Showing papers by "Detlef Weigel published in 2006"

Highly specific gene silencing by artificial microRNAs in Arabidopsis.

Abstract: Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).

A divergent external loop confers antagonistic activity on floral regulators FT and TFL1

Abstract: The Arabidopsis genes FT and TERMINAL FLOWER1 (TFL1) encode related proteins with similarity to human Raf kinase inhibitor protein. FT, and likely also TFL1, is recruited to the promoters of floral genes through interaction with FD, a bZIP transcription factor. FT, however, induces flowering, while TFL1 represses flowering. Residues responsible for the opposite activities of FT and TFL1 were mapped by examining plants that overexpress chimeric proteins. A region important in vivo localizes to a 14-amino-acid segment that evolves very rapidly in TFL1 orthologs, but is almost invariant in FT orthologs. Crystal structures show that this segment forms an external loop of variable conformation. The only residue unambiguously distinguishing the FT and TFL1 loops makes a hydrogen bond with a residue near the entrance of a potential ligand-binding pocket in TFL1, but not in FT. This pocket is contacted by a C-terminal peptide, which also contributes to the opposite FT and TFL1 activities. In combination, these results identify a molecular surface likely to be recognized by FT- and/or TFL1-specific interactors.

Transformation of Agrobacterium Using the Freeze-Thaw Method

Abstract: INTRODUCTIONThis protocol describes a method for transforming Agrobacterium with plasmid DNA using a freeze-thaw technique. Although the transformation efficiency for Agrobacterium is lower than that for Escherichia coli, it is possible to obtain adequate numbers of transformants with this technique.

The PHYTOCHROME C photoreceptor gene mediates natural variation in flowering and growth responses of Arabidopsis thaliana

Abstract: Light has an important role in modulating seedling growth and flowering time. We show that allelic variation at the PHYTOCHROME C (PHYC) photoreceptor locus affects both traits in natural populations of A. thaliana. Two functionally distinct PHYC haplotype groups are distributed in a latitudinal cline dependent on FRIGIDA, a locus that together with FLOWERING LOCUS C explains a large portion of the variation in A. thaliana flowering time. In a genome-wide scan for association of 65 loci with latitude, there was an excess of significant P values, indicative of population structure. Nevertheless, PHYC was the most strongly associated locus across 163 strains, suggesting that PHYC alleles are under diversifying selection in A. thaliana. Our work, together with previous findings, suggests that photoreceptor genes are major agents of natural variation in plant flowering and growth response.

Identification of plant microRNA homologs

Abstract: Summary: MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs that regulate gene and protein expression in plants and animals. MiRNAs have so far been identified mostly by specific cloning of small RNA molecules, complemented by computational methods. We present a computational identification approach that is able to identify candidate miRNA homologs in any set of sequences, given a query miRNA. The approach is based on a sequence similarity search step followed by a set of structural filters. Availability: microHARVESTER is offered as a web-service and additionally as source code upon request at http://www-ab.informatik.uni-tuebingen.de/software/microHARVESTER Contact: dezulian@informatik.uni-tuebingen.de

NUBBIN and JAGGED define stamen and carpel shape in Arabidopsis.

Abstract: Differential growth of tissues during lateral organ development is essential for producing variation in shape and size. Previous studies have identified JAGGED ( JAG ), a gene that encodes a putative C 2 H 2 zinc-finger transcription factor, as a key regulator of shape that promotes growth in lateral organs. Although JAG expression is detected in all floral organs, loss-of-function jag alleles have their strongest effects on sepal and petal development, suggesting that JAG may act redundantly with other factors in stamens and carpels. Here, we show that NUBBIN ( NUB ), a gene closely related to JAG , is responsible for this redundancy. Unlike JAG, NUB is exclusively expressed in leaves, stamens and carpels, and briefly in petal primordia. Furthermore, whereas JAG expression extends into all cell layers of lateral organs, NUB is restricted to the interior adaxial side. Our analysis focuses on stamen and gynoecium development, where we find that NUB acts redundantly with JAG to promote the growth of the pollen-bearing microsporangia of the anthers and the carpel walls of the gynoecium, which enclose the ovules. JAG and NUB also act redundantly to promote the differentiation of adaxial cell types in the carpel walls, and in the establishment of the correct number of cell layers. The important role these two factors play in regulating organ growth is further demonstrated by gain-of-function experiments showing that ectopic NUB expression is sufficient to drive the proliferation of tissues and the amplification of cell-layer number.

Transformation of Agrobacterium Using Electroporation

Abstract: INTRODUCTIONThis protocol describes a method for transforming Agrobacterium with plasmid DNA using electroporation in a manner similar to that commonly used for Escherichia coli. Although the transformation efficiency for Agrobacterium is lower than that for E. coli, it is possible to obtain adequate numbers of Agrobacterium transformants with this technique.

In planta transformation of Arabidopsis.

Abstract: INTRODUCTIONA breakthrough in Arabidopsis research was the invention of the vacuum-infiltration procedure, a simple and reliable method of obtaining transformants at high efficiency while avoiding the use of tissue culture. The plant transformation procedures described here involve floral dip, vacuum infiltration, and spraying. They yield transformants at frequencies ranging up to several percent, with the most common frequency being 0.1%-1%.

Temperature Induced Flowering in Arabidopsis thaliana.

Abstract: The formation of flowers, a tightly controlled process, is modulated by developmental as well as environmental cues. Among the environmental factors that influence flowering, our knowledge of the effects of growth temperature is relatively less. In the July issue of PLoS Genetics, we have shown that higher temperatures can induce flowering by passing the requirement for long photoperiods for floral transition in Arabidopsis thaliana. By exploiting natural variation in combination with mutant analysis and genome wide expression profiling, we have shown that this floral induction has a novel genetic basis and is possibly associated with genome wide alterations in splicing patterns of several transcripts including FLOWERING LOCUS M, which we have shown to be a major effect QTL for thermo-sensitivity. Our study nicely demonstrates the power of a combinatorial approach to understand the molecular genetic basis of complex traits. In this addendum, we propose a testable hypothesis for FLM function in regulation of temperature mediated floral transition as well as the function of FLM in flowering time regulation.

EMS Mutagenesis of Arabidopsis Seed.

Abstract: INTRODUCTIONEMS-mutagenized Arabidopsis seed collections can be purchased from companies or acquired from other researchers. But in some cases (e.g., mutagenesis of a specific genotype), it is necessary for an investigator to generate EMS-mutagenized seed. In this protocol, EMS is used at concentrations that induce multiple point mutations in each plant, such that mutant alleles of a specific locus are found at a rate of ~1 in 2000-5000 M(2) plants. This high rate of mutagenesis makes possible the screening of relatively few plants to find those with the phenotype of interest, a particular advantage if the screen is laborious or if only a small number of genes mutate to the required phenotype.

Setting Up Arabidopsis Crosses

Abstract: INTRODUCTIONThe inheritance of mutant phenotypes is most often analyzed in the progeny resulting from crosses between different parents. Because Arabidopsis naturally self-pollinates, the generation of cross-progeny requires some intervention by the investigator. This protocol describes the generation and collection of seeds by crossing suitable Arabidopsis parent plants.

In vitro culture of embryos of the guppy, Poecilia reticulata.

Abstract: The rich variation in adult color patterns of male guppies (Poecilia reticulata) has attracted the attention of geneticists and ecologists for almost a century. Studies on their embryogenesis, however, have been limited by the fact that guppies are live bearers. We have observed normal development after explantation of guppy embryos from the ovary of pregnant females at various times after last parturition, and found that development of each batch of eggs is slightly asynchronous, most likely due to asynchronous fertilization. We have cultured explanted embryos in vitro and continuously observed their development. Although embryos explanted a few days after fertilization survived up to 4 weeks in culture, they did not complete their development. In contrast, embryos explanted at late stages of gestation could hatch and develop to fertile adults. Our embryo culture techniques overcome some of the limitations of using livebearers as study objects, and they allow continuous observation of and accessibility to live embryos at all stages.

Evolutionary divergence of LFY function in the mustards Arabidopsis thaliana and Leavenworthia crassa.

