Endosperm Weakening

Established and emerging seed model systems for endosperm weakening

• In the case of endosperm-limited germination, the endosperm acts as a mechanical barrier for radicle protrusion. A decline in the mechanical resistance of the micropylar endosperm (the endosperm covering the radicle tip) appears to be a prerequisite for radicle protrusion (selected reviews: Bewley, 1997, Leubner-Metzger, 2003, Kucera et al., 2005, Finch-Savage and Leubner-Metzger, 2006).
• Direct and indirect experimental evidence for endosperm weakening has been obtained in several species shown in the figure below. Direct evidence for endosperm weakening has been obtained by puncture-force measurement, i.e. the direct quantification of the force needed for puncturing the micropylar endosperm by a metal pin that has the shape of the radicle. Seeds of Arabidopsis or tobacco are too small for punture-force experiments. However, indirect evidence including microscopically visible early reserve breakdown in the micropylar endosperm or altered seed responses of Arabidopsis mutants and transgenic tobacco seeds, strongly supports the view that endosperm weakening is a widespread phenomenon.
• Solanaceae species like tomato, tobacco, pepper and Datura have become model species for endosperm weakening. Puncture force measurements of tomato, including the GA-deficient gib1 mutant, demonstrated that endosperm weakening is a prerequisite to germination. Endosperm weakening is regulated by plant hormones and by environmental factors. Endosperm weakening can be promoted by GA and, at least in part, inhibited by ABA.
• Until 2005 direct evidence for endosperm weakening and it's promotion by GA was only obtained for the established model systems of the Asterid clade (see figure): tomato and pepper (Solanaceae), Syringa and Fraxinus (Oleaceae), coffee (Rubiaceae), and lettuce (Asteraceae). Almost nothing was known for other clades including the highly developed Rosid clade (perisperm weakening of Cucumis was known, but the hormonal regulation has not been investigated so far). Endosperm weakening of tomato (Toorop et al., 2000) and coffee (da Silva et al., 2004) appears to be biphasic with respect to abscisic acid (ABA): The first phase is not inhibited by ABA, i.e. is ABA-insensitive; but the second phase leading to radicle protrusion is inhibited by ABA.
• The publication "Endosperm-limited Brassicaceae seed germination: Abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana" by Müller, Tintelnot and Leubner-Metzger (2006) provides the first direct evidence for endosperm weakening of the Rosid clade by using seeds of Lepidium sativum, a close relative of the model plant Arabidopsis thaliana. Lepidium and Arabidopsis endosperm rupture is promoted by GA and inhibited by ABA and Lepidium endosperm weakening is promoted by GA and inhibited by ABA. These results support the hypothesis that the GA-ABA control of endosperm rupture is mediated, at least in part, by the antagonistic hormonal effects on endosperm weakening. The molecular mechanisms that control endosperm weakening might differ among seeds from distinct angiosperm clades. A “one-phase” ABA-inhibited endosperm weakening is evident in Lepidium seeds. We speculate that during evolution the endospermic Brassicaceae seeds have retained ABA-inhibitable molecular mechanisms also found in asterid seeds (second phase of endosperm weakening), whereas the ABA-insensitive phase of endosperm weakening was lost.

Endosperm weakening from a seed evolutionary viewpoint. Clades with experimental evidence for endosperm-limited germination and endosperm weakening are in bold and numbered (1) to (10). Puncture force measurements: C = Endosperm weakening prior to endosperm rupture; GA = Endosperm weakening promoted by GA; ABA = Endosperm weakening inhibited by ABA.The experimental evidence can be summarised as follows (for references see Finch-Savage and Leubner-Metzger, 2006):

Rosid clade:
(1) Cucurbitaceae: Cucumis C (perisperm weakening).
(2) Brassicaceae: Lepidium C GA ABA (Müller et al., 2006), Arabidopsis.
Asterid clade:
(3) Oleaceae: Syringa P GA (Junttila, 1973), Fraxinus C GA (Fig. 6, Finch-Savage & Clay, 1997).
(4) Solanaceae (Solanoideae): Lycopersicon C GA ABA (Groot & Karssen, 1987, 1992; Toorop et al., 2000; Wu et al., 2000), Capsicum C GA (Watkins & Cantliffe, 1983; Petruzzelli et al., 2003), Datura (Sanchez & Mella, 2004).
(4) Solanaceae (Cestroideae): Nicotiana (reviewed in Leubner-Metzger, 2003), Petunia (Petruzzelli et al., 2003).
(5) Rubiaceae: Coffea C GA ABA (da Silva et al., 2004; da Silva et al., 2005).
(6) Asteraceae: Lactuca C GA (Tao & Khan, 1979).
(7) Apiaceae: Apium (Jacobsen & Pressman, 1979).
Other clades:
(8) Amaranthaceae: Chenopodium (Karssen, 1976).
(9) Ranunculaceae: Trollius (Hepher & Roberts, 1985).
(10) Iridaceae: Iris C (Blumenthal et al., 1986), Poaceae: Triticum C (Benech-Arnold, 2004).

