Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants

<p>Abstract</p> <p>Background</p> <p><it>Arabidopsis </it>ovules comprise four morphologically distinct parts: the nucellus, which contains the embryo sac, two integuments that become the seed coat, and the funiculus that anchors the ovule within the carpel. Analysis of developmental mutants has shown that ovule morphogenesis relies on tightly regulated genetic interactions that can serve as a model for developmental regulation. Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes. Expression-based gene discovery can be used access such obscured genes.</p> <p>Results</p> <p>Affymetrix microarrays were used for expression-based gene discovery to identify sets of genes expressed in either or both integuments. The genes were identified by comparison of pistil mRNA from wild type with mRNA from two mutants; <it>inner no outer </it>(<it>ino</it>, which lacks the outer integument), and <it>aintegumenta </it>(<it>ant</it>, which lacks both integuments). Pools of pistils representing early and late stages of ovule development were evaluated and data from the three genotypes were used to designate genes that were predominantly expressed in the integuments using pair-wise and cluster analyses. Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and <it>in situ </it>hybridization.</p> <p>Conclusion</p> <p>The results showed that it was possible to use a mutant, <it>ant</it>, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, <it>ino</it>. Robust microarray averaging (RMA) analysis of array data provided the most reliable comparisons, especially for weakly expressed genes. The studies yielded an over-abundance of transcriptional regulators in the identified genes, and these form a set of candidate genes for evaluation of roles in ovule development using reverse genetics.</p

Recent advances in understanding female gametophyte development [version 1; referees: 2 approved]

The haploid female gametophyte (embryo sac) is an essential reproductive unit of flowering plants, usually comprising four specialized cell types, including the female gametes (egg cell and central cell). The differentiation of these cells relies on spatial signals which pattern the gametophyte along a proximal-distal axis, but the molecular and genetic mechanisms by which cell identities are determined in the embryo sac have long been a mystery. Recent identification of key genes for cell fate specification and their relationship to hormonal signaling pathways that act on positional cues has provided new insights into these processes. A model for differentiation can be devised with egg cell fate as a default state of the female gametophyte and with other cell types specified by the action of spatially regulated factors. Cell-to-cell communication within the gametophyte is also important for maintaining cell identity as well as facilitating fertilization of the female gametes by the male gametes (sperm cells)

ABERRANT TESTA SHAPE encodes a KANADI family member, linking polarity determination to separation and growth of Arabidopsis ovule integuments

The Arabidopsis aberrant testa shape (ats) mutant produces a single integument instead of the two integuments seen in wild-type ovules. Cellular anatomy and patterns of marker gene expression indicate that the single integument results from congenital fusion of the two integuments of the wild type. Isolation of the ATS locus showed it to encode a member of the KANADI (KAN) family of putative transcription factors, previously referred to as KAN4. ATS was expressed at the border between the two integuments at the time of their initiation, with expression later confined to the abaxial layer of the inner integument. In an inner no outer (ino) mutant background, where an outer integument does not form, the ats mutation led to amorphous inner integument growth. The kan1 kan2 double mutant exhibits a similar amorphous growth of the outer integument without affecting inner integument growth. We hypothesize that ATS and KAN1/KAN2 play similar roles in the specification of polarity in the inner and outer integuments, respectively, that parallel the known roles of KAN proteins in promoting abaxial identity during leaf development. INO and other members of the YABBY gene family have been hypothesized to have similar parallel roles in outer integument and leaf development. Together, these two hypotheses lead us to propose a model for normal integument growth that also explains the described mutant phenotypes

Functional conservation of the grapevine candidate gene INNER NO OUTER for ovule development and seed formation

Seedlessness represents a highly appreciated trait in table grapes. Based on an interesting case of seedless fruit production described in the crop species Annona squamosa, we focused on the Vitis vinifera INNER NO OUTER (INO) gene as a candidate. This gene encodes a transcription factor belonging to the YABBY family involved in the determination of abaxial identity in several organs. In Arabidopsis thaliana, this gene was shown to be essential for the formation and asymmetric growth of the ovule outer integument and its mutation leads to a phenotypic defect of ovules and failure in seed formation. In this study, we identified in silico the V. vinifera orthologue and investigated its phylogenetic relationship to INO genes from other species and its expression in different organs in seeded and seedless varieties. Applying cross-species complementation, we have tested its functionality in the Arabidopsis ino-1 mutant. We show that the V. vinifera INO successfully rescues the ovule outer integument growth and seeds set and also partially complements the outer integument asymmetric growth in the Arabidopsis mutant, differently from orthologues from other species. These data demonstrate that VviINO retains similar activity and protein targets in grapevine as in Arabidopsis. Potential implications for grapevine breeding are discussed

Geologic Interpretation of Data Sets Collected by Planetary Analog Geology Traverses and by Standard Geologic Field Mapping

