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.

Marker gene tethering by nucleoporins affects gene expression in plants.

In non-plant systems, chromatin association with the nuclear periphery affects gene expression, where interactions with nuclear envelope proteins can repress and interactions with nucleoporins can enhance transcription. In plants, both hetero- and euchromatin can localize at the nuclear periphery, but the effect of proximity to the nuclear periphery on gene expression remains largely unknown. This study explores the putative function of Seh1 and Nup50a nucleoporins on gene expression by using the Lac Operator / Lac Repressor (LacI-LacO) system adapted to Arabidopsis thaliana. We used LacO fused to the luciferase reporter gene (LacO:Luc) to investigate whether binding of the LacO:Luc transgene to nucleoporin:LacI protein fusions alters luciferase expression. Two separate nucleoporin-LacI-YFP fusions were introduced into single insert, homozygous LacO:Luc Arabidopsis plants. Homozygous plants carrying LacO:Luc and a single insert of either Seh1-LacI-YFP or Nup50a-LacI-YFP were tested for luciferase activity and compared to plants containing LacO:Luc only. Seh1-LacI-YFP increased, while Nup50a-LacI-YFP decreased luciferase activity. Seh1-LacI-YFP accumulated at the nuclear periphery as expected, while Nup50a-LacI-YFP was nucleoplasmic and was not selected for further study. Protein and RNA levels of luciferase were quantified by western blotting and RT-qPCR, respectively. Increased luciferase activity in LacO:Luc+Seh1-LacI-YFP plants was correlated with increased luciferase protein and RNA levels. This change of luciferase expression was abolished by disruption of LacI-LacO binding by treating with IPTG in young seedlings, rosette leaves and inflorescences. This study suggests that association with the nuclear periphery is involved in the regulation of gene expression in plants.

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

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.

Cardamine hirsuta: a versatile genetic system for comparative studies.

A major goal in biology is to identify the genetic basis for phenotypic diversity. This goal underpins research in areas as diverse as evolutionary biology, plant breeding and human genetics. A limitation for this research is no longer the availability of sequence information but the development of functional genetic tools to understand the link between changes in sequence and phenotype. Here we describe Cardamine hirsuta, a close relative of the reference plant Arabidopsis thaliana, as an experimental system in which genetic and transgenic approaches can be deployed effectively for comparative studies. We present high-resolution genetic and cytogenetic maps for C. hirsuta and show that the genome structure of C. hirsuta closely resembles the eight chromosomes of the ancestral crucifer karyotype and provides a good reference point for comparative genome studies across the Brassicaceae. We compared morphological and physiological traits between C. hirsuta and A. thaliana and analysed natural variation in stamen number in which lateral stamen loss is a species characteristic of C. hirsuta. We constructed a set of recombinant inbred lines and detected eight quantitative trait loci that can explain stamen number variation in this population. We found clear phylogeographic structure to the genetic variation in C. hirsuta, thus providing a context within which to address questions about evolutionary changes that link genotype with phenotype and the environment.

A PHABULOSA/cytokinin feedback loop controls root growth in Arabidopsis.

The hormone cytokinin (CK) controls root length in Arabidopsis thaliana by defining where dividing cells, derived from stem cells of the root meristem, start to differentiate [1-6]. However, the regulatory inputs directing CK to promote differentiation remain poorly understood. Here, we show that the HD-ZIPIII transcription factor PHABULOSA (PHB) directly activates the CK biosynthesis gene ISOPENTENYL TRANSFERASE 7 (IPT7), thus promoting cell differentiation and regulating root length. We further demonstrate that CK feeds back to repress both PHB and microRNA165, a negative regulator of PHB. These interactions comprise an incoherent regulatory loop in which CK represses both its activator and a repressor of its activator. We propose that this regulatory circuit determines the balance of cell division and differentiation during root development and may provide robustness against CK fluctuations.

Arabidopsis PLETHORA transcription factors control phyllotaxis.

