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SummaryExploding seed pods of the common weedCardamine hirsutahave the remarkable ability to launch seeds far from the plant. The energy for this explosion comes from tension that builds up in the fruit valves. Above a critical threshold, the fruit fractures along its dehiscence zone and the two valves coil explosively, ejecting the seeds. Tension is commonly generated as seed pods dry, causing fruit tissues to deform. However, this does not happen inC. hirsuta.Instead, tension is produced by active contraction of growing exocarp cells in the outer layer of the fruit valves. Exactly how growth leads to contraction in these cells is unknown. Here we show that microtubule dynamics in the exocarp cell cortex control the specific orientation of cellulose microfibrils in the cell wall, and the consequent cellular growth pattern, which together drive contraction. We used mechanical modeling and simulations to show how tension emerges through the general process of plant cell growth, due to the highly anisotropic orientation of load-bearing cellulose microfibrils and their effect on cell shape. By explicitly defining the cell wall as multi-layered in our model, we discovered that a cross-lamellate pattern of cellulose microfibrils further enhances the developing tension in growing cells. Therefore, the interplay of cell wall properties with turgor-driven growth enables the fruit exocarp layer to develop sufficient tension to explode.

Exploding seed pods evolved in the Arabidopsis relative, Cardamine hirsuta, via morphomechanical innovations that allow the storage and rapid release of elastic energy. Asymmetric lignin deposition within endocarpb cell walls is one such innovation that is required for explosive seed dispersal and evolved in association with the trait. However, the genetic control of this novel lignin pattern is unknown. Here, we identify three lignin-polymerizing laccases, LAC4, 11 and 17, that precisely co-localize with, and are redundantly required for, asymmetric lignification of endocarpb cells. By screening for C. hirsuta mutants with less lignified fruit valves, we found that loss of function of the transcription factor gene SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 7 (SPL7) caused a reduction in endocarpb cell wall lignification and a consequent reduction in seed dispersal range. SPL7 is a conserved regulator of copper homeostasis and is both necessary and sufficient for copper to accumulate in the fruit. Laccases are copper-requiring enzymes. We discovered that laccase activity in endocarpb cell walls depends on the SPL7 pathway to acclimate to copper deficiency and provide sufficient copper for lignin polymerization. Hence, SPL7 links mineral nutrition to efficient dispersal of the next generation.

Quantitative live-cell imaging is an exciting approach to study floral organs. In this review, we highlight recent advances using this approach to understand floral organ initiation and cell fate acquisition., The chance to watch floral organs develop live is not to be missed! Here, we outline reasons why quantitative, live-cell imaging is an important approach to study floral morphogenesis, and provide a basic workflow of how to get started. We highlight key advances in morphodynamics of lateral organ development, and discuss recent work that uses live confocal imaging to address the regulation of floral organ number, its robustness, and patterning mechanisms that exploit stochasticity.

Different patterns of lignified cell walls are associated with diverse functions in a variety of plant tissues. These functions rely on the stiffness and hydrophobicity that lignin polymers impart to the cell wall. The precise pattern of subcellular lignin deposition is critical for the structure–function relationship in each lignified cell type. Here, we describe the role of xylem vessels as water pipes, Casparian strips as apoplastic barriers, and the role of asymmetrically lignified endocarp b cells in exploding seed pods. We highlight similarities and differences in the genetic mechanisms underpinning local lignin deposition in these diverse cell types. By bringing together examples from different developmental contexts and different plant species, we propose that comparative approaches can benefit our understanding of lignin patterning mechanisms.

Background and AimsSeeds are dispersed by explosive coiling of the fruit valves in Cardamine hirsuta. This rapid coiling launches the small seeds on ballistic trajectories to spread over a 2 m radius around the parent plant. The seed surface interacts with both the coiling fruit valve during launch and subsequently with the air during flight. We aim to identify features of the seed surface that may contribute to these interactions by characterizing seed coat differentiation.MethodsDifferentiation of the outermost seed coat layers from the outer integuments of the ovule involves dramatic cellular changes that we characterize in detail at the light and electron microscopical level including immunofluorescence and immunogold labelling.Key ResultsWe found that the two outer integument (oi) layers of the seed coat contributed differently to the topography of the seed surface in the explosively dispersed seeds of C. hirsuta vs. the related species Arabidopsis thaliana where seed dispersal is non-explosive. The surface of A. thaliana seeds is shaped by the columella and the anticlinal cell walls of the epidermal oi2 layer. In contrast, the surface of C. hirsuta seeds is shaped by a network of prominent ridges formed by the anticlinal walls of asymmetrically thickened cells of the sub-epidermal oi1 layer, especially at the seed margin. Both the oi2 and oi1 cell layers in C. hirsuta seeds are characterized by specialized, pectin-rich cell walls that are deposited asymmetrically in the cell.ConclusionsThe two outermost seed coat layers in C. hirsuta have distinct properties: the sub-epidermal oi1 layer determines the topography of the seed surface, while the epidermal oi2 layer accumulates mucilage. These properties are influenced by polar deposition of distinct pectin polysaccharides in the cell wall. Although the ridged seed surface formed by oi1 cell walls is associated with ballistic dispersal in C. hirsuta, it is not restricted to explosively dispersed seeds in the Brassicaceae.

