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Seed Germination


Seed germination: definition and reviews
One-step seed germination of Brassica and pea seeds: testa rupture and initial radicle elongation
Two-step seed germination of Lepidium and Arabidopsis (Brassicaceae): testa and endosperm rupture
Two-step seed germination of Nicotiana spp. (Solanaceae): testa and endosperm rupture

     

Lab work: Dissecting seeds     
Lab work: Xiaofeng is dissecting seeds. Photographer: Johannes Fehrle




Seed germination: definition and reviews





One-step seed germination of Brassica and pea seeds: testa rupture and initial radicle elongation

  • The endosperm is completele obliterated during the seed development of Brassica spp. (see figure below) or pea and the mature seeds of these species are therefore non-endospermic. Uptake of water by a seed is triphasic with a rapid initial uptake (phase I, i.e. imbibition) followed by a plateau phase (phase II). A further increase in water uptake (phase III) occurs only when germination is completed, as the embryo axes elongates and breaks through the testa. Thus, besides radicle elongation, testa rupture is the only visible landmark during Brassica spp. and pea seed germination.
  • Abscisic acid (ABA) does not inhibit imbibition and testa rupture (see figure below), but ABA inhibits phase III water uptake and the transition from germination to postgermination growth (e.g. Schopfer and Plachy, 1984; Manz et al., 2005).

    Brassica seed germination

    Brassica napus seed germination is one-step. The mature seeds of these species are without endosperm and so testa rupture plus initial radicle elongation result in the completion of germination. ABA does not inhibit testa rupture, but inhibits subsequent radicle growth (Schopfer & Plachy, 1984). Review: Finch-Savage and Leubner-Metzger (2006).





Two-step seed germination of Lepidium and Arabidopsis (Brassicaceae): testa and endosperm rupture

  • For the Lepidium and Arabidopsis seed anatomy see the webpage "Seed Structure".
  • Rupture of the testa (seed coat) and rupture of the endosperm are separate events in the germination of Lepidium and Arabidopsis seeds (see figures below). Arabidopsis (Liu et al., Plant J 41:936-944, 2005) and Lepidium (Müller et al., 2006) exhibit a two-step germination, in which testa rupture and endosperm rupture are sequential events.
  • Such two-step germination is widespread over the entire phylogenetic tree and has been described for many species, e.g. for Trollius (Ranunculaceae; Hepher and Roberts 1985), Chenopodium (Amaranthaceae; Karssen 1968; Karssen 1976), Nicotiana and Petunia (Cestroideae subfamily of the Solanaceae, Krock et al. 2002; Leubner-Metzger et al. 1995; Petruzzelli et al. 2003).
  • We found that the plant hormone ABA inhibits endosperm rupture, but not testa rupture, of Arabidopsis and Lepidium (Müller et al., 2006). This inhibitory effect of ABA is counteracted by GA, supporting the view that endosperm rupture is under the control of an ABA-GA antagonism (Kucera et al. 2005).
  • We found that ABA inhibits endosperm weakening of Lepidium, and this inhibitory effect is counteracted by GA (Müller et al., 2006). This supports the view that weakening of the micropylar endosperm occurs in Arabidopsis and Lepidium seeds (Brassicaceae, Rosid clade), is under ABA-GA control, and is functioning in controlling the germination of endospermic Brassicaceae seeds.
  • We show that ethylene promotes endosperm cap weakening of Lepidium sativum and endosperm rupture of the close Brassicaceae relatives Lepidium sativum and Arabidopsis thaliana and that it counteracts the inhibitory action of abscisic acid (ABA) on these two processes (Linkies et al., 2009). Cross-species microarrays of the Lepidium micropylar endosperm cap and the radicle show that the ethylene-ABA antagonism involves both tissues and has the micropylar endosperm cap as a major target. Ethylene counteracts the ABA-induced inhibition without affecting seed ABA levels. The Arabidopsis loss-of-function mutants ACC oxidase2 (aco2; ethylene biosynthesis) and constitutive triple response1 (ctr1; ethylene signaling) are impaired in the 1-aminocyclopropane-1-carboxylic acid (ACC)-mediated reversion of the ABA-induced inhibition of seed germination. Ethylene production by the ACC oxidase orthologs Lepidium ACO2 and Arabidopsis ACO2 appears to be a key regulatory step. Endosperm cap weakening and rupture are promoted by ethylene and inhibited by ABA to regulate germination in a process conserved across the Brassicaceae (Linkies et al., 2009).

