Transposon based activation tagging in diploid strawberry and monoploid derivatives of potato

Key message

Diploid strawberry and potato transformed with a transposon tagging construct exhibited either global (strawberry) or local transposition (potato). An activation tagged, compact-sized strawberry mutant overexpressed the gene adjacent to Ds.

Abstract

As major fruit and vegetable crops, respectively, strawberry and potato are among the first horticultural crops with draft genome sequences. To study gene function, we examined transposon-tagged mutant strategies in model populations for both species, Fragaria vesca and Solanum tuberosum Group Phureja, using the same Activation/Dissociation (Ac/Ds) construct. Early somatic transposition during tissue culture occurred at a frequency of 18.5 % in strawberry but not in potato transformants. Green fluorescent protein under a monocot promoter was a more reliable selectable marker in strawberry compared to potato. BASTA (gluphosinate herbicide) resistance served as an effective selectable marker for both species (80 and 85 % reliable in strawberry and potato, respectively), although the effective concentration differed (0.5 % for strawberry and 0.03 % for potato). Transposons preferentially reinserted within genes (exons and introns) in both species. Real-time quantitative PCR revealed enhanced gene expression (670 and 298-fold expression compared to wild type in petiole and leaf tissue, respectively) for an activation tagged strawberry mutant with Ds inserted about 0.6 kb upstream from a gene coding for an epidermis-specific secreted glycoprotein EP1. Our data also suggested that endopolyploid (diploid) cells occurring in leaf explants of monoploid potato were the favored targets of T-DNA integration during transformation. Mutants obtained in these studies provide a useful resource for future genetic studies.     

High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics

Fragaria vesca L., a diploid (2n=2x=14) relative of the commercial octoploid strawberry, is an attractive model for functional genomics research in Rosaceae. Its small genome size, short reproductive cycle, and facile vegetative and seed propagation make F. vesca a promising candidate for forward and reverse genetics experiments. However, the lack of a high-efficiency transformation protocol required for systematic production of thousands of T-DNA insertional mutant lines and high-throughput gene validation is a major bottleneck. We describe a new transformation procedure that uses leaf explants from newly unfolded trifoliate leaves obtained from stock plants 6–7 weeks after seed germination, co-cultivation with Agrobacterium strain GV3101, and stringent selection on MS medium containing 4 mg l⁻¹ hygromycin. Using this protocol we achieved 100% transformation efficiency for 6 of 14 F. vesca accessions tested. Accession PI 551572 was determined to be the best candidate for a model in F. vesca functional genomics research, as it showed the greatest propensity for callus formation, transformation, shoot regeneration, ex vitro establishment, and plant growth, requiring only 14–15 weeks to complete its life cycle in different seasons in the greenhouse.     

