Nese Sreenivasulu

PageMan: An interactive ontology tool to generate, display, and annotate overview graphs for profiling experiments

BackgroundMicroarray technology has become a widely accepted and standardized tool in biology. The first microarray data analysis programs were developed to support pair-wise comparison. However, as microarray experiments have become more routine, large scale experiments have become more common, which investigate multiple time points or sets of mutants or transgenics. To extract biological information from such high-throughput expression data, it is necessary to develop efficient analytical platforms, which combine manually curated gene ontologies with efficient visualization and navigation tools. Currently, most tools focus on a few limited biological aspects, rather than offering a holistic, integrated analysis.ResultsHere we introduce PageMan, a multiplatform, user-friendly, and stand-alone software tool that annotates, investigates, and condenses high-throughput microarray data in the context of functional ontologies. It includes a GUI tool to transform different ontologies into a suitable format, enabling the user to compare and choose between different ontologies. It is equipped with several statistical modules for data analysis, including over-representation analysis and Wilcoxon statistical testing. Results are exported in a graphical format for direct use, or for further editing in graphics programs.PageMan provides a fast overview of single treatments, allows genome-level responses to be compared across several microarray experiments covering, for example, stress responses at multiple time points. This aids in searching for trait-specific changes in pathways using mutants or transgenics, analyzing development time-courses, and comparison between species. In a case study, we analyze the results of publicly available microarrays of multiple cold stress experiments using PageMan, and compare the results to a previously published meta-analysis.PageMan offers a complete users guide, a web-based over-representation analysis as well as a tutorial, and is freely available at http://mapman.mpimp-golm.mpg.de/pageman/.ConclusionPageMan allows multiple microarray experiments to be efficiently condensed into a single page graphical display. The flexible interface allows data to be quickly and easily visualized, facilitating comparisons within experiments and to published experiments, thus enabling researchers to gain a rapid overview of the biological responses in the experiments.

Haplotyping, linkage mapping and expression analysis of barley genes regulated by terminal drought stress influencing seed quality.

BackgroundThe increasingly narrow genetic background characteristic of modern crop germplasm presents a challenge for the breeding of cultivars that require adaptation to the anticipated change in climate. Thus, high priority research aims at the identification of relevant allelic variation present both in the crop itself as well as in its progenitors. This study is based on the characterization of genetic variation in barley, with a view to enhancing its response to terminal drought stress.ResultsThe expression patterns of drought regulated genes were monitored during plant ontogeny, mapped and the location of these genes was incorporated into a comprehensive barley SNP linkage map. Haplotypes within a set of 17 starch biosynthesis/degradation genes were defined, and a particularly high level of haplotype variation was uncovered in the genes encoding sucrose synthase (types I and II) and starch synthase. The ability of a panel of 50 barley accessions to maintain grain starch content under terminal drought conditions was explored.ConclusionThe linkage/expression map is an informative resource in the context of characterizing the response of barley to drought stress. The high level of haplotype variation among starch biosynthesis/degradation genes in the progenitors of cultivated barley shows that domestication and breeding have greatly eroded their allelic diversity in current elite cultivars. Prospective association analysis based on core drought-regulated genes may simplify the process of identifying favourable alleles, and help to understand the genetic basis of the response to terminal drought.

Barley grain maturation and germination: Metabolic pathway and regulatory network commonalities and differences highlighted by new MapMan/PageMan profiling tools

Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley (Hordeum vulgare) grain maturation, desiccation, and germination in two tissue fractions (starchy endosperm/aleurone and embryo/scutellum) using the Affymetrix Barley1 GeneChip. To aid data evaluation, Arabidopsis thaliana MapMan and PageMan tools were adapted to barley. The analyses allow a number of conclusions: (1) Cluster analysis revealed a smooth transition in transcription programs between late seed maturation and germination within embryo tissues, but not in the endosperm/aleurone. (2) More than 12,000 transcripts are stored in the embryo of dry barley grains, many of which are presumably activated during germination. (3) Transcriptional activation of storage reserve mobilization events occurs at an early stage of germination, well before radicle protrusion. (4) Key genes of gibberellin (GA) biosynthesis are already active during grain maturation at a time when abscisic acid peaks suggesting the formation of an endogenous store of GA in the aleurone. This GA probably acts later during germination in addition to newly synthesized GA. (5) Beside the well-known role of GA in gene activation during germination spatiotemporal expression data and cis-element searches in homologous rice promoters confirm an equally important gene-activating role of abscisic acid during this developmental period. The respective regulatory webs are linked to auxin and ethylene controlled networks. In summary, new bioinformatics PageMan and MapMan tools developed in barley have been successfully used to investigate in detail the transcriptome relationships between seed maturation and germination in an important crop plant.