Geologic maps integrate the distributions, contacts, and compositions of rock and sediment bodies as a means to interpret local to regional formative histories. Applying terrestrial mapping techniques to other planets is challenging because data is collected primarily by orbiting instruments, with infrequent, spatiallylimited in situ human and robotic exploration. Although geologic maps developed using remote data sets and limited "Apollo-style" field access likely contain inaccuracies, the magnitude, type, and occurrence of these are only marginally understood. This project evaluates the interpretative and cartographic accuracy of both field- and remote-based mapping approaches by comparing two 1:24,000 scale geologic maps of the San Francisco Volcanic Field (SFVF), north-central Arizona. The first map is based on traditional field mapping techniques, while the second is based on remote data sets, augmented with limited field observations collected during NASA Desert Research & Technology Studies (RATS) 2010 exercises. The RATS mission used Apollo-style methods not only for pre-mission traverse planning but also to conduct geologic sampling as part of science operation tests. Cross-comparison demonstrates that the Apollo-style map identifies many of the same rock units and determines a similar broad history as the field-based map. However, field mapping techniques allow markedly improved discrimination of map units, particularly unconsolidated surficial deposits, and recognize a more complex eruptive history than was possible using Apollo-style data. Further, the distribution of unconsolidated surface units was more obvious in the remote sensing data to the field team after conducting the fieldwork. The study raises questions about the most effective approach to balancing mission costs with the rate of knowledge capture, suggesting that there is an inflection point in the "knowledge capture curve" beyond which additional resource investment yields progressively smaller gains in geologic knowledge

Marker development for quality protein maize breeding and an interaction study between Opaque-2 and Ask2 genes

Quality Protein Maize (QPM) kernels contain twice the amounts of lysine and tryptophan compared to normal corn kernels. Although the opaque-2 (o2) mutation is the underlying cause of this beneficial change, other genes such as Aspartate kinase-2 (Ask2) affect the amino acid content in the endosperm to a lesser degree. To date, reports on the interaction between both loci are scarce and there are no high-throughput assays for the identification of the alleles of these genes. The objectives of this research were: 1) to study the interaction between the o2 and Ask2 genes with respect to the accumulation of amino acids in the endosperm in an F2 population, 2) to identify conserved SNPs into the o2 gene that can be used as markers, 3) to estimate the frequency of an SNP of Ask2 associated with the accumulation of lysine in the endosperm in CIMMYT germplasm, and 4) to develop high-throughput marker assays for these SNPs. The interaction study showed a preponderant effect of o2 on the accumulation of 11 amino acids (P ≤ 0.01). Ask2 appeared only to act with o2 to enhance marginally lysine, histidine and methionine levels in the double recessive homozygotes. Sequencing of amplicons at the o2 locus led to the identification of an SNP in exon 1 that discriminated all QPM (C) genotypes from non-QPM (T) genotypes. Validation of this SNP through KASP™ assays indicated that it was 92 % assertive in differentiating the o2 genotypes. In contrast, the frequency of the Ask2 SNP in CIMMYT QPM germplasm was low; however, an SSCP marker developed using this SNP detected five variants indicating that other unknown base changes may confer positive lysine-increasing responses. These markers could aid the marker-assisted selection of QPM cultivars

Burnout and use of HIV services among health care workers in Lusaka District, Zambia: a cross-sectional study

BACKGROUND: Well-documented shortages of health care workers in sub-Saharan Africa are exacerbated by the increased human resource demands of rapidly expanding HIV care and treatment programmes. The successful continuation of existing programmes is threatened by health care worker burnout and HIV-related illness. METHODS: From March to June 2007, we studied occupational burnout and utilization of HIV services among health providers in the Lusaka public health sector. Providers from 13 public clinics were given a 36-item, self-administered questionnaire and invited for focus group discussions and key-informant interviews. RESULTS: Some 483 active clinical staff completed the questionnaire (84% response rate), 50 staff participated in six focus groups, and four individuals gave interviews. Focus group participants described burnout as feeling overworked, stressed and tired. In the survey, 51% reported occupational burnout. Risk factors were having another job (RR 1.4 95% CI 1.2-1.6) and knowing a co-worker who left in the last year (RR 1.6 95% CI 1.3-2.2). Reasons for co-worker attrition included: better pay (40%), feeling overworked or stressed (21%), moving away (16%), death (8%) and illness (5%). When asked about HIV testing, 370 of 456 (81%) reported having tested; 240 (50%) tested in the last year. In contrast, discussion groups perceived low testing rates. Both discussion groups and survey respondents identified confidentiality as the prime reason for not undergoing HIV testing. CONCLUSION: In Lusaka primary care clinics, overwork, illness and death were common reasons for attrition. Programmes to improve access, acceptability and confidentiality of health care services for clinical providers and to reduce workplace stress could substantially affect workforce stability

Conservation of the role of INNER NO OUTER in development of unitegmic ovules of the Solanaceae despite a divergence in protein function

The P-SlINO::SlINO-GFP transgene continues to be expressed after fertilization during the onset of fruit development. A-C: Ovules from P-SlINO::SlINO-GFP plants. D, E: Ovules from control plants. Images A (confocal) and B (DIC overlaid with GFP channel) show expression in the outer cell layer in an ovule post-anthesis. C-E are images of the surface cells of the integument of ovules taken from 3–4 mm fruits. C and D are images taken on an epifluorescence microscope (Axioplan) using a Chroma GFP filter set 41017 (Chroma, Bellows Falls, VT). E is a dark-field image of the same ovule in D. These images show expression is present in developing fruit. Scale bar in B represents 20 μm, scale bar in E represents 20 μm in C-E. (TIF 4435 kb

Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic Medicine

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine