Proteomic analysis of arabidopsis seed germination and priming

Abstract

To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-regulation) of 74 proteins were observed during germination sensu stricto (i.e. prior to radicle emergence) and the radicle protrusion step. This approach was also used to analyze protein changes occurring during industrial seed pretreatments such as priming that accelerate seed germination and improve seedling uniformity. Several proteins were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Some of them had previously been shown to play a role during germination and/or priming in several plant species, a finding that underlines the usefulness of using Arabidopsis as a model system for molecular analysis of seed quality. Furthermore, the present study, carried out at the protein level, validates previous results obtained at the level of gene expression (e.g. from quantitation of differentially expressed mRNAs or analyses of promoter/reporter constructs). Finally, this approach revealed new proteins associated with the different phases of seed germination and priming. Some of them are involved either in the imbibition process of the seeds (such as an actin isoform or a WD-40 repeat protein) or in the seed dehydration process (e.g. cytosolic glyceraldehyde-3-phosphate dehydrogenase). These facts highlight the power of proteomics to unravel specific features of complex developmental processes such as germination and to detect protein markers that can be used to characterize seed vigor of commercial seed lots and to develop and monitor priming treatments.

Figure 1

2-D analysis of the Arabidopsis proteome during seed germination. 2-D gel profiles of total proteins from: A, dry mature seeds (1,272 proteins detected in 2-D gels); B, 1-d imbibed seeds (1,338 proteins detected); C, 2-d imbibed seeds (1,461 proteins detected); and D, 3-d imbibed seeds (1,133 proteins detected). An equal amount (200 μg) of total protein extracts was loaded in each gel.

Figure 2

Characterization of some Arabidopsis seed proteins whose abundance vary during germination and priming. A, Dry mature seeds; B, 1-d imbibed seeds (germination sensu stricto); C, redried 1-d imbibed seeds (hydroprimed seeds); D, 2-d imbibed seeds (radicle emergence step). Proteins shown in each top window are within the pI and size ranges: 6.2 < pI < 7.0 and 40.0 kD < M_r < 58.0 kD; labeled proteins (experimental molecular mass in kD, experimental pI) are type-4 protein number 69 (43.67 and 6.29), type-6 protein number 37 (40.29 and 6.49), type-3 protein number 73 (42.28 and 6.47); and type-11 protein number 23 (56.48 and 6.64). Proteins shown in each bottom window are within the pI and size ranges: 4.8 < pI < 5.5 and 41.0 kD < M_r < 57.0 kD; labeled proteins (experimental molecular mass in kD, experimental pI) are type-5 protein number 24 (42.94 and 5.06), type-11 protein number 6 (54.71 and 5.06), and type-0 protein number 128 (47.55 and 5.44). The protein types are depicted in Tables II through IV. Identified proteins (by MALDI-TOF and/or post source decay [PSD]) are listed in Tables V through IX. Protein spot quantitation was carried out as described in “Materials and Methods,” from at least three gels for each condition. For example, in C the following spot volumes were measured: spot 23 (435, 429, and 466; 444 ± 16); spot 69 (575, 546, and 540; 554 ± 15); spot 37 (1,141, 1,054, and 1,097; 1097 ± 36); spot 6 (78, 64, and 74; 73 ± 6); and spot 128 (244, 254, and 259; 252 ± 6).