Leaves display a remarkable range of forms, from flat sheets with simple outlines to cup-shaped traps. Although much progress has been made in understanding the mechanisms of planar leaf development, it is unclear whether similar or distinctive mechanisms underlie shape transformations during development of more complex curved forms. Here, we use 3D imaging and cellular and clonal analysis, combined with computational modelling, to analyse the development of cup-shaped traps of the carnivorous plant Utricularia gibba. We show that the transformation from a near-spherical form at early developmental stages to an oblate spheroid with a straightened ventral midline in the mature form can be accounted for by spatial variations in rates and orientations of growth. Different hypotheses regarding spatiotemporal control predict distinct patterns of cell shape and size, which were tested experimentally by quantifying cellular and clonal anisotropy. We propose that orientations of growth are specified by a proximodistal polarity field, similar to that hypothesised to account for Arabidopsis leaf development, except that in Utricularia, the field propagates through a highly curved tissue sheet. Independent evidence for the polarity field is provided by the orientation of glandular hairs on the inner surface of the trap. Taken together, our results show that morphogenesis of complex 3D leaf shapes can be accounted for by similar mechanisms to those for planar leaves, suggesting that simple modulations of a common growth framework underlie the shaping of a diverse range of morphologies.

Funding: This work was supported by ERC (grant no. Carnomorph, 323028) and grants from the Biotechnology and Biological Sciences Research Council (grant nos. BB/M023117/1, BB/L008920/1, BB/P020747/1 and BBS/E/J/000PR9787) awarded to EC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we develop and apply these approaches to analyse trap morphogenesis in U. gibba. We show that after forming a near-spherical shape, U. gibba traps undergo defined changes in shape and curvature. By measuring 3D snapshots of traps at various developmental stages and exploring computational growth models, we show that differential rates and orientations of growth are both likely involved in the observed shape transformations. This hypothesis is further tested by marking cells with green fluorescent protein (GFP) and testing the resulting cell and clone shapes against model predictions. To account for oriented growth, the computational model invokes a proximodistal polarity field that is comparable to that proposed to account for Arabidopsis leaf development, except that it propagates within a curved sheet. The proposed polarity field is supported through analysis of quadrifid gland orientations. Our findings thus suggest that simple modulation of mechanisms underlying planar leaf development can account for shaping of more complex 3D leaf shapes, providing a unified explanation for diverse leaf forms.

Snapshots, scanning electron micrographs, and drawings of Utricularia traps at various developmental phases have been described [ 18 , 32 – 35 ]. However, quantitative growth and cellular analysis of morphogenesis have not been carried out. Such studies require the development of transformation methods for introducing fluorescent proteins to mark cell membranes or clones in Utricularia, followed by 3D imaging at different developmental stages. Moreover, models need to be developed for how changes in three-dimensional shape and curvature arise, and predictions of these models need to be tested against experimental data.

In the genus Utricularia (Lentibulariaceae), epiascidiate leaves, termed traps, use suction to catch prey, requiring highly coordinated morphogenesis to ensure that the opening and closing mechanisms operate effectively. Utricularia has several advantages for analysis of epiascidiate leaf development [ 17 ]. The traps are transparent and only a few millimetres long, making them convenient for imaging. Much of the trap comprises only two cell layers [ 18 , 19 ], compared to approximately seven cells for Arabidopsis leaves, simplifying growth analysis. The genome of Utricularia gibba is among the smallest in plants (100 Mb) and has been fully sequenced, providing a resource for molecular genetic and evolutionary studies [ 20 – 27 ]. Utricularia is also a large genus, comprising about 235 species with varying trap shapes, allowing for comparative analysis [ 28 – 31 ].

Epiascidiate leaves have evolved four times independently: in the families Nepenthaceae, Sarraceniaceae, Cephalotaceae, and Lentibulariaceae [ 13 , 14 ]. In all these cases, the epiascidiate form is associated with nutrient acquisition from animals (carnivory). Based on comparative anatomy, the inner surface of the epiascidiate leaf is believed to be equivalent to the adaxial surface of a planar leaf, whereas the outer surface is equivalent to the abaxial surface [ 4 , 13 , 15 , 16 ]. The petiole of the epiascidiate leaf inserts on the abaxial side, similar to the situation for a peltate leaf [ 13 ]. However, the mechanism by which the epiascidiate leaf is initially formed and then shaped during development is poorly understood. Here, we address the developmental mechanisms controlling the second aspect, involving shaping of a highly curved sheet.

Many plant and animal organs, such as leaves, flowers, hearts, and wings, derive from tissue sheets. A general question in developmental and evolutionary biology is how tissue sheets are shaped to create such a diversity of forms. A good illustration is leaf development. Leaves exhibit remarkable variation in shape, from simple or compound planar forms to convoluted three-dimensional forms such as those of pitcher plants. The molecular genetic control of leaf shape has been extensively studied for planar forms, with key genes modifying leaf shape identified [ 1 – 6 ]. Variation in patterns of gene activity has also been shown to underlie variation in leaf shape between species [ 7 – 10 ]. Clonal analysis and tracking and monitoring cell division have further revealed spatiotemporal variation in patterns of division and growth, and led to the formulation of models for how shape arises through local variations in rates and orientations of growth [ 11 , 12 ]. However, it is unclear how these models for planar leaf development are related to morphogenetic changes in highly curved 3D leaf forms, such as epiascidiate (cup or tubular-shaped) leaves.

Results

Growth rates during trap development To explore hypotheses for how transformations in trap shape arise, we used a computational modelling approach. Such an approach is more powerful if constrained by known growth rate measurements. To obtain these measurements, we first established a temporal framework for trap development by following the growth of individual traps. At very early phases of development, traps were hidden from view because they were held within a spiral structure, termed the circinate apex [39]. We imaged traps at daily intervals from when they emerged from the spiral until they reached maturity. Trap length was estimated according to the distance from the dorsal lip (Fig 1C, red square) to the furthest point at the back of the trap (Fig 1A, red line). Plotting log of trap length against time gave an estimated strain rate (relative growth rate) of 1.8% h−1 ± 0.13 (Fig 3J, S3 Data, all measured rate estimates are given with ± twice the standard deviation). The growth curve was extrapolated back in time (Fig 3J, dotted line) to define an initiation time (0 days after initiation [DAI]), corresponding to a length of 10 μm (i.e., approximately 1–2 cells). Using this growth curve, a standard time in DAI could be assigned to any trap based on its length (blue region, Fig 3J, S4 Data). The above framework allowed us to determine strain rates for various trap domains that could later be used to constrain parameters in growth models. We first measured circumferences in the three section planes at different stages of development (Fig 3K, S3 Data). The strain rate was higher along the sagittal circumference (1 .65% h−1 ± 0.06) compared to the other circumferences (1.52% h−1 ± 0.07 and 1.39% h−1 ± 0.06). The sagittal section was further divided into three subdomains based on three landmarks that could be identified throughout development (Fig 1C and Fig 3A–3I, green, magenta, and red squares). These landmarks allowed three domains to be defined: ventral midline, dorsal midline, and stalk diameter (Fig 1C, magenta, red, and green domains). Strain rates for these regions were then estimated from the staged traps (Fig 3L, S3 Data). The ventral midline grew faster (2.07% h−1 ± 0.09) than the dorsal midline (1.71% h−1 ± 0.09), and the stalk diameter grew the slowest (0.77% h−1 ± 0.13).