Abstract: LEAFY (LFY), a transcription factor involved in the regulation of flower development in Arabidopsis thaliana, has been identified as a candidate gene in the diversification of plant architecture in Brassicaceae. Previous research with Leavenworthia crassa, which produces solitary flowers in the axils of rosette leaves, has shown that the L. crassa LFY ortholog, LcrLFY, rescues most aspects of flower development in A. thaliana but showed two novel traits: flowers produced additional petals and inflorescences produced terminal flowers. In this paper, we explore the molecular mechanisms responsible for these novel phenotypes. We used microarray hybridizations to identify 32 genes differentially expressed between a transgenic LcrLFY line and a control transgenic LFY line. Of particular interest, TERMINAL FLOWER 1 (TFL1) transcripts were found at elevated levels in LcrLFY lines. To distinguish regulatory versus functional changes within the LcrLFY locus, reciprocal chimeric transgenes between LcrLFY and LFY were constructed. These lines implicate divergence of LcrLFY cis-regulation as the primary cause of both novel transgenic phenotypes but implicate divergence of LcrLFY protein function as the primary cause of elevated TFL1 levels. Taken together these results show that LcrLFY has diverged from A. thaliana in both the cis-regulatory and protein-coding regions and imply that molecular coevolution of LcrLFY and the L. crassa TFL1 ortholog, LcrTFL1, contributed to the evolution of rosette flowering.

Root transformation of Arabidopsis.

Abstract: INTRODUCTIONArabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. We describe the generation of transgenic plants via root transformation in tissue culture, which can be useful for transforming sterile mutants.

Glufosinate ammonium selection of transformed Arabidopsis.

Abstract: INTRODUCTIONOne of the most commonly used markers for the selection of transgenic Arabidopsis is resistance to glufosinate ammonium, an herbicide that is sold under a variety of trade names including Basta and Finale. Resistance to glufosinate ammonium is conferred by the bacterial bialophos resistance gene (BAR) encoding the enzyme phosphinotricin acetyl transferase (PAT). This protocol describes the use of glufosinate ammonium to select transformed Arabidopsis plants. The major advantage of glufosinate ammonium selection is that it can be performed on plants growing in soil and does not require the use of sterile techniques.

Vectors and agrobacterium hosts for Arabidopsis transformation.

Abstract: INTRODUCTIONArabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. A. tumefaciens is a soil-dwelling bacterium that transforms normal plant cells into tumor-forming cells by inserting a piece of bacterial DNA (the transfer, or "T," DNA) into the plant cell genome. The T-DNA, which is flanked by left- and right-border (LB and RB) sequences, resides on a tumor-inducing (Ti) plasmid. The Ti plasmid also carries many of the transfer functions for mobilizing the T-DNA. This article provides a brief discussion of the principles of T-DNA transformation, including consideration of T-DNA vectors and their hosts.

Kanamycin selection of transformed Arabidopsis.

Abstract: INTRODUCTIONThe most commonly used markers for selection of transgenic Arabidopsis are resistance to the antibiotic kanamycin and to the herbicide glufosinate ammonium. Resistance to kanamycin is conferred by a bacterial gene encoding the enzyme neomycin phosphotransferase (NPT). In this protocol, kanamycin-resistant seedlings are selected on solid medium.

CrossLink: visualization and exploration of sequence relationships between (micro) RNAs

Abstract: CrossLink is a versatile tool for the exploration of relationships between RNA sequences. After a parametrization phase, CrossLink delegates the determination of sequence relationships to established tools (BLAST, Vmatch and RNAhybrid) and then constructs a network. Each node in this network represents a sequence and each link represents a match or a set of matches. Match attributes are reflected by graphical attributes of the links and corresponding alignments are displayed on a mouse-click. The distributions of match attributes such as E-value, match length and proportion of identical nucleotides are displayed as histograms. Sequence sets can be highlighted and visibility of designated matches can be suppressed by real-time adjustable thresholds for attribute combinations. Powerful network layout operations (such as spring-embedding algorithms) and navigation capabilities complete the exploration features of this tool. CrossLink can be especially useful in a microRNA context since Vmatch and RNAhybrid are suitable tools for determining the antisense and hybridization relationships, which are decisive for the interaction between microRNAs and their targets. CrossLink is available both online and as a standalone version at http://www-ab.informatik.uni-tuebingen.de/software.

Screening DNA Pools for T-DNA Insertions in Arabidopsis Genes.

Abstract: INTRODUCTIONMany investigators are interested in studying the functions of genes whose sequences or expression patterns suggest roles in particular biological processes. A powerful way to identify a mutation in the gene of interest and to test mutant plants for phenotypes that are predicted to result from loss of function of that gene is by PCR screening. Pools of insertion lines are screened using one primer corresponding to the gene of interest and one primer corresponding to the end of the insertion element. The synthesis of a product indicates the presence of an insertion in the gene of interest. The pool is then repeatedly subdivided until a single plant carrying the desired insertion is identified.