Daniel Kierzkowski

Elastic Domains Regulate Growth and Organogenesis in the Plant Shoot Apical Meristem

Shape-Shifting Signals Although orthogonal signaling systems seem to direct various developmental processes, few tissues remain in the same shape as they are at initiation to that of the final form. Arabidopsis leaves are free of the cell migrations that complicate animal development, and thus allowed Kuchen et al. (p. 1092) to track and model the trajectory of leaf growth under a variety of perturbations. Varying the values of parameters in their model produced outputs of different leaf shapes ranging from obcordate, ovate, and oval to elliptic, and offered predictions for genes that regulate the developmental process. The meristem at the growing tip of plants is home to stem cells and is the source of newly differentiating shoots and leaves. New leaves make their first appearance as bulges at the side of the dome-shaped meristem. Although these developmental events are under hormonal control, they also seem to be constrained by the physical properties of the meristem. Kierzkowski et al. (p. 1096) tested physical effects acting on the shoot apical meristem of growing tomato shoots that alter turgor pressure. Again, mathematical modeling combined with observations of plant tissue helped to define the different zones in the meristem that respond to diverse mechanical stimuli. New leaves emerge where they are allowed. Although genetic control of morphogenesis is well established, elaboration of complex shapes requires changes in the mechanical properties of cells. In plants, the first visible sign of leaf formation is a bulge on the flank of the shoot apical meristem. Bulging results from local relaxation of cell walls, which causes them to yield to internal hydrostatic pressure. By manipulation of tissue tension in combination with quantitative live imaging and finite-element modeling, we found that the slow-growing area at the shoot tip is substantially strain-stiffened compared with surrounding fast-growing tissue. We propose that strain stiffening limits growth, restricts organ bulging, and contributes to the meristems functional zonation. Thus, mechanical signals are not just passive readouts of gene action but feed back on morphogenesis.

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 softwares 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. DOI: http://dx.doi.org/10.7554/eLife.05864.001

Leaf shape evolution through duplication, regulatory diversification, and loss of a homeobox gene.

The evolutionary trajectory leading to crucifer leaf shape in Cardamine hirsuta plants is elucidated. In this work, we investigate morphological differences between Arabidopsis thaliana, which has simple leaves, and its relative Cardamine hirsuta, which has dissected leaves comprising distinct leaflets. With the use of genetics, interspecific gene transfers, and time-lapse imaging, we show that leaflet development requires the REDUCED COMPLEXITY (RCO) homeodomain protein. RCO functions specifically in leaves, where it sculpts developing leaflets by repressing growth at their flanks. RCO evolved in the Brassicaceae family through gene duplication and was lost in A. thaliana, contributing to leaf simplification in this species. Species-specific RCO action with respect to its paralog results from its distinct gene expression pattern in the leaf base. Thus, regulatory evolution coupled with gene duplication and loss generated leaf shape diversity by modifying local growth patterns during organogenesis. Developmental Complexity Although related, the plants Arabidopsis thaliana and Cardamine hirsuta have different sorts of leaves—one, a rather plain oval and the other, a complicated multipart construction. Comparing the development of the two leaf types, Vlad et al. (p. 780) uncovered a gene that regulates developmental growth. The C. hirsuta gene encoding the REDUCED COMPLEXITY (RCO) homeodomain protein arose through gene duplication and neofunctionalization, but was lost in the A. thaliana lineage. In C. hirsuta, RCO suppresses growth in domains around the perimeter of the developing leaf, yielding complex-shaped leaves. A. thaliana, lacking RCO, produces simple leaves. When RCO was expressed in A. thaliana, the leaves became more complex. Thus, the capacity to produce complex leaves remains, despite loss of the initiator.

PIN1-Independent Leaf Initiation in Arabidopsis

Phyllotaxis, the regular arrangement of leaves and flowers around the stem, is a key feature of plant architecture. Current models propose that the spatiotemporal regulation of organ initiation is controlled by a positive feedback loop between the plant hormone auxin and its efflux carrier PIN-FORMED1 (PIN1). Consequently, pin1 mutants give rise to naked inflorescence stalks with few or no flowers, indicating that PIN1 plays a crucial role in organ initiation. However, pin1 mutants do produce leaves. In order to understand the regulatory mechanisms controlling leaf initiation in Arabidopsis (Arabidopsis thaliana) rosettes, we have characterized the vegetative pin1 phenotype in detail. We show that although the timing of leaf initiation in vegetative pin1 mutants is variable and divergence angles clearly deviate from the canonical 137° value, leaves are not positioned at random during early developmental stages. Our data further indicate that other PIN proteins are unlikely to explain the persistence of leaf initiation and positioning during pin1 vegetative development. Thus, phyllotaxis appears to be more complex than suggested by current mechanistic models.

