In other words, how do mutant (emb) gametophytes survive and participate in fertilization when an essential cellular function is disrupted? Furthermore, why do some mutant embryos proceed further in development than others? To address these questions, we first established a curated dataset of genes required for gametophyte development in Arabidopsis based on information extracted from the literature. This provided a basis for comparison with EMB genes obtained from the SeedGenes dataset. We also identified genes that exhibited both embryo and gametophyte defects when disrupted by a loss-of-function mutation. We then evaluated the relationship between mutant phenotype, gene redundancy, mutant allele strength, gene expression pattern, protein function, and intracellular protein localization to determine what factors influence the phenotypes of lethal mutants in Arabidopsis. After removing cases where continued development potentially resulted from gene redundancy or residual function of a weak mutant allele, we identified numerous examples of viable mutant (emb) gametophytes that required further explanation. We propose that the presence of gene products derived from transcription in diploid (heterozygous) sporocytes often enables mutant gametophytes to survive the loss of an essential gene in Arabidopsis. Whether gene disruption results in embryo or gametophyte lethality therefore depends in part on the ability of residual, parental gene products to support gametophyte development. We also highlight here 70 preglobular embryo mutants with a zygotic pattern of inheritance, which provide valuable insights into the maternal-to-zygotic transition in Arabidopsis and the timing of paternal gene activation during embryo development

Molecular cytogenetics (FISH, GISH) of Coccinia grandis: A ca. 3 myr-old species of Cucurbitaceae with the largest Y/autosome divergence in flowering plants

The independent evolution of heteromorphic sex chromosomes in 19 species from 4 families of flowering plants permits studying X/Y divergence after the initial recombination suppression. Here, we document autosome/Y divergence in the tropical Cucurbitaceae Coccinia grandis, which is ca. 3 myr old. Karyotyping and C-value measurements show that the C. grandis Y chromosome has twice the size of any of the other chromosomes, with a male/female C-value difference of 0.094 pg or 10% of the total genome. FISH staining revealed 5S and 45S rDNA sites on autosomes but not on the Y chromosome, making it unlikely that rDNA contributed to the elongation of the Y chromosome; recent end-to-end fusion also seems unlikely given the lack of interstitial telomeric signals. GISH with different concentrations of female blocking DNA detected a possible pseudo-autosomal region on the Y chromosome, and C-banding suggests that the entire Y chromosome in C. grandis is heterochromatic. During meiosis, there is an end-to-end connection between the X and the Y chromosome, but the X does not otherwise differ from the remaining chromosomes. These findings and a review of plants with heteromorphic sex chromosomes reveal no relationship between species age and degree of sex chromosome dimorphism. Its relatively small genome size (0.943 pg/2C in males), large Y chromosome, and phylogenetic proximity to the fully sequenced Cucumis sativus make C. grandis a promising model to study sex chromosome evolution. Copyright © 2012 S. Karger AG, Base

Formin homology 2 domains occur in multiple contexts in angiosperms

BACKGROUND: Involvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions. RESULTS: In order to identify possible plant-specific sequence features within the FH2 protein family, we performed a detailed analysis of angiosperm formin-related sequences available in public databases, with particular focus on the complete Arabidopsis genome and the nearly finished rice genome sequence. This has led to revision of the current annotation of half of the 22 Arabidopsis formin-related genes. Comparative analysis of the two plant genomes revealed a good conservation of the previously described two subfamilies of plant formins (Class I and Class II), as well as several subfamilies within them that appear to predate the separation of monocot and dicot plants. Moreover, a number of plant Class II formins share an additional conserved domain, related to the protein phosphatase/tensin/auxilin fold. However, considerable inter-species variability sets limits to generalization of any functional conclusions reached on a single species such as Arabidopsis. CONCLUSIONS: The plant-specific domain context of the conserved FH2 domain, as well as plant-specific features of the domain itself, may reflect distinct functional requirements in plant cells. The variability of formin structures found in plants far exceeds that known from both fungi and metazoans, suggesting a possible contribution of FH2 proteins in the evolution of the plant type of multicellularity