Mathematical Analysis and Computational Integration of Massive Heterogeneous Data from the Human Retina

Modern epidemiology integrates knowledge from heterogeneous collections of data consisting of numerical, descriptive and imaging. Large-scale epidemiological studies use sophisticated statistical analysis, mathematical models using differential equations and versatile analytic tools that handle numerical data. In contrast, knowledge extraction from images and descriptive information in the form of text and diagrams remain a challenge for most fields, in particular, for diseases of the eye. In this article we provide a roadmap towards extraction of knowledge from text and images with focus on forthcoming applications to epidemiological investigation of retinal diseases, especially from existing massive heterogeneous collections of data distributed around the globe.Comment: 9 pages, 3 figures, submitted and accepted in Damor2012 conference: http://www.uninova.pt/damor2012/index.php?page=author

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

Life Sciences Program Tasks and Bibliography for FY 1997

This document includes information on all peer reviewed projects funded by the Office of Life and Microgravity Sciences and Applications, Life Sciences Division during fiscal year 1997. This document will be published annually and made available to scientists in the space life sciences field both as a hard copy and as an interactive internet web page

MADS specificity : Unravelling the dual function of the MADS domain protein FRUITFULL

Encrypted in the DNA lays most information needed for the development of an organism. The transcription of this information into precise patterns of gene activity results in the development of different cell types, organs, and developmental structures. Moreover, transcriptional regulation enables an organism to respond to changing environmental conditions. Essential for the regulation of transcription are DNA-binding transcription factors (TFs). TFs bind the DNA in a sequence-specific fashion. Upon binding of a TF to its DNA binding site, TFs typically activate or repress the transcription of nearby genes. To better understand transcriptional regulation it is essential to study DNA binding specificity of TFs. In the last decades, technological advances allowed the development of high-throughput methods to study protein-DNA interactions. Traditional in vitro methods study one or a few interactions, while new high-throughput methods can determine TF specificity by measuring relative DNA-binding affinities against a large collection or even all possible binding sites. Several high-throughput techniques to study TF-DNA interactions are discussed in Chapter 1 of this thesis. These new technologies and methods resulted in a fast growing number of studies on DNA binding specificities of TFs, expanding the knowledge about TF specificity. A review on the current knowledge of TF DNA binding specificity is described in Chapter 1. One aspect that influences DNA binding of TFs are differences in ability to form protein-protein interactions. The aim of this thesis was to study the role of protein-protein interactions in determining DNA binding specificity of a developmental regulatory MADS domain TF in Arabidopsis thaliana. While the members of the MADS-box protein family have many, diverse in vivo functions, all members bind in vitro to a 10-bp motif called the CArG-box. Moreover, studies demonstrated that closely related MADS proteins are expressed in the same cells, therefore encountering the same DNA accessibility and DNA methylation patterns, but bind different in vivo targets. Interestingly, MADS domain proteins bind DNA obligatorily as homo- and heterodimers and the interactions between MADS domain proteins are highly protein specific. Hence, MADS domain proteins are a perfect model system to study the influence of intra-family protein interactions on DNA binding specificity. To study the influence of protein-protein interactions on DNA-binding specificity this work focusses on one specific MADS domain protein, FRUITFULL (FUL). FUL is expressed at two stages during flower development and, in both stages FUL has highly diverse functions. In Chapter 2 we demonstrate using RNA-seq that FUL regulates different sets of target genes in the two stages. Moreover, using ChIP-seq we show that FUL genomic DNA binding is partly tissue-specific. These tissue-specifically bound and regulated genes are in line with the known dual functions of FUL during