67 research outputs found
MADS-complexes regulate transcriptome dynamics during pollen maturation
Pollen transcript profiling of mutants defective in MADS-domain MIKC* protein complexes suggests they control a transcriptional network directing cellular differentiation during pollen maturation
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The role of D3-type cyclins is related to cytokinin and the bHLH transcription factor SPATULA in Arabidopsis gynoecium development.
We studied the D3-type cyclin function during gynoecium development in Arabidopsis and how they are related to the hormone cytokinin and the transcription factor SPATULA. Growth throughout the life of plants is sustained by cell division and differentiation processes in meristematic tissues. In Arabidopsis, gynoecium development implies a multiphasic process where the tissues required for pollination, fertilization, and seed development form. The Carpel Margin Meristem (CMM) is a mass of undifferentiated cells that gives rise to the gynoecium internal tissues, such as septum, ovules, placenta, funiculus, transmitting tract, style, and stigma. Different genetic and hormonal factors, including cytokinin, control the CMM function. Cytokinin regulates the cell cycle transitions through the activation of cell cycle regulators as cyclin genes. D3-type cyclins are expressed in proliferative tissues, favoring the mitotic cell cycle over the endoreduplication. Though the role of cytokinin in CMM and gynoecium development is highly studied, its specific role in regulating the cell cycle in this tissue remains unclear. Additionally, despite extensive research on the relationship between CYCD3 genes and cytokinin, the regulatory mechanism that connects them remains elusive. Here, we found that D3-type cyclins are expressed in proliferative medial and lateral tissues. Conversely, the depletion of the three CYCD3 genes showed that they are not essential for gynoecium development. However, the addition of exogenous cytokinin showed that they could control the division/differentiation balance in gynoecium internal tissues and outgrowths. Finally, we found that SPATULA can be a mechanistic link between cytokinin and the D3-type cyclins. The data suggest that the role of D3-type cyclins in gynoecium development is related to the cytokinin response, and they might be activated by the transcription factor SPATULA
Tagging of MADS domain proteins for chromatin immunoprecipitation
<p>Abstract</p> <p>Background</p> <p>Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow <it>in vivo </it>identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene.</p> <p>Results</p> <p>Here, we present the results of experiments aiming at the development of a generic tagging approach for the Arabidopsis MADS domain proteins AGAMOUS, SEPALLATA3, and FRUITFULL. For this, Arabidopsis wild type plants were transformed with constructs containing a MADS-box gene fused to either a double <it>Strep</it>-tag<sup>® </sup>II-FLAG-tag, a triple HA-tag, or an eGFP-tag, all under the control of the constitutive double 35S Cauliflower Mosaic Virus (CaMV) promoter. Strikingly, in all cases, the number of transformants with loss-of-function phenotypes was much larger than those with an overexpression phenotype. Using endogenous promoters in stead of the 35S CaMV resulted in a dramatic reduction in the frequency of loss-of-function phenotypes. Furthermore, pleiotropic defects occasionally caused by an overexpression strategy can be overcome by using the native promoter of the gene. Finally, a ChAP result is presented using GFP antibody on plants carrying a genomic fragment of a MADS-box gene fused to GFP.</p> <p>Conclusion</p> <p>This study revealed that MADS-box proteins are very sensitive to fusions with small peptide tags and GFP tags. Furthermore, for the expression of chimeric versions of MADS-box genes it is favorable to use the entire genomic region in frame to the tag of choice. Interestingly, though unexpected, it appears that the use of chimeric versions of MADS-box genes under the control of the strong 35S CaMV promoter is a very efficient method to obtain dominant-negative mutants, either caused by cosuppression or by alteration of the activity of the recombinant protein. Finally, we were able to demonstrate AGAMOUS binding to one of its targets by ChAP.</p
In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana
BACKGROUND: MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). RESULTS: We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. CONCLUSION: This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these proteins in the regulatory processes that drive floral development and leads to new hypotheses
An efficient method for miRNA detection and localization in crop plants
MiRNAs are a class of non-coding small RNAs (sRNAs) that are important regulators of gene expression at the post-transcriptional level by mRNA cleavage or translation inhibition. Another class of sRNAs are siRNAs, which also regulate gene expression but by causing DNA methylation. This epigenetic regulatory role has been observed for some miRNAs as well. The use of sRNAs allows the development of biotechnological applications in plants. To develop these types of applications, and to better understand the natural roles they play, it is important to be able to detect and to localize these sRNAs at the plant tissue level. Sometimes, in crop plants this can be challenging. Therefore, we developed a tissue printing hybridization protocol for easy and efficient detection of sRNAs and demonstrate this by the analysis of the spatio-temporal expression patterns of the miRNAs miR159 and miR164 in fruits of various crop plants. Moreover, we show the possibility to also detect the expression of miRNAs in fruit juice using a dot blot hybridization approach
SEPALLATA3: the 'glue' for MADS box transcription factor complex formation
A yeast 3-hybrid screen in Arabidopsis reveals MADS box protein complexes: SEP3 is shown to mediate complex formation and floral timing
Non-destructive Plant Morphometric and Color Analyses Using an Optoelectronic 3D Color Microscope
Gene function discovery in plants, as other plant science quests, is aided by tools that image, document, and measure plant phenotypes. Tools that acquire images of plant organs and tissues at the microscopic level have evolved from qualitative documentation tools, to advanced tools where software-assisted analysis of images extracts quantitative information that allows statistical analyses. They are useful to perform morphometric studies that describe plant physical characteristics and quantify phenotypes, aiding gene function discovery. In parallel, non-destructive, versatile, robust, and user friendly technologies have also been developed for surface topography analysis and quality control in the industrial manufacture sector, such as optoelectronic three-dimensional (3D) color microscopes. These microscopes combine optical lenses, electronic image sensors, motorized stages, graphics engines, and user friendly software to allow the visualization and inspection of objects of diverse sizes and shapes from different angles. This allow the integration of different automatically obtained images along the Z axis of an object, into a single image with a large depth-of-field, or a 3D model in color. In this work, we explored the performance of an optoelectronic microscope to study plant morphological phenotypes and plant surfaces in different model species. Furthermore, as a “proof-of-concept,” we included the phenotypic characterization (morphometric analyses at the organ level, color, and cell size measurements) of Arabidopsis mutant leaves. We found that the microscope tested is a suitable, practical, and fast tool to routinely and precisely analyze different plant organs and tissues, producing both high-quality, sharp color images and morphometric and color data in real time. It is fully compatible with live plant tissues (no sample preparation is required) and does not require special conditions, high maintenance, nor complex training. Therefore, though barely reported in plant scientific studies, optoelectronic microscopes should emerge as convenient and useful tools for phenotypic characterization in plant sciences
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