A Growth-Based Framework for Leaf Shape Development and Diversity.

How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.

Morphomechanical Innovation Drives Explosive Seed Dispersal.

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.

A deep learning-based toolkit for 3D nuclei segmentation and quantitative analysis in cellular and tissue context.

We present a new set of computational tools that enable accurate and widely applicable 3D segmentation of nuclei in various 3D digital organs. We have developed an approach for ground truth generation and iterative training of 3D nuclear segmentation models, which we applied to popular CellPose, PlantSeg and StarDist algorithms. We provide two high-quality models trained on plant nuclei that enable 3D segmentation of nuclei in datasets obtained from fixed or live samples, acquired from different plant and animal tissues, and stained with various nuclear stains or fluorescent protein-based nuclear reporters. We also share a diverse high-quality training dataset of about 10,000 nuclei. Furthermore, we advanced the MorphoGraphX analysis and visualization software by, among other things, providing a method for linking 3D segmented nuclei to their surrounding cells in 3D digital organs. We found that the nuclear-to-cell volume ratio varies between different ovule tissues and during the development of a tissue. Finally, we extended the PlantSeg 3D segmentation pipeline with a proofreading tool that uses 3D segmented nuclei as seeds to correct cell segmentation errors in difficult-to-segment tissues.

A CUC1/auxin genetic module links cell polarity to patterned tissue growth and leaf shape diversity in crucifer plants.

How tissue-level information encoded by fields of regulatory gene activity is translated into the patterns of cell polarity and growth that generate the diverse shapes of different species remains poorly understood. Here, we investigate this problem in the case of leaf shape differences between Arabidopsis thaliana, which has simple leaves, and its relative Cardamine hirsuta that has complex leaves divided into leaflets. We show that patterned expression of the transcription factor CUP-SHAPED COTYLEDON1 in C. hirsuta (ChCUC1) is a key determinant of leaf shape differences between the two species. Through inducible genetic perturbations, time-lapse imaging of growth, and computational modeling, we find that ChCUC1 provides instructive input into auxin-based leaf margin patterning. This input arises via transcriptional regulation of multiple auxin homeostasis components, including direct activation of WAG kinases that are known to regulate the polarity of PIN-FORMED auxin transporters. Thus, we have uncovered a mechanism that bridges biological scales by linking spatially distributed and species-specific transcription factor expression to cell-level polarity and growth, to shape diverse leaf forms.

Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.

LMI1 homeodomain protein regulates organ proportions by spatial modulation of endoreduplication.

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.

Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation.

Shade caused by the proximity of neighboring vegetation triggers a set of acclimation responses to either avoid or tolerate shade. Comparative analyses between the shade-avoider Arabidopsis thaliana and the shade-tolerant Cardamine hirsuta revealed a role for the atypical basic-helix-loop-helix LONG HYPOCOTYL IN FR 1 (HFR1) in maintaining the shade tolerance in C. hirsuta, inhibiting hypocotyl elongation in shade and constraining expression profile of shade-induced genes. We showed that C. hirsuta HFR1 protein is more stable than its A. thaliana counterpart, likely due to its lower binding affinity to CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), contributing to enhance its biological activity. The enhanced HFR1 total activity is accompanied by an attenuated PHYTOCHROME INTERACTING FACTOR (PIF) activity in C. hirsuta. As a result, the PIF-HFR1 module is differently balanced, causing a reduced PIF activity and attenuating other PIF-mediated responses such as warm temperature-induced hypocotyl elongation (thermomorphogenesis) and dark-induced senescence. By this mechanism and that of the already-known of phytochrome A photoreceptor, plants might ensure to properly adapt and thrive in habitats with disparate light amounts.

The trichome pattern diversity of Cardamine shares genetic mechanisms with Arabidopsis but differs in environmental drivers.