Integrated genomics, physiology and breeding approaches for improving drought tolerance in crops

Drought is one of the most serious production constraint for world agriculture and is projected to worsen with anticipated climate change. Inter-disciplinary scientists have been trying to understand and dissect the mechanisms of plant tolerance to drought stress using a variety of approaches; however, success has been limited. Modern genomics and genetic approaches coupled with advances in precise phenotyping and breeding methodologies are expected to more effectively unravel the genes and metabolic pathways that confer drought tolerance in crops. This article discusses the most recent advances in plant physiology for precision phenotyping of drought response, a vital step before implementing the genetic and molecular-physiological strategies to unravel the complex multilayered drought tolerance mechanism and further exploration using molecular breeding approaches for crop improvement. Emphasis has been given to molecular dissection of drought tolerance by QTL or gene discovery through linkage and association mapping, QTL cloning, candidate gene identification, transcriptomics and functional genomics. Molecular breeding approaches such as marker-assisted backcrossing, marker-assisted recurrent selection and genome-wide selection have been suggested to be integrated in crop improvement strategies to develop drought-tolerant cultivars that will enhance food security in the context of a changing and more variable climate.

Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue

Proline has been recognized as a multi-functional molecule, accumulating in high concentrations in response to a variety of abiotic stresses. It is able to protect cells from damage by acting as both an osmotic agent and a radical scavenger. Proline accumulated during a stress episode is degraded to provide a supply of energy to drive growth once the stress is relieved. Proline homeostasis is important for actively dividing cells as it helps to maintain sustainable growth under long-term stress. It also underpins the importance of the expansion of the proline sink during the transition from vegetative to reproductive growth and the initiation of seed development. Its role in the reproductive tissue is to stabilize seed set and productivity. Thus, to cope with abiotic stress, it is important to develop strategies to increase the proline sink in the reproductive tissue. We give a holistic account of proline homeostasis, taking into account the regulation of proline synthesis, its catabolism, and intra- and intercellular transport, all of which are vital components of growth and development in plants challenged by stress.

ABA biosynthesis and degradation contributing to ABA homeostasis during barley seed development under control and terminal drought-stress conditions

Drought is one of the most severe environmental stress factors limiting crop yield especially when occurring during anthesis and seed filling. This terminal drought is characterized by an excess production of the phytohormone abscisic acid (ABA) which plays an important role during seed development and dormancy. All the genes putatively involved in ABA biosynthesis and inactivation in barley were identified and their expression studied during plant ontogeny under standard and drought-stress conditions to learn more about ABA homeostasis and the possible mode of cross-talk between source and sink tissues. Out of 41 genes related to ABA biosynthesis and inactivation 19 were found to be differentially regulated under drought stress in both flag leaves and developing seed during seed filling. Transcripts of plastid-located enzymes are regulated similarly in flag leaf and seed under terminal drought whereas transcripts of cytosolic enzymes are differentially regulated in the two tissues. Detailed information on the expression of defined gene family members is supplemented by measurements of ABA and its degradation and conjugation products, respectively. Under drought stress, flag leaves in particular contain high concentrations of both ABA and the ABA degradation products phaseic acid (PA) and diphaseic acid (DPA); whereas, in seeds, besides ABA, DPA was mainly found. The measurements also revealed a positive correlation between ABA level and starch content in developing seeds for the following reasons: (i) genes of the ABA controlled SnRK2.6 and RCAR/PP2C-mediated signal transduction pathway to the ABF transcription factor HvABI5 are activated in the developing grain under drought, (ii) novel ABA- and dehydration-responsive cis-elements have been found in the promoters of key genes of starch biosynthesis (HvSUS1, HvAGP-L1) and degradation (HvBAM1) and these transcripts/activity are prominently induced in developing seeds during 12 and 16 DAF, (iii) spraying of fluridone (an ABA biosynthesis inhibitor) to drought-stressed plants results in severely impaired starch content and thousand grain weight of mature seeds.

Contrapuntal role of ABA: Does it mediate stress tolerance or plant growth retardation under long-term drought stress?