Angiosperm seed evolution depicted in a phylogenetic tree of the Angiosperm Phylogeny Group II (2003). For each family, pictographs of the seed type and symbols for the dormancy class are placed next to the corresponding clade (see figure above for symbols and data source). The number of pictographs or symbols is equal to the number of families with the corresponding seed type or dormancy class, respectively. Numbers in the phylogenetic tree represent "embryo to seed" (E:S) ratios expressed as GSL (generalized least squares) values from Forbis et al. (2002, personal communication). Phylogenetic tree modified from Judd et al. (2002); Angiosperm Phylogeny Group (2003); Soltis and Soltis (2003); Stevens (2006). Figure published in the Tansley review "Seed dormancy and the control of germination" by Finch-Savage WE and Leubner-Metzger G (New Phytologist 171: 2006). A PDF file of this review is available for download from either "The Seed Biology Place" (Finch-Savage and Leubner-Metzger, 2006) or the New Phytologist Trust website at New Phytologist Trust - Tansley Reviews - © Blackwell Science - http://www.blackwell-science.com/

"Hatching enzymes" - Endosperm weakening and endosperm cell wall hydrolases

• The "hatching enzyme" hypothesis in seed biology: Ikuma and Thiman (Plant & Cell Physiol 4: 169-185, 1963) first proposed proposed for lettuce that "... the final step in the germination control process is the production of an enzyme whose action enables the tip of the radicle to penetrate through the coat".
• Endo-ß-mannanase (excellent review by Bewley, 1997) and endo-ß-1,3-glucanase were proposed to be the cause of endosperm weakening.
• In addition numerous other cell-wall modifying proteins, e.g. ß-mannosidase, a-galactosidase, cellulase, pectin methylesterase, polygalacturonase, xyloglucan endo-transglycosylase, chitinase, peroxidase and expansin were examined. Several of these studies provided evidence for the possible contribution of a specific cell wall hydrolase in a certain species and unraveled some of the complexity of the hormonal regulation of seed germination and dormancy. However, conclusive evidence for a single "germination enzyme" has not yet been found.
• Endosperm weakening and hydrolase action in lettuce, tomato, tobacco, Datura seeds seems to be promoted by gibberellins (GA) and red light (via phytochrome) and inhibited by far-red light and often by abscisic acid (ABA).
• While endo-ß-mannanase appears to be necessary for tomato endosperm weakening, it is not sufficient for the completion of germination (e.g. Toorop et al., Planta 200, 153-158, 1996; Wu et al., 2001). Abscisic acid (ABA) clearly controls the final step of radicle protrusion, but it does not inhibit tomato endosperm weakening caused by endo-ß-mannanase (e.g. Toorop et al., J Exptl Bot 51, 1371-1379, 2000; Wu et al., 2001).
• A two-phase endosperm weakening process was proposed for tomato by Toorop et al. (2000). The first phase is not inhibited by ABA and involves endo-ß-mannanase-mediated weakening. The second phase is inhibited by ABA and correlates with the expression of class I ß-1,3-glucanase in the micropylar cap of tomato.
• Tobacco seeds are too small for puncture force experiments. We proposed that in the case of tobacco, class I ß-1,3-glucanase, which is induced in the micropylar endosperm just prior to its rupture and is tightly linked with altered endosperm rupture in response to light, gibberellins (GA), ABA, and ethylene, is involved in regulating endosperm weakening (Leubner-Metzger and Meins, 1999).
• Considerable evidence from work with transgenic seeds suggests that ß-1,3-glucanase substantially contributes to the regulation of endosperm rupture, dormancy release and after-ripening of dicot seeds (Leubner-Metzger and Meins 2000; Leubner-Metzger 2002; Leubner-Metzger 2003).
• Proteomic analysis of Arabidopsis showed that ß-1,3-glucanases are among the group of glycosylphosphatidylinositol-anchored membrane proteins (GPI-APs, Elortza et al. 2003). GPI-APs are proposed to be involved as enzymes and receptors in cell adhesion, cell separation and differentiation processes. This could be a new function of ß-1,3-glucanases during endosperm weakening. In sea urchins ß-1,3-glucanases are reported as part of the embryo hatching enzyme (Bachman and McClay 1996).
• ABA inhibition of ß-1,3-glucanase in the endosperm is widespread among Solanaceae seeds (Petruzelli et al. 2003).
• ABA inhibition of ß-1,3-glucanase is also evident in perisperm weakening of Cucurbitaceae seeds where a callose layer contributes to the semipermeability of the perisperm (Yim and Bradford 1998, Amritphale et al. 2005, Ramakrishna and Amritphale 2005).
• Taken together, these findings support the view that germination control by the seed covering layers is achieved by the collaborative or successive action of several cell-wall-modifying proteins and by several different molecular mechanisms (Leubner-Metzger 2003).

Gibberellins (GA) promotes and abscisic acid (ABA) inhibits endosperm weakening

Established Asterid model systems: Direct evidence for endosperm weakening and it's hormonal control - Two-phase endosperm weakening of tomato and coffee - ABA inhibits the second phase

Emerging Rosid model systems: Hormonal regulation of Brassicaceae endosperm weakening and endosperm rupture - GA promotes and ABA inhibits endosperm rupture of Lepidium sativum (garden cress)

Reactive oxygen species (ROS) as a molecular mechanism of endosperm weakening and radicle growth