Samantha Fox

Background: Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like Arabidopsis thaliana, they have not yet been implemented in grasses. Results: Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C). Conclusions: This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.

Background: Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like Arabidopsis thaliana , they have not yet been implemented in grasses. Results: Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C). Conclusions: This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.

A developing plant organ exhibits complex spatiotemporal patterns of growth, cell division, cell size, cell shape, and organ shape. Explaining these patterns presents a challenge because of their dynamics and cross-correlations, which can make it difficult to disentangle causes from effects. To address these problems, we used live imaging to determine the spatiotemporal patterns of leaf growth and division in different genetic and tissue contexts. In the simplifying background of the speechless (spch) mutant, which lacks stomatal lineages, the epidermal cell layer exhibits defined patterns of division, cell size, cell shape, and growth along the proximodistal and mediolateral axes. The patterns and correlations are distinctive from those observed in the connected subepidermal layer and also different from the epidermal layer of wild type. Through computational modelling we show that the results can be accounted for by a dual control model in which spatiotemporal control operates on both growth and cell division, with cross-connections between them. The interactions between resulting growth and division patterns lead to a dynamic distributions of cell sizes and shapes within a deforming leaf. By modulating parameters of the model, we illustrate how phenotypes with correlated changes in cell size, cell number, and organ size may be generated. The model thus provides an integrated view of growth and division that can act as a framework for further experimental study. Organ morphogenesis involves two coordinated processes: growth of tissue and increase in cell number through cell division. Both processes have been analysed individually in many systems and shown to exhibit complex patterns in space and time. However, it is unclear how these patterns of growth and cell division are coordinated in a growing leaf that is undergoing shape changes. We have addressed this problem using live imaging to track growth and cell division in the developing leaf of the mustard plant Arabidopsis thaliana. Using subsequent computational modelling, we propose an integrated model of leaf growth and cell division, which generates dynamic distributions of cell size and shape in different tissue layers, closely matching those observed experimentally. A key aspect of the model is dual control of spatiotemporal patterns of growth and cell division parameters. By modulating parameters in the model, we illustrate how phenotypes may correlate with changes in cell size, cell number, and organ size.

Guard cells (GCs) are specialised cells within the plant epidermis which form stomatal pores, through which gas exchange can occur. The GCs derive through a specialised lineage of cell divisions which is specified by the transcription factor SPEECHLESS (SPCH), the expression of which can be detected in undifferentiated epidermal cells prior to asymmetric division. Other transcription factors may act before GC specification and be required for correct GC patterning. Previously, the DOF transcription factor STOMATAL CARPENTER 1 (SCAP1) was shown to be involved in GC function, by activating a set of GC–specific genes required for GC maturation and activity. It is thus far unknown whether SCAP1 can also affect stomatal development. Here we show that SCAP1 expression can also be observed in young leaf primordia, before any GC differentiation occurs. The study of transgenic plants carrying a proSCAP1:GUS-GFP transcriptional fusion, coupled with qPCR analyses, indicate that SCAP1 expression peaks in a temporal window which is coincident with expression of stomatal patterning genes. Independent scap1 loss-of-function mutants have a reduced number of GCs whilst SCAP1 over expression lines have an increased number of GCs, in addition to altered GC distribution and spacing patterns. The study of early markers for stomatal cell lineage in a background carrying gain–of–function alleles of SCAP1 revealed that, compared to the wild type, an increased number of protodermal cells are recruited in the GC lineage, which is reflected in an increased number of meristemoids. Our results suggest an early role for SCAP1 in GC differentiation. We propose that a function of SCAP1 is to integrate different aspects of GC biology including specification, spacing, maturation and function.

Summary: Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease‐mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

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.

Plant-cell expansion is controlled by cellulose microfibrils in the wall¹ with microtubules providing tracks for cellulose synthesizing enzymes². Microtubules can be reoriented experimentally^{3,4,5,6,7,8,9,10,11} and are hypothesized to reorient cyclically in aerial organs^12,13,14, but the mechanism is unclear. Here, Arabidopsis hypocotyl microtubules wershee labelled with AtEB1a–GFP (Arabidopsis microtubule end-binding protein 1a) or GFP–TUA6 (Arabidopsis α-tubulin 6) to record long cycles of reorientation. This revealed microtubules undergoing previously unseen clockwise or counter-clockwise rotations. Existing models emphasize selective shrinkage and regrowth¹⁵ or the outcome of individual microtubule encounters to explain realignment¹⁶. Our higher-order view emphasizes microtubule group behaviour over time. Successive microtubules move in the same direction along self-sustaining tracks. Significantly, the tracks themselves migrate, always in the direction of the individual fast-growing ends, but twentyfold slower. Spontaneous sorting of tracks into groups with common polarities generates a mosaic of domains. Domains slowly migrate around the cell in skewed paths, generating rotations whose progressive nature is interrupted when one domain is displaced by collision with another. Rotary movements could explain how the angle of cellulose microfibrils can change from layer to layer in the polylamellate cell wall.

A key challenge in evolutionary biology is to understand how new morphologies can arise through changes in gene regulatory networks. For example, floral asymmetry is thought to have evolved many times independently from a radially symmetrical ancestral condition, yet the molecular changes underlying this innovation are unknown. Here, we address this problem by investigating the action of a key regulator of floral asymmetry, CYCLOIDEA (CYC), in species with asymmetric and symmetric flowers. We show that CYC encodes a DNA-binding protein that recognises sites in a downstream target gene RADIALIS (RAD)in Antirrhinum. The interaction between CYC and RADcan be reconstituted in Arabidopsis, which has radially symmetrical flowers. Overexpression of CYC in Arabidopsis modifies petal and leaf development, through changes in cell proliferation and expansion at various stages of development. This indicates that developmental target processes are influenced by CYC in Arabidopsis, similar to the situation in Antirrhinum. However, endogenous RAD-like genes are not activated by CYC in Arabidopsis, suggesting that co-option of RAD may have occurred specifically in the Antirrhinum lineage. Taken together, our results indicate that floral asymmetry may have arisen through evolutionary tinkering with the strengths and pattern of connections at several points in a gene regulatory network.