Post-germination changes in Hevea brasiliensis seeds proteome

Plant Science 169 (2005) 303–311 www.elsevier.com/locate/plantsci

Post-germination changes in Hevea brasiliensis seeds proteome Pooi-Fong Wong, Sazaly Abubakar * Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Received 9 August 2004; received in revised form 19 January 2005; accepted 24 January 2005 Available online 2 March 2005

Abstract Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry methods were established and utilized to examine the changes in protein expressions associated with post-germination of Hevea brasiliensis seed. No significant differences in the total number of proteins were observed but characteristic protein spots were present in both proteomes. The mature dry seed proteome contained clusters of proteins of about 36.5 and 23 kDa at pH 4–7 and a group of basic proteins at pH 10. The presence of the 23 kDa proteins was markedly reduced in the post-germinated seed proteome. Approximately 60% of the proteins noted in the germinated seed proteome matched those of the mature dry seeds, with the remaining 40% protein spots as either unmatched or unique to the germinated seed proteome. Of the proteins detected, a putative beta-glucosidase, starch branching enzyme IIb and a MutT/nudix family of protein that were decreased in abundance in the germinated seed proteome and two proteins, acidic lectin and gibberellin 20-oxidase found unique to the mature dry seed proteome were identified by mass spectrometry. This report highlights the potential of using 2D-PAGE and mass spectrometry as means for identification of H. brasiliensis proteins and studying its seeds germination processes. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Germination; Hevea brasiliensis; Proteome; Rubber; Seed

1. Introduction It is well recognized that the dynamics of a protein in a system is influenced by many factors. These include among others, the cell type, temperature, pH, ion concentrations, drugs and disease. These epigenetic factors also determine the protein’s post-translational modifications, secretion and function in the system. In addition, protein conformation, longevity and biomolecular interactions add further complexity to the protein in vivo [1]. These specific manifestations of protein dynamics, structures and functions in a system cannot be derived simply from the genome sequences nor can it be determined by simple cloning and expressing genes in expression systems but requires that one studies the protein itself. Attempts to map and characterize the whole protein networks in a system lead to the

* Corresponding author. Tel.: +60 3 79675756; fax: +60 3 79675757. E-mail addresses: [email protected], [email protected] (S. Abubakar).

recognition of proteome science as an important research area that complement genome studies [2]. Hevea brasiliensis or known commonly as rubber tree is the main source of natural rubber. The tree is cultivated in large commercial scale in several countries in the tropics amounting to 9.485 million ha worldwide [3]. Apart from its latex, rubber tree has also been harnessed for its wood for making furniture and the seeds for para rubber seed oil used for manufacturing soap, paint, varnishes, fertilizer and animal feeds [4]. Exploitation of these other components of the rubber trees added further value to the planting of rubber trees. However, to improve the yield and quality of latex and these other value added component of rubber trees, it is necessary to choose and plant the most suitable varieties, and this has been accomplished mostly through conventional selective breeding. This method is laborious and time consuming, but it is presently the method of choice as there is very little knowledge of the genetics and molecular biology of rubber trees. As an initial attempt to provide basic knowledge of the genome encoding the rubber tree proteins, in the present study, methods were established to enable

0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.01.018

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examination of the rubber seed proteome and changes in the proteome following rubber seed germination.

