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 PIN-FORMED1 (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.L eaf margin morphology is commonly used to distinguish different plant species and often evolves in close correspondence with the environment. For example, the degree of leaf serration is a good predictor of mean annual temperature of landmasses over geological timescales (1). Variations in margin morphology were first documented in antiquity (2) and were among the first heritable traits studied in plants (3). Nonetheless, a predictive model of leaf margin shape acquisition is lacking. Recent genetic analyses have revealed two key processes required for serration formation: regulated auxin transport by the efflux carrier PINFORMED1 (PIN1) (4) and activity of the growth repressor CUP-SHAPED COTYLEDON2 (CUC2), which is negatively regulated by miR164 (5). PIN1 has a polar subcellular localization and forms convergence points at the margins of leaves, creating localized auxin activity maxima that are required for the outgrowth of serrations (4, 6). Leaves of both pin1 and cuc2 mutants fail to initiate serrations and have smooth margins, highlighting the importance of these gene products for leaf morphogenesis (4, 5). Here, we show how CUC2 activity and auxin transport and signaling are regulated and integrated to sculpt leaf margin serrations.

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.

Summary 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

The hormone auxin plays a crucial role in plant morphogenesis. In the shoot apical meristem, the PIN-FORMED1 (PIN1) efflux carrier concentrates auxin into local maxima in the epidermis, which position incipient leaf or floral primordia. From these maxima, PIN1 transports auxin into internal tissues along emergent paths that pattern leaf and stem vasculature. In Arabidopsis thaliana, these functions are attributed to a single PIN1 protein. Using phylogenetic and gene synteny analysis we identified an angiosperm PIN clade sister to PIN1, here termed Sister-of-PIN1 (SoPIN1), which is present in all sampled angiosperms except for Brassicaceae, including Arabidopsis. Additionally, we identified a conserved duplication of PIN1 in the grasses: PIN1a and PIN1b. In Brachypodium distachyon, SoPIN1 is highly expressed in the epidermis and is consistently polarized toward regions of high expression of the DR5 auxin-signaling reporter, which suggests that SoPIN1 functions in the localization of new primordia. In contrast, PIN1a and PIN1b are highly expressed in internal tissues, suggesting a role in vascular patterning. PIN1b is expressed in broad regions spanning the space between new primordia and previously formed vasculature, suggesting a role in connecting new organs to auxin sinks in the older tissues. Within these regions, PIN1a forms narrow canals that likely pattern future veins. Using a computer model, we reproduced the observed spatio-temporal expression and localization patterns of these proteins by assuming that SoPIN1 is polarized up the auxin gradient, and PIN1a and PIN1b are polarized to different degrees with the auxin flux. Our results suggest that examination and modeling of PIN dynamics in plants outside of Brassicaceae will offer insights into auxin-driven patterning obscured by the loss of the SoPIN1 clade in Brassicaceae.