The shoot apical meristem (SAM) is a pluripotent group of cells that gives rise to the aerial parts of higher plants. Class-I KNOTTED1-like homeobox (KNOX) transcription factors promote meristem function partly through repression of biosynthesis of the growth regulator gibberellin (GA). However, regulation of GA activity cannot fully account for KNOX action. Here, we show that KNOX function is also mediated by cytokinin (CK), a growth regulator that promotes cell division and meristem function. We demonstrate that KNOX activity is sufficient to rapidly activate both CK biosynthetic gene expression and a SAM-localized CK-response regulator. We also show that CK signaling is necessary for SAM function in a weak hypomorphic allele of the KNOX gene SHOOTMERISTEMLESS (STM). Additionally, we provide evidence that a combination of constitutive GA signaling and reduced CK levels is detrimental to SAM function. Our results indicate that CK activity is both necessary and sufficient for stimulating GA catabolic gene expression, thus reinforcing the low-GA regime established by KNOX proteins in the SAM. We propose that KNOX proteins may act as general orchestrators of growth-regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity.

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.

SummaryKnotted1-like homeobox (KNOX) proteins are homeodomain transcription factors that maintain an important pluripotent cell population called the shoot apical meristem, which generates the entire above-ground body of vascular plants. KNOX proteins regulate target genes that control hormone homeostasis in the meristem and interact with another subclass of homeodomain proteins called the BELL family. Studies in novel genetic systems, both at the base of the land plant phylogeny and in flowering plants, have uncovered novel roles for KNOX proteins in sculpting plant form and its diversity. Here, we discuss how KNOX proteins influence plant growth and development in a versatile context-dependent manner.

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.DOI: http://dx.doi.org/10.7554/eLife.05864.001

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.