2. Materials and methods 2.1. Extraction of rubber seed proteins Rubber seeds were collected from a nearby rubber plantation, washed and kept dry at room temperature. Seeds were allowed to germinate in a typical garden soil for up to 2 weeks. The seeds were regarded as germinated when the radicle protruded through the seed coat. For protein extraction, both the mature dry and germinated seeds were cleaned thoroughly paying special attention to avoid any possible contaminations of the samples. The seed coat was peeled off using sterilized scalpel. The seed tissues were then chopped into small cubes and rapidly frozen in liquid nitrogen. The tissues were homogenized into fine powder in liquid nitrogen using a mortar and pestle. Total proteins were extracted using two methods, i.e. phenol–guanidine– isothiocyanate–chloroform method according to the protocol provided by the manufacturer (TRI reagent; Molecular Research Center Inc., USA) and urea buffer method. For phenol–guanidine–isothiocyanate–chloroform method, TRI reagent (1 ml) was added to 50 mg of the seed powder and the mixture was incubated for 15 min at room temperature. The suspension was then sedimented by centrifugation at 12,000  g for 10 min to remove insoluble materials. The clear phenol extract was transferred into a new tube and chloroform (200 ml) was added followed by vigorous shaking (15 s) and incubation (10 min) at room temperature. Following centrifugation (12,000  g) for 15 min at 4 8C, the mixture was separated into a lower red phenol– chloroform phase, interphase and the colorless upper aqueous phase. The RNA containing colorless aqueous phase was discarded while the DNA and protein containing interphase and organic phase was subjected to DNA precipitation process. Approximately 300 ml of absolute ethanol was used to precipitate DNA and the mixture was centrifuged at 2000  g for 5 min at 4 8 C. The resulting phenol–ethanol supernatant, which contained protein, was transferred into a new tube. Proteins were precipitated after adding 1.5 ml of isopropanol to the tube and sedimenting the samples at 12,000  g for 10 min at 4 8C following a 30 min incubation period at room temperature. The protein pellets were then washed three times with a washing solution (0.3 M guanidine hydrochloride in 95% ethanol) followed by another round of washing in absolute ethanol. The protein pellets were allowed to dry and then resolubilized in SDS buffer containing 10% SDS, 20 mM DTT and 40 mM Trisbase pH 8.8. The mixture was incubated at 95 8C for 15 min followed by a brief spin to remove insoluble materials. The solubilized protein was stored at 70 8C in 10% glycerol until it is needed. For urea buffer extraction, 50 mg of the seed powder were pre-incubated in 1% SDS prepared in

40 mM Tris and 65 mM dithioethreitol; DTT (Amersham Biosciences, Sweden) for 10 min at 95 8C. The mixture was then centrifuged at 40,000  g for 20 min. The supernatant was mixed with 40 mM Tris buffer containing 8 M urea, 4% 3-[(3-cholamidopropyl)dimethy-ammonio]-1-propanesulfonate (CHAPS; Pierce, USA) and 2 mM tributylphosphine (TBP; Bio-Rad Laboratories, USA) and incubated on ice for 30 min prior to centrifugation at 40,000  g for 1 h. The resulting protein supernatant was then transferred into a new tube and stored in 70 8C in 10% glycerol. Proteins were quantitated using the Micro BCA Protein Assay System (Pierce, USA). 2.2. Two-dimensional (2D) polyacrylamide gel electrophoresis Isoelectric focusing (IEF) was performed on immobilized pH-gradients (IPG) strips (pH 3–10, 4–7 and 5–8; 7 and 18 cm) with IPGphor (Amersham Biosciences, Sweden). A total of 200 mg of protein was used for the 18 cm IPG strips in a total volume of 350 ml rehydrating buffer (8 M urea, 2% CHAPS, 0.0007% of bromophenol blue, 18 mM DTT, 0.8% ampholytes of pH 3–10 (Amersham Biosciences, Sweden) and 50–100 mg for the 7 cm IPG strips in a total volume of 160 ml of rehydrating buffer. IPG strips were rehydrated overnight at 50 V on IPGphor System (Amersham Biosciences, Sweden) at 20 8C. IEF was performed at 20 8C using the following parameters: 200 V for 200 V/h, 500 V for 500 V/h, 1000 V for 1000 V/h at gradient mode, 8000 V for 4000 V/h at step and hold mode and 8000 V for 40,000 V/h at step and hold. After IEF, the IPG strips were equilibrated in equilibration buffer I (6 M urea, 2% SDS, 0.375 M Tris–HCl, 20% glycerol and 65 mM DTT) followed by equilibration buffer II (6 M urea, 2% SDS, 0.375 M Tris–HCl, 20% glycerol and 260 mM iodoacetamide) for 15 min each at room temperature. The gel strips were then rinsed in water and equilibrated for 2 min in SDSPAGE running buffer. After equilibration, excess liquid on the gel strips were blotted dry with a hand towel. Gel strip was positioned on top of a vertical 15% polyacrylamide gel, held in place by 1% agarose gel and electrophoresed at a constant current of 15 mA for 1 h, 17.5 mA for 1 h and finally 20 mA per gel for 6.5 h. Analytical gels were stained with silver nitrate as described previously [5]. Triplicates gels for each sample were prepared and the stained gels were imaged with an Epson 1600 Pro Scanner. PDQUEST software version 7.1.1 (Bio-Rad Laboratories, USA) was used for imaging and analysis of all the 2D gels. Prior to spot detection, the images were subjected to a median filter, which replaced the value of the pixel being processed with median value of pixels within the filtering window to remove outliers’ noise caused by dust and lint. Vertical and horizontal streakings were also removed and background intensity was subtracted during spot detection. The sensitivity level was adjusted to ensure detection of even the faintest spots present in the gel images. To ensure better

