Scan, extract, wrap, compute-a 3D method to analyse morphological shape differences.

Quantitative analysis of shape and form is critical in many biological disciplines, as context-dependent morphotypes reflect changes in gene expression and physiology, e.g., in comparisons of environment-dependent phenotypes, forward/reverse genetic assays or shape development during ontogenesis. 3D-shape rendering methods produce models with arbitrarily numbered, and therefore non-comparable, mesh points. However, this prevents direct comparisons. We introduce a workflow that allows the generation of comparable 3D models based on several specimens. Translocations between points of modelled morphotypes are plotted as heat maps and statistically tested. With this workflow, we are able to detect, model and investigate the significance of shape and form alterations in all spatial dimensions, demonstrated with different morphotypes of the pond-dwelling microcrustacean Daphnia. Furthermore, it allows the detection even of inconspicuous morphological features that can be exported to programs for subsequent analysis, e.g., streamline- or finite-element analysis.

A Novel RGL2-DOF6 Complex Contributes to Primary Seed Dormancy in Arabidopsis thaliana by Regulating a GATA Transcription Factor.

The DELLA protein RGA-LIKE2 (RGL2) is a key transcriptional repressor of gibberellic acid (GA) signaling that regulates seed germination. We identified GATA12, a gene encoding a GATA-type zinc finger transcription factor, as one of the downstream targets of RGL2 in Arabidopsis thaliana. Our data show that freshly harvested (unstratified) seeds of GATA12 antisense suppression lines have reduced dormancy compared with the wild-type, while ectopic expression lines show enhanced seed dormancy. We show that GATA12 expression is negatively regulated by GA, and its transcript levels decline dramatically under dormancy-breaking conditions such as dry storage and cold stratification of seeds. GATA12 promoter has several GAMYB- and DOF-associated motifs that are known to be GA- and RGL2-responsive, respectively. Chromatin immunoprecipitation assay showed that a protein complex containing RGL2 can bind to GATA12 promoter and thereby regulate its expression. RGL2 lacks a DNA binding domain and requires a transcription factor to induce GATA12 expression. Our data show that this RGL2-containing protein complex includes DNA BINDING1 ZINC FINGER6 (DOF6), which is a known negative regulator of germination in freshly harvested seeds. We further show that this novel RGL2-DOF6 complex is required for activating GATA12 expression, thus revealing a molecular mechanism to enforce primary seed dormancy.

Topological analysis of multicellular complexity in the plant hypocotyl.

Multicellularity arose as a result of adaptive advantages conferred to complex cellular assemblies. The arrangement of cells within organs endows higher-order functionality through a structure-function relationship, though the organizational properties of these multicellular configurations remain poorly understood. We investigated the topological properties of complex organ architecture by digitally capturing global cellular interactions in the plant embryonic stem (hypocotyl), and analyzing these using quantitative network analysis. This revealed the presence of coherent conduits of reduced path length across epidermal atrichoblast cell files. The preferential movement of small molecules along this cell type was demonstrated using fluorescence transport assays. Both robustness and plasticity in this higher order property of atrichoblast patterning was observed across diverse genetic backgrounds, and the analysis of genetic patterning mutants identified the contribution of gene activity towards their construction. This topological analysis of multicellular structural organization reveals higher order functions for patterning and principles of complex organ construction.

In Silico Methods for Cell Annotation, Quantification of Gene Expression, and Cell Geometry at Single-Cell Resolution Using 3DCellAtlas.

A comprehensive understanding of plant growth and development requires the integration of the spatial and temporal dynamics of gene regulatory networks with changes in cellular geometry during 3D organ growth. 3DCellAtlas is an integrative computational pipeline that semi-automatically identifies cell type and position within radially symmetric plant organs, and simultaneously quantifies 3D cell anisotropy and reporter abundance at single-cell resolution. It is a powerful tool that generates digital single-cell cellular atlases of plant organs and enables 3D cell geometry and reporter abundance (gene/protein/biosensor) from multiple samples to be integrated at single-cell resolution across whole organs. Here we describe how to use 3DCellAtlas to process and analyze radially symmetric organs, and to identify cell types and extract geometric cell data within these 3D cellular datasets. We detail how to use two statistical tools in 3DCellAtlas to compare cellular geometries, and to analyze reporter abundance at single-cell resolution.

The Transcription Factor ATHB5 Affects GA-Mediated Plasticity in Hypocotyl Cell Growth during Seed Germination.

