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A pea seed mutant affected in the differentiation of the embryonic epidermis is impaired in embryo growth and seed maturation

¹ Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany
² Department of Applied Genetics, John Innes Centre, Colney, Norwich NR4 7UH, UK

View larger version (42K): [in a new window]   Fig. 1. Hand cut sections of E2748 mutant and wild-type seeds harvested from the same pod at different developmental stages. e, embryo; v, endospermal vacuole; sc, seed coat. Scale bars: 3 mm.

View larger version (13K): [in a new window]   Fig. 2. Developmental parameters of E2748 mutant and wild-type seeds. Fresh weight accumulation of seed coats (A) and embryos (B), and endospermal vacuole volumes (C). Data points represent single measurements. See also legend of Fig. 13, for staging of pea seed development.

View larger version (16K): [in a new window]   Fig. 3. Concentrations of hexoses (sum of glucose and fructose) and sucrose within endospermal vacuoles of E2748 mutant and wild-type seeds during development. Data points represent single measurements.

View larger version (14K): [in a new window]   Fig. 4. Levels of sucrose (A) and starch (B) in embryos of E2748 mutant (black symbols) and wild-type (white symbols) seeds during development. Data points in A represent single measurements. Data points in B represent means ± s.d. of 3-5 measurements.

View larger version (120K): [in a new window]   Fig. 5. Epidermal and transfer cell structure in wild-type pea embryos stained with Toluidine Blue in A,E,I and viewed by transmission electron microscopy in B-D,F-H,J-L. (A) Young embryo at late heart stage (50 mg seed weight) with single epidermal cell layer (ep) and underlying parenchyma cells (p). (B) Ultrastructure of the epidermal layer and parenchyma cells seen in A. (C) Morphology of outer wall of epidermal cell (upper frame in B). (D) Morphology of inner wall of epidermal cell, facing the parenchyma (lower frame in B). (E) Embryo from a 160 mg seed with single-celled transfer layer (ep) and underlying parenchyma cells (p). (F) Ultrastructure of the transfer cell layer and underlying parenchyma cells seen in E. (G,H) Morphology of (G) outer cell wall (upper frame in F), (H) inner cell wall of transfer cell (lower frame in E). (I) Embryo from a 260 mg seed showing single-celled transfer layer (arrowheads) and underlying parenchyma cells (p). (J) Ultrastructure of transfer layer and underlying parenchyma cells seen in I. (K) Higher magnification of upper frame in J showing cell wall ingrowth (arrowhead). (L) Higher magnification of lower frame in J showing branched-type plasmodesmata (arrowhead). ap, amyloplast; ep, epidermis; er, endoplasmic reticulum; m, mitochondrion; n, nucleus; p, parenchyma; v, vacuole. Scale bars: 10 µm (A), 5 µm (B), 1.25 µm (C,D), 6 µm (E,F,I), 0.4 µm (G, H), 4.5 µm (J), 0.56 µm (K, L).

View larger version (83K): [in a new window]   Fig. 6. Epidermis and cell structure in a single E2748 embryo (60 mg seed, transition state), Toluidine Blue stained in A-D and transmission electron microscopy in E-G. (A) Young embryo at the early cotyledon stage with single epidermal cell layer having wild-type-like character (arrowheads). (B) As in A but note changed cell shape (arrowheads). (C) Highly vacuolated modified epidermal cells with altered surface. Dashed line indicates increasing morphological aberrations. (D) Modified epidermal cell with periclinal as well as anticlinal cell divisions (arrowheads). (E) Contact zone of modified epidermal cell and endosperm; note the tight contact zone with an increase in mitochondria and membrane structures. (F) Outer region of modified epidermal cell. (G) Inner region of modified epidermal cell (arrowhead indicates cell wall). ch, chloroplast; em, embryo; en, endosperm; er, endoplasmic reticulum; m, mitochondrion; n, nucleus; v, vacuole. Scale bars, 10 µm (A-D), 3 µm (E), 1.1 µm (F,G).

View larger version (124K): [in a new window]   Fig. 7. Cell structure in an E2748 seed (160 mg seed in A-C and 260 mg seed in D-E), Toluidine Blue staining in A,D and transmission electron microscopy in B,C,E. (A) Contact zone of cotyledon with seed coat (arrowhead 1) and with the endosperm (arrowhead 2). (B) Ultrastructure of the cotyledon to seed coat contact zone. (C) Ultrastructure of the cotyledon with endosperm contact zone, note the irregularly thickened cell wall (arrowheads). (D) Contact zone of cotyledon with seed coat (260 mg seed). (E) Ultrastructure of the cotyledon to seed coat contact zone. Note the large differences in cell size of the outermost cells. ch, chloroplast; en, endosperm; er, endoplasmic reticulum; n, nucleus; v, vacuole. Scale bars, 40 µm (A,B,D,E), 5 µm (C).

View larger version (92K): [in a new window]   Fig. 8. Cotyledon cell morphology and starch deposition in wild-type (A,C,E) and mutant embryos (B,D,F) at mid-cotyledon stage. The section through the abaxial region of a mutant embryo shows highly vacuolated cells (arrowhead) on the surface and in the outer tissue layers of the cotyledon, whereas cell sizes increase toward the interior (B). After iodine staining in wild-type cotyledons, starch grains are barely recognisable because of the low resolution (C). Starch deposition in the outer tissue layers of the mutant cotyledon (D). In the seed coats of both wild-type and mutant seeds starch accumulation is similar (C,D). Accumulation of starch grains during maturation coincides with the different cell size gradients in both wild-type (E) and mutant cotyledons (F). Scale bars: 125 µm (A,B); 450 µm (C,D); 200 µm (E,F).

