The role of the testa during the establishment of physical dormancy in the pea seed

Abstract

Background: A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing dormancy. The physical and functional properties of the macrosclereids are thought to be critical determinants of dormancy; however, the mechanisms underlying the maintenance of and release from dormancy in pea are not well understood.

Methods: Seeds of six pea accessions of contrasting dormancy type were tested for their ability to imbibe and the permeability of their testa was evaluated. Release from dormancy was monitored following temperature oscillation, lipid removal and drying. Histochemical and microscopic approaches were used to characterize the structure of the testa.

Key results: The strophiole was identified as representing the major site for the entry of water into non-dormant seeds, while water entry into dormant seeds was distributed rather than localized. The major barrier for water uptake in dormant seeds was the upper section of the macrosclereids, referred to as the 'light line'. Dormancy could be released by thermocycling, dehydration or chloroform treatment. Assays based on either periodic acid or ruthenium red were used to visualize penetration through the testa. Lipids were detected within a subcuticular waxy layer in both dormant and non-dormant seeds. The waxy layer and the light line both formed at the same time as the establishment of secondary cell walls at the tip of the macrosclereids.

Conclusions: The light line was identified as the major barrier to water penetration in dormant seeds. Its outer border abuts a waxy subcuticular layer, which is consistent with the suggestion that the light line represents the interface between two distinct environments - the waxy subcuticular layer and the cellulose-rich secondary cell wall. The mechanistic basis of dormancy break includes changes in the testa's lipid layer, along with the mechanical disruption induced by oscillation in temperature and by a decreased moisture content of the embryo.

Keywords: Pisum sativum seed; Hardseedness; light line; macrosclereid; physical dormancy; seed coat; subcuticular lipids; testa; water permeability.

Fig. 1.

Control of water entry during the germination of non-dormant seeds of Terno, Cameor and J192. The hilum (H), strophiole (S) and hilum + strophiole + micropyle (HSM) were sealed with warm lanolin; control seeds (C) were not treated with lanolin. Statistically significant within-treatment differences are indicated by different letters at the head of each column.

Fig. 2.

Testa surface of dormant seeds of JI64, L100 and VIR320 before (A, C, E) and after (B, D, F) thermocycling treatment applied to (A, B) JI64, (C, D) L100 and (E, F) VIR320 seeds. Fissures (arrowed) developed on the seed surface of JI64 and L100, but not on the surface of the other seeds. Arrowheads indicate gritty structures on the surface of JI64 and L100 seeds. Scale bars = 0.5 mm (50 μm in the insets).

Fig. 3.

Variation in testa permeability between dormant (JI64: A, D, G, J, M), non-dormant (JI92: B, E, H, K, N) and Terno (C, F, I, L, O) seeds. Permeability was assayed either using periodic acid–Schiff solution (violet staining) or ruthenium red (red staining). (A–C) Schiff solution staining without periodic acid treatment. Scale bars = 50 μm. (D–F) Periodic acid treatment. Scale bars = 50 μm (12.5 μm in the insets). (G–I) Ruthenium red staining. Scale bars = 50 μm. (J–L) Ruthenium red staining around the hilum. Scale bars = 100 μm. (M–O) Ruthenium red penetrating the testa. Arrows in (A–L) indicate the LL.

Fig. 4.

The presence of pectin in the testa cell wall, as shown by ruthenium red staining of paradermal sections. (A) In JI92, the macrosclereid cavity (arrow) branches into fine channels and the cavity is filled with extracellular waxy material. (B) An oblique paradermal section of Terno demonstrates the arrangement of the central cavity of the macrosclereids at various distances from the cuticle; fine channels near the cuticle are indicated by arrows, and larger ones at a greater distance from the cuticle by asterisks. (C) At the surface of a JI64 seed, ruthenium red stains only the tops of the gritty surface. (A–C) Macrosclereid cavities are indicated by arrows. Compound middle lamellae are indicated by arrowheads. Scale bars = 25 μm.

Fig. 5.