How traits evolve to produce novelty or stasis is an open question in biology. We investigate this question in Cardamine hirsuta, a relative of Arabidopsis thaliana that employs explosive fracture to disperse its seeds. This trait evolved through key morphomechanical innovations that distinguish the otherwise very similar, dehiscent fruit of these two species. Using CRISPR/Cas9, we show that dehiscence zone formation is absolutely required for explosive fracture in C. hirsuta, and is controlled by the bHLH transcription factor INDEHISCENT (IND). Using mutant screens, we identified the MADS-box transcription factor FRUITFULL (FUL) as a negative regulator of IND in C. hirsuta. Although FUL function is conserved in C. hirsuta, the consequences of IND mis-expression differ in ful mutants of C. hirsuta versus A. thaliana. In ful mutants of both species, valve tissue is replaced by dehiscence zone tissue, which comprises two distinct cell types: lignified layer and separation layer cells. While A. thaliana ful mutants develop stunted fruit with ectopic lignified layer cells, C. hirsuta ful mutants have elongated fruit with ectopic separation layer cells. We show that IND dose determines the proportion of these two cell types in ectopic dehiscence zones. We also show that the extent of ectopic lignification caused by IND mis-expression determines fruit length. Our findings indicate developmental system drift in the conserved gene network patterning dehiscent fruit in two related Brassicaceae species.

Summary Polyploidization is pervasive in plants, but little is known about the niche divergence of wild allopolyploids (species that harbor polyploid genomes originating from different diploid species) relative to their diploid progenitor species and the gene expression patterns that may underlie such ecological divergence. We conducted a fine‐scale empirical study on habitat and gene expression of an allopolyploid and its diploid progenitors.We quantified soil properties and light availability of habitats of an allotetraploid Cardamine flexuosa and its diploid progenitors Cardamine amara and Cardamine hirsuta in two seasons. We analyzed expression patterns of genes and homeologs (homeologous gene copies in allopolyploids) using RNA sequencing.We detected niche divergence between the allopolyploid and its diploid progenitors along water availability gradient at a fine scale: the diploids in opposite extremes and the allopolyploid in a broader range between diploids, with limited overlap with diploids at both ends. Most of the genes whose homeolog expression ratio changed among habitats in C. flexuosa varied spatially and temporally.These findings provide empirical evidence for niche divergence between an allopolyploid and its diploid progenitor species at a fine scale and suggest that divergent expression patterns of homeologs in an allopolyploid may underlie its persistence in diverse habitats.

Contemporary speciation provides a unique opportunity to directly observe the traits and environmental responses of a new species. Cardamine insueta is an allotriploid species that appeared within the past 150 years in a Swiss village, Urnerboden. In contrast to its two progenitor species, C. amara and C. rivularis that live in wet and open habitats, respectively, C. insueta is found in-between their habitats with temporal water level fluctuation. This triploid species propagates clonally and serves as a triploid bridge to form higher ploidy species. Although niche separation is observed in field studies, the mechanisms underlying the environmental robustness of C. insueta are not clear. To characterize responses to a fluctuating environment, we performed a time-course analysis of homeolog gene expression in C. insueta in response to submergence treatment. For this purpose, the two parental (C. amara and C. rivularis) genome sequences were assembled with a reference-guided approach, and homeolog-specific gene expression was quantified by using HomeoRoq software. We found that C. insueta and C. rivularis initiated vegetative propagation by forming ectopic meristems on leaves, while C. amara did not. We examined homeolog-specific gene expression of three species at nine time points during the treatment. The genome-wide expression ratio of homeolog pairs was 2:1 over the time-course, consistent with the ploidy number. By searching the genes with high coefficient of variation of expression over time-course transcriptome data, we found many known key transcriptional factors related to meristem development and formation upregulated in both C. rivularis and rivularis-homeolog of C. insueta, but not in C. amara. Moreover, some amara-homeologs of these genes were also upregulated in the triploid, suggesting trans-regulation. In turn, Gene Ontology analysis suggested that the expression pattern of submergence tolerant genes in the triploid was inherited from C. amara. These results suggest that the triploid C. insueta combined advantageous patterns of parental transcriptomes to contribute to its establishment in a new niche along a water-usage gradient.

