Suspensor-derived somatic embryogenesis in Arabidopsis.

In many flowering plants, asymmetric division of the zygote generates apical and basal cells with different fates. In Arabidopsis thaliana, the apical cell generates the embryo while the basal cell divides anticlinally, leading to a suspensor of six to nine cells that remain extra-embryonic and eventually senesce. In some genetic backgrounds, or upon ablation of the embryo, suspensor cells can undergo periclinal cell divisions and eventually form a second twin embryo. Likewise, embryogenesis can be induced from somatic cells by various genes, but the relationship with suspensor-derived embryos is unclear. Here, we addressed the nature of the suspensor to embryo fate transformation and its genetic triggers. We expressed most known embryogenesis-inducing genes specifically in suspensor cells. We next analyzed morphology and fate-marker expression in embryos in which suspensor division was activated by different triggers to address the developmental paths towards reprogramming. Our results show that reprogramming of Arabidopsis suspensor cells towards embryonic identity is a specific cellular response that is triggered by defined regulators, follows a conserved developmental trajectory and shares similarity to the process of somatic embryogenesis from post-embryonic tissues.

A Robust Auxin Response Network Controls Embryo and Suspensor Development through a Basic Helix Loop Helix Transcriptional Module.

Land plants reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. Thus, a key question is how embryo identity in plants is controlled, and how this process is modified during nonzygotic embryogenesis. The Arabidopsis (Arabidopsis thaliana) zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We found that reprogramming is complex and accompanied by large transcriptomic changes before anatomical changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon the re-establishment of cellular auxin levels or response. This finding points to a remarkable degree of feedback regulation to create resilience in the auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identified an auxin-dependent basic Helix Loop Helix transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor.

Molecular Characterization of Arabidopsis GAL4/UAS Enhancer Trap Lines Identifies Novel Cell-Type-Specific Promoters.

Cell-type-specific gene expression is essential to distinguish between the numerous cell types of multicellular organism. Therefore, cell-type-specific gene expression is tightly regulated and for most genes RNA transcription is the central point of control. Thus, transcriptional reporters are broadly used markers for cell identity. In Arabidopsis (Arabidopsis thaliana), a recognized standard for cell identities is a collection of GAL4/UAS enhancer trap lines. Yet, while greatly used, very few of them have been molecularly characterized. Here, we have selected a set of 21 frequently used GAL4/UAS enhancer trap lines for detailed characterization of expression pattern and genomic insertion position. We studied their embryonic and postembryonic expression domains and grouped them into three groups (early embryo development, late embryo development, and embryonic root apical meristem lines) based on their dominant expression. We show that some of the analyzed lines are expressed in a domain often broader than the one that is reported. Additionally, we present an overview of the location of the T-DNA inserts of all lines, with one exception. Finally, we demonstrate how the obtained information can be used for generating novel cell-type-specific marker lines and for genotyping enhancer trap lines. The knowledge could therefore support the extensive use of these valuable lines.

A PARTHENOGENESIS allele from apomictic dandelion can induce egg cell division without fertilization in lettuce.

Apomixis, the clonal formation of seeds, is a rare yet widely distributed trait in flowering plants. We have isolated the PARTHENOGENESIS (PAR) gene from apomictic dandelion that triggers embryo development in unfertilized egg cells. PAR encodes a K2-2 zinc finger, EAR-domain protein. Unlike the recessive sexual alleles, the dominant PAR allele is expressed in egg cells and has a miniature inverted-repeat transposable element (MITE) transposon insertion in the promoter. The MITE-containing promoter can invoke a homologous gene from sexual lettuce to complement dandelion LOSS OF PARTHENOGENESIS mutants. A similar MITE is also present in the promoter of the PAR gene in apomictic forms of hawkweed, suggesting a case of parallel evolution. Heterologous expression of dandelion PAR in lettuce egg cells induced haploid embryo-like structures in the absence of fertilization. Sexual PAR alleles are expressed in pollen, suggesting that the gene product releases a block on embryogenesis after fertilization in sexual species while in apomictic species PAR expression triggers embryogenesis in the absence of fertilization.

Design principles of a minimal auxin response system.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1-3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.

Evolution, Initiation, and Diversity in Early Plant Embryogenesis.