Gibberellic acid (GA)-mediated cell expansion initiates the seed-to-seedling transition in plants and is repressed by DELLA proteins. Using digital single-cell analysis, we identified a cellular subdomain within the midhypocotyl, whose expansion drives the final step of this developmental transition under optimal conditions. Using network inference, the transcription factor ATHB5 was identified as a genetic factor whose localized expression promotes GA-mediated expansion specifically within these cells. Both this protein and its putative growth-promoting target EXPANSIN3 are repressed by DELLA, and coregulated at single-cell resolution during seed germination. The cellular domains of hormone sensitivity were explored within the Arabidopsis (Arabidopsis thaliana) embryo by putting seeds under GA-limiting conditions and quantifying cellular growth responses. The middle and upper hypocotyl have a greater requirement for GA to promote cell expansion than the lower embryo axis. Under these conditions, germination was still completed following enhanced growth within the radicle and lower axis. Under GA-limiting conditions, the athb5 mutant did not show a phenotype at the level of seed germination, but it did at a cellular level with reduced cell expansion in the hypocotyl relative to the wild type. These data reveal that the spatiotemporal cell expansion events driving this transition are not determinate, and the conditional use of GA-ATHB5-mediated hypocotyl growth under optimal conditions may be used to optionally support rapid seedling growth. This study demonstrates that multiple genetic and spatiotemporal cell expansion mechanisms underlie the seed to seedling transition in Arabidopsis.

Re-induction of the cell cycle in the Arabidopsis post-embryonic root meristem is ABA-insensitive, GA-dependent and repressed by KRP6.

Seeding establishment following seed germination requires activation of the root meristem for primary root growth. We investigated the hormonal and genetic regulation of root meristem activation during Arabidopsis seed germination. In optimal conditions, radicle cell divisions occur only after the completion of germination and require de novo GA synthesis. When the completion of germination is blocked by ABA, radicle elongation and cell divisions occurred in these non-germinating seeds. Conversely under GA-limiting conditions, ABA-insensitive mutants complete germination in the absence of radicle meristem activation and growth. Radicle meristem activation and extension can therefore occur independently of completion of the developmental transition of germination. The cell cycle regulator KRP6 partially represses GA-dependent activation of the cell cycle. Germination of krp6 mutant seeds occurs more rapidly, is slightly insensitive to ABA in dose-response assays, but also hypersensitive to the GA synthesis inhibitor PAC. These conflicting phenotypes suggest the cell cycle uncouples GA and ABA responses in germinating Arabidopsis seeds, and that KRP6 acts downstream of GA to inhibit mitotic cell cycle activation during germination.

Digital Single-Cell Analysis of Plant Organ Development Using 3DCellAtlas.

Diverse molecular networks underlying plant growth and development are rapidly being uncovered. Integrating these data into the spatial and temporal context of dynamic organ growth remains a technical challenge. We developed 3DCellAtlas, an integrative computational pipeline that semiautomatically identifies cell types and quantifies both 3D cellular anisotropy and reporter abundance at single-cell resolution across whole plant organs. Cell identification is no less than 97.8% accurate and does not require transgenic lineage markers or reference atlases. Cell positions within organs are defined using an internal indexing system generating cellular level organ atlases where data from multiple samples can be integrated. Using this approach, we quantified the organ-wide cell-type-specific 3D cellular anisotropy driving Arabidopsis thaliana hypocotyl elongation. The impact ethylene has on hypocotyl 3D cell anisotropy identified the preferential growth of endodermis in response to this hormone. The spatiotemporal dynamics of the endogenous DELLA protein RGA, expansin gene EXPA3, and cell expansion was quantified within distinct cell types of Arabidopsis roots. A significant regulatory relationship between RGA, EXPA3, and growth was present in the epidermis and endodermis. The use of single-cell analyses of plant development enables the dynamics of diverse regulatory networks to be integrated with 3D organ growth.

Mechanical constraints imposed by 3D cellular geometry and arrangement modulate growth patterns in the Arabidopsis embryo.

Morphogenesis occurs in 3D space over time and is guided by coordinated gene expression programs. Here we use postembryonic development in Arabidopsis plants to investigate the genetic control of growth. We demonstrate that gene expression driving the production of the growth-stimulating hormone gibberellic acid and downstream growth factors is first induced within the radicle tip of the embryo. The center of cell expansion is, however, spatially displaced from the center of gene expression. Because the rapidly growing cells have very different geometry from that of those at the tip, we hypothesized that mechanical factors may contribute to this growth displacement. To this end we developed 3D finite-element method models of growing custom-designed digital embryos at cellular resolution. We used this framework to conceptualize how cell size, shape, and topology influence tissue growth and to explore the interplay of geometrical and genetic inputs into growth distribution. Our simulations showed that mechanical constraints are sufficient to explain the disconnect between the experimentally observed spatiotemporal patterns of gene expression and early postembryonic growth. The center of cell expansion is the position where genetic and mechanical facilitators of growth converge. We have thus uncovered a mechanism whereby 3D cellular geometry helps direct where genetically specified growth takes place.

Auxin and gibberellin responsive Arabidopsis SMALL AUXIN UP RNA36 regulates hypocotyl elongation in the light.