PlantMetabolomics.org: A Web Portal for Plant Metabolomics Experiments

PlantMetabolomics.org (PM) is a web portal and database for exploring, visualizing, and downloading plant metabolomics data. Widespread public access to well-annotated metabolomics datasets is essential for establishing metabolomics as a functional genomics tool. PM integrates metabolomics data generated from different analytical platforms from multiple laboratories along with the key visualization tools such as ratio and error plots. Visualization tools can quickly show how one condition compares to another and which analytical platforms show the largest changes. The database tries to capture a complete annotation of the experiment metadata along with the metabolite abundance databased on the evolving Metabolomics Standards Initiative. PM can be used as a platform for deriving hypotheses by enabling metabolomic comparisons between genetically unique Arabidopsis (Arabidopsis thaliana) populations subjected to different environmental conditions. Each metabolite is linked to relevant experimental data and information from various annotation databases. The portal also provides detailed protocols and tutorials on conducting plant metabolomics experiments to promote metabolomics in the community. PM currently houses Arabidopsis metabolomics data generated by a consortium of laboratories utilizing metabolomics to help elucidate the functions of uncharacterized genes. PM is publicly available at http://www.plantmetabolomics.org.In the post genomics era, metabolomics is fast emerging as a vital source of information to aid in solving systems biology puzzles with an emphasis on metabolic solutions. Metabolomics is the science of measuring the pool sizes of metabolites (small molecules of M_r ≤ 1,000 D), which collectively define the metabolome of a biological sample (Fiehn et al., 2000; Hall et al., 2002). Coverage of the entire plant metabolome is a daunting task as it is estimated that there are over 200,000 different metabolites within the plant kingdom (Goodacre et al., 2004). Although technology is rapidly advancing, there are still large gaps in our knowledge of the plant metabolome.Despite this lack of complete knowledge and the immense metabolic diversity among plants, metabolomics has become a key analytical tool in the plant community (Hall et al., 2002). This has led to the emergence of multiple experimental and analytical platforms that collectively generate millions of metabolite data points. Because of this vast amount of data, the development of public databases to capture information from metabolomics experiments is vital to provide the scientific community with comprehensive knowledge about metabolite data generation, annotation, and integration with metabolic pathway data. Some examples of these public databases are given below. The Human Metabolome Project contains comprehensive data for more than 2,000 metabolites found within the human body (Wishart et al., 2007). The Golm Database is a repository that provides access to mass spectrometry (MS) libraries, metabolite profiling experiments, and related information from gas chromatography (GC)-MS experimental platforms, along with tools to integrate this information with other systems biology knowledge (Kopka et al., 2005). The Madison Metabolomics Consortium Database contains primarily NMR spectra for Arabidopsis (Arabidopsis thaliana) and features thorough NMR search tools (Cui et al., 2008). SetupX and Binbase provide a framework that combines MS data and biological metadata for steering laboratory work flows and employs automated metabolite annotation (Scholz and Fiehn, 2007).A single analytical technique cannot identify and quantify all the metabolites found in plants. Thus, PlantMetabolomics.org (PM) was developed to provide a portal for accessing publicly available MS-based plant metabolomics experimental results from multiple analytical and separation techniques. PM also follows the emerging metabolomics standards for experiment annotation. PM has extensive annotation links between the identified metabolites and metabolic pathways in AraCyc (Mueller et al., 2003) at The Arabidopsis Information Resource (Rhee et al., 2003) and the Plant Metabolic Network (www.plantcyc.org), the Kyoto Encyclopedia of Genes and Genomes (KEGG; Kanehisa et al., 2004), and MetNetDB (Wurtele et al., 2007).Standards for the annotation of metabolomics experiments are still under active development and the metadata types collected in PM are based on the recommendations of the Metabolomics Standards Initiative (MSI; Fiehn et al., 2007a) and the Minimal Information for a Metabolomic Experiment (Bino et al., 2004) standards. MSI attempts to capture the complete annotation of metabolomics experiments and includes metadata of the experiments along with the metabolite abundance data. The initial database schema design was guided by the schema proposed in the Architecture for Metabolomics project (Jenkins et al., 2004).     

Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMECs), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5) were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrated that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. Whereas normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMECs under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. We discuss some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from normal mammary epithelial cells as regards the response to acute oxidative stress.     

Transposon tagging in diploid strawberry

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T(1) progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T(0) launch pads, putative transposants in the T(1) generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T(1) plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T(1) plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T(0) generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T(2) transposon-tagged plants. The mutant collection has been catalogued in an on-line database.     

Inhibition of Programmed Cell Death in Tobacco Plants during a Pathogen-Induced Hypersensitive Response at Low Oxygen Pressure

The hypersensitive response (HR) of plants to invading pathogens is thought to involve a coordinated activation of plant defense mechanisms and programmed cell death (pcd). To date, little is known about the mechanism underlying death of plant cells during this response. In addition, it is not known whether suppression of pcd affects the induction of other defense mechanisms during the HR. Here, we report that death of tobacco cells (genotype NN) infected with tobacco mosaic virus (TMV) is inhibited at low oxygen pressure. In contrast, virus replication and activation of defense mechanisms, as measured by synthesis of the pathogenesis-related protein PR-1a, were not inhibited at low oxygen pressure. Bacterium-induced pcd was also inhibited at low oxygen pressure. However, pcd induced by TMV or bacteria was not inhibited in transgenic tobacco plants expressing the mammalian anti-pcd protein Bcl-XL. Our results suggest that ambient oxygen levels are required for efficient pcd induction during the HR of plants and that activation of defense responses can be uncoupled from cell death. Furthermore, pcd that occurs during the interaction of tobacco with TMV or bacteria may be distinct from some cases of pcd or apoptosis in animals that are insensitive to low oxygen or inhibited by the Bcl-XL protein.     

A systems biology view of cancer

In order to understand how a cancer cell is functionally different from a normal cell it is necessary to assess the complex network of pathways involving gene regulation, signaling, and cell metabolism, and the alterations in its dynamics caused by the several different types of mutations leading to malignancy. Since the network is typically complex, with multiple connections between pathways and important feedback loops, it is crucial to represent it in the form of a computational model that can be used for a rigorous analysis. This is the approach of systems biology, made possible by new -omics data generation technologies. The goal of this review is to illustrate this approach and its utility for our understanding of cancer. After a discussion of recent progress using a network-centric approach, three case studies related to diagnostics, therapy, and drug development are presented in detail. They focus on breast cancer, B-cell lymphomas, and colorectal cancer. The discussion is centered on key mathematical and computational tools common to a systems biology approach.