During embryogenesis in plants, cell identities are specified de novo, starting from a single cell. By combining imaging, genomic profiling, and genetics, principles of early plant development have been unraveled in the dicotyledonous plant Arabidopsis. A central emerging question, however, is how well zygotic embryogenesis in Arabidopsis reflects homologous processes in other plant species, including early diverging, non-flowering, and non-seed plants. Here, we consider plant embryogenesis with an emphasis on its evolutionary history, the diverse modes of its initiation, and the concepts in pattern formation among morphologically distinct plant groups. Furthermore, we explore challenges and future directions in plant embryogenesis research.

Cell-Type-Specific Promoter Identification Using Enhancer Trap Lines.

Many developmental processes involve transitions between different cell identities as cells differentiate or undergo reprogramming. Cell identity specifications are generally associated with the activation and suppression of specific sets of genes mediated by transcription factors. Therefore, transcriptional reporters, such as promoters of cell-type-specific genes, are broadly used as cell identity markers in developmental biology. In Arabidopsis (Arabidopsis thaliana), a collection of GAL4/UAS enhancer trap lines is an established standard for inferring cell identity. However, only a few of these enhancer trap lines have been molecularly characterized, which limits their potential. Here, we describe an approach for a detailed characterization of expression and mapping of T-DNA insert location of GAL4/UAS enhancer trap lines. Additionally, we demonstrate how the acquired information can be further used for the generation of novel cell-type-specific promoters as well as for genotyping of enhancer trap lines.

FRET-FLIM for Visualizing and Quantifying Protein Interactions in Live Plant Cells.

Proteins are the workhorses that control most biological processes in living cells. Although proteins can accomplish their functions independently, the vast majority of functions require proteins to interact with other proteins or biomacromolecules. Protein interactions can be investigated through biochemical assays such as co-immunoprecipitation (co-IP) or Western blot analysis, but such assays lack spatial information. Here we describe a well-developed imaging method, Förster resonance energy transfer (FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM), that can be used to visualize protein interactions with both spatial and temporal resolution in live cells. We demonstrate its use in plant developmental research by visualizing in vivo dimerization of AUXIN RESPONSE FACTOR (ARF) proteins, mediating auxin responses.

A set of domain-specific markers in the Arabidopsis embryo.

KEY MESSAGE: We describe a novel set of domain-specific markers that can be used in genetic studies, and we used two examples to show loss of stem cells in a monopteros background. Multicellular organisms can be defined by their ability to establish distinct cell identities, and it is therefore of critical importance to distinguish cell types. One step that leads to cell identity specification is activation of unique sets of transcripts. This property is often exploited in order to infer cell identity; the availability of good domain-specific marker lines is, however, poor in the Arabidopsis embryo. Here we describe a novel set of domain-specific marker lines that can be used in Arabidopsis (embryo) research. Based on transcriptomic data, we selected 12 genes for expression analysis, and according to the observed expression domain during embryogenesis, we divided them into four categories (1-ground tissue; 2-root stem cell; 3-shoot apical meristem; 4-post-embryonic). We additionally show the use of two markers from the "stem cell" category in a genetic study, where we use the absence of the markers to infer developmental defects in the monopteros mutant background. Finally, in order to judge whether the established marker lines also play a role in normal development, we generated loss-of-function resources. None of the analyzed T-DNA insertion, artificial microRNA, or misexpression lines showed any apparent phenotypic difference from wild type, indicating that these genes are not nonredundantly required for development, but also suggesting that marker activation can be considered an output of the patterning process. This set of domain-specific marker lines is therefore a valuable addition to the currently available markers and will help to move toward a generic set of tissue identity markers.

A roadmap to embryo identity in plants.

Although plant embryogenesis is usually studied in the context of seed development, there are many alternative roads to embryo initiation. These include somatic embryogenesis in tissue culture and microspore embryogenesis, both widely used in breeding and crop propagation, but also include other modes of ectopic embryo initiation. In the past decades several genes, mostly transcription factors, were identified that can induce embryogenesis in somatic cells. Because the genetic networks in which such regulators operate to promote embryogenesis are largely unknown, a key question is how their activity relates to zygotic and alternative embryo initiation. We describe here the many roads to plant embryo initiation and discuss a framework for defining the developmental roles and mechanisms of plant embryogenesis regulators.