Fruit fracture biomechanics and the release of Lepidium didymum pericarp-imposed mechanical dormancy by fungi

Abstract

The biomechanical and ecophysiological properties of plant seed/fruit structures are fundamental to survival in distinct environments. Dispersal of fruits with hard pericarps (fruit coats) encasing seeds has evolved many times independently within taxa that have seed dispersal as their default strategy. The mechanisms by which the constraint of a hard pericarp determines germination timing in response to the environment are currently unknown. Here, we show that the hard pericarp of Lepidium didymum controls germination solely by a biomechanical mechanism. Mechanical dormancy is conferred by preventing full phase-II water uptake of the encased non-dormant seed. The lignified endocarp has biomechanically and morphologically distinct regions that serve as predetermined breaking zones. This pericarp-imposed mechanical dormancy is released by the activity of common fungi, which weaken these zones by degrading non-lignified pericarp cells. We propose that the hard pericarp with this biomechanical mechanism contributed to the global distribution of this species in distinct environments.

Fig. 1

The dispersal units of Lepidium didymum are fruit valves each harbouring a single seed encased in a hard pericarp (fruit coat). a Oblate spheroid-shaped fruits each consisting of two fruit valves attached to the replum. b Detachment from the fruit’s replum at maturity provides two dispersed fruit valves. The mature fruit valves have a hard pericarp and a ‘Natural Pericarp Opening’ (NPO) that permits water uptake and gas exchange. c Dry fruit valve, cracked open, exposing the encased single dry seed that fills almost the entire pericarp cavity. The seed is always positioned inside the cavity with the radicle (embryonic root) directed towards the distal pericarp region adjacent to the NPO. The pericarp layers and the ‘Predetermined Breaking Zone’ (PBZ) along the distal and dorsal pericarp regions are indicated. d Germinating fruit valve with pericarp rupture initiated in the distal pericarp region (crack initiation point) adjacent to the NPO. e Germinated fruit valve opened like a shell by progressed pericarp rupture to facilitate radicle emergence

Fig. 2

The comparative germination of Lepidium didymum fruit valves and extracted seeds reveals the mechanical constraint of the pericarp to full phase-II seed water content. a Time course of visible germination of surface-sterilised, intact and scarified fruit valves compared to extracted seeds (manually removed from cracked open fruit valves). Note that extracted seeds are non-dormant (ND) and germinate readily in the fresh mature state. Mean values ± SE (N = 3 × 50) of accession KM2423 at conditions identified to be optimal for the germination of fresh, mature seeds (Supplementary Fig. 2a–e) and therefore used as ‘standard conditions’ (15/5 °C day/night with 12 h photoperiod, white light at ~100 µmol/m²/s¹). The timing of testa rupture (TR; see e) of seeds within fruit valves is indicated (brown arrows) as percentage of the population (onset at T_1% until maximum at T_100%). Note that TR is completed prior to the onset of pericarp rupture, and that the TR confirms the ND state of the seeds. b Water uptake patterns of extracted seeds compared to seeds within fruit valves. Seed extracted from dry (day 0) fruit valves exhibit a typical three-phasic pattern of water uptake: phase-I (imbibition) is followed by the plateau phase-II (metabolic activation) and upon endosperm rupture the radicle emergence is associated with phase-III water uptake. The water content of seeds within fruit valves without pericarp rupture remained in phase-II. The phase-II water content of seeds within fruit valves was significantly lower compared to the phase-II water content of extracted seeds. Pericarp rupture and radicle emergence were associated with phase-III water uptake of seeds within fruit valves; see Supplementary Fig. 4 for further details and statistical analyses. c Extracted seeds germinate with testa rupture followed by endosperm rupture as two visible steps; see Supplementary Fig. 2a–e for a detailed analysis of seed germination. d Fruit valves germinate with pericarp rupture as visible step. e Fruit valve, cracked open, with seed exhibiting testa rupture, which occurred inside the fruit valve during imbibition. For NPO and PBZ see Fig. 1

Fig. 3

The promotional effect of fungal activity on the germination kinetics of Lepidium didymum fruit valves. a Time course of visible germination of ‘untreated’ (fungi) compared to ‘surface-sterilised’ (no fungi) fruit valves. Note that the promotional effect on pericarp rupture was also evident when surface-sterilised fruit valves were re-inoculated with the fungi. The germination kinetics of extracted seeds was not affected by the fungi, nor were the phase-II water content, seed viability or seedling growth (Supplementary Figs. 2 and 4). Mean values ± SE (N = 3 × 50) of accession KM2423 at standard conditions. The time for the pericarp biomechanics (Fig. 5) is indicated (pink arrow). b Mean values ± SE of the effect of fungi on the germination of fruit valves and extracted seeds from ten independent L. didymum accessions. The individual time courses are presented in Supplementary Fig. 3 and the origin of the ten accessions is listed in Supplementary Table 1