The pattern of plant organ initiation at the shoot apical meristem (SAM), termed phyllotaxis, displays regularities that have long intrigued botanists and mathematicians alike. In the SAM, the central zone (CZ) contains a population of stem cells that replenish the surrounding peripheral zone (PZ), where organs are generated in regular patterns. These patterns differ between species and may change in response to developmental or environmental cues [1]. Expression analysis of auxin efflux facilitators of the PIN-FORMED (PIN) family combined with modeling of auxin transport has indicated that organ initiation is associated with intracellular polarization of PIN proteins and auxin accumulation [2-10]. However, regulators that modulate PIN activity to determine phyllotactic patterns have hitherto been unknown. Here we reveal that three redundantly acting PLETHORA (PLT)-like AP2 domain transcription factors control shoot organ positioning in the model plant Arabidopsis thaliana. Loss of PLT3, PLT5, and PLT7 function leads to nonrandom, metastable changes in phyllotaxis. Phyllotactic changes in plt3plt5plt7 mutants are largely attributable to misregulation of PIN1 and can be recapitulated by reducing PIN1 dosage, revealing that PLT proteins are key regulators of PIN1 activity in control of phyllotaxis.

Model for the regulation of Arabidopsis thaliana leaf margin development.

Biological shapes are often produced by the iterative generation of repeated units. The mechanistic basis of such iteration is an area of intense investigation. Leaf development in the model plant Arabidopsis is one such example where the repeated generation of leaf margin protrusions, termed serrations, is a key feature of final shape. However, the regulatory logic underlying this process is unclear. Here, we use a combination of developmental genetics and computational modeling to show that serration development is the morphological read-out of a spatially distributed regulatory mechanism, which creates interspersed activity peaks of the growth-promoting hormone auxin and the cup-shaped cotyledon2 (CUC2) transcription factor. This mechanism operates at the growing leaf margin via a regulatory module consisting of two feedback loops working in concert. The first loop relates the transport of auxin to its own distribution, via polar membrane localization of the pinformed1 (PIN1) efflux transporter. This loop captures the potential of auxin to generate self-organizing patterns in diverse developmental contexts. In the second loop, CUC2 promotes the generation of PIN1-dependent auxin activity maxima while auxin represses CUC2 expression. This CUC2-dependent loop regulates activity of the conserved auxin efflux module in leaf margins to generate stable serration patterns. Conceptualizing leaf margin development via this mechanism also helps to explain how other developmental regulators influence leaf shape.

Weeds of change: Cardamine hirsuta as a new model system for studying dissected leaf development.

Cardamine hirsuta, a small crucifer closely related to the model organism Arabidopsis thaliana, offers high genetic tractability and has emerged as a powerful system for studying the genetic basis for diversification of plant form. Contrary to A. thaliana, which has simple leaves, C. hirsuta produces dissected leaves divided into individual units called leaflets. Leaflet formation requires activity of Class I KNOTTED1-like homeodomain (KNOX) proteins, which also promote function of the shoot apical meristem (SAM). In C. hirsuta, KNOX genes are expressed in the leaves whereas in A. thaliana their expression is confined to the SAM, and differences in expression arise through cis-regulatory divergence of KNOX regulation. KNOX activity in C. hirsuta leaves delays the transition from proliferative growth to differentiation thus facilitating the generation of lateral growth axes that give rise to leaflets. These axes reflect the sequential generation of cell division foci across the leaf proximodistal axis in response to auxin activity maxima, which are generated by the PINFORMED1 (PIN1) auxin efflux carriers in a process that resembles organogenesis at the SAM. Delimitation of C. hirsuta leaflets also requires the activity of CUP SHAPED COTYLEDON (CUC) genes, which direct formation of organ boundaries at the SAM. These observations show how species-specific deployment of fundamental shoot development networks may have sculpted simple versus dissected leaf forms. These studies also illustrate how extending developmental genetic studies to morphologically divergent relatives of model organisms can greatly help elucidate the mechanisms underlying the evolution of form.

Hormonal input in plant meristems: A balancing act.