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spots resolution, Gaussian modeling was selected for spots detection. Similar spot detection parameters were applied for all gel replicates. The gels were then matched by landmarking common spots found in the different gels. Normalization was performed to correct variations in spot size and intensity betweens gels that is not due to differential protein expression by applying the normalization formula that divides the raw quantity of each spot in a gel by total intensity value of all the pixels in the image. Spot intensity quantitation of the PDQUEST software is based on the area under the Gaussian representation of the spot and the average spot quantities for gel replicates were calculated and used for differential expression analysis. Quantitative analyses were performed by comparing reduction or increment of spots intensities in different gels by both two- and five-fold difference. Statistical analysis (Student’s t-test, 95% confidence) was then applied to these spots to check the significance. Qualitative analyses were also performed to detect the presence of unique spots in the germinated seed gels as compared to the dry seed gels. 2.3. Peptide mass fingerprinting Silver-stained protein spots were excised from the 2DPAGE gels and the gel plugs were destained with freshly prepared destaining solution (30 mM potassium ferricyanide and 100 mM Na2O3S2
5H2O in 1:1 ratio). The gel plugs were then reduced and alkylated in 10 mM DTT prepared in 50 mM NH4HCO3 at room temperature for 3 h, followed by 30 min incubation in the dark with 80 mM of monoiodoacetic acid prepared in 50 mM NH4HCO3 at half of the initial volume used for the 10 mM DTT. The gel plugs were then digested with trypsin (0.02 mg/ml prepared in 50 mM NH4HCO3; Promega, USA) at 37 8 C for 12 h. After the

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incubation, trypsin solution was removed and saved into a new 0.5 ml microcentrifuge tube. Peptides were extracted from the gel plugs by three cycles of sonication in water followed by acetonitrile for 15 min. The extracted peptides were concentrated using vacuum centrifugation and then mixed with saturated matrix (a-cyano-4-hydroxycinnamic acid prepared in 0.5% trifluoro-acetic acid and 50% acetonitrile) at 1:1 ratio. The mixture (0.5 ml) was spotted onto slides for matrix assisted laser desorption/ionizing-time of flight (MALDI-ToF) mass spectrometry analysis (Amersham Biosciences, Sweden). Prior to each analysis, the instrument was calibrated with two known synthetic peptides. The MALDI-ToF-generated peptide mass values were searched against the NCBInr protein databases, mainly using ProFound; http://129.85.19.192/profound_bin/ WebProFound.exe. Searches were performed against the Viridiplantae (green plants) database. The following parameters were used during the search: monoisotopic masses, iodoacetic acid modification of cysteine, oxidation of methionine, mass tolerance of 0.5 and one missed cleavage for trypsin. A protein is denoted as unambiguously identified if the matching peptide coverage were at least 12% with a probability close to one.