How the interplay between cell- and tissue-level processes produces correctly proportioned organs is a key problem in biology. In plants, the relative size of leaves compared with their lateral appendages, called stipules, varies tremendously throughout development and evolution, yet relevant mechanisms remain unknown. Here we use genetics, live imaging, and modeling to show that in Arabidopsis leaves, the LATE MERISTEM IDENTITY1 (LMI1) homeodomain protein regulates stipule proportions via an endoreduplication-dependent trade-off that limits tissue size despite increasing cell growth. LM1 acts through directly activating the conserved mitosis blocker WEE1, which is sufficient to bypass the LMI1 requirement for leaf proportionality.

Objective: The aim of the study was to explore the range of psychiatric diagnoses seen in pregnant women who score above the ‘cut-off’ on the Edinburgh Postnatal Depression Scale when this is used as a routine screening instrument in the antenatal period. Method: Subjects were all pregnant women referred to and seen by the Perinatal Consultation-Liaison Psychiatry Team of a tertiary public hospital over a 14-month period. Edinburgh Postnatal Depression Scale score at maternity ‘booking-in’ visit, demographic and clinical data were recorded and diagnoses were made according to Diagnostic and Statistical Manual of Mental Disorders (5th ed.) criteria following clinical interview(s) and review of documented past history. Data were analysed using descriptive statistics. Results: A total of 200 patients who had completed the Edinburgh Postnatal Depression Scale were seen for assessment; 86 (43%) scored ⩾13 on Edinburgh Postnatal Depression Scale. Of those scoring 13 or more on Edinburgh Postnatal Depression Scale, 22 (25.6%) had a depressive disorder. In total, 12 patients (14%) had an anxiety disorder, 14 (16.3%) had borderline personality disorder and 13 (15.1%) had a substance use disorder. An additional 23 women (26.7%) had two or more borderline personality traits. Conclusion: Psychiatric assessment of women who scored 13 or more on the Edinburgh Postnatal Depression Scale at routine antenatal screening identified a significant number with borderline personality disorder or borderline personality traits rather than depressive or anxiety disorders. Clinical Practice Guidelines note the importance of further assessment for all women who score 13 or more on the Edinburgh Postnatal Depression Scale. The findings here suggest that this assessment should be made by a clinician able to identify personality pathology and organise appropriate and timely interventions.

Here, Vuolo et al. investigated the mechanisms controlling the relative size of leaves compared with their lateral appendages (stipules). Using genetics, live imaging, and modeling, they demonstrate that the LATE MERISTEM IDENTITY1 (LMI1) homeodomain protein regulates stipule proportions via an endoreduplication-dependent trade-off that limits tissue size despite increasing cell growth.

International audience; 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.

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., eLife digest Many plants produce flowers that attract insects to land on them. Different insects are attracted to flowers of different shapes and colors. Therefore, it is generally advantageous for plants of the same species to produce flowers that look very similar. For example, a small weed known as Arabidopsis – which is often used in research studies – produces little white flowers that all have four petals. Thus, the number of petals in Arabidopsis flowers is said to be a ‘robust’ trait. However, a closely-related plant called hairy bittercress produces flowers with any number of petals between zero and four. Studying the genetic differences between Arabidopsis and hairy bittercress can help to reveal why the numbers of petals on hairy bittercress flowers vary. A gene called APETALA1 helps to control how petals form. Monniaux, Pieper et al. found that Arabidopsis and hairy bittercress have different versions of this gene that determine whether the number of petals may vary between individual flowers. Inserting the Arabidopsis version of APETALA1 into hairy bittercress plants caused the plants to produce flowers that had more similar numbers of petals to each other, that is, the petal number became more robust. Monniaux, Pieper et al. then used a statistical method called quantitative trait locus analysis to identify the precise location of regions in the hairy bittercress genome that control petal number. This showed that the Arabidopsis version of APETALA1, but not the hairy bittercress version, conceals the action of these genes that could alter petal number. These findings reveal that evolutionary change in a single gene of hairy bittercress unmasked the action of other genes that caused petal number to vary. A next step will be to identify some of these genes and understand how they control petal number.

