Analysis of central Hox protein types across bilaterian clades: on the diversification of central Hox proteins from an Antennapedia/Hox7-like protein.

Hox proteins are among the most intensively studied transcription factors and represent key factors in establishing morphological differences along the anterior-posterior axis of animals. They are generally regarded as highly conserved in function, a view predominantly based on experiments comparing a few (anterior) Hox proteins. However, the extent to which central or abdominal Hox proteins share conserved functions and sequence signatures remains largely unexplored. To shed light on the functional divergence of the central Hox proteins, we present an easy to use resource aimed at predicting the functional similarities of central Hox proteins using sequence elements known to be relevant to Hox protein functions. We provide this resource both as a stand-alone download, including all information, as well as via a simplified web-interface that facilitates an accurate and fine-tuned annotation of novel Hox sequences. The method used in the manuscript is, so far, the only published sequence-based method capable of differentiating between the functionally distinct central Hox proteins with near-identical homeodomains (such as the Drosophila Antp, Ubx and Abd-A Hox proteins). In this manuscript, a pairwise-sequence-similarity based approach (using the bioinformatics tool CLANS) is used to analyze all available central Hox protein sequences. The results are combined with a large-scale species phylogeny to depict the presence/absence of central Hox sequence-types across the bilaterian lineage. The obtained pattern of distribution of the Hox sequence-types throughout the species tree enables us to infer at which branching point a specific type of central Hox protein was present. Based on the Hox sequences currently available in public databases, seven sequence-similarity groups could be identified for the central Hox proteins, two of which have never been described before (Echi/Hemi7 and Echi/Hemi8). Our work also shows, for the first time, that Antp/Hox7-like sequences are present throughout all bilaterian clades and that all other central Hox protein groups are specific to sub-lineages in the protostome or deuterostome branches only.

microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula.

Plant root architecture is regulated by the initiation and modulation of cell division in regions containing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organogenesis at the site of lateral root or, in legumes, nodule formation. Root meristems can also be generated in vitro from leaf explants cultures supplemented with auxin. microRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root development. To identify key miRNAs involved in root meristem formation in Medicago truncatula, we used deep sequencing to compare miRNA populations. Comparisons were made between: (1) the root tip (RT), containing the RAM and the elongation zone (EZ) tissue and (2) root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families miR165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Selected miRNAs and targets were validated using Taqman miRNA assays and 5' RACE. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Target prediction revealed a number of miRNAs to target genes previously shown to be differentially expressed between RT and EZ or RFC and NRFC and important in root development. Additionally, we predict the miRNA/target relationships for miR397 and miR160 to be conserved in M. truncatula. Amongst the predictions, were AUXIN RESPONSE FACTOR 10, targeted by miR160 and a LACCASE-like gene, targeted by miR397, both are miRNA/target pairings conserved in other species.

Comparative proteomic profiles of the soybean (Glycine max) root apex and differentiated root zone.

The root apical meristem (RAM) is responsible for the growth of the plant root system. Because of the importance of root architecture in the performance of crop plants, we established a proteome reference map of the soybean root apex and compared this with the proteome of the differentiated root zone. The root apex samples contained the apical 1 mm of the root, comprising the RAM, quiescent center and root cap. We identified 342 protein spots from 550 excised proteins (∼62%) of root apex samples by MALDI-TOF MS/MS analysis. All these proteins were also present in the differentiated root, but differed in abundance. Functional classification showed that the most numerous protein categories represented in the root were those of stress response, glycolysis, redox homeostasis and protein processing. Using DIGE, we identified 73 differentially accumulated proteins between root apex and differentiated root. Proteins overrepresented in the root apex belonged primarily to the pathways for protein synthesis and processing, cell redox homeostasis and flavonoid biosynthesis. Proteins underrepresented in the root apex were those of glycolysis, tricarboxylic acid metabolism and stress response. Our results highlight the importance of stress and defense response, redox control and flavonoid metabolism in the root apex.

Improving Hox protein classification across the major model organisms.