3. Results The proteome profile of rubber seeds proteins extracted using the typical 2D-PAGE protein extraction buffer containing urea, CHAPS and TBP showed the presence of 107 protein spots (Fig. 1a). The 2D-PAGE gels of the proteins had numerous vertical streaking and dark patches perhaps due to incomplete focusing during IEF caused by

Fig. 1. Two-dimensional gels of rubber seed proteins. Rubber seed proteins were extracted by direct lysis method using urea buffer (a) and phenol/chloroform method followed by solubilization with 10% SDS (b). Isoelectric focusing (IEF) was performed on 7 cm IPG strips of pI 3–10 using 50 mg proteins.

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impurities present in the samples. In contrast, the 2DPAGE gels of the rubber seed proteins extracted using the phenol–guanidine–isothiocyanate–chloroform extraction method followed by solubilization in 10% SDS (phenol/ chloroform–SDS) showed the presence of 244 wellresolved protein spots with very little streaking (Fig. 1b). There were also more protein spots detected towards the basic end of the 2D-PAGE gels, suggesting that the phenol/

chloroform–SDS method enhanced recovery of the basic proteins. In addition, the rubber seed proteins prepared using phenol/chloroform–SDS method were well resolved on the 18 cm IPG strips with pI range 4–7 (Fig. 2a and b) and 5–8 (Fig. 2c and d). Fig. 2a and b represents 2D-PAGE gels of the mature dry and germinated rubber seeds at pI range 4–7, while Fig. 2c and d represents 2D-PAGE gels of the mature dry and germinated rubber seeds at pI range

Fig. 2. Two-dimensional gels of mature dry and germinated rubber seed proteins. Mature dry and germinating rubber seed proteins were electrophoresed on 17 cm IPG strips of pI 4–7 (a, b); pI 5–8 (c, d), respectively.

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Fig. 3. A composite image of the 2D gels of mature dry and germinated rubber seed proteins. Proteins present in both gels are circled in green, protein spots unique to mature dry rubber seed gels are circled in red and the germinated rubber seed gels in blue. Identification of the protein spots was performed using PDQUEST version 7.1.1 (Bio-Rad, USA). Arrows indicate protein spots with identification. The spot numbers correspond to spot numbers in Fig. 5 and Table 1.

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5–8. Hence, the phenol/chloroform–SDS method was used throughout the subsequent study. At least 282 (9.6 S.D.) and 261 (9.8 S.D.) protein spots were identified from the 2D-PAGE gels of the mature dry and germinated rubber seeds, respectively (Fig. 2a and b). The mature dry rubber seed proteome showed two major clusters of protein spots at the molecular mass of 36.5 to 40 kDa from pH range 4.8–6.0 and 26.6–23 kDa from pH range 4.6–5.4 (Fig. 2a). Although most proteins were clustered, individual proteins within these clusters were resolved. The germinated rubber seed proteome, on the other hand, showed more evenly distributed protein spots and the presence of protein spots with molecular mass of 36.5 to 40 kDa pH range 4.8–6.0 (Fig. 2b). The 26.6–23 kDa protein clusters noted in the mature dry seed proteome, however, were absent. Comparisons made between the germinated and mature dry rubber seed proteomes did not reveal significant differences (Student’s t-test, p > 0.05) in the number of total protein spots. However, only 60% of the proteins in the mature dry seed proteome matched that of the germinated seed proteome and most of which were of the 36.5 to 40 kDa group of proteins (Fig. 3). At least 56 proteins spots found in the mature dry seed samples were absent from the germinated seed proteome and a total of 56 different protein spots were found unique to the germinated seed (Fig. 4). Amongst the rubber seed proteins found common to both the germinated and the mature dry seeds proteomes, 24 spots were found increased in protein abundance in the germinated seed proteome, nine of which by at least five-fold and the remaining by at least two-fold.

Fig. 4. Identification of differentially expressed rubber seed proteins. Protein spots unique to the mature dry rubber seed proteome (a) and those that appeared post-germination (b) were located using PDQUEST version 7.1.1 (Bio-Rad, USA). Each spot was labeled with its unique identification number.