There is an increasing appreciation for the role of physical forces in plant development. Mechanics are fundamental to how explosive fruit eject their seeds, and recent studies have successfully combined mechanics with developmental genetics to help explain how these dispersal traits are produced and how they evolved. Computational modeling is used more and more to address developmental questions, and explosive fruit are particularly good systems for combining biology and modeling approaches. Finite element models have been recently used to explore questions such as: Why do touch-me-not species with similar fruits, differ so much in how efficiently they transfer stored energy to eject seeds? And how do popping cress fruits use the expansive force of turgor pressure for tissue contraction?

The shape and function of plant cells are often highly interdependent. The puzzle-shaped cells that appear in the epidermis of many plants are a striking example of a complex cell shape, however their functional benefit has remained elusive. We propose that these intricate forms provide an effective strategy to reduce mechanical stress in the cell wall of the epidermis. When tissue-level growth is isotropic, we hypothesize that lobes emerge at the cellular level to prevent formation of large isodiametric cells that would bulge under the stress produced by turgor pressure. Data from various plant organs and species support the relationship between lobes and growth isotropy, which we test with mutants where growth direction is perturbed. Using simulation models we show that a mechanism actively regulating cellular stress plausibly reproduces the development of epidermal cell shape. Together, our results suggest that mechanical stress is a key driver of cell-shape morphogenesis., eLife digest Cells with complex interlocking shapes, similar to pieces of a jigsaw puzzle, cover the surface of many leaves. Why do these curious shapes form, and what benefit do they provide to the plant? Plant cells are like small balloons surrounded by a strong cell wall. Their internal pressure can be higher than the pressure in a car tire. It is this pressure that gives non-woody plant tissue its shape. Take away the pressure, and the plant wilts. The pressure inside a cell creates a lot of mechanical stress on the epidermal cell walls – those that make up the surface of the plant. The extent of the stress depends on the shape and size of the cells; for example, large cells bulge out and experience more stress than small cells. This could mean that the shape of puzzle cells is an adaptation used by plants to reduce the stress on their surface. To investigate this possibility, Sapala, Runions et al. developed a computer simulation that models how a plant grows and re-creates a variety of realistic puzzle cell shapes. The simulations show that ‘paving’ the leaf surface with puzzle shaped cells instead of more regularly shaped cells reduces the stress in the epidermal cell walls. Counterintuitively, the simulations also show that complex puzzle shapes develop in parts of the plant that grow isotropically (uniformly in all directions), such as leaves. If a plant organ grows mostly in one direction, like in a root or stem, long thin cells are sufficient to reduce the stress on the epidermal cell wall. Sapala, Runions et al. tested this idea by analyzing the shape of organs and cells in many plant species and by genetically modifying growth directions in Arabidopsis thaliana plants. This confirmed that puzzle cell shape is related to both organ shape and how isotropically the plant grows. It had previously been proposed that mobile chemical signals passed between cells coordinate the process by which a lobe in one puzzle cell matches an indentation in its neighbor. However, the model developed by Sapala, Runions et al. does not require such chemical signaling. Instead, mechanical forces and the shape the puzzle cells themselves may transmit this information. Mechanical forces are known to have important effects on the shape and behavior of cells from other species too. For example, animal cells can develop into different cell types depending on the stiffness of the surface they are placed on. Now that Sapala, Runions et al. have highlighted that plant cell shapes also adapt to mechanical forces, further research is needed to uncover how these forces are sensed.

International audience; Four petals characterize the flowers of most species in the Brassicaceae family, and this phenotype is generally robust to genetic and environmental variation. A variable petal number distinguishes the flowers of Cardamine hirsuta from those of its close relative Arabidopsis (Arabidopsis thaliana), and allelic variation at many loci contribute to this trait. However, it is less clear whether C. hirsuta petal number varies in response to seasonal changes in environment. To address this question, we assessed whether petal number responds to a suite of environmental and endogenous cues that regulate flowering time in C. hirsuta. We found that petal number showed seasonal variation in C. hirsuta, such that spring flowering plants developed more petals than those flowering in summer. Conditions associated with spring flowering, including cool ambient temperature, short photoperiod, and vernalization, all increased petal number in C. hirsuta. Cool temperature caused the strongest increase in petal number and lengthened the time interval over which floral meristems matured. We performed live imaging of early flower development and showed that floral buds developed more slowly at 15°C versus 20°C. This extended phase of floral meristem formation, coupled with slower growth of sepals at 15°C, produced larger intersepal regions with more space available for petal initiation. In summary, the growth and maturation of floral buds is associated with variable petal number in C. hirsuta and responds to seasonal changes in ambient temperature.

