Morphological characterization of seed capsules

Figure 1 summarizes the hierarchical structure of the seed capsule from D. nakurense in the dry and hydrated state and provides a schematic representation of the three-dimensional structure at each level. The axes in the schematics are defined relative to the position and orientation of the keel and its cells. In the dry state, five protective valves cover the seed compartments, preventing premature dispersion of seeds (Fig. 1a). When hydrated with liquid water, each valve unfolds outwards and backwards over an angle of ∼150° within minutes (Fig. 1a–c; Supplementary Movie 1). Seed compartments are partitioned by five septa, which lie beneath the centre of each valve in the closed dry state. The schematic in Figure 1b is a 3D illustration of the whole capsule in which each valve is depicted at a different stage of opening, to provide a more intuitive representation of the subsequent steps involved in the unfolding process. The hygroscopic keel, a prominent tissue attached along the centre of the inner valve surface can clearly be seen in Figure 1c and was previously suggested to actuate this water-dependent movement8,9. The reversible water-actuated flexing movement of the keel can still be observed even when it is dissected from the backing (Fig. 1d–f; Supplementary Movies 2 and 3). The schematic in Figure 1e further illustrates the progression of this movement, which occurs over a time scale of a couple of minutes.

The keel consists of two separated halves with single cells of different lengths running from the top to the bottom of the keel (the longitudinal axis of the cell is assigned as the Z-direction) (Fig. 1e,g,h). The two halves of the keel are separated in the dry state, but come into contact by swelling when hydrated, creating a bridge-like structure with an empty space between the upper portion of the keel and valve backing (Fig. 1g,h). Additionally, changes in the hydration state appear to result in a curvature of the valve backing in the dry state and straightening in the wet state (Supplementary Movie 4).

Transverse sections of keels reveal a lattice structure with hexagonal/elliptical shaped cells in the wet state. When dried, the elliptical shaped cells are collapsed preferentially in the shorter axis of the transverse cell cross-section, which we assign as the Y-direction (Fig. 1i–k). Consequently, the longer axis of the transverse cell cross-section is assigned the X-direction. The orientation of the cells within the larger keel structure, as well as the hydration-dependent changes in shape, is depicted in the schematic in Figure 1j.

The stepwise changes in the keel during drying were examined in more detail using image correlation to track the progressive movements (Fig. 2). From the plot in Figure 2d, it is apparent that the tip of the keel (point A) is translated about the base (point E) through a large angle. Additionally, there is a point of maximum bending in the keel in the dry state, located between points C and D in Figure 2c, that we define as the 'hinge' of the flexing movement. It is also clear from the insets in Figure 2a,c (cut made through the keel between points D and E) that the gap between the two keel halves in the dry state allows them to fit tightly around the septum as they close, without being obstructed. Because the valves are closed during development, the gap between the keels in the dry state represents the initial conformation and seems to be a prerequisite for a tight packing of the valve.

Figure 2: Detailed analysis of keel movement. Image correlation was used to track the movement of a dissected keel during drying. A keel attached to the septum with the backing dissected away can be seen in the wet state (a), an intermediate state (b), and the dry state (c). The red spots labelled A–E were followed in consecutive frames to observe the relative movements of the keel. Insets (a, c) show the tight packing of the keel around the septum in the dry state. (d) plot of relative movements of points A–E during drying. Full size image

Morphology and composition of hygroscopic keel cells

The large and directed change in the Y-direction of keel cells upon hydration indicates that the flexing movement of the keel originates at the cell and cell wall level. In fact, cells show a highly anisotropic deformation with a fourfold increase in length along the Y-direction (short cross-sectional cell axis) whereas the X-direction (long cross-sectional cell axis) length shows only minor changes (Table 1). This is accompanied by a nearly fourfold enlargement of the cross-sectional area and a modest increase in the perimeter (Table 1). Additionally, it was observed that the cell length in the Y-direction of the expanded wet state was 20% lower in keel cells attached to the backing (Fig. 1e,g,h) than in those near the ridge of the keel (T-test P<0.005). This asymmetrical deformation facilitates the bending of the keel during cell swelling.

