An Automated Confocal Micro-Extensometer Enables in Vivo Quantification of Mechanical Properties with Cellular Resolution.

How complex developmental-genetic networks are translated into organs with specific 3D shapes remains an open question. This question is particularly challenging because the elaboration of specific shapes is in essence a question of mechanics. In plants, this means how the genetic circuitry affects the cell wall. The mechanical properties of the wall and their spatial variation are the key factors controlling morphogenesis in plants. However, these properties are difficult to measure and investigating their relation to genetic regulation is particularly challenging. To measure spatial variation of mechanical properties, one must determine the deformation of a tissue in response to a known force with cellular resolution. Here, we present an automated confocal micro-extensometer (ACME), which greatly expands the scope of existing methods for measuring mechanical properties. Unlike classical extensometers, ACME is mounted on a confocal microscope and uses confocal images to compute the deformation of the tissue directly from biological markers, thus providing 3D cellular scale information and improved accuracy. Additionally, ACME is suitable for measuring the mechanical responses in live tissue. As a proof of concept, we demonstrate that the plant hormone gibberellic acid induces a spatial gradient in mechanical properties along the length of the Arabidopsis thaliana hypocotyl.

Toward a 3D model of phyllotaxis based on a biochemically plausible auxin-transport mechanism.

Polar auxin transport lies at the core of many self-organizing phenomena sustaining continuous plant organogenesis. In angiosperms, the shoot apical meristem is a potentially unique system in which the two main modes of auxin-driven patterning-convergence and canalization-co-occur in a coordinated manner and in a fully three-dimensional geometry. In the epidermal layer, convergence points form, from which auxin is canalized towards inner tissue. Each of these two patterning processes has been extensively investigated separately, but the integration of both in the shoot apical meristem remains poorly understood. We present here a first attempt of a three-dimensional model of auxin-driven patterning during phyllotaxis. We base our simulations on a biochemically plausible mechanism of auxin transport proposed by Cieslak et al. (2015) which generates both convergence and canalization patterns. We are able to reproduce most of the dynamics of PIN1 polarization in the meristem, and we explore how the epidermal and inner cell layers act in concert during phyllotaxis. In addition, we discuss the mechanism by which initiating veins connect to the already existing vascular system.

Novel tools for quantifying secondary growth.

Secondary growth occurs in dicotyledons and gymnosperms, and results in an increased girth of plant organs. It is driven primarily by the vascular cambium, which produces thousands of cells throughout the life of several plant species. For instance, even in the small herbaceous model plant Arabidopsis, manual quantification of this massive process is impractical. Here, we provide a comprehensive overview of current methods used to measure radial growth. We discuss the issues and problematics related to its quantification. We highlight recent advances and tools developed for automated cellular phenotyping and its future applications.

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.

Cryptic variation between species and the basis of hybrid performance.

Crosses between closely related species give two contrasting results. One result is that species hybrids may be inferior to their parents, for example, being less fertile [1]. The other is that F1 hybrids may display superior performance (heterosis), for example with increased vigour [2]. Although various hypotheses have been proposed to account for these two aspects of hybridisation, their biological basis is still poorly understood [3]. To gain further insights into this issue, we analysed the role that variation in gene expression may play. We took a conserved trait, flower asymmetry in Antirrhinum, and determined the extent to which the underlying regulatory genes varied in expression among closely related species. We show that expression of both genes analysed, CYC and RAD, varies significantly between species because of cis-acting differences. By making a quantitative genotype-phenotype map, using a range of mutant alleles, we demonstrate that the species lie on a plateau in gene expression-morphology space, so that the variation has no detectable phenotypic effect. However, phenotypic differences can be revealed by shifting genotypes off the plateau through genetic crosses. Our results can be readily explained if genomes are free to evolve within an effectively neutral zone in gene expression space. The consequences of this drift will be negligible for individual loci, but when multiple loci across the genome are considered, we show that the variation may have significant effects on phenotype and fitness, causing a significant drift load. By considering these consequences for various gene-expression-fitness landscapes, we conclude that F1 hybrids might be expected to show increased performance with regard to conserved traits, such as basic physiology, but reduced performance with regard to others. Thus, our study provides a new way of explaining how various aspects of hybrid performance may arise through natural variation in gene activity.

Constraints of space in plant development.

Like all forms in nature, plants are subject to the properties of space. On the one hand, space prevents configurations that would place more than one component in the same location at the same time. A generalization of this constraint limits proximity and density of organs. On the other hand, space provides a means for a plant to create three-dimensional forms by differentially controlling their growth. This results from a connection between the metric properties of surfaces and their Gaussian curvature. Three strategies used by plants to develop within the constraints of space are presented: expansion to another dimension, egalitarian partitioning of space, and competition for space. These strategies are illustrated with examples of curved surfaces of leaves and petals, self-similar branching structures of compound leaves and inflorescences, and tree architecture. The examples highlight the fundamental role of the constraints of space in plant development, and the complementary role of genetic regulation and space-dependent emergent phenomena in shaping a plant.

Panicle Angle is an Important Factor in Tef Lodging Tolerance.

Tef [Eragrostis tef (Zucc.) Trotter] is an important crop in the Horn of Africa, particularly in Ethiopia, where it is a staple food for over 60 million people. However, the productivity of tef remains extremely low in part due to its susceptibility to lodging. Lodging is the displacement of the plant from the upright position, and it is exacerbated by rain, wind and the application of fertilizer. In order to address the issue of global food security, especially in the Horn of Africa, greater insight into the causes of tef lodging is needed. In this study, we combine modeling and biomechanical measurements to compare the properties relating to lodging tolerance in high yielding, improved tef genotypes, and lower yielding natural landraces. Our results indicate that the angle of the panicle contributes to the likelihood of lodging in tef. Varieties with compact panicles and reduced height had increased lodging resistance compared to the other varieties. By comparing different varieties, we found that overall, the landraces of tef lodged less than improved varieties. We constructed a model of stem bending and found that panicle angle was an important determinant of the amount of lodging. The findings from this study provide key information to those involved in tef improvement, especially those interested in lodging tolerance.

Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots.

Vascular cambium, a lateral plant meristem, is a central producer of woody biomass. Although a few transcription factors have been shown to regulate cambial activity1, the phenotypes of the corresponding loss-of-function mutants are relatively modest, highlighting our limited understanding of the underlying transcriptional regulation. Here, we use cambium cell-specific transcript profiling followed by a combination of transcription factor network and genetic analyses to identify 62 new transcription factor genotypes displaying an array of cambial phenotypes. This approach culminated in virtual loss of cambial activity when both WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNOTTED-like from Arabidopsis thaliana 1 (KNAT1; also known as BREVIPEDICELLUS) were mutated, thereby unlocking the genetic redundancy in the regulation of cambium development. We also identified transcription factors with dual functions in cambial cell proliferation and xylem differentiation, including WOX4, SHORT VEGETATIVE PHASE (SVP) and PETAL LOSS (PTL). Using the transcription factor network information, we combined overexpression of the cambial activator WOX4 and removal of the putative inhibitor PTL to engineer Arabidopsis for enhanced radial growth. This line also showed ectopic cambial activity, thus further highlighting the central roles of WOX4 and PTL in cambium development.

Patterns of Stem Cell Divisions Contribute to Plant Longevity.

The lifespan of plants ranges from a few weeks in annuals to thousands of years in trees. It is hard to explain such extreme longevity considering that DNA replication errors inevitably cause mutations. Without purging through meiotic recombination, the accumulation of somatic mutations will eventually result in mutational meltdown, a phenomenon known as Muller's ratchet. Nevertheless, the lifespan of trees is limited more often by incidental disease or structural damage than by genetic aging. The key determinants of tree architecture are the axillary meristems, which form in the axils of leaves and grow out to form branches. The number of branches is low in annual plants, but in perennial plants iterative branching can result in thousands of terminal branches. Here, we use stem cell ablation and quantitative cell-lineage analysis to show that axillary meristems are set aside early, analogous to the metazoan germline. While neighboring cells divide vigorously, axillary meristem precursors maintain a quiescent state, with only 7-9 cell divisions occurring between the apical and axillary meristem. During iterative branching, the number of branches increases exponentially, while the number of cell divisions increases linearly. Moreover, computational modeling shows that stem cell arrangement and positioning of axillary meristems distribute somatic mutations around the main shoot, preventing their fixation and maximizing genetic heterogeneity. These features slow down Muller's ratchet and thereby extend lifespan.

MorphoGraphX: A platform for quantifying morphogenesis in 4D.

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX ( www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The software's modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.

Genetic control of plant development by overriding a geometric division rule.

Formative cell divisions are critical for multicellular patterning. In the early plant embryo, such divisions follow from orienting the division plane. A major unanswered question is how division plane orientation is genetically controlled, and in particular whether this relates to cell geometry. We have generated a complete 4D map of early Arabidopsis embryogenesis and used computational analysis to demonstrate that several divisions follow a rule that uses the smallest wall area going through the center of the cell. In other cases, however, cell division clearly deviates from this rule, which invariably leads to asymmetric cell division. By analyzing mutant embryos and through targeted genetic perturbation, we show that response to the hormone auxin triggers a deviation from the "shortest wall" rule. Our work demonstrates that a simple default rule couples division orientation to cell geometry in the embryo and that genetic regulation can create patterns by overriding the default rule.

A bHLH complex controls embryonic vascular tissue establishment and indeterminate growth in Arabidopsis.

Plants have a remarkable potential for sustained (indeterminate) postembryonic growth. Following their specification in the early embryo, tissue-specific precursor cells first establish tissues and later maintain them postembryonically. The mechanisms underlying these processes are largely unknown. Here we define local control of oriented, periclinal cell division as the mechanism underlying both the establishment and maintenance of vascular tissue. We identify an auxin-regulated basic helix-loop-helix (bHLH) transcription factor dimer as a critical regulator of vascular development. Due to a loss of periclinal divisions, vascular tissue gradually disappears in bHLH-deficient mutants; conversely, ectopic expression is sufficient for triggering periclinal divisions. We show that this dimer operates independently of tissue identity but is restricted to a small vascular domain by integrating overlapping transcription patterns of the interacting bHLH proteins. Our work reveals a common mechanism for tissue establishment and indeterminate vascular development and provides a conceptual framework for developmental control of local cell divisions.

Generation of spatial patterns through cell polarity switching.

The mechanisms that generate dynamic spatial patterns within proliferating tissues are poorly understood, largely because of difficulties in unravelling interactions between cell specification, polarity, asymmetric division, rearrangements, and growth. We address this problem for stomatal spacing in plants, which offer the simplifying advantage that cells do not rearrange. By tracking lineages and gene activities over extended periods, we show that limited stem cell behavior of stomatal precursors depends on maintenance of the SPEECHLESS (SPCH) transcription factor in single daughter cells. Modeling shows how this property can lead to observed stereotypical stomata lineages through a postmitotic polarity-switching mechanism. The model predicts the location of a polarity determinant BASL over multiple divisions, which we validate experimentally. Our results highlight the dynamic two-way interactions between stem cells and their neighborhood during developmental patterning.