Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

Plasma Membranes Are Subcompartmentalized into a Plethora of Coexisting and Diverse Microdomains in Arabidopsis and Nicotiana benthamiana.

Eukaryotic plasma membranes are highly compartmentalized structures. So far, only a few individual proteins that function in a wide range of cellular processes have been shown to segregate into microdomains. However, the biological roles of most microdomain-associated proteins are unknown. Here, we investigated the heterogeneity of distinct microdomains and the complexity of their coexistence. This diversity was determined in living cells of intact multicellular tissues using 20 different marker proteins from Arabidopsis thaliana, mostly belonging to the Remorin protein family. These proteins associate with microdomains at the cytosolic leaflet of the plasma membrane. We characterized these membrane domains and determined their lateral dynamics by extensive quantitative image analysis. Systematic colocalization experiments with an extended subset of marker proteins tested in 45 different combinations revealed the coexistence of highly distinct membrane domains on individual cell surfaces. These data provide valuable tools to study the lateral segregation of membrane proteins and their biological functions in living plant cells. They also demonstrate that widely used biochemical approaches such as detergent-resistant membranes cannot resolve this biological complexity of membrane compartmentalization in vivo.

Evaluating the predictive value of doublecortin as a marker for adult neurogenesis in canaries (Serinus canaria).

Doublecortin (DCX) is an important microtubule-associated protein involved in the migration of young neurons into the cortical layers of the brain during early human development. The continued expression of DCX in brain areas with protracted neuron recruitment has promoted this endogenous protein as a popular indirect tool to monitor adult neurogenesis in a variety of species. However, little is known about its possible involvement in other cellular processes and a thorough validation of DCX as a quantitative measure for neurogenesis is generally lacking. Here we investigated the relationship between DCX expression and neuron recruitment in the brains of adult canaries (Serinus canaria), a species well-known for its adult neurogenesis. We examined the age and functional state of DCX-labeled cells by using mitotic and neuron-specific markers, retrograde tracings, and immediate early gene colocalizations. Although DCX expression was high in brain areas implicated in adult neurogenesis, DCX-expressing neurons were also abundant in regions that do not recruit new neurons. Moreover, DCX expression was observed in adult, active neurons, differentiated projection neurons, and birth-dated neurons of up to 1 year of age. Season and testosterone treatment affected DCX expression in two song control nuclei, HVC and Area X, but did not correlate with known patterns of neuron recruitment. Together, these results demonstrate that DCX expression is not exclusive to young migrating neurons, and does not predict neuron recruitment equally throughout the canary brain. Therefore, DCX labeling needs careful validation for each brain region separately in each species analyzed when used to quantify adult neurogenesis.

Plasticity of plasma membrane compartmentalization during plant immune responses.

Plasma membranes require high levels of plasticity to modulate the perception and transduction of extracellular and intracellular signals. Dynamic lateral assembly of protein complexes combined with an independent compositional lipid patterning in both membrane leaflets provide cells the opportunity to decorate this interface with specific proteins in an organized but dynamic manner. Such ability to dynamically reorganize the protein content of the plasma membrane is essential for the regulation of processes such as polarity of transport, development, and microbial infection. While the plant cell wall represents the first physical and mostly unspecific barrier for invading microbes, the plasma membrane is at the forefront of microbial recognition and initiation of defense responses. Accumulating evidence indicating dynamic compartmentalization of plasma membranes in response to environmental cues has increased the interest in the compositional heterogeneity of this bilayer. Here, we elucidate the recruitment of specific proteins into defined membrane structures that ensure functional compartmentalization of the bilayer during infection processes.

Investigation of MADS domain transcription factor dynamics in the floral meristem.

To study the importance of intercellular transport for MADS domain transcription factor functioning during floral development, we analyzed the dynamic behavior of fluorescently-tagged MADS domain proteins in transgenic plants by Confocal Laser Scanning Microscopy. These analyses, described in a recent paper in The Plant Journal, provided proof for previous suggestions that the Arabidopsis thaliana C-type protein AGAMOUS has a non-cell-autonomous role in floral meristem integrity. Furthermore, it indicated a possible non-cell-autonomous role for the B-type proteins APETALA3 and PISTILLATA, and the E-type protein SEPALLATA3, through lateral intercellular movement in the floral meristem. In this addendum we compare some of the available fluorescent protein-based technologies for the investigation of transcription factor movements and dynamics.

Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition.

During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins.

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.

Tagging of MADS domain proteins for chromatin immunoprecipitation.

BACKGROUND: 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 in vivo 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. RESULTS: 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 Strep-tag 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. CONCLUSION: 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.