The family of Hox-proteins has been a major focus of research for over 30 years. Hox-proteins are crucial to the correct development of bilateral organisms, however, some uncertainty remains as to which Hox-proteins are functionally equivalent across different species. Initial classification of Hox-proteins was based on phylogenetic analysis of the 60 amino acid homeodomain. This approach was successful in classifying Hox-proteins with differing homeodomains, but the relationships of Hox-proteins with nearly identical homeodomains, yet distinct biological functions, could not be resolved. Correspondingly, these 'problematic' proteins were classified into one large unresolved group. Other classifications used the relative location of the Hox-protein coding genes on the chromosome (synteny) to further resolve this group. Although widely used, this synteny-based classification is inconsistent with experimental evidence from functional equivalence studies. These inconsistencies led us to re-examine and derive a new classification for the Hox-protein family using all Hox-protein sequences available in the GenBank non-redundant protein database (NCBI-nr). We compare the use of the homeodomain, the homeodomain with conserved flanking regions (the YPWM and linker region), and full length Hox-protein sequences as a basis for classification of Hox-proteins. In contrast to previous attempts, our approach is able to resolve the relationships for the 'problematic' as well as ABD-B-like Hox-proteins. We highlight differences to previous classifications and clarify the relationships of Hox-proteins across the five major model organisms, Caenorhabditis elegans, Drosophila melanogaster, Branchiostoma floridae, Mus musculus and Danio rerio. Comparative and functional analysis of Hox-proteins, two fields crucial to understanding the development of bilateral organisms, have been hampered by difficulties in predicting functionally equivalent Hox-proteins across species. Our classification scheme offers a higher-resolution classification that is in accordance with phylogenetic as well as experimental data and, thereby, provides a novel basis for experiments, such as comparative and functional analyses of Hox-proteins.

Dissection of symbiosis and organ development by integrated transcriptome analysis of lotus japonicus mutant and wild-type plants.

Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful gene expression data set.

Computational prediction of candidate miRNAs and their targets from Medicago truncatula non-protein-coding transcripts.

Identification and analysis of miRNAs enhances our understanding of the important roles that small RNAs play in complex regulatory networks. It is often difficult to perform large-scale validation of miRNA expression that is predicted from genomic regions. Expressed transcripts provide an alternative resource to facilitate identification of miRNAs and their targets. We developed a computational pipeline to scan for miRNA genes from polyadenylated transcripts that were associated with limited protein coding potentials, corresponding to the intergenic regions of Medicago truncatula genomic sequences. Each predicted miRNA was required to have a near perfect match with target genes. We also searched for miRNA conservation in other plant species, clustered highly similar miRNAs, and provided a functional classification of target genes.

Detecting genetic recombination.

Recombination is the major motor of evolution. While mutations result in gradual changes, recombination reshuffles entire functional modules and thus progresses evolution in leaps and bounds. We need to identify recombination breakpoints in sequences to understand the evolutionary process, the impact of recombination, and to reconstruct the phylogenetic history of genes and genomes. This chapter provides a step by step guide for detecting recombination even in large and complex sequence alignments.

Transcriptional profiling of Medicago truncatula meristematic root cells.

BACKGROUND: The root apical meristem of crop and model legume Medicago truncatula is a significantly different stem cell system to that of the widely studied model plant species Arabidopsis thaliana. In this study we used the Affymetrix Medicago GeneChip(R) to compare the transcriptomes of meristem and non-meristematic root to identify root meristem specific candidate genes. RESULTS: Using mRNA from root meristem and non-meristem we were able to identify 324 and 363 transcripts differentially expressed from the two regions. With bioinformatics tools developed to functionally annotate the Medicago genome array we could identify significant changes in metabolism, signalling and the differentially expression of 55 transcription factors in meristematic and non-meristematic roots. CONCLUSION: This is the first comprehensive analysis of M. truncatula root meristem cells using this genome array. This data will facilitate the mapping of regulatory and metabolic networks involved in the open root meristem of M. truncatula and provides candidates for functional analysis.

Bioinformatic analysis of the CLE signaling peptide family.