Maize leaves have distinct tissues that serve specific purposes. The blade tilts back to photosynthesize and the sheath wraps around the stem to provide structural support and protect young leaves. At the junction between blade and sheath are the ligule and auricles, both of which are absent in the recessive liguleless1 (lg1) mutant. Using an antibody against LG1, we reveal LG1 accumulation at the site of ligule formation and in the axil of developing tassel branches. The dominant mutant Wavy auricle in blade1 (Wab1-R) produces ectopic auricle tissue in the blade and increases the domain of LG1 accumulation. We determined that wab1 encodes a TCP transcription factor by positional cloning and revertant analysis. Tassel branches are few and upright in the wab1 revertant tassel and have an increased branch angle in the dominant mutant. wab1 mRNA is expressed at the base of branches in the inflorescence and is necessary for LG1 expression. wab1 is not expressed in leaves, except in the dominant mutant. The domain of wab1 expression in the Wab1-R leaf closely mirrors the accumulation of LG1. Although wab1 is not needed to induce lg1 expression in the leaf, LG1 is needed to counteract the severe phenotype of the dominant Wab1-R mutant. The regulatory interaction of LG1 and WAB1 reveals a link between leaf shape and tassel architecture, and suggests the ligule is a boundary similar to that at the base of lateral organs.

International audience; A key question in biology is how the endless diversity of forms found in nature evolved. Understanding the cellular basis of this diversity has been aided by advances in non-model experimental systems, quantitative image analysis tools, and modeling approaches. Recent work in plants highlights the importance of cell wall and cuticle modifications for the emergence of diverse forms and functions. For example, explosive seed dispersal in Cardamine hirsuta depends on the asymmetric localization of lignified cell wall thickenings in the fruit valve. Similarly, the iridescence of Hibiscus trionum petals relies on regular striations formed by cuticular folds. Moreover, NAC transcription factors regulate the differentiation of lignified xylem vessels but also the water-conducting cells of moss that lack a lignified secondary cell wall, pointing to the origin of vascular systems. Other novel forms are associated with modified cell growth patterns, including oriented cell expansion or division, found in the long petal spurs of Aquilegia flowers, and the Sarracenia purpurea pitcher leaf, respectively. Another good example is the regulation of dissected leaf shape in C. hirsuta via local growth repression, controlled by the REDUCED COMPLEXITY HD-ZIP class I transcription factor. These studies in non-model species often reveal as much about fundamental processes of development as they do about the evolution of form. Address

The maize leaf consists of four distinct tissues along its proximodistal axis: sheath, ligule, auricle and blade. liguleless1 (lg1)functions cell autonomously to specify ligule and auricle, and may propagate a signal that correctly positions the blade-sheath boundary. The dominant Wavy auricle in blade (Wab1) mutation disrupts both the mediolateral and proximodistal axes of the maize leaf. Wab1 leaf blades are narrow and ectopic auricle and sheath extend into the blade. The recessive lg1-R mutation exacerbates the Wab1 phenotype; in the double mutants, most of the proximal blade is deleted and sheath tissue extends along the residual blade. We show that lg1 is misexpressed in Wab1 leaves. Our results suggest that the Wab1 defect is partially compensated for by lg1 expression. A mosaic analysis of Wab1 was conducted in Lg1+ and lg1-R backgrounds to determine if Wab1 affects leaf development in a cell-autonomous manner. Normal tissue identity was restored in all wab1+/–sectors in a lg1-R mutant background, and in three quarters of sectors in a Lg1+ background. These results suggest that lg1can influence the autonomy of Wab1. In both genotypes, leaf-halves with wab1+/– sectors were significantly wider than non-sectored leaf-halves, suggesting that Wab1 acts cell-autonomously to affect lateral growth. The mosaic analysis, lg1 expression data and comparison of mutant leaf shapes reveal previously unreported functions of lg1 in both normal leaf development and in the dominant Wab1mutant.

Plant hormones are signalling molecules that control growth and development. Growth of the aerial parts of higher plants requires the continuous activity of the shoot apical meristem, a small mound of cells at the apex of a plant. KNOTTED1-like HOMEOBOX (KNOX) genes are involved in regulating meristem activity, however, little is known about how this regulation is mediated. Recent evidence suggests that KNOX transcription factors may control meristem development by regulating the balance of activities of multiple hormones.

