In-depth proteomic analyses of <Emphasis Type="Italic">Haliotis laevigata</Emphasis> (greenlip abalone) nacre and prismatic organic shell matrix

Background

The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date.

Methods

The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing.

Results

We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins.

Conclusions

The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.

     

Agrobacterium-mediated transformation of tomato elicits unexpected flower phenotypes with similar gene expression profiles

Background

Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in alterred floral structure.

Methodology/Principal Findings

Previously we identified two genes (LeTGA1 and SOLly GLB1) induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each transformed into Micro-Tina tomato plants, one primary transformant with similar mutant flower phenotypes was identified from transformation of each construct. Microarray analysis shows that a similar set of genes were up- or downregulated in both mutants. Sequencing of insertion sites indicates that each is inserted into a repetitive region which could impact expression of affected genes but direct alteration of floral homeotic gene sequences was not detected.

Conclusion

This is the first report that dominant flower mutations could be caused by genetic transformation designed to knock out two nutrient stress related genes.     

Expression of genes responsible for biomineralization of Pinctada fucata during development

This study compares the expression levels of nacrein, N16, MSI60, Prismalin-14, aspein and MSI31 genes during the ontogeny of Pinctada fucata. Several novel findings were obtained: 1) The early calcitic prismatic layer was distinguished as a thin membrane-like structure. 2) Initial formation of the nacreous layer started from the mantle pallial region at the age of 31 days. 3) 18S rRNA of P. fucata was determined to be more suitable as a real-time PCR reference gene compared with GAPDH and β-actin genes. 4) A relationship was recognized between the expression levels of the above six organic matrix genes and biomineralization of the larval shell. The lack of calcite in the shells of the veliger and pediveliger stages, when MSI31 and Prismalin-14 genes were expressed, makes a role of polymorph control by these genes less likely. The hypothetical involvement of N16 and MSI60 proteins in aragonitic nacreous layer formation was corroborated by the expression levels of N16 and MSI60 genes during ontogeny. Our results are important with respect to the control of CaCO₃ crystal polymorphism and shell microstructures in P. fucata.     

Expression analysis of asthma candidate genes during human and murine lung development

Background

Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.

Objective

To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.

Methods

Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.

Results

In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.

Conclusions

Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.     

A high-density SNP genetic linkage map for the silver-lipped pearl oyster,Pinctada maxima: a valuable resource for gene localisation and marker-assisted selection

Background

The silver-lipped pearl oyster, Pinctada maxima, is an important tropical aquaculture species extensively farmed for the highly sought "South Sea" pearls. Traditional breeding programs have been initiated for this species in order to select for improved pearl quality, but many economic traits under selection are complex, polygenic and confounded with environmental factors, limiting the accuracy of selection. The incorporation of a marker-assisted selection (MAS) breeding approach would greatly benefit pearl breeding programs by allowing the direct selection of genes responsible for pearl quality. However, before MAS can be incorporated, substantial genomic resources such as genetic linkage maps need to be generated. The construction of a high-density genetic linkage map for P. maxima is not only essential for unravelling the genomic architecture of complex pearl quality traits, but also provides indispensable information on the genome structure of pearl oysters.

Results

A total of 1,189 informative genome-wide single nucleotide polymorphisms (SNPs) were incorporated into linkage map construction. The final linkage map consisted of 887 SNPs in 14 linkage groups, spans a total genetic distance of 831.7 centimorgans (cM), and covers an estimated 96% of the P. maxima genome. Assessment of sex-specific recombination across all linkage groups revealed limited overall heterochiasmy between the sexes (i.e. 1.15:1 F/M map length ratio). However, there were pronounced localised differences throughout the linkage groups, whereby male recombination was suppressed near the centromeres compared to female recombination, but inflated towards telomeric regions. Mean values of LD for adjacent SNP pairs suggest that a higher density of markers will be required for powerful genome-wide association studies. Finally, numerous nacre biomineralization genes were localised providing novel positional information for these genes.

Conclusions

This high-density SNP genetic map is the first comprehensive linkage map for any pearl oyster species. It provides an essential genomic tool facilitating studies investigating the genomic architecture of complex trait variation and identifying quantitative trait loci for economically important traits useful in genetic selection programs within the P. maxima pearling industry. Furthermore, this map provides a foundation for further research aiming to improve our understanding of the dynamic process of biomineralization, and pearl oyster evolution and synteny.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-810) contains supplementary material, which is available to authorized users.     

Characterization of MORE AXILLARY GROWTH Genes in Populus

Background

Strigolactones are a new class of plant hormones that play a key role in regulating shoot branching. Studies of branching mutants in Arabidopsis, pea, rice and petunia have identified several key genes involved in strigolactone biosynthesis or signaling pathway. In the model plant Arabidopsis, MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3 and MAX4 are four founding members of strigolactone pathway genes. However, little is known about the strigolactone pathway genes in the woody perennial plants.

Methodology/Principal Finding

Here we report the identification of MAX homologues in the woody model plant Populus trichocarpa. We identified the sequence homologues for each MAX protein in P. trichocarpa. Gene expression analysis revealed that Populus MAX paralogous genes are differentially expressed across various tissues and organs. Furthermore, we showed that Populus MAX genes could complement or partially complement the shoot branching phenotypes of the corresponding Arabidopsis max mutants.

