Discussion

The two dark-stratification treatments produced seeds with contrasting levels of dormancy. Those stratified for 2 days had a high level of dormancy because they

Table 23.2. Identification of peptides derived from light-insensitive and light-sensitive embryos obtained by MS/MS analysis. Protein spots from Fig. 23.2 were excised, digested with trypsin and multiply charged peptides fragmented by MS/MS.

M/z

Mass

Delta

MOWSE

Peptide

Match

Spot 1

435.2

868.5

-0.03

9

VLPELNGK

gi|46507768|gb|

581.3

1160.6

-0.05

47

AGIALNDNFVK

Mass: 21,613

717.9

1433.7

-0.05

12

AASFNIIPSSTGAAK

Lolium multiflorum Glyceraldehyde 3-phosphate dehydrogenase

Spot 2

581.3

1160.6

-0.04

31

AGIALNDNFVK

gi|46507768|gb|

717.9

1433.7

-0.04

11

AASFNIIPSSTGAAK

Mass: 21,613 Lolium multiflorum Glyceraldehyde 3-phosphate dehydrogenase

Spot 4

429.2

856.4

-

-

(Q/K)(L/I)WASPR

495.8

989.5

-

-

ATDS(L/I)(L/I)TAAK

The mass/charge ratio (M/z), the predicted mass of the peptides (mass), the mass difference to matched sequence (Delta), the molecular weight search (MOWSE) score and the peptide sequence matched are shown for spots 1 and 2. The mass/charge ratio (M/z), the predicted mass of the peptides (mass) and the de novo sequence deduced are shown for spot 4. Leucine and isoleucine residues have identical mass and are shown as alternatives. Lysine (K) and glutamine (Q) residues only differ in mass by 0.05 Da, given that a tryptic peptide would not be expected to contain an N-terminal K, Q is preferred but it is designated Q/K.

were unresponsive to germination stimulation by light. In contrast, those stratified for 35 days did respond when light was provided. These cannot be termed 'non-dormant' because they were unable to germinate without additional germination stimulants (Vleeshouwers et al., 1995). In L. rigidum seeds, a non-dormant state is not reached by dark stratification, but appears to be possible only by extended periods of dry after-ripening (Steadman et al., 2003).

Both the light-insensitive and light-sensitive embryos can be expected not to begin germination-related biochemistry. Seeds that were exposed to a light flash, similar to that experienced during embryo excision, did not subsequently germinate in darkness, and embryos that were excised were quickly placed into subzero temperatures to stop any biochemical changes. Furthermore, proteomic analysis established that very few proteins exhibited significant differential abundance between imbibed light-insensitive (i.e. dormant) and light-sensitive seeds. Large numbers of proteins change in abundance during the early stages of germination (Gallardo et al., 2001), so germination-related events can be presumed not to have begun in L. rigidum. Thus, with L. rigidum we can easily excise the embryo from the rest of the seed at a variety of dormancy levels without germination-related biochemistry complicating the picture.

Proteomic analysis indicated that GAPDH was undetectable in light-insensitive seed embryos but present in light-sensitive seeds. Previously, an increase in glycolysis was the evidence of the start of germination. For example, a rise in fructose 2,6-bisphosphate is one of the earliest metabolic events in the germination of Avena sativa L. seeds (Larondelle et al., 1987). Similarly, GAPDH and other metabolic enzymes increase upon thidiazuron-induced dormancy release in apple (Malus pumila Mill.) buds, correlating with the initial stages of resumption of growth (Wang et al., 1991). In Arabidopsis seeds, GAPDH is one of the proteins upregulated in seeds imbibed for 1 day, prior to initiation of radicle growth (Gallardo et al., 2002). In this case, the seeds appear to be preparing for germination by increasing the capacity for energy production even before they switch into a germinative mode. GAPDH is commonly observed to be stress-responsive (Velasco et al., 1994; Laxalt et al., 1996), raising the possibility that the 35-day period of dark stratification was a stressful event that elicited the increase in abundance over that of the 2-day stratified seeds, rather than the change in dormancy status per se.

This initial study has shown that there are some differences in the protein profile between the embryos of light-insensitive and light-sensitive seeds. This work needs to be extended in a number of ways:

1. Currently, only the more abundant portion of the proteins present in the seeds has been visualized, and there may be many changes occurring in the less abundant proteins, so protein loading on the gels should be increased so that they can be detected.

2. Membrane-bound proteins have been hypothesized, which may be involved in the dormancy mechanism (Hilhorst, 1998; Hallett and Bewley, 2002; Steadman, 2004), so separation of membrane-bound and cytosolic proteins is required.

3. The endosperm in the region of the radicle tip has an important role in dormancy in a number of species (Leubner-Metzger, 2003), so particular emphasis on these tissues is necessary.

4. Light (red or white) can inhibit dormancy release during warm stratification, while darkness and far-red light are permissive for dormancy release (Steadman, 2004), presenting an alternative control treatment that would allow light-insensitive and light-sensitive seeds to be imbibed for equal periods of time, avoiding the potential for differences in the imbibition period to influence protein changes.

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