Expression of KNOX(KNOTTED1-like homeobox) genes in the shoot apical meristem of Arabidopsis is required for maintenance of a functional meristem, whereas exclusion of KNOX gene expression from leaf primordia is required for the elaboration of normal leaf morphology. We have constructed a steroid-inducible system to regulate both the amount and timing of KN1(KNOTTED1) misexpression in Arabidopsis leaves. We demonstrate that lobed leaf morphology is produced in a dose-dependent manner, indicating that the amount of KN1 quantitatively affects the severity of lobing. The KN1-glucocorticoid receptor fusion protein is not detected in leaves in the absence of steroid induction, suggesting that it is only stable when associated with steroid in an active state. By using a second inducible fusion protein to mark exposure of leaf primordia to the steroid, we determined the stage of leaf development that produces lobed leaves in response to KN1. Primordia as old as plastochron 7 and as young as plastochron 2 were competent to respond to KN1.

Leaves of flowering plants are determinate organs produced by pluripotent structures termed shoot apical meristems. Once specified, leaves differentiate an adaxial (upper) side specialized for light capture, and an abaxial (lower) side specialized for gas exchange. A functional relationship between meristem activity and the differentiation of adaxial leaf fate has been recognized for over fifty years, but the molecular basis of this interaction is unclear. In Arabidopsis thaliana, activity of the class I KNOX (KNOTTED1-like homeobox) genes SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) is required for meristem function but excluded from leaves, whereas members of the HD-Zip III (class III homeodomain leucine zipper) protein family function to promote both meristem activity and adaxial leaf fate. Here we show that the zinc-finger protein SERRATE acts in a microRNA (miRNA) gene-silencing pathway to regulate expression of the HD-Zip III gene PHABULOSA (PHB) while also limiting the competence of shoot tissue to respond to KNOX expression. Thus, SERRATE acts to coordinately regulate meristem activity and leaf axial patterning.

Knotted1 defined the first homeobox gene family to be isolated in plants and was identified from dominant maize mutants that produced 'knots' of mis-specified tissue on the leaf. The Knotted1-like homeobox (KNOX) gene family expanded last year to include members lacking the defining homeobox with exciting implications for KNOX gene regulation and function. Recent evidence for direct KNOX regulation by myb-related ARP proteins and epigenetic silencing by polycomb repressive complexes have also shed light on the mechanisms defining KNOX gene expression.

Summary How mechanical and biological processes are coordinated across cells, tissues, and organs to produce complex traits is a key question in biology. Cardamine hirsuta, a relative of Arabidopsis thaliana, uses an explosive mechanism to disperse its seeds. We show that this trait evolved through morphomechanical innovations at different spatial scales. At the organ scale, tension within the fruit wall generates the elastic energy required for explosion. This tension is produced by differential contraction of fruit wall tissues through an active mechanism involving turgor pressure, cell geometry, and wall properties of the epidermis. Explosive release of this tension is controlled at the cellular scale by asymmetric lignin deposition within endocarp b cells—a striking pattern that is strictly associated with explosive pod shatter across the Brassicaceae plant family. By bridging these different scales, we present an integrated mechanism for explosive seed dispersal that links evolutionary novelty with complex trait innovation. Video Abstract, Graphical Abstract, Highlights • Fruits explode while turgid, not dry, due to active contraction of epidermal cells • Asymmetric deposition of lignin in endocarp b cells drives explosive energy release • Endocarp b asymmetry is an evolutionary novelty underlying explosive dispersal • Explosive dispersal emerges from cell and tissue interactions in multi-scale model, The violent explosion of seed pods is one of the fastest movements in the plant kingdom. Using interactions between cell and tissue-level processes, a multi-scale model reproduces the explosive seed dispersal in popping cress.

International audience; Background and Aims Floral development is remarkably robust in terms of the identity and number of floral organs in each whorl, whereas vegetative development can be quite plastic. This canalization of flower development prevents the phenotypic expression of cryptic genetic variation, even in fluctuating environments. A cruciform peri-anth with four petals is a hallmark of the Brassicaceae family, typified in the model species Arabidopsis thaliana. However, variable petal loss is found in Cardamine hirsuta, a genetically tractable relative of A. thaliana. Cardamine hirsuta petal number varies in response to stochastic, genetic and environmental perturbations, which makes it an interesting model to study mechanisms of decanalization and the expression of cryptic variation. Methods Multitrait quantitative trait locus (QTL) analysis in recombinant inbred lines (RILs) was used to identify whether the stochastic variation found in C. hirsuta petal number had a genetic basis. Key Results Stochastic variation (standard error of the average petal number) was found to be a heritable phenotype , and four QTL that influenced this trait were identified. The sensitivity to detect these QTL effects was increased by accounting for the effect of ageing on petal number variation. All QTL had significant effects on both average petal number and its standard error, indicating that these two traits share a common genetic basis. However, for some QTL, a degree of independence was found between the age of the flowers where allelic effects were significant for each trait. Conclusions Stochastic variation in C. hirsuta petal number has a genetic basis, and common QTL influence both average petal number and its standard error. Allelic variation at these QTL can, therefore, modify petal number in an age-specific manner via effects on the phenotypic mean and stochastic variation. These results are discussed in the context of trait evolution via a loss of robustness.