Natural variation in trichome pattern (amount and distribution) is prominent among populations of many angiosperms. However, the degree of parallelism in the genetic mechanisms underlying this diversity and its environmental drivers in different species remain unclear. To address these questions, we analyzed the genomic and environmental bases of leaf trichome pattern diversity in Cardamine hirsuta, a relative of Arabidopsis (Arabidopsis thaliana). We characterized 123 wild accessions for their genomic diversity, leaf trichome patterns at different temperatures, and environmental adjustments. Nucleotide diversities and biogeographical distribution models identified two major genetic lineages with distinct demographic and adaptive histories. Additionally, C. hirsuta showed substantial variation in trichome pattern and plasticity to temperature. Trichome amount in C. hirsuta correlated positively with spring precipitation but negatively with temperature, which is opposite to climatic patterns in A. thaliana. Contrastingly, genetic analysis of C. hirsuta glabrous accessions indicated that, like for A. thaliana, glabrousness is caused by null mutations in ChGLABRA1 (ChGL1). Phenotypic genome-wide association studies (GWAS) further identified a ChGL1 haplogroup associated with low trichome density and ChGL1 expression. Therefore, a ChGL1 series of null and partial loss-of-function alleles accounts for the parallel evolution of leaf trichome pattern in C. hirsuta and A. thaliana. Finally, GWAS also detected other candidate genes (e.g. ChETC3, ChCLE17) that might affect trichome pattern. Accordingly, the evolution of this trait in C. hirsuta and A. thaliana shows partially conserved genetic mechanisms but is likely involved in adaptation to different environments.

Gene expression evolution in pattern-triggered immunity within Arabidopsis thaliana and across Brassicaceae species.

Plants recognize surrounding microbes by sensing microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). Despite their significance for microbial control, the evolution of PTI responses remains largely uncharacterized. Here, by employing comparative transcriptomics of six Arabidopsis thaliana accessions and three additional Brassicaceae species to investigate PTI responses, we identified a set of genes that commonly respond to the MAMP flg22 and genes that exhibit species-specific expression signatures. Variation in flg22-triggered transcriptome responses across Brassicaceae species was incongruent with their phylogeny, while expression changes were strongly conserved within A. thaliana. We found the enrichment of WRKY transcription factor binding sites in the 5'-regulatory regions of conserved and species-specific responsive genes, linking the emergence of WRKY-binding sites with the evolution of gene expression patterns during PTI. Our findings advance our understanding of the evolution of the transcriptome during biotic stress.

Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity.

Here we investigate mechanisms underlying the diversification of biological forms using crucifer leaf shape as an example. We show that evolution of an enhancer element in the homeobox gene REDUCED COMPLEXITY (RCO) altered leaf shape by changing gene expression from the distal leaf blade to its base. A single amino acid substitution evolved together with this regulatory change, which reduced RCO protein stability, preventing pleiotropic effects caused by its altered gene expression. We detected hallmarks of positive selection in these evolved regulatory and coding sequence variants and showed that modulating RCO activity can improve plant physiological performance. Therefore, interplay between enhancer and coding sequence evolution created a potentially adaptive path for morphological evolution.

The annotation and analysis of complex 3D plant organs using 3DCoordX.

A fundamental question in biology concerns how molecular and cellular processes become integrated during morphogenesis. In plants, characterization of 3D digital representations of organs at single-cell resolution represents a promising approach to addressing this problem. A major challenge is to provide organ-centric spatial context to cells of an organ. We developed several general rules for the annotation of cell position and embodied them in 3DCoordX, a user-interactive computer toolbox implemented in the open-source software MorphoGraphX. 3DCoordX enables rapid spatial annotation of cells even in highly curved biological shapes. Using 3DCoordX, we analyzed cellular growth patterns in organs of several species. For example, the data indicated the presence of a basal cell proliferation zone in the ovule primordium of Arabidopsis (Arabidopsis thaliana). Proof-of-concept analyses suggested a preferential increase in cell length associated with neck elongation in the archegonium of Marchantia (Marchantia polymorpha) and variations in cell volume linked to central morphogenetic features of a trap of the carnivorous plant Utricularia (Utricularia gibba). Our work demonstrates the broad applicability of the developed strategies as they provide organ-centric spatial context to cellular features in plant organs of diverse shape complexity.

Alternate wiring of a KNOXI genetic network underlies differences in leaf development of A. thaliana and C. hirsuta.

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.

Photoreceptor Activity Contributes to Contrasting Responses to Shade in Cardamine and Arabidopsis Seedlings.

Plants have evolved two major ways to deal with nearby vegetation or shade: avoidance and tolerance. Moreover, some plants respond to shade in different ways; for example, Arabidopsis (Arabidopsis thaliana) undergoes an avoidance response to shade produced by vegetation, but its close relative Cardamine hirsuta tolerates shade. How plants adopt opposite strategies to respond to the same environmental challenge is unknown. Here, using a genetic strategy, we identified the C. hirsuta slender in shade1 mutants, which produce strongly elongated hypocotyls in response to shade. These mutants lack the phytochrome A (phyA) photoreceptor. Our findings suggest that C. hirsuta has evolved a highly efficient phyA-dependent pathway that suppresses hypocotyl elongation when challenged by shade from nearby vegetation. This suppression relies, at least in part, on stronger phyA activity in C. hirsuta; this is achieved by increased ChPHYA expression and protein accumulation combined with a stronger specific intrinsic repressor activity. We suggest that modulation of photoreceptor activity is a powerful mechanism in nature to achieve physiological variation (shade tolerance versus avoidance) for species to colonize different habitats.