Fig. 4

Colonisation of Lepidium didymum fruit valves with fungi leads to degradation of the outer pericarp layers without visible effects on the lignified endocarp. a Light microscopy (LM) of a full longitudinal fruit valve section with safranin-astrablue illustrates the distinct pericarp layers (see Fig. 6a detailing the location of the section). For the endocarp, the intense red staining indicates highly lignified thick secondary cell walls of dead cells. For the exocarp and mesocarp the blue staining of the non-lignified primary cell walls indicates living parenchymatic cells. b–d Scanning electron microscopy (SEM) of fruit valves visualising the colonisation of the outer pericarp by fungi during incubation at standard conditions. e–g LM of pericarp demonstrating the degradation of the exocarp and mesocarp layers by fungal activity after 7 days of incubation while the lignified endocarp and the seed’s testa did not show any visible degradation. The surface-sterilised control (no fungi) shows no pericarp degradation. h Effect of localised mechanical abrasion of either distal or proximal outer pericarp on the time course of pericarp rupture of surface-sterilised fruit valves (no fungi). Note that the localised mechanical abrasion was conducted in a way that the endocarp remained intact (see Supplementary Fig. 6 for details)

Fig. 5

Fracture biomechanics of the pericarp and the effect of fungal degradation on the mechanical resistance of distinct Lepidium didymum fruit valve regions. a Comparative puncture force analysis of the distal and proximal pericarp regions. Fungal activity caused a drastic decrease by ~52% in the mechanical resistance (breaking strength) of the distal pericarp (p-value < 0.001) where the pericarp rupture is initiated and the radicle will emerge. A smaller but significant decrease by fungal activity of ~30% was evident in the proximal (p-value = 0.04) and dorsal (Supplementary Fig. 2c) pericarp regions. To conduct the biomechanical analyses, fruit valves of accession KM2423 were incubated at standard conditions for the time indicated either untreated (fungi colonise the pericarp) or surface-sterilised (no fungi). No decrease in the mechanical resistance was observed upon surface-sterilisation (distal p-value = 0.89; proximal p-value > 0.99). Mean values ± SE (N ≥ 26). b Comparative force–displacement curves revealing distinct fracture biomechanical properties, namely, sudden complete failure (fatal ‘brittle’ failure) for the distal pericarp, and slower gradual failure (‘composite’ failure) for the proximal pericarp. This breaking behaviour clearly identifies this distal region mechanically as the PBZ crack initiation zone (iPBZ) for pericarp rupture, which upon mechanical failure causes the fruit valve to split into half. In contrast to this, in the proximal pericarp the measuring needle was driven through the proximal pericarp, layer by layer until the fruit valve finally split in half. Examples presented are from surface-sterilised fruit valves (35 days); the same difference in breaking behaviours between distal and proximal pericarps were evident for the other conditions. c Light microscopy of a full centric longitudinal fruit valve section exhibiting the distinct pericarp layers (toluidinblue histostaining). Arrows indicate force directions for the biomechanical analyses

Fig. 6

Microscopy of the L. didymum fruit valve reveals functional–morphological and micromechanical distinct endocarp regions. a Schematic drawing of the fruit valve to illustrate the orientation of imaged sections. The seed is always positioned inside the cavity with the radicle (embryonic root) directed towards the distal pericarp region adjacent to the ‘Natural Pericarp Opening’ (NPO) where the pericarp rupture initiates (iPBZ, crack initiation zone). b Scanning electron microscopy (SEM) top view onto the inside wall and the smooth breaking edge along the PBZ. The pericarp rupture spreads from the iPBZ, along the distal and dorsal pericarp as indicated (arrow). c SEM top view onto the smooth breaking edge of the lateral pericarp region. d SEM view onto the inside valve endocarp at the PBZ. e SEM top view onto the pericarp at the distal NPO border. f Light microscopy (LM) cross section of the pericarp at the distal NPO border. Red safranin staining indicates intense lignification of the dead thick-walled endocarp cells. Astrablue staining indicates non-lignified primary cell walls of the living parenchymatic exocarp and mesocarp cells. g SEM top view onto the pericarp at the proximal NPO border. h LM cross section of the pericarp at the proximal NPO border. i LM longitudinal section (safranin-astrablue histostain) of the distal pericarp with the iPBZ (crack initiation zone). Reduced lignification of the iPBZ endocarp compared to the adjacent endocarp is evident. j, k Fluorescence microscopy of the distal pericarp with the iPBZ. j Red fluorescence in the iPBZ due to binding of wheat germ agglutinin (WGA, conjugated with Alexa Fluor 633 nm) indicates distinct glycoprotein or hemicellulose composition of the iPBZ endocarp compared to the adjacent endocarp. k Autofluorescence (control without WGA) supports reduced lignification of the iPBZ. c, e–h, Red arrows, parallel oriented long endocarp cells. Yellow arrows, cross-sectioned endocarp cells