Conclusion/Significance

This study provides genetic evidence that strigolactone pathway genes are likely conserved in the woody perennial plants and lays a foundation for further characterization of strigolactone pathway and its functions in the woody perennial plants.     

Unacylated Ghrelin Rapidly Modulates Lipogenic and Insulin Signaling Pathway Gene Expression in Metabolically Active Tissues of GHSR Deleted Mice

Background

There is increasing evidence that unacylated ghrelin (UAG) improves insulin sensitivity and glucose homeostasis; however, the mechanism for this activity is not fully understood since a UAG receptor has not been discovered.

Methodology/Principal Findings

To assess potential mechanisms of UAG action in vivo, we examined rapid effects of UAG on genome-wide expression patterns in fat, muscle and liver of growth hormone secretagogue receptor (GHSR)-ablated mice using microarrays. Expression data were analyzed using Ingenuity Pathways Analysis and Gene Set Enrichment Analysis. Regulation of subsets of these genes was verified by quantitative PCR in an independent experiment. UAG acutely regulated clusters of genes involved in glucose and lipid metabolism in all three tissues, consistent with enhancement of insulin sensitivity.

Conclusions/Significance

Fat, muscle and liver are central to the control of lipid and glucose homeostasis. UAG rapidly modulates the expression of metabolically important genes in these tissues in GHSR-deleted mice indicating a direct, GHSR-independent, action of UAG to improve insulin sensitivity and metabolic profile.     

Functional significance of allelic variation at methuselah, an aging gene in Drosophila

Background

Longevity and age-specific patterns of mortality are complex traits that vary within and among taxa. Multiple candidate genes for aging have been identified in model systems by extended longevity mutant phenotypes, including the G-protein coupled receptor methuselah (mth) in D. melanogaster. These genes offer important insights into the mechanisms of lifespan determination and have been major targets of interest in the biology of aging. However, it is largely unknown whether these genes contribute to genetic variance for lifespan in natural populations, and consequently contribute to lifespan evolution.

Methodology/Principle Findings

For a gene to contribute to genetic variance for a particular trait, it must meet two criteria: natural allelic variation and functional differences among variants. Previous work showed that mth varies significantly among wild populations; here we assess the functional significance of wild-derived mth alleles on lifespan, fecundity and stress resistance using a quantitative complementation scheme. Our results demonstrate that mth alleles segregating in nature have a functional effect on all three traits.

Conclusions/Significance

These results suggest that allelic variation at mth contributes to observed differences in lifespan and correlated phenotypes in natural populations, and that evaluation of genetic diversity at candidate genes for aging can be a fruitful approach to identifying loci contributing to lifespan evolution.     

Genome-Wide Survey and Expression Analysis of Amino Acid Transporter Gene Family in Rice (Oryza sativa L.)

Background

Amino acid transporters (AATs) that transport amino acids across cellular membranes are essential for plant growth and development. To date, a genome-wide overview of the AAT gene family in rice is not yet available.

Methodology/Principal Findings

In this study, a total of 85 AAT genes were identified in rice genome and were classified into eleven distinct subfamilies based upon their sequence composition and phylogenetic relationship. A large number of OsAAT genes were expanded via gene duplication, 23 and 24 OsAAT genes were tandemly and segmentally duplicated, respectively. Comprehensive analyses were performed to investigate the expression profiles of OsAAT genes in various stages of vegetative and reproductive development by using data from EST, Microarrays, MPSS and Real-time PCR. Many OsAAT genes exhibited abundant and tissue-specific expression patterns. Moreover, 21 OsAAT genes were found to be differentially expressed under the treatments of abiotic stresses. Comparative analysis indicates that 26 AAT genes with close evolutionary relationships between rice and Arabidopsis exhibited similar expression patterns.

Conclusions/Significance

This study will facilitate further studies on OsAAT family and provide useful clues for functional validation of OsAATs.     

Secretome analysis reveals effector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum

Background

The white mold fungus Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a remarkably broad host range. The interaction of necrotrophs with their hosts is more complex than initially thought, and still poorly understood.

Results

We combined bioinformatics approaches to determine the repertoire of S. sclerotiorum effector candidates and conducted detailed sequence and expression analyses on selected candidates. We identified 486 S. sclerotiorum secreted protein genes expressed in planta, many of which have no predicted enzymatic activity and may be involved in the interaction between the fungus and its hosts. We focused on those showing (i) protein domains and motifs found in known fungal effectors, (ii) signatures of positive selection, (iii) recent gene duplication, or (iv) being S. sclerotiorum-specific. We identified 78 effector candidates based on these properties. We analyzed the expression pattern of 16 representative effector candidate genes on four host plants and revealed diverse expression patterns.

Conclusions

These results reveal diverse predicted functions and expression patterns in the repertoire of S. sclerotiorum effector candidates. They will facilitate the functional analysis of fungal pathogenicity determinants and should prove useful in the search for plant quantitative disease resistance components active against the white mold.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-336) contains supplementary material, which is available to authorized users.