Lepidium seed germination

Two-step germination of Lepidium sativum. (C-F) During the two-step germination of Lepidium testa rupture (C,D) is followed by endosperm rupture, which occurs after 16 h under control conditions (E). Due to the microphotographic settings the transparent outer mucilage layer is not visible. (F) ABA specifically inhibits endosperm rupture, the radicle remains covered by the micropylar endosperm even after 60 h incubation in the presence of ABA. (G) Drawing of a mature Arabidopsis seed; the seed anatomy that is very similar to that of Lepidium. (H-J) Arabidopsis seeds also germinate with testa rupture (H) preceding endosperm rupture (I). Also during the two-step germination process of Arabidopsis, ABA specifically inhibits endosperm rupture (J). Seeds were incubated in continuous light without (control) or with 10 µM ABA added to the medium. From Müller et al., (2006).

Arabidopsis seed germination

Two-step germination of Arabidopsis thaliana. (G) Drawing of a mature Arabidopsis seed; the seed anatomy that is very similar to that of Lepidium. (H-J) Arabidopsis seeds also germinate with testa rupture (H) preceding endosperm rupture (I). Also during the two-step germination process of Arabidopsis, ABA specifically inhibits endosperm rupture (J). Seeds were incubated in continuous light without (control) or with 10 µM ABA added to the medium. From Müller et al., (2006).

A comprehensive table of Arabidopsis hormone mutants summarizes the altered phenotypes regarding germination and dormancy.

 





Two-step seed germination of Nicotiana spp. (Solanaceae): testa and endosperm rupture

  • For the tobacco seed anatomy see the webpage "Seed Structure".
  • Rupture of the testa (seed coat) and rupture of the endosperm are separate events in the germination of tobacco seeds and there is strong evidence that both, testa rupture and endosperm rupture are the limiting factors in the germination of these seeds (reviewed by Leubner-Metzger 2003).

Nicotiana two-step germination



  • Electron microscopic studies support the view that the endospermic hole of the germinated tobacco seed, which is always at the micropylar end, is formed by "dissolution" rather than by "pushing" action (Arcila and Mohapatra, Tobacco Science 27: 35-40, 1983). Endosperm weakening is a pre-requisite for the completion of germination of many Solanaceous seeds.
  • In photodormant varieties of tobacco that do not germinate in darkness, both the testa rupture and endosperm remain intact in the photodormant seeds. However, when the testa and endosperm are mechanically removed, there is radicle growth even in the absence of light. This shows that in tobacco coat-imposed dormancy is more important than embryo dormancy.
  • Treatment of tobacco seeds with 10 µM abscisic acid (ABA) greatly delays endosperm rupture, but not testa rupture, and results in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue.

Figure 2:

Leubner-Metzger et al. (1995
)

Testa rupture (b) and endosperm rupture (c) are separate events during the germination of tobacco seeds.

ABA delays endosperm rupture, but not testa rupture (e and h).

Figure 2: Stages in the germination of tobacco seed homozygous for the GLB-GUS transgene. Seeds were germinated in continuous light at 24 °C with 10 µM ABA (ABA) and without ABA (control) added to the medium. At the times indicated after the start of imbibition, seeds were stained for GUS activity. The blue staining is indicative of transcriptional activity of the class I ß-1,3-glucanase B promoter. (a) Stage I (control, 3 h): intact seed prior to seed-coat rupture. (b) Stage II (control, 60 h): seed with ruptured seed coat and protruding endosperm. (c) Stage III (control, 72 h): seeds with ruptured endosperm showing GUS staining at the rupture site and emerging radicles, which do not stain for GUS. (d) Stage III (control, 96 h): seed with ruptured endosperm and elongating radicle. GUS staining is localized in a collar of endosperm tissue at the site of radicle penetration. (e) Stage II (ABA, 144 h): ABA treatment markedly delays endosperm rupture and results in a novel structure consisting of the enlarging radicle completely enclosed in a sheath of intact endosperm, which does not stain for GUS. (f) Stage II (control, 60 h): endosperm dissected prior to rupture showing GUS stain localized in the micropylar region. (g) Stage III (control, 96 h): endosperm dissected after rupture showing that the radicle penetrates the region which stains for GUS. (h) Stage II (ABA, 144 h): a seed arrested in stage II by ABA treatment dissected to show that the elongated radicle is enclosed in a sheath of endosperm. Magnification: 40X.


 


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