BACKGROUND: Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases. RESULTS: We identified 114 new members of the CLE protein family from various plant species, as well as five protein sequences containing multiple CLE domains. We were able to cluster the CLE domain proteins into 13 distinct groups based on their pairwise similarities in the primary CLE motif. In addition, we identified secondary motifs that coincide with our sequence clusters. The groupings based on the CLE motifs correlate with known biological functions of CLE signaling peptides and are analogous to groupings based on phylogenetic analysis and ectopic overexpression studies. We tested the biological function of two of the predicted CLE signaling peptides in the legume Medicago truncatula. These peptides inhibit the activity of the root apical and lateral root meristems in a manner consistent with our functional predictions based on other CLE signaling peptides clustering in the same groups. CONCLUSION: Our analysis provides an identification and classification of a large number of novel potential CLE signaling peptides. The additional motifs we found could lead to future discovery of recognition sites for processing peptidases as well as predictions for receptor binding specificity.

In Silico identification and characterization of mRNA-like noncoding transcripts in Medicago truncatula.

Accumulating evidence suggests that that non-coding RNAs (ncRNAs) play key roles in gene regulation and may form the basis of an inter-gene communication system. Many ncRNAs are synthesized similar to mRNAs and can be detected through screening of polyA-rich EST or cDNA libraries. We developed a computational pipeline to screen EST and genomic sequence data for those transcribed genes with limited protein coding potential and applied this pipeline to the model legume Medicago truncatula. This process identified a set of 503 mRNA-like transcripts that appear not to encode proteins. Further computational analysis showed that many of these ncRNA candidates share structural similarities to known ncRNAs and that they clearly differ from protein coding genes and non-transcribed regions in their base and oligonucleotide compositions, as well as in aspects of secondary structure. By using a machine learning approach, we show that the distinctive ncRNA features presented in this study can be used to discriminate most ncRNAs and may thus be useful for improving ncRNA prediction. Computational analysis of EST isolation frequencies in various plant tissues showed that the expression levels and expression profiles of the putative ncRNAs and mRNAs differ - most interestingly, the putative ncRNAs are highly expressed relative to mRNAs in the root nodule tissue and conserved only in closely related plants. The work presented here constitutes the first large-scale prediction and characterization of ncRNAs in legumes, and provides a basis for further research on elucidating ncRNA function in legume genomics.

Computational analyses show A-to-G mutations correlate with nascent mRNA hairpins at somatic hypermutation hotspots.

Activation-induced cytidine deaminase (AID) initiates Phase I somatic hypermutation (SHM) of antibody genes by deaminating deoxy-cytosine to deoxy-uracil (C-to-U). These lesions trigger Phase II, a poorly understood process of error-prone repair targeting A-T pairs by DNA polymerase eta (Pol eta). Since Pol eta is also a reverse transcriptase, Phase II could involve copying off RNA as well as DNA templates. We explore this idea further since in an RNA-based pathway it is conceivable that adenosine-to-inosine (A-to-I) RNA editing causes A-to-G transitions since I like G pairs with C. Adenosine deaminases (ADARs) are known to preferentially edit A nucleotides that are preceded by an A or U (W) in double-stranded RNA substrates. On this assumption and using a theoretical bioinformatics approach we show that a significant and specific correlation (P<0.002) exists between the frequency of WA-to-WG mutations and the number of mRNA hairpins that could potentially form at the mutation site. This implies roles for both RNA editing and reverse transcription during SHM in vivo and suggests definitive genetic experiments targeting the appropriate ADAR1 isoform (gammaINF-ADAR1) and/or Ig pre-mRNA templates.

The modal distribution of protein isoelectric points reflects amino acid properties rather than sequence evolution.

Two-dimensional gel electrophoresis, a routine application in proteomics, separates proteins according to their molecular mass (M(r)) and isoelectric point (pI). As the genomic sequences for more and more organisms are determined, the M(r) and pI of all their proteins can be estimated computationally. The examination of several of these theoretical proteome plots has revealed a multimodal pI distribution, however, no conclusive explanation for this unusual distribution has so far been presented. We examined the pI distribution of 115 fully sequenced genomes and observed that the modal distribution does not reflect phylogeny or sequence evolution, but rather the chemical properties of amino acids. We provide a statistical explanation of why the observed distributions of pI values are multimodal.

A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.