Model-based reconstruction of whole organ growth dynamics reveals invariant patterns in leaf morphogenesis.

Plant organ morphogenesis spans several orders of magnitude in time and space. Because of limitations in live-imaging, analysing whole organ growth from initiation to mature stages typically rely on static data sampled from different timepoints and individuals. We introduce a new model-based strategy for dating organs and for reconstructing morphogenetic trajectories over unlimited time windows based on static data. Using this approach, we show that Arabidopsis thaliana leaves are initiated at regular 1-day intervals. Despite contrasted adult morphologies, leaves of different ranks exhibited shared growth dynamics, with linear gradations of growth parameters according to leaf rank. At the sub-organ scale, successive serrations from same or different leaves also followed shared growth dynamics, suggesting that global and local leaf growth patterns are decoupled. Analysing mutants leaves with altered morphology highlighted the decorrelation between adult shapes and morphogenetic trajectories, thus stressing the benefits of our approach in identifying determinants and critical timepoints during organ morphogenesis.

De novo stem cell establishment in meristems requires repression of organ boundary cell fate.

Stem cells play important roles in animal and plant biology, as they sustain morphogenesis and tissue replenishment following aging or injury. In plants, stem cells are embedded in multicellular structures called meristems. The formation of new meristems is essential for the plastic expansion of the highly branched shoot and root systems. In particular, axillary meristems (AMs) that produce lateral shoots arise from the division of boundary domain cells at the leaf base. The CUP-SHAPED COTYLEDON (CUC) genes are major determinants of the boundary domain and are required for AM initiation. However, how AMs get structured and how stem cells become established de novo remain elusive. Here, we show that two NGATHA-LIKE (NGAL) transcription factors, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2, redundantly repress CUC expression in initiating AMs of Arabidopsis thaliana. Ectopic boundary fate leads to abnormal growth and organization of the AM and prevents de novo stem cell establishment. Floral meristems of the dpa4 sod7 double mutant show a similar delay in de novo stem cell establishment. Altogether, while boundary fate is required for the initiation of AMs, our work reveals how it is later repressed to allow proper meristem establishment and de novo stem cell niche formation.

Frizzled-Dependent Planar Cell Polarity without Secreted Wnt Ligands.

Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here, we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.

Dissecting the pathways coordinating patterning and growth by plant boundary domains.

Boundary domains play important roles during morphogenesis in plants and animals, but how they contribute to patterning and growth coordination in plants is not understood. The CUC genes determine the boundary domains in the aerial part of the plants and, in particular, they have a conserved role in regulating leaf complexity across Angiosperms. Here, we used tooth formation at the Arabidopsis leaf margin controlled by the CUC2 transcription factor to untangle intertwined events during boundary-controlled morphogenesis in plants. Combining conditional restoration of CUC2 function with morphometrics as well as quantification of gene expression and hormone signaling, we first established that tooth morphogenesis involves a patterning phase and a growth phase. These phases can be separated, as patterning requires CUC2 while growth can occur independently of CUC2. Next, we show that CUC2 acts as a trigger to promote growth through the activation of three functional relays. In particular, we show that KLUH acts downstream of CUC2 to modulate auxin response and that expressing KLUH can compensate for deficient CUC2 expression during tooth growth. Together, we reveal a genetic and molecular network that allows coordination of patterning and growth by CUC2-defined boundaries during morphogenesis at the leaf margin.

Getting leaves into shape: a molecular, cellular, environmental and evolutionary view.

Leaves arise from groups of undifferentiated cells as small primordia that go through overlapping phases of morphogenesis, growth and differentiation. These phases are genetically controlled and modulated by environmental cues to generate a stereotyped, yet plastic, mature organ. Over the past couple of decades, studies have revealed that hormonal signals, transcription factors and miRNAs play major roles during leaf development, and more recent findings have highlighted the contribution of mechanical signals to leaf growth. In this Review, we discuss how modulating the activity of some of these regulators can generate diverse leaf shapes during development, in response to a varying environment, or between species during evolution.

GDP-L-fucose is required for boundary definition in plants.

The CUP-SHAPED COTYLEDON (CUC) transcription factors control plant boundary formation, thus allowing the emergence of novel growth axes. While the developmental roles of the CUC genes in different organs and across species are well characterized, upstream and downstream events that contribute to their function are still poorly understood. To identify new players in this network, we performed a suppressor screen of CUC2g-m4, a line overexpressing CUC2 that has highly serrated leaves. We identified a mutation that simplifies leaf shape and affects MURUS1 (MUR1), which is responsible for GDP-L-fucose production. Using detailed morphometric analysis, we show that GDP-L-fucose has an essential role in leaf shape acquisition by sustaining differential growth at the leaf margins. Accordingly, reduced CUC2 expression levels are observed in mur1 leaves. Furthermore, genetic analyses reveal a conserved role for GDP-L-fucose in different developmental contexts where it contributes to organ separation in the same pathway as CUC2. Taken together, our results reveal that GDP-L-fucose is necessary for proper establishment of boundary domains in various developmental contexts.

Multiscale quantification of morphodynamics: MorphoLeaf software for 2D shape analysis.

A major challenge in morphometrics is to analyse complex biological shapes formed by structures at different scales. Leaves exemplify this challenge as they combine differences in their overall shape with smaller shape variations at their margin, leading to lobes or teeth. Current methods based on contour or on landmark analysis are successful in quantifying either overall leaf shape or leaf margin dissection, but fail in combining the two. Here, we present a comprehensive strategy and its associated freely available platform for the quantitative, multiscale analysis of the morphology of leaves with different architectures. For this, biologically relevant landmarks are automatically extracted and hierarchised, and used to guide the reconstruction of accurate average contours that properly represent both global and local features. Using this method, we establish a quantitative framework of the developmental trajectory of Arabidopsis leaves of different ranks and retrace the origin of leaf heteroblasty. When applied to different mutant forms, our method can contribute to a better understanding of gene function, as we show here for the role of CUC2 during Arabidopsis leaf serration. Finally, we illustrate the wider applicability of our tool by analysing hand morphometrics.