Recent developments in defining the functional basis of abscisic acid in regulating growth, development and stress response have provided essential components for its actions. We are yet to envision the impact of how differential levels of ABA influence plant growth across life cycle. Here we reviewed the information arising from the recent unprecedented advancement made in the field of ABA signaling operative under calcium-dependent and calcium-independent pathways mediating the transcriptional reprogramming under short-term stress response. Advancement made in the field of ABA receptors and transporters has started to fill major gaps in our understanding of the ABA action. However, ABA just not only regulates guard cell movement but impacts other reproductive tissue development through massive transcriptional reprogramming events affecting various stages of the plant life cycle. Therefore many questions still remain unanswered. One such intriguing question is the contradictory role of ABA known to mediate two opposite faces of the coin: regulating abiotic stress tolerance and imparting growth retardation. In this review, we critically assessed the impact of substantial elevated levels of ABA on impairment of photosynthesis and growth alteration and its subsequent influence on seed yield formation. Excess biosynthesis of ABA under stress may deprive the same precursor pool necessary for chlorophyll biosynthesis pathway, thereby triggering growth retardation. Further, we emphasized the importance of ABA homeostasis for integrating stress cues towards coordinating sustainable plant growth. Also we provided a pertinent background on ABA biosynthesis and degradation pathway manipulation to highlight the genes and processes used in genetic engineering of plants for changed ABA content.

Spatiotemporal Profiling of Starch Biosynthesis and Degradation in the Developing Barley Grain

Barley (Hordeum vulgare) grains synthesize starch as the main storage compound. However, some starch is degraded already during caryopsis development. We studied temporal and spatial expression patterns of genes coding for enzymes of starch synthesis and degradation. These profiles coupled with measurements of selected enzyme activities and metabolites have allowed us to propose a role for starch degradation in maternal and filial tissues of developing grains. Early maternal pericarp functions as a major short-term starch storage tissue, possibly ensuring sink strength of the young caryopsis. Gene expression patterns and enzyme activities suggest two different pathways for starch degradation in maternal tissues. One pathway possibly occurs via α-amylases 1 and 4 and β-amylase 1 in pericarp, nucellus, and nucellar projection, tissues that undergo programmed cell death. Another pathway is deducted for living pericarp and chlorenchyma cells, where transient starch breakdown correlates with expression of chloroplast-localized β-amylases 5, 6, and 7, glucan, water dikinase 1, phosphoglucan, water dikinase, isoamylase 3, and disproportionating enzyme. The suite of genes involved in starch synthesis in filial starchy endosperm is much more complex than in pericarp and involves several endosperm-specific genes. Transient starch turnover occurs in transfer cells, ensuring the maintenance of sink strength in filial tissues and the reallocation of sugars into more proximal regions of the starchy endosperm. Starch is temporally accumulated also in aleurone cells, where it is degraded during the seed filling period, to be replaced by storage proteins and lipids.

Seed-development programs: a systems biology-based comparison between dicots and monocots.

Seeds develop differently in dicots and monocots, especially with respect to the major storage organs. High-resolution transcriptome data have provided the first insights into the molecular networks and pathway interactions that function during the development of individual seed compartments. Here, we review mainly recent data obtained by systems biology-based approaches, which have allowed researchers to construct and model complex metabolic networks and fluxes and identify key limiting steps in seed development. Comparative coexpression network analyses define evolutionarily conservative (FUS3/ABI3/LEC1) and divergent (LEC2) networks in dicots and monocots. Finally, we discuss the determination of seed size--an important yield-related characteristic--as mediated by a number of processes (maternal and epigenetic factors, fine-tuned regulation of cell death in distinct seed compartments, and endosperm growth) and underlying genes defined through mutant analyses. Altogether, systems approaches can make important contributions toward a more complete and holistic knowledge of seed biology and thus support strategies for knowledge-based molecular breeding.

Robin: An Intuitive Wizard Application for R-Based Expression Microarray Quality Assessment and Analysis

The wide application of high-throughput transcriptomics using microarrays has generated a plethora of technical platforms, data repositories, and sophisticated statistical analysis methods, leaving the individual scientist with the problem of choosing the appropriate approach to address a biological question. Several software applications that provide a rich environment for microarray analysis and data storage are available (e.g. GeneSpring, EMMA2), but these are mostly commercial or require an advanced informatics infrastructure. There is a need for a noncommercial, easy-to-use graphical application that aids the lab researcher to find the proper method to analyze microarray data, without this requiring expert understanding of the complex underlying statistics, or programming skills. We have developed Robin, a Java-based graphical wizard application that harnesses the advanced statistical analysis functions of the R/BioConductor project. Robin implements streamlined workflows that guide the user through all steps of two-color, single-color, or Affymetrix microarray analysis. It provides functions for thorough quality assessment of the data and automatically generates warnings to notify the user of potential outliers, low-quality chips, or low statistical power. The results are generated in a standard format that allows ready use with both specialized analysis tools like MapMan and PageMan and generic spreadsheet applications. To further improve user friendliness, Robin includes both integrated help and comprehensive external documentation. To demonstrate the statistical power and ease of use of the workflows in Robin, we present a case study in which we apply Robin to analyze a two-color microarray experiment comparing gene expression in tomato (Solanum lycopersicum) leaves, flowers, and roots.