View larger version (24K): [in a new window]   Fig. 9. Transcript levels of transport- and storage-associated genes in embryos (A,C,E,G,I,K) and seed coats (B,D,F,H,J,L) of E2748 and wild-type cotyledons during development. The experiment had been repeated twice with similar results. The figure shows representative results from a single experiment. Identical blots were hybridised by sequential rounds of northern hybridisations with probes detecting the following mRNAs: sucrose transporter (PsSUT1; A,B); potassium channel (PsKT1; C,D); H+-ATPase (PsATP1; E,F); sucrose synthase (PsSUS1; G,H); ADP glucose pyrophosphorylase large subunit (PsAGPL; I,J); plastidial phosphoglucomutase (PspPGM; K,L). The values on the x axis refer to seed fresh weight of wild-type seeds. Black symbols: mutant; white symbols: wild type.

View larger version (147K): [in a new window]   Fig. 10. Localisation of Sut1 mRNA in developing cotyledons of wild-type and mutant phenotype. Toluidine Blue stained sections are shown under bright-field illumination (A,C,D,F,H,J,K,M) and the same section after in situ hybridisation under dark-field illumination (B,E,G,I,L,N): signals are seen as white grains. (A) Wild-type seed of 60 mg weight and (B,C) its cotyledons (see frame in A). (D) Mutant seed of 60 mg weight and (E,F) its cotyledons (see frame in D). (G,H) Mutant cotyledon of a 160 mg seed. Note the low signal intensity in certain regions (arrowheads). (I,J) High signal intensity of SUT1 in vacuolated cells of the outer boundary (frame in G). (K) Wild-type seed at mid-cotyledon stage. Localisation of abaxial transfer cells is indicated by an arrowhead. (L) Cotyledons (see frame in K) after in situ hybridisation (arrowhead indicates abaxial transfer cells). (M,N) Mutant seed at mid-cotyledon stage, arrowheads indicate the cotyledon to seed coat boundary. (N) Note the completely irregular pattern of the in situ hybridisation signal. ab, abaxial; ad, adaxial; ax, embryo axis; co, cotyledon; e, endosperm; ev, endospermal vacuole; sc, seed coat. Scale bars, 0.7 mm (A,D), 0.1 mm (C,F), 0.4 mm (H,L), 0.2 mm (J), 1.5 mm (K,M,N).

View larger version (79K): [in a new window]   Fig. 11. Distribution of fluorescent symplasmic tracer in embryo tissues of E2748 and wild type. Optical sections of wild-type (A-C) and mutant (D-F) embryos at the early cotyledon stage. The tissues were incubated with CFDA for 10 minutes (A,D) and 1 hour (B,C,E,F). Arrowheads in A,B and G indicate the boundaries of the outer cell layer and point towards the interior of the cotyledon parenchyma. In mutant embryos the tracer accumulated only in distinct groups of cells (E,F). (G-I) Tissue was treated for 15 minutes with CFDA treatment following a 1-hour chase period without CFDA. Symplasmic tracer (arrowheads) is visible only in the outer region of the wild-type cotyledon (G), whereas in the E2748 mutant small groups of cells inside the parenchyma are labelled (H, arrowheads). (I) A seed of 200 mg where the embryo was tightly adhered to the inner layers of the seed coats was cut off at the chalazal part without injuring the embryo and the wounded seed coat surface was brought in contact to CFDA. Tracer is present within the seed coat parenchyma (sc p) and the endospermal tissue (en) but not within embryonic tissue (e) which appears as a black layer. Scale bars: 200 µm (A,B,D,E); 1 mm (C,F).

View larger version (53K): [in a new window]   Fig. 12. Local concentration of sucrose within a wild-type (A) and mutant seed (B) determined by quantitative bioluminescence imaging. Sections used for the experiment are shown in C and D for the wild type and the mutant, respectively. In wild-type cotyledons higher sucrose is present within the adaxial region (ad) and lower values within abaxial regions (ab). In mutant cotyledons sucrose is generally lower but concentrations are relatively higher in the abaxal regions adjacent to the seed coat (sc). Scale bar: 1 mm.

View larger version (43K): [in a new window]   Fig. 13. Schematic view of major histological changes in E2748 (1m-5m) and wild-type (1-5) embryos. The underlying colour indicates the sugar status from a high hexose to sucrose ratio (H>S) to a low hexose to sucrose ratio (H<S) changing during development. The E2748 mutation blocks epidermal differentiation. The mutant phenotype is not recognisable in cotyledons of the early stage (1, <50 mg seed weight). Primary differences in epidermal morphology occur during early cotyledon stage (transition from 2m to 3m, 60 mg seed weight) when cells of the outer cell layer of the mutant become vacuolated (3m) instead forming transfer cells (3). Stages 4 (160 mg seed weight) to 5 (260 mg seed weight) represent the main storage phase. Development is arrested in the mutant (4m – 5m). For further details see text.

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