The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana

How alternative splicing (AS) is regulated in plants has not yet been elucidated. Previously, we have shown that the nuclear cap-binding protein complex (AtCBC) is involved in AS in Arabidopsis thaliana. Here we show that both subunits of AtCBC (AtCBP20 and AtCBP80) interact with SERRATE (AtSE), a protein involved in the microRNA biogenesis pathway. Moreover, using a high-resolution reverse transcriptase-polymerase chain reaction AS system we have found that AtSE influences AS in a similar way to the cap-binding complex (CBC), preferentially affecting selection of 5′ splice site of first introns. The AtSE protein acts in cooperation with AtCBC: many changes observed in the mutant lacking the correct SERRATE activity were common to those observed in the cbp mutants. Interestingly, significant changes in AS of some genes were also observed in other mutants of plant microRNA biogenesis pathway, hyl1-2 and dcl1-7, but a majority of them did not correspond to the changes observed in the se-1 mutant. Thus, the role of SERRATE in AS regulation is distinct from that of HYL1 and DCL1, and is similar to the regulation of AS in which CBC is involved.

Interaction between Meristem Tissue Layers Controls Phyllotaxis

Phyllotaxis and vein formation are among the most conspicuous patterning processes in plants. The expression and polarization of the auxin efflux carrier PIN1 is the earliest marker for both processes, with mathematical models indicating that PIN1 can respond to auxin gradients and/or auxin flux. Here, we use cell-layer-specific PIN1 knockouts and partial complementation of auxin transport mutants to examine the interaction between phyllotactic patterning, which occurs primarily in the L1 surface layer of the meristem, and midvein specification in the inner tissues. We show that PIN1 expression in the L1 is sufficient for correct organ positioning, as long as the L1-specific influx carriers are present. Thus, differentiation of inner tissues can proceed without PIN1 or any of the known polar transporters. On theoretical grounds, we suggest that canalization of auxin flux between an auxin source and an auxin sink may involve facilitated diffusion rather than polar transport.

Clones of cells switch from reduction to enhancement of size variability in Arabidopsis sepals

Organs form with remarkably consistent sizes and shapes during development, whereas a high variability in growth is observed at the cell level. Given this contrast, it is unclear how such consistency in organ scale can emerge from cellular behavior. Here, we examine an intermediate scale, the growth of clones of cells in Arabidopsis sepals. Each clone consists of the progeny of a single progenitor cell. At early stages, we find that clones derived from a small progenitor cell grow faster than those derived from a large progenitor cell. This results in a reduction in clone size variability, a phenomenon we refer to as size uniformization. By contrast, at later stages of clone growth, clones change their growth pattern to enhance size variability, when clones derived from larger progenitor cells grow faster than those derived from smaller progenitor cells. Finally, we find that, at early stages, fast growing clones exhibit greater cell growth heterogeneity. Thus, cellular variability in growth might contribute to a decrease in the variability of clones throughout the sepal. Summary: Growth analyses of Arabidopsis sepals identify a tipping point in organ development, at which clones of cells change their growth pattern from size uniformization to size variability enhancement.

Coping with stress: mechanics of the expanding leaf

The precise control of physical properties of growing tissues is crucial for plant morphogenesis. Sahaf and Sharon (pages 5509–5515 in this issue) examined the mechanics of the expanding leaf and showed that plant tissues respond to stress by changing their mechanical properties. A new method is proposed to distinguish reversible and irreversible tissue deformation, an important step in understanding the physics of a growing cell wall. Leaf blades could hold the key to understanding how plants regulate their growth in different directions.

Cellular basis of growth in plants: geometry matters

The growth of individual cells underlies the development of biological forms. In plants, cells are interconnected by rigid walls, fixing their position with respect to one another and generating mechanical feedbacks between cells. Current research is shedding new light on how plant growth is controlled by physical inputs at the level of individual cells and growing tissues. In this review, we discuss recent progress in our understanding of the cellular basis of growth from a biomechanical perspective. We describe the role of the cell wall and turgor pressure in growth and highlight the often-overlooked role of cell geometry in this process. It is becoming apparent that a combination of experimental and theoretical approaches is required to answer new emerging questions in the biomechanics of plant morphogenesis. We summarise how this multidisciplinary approach brings us closer to a unified understanding of the generation of biological forms in plants.

The role of APETALA1 in petal number robustness

Invariant floral forms are important for reproductive success and robust to natural perturbations. Petal number, for example, is invariant in Arabidopsis thaliana flowers. However, petal number varies in the closely related species Cardamine hirsuta, and the genetic basis for this difference between species is unknown. Here we show that divergence in the pleiotropic floral regulator APETALA1 (AP1) can account for the species-specific difference in petal number robustness. This large effect of AP1 is explained by epistatic interactions: A. thaliana AP1 confers robustness by masking the phenotypic expression of quantitative trait loci controlling petal number in C. hirsuta. We show that C. hirsuta AP1 fails to complement this function of A. thaliana AP1, conferring variable petal number, and that upstream regulatory regions of AP1 contribute to this divergence. Moreover, variable petal number is maintained in C. hirsuta despite sufficient standing genetic variation in natural accessions to produce plants with four-petalled flowers.