Current advances in developmental genetics are increasingly underpinned by comparative approaches as more powerful experimental tools become available in non-model organisms. Cardamine hirsuta is related to the model plant Arabidopsis thaliana and comparisons between these two experimentally tractable species have advanced our understanding of development and diversity. The power of forward genetics to uncover new biology was evident in the isolation of REDUCED COMPLEXITY, a gene which is present in C. hirsuta but lost in A. thaliana, and shapes crucifer leaf diversity. Transferring two Knotted1-like homeobox genes between C. hirsuta and A. thaliana revealed a constraint imposed by pleiotropy on the evolutionary potential of cis regulatory change to modify leaf shape. FLOWERING LOCUS C was identified as a heterochronic gene that underlies natural leaf shape variation in C. hirsuta.

International audience; Invariant petal number is a characteristic of most flowers and is generally robust to genetic and environmental variation. We took advantage of the natural variation found in Cardamine hirsuta petal number to investigate the genetic basis of this trait in a case where robustness was lost during evolution. We used quantitative trait locus (QTL) analysis to characterize the genetic architecture of petal number. Αverage petal number showed transgressive variation from zero to four petals in five C. hirsuta mapping populations, and this variation was highly heritable. We detected 15 QTL at which allelic variation affected petal number. The effects of these QTL were relatively small in comparison with alleles induced by mutagenesis, suggesting that natural selection may act to maintain petal number within its variable range below four. Petal number showed a temporal trend during plant ageing, as did sepal trichome number, and multi-trait QTL analysis revealed that these age-dependent traits share a common genetic basis. Our results demonstrate that petal number is determined by many genes of small effect, some of which are age-dependent, and suggests a mechanism of trait evolution via the release of cryptic variation.

The diversity of flowering structures dazzles the eye, dominates the landscape, and invites evolutionary questions regarding the development of such variety. Comparative work in a number of genetically tractable plant species has addressed how diverse floral architectures develop, and started to reveal the balance between conservation and divergence of the patterning mechanisms responsible for when and where flowers form on a plant. We highlight findings from Petunia where conserved LFY/UFO function is under species-specific regulation, and a novel mechanism involving WOX homeodomain proteins for modulating cyme development in diverse nightshades. We also draw attention to recent findings in Arabidopsis of miRNA and chromatin-based timing mechanisms controlling floral development, and illustrate how genetic studies in Arabidopsis relatives can reveal how evolutionary changes in such mechanisms generate diversity in form.

The developmental basis for the generation of divergent leaf forms is largely unknown. Here we investigate this problem by studying processes that distinguish development of two related species: Arabidopsis thaliana, which has simple leaves, and Cardamine hirsuta, which has dissected leaves with individual leaflets. Using genetics, expression studies and cell lineage tracing, we show that lateral leaflet formation in C. hirsuta requires the establishment of growth foci that form after leaf initiation. These growth foci are recruited at the leaf margin in response to activity maxima of auxin, a hormone that polarizes growth in diverse developmental contexts. Class I KNOTTED1-like homeobox (KNOX) proteins also promote leaflet initiation in C. hirsuta, and here we provide evidence that this action of KNOX proteins is contingent on the ability to organize auxin maxima via the PINFORMED1 (PIN1) auxin efflux transporter. Thus, differential deployment of a fundamental mechanism polarizing cellular growth contributed to the diversification of leaf form during evolution.

A key problem in biology is whether the same processes underlie morphological variation between and within species. Here, by using plant leaves as an example, we show that the causes of diversity at these two evolutionary scales can be divergent. Some species like the model plant Arabidopsis thaliana have simple leaves, whereas others like the A. thaliana relative Cardamine hirsuta bear complex leaves comprising leaflets. Previous work has shown that these interspecific differences result mostly from variation in local tissue growth and patterning. Now, by cloning and characterizing a quantitative trait locus (QTL) for C. hirsuta leaf shape, we find that a different process, age-dependent progression of leaf form, underlies variation in this trait within species. This QTL effect is caused by cis-regulatory variation in the floral repressor ChFLC, such that genotypes with low-expressing ChFLC alleles show both early flowering and accelerated age-dependent changes in leaf form, including faster leaflet production. We provide evidence that this mechanism coordinates leaf development with reproductive timing and may help to optimize resource allocation to the next generation.