development. Interestingly, using protein complex immunoprecipitation for the two studied tissues/stages we show that the interactions of FUL with other MADS domain proteins are also tissue-specific. To determine whether the tissue-specific in vivo binding pattern are due to differences in DNA binding specificity of the FUL-MADS dimers, we studied the DNA binding specificities of the different protein complexes using SELEX-seq. The SELEX-seq results show that although all tested dimers preferably bind the canonical binding motif of MADS domain proteins, different dimers have different preferences for nucleotides within and surrounding the canonical binding site. Hence, different MADS domain dimers have different in vitro DNA binding specificities. By mapping the SELEX-seq affinities to the genome we were able to compare these results with in vivo tissue-specific ChIP-seq data. This analysis revealed a strong correlation between tissue-specific dimer affinities and tissue-specific genomic binding sites of FUL. Hence, we show that the choice of MADS dimerization partner influences DNA binding specificity, highlighting the role of intra-family protein interactions in defining DNA binding specificity. To allow other researchers to determine genome-wide DNA binding of TFs Chapter 3 provides a step-by-step guide for ChIP-seq experiments and computational analysis. The protocol is designed for wet-lab biologists to perform ChIP-seq experiments and analyse their own ChIP-seq data. Using the genome-wide DNA binding patterns determined by ChIP-seq, Chapter 4 and Chapter 5 take a more detailed look at some of the genes directly bound by FUL. In Chapter 4, we demonstrate a connection between developmentally and environmentally regulated growth programs. We studied a gene directly bound by FUL in pistil tissue, SMALL AUXIN UPREGULATED RNA 10 (SAUR10). SAUR10 expression is regulated by FUL in multiple tissues, among others cauline leaves, stems, and branches. The results show that the expression of SAUR10 at the abaxial side of branches is influenced by a combination of environmental and developmental regulated growth programs: hormones, light conditions, and FUL binding. This spatial regulation possibly affects the angle between the side branches and the main inflorescence stem. Additionally, we discuss several other FUL target genes involved in hormone pathways and light conditions. Chapter 5 focusses on the putative direct targets of FUL in IM tissue. Among the putative direct targets two genes involved in flavonoid synthesis were identified, FLAVONOID SYNTHESE 1 (FLS1) and UDP-GLUCOSYL TRANSFERASE 78D3 (UGT78D3). Interestingly, similar to the ful-7 mutant, the fls1 mutant is late flowering. Moreover, expression data exposed an increased gene expression for both FLS1 and UGT78D3 in developing meristems and showed FLS1 expression to be influenced by light conditions. We report the first link between the MADS domain protein FUL and flavonoid synthesis in Arabidopsis. Moreover, our results indicate a possible link between flavonoids and flowering time. In Chapter 6 I discuss the findings of this thesis and make suggestions for further research. Taken together, the work in this thesis shows that intra-family protein interactions can influence DNA-binding specificity of a protein. Thereby these protein-protein interactions can influence genome-wide binding patterns and, as a result, the function of a protein. Moreover, by studying several putative direct targets of FUL in more detail, we demonstrated a connection between development and environment in growth-regulated programs. Interestingly, the FUL target SAUR10 is repressed by FUL in several tissues, including cauline leaves, inflorescence stems, and branches. However, no influence of FUL on SAUR10 expression could be detected in the pistil. So, despite the binding of FUL to the promotor of SAUR10 in the pistil, this binding does not result in gene regulation. This finding reflects the complex relation between TF occupancy and gene regulation, further research is needed to better understand this relation. Moreover, besides MADS domain protein interactions, we found FUL to interact with several proteins of other families. The role of these cross-family protein interactions in cooperative gene regulation is not fully understood and will be an important research topic in the coming years.</p

Callose-mediated regulation of Plasmodesmata during the establishment of Medicago Truncatula-Sinorhizobium Meliloti Symbiotic interaction