Micrographs showing the detailed structure of the macrosclereids. (A) Longitudinal section of the JI64 testa, showing the outer part of macrosclereids, including their terminal caps, composed of compact extracellular material, and the subcuticular waxy layer. Arrows indicate the lower surface of the LL. Scale bar = 10 μm. (B) Individual macrosclereids of JI64 released from the testa after pectinase treatment and stained with calcofluor white. Arrows indicate the lower surface of the LL and asterisks the macrosclereids’ internal channels. The inset box indicates enlarged portion of the macrosclereids presented on the left. Scale bar = 10 μm. (C) Three-dimensional projection illustrating deposition of waxy material within the branched internal macrosclereid cavity. (D) Autofluorescence (two-photon 720-nm excitation) due to the waxy content of the branched internal macrosclereid cavity, as seen from the testa surface. Scale bar = 10 μm. (E) Surface paradermal section showing differences in the macrosclereid cavity at various distances from the cuticle. The macrosclereid cavity is branched at the terminal cap; the branches are fine in the vicinity of the LL and more extensive in the basal part of the macrosclereid. Scale bar = 25 μm.

Fig. 6.

Lipids in the testa visualized in cross-sections of JI64 (A, C, E, F) and JI92 (B, D). The testas were stained with Sudan red for 2 h (A, B), treated with hexane before staining with Sudan red (C, D) or treated with sulphuric acid (E, F). The sections were photographed either in bright-field illumination (A–E) or with UV excitation (F) to generate autofluorescence. The LL is indicated by arrows, the subcuticular waxy layer by stars and the position of cuticle by arrowheads. Scale bars = 25 μm.

Fig. 7.

Effect of thermocycling on testa permeability. Surface permeability of VIR320 (A–C, M–O), L100 (D–F, P–R) and JI64 (G–I, S–U) seeds after 2 weeks of thermocycling as assayed with periodic acid (A–I), with Schiff’s solution without periodic acid (J–L) or with ruthenium red (M–U). The arrows point to the LL. (A, D, G, J, M, P, S) Seed side. Scale bars = 50 μm. (B, E, H, K, N, Q, T) Hilum. Scale bars (B, E, H) = 200 μm, (K, Q) = 100 μm, (N, T) = 50 μm. (C, F, I, L, O, R, U) Strophiole. Scale bars (C, I) = 200 μm, (F, L, O, R, U) = 100 μm.

Fig. 8.

Changes in the permeability of the testa of the dormant entry JI64 following chloroform treatment. Surface permeability was assayed using periodic acid (A–C), ruthenium red (D–F) or Schiff’s solution without periodic acid (G–I). The LL is indicated by an arrow. (A, D, G) Seed side. Scale bar = 50 μm. (B, E, H) Hilum. Scale bar = 100 μm. (C, F, H) Strophiole. Scale bar = 100 μm. (J–L) Macrographs of intact JI64 seeds following ruthenium red staining of chloroform-treated material. (J) Seed side, (K) hilum, (L) strophiole.

Fig. 9.

Cracking of the testa after freeze-drying and chloroform treatment (A, B). Control (no treatment) (C, D). Immediately after freeze-drying treatment. Isolated testas are shown on the left and testas with embryo on the right (E, F). Rehydration following freeze-drying treatment. Isolated testas are shown on the left and testas with embryo on the right (G, H). Thermocycling treatment (A, C) J192, (B, D, F, G, H) J164.

Fig. 10.

Development of the cuticle, subcuticular waxy layer and the LL. Cross-sections of seeds of JI92 harvested at 10, 14 and 17 d post-anthesis (DPA) (A–F) and JI64 harvested at 9, 14, 15 and 17 DPA (G–N) stained with Sudan red (A–C, G–J) or toluidine blue (D–F, K–N). The LL is indicated by arrows, the subcuticular waxy layer by stars and the cuticle by arrowheads. (A, D) 10 DPA, (B, E) 14 DPA, (C, F) 17 DPA; (G, K) 9 DPA, (H, L) 14 DPA, (I, M) 15 DPA, (J, N) 17 DPA. Scale bars = 50 μm (20 μm in inset).

Fig. 11.

The hilum aperture responds to water entry. Cross-sections of JI64 (A, C) and JI92 (B, D) seeds taken before (A, B) and after (C, D) imbibition. Arrows indicate the aperture. Scale bar = 200 μm.