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., eLife, 4, ISSN:2050-084X

Two interrelated problems in biology are understanding the regulatory logic and predictability of morphological evolution. Here, we studied these problems by comparing Arabidopsis thaliana, which has simple leaves, and its relative, Cardamine hirsuta, which has dissected leaves comprising leaflets. By transferring genes between the two species, we provide evidence for an inverse relationship between the pleiotropy of SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) homeobox genes and their ability to modify leaf form. We further show that cis-regulatory divergence of BP results in two alternative configurations of the genetic networks controlling leaf development. In C. hirsuta, ChBP is repressed by the microRNA164A (MIR164A)/ChCUP-SHAPED COTYLEDON (ChCUC) module and ChASYMMETRIC LEAVES1 (ChAS1), thus creating cross-talk between MIR164A/CUC and AS1 that does not occur in A. thaliana. These different genetic architectures lead to divergent interactions of network components and growth regulation in each species. We suggest that certain regulatory genes with low pleiotropy are predisposed to readily integrate into or disengage from conserved genetic networks influencing organ geometry, thus rapidly altering their properties and contributing to morphological divergence.

A key question in biology is how differences in gene function or regulation produce new morphologies during evolution. Here we investigate the genetic basis for differences in leaf form between two closely related plant species, Arabidopsis thaliana and Cardamine hirsuta. We report that in C. hirsuta, class I KNOTTED1-like homeobox (KNOX) proteins are required in the leaf to delay cellular differentiation and produce a dissected leaf form, in contrast to A. thaliana, in which KNOX exclusion from leaves results in a simple leaf form. These differences in KNOX expression arise through changes in the activity of upstream gene regulatory sequences. The function of ASYMMETRIC LEAVES1/ROUGHSHEATH2/PHANTASTICA (ARP) proteins to repress KNOX expression is conserved between the two species, but in C. hirsuta the ARP-KNOX regulatory module controls new developmental processes in the leaf. Thus, evolutionary tinkering with KNOX regulation, constrained by ARP function, may have produced diverse leaf forms by modulating growth and differentiation patterns in developing leaf primordia.

The Society for Experimental Biology organised a `Plant Frontier' meeting,which was recently held at the University of Sheffield, UK. One of the sessions of this broad meeting was on plant meristems, which covered a range of topics, including stem cells, patterning, long distance signalling and epigenetic regulation of meristem development.

A key problem in developmental biology is understanding the origin of morphological innovations. Comparative studies in plants with different leaf morphologies indicate that the developmental pathway defined by KNOTTED1-type homeodomain proteins could be involved in generating different leaf forms. The differential expression of regulatory proteins has emerged as an important factor in driving morphological innovations in the plant kingdom--an idea that is well supported by quantitative trait locus analyses.

Background: The shoot apical meristem (SAM) is an indeterminate structure that gives rise to the aerial parts of higher plants. Leaves arise from the differentiation of cells at the flanks of the SAM. Current evidence suggests that the precise regulation of KNOTTED1-like homeobox (KNOX) transcription factors is central to the acquisition of leaf versus meristem identity in a wide spectrum of plant species. Factors required to repress KNOX gene expression in leaves have recently been identified. Additional factors such as the CHD3 chromatin remodeling factor PICKLE (PKL) act to restrict meristematic activity in Arabidopsis leaves without repressing KNOX gene expression. Less is known regarding downstream targets of KNOX function. Recent evidence, however, has suggested that growth regulators may mediate KNOX activity in a variety of plant species. Results: Here we show that reduced activity of the gibberellin (GA) growth regulator pathway promotes meristematic activity, both in the natural context of KNOX function in the SAM and upon ectopic KNOX expression in Arabidopsis leaves. We show that constitutive signaling through the GA pathway is detrimental to meristem maintenance. Furthermore, we provide evidence that one of the functions of the KNOX protein SHOOTMERISTEMLESS (STM) is to exclude transcription of the GA-biosynthesis gene AtGA20ox1 from the SAM. We also demonstrate that AtGA20ox1 transcript is reduced in the pkl mutant in a KNOX-independent manner. Moreover, we show a similar interaction between KNOX proteins and GA-biosynthesis gene expression in the tomato leaf and implicate this interaction in regulation of the dissected leaf form. Conclusions: We suggest that repression of GA activity by KNOX transcription factors is a key component of meristem function. Transfer of the KNOX/GA regulatory module from the meristem to the leaf may have contributed to the generation of the diverse leaf morphologies observed in higher plants.