Differential spatial distribution of miR165/6 determines variability in plant root anatomy.

A clear example of interspecific variation is the number of root cortical layers in plants. The genetic mechanisms underlying this variability are poorly understood, partly because of the lack of a convenient model. Here, we demonstrate that Cardamine hirsuta, unlike Arabidopsis thaliana, has two cortical layers that are patterned during late embryogenesis. We show that a miR165/6-dependent distribution of the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) transcription factor PHABULOSA (PHB) controls this pattern. Our findings reveal that interspecies variation in miRNA distribution can determine differences in anatomy in plants.

Fine-scale empirical data on niche divergence and homeolog expression patterns in an allopolyploid and its diploid progenitor species.

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.

Phyllotaxis: is the golden angle optimal for light capture?

Phyllotactic patterns are some of the most conspicuous in nature. To create these patterns plants must control the divergence angle between the appearance of successive organs, sometimes to within a fraction of a degree. The most common angle is the Fibonacci or golden angle, and its prevalence has led to the hypothesis that it has been selected by evolution as optimal for plants with respect to some fitness benefits, such as light capture. We explore arguments for and against this idea with computer models. We have used both idealized and scanned leaves from Arabidopsis thaliana and Cardamine hirsuta to measure the overlapping leaf area of simulated plants after varying parameters such as leaf shape, incident light angles, and other leaf traits. We find that other angles generated by Fibonacci-like series found in nature are equally optimal for light capture, and therefore should be under similar evolutionary pressure. Our findings suggest that the iterative mechanism for organ positioning itself is a more likely target for evolutionary pressure, rather than a specific divergence angle, and our model demonstrates that the heteroblastic progression of leaf shape in A. thaliana can provide a potential fitness benefit via light capture.

Resolving the backbone of the Brassicaceae phylogeny for investigating trait diversity.

The Brassicaceae family comprises c. 4000 species including economically important crops and the model plant Arabidopsis thaliana. Despite their importance, the relationships among major lineages in the family remain unresolved, hampering comparative research. Here, we inferred a Brassicaceae phylogeny using newly generated targeted enrichment sequence data of 1827 exons (> 940 000 bases) representing 63 species, as well as sequenced genome data of 16 species, together representing 50 of the 52 currently recognized Brassicaceae tribes. A third of the samples were derived from herbarium material, facilitating broad taxonomic coverage of the family. Six major clades formed successive sister groups to the rest of Brassicaceae. We also recovered strong support for novel relationships among tribes, and resolved the position of 16 taxa previously not assigned to a tribe. The broad utility of these phylogenetic results is illustrated through a comparative investigation of genome-wide expression signatures that distinguish simple from complex leaves in Brassicaceae. Our study provides an easily extendable dataset for further advances in Brassicaceae systematics and a timely higher-level phylogenetic framework for a wide range of comparative studies of multiple traits in an intensively investigated group of plants.

MpTCP1 controls cell proliferation and redox processes in Marchantia polymorpha.

TCP transcription factors are key regulators of angiosperm cell proliferation processes. It is unknown whether their regulatory growth capacities are conserved across land plants, which we examined in liverworts, one of the earliest diverging land plant lineages. We generated knockout mutants for MpTCP1, the single TCP-P clade gene in Marchantia polymorpha, and characterized its function by conducting cell proliferation and morphological analyses as well as messenger RNA expression, transcriptome, chemical, and DNA binding studies. Mptcp1ge lines show a reduced vegetative thallus growth and extra tissue formation in female reproductive structures. Additionally, mutant plants reveal increased hydrogen peroxide (H2 O2 ) levels and an enhanced pigmentation in the thallus caused by formation of secondary metabolites, such as aminochromes. MpTCP1 proteins interact redox dependently with DNA and regulate the expression of a comprehensive redox network, comprising enzymes involved in H2 O2 metabolism. MpTCP1 regulates Marchantia growth in a context-dependent manner. Redox sensitivity of the DNA binding capacity of MpTCP1 proteins provides a mechanism to respond to altered redox conditions. Our data suggest that MpTCP1 activity could thereby have contributed to diversification of land plant morphologies and to adaptations to abiotic and biotic challenges, as experienced by liverworts during early land plant colonization.