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Table 1 Rubber seed proteins identified by MALDI-ToF mass spectrometry Exp. molecular mass (kDa)

Exp. pI

Protein name

Protein abundance

No. of matched peptides

Predicted peptides masses

1

23.7

5.23

decreased after germination

7

2

27.1

5.78

Beta-glucosidase (putative)/glycosyl hydrolase family 1 protein MuT/nudix family protein

decreased after germination

4

1567.32, 2384.57, 2667.56, 2713.28 1150.56, 1804.88,

3

26.0

5.77

Starch branching enzyme IIb

decreased after germination

3

1040.40, 1150.31, 1804.35

4

27.1

5.58

Acidic lectin

absent after germination

3

932.55, 1298.38, 2611.07

5

35.3

5.65

Gibberellin 20-oxidase

absent after germination

4

1244.41, 1298.52, 1861.51, 2570.06

6

35.6

5.30

conserved

4

957.36, 1377.45, 2002.21, 2396.72

7

37.9

5.41

Ribulose bisphosphate carboxylase small chain, chloroplast precursor (RuBisCO small subunit) Tyrosine decarboxylase

conserved

8

932.64, 944.55, 997.65, 1063.50, 1080.47, 1244.40, 1862.32, 1873.59

8

39.0

5.76

1-Aminocyclopropane-1carboxylate synthase (ACC synthase)

conserved

5

1297.64, 1531.77, 2191.30, 3055.75, 3208.42

The spot numbers correspond to spot numbers in Figs. 3 and 5.

1584.33, 2396.43, 2697.62, 1297.52, 2159.13

Observed peptides masses

Coverage (%)

Theoretical molecular mass (kDa)

Theoretical pI

NCBI accession no.

1040.56, 1568.41, 1585.53, 2386.04, 2397.43, 2668.39, 2698.61, 2715.08 1151.45, 1298.53, 1805.66, 2159.94, 2612.42, 3040.21 1041.40, 1151.31, 1791.14, 1493.61, 1805.36, 2384.11 933.56, 1299.39, 1806.63, 2159.60, 2612.08 1245.42, 1299.53, 1862.52, 2571.07, 1970.50, 2151.02 958.37, 1040.44, 1378.46, 1573.32, 1585.40, 2003.21, 2397.72 933.65, 945.55, 998.66, 1043.47, 1064.51, 1081.47, 1245.41, 1298.63, 1863.33, 1874.60 1298.64, 1532.78, 1212.44, 1805.92, 2192.31, 2612.63, 3056.76, 3209.43

19

60.91

6.8

15218992

26

21.32

5.5

21595695

21

13.01

5.2

31415032

15

26.42

4.8

6018681

16

40.90

5.6

13625525

28

20.25

9.2

132161

19

49.17

7.67

169669

21

53.04

6.8

1244716

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Conversely, protein abundance of 13 spots decreased by at least two-fold and another 15 decreased by at least five-fold. As an early attempt to identify proteins encoded by H. brasiliensis, some of the protein spots were picked for identification using mass spectrometry. Of the proteins identified were three spots with decreased protein abundance in the germinated rubber seed proteomes, i.e. proteins with peptide mass values that matched a putative beta-glucosidase of Arabidopsis thaliana (Table 1; Fig. 3; spot 1 in Fig. 5a), MutT/nudix family protein of Arabidopsis thaliana (Table 1; Figs. 3; spot 2 in Fig. 5a) and starch branching enzyme IIb of Zea mays (Table 1; Fig. 3; spot 3 in Fig. 5a). The peptide mass values of two unique protein spots present only in the mature dry seed proteome matched the peptide mass values of acidic lectin (Table 1; Fig. 3; spot 4 in Fig. 5b) and gibberellin 20-oxidase (Table 1; Fig. 3, spot 5 in Fig. 5b) of Psophocarpus tetragonolobus and Lolium perenne, respectively. Three protein spots found conserved in both the mature dry and germinating seeds had peptide mass values that matched the ribulose bisphosphate carboxylase small chain (RuBisCO small subunit) (Table 1; Fig. 3; spot 6 in Fig. 5c), tyrosine decarboxylase (