Table 1 Transverse cell shape changes during wetting and drying cycles. Full size table

Cell wall organization and composition were investigated by staining with a fuchsin-chrysoidin-astra blue mixture (FCA), which revealed lignified cell walls (red staining) and a non-lignified cellulose structure filling the lumina (bright blue staining) (Fig. 3a). Further characterization with confocal Raman spectroscopy (Fig. 3b–e) corroborated these observations. The main Raman peaks for cellulose appear in both the cell wall and the inner layer, whereas the lignin peak appears only in the cell wall and between cells (Fig. 3b–d). Whereas the inner layer, which we will hereby refer to as the cellulosic inner layer (CIL), may contain additional biomacromolecules such as pectin or hemicelluloses in smaller amounts10,11, it is most important for this study that the CIL can apparently absorb large amounts of water (Fig. 3e). Additionally, the CIL appears to be organized as thin lamellae running parallel to the X-direction (Fig. 3c). Consecutive laser scanning confocal microscopy images of drying keel cells in which the cell wall and CIL are differentially stained reveal that, during cell collapse, the CIL retracts towards and remains attached to the surrounding cell wall (Fig. 3f–h; Supplementary Movie 5), suggesting the CIL may have an active role in the reversible actuated cell deformation.

Figure 3: Biomolecular composition of hygroscopic keel cells. (a) FCA staining reveals a lignified thin cell wall (red) and a CIL in the cell lumen (blue). (b–e) Confocal Raman spectroscopic images of keel cells integrated for cellulose (b and c, 1,067–1,143 cm−1) at two different intensity levels, lignin (d, 1,546–1,643 cm−1), and water (e, 2,223–2,725 cm−1). (f–h) Consecutive confocal microscopy images of safranine-stained keel cells during drying, coloured to differentiate between the cell wall (red) and CIL (green-yellow). (i) Schematic of changes in keel cell cross-sectional shape following treatments. Relative cell shapes are represented as idealized hexagons with the appropriate ratios of X to Y lengths. Thick black arrows represent the direction of change in the X and Y lengths of the cell following the respective treatment. Scale bars are defined as follows: a=25 μm; b–d=20 μm; f–h=50 μm. CCD, cts, Full size image

Mechanics of hygroscopic keel cell swelling

Following enzymatic removal of the CIL, cells largely lose the ability to close during drying (Fig. 3i and Table 2). In addition, removal of the CIL in the wet state results in a cell-shape relaxation in which the Y-length decreases and the X-length increases (Fig. 3i and Table 2). These observations suggest that the CIL has a role in both closing and opening of the cells. Specifically, the hydrated CIL may generate outward stresses on the cell wall through an internal pressure during swelling, thus facilitating the cell opening. For this particular mechanism to function, it would be necessary for the cells to deform more easily in the Y- than X-direction. In fact, calculations of elastic deformability based purely on geometrical considerations support this assumption. If we consider keel cells as an ideal cellular solid with a hexagonal shape as depicted in Figure 3i, the ratio of stiffness in the X- and Y-direction can be calculated according to equation (1) derived from Gibson & Ashby12.

Table 2 Relative changes in cell dimensions due to hydration changes and enzymatic removal of CIL. Full size table

Based on the anatomical cell parameters measured in the dry keel (Fig. 3i and Table 1) and under the simplifying assumption of isotropic cell wall material, the keel tissue would be ∼5,500 times stiffer in the X-direction than in the Y-direction. When the cells are in the wet open state, this factor decreases to a value of only ∼30. This mechanical and geometrical anisotropy is consistent with a CIL-driven mechanism of keel cell opening. Additionally, the apparent lamellar structure of the CIL may lead to an anisotropic swelling which would further facilitate the deformation in the Y-direction; however, based on our calculations, this is not necessarily required.

Characterization of valve curvature

In addition to the flexing deformation of the valve described above, we also observe a change in the curvature of the valve backing in the XZ-plane, which may be important for the opening mechanism (Fig. 4). In the dry state, the backing shows a concave-curved morphology, which appears to be related to the gap formed between the two keel halves and thus important for tight valve packing (Figs 2c and 4). The curvature exists from the tip of the valve down to the hinge, with a graded reduction in the degree of curvature as one approaches the hinge. Figure 4a,b depicts the curvature in the wet and dry state of the keel backing at the hinge (cut between points C and D as defined in Fig. 2c). On wetting, the valve backing straightens and the tips of the keel halves move towards each other (Fig. 4a–d; Supplementary Movie 4). When keels have been dissected away from the backing, this change in curvature of the valve is not observed (Supplementary Movie 6), suggesting that the large deformation of the keel in the Y-direction may not only actuate the flexing movement, but may also additionally influence the valve curvature. This effect can be simulated by simple finite element modelling of contractile elements (keels) on top of a shell (backing) (Fig. 4e,f). Contraction of the model 'keels' in the Y-direction results in the development of elastic stresses in the shell which are released via a curvature of the shell in the XZ-plane in the expected direction. The magnitude of the curvature varies with shape, thickness and elastic properties of the backing and the keel. Comparable deformation mechanisms have been identified in other shell-like materials such as leaves where stress gradients arising from differences in growth rates within the leaf plane can be relieved by out-of-plane bending13,14.