Legumes, such as Medicago truncatula, can fix atmospheric nitrogen by forming symbiotic associations with soil-borne bacteria collectively called rhizobia. As a result of this relationship, specific roots organs called nodules, are developed that houses rhizobia and where the nitrogen fixation process occurs. Nodule formation is tightly regulated by complex signalling mechanisms and environmental cues, such as nitrate availability. Molecular signals move between the site of infection and the cortex/pericycle to coordinate nodule organogenesis and also systemically along the vascular system to coordinate root and shoot responses. Despite recent progress in the identification of some of these signals very little is known about the pathways for intercellular transport. In this project, the role of the cell-wall polysaccharide callose in the establishment of symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti was addressed. Callose metabolism regulates transport through plasmodesmata: intercellular channels that form a symplastic path for transport. Using immuno-histochemistry we found that callose is downregulated as early as 16 hours post-bacterial inoculation. Concomitantly, the expression of a plasmodesmata located callose degrading enzyme (Medtr3g083580), identified using phylogeny, was induced. Roots constitutively expressing either Medtr3g083580 or its Arabidopsis orthologue PdBG1, showed reduced callose levels and a higher rate of infection and nodulation, even when grown in nitrate concentrations that inhibit nodulation. The effects were stronger when using a promoter active early after rhizobial infection and were mimicked, in high nitrate conditions, by the ectopic expression of a novel plasmodesmata receptor-like kinase (Medtr1g073320). The results suggest an important role for callose in the control of nodulation, both under nitrate deprived or sufficient conditions, likely associated with the regulation of transport via plasmodesmata. The relevance of the findings is discussed in light of potential applications in crop improvement and in reducing the use of nitrogen fertilizers

Diagnostics in Plant Breeding

“Diagnostics in Plant Breeding” is systematically organizing cutting-edge research reviews on the development and application of molecular tools for the prediction of plant performance. Given its significance for mankind and the available research resources, medical sciences are leading the area of molecular diagnostics, where DNA-based risk assessments for various diseases and biomarkers to determine their onset become increasingly available. So far, most research in plant genomics has been directed towards understanding the molecular basis of biological processes or phenotypic traits. From a plant breeding perspective, however, the main interest is in predicting optimal genotypes based on molecular information for more time- and cost-efficient breeding schemes. It is anticipated that progress in plant genomics and in particular sequence technology made recently will shift the focus from “explanatory” to “predictive” in crop science. This book assembles chapters on all areas relevant to development and application of predictive molecular tools in plant breeding by leading authorties in the respective areas

Modelling Plant Variety Dependent Least Limiting Water Range (LLWR)

Drought stress is a major limiting factor for yield on a global scale (Solh and van Ginkel, 2014), with drought effects being predicted to become more severe with increasing global temperatures (IPCC, 2014). Climate change is also expected to increase the frequency and severity of floods leading to root oxygen stress (Trenberth, 2011). At the same time, current agricultural practises are increasingly relying on heavy machinery leading to soil compaction and changes in soil structure (Chamen et al., 2003), reducing the rate of cell division in the root meristem, and decreasing cell expansion (Bengough and Mullins, 1990). As such, in order to reduce yield losses it is essential to understand the complex interaction between oxygen stress, water stress and mechanical stress (Mohammadi et al., 2010). The least limiting water range (LLWR) is one such model which relates the above-mentioned soil stressors in order to estimate the soil moisture range in a particular soil for which plants should be less limited in terms of growth. However, the extent to which the LLWR considers the influence of root traits in changing its boundaries is currently limited. In order to be able to assess the effects of root trait variability on the LLWR boundaries while manipulating the LLWR soil stressors a minirhizotron based system (RS) was developed. This cheap (~£10 per unit), acrylic based, A3 sized system enabled in situ imaging of roots and root hairs. Destructive sampling methods were also used to determine root border cell numbers and root tip geometry. To further optimise the process of data collection, Rcpp based image processing algorithms were developed to obtain automated estimates of the root traits of root length, root hair, root border cells and root tip eccentricity to further increase the efficiency of the RS phenotyping platform. To test how contrasting root traits influence the LLWR a plant phenotyping experiment was performed comparing four spring barley (Hordeum vulgare L.) varieties, Optic, KWS Sassy, Derkado and Golden Promise. Root growth rates both in the vertical and horizontal directions all increased with increasing water availability and decreasing substrate density. Root hair area did not vary significantly among treatments and between variaties. Root border cell count and root tip eccentricity increased with increasing substrate density but did not vary significantly across varieties. A root micro-trait based linear interaction model was developed to describe average root growth rates and it was demonstrated that root growth rates on average follow a linear patern for values >= 8 mm day-1. Root micro-traits mostly failed to correlate well with root growth rates except for a negative assosiation with root tip geometry (cor = -0.4192, p = 2e-05**)