KEY MESSAGE: The Arabidopsis SAUR36, renamed RAG1, integrates auxin and gibberellin signals to regulate apical hook maintenance in etiolated seedlings, hypocotyl elongation in the light and fertility. Phytohormone signalling intermediates integrate responses to developmental cues and the variety of environmental inputs thereby governing all aspects of plant growth and development. At the genetic level, interactions of different phytohormone signalling pathways lead to the regulation of overlapping sets of target genes. We have characterised SMALL AUXIN UP RNA 36 (SAUR36, At2g45210) whose expression is induced by auxins and repressed by gibberellins. Its expression appears to be restricted to elongating tissues. Germination responses to treatments with paclobutrazol and exogenous abscisic acid were affected in knock-out, knock-down as well as ectopic expression lines. At later stages of development, however, transgenic plants with reduced levels of SAUR36 expression appeared similar to wild-type plants, while ectopic expression of SAUR36 led to the absence of apical hooks in etiolated seedlings and longer hypocotyls in light-grown seedlings. Mature plants ectopically expressing SAUR36 further displayed strongly reduced fertility and wavy growth of inflorescence axes, the latter of which could be linked to defects in auxin transport. Taken together, our data suggest that SAUR36 plays a role in the regulation of seed germination by gibberellins and abscisic acid, light-dependent hypocotyl elongation as well as apical hook formation or maintenance. Therefore, we propose that it could act as one of the converging points of auxin and gibberellin signal integration in controlling key plant developmental events. Hence, we named the gene RESPONSE TO AUXINS AND GIBBERELLINS 1 (RAG1).

Manipulation of plant architecture to enhance lignocellulosic biomass.

BACKGROUND: Biofuels hold the promise to replace an appreciable proportion of fossil fuels. Not only do they emit significantly lower amounts of greenhouse gases, they are much closer to being 'carbon neutral', since the source plants utilize carbon dioxide for their growth. In particular, second-generation lignocellulosic biofuels from agricultural wastes and non-food crops such as switchgrass promise sustainability and avoid diverting food crops to fuel. Currently, available lignocellulosic biomass could yield sufficient bioethanol to replace ∼10 % of worldwide petroleum use. Increasing the biomass used for biofuel production and the yield of bioethanol will thus help meet global energy demands while significantly reducing greenhouse gas emissions. SCOPE: We discuss the advantages of various biotechnological approaches to improve crops and highlight the contribution of genomics and functional genomics in this field. Current knowledge concerning plant hormones and their intermediates involved in the regulation of plant architecture is presented with a special focus on gibberellins and cytokinins, and their signalling intermediates. We highlight the potential of information gained from model plants such as Arabidopsis thaliana and rice (Oryza sativa) to accelerate improvement of fuel crops.

Insights into the molecular mechanism of RGL2-mediated inhibition of seed germination in Arabidopsis thaliana.

BACKGROUND: Seed germination is of immense significance for agriculture and has been studied for centuries. Yet, our understanding of the molecular mechanisms underlying regulation of dormancy and germination is still in its infancy. Gibberellins are the key phytohormones that promote germination, and the DELLA protein RGL2 is the main signalling intermediate involved in this response. Germination is completely inhibited if functional RGL2 is overexpressed and/or stabilized; however, the molecular mechanisms of RGL2 function are still largely unknown. We therefore attempted to shed light onto some of the genetic events downstream of RGL2. RESULTS: Gene ontology of the transcriptome differentially regulated by RGL2, as well as extensive cross-comparison with other available microarray data indicates that RGL2-mediated inhibition of germination causes seeds to enter a state of dormancy. RGL2 also appears to differentially regulate a number of transcription factors, many of which are known to be involved in light- or phytohormone-mediated aspects of germination. A promoter analysis of differentially expressed genes identified an enrichment of several motifs that can be bound by specific transcription factors, for example GAMYB, ARF1, or Dof-type zinc fingers. We show that Dof-binding motifs indeed play a role in RGL2-mediated transcription. Using Chromatin Immunoprecipitation (ChIP), we show that RGL2 directly downregulates at least one cell wall modifying enzyme, which is predicted to constrain cell growth thereby leading to inhibition of seed germination. CONCLUSIONS: Our results reveal that RGL2 controls various aspects of germination. Through the repression of cell wall modifying enzymes, cell growth is directly constrained to inhibit germination. Furthermore, RGL2 likely interacts with various types of proteins to regulate transcription, and differentially regulates several transcription factors. Collectively, our data indicate that gibberellins, acting via RGL2, control several aspects of seed germination.

The phytohormone signal network regulating elongation growth during shade avoidance.

In contrast to animals, plants maintain highly plastic growth and development throughout their life, which enables them to adapt to environmental fluctuations. Phytohormones coordinately regulate these adaptations by integrating environmental inputs into a complex signalling network. In this review, the focus is on the rapid elongation that occurs in response to canopy shading or submergence, and current knowledge and recent advances in deciphering the network of phytohormone signalling that regulates this response are explored. The review concentrates on the involvement of the phytohormones auxins, gibberellins, cytokinins, and ethylene. Despite the occurrence of considerable gaps in current understanding of the underlying molecular mechanisms, it was possible to identify a network of phytohormone signalling intermediates at multiple levels that regulates elongation growth in response to canopy shade or submergence. Based on the observations that there are spatial and temporal differences in the interactions of phytohormones, the importance of more integrative approaches for future studies is highlighted.