Proteome analysis of grain filling and seed maturation in barley

Abstract

In monocotyledonous plants, the process of seed development involves the deposition of reserves in the starchy endosperm and development of the embryo and aleurone layer. The final stages of seed development are accompanied by an increase in desiccation tolerance and drying out of the mature seed. We have used two-dimensional gel electrophoresis for a time-resolved study of the changes in proteins that occur during seed development in barley (Hordeum vulgare). About 1,000 low-salt extractable protein spots could be resolved on the two-dimensional gels. Protein spots were divided into six categories according to the timing of appearance or disappearance during the 5-week period of comparison. Nineteen different proteins or protein fragments in 36 selected spots were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry (MS) or nano-electrospray tandem MS/MS. Some proteins were present throughout development (for example, cytosolic malate dehydrogenase), whereas others were associated with the early grain filling (ascorbate peroxidase) or desiccation (Cor14b) stages. Most noticeably, the development process is characterized by an accumulation of low-M(r) alpha-amylase/trypsin inhibitors, serine protease inhibitors, and enzymes involved in protection against oxidative stress. We present examples of proteins not previously experimentally observed, differential extractability of thiol-bound proteins, and possible allele-specific spot variation. Our results both confirm and expand on knowledge gained from previous analyses of individual proteins involved in grain filling and maturation.

Figure 1

Two-dimensional gel protein patterns of developing barley seeds. Two-dimensional gel patterns obtained from extracts of water-soluble proteins of barley cv Barke. Approximately 40 μg of protein was loaded on each gel. Sizes of molecular mass markers (in kD) and the pI range of the first dimension (pI 4–7) are indicated. Development stages 80 and 87 according to the Zadoks scale are shown. Numbered boxes indicate regions 1 through 6 of the two-dimensional gels from which close-up views are shown in Figures 2 and 3. Numbered spots refer to Table I.

Figure 2

Variations in protein spots during seed development. Close-up views of A, region 2 (22–30 kD, pI 5.0–5.9); B, region 3 (30–45 kD, pI 4.7–5.7); C, region 4 (10–20 kD, pI 6.2–7.0.); D, region 5 (20–30 kD, pI 5.1–5.6); and E, region 6 (22–30 kD, pI 6.25–6.6.). Numbered spots correspond to Table I. For ease of comparison in C through E, some reference spots have been circled. A, This region shows a group of spots that is abundant at the early and late stages of dough formation but less abundant at the middle stage (upwards arrows). Another spot remains constant throughout the process (horizontal arrow) and yet another spot is present at all stages of development, but most abundant at the final stage (downwards arrow). Spots 7, 9 (Fig. 1), 95, 120 through 122, and 138 have been identified as triose phosphate isomerase. An as yet unidentified spot that has expression pattern III is marked (a). B, Group of spots (spots 84–87) accumulates transiently at the middle stages of development. These have been identified as C-terminal fragments of protein disulfide isomerase (PDI). Another group of spots, including spots 22 and 24, increases gradually both in intensity and number throughout development and are identified as protein Z4 (a Ser protease inhibitor [serpin]). Below the serpin spots, glyoxalase I (spot 6) is present throughout and is particularly abundant at the latest stage. A fragment of this protein is also present (spot 149). C, Spot 140 is probably a degradation product of spot 139. These spots decrease gradually in intensity during development (group I). Both have been identified as the small subunit of ribulose bisphosphate carboxylase. Spot 20 (group IV, desiccation related) is a glyoxalase I-related protein (see text). Another, unidentified protein (spot b) has the same pattern of appearance. D, Spot 149 (glyoxalase I fragment) appears to alter position during development. An as yet unidentified spot with expression pattern I (c) is marked. E, Spot 79, also with expression pattern I, has been identified as cytosolic ascorbate peroxidase (APX). Spots with expression pattern IV (d) and O (e) are marked. Spot d has been identified as a 1cys peroxiredoxin (O. Østergaard, C. Finnie, S. Melchior, P. Roepstorff, and B. Svensson, unpublished data), whereas spot e is not yet identified.

Figure 3

Protein identification by matrix-assisted laser-desorption ionization (MALDI)-time of flight (TOF) peptide mapping. A, Alignment of PDI sequences from barley (P80284) and wheat (P52589). Only residues differing from the barley sequence are shown and identities are indicated by dashes. Underscores indicate gaps introduced into the alignment. The predicted N-terminal signal peptide is shown in italics. Boxes indicate tryptic peptides identified from MALDI-TOF spectra obtained from spot 124 (clear) and spot 87 (shaded). One peptide from spot 124 that is specific for the wheat sequence is boxed with a thick line. B, Section of the MALDI-TOF mass spectrum obtained for spot 140 (the same peaks were observed in the spectrum for spot 139), together with the sequence of ribulose bisphosphate carboxylase small subunit. The predicted N-terminal chloroplast targeting sequence is shown in italics. Tryptic peptides identified from the spectra of both spot 139 and 140 are boxed. The C-terminal tryptic peptide, observed in the spectrum from spot 139 but not from spot 140, is shaded. The N-terminal peptide of the mature protein is shown underlined and in bold. Two peaks in the mass spectrum that have [M + H] 14 D higher than the predicted mass of this peptide with no missed cleavages (*, predicted [M + H] = 1,216.64) or a single missed cleavage (**, predicted [M + H] = 1,344.73) are indicated. The [M + H] = 1,214.7 peak was confirmed by MS/MS peptide fragmentation to correspond to the N-methylated peptide. Other peaks in the spectrum that originate from ribulose bisphosphate carboxylase small subunit are also labeled with [M + H] values.

Figure 4

Changes in α-amylase/trypsin inhibitor proteins. A, Close-up view of region 1 of the two-dimensional gels, spanning approximately 6 to 20 kD and pI 4.5 to 5.8. Numbered spots correspond to Table I. This shows a group of spots that become gradually more well defined during seed development, and are highly abundant during the later stages. Many of these spots have been identified as α-amylase/trypsin inhibitors. An as yet unidentified spot that has transient expression pattern III is marked (f). B, Comparison of α-amylase/trypsin inhibitor proteins in barley cv Barke, cv Mentor, and cv Meltan, at the latest timepoint analyzed, at which the cultivars were at the indicated stages of development (top row), and in the mature seeds (M, middle and bottom rows). Proteins that are released into the supernatant by dithiothreitol (DTT) extraction of mature seeds (bottom row) are circled.

Figure 5

Putative allele-specific protein spots. A, Comparison of barley cv Barke, cv Mentor, cv Meltan, and cv Morex at an intermediate stage of seed development, in a region of the two-dimensional gels spanning approximately 20 to 30 kD and pI 4.5 to 5.0. Numbered spots correspond to Table I. For ease of comparison, reference spots are circled. Vertical arrows indicate spots 80 and 81 identified as fragments of β-amylase. B, Detail of MALDI-TOF mass spectra for spots 80 and 81. The peak with [M + H] = 2,086.1 corresponds to a tryptic peptide from β-amylase covering residues 129 through 146 that is identical in both forms of the protein. Peaks can also be seen corresponding to tryptic peptides from β-amylases covering residues 109 through 126, and containing either Cys ([M + H] = 2025.8) or Arg ([M + H] = 2021.9) at position 115.