Proteomics of Arabidopsis seed germination. A comparative study of wild-type and gibberellin-deficient seeds

Abstract

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA biosynthesis inhibitor. With both systems, radicle protrusion was strictly dependent on exogenous GAs. The proteomic analysis indicated that GAs do not participate in many processes involved in germination sensu stricto (prior to radicle protrusion), as, for example, the initial mobilization of seed protein and lipid reserves. Out of 46 protein changes detected during germination sensu stricto (1 d of incubation on water), only one, corresponding to the cytoskeleton component alpha-2,4 tubulin, appeared to depend on the action of GAs. An increase in this protein spot was noted for the wild-type seeds but not for the ga1 seeds incubated for 1 d on water. In contrast, GAs appeared to be involved, directly or indirectly, in controlling the abundance of several proteins associated with radicle protrusion. This is the case for two isoforms of S-adenosyl-methionine (Ado-Met) synthetase, which catalyzes the formation of Ado-Met from Met and ATP. Owing to the housekeeping functions of Ado-Met, this event is presumably required for germination and seedling establishment, and might represent a major metabolic control of seedling establishment. GAs can also play a role in controlling the abundance of a beta-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.

Figure 1

Characterization of Arabidopsis proteins whose abundance differed in dry mature seeds of WT and ga1 mutant. An equal amount (200 μg) of total protein extracts was loaded in each gel. A, Silver-stained two-dimensional gel of total proteins from dry mature seeds of ga1 mutant. The indicated portions of the gel, a through c, are reproduced in B. B, Enlarged windows, a through c, of two-dimensional gels as shown in A for WT seeds (left) and ga1 mutant seeds (right). The six labeled protein spots (protein nos. 151, 177, 69, 169, 70, and 71) were identified by matrix-assisted laser desorption-ionization-time-of-flight (MALDI-TOF) analysis (see Table II). The figure shows representative experiments carried out at least five times. Protein spot quantitation was carried out as described in “Materials and Methods” from at least three gels for each seed sample.

Figure 2

Reference maps for WT Arabidopsis seed proteins whose abundance specifically vary during germination (1 d of imbibition in water) and radicle protrusion (2 d of imbibition in water). An equal amount (200 μg) of total protein extracts was loaded in each gel. The figure shows representative experiments carried out at least five times. A, Silver-stained two-dimensional gel of total proteins from dry mature seeds showing the type-2 proteins (labeled) whose abundance specifically decreased during germination. The labeled proteins are listed in Table III. B, Silver-stained two-dimensional gel of total proteins from 1-d imbibed seeds showing the type-1 proteins (labeled) whose abundance specifically increased during germination. The labeled proteins are listed in Table III. C, Silver-stained two-dimensional gel of total proteins from 2-d imbibed seeds showing the type-3 proteins (labeled) whose abundance specifically increased during radicle protrusion. The labeled proteins are listed in Table IV.

Figure 3

Mobilization of seed storage proteins during 1 d of imbibition. An equal amount (200 μg) of total protein extracts was loaded in each gel. The figure shows representative experiments carried out at least five times. A, Silver-stained two-dimensional gel of total proteins from dry mature WT seeds. The indicated portion of the gel is reproduced in B through F for the following seed samples. B, Dry mature WT seeds. C, Dry mature ga1 seeds. D, WT seeds incubated for 1 d in 100 μm PAC (none of the seeds germinated under these conditions; see Table I). E, WT seeds incubated for 1 d in water (none of the seeds germinated under these conditions; see Table I). F, ga1 mutant seeds incubated for 1 d in water (none of the seeds germinated under these conditions; see Table I). The labeled type-1 proteins (whose abundance increased during 1 d of imbibition for the three seed samples) are listed in Table III. They correspond to fragments of 12S cruciferin α-subunits (protein nos. 33, 76, and 77) and β-subunits (protein nos. 12, 32, and 89).

Figure 4

Characterization of some cytoskeleton protein components whose abundance increased during 1 d of imbibition (type-1 proteins). An equal amount (200 μg) of total protein extracts was loaded in each gel. The figure shows representative experiments carried out at least five times. A, Silver-stained two-dimensional gel of total proteins from dry mature WT seeds. The indicated portion of the gel is reproduced in B through F for the following seed samples. B, Dry mature WT seeds. C, Dry mature ga1 seeds. D, WT seeds incubated for 1 d in 100 μm PAC (none of the seeds germinated under these conditions; see Table I). E, WT seeds incubated for 1 d in water (none of the seeds germinated under these conditions; see Table I). F, ga1 mutant seeds incubated for 1 d in water (none of the seeds germinated under these conditions; see Table I). The labeled proteins are listed in Table III. They correspond to type-1 protein numbers 4 (β-tubulin), 5 (α-3, 5 tubulin), and 24 (actin 7).

Figure 5

Quantitation of the accumulation level of α-2,4 tubulin (type-1 protein no. 6 in Table III) during 1 d of imbibition. The theoretical molecular mass and pI of α-2,4-tubulin are 49.54 kD and 4.93, respectively (Table III). The results are expressed as normalized volumes of the α-2,4 tubulin spot (± sd; n = 3) after 1 d of imbibition in water or in the presence of 100 μm PAC or 100 μm GA₄₊₇. A portion of an area of silver-stained two-dimensional gels is shown under the graph. The arrows point to the position of the α-2,4 tubulin spot.

Figure 6

Evolution of the seed proteome in WT and ga1 mutant seeds incubated for up to 2 d in water and in WT seeds incubated for up to 2d in PAC, which period corresponded to radicle protrusion for the WT seeds incubated on water (see Table I). An equal amount (200 μg) of total protein extracts was loaded in each gel. The figure shows representative experiments carried out at least five times. A, Silver-stained two-dimensional gel of total proteins from WT seeds incubated for 2 d in water showing the type-3 proteins (labeled) whose abundance specifically increased during radicle protrusion. The labeled proteins are listed in Table IV. The indicated portions of the gel, a through d, are reproduced in B. B, enlarged windows, a through d, of two-dimensional gels as shown in A for WT seeds incubated for 2 d in water (left), ga1 mutant seeds incubated for 2 d in water (middle; none of the seeds germinated under these conditions; see Table I), and WT seeds incubated for 2 d in 100 μm PAC (right; none of the seeds germinated under these conditions; see Table I). The labeled proteins are listed in Table IV.

Figure 7

Evolution of the seed proteome in WT and ga1 mutant seeds incubated for up to 3 d in water and in WT seeds incubated for up to 3 d in PAC. An equal amount (200 μg) of total protein extracts was loaded in each gel. The figure shows representative experiments carried out at least five times. A, Silver-stained two-dimensional gel of total proteins from dry mature WT seeds. B, Silver-stained two-dimensional gel of total proteins from WT seeds incubated in water for 3 d corresponding to seedling establishment. C, Silver-stained two-dimensional gel of total proteins from ga1 mutant seeds incubated for 3 d in water. D, Silver-stained two-dimensional gel of total proteins from WT seeds incubated in 100 μm PAC for 3 d. The figure shows that the proteome of the ga1 mutant seeds incubated for 3 d in water (C) and that of the WT seeds incubated for 3 d in PAC (D) did not exhibit the characteristic changes observed with the WT seeds incubated in water (B). Note in particular the massive loss of low-M_r proteins in the 5.9 to 8.7 pI range observed during the 3 d of imbibition in water of the WT seeds (compare A and B), but not with the ga1 mutant seeds (C) and the WT seeds incubated in PAC (D).