Plant hormones are a group of chemically diverse molecules that control virtually all aspects of plant development. Classical plant hormones were identified many decades ago in physiology studies that addressed plant growth regulation. In recent years, biochemical and genetic approaches led to the identification of many molecular components that mediate hormone activity, such as hormone receptors and hormone-regulated genes. This has greatly contributed to the understanding of the mechanisms underlying hormone activity and highlighted the intricate crosstalk and integration of hormone signalling and developmental pathways. Here we review and discuss recent findings on how hormones regulate the activity of shoot and root apical meristems.

Repression of apical homeobox genes is required for embryonic root development in Arabidopsis.

Development of seed plant embryos is polarized along the apical-basal axis. This polarization occurs in the absence of cell migration and culminates in the establishment of two distinct pluripotent cell populations: the shoot apical meristem (SAM) and root meristem (RM), which postembryonically give rise to the entire shoot and root systems of the plant. The acquisition of genetic pathways that delimit root from shoot during embryogenesis must have played a pivotal role during land plant evolution because roots evolved after shoots in ancestral vascular plants and may be shoot-derived organs. However, such pathways are very poorly understood. Here we show that RM establishment in the model plant Arabidopsis thaliana requires apical confinement of the Class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) proteins PHABULOSA (PHB) and PHAVOLUTA (PHV), which direct both SAM development and shoot lateral organ polarity. Failure to restrict PHB and PHV expression apically via a microRNA-dependent pathway prevents correct elaboration of the embryonic root development program and results in embryo lethality. As such, repression of a fundamental shoot development pathway is essential for correct root development. Additionally, our data suggest that a single patterning process, based on HD-ZIP III repression, mediates both apical-basal and radial polarity in the embryo and lateral organ polarity in the shoot.

PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development.

Factors with a graded distribution can program fields of cells in a dose-dependent manner, but no evidence has hitherto surfaced for such mechanisms in plants. In the Arabidopsis thaliana root, two PLETHORA (PLT) genes encoding AP2-domain transcription factors have been shown to maintain the activity of stem cells. Here we show that a clade of four PLT homologues is necessary for root formation. Promoter activity and protein fusions of PLT homologues display gradient distributions with maxima in the stem cell area. PLT activities are largely additive and dosage dependent. High levels of PLT activity promote stem cell identity and maintenance; lower levels promote mitotic activity of stem cell daughters; and further reduction in levels is required for cell differentiation. Our findings indicate that PLT protein dosage is translated into distinct cellular responses.

From genes to shape: regulatory interactions in leaf development.

In the past two years novel connections were described between auxin function and transcription factor patterning systems involved in both leaf initiation and elaboration of leaf axial patterning. A cascade of small RNA-based regulatory steps was suggested to facilitate delimitation of cell types comprising the upper versus lower parts of the leaf. Developmental regulation of cellular growth emerged as a crucial component in regulation of leaf form with TCP and CUC2 transcription factors playing a key role in this process. Finally, cis-regulatory evolution of developmental genes emerged as a process that likely contributed to diversification of leaf form, while studies in seedless land plants have begun to elucidate the ancestral and derived aspects of leaf development pathways.

The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche.

A small organizing center, the quiescent center (QC), maintains stem cells in the Arabidopsis root and defines the stem cell niche. The phytohormone auxin influences the position of this niche by an unknown mechanism. Here, we identify the PLETHORA1 (PLT1) and PLT2 genes encoding AP2 class putative transcription factors, which are essential for QC specification and stem cell activity. The PLT genes are transcribed in response to auxin accumulation and are dependent on auxin response transcription factors. Distal PLT transcript accumulation creates an overlap with the radial expression domains of SHORT-ROOT and SCARECROW, providing positional information for the stem cell niche. Furthermore, the PLT genes are activated in the basal embryo region that gives rise to hypocotyl, root, and root stem cells and, when ectopically expressed, transform apical regions to these identities. Thus, the PLT genes are key effectors for establishment of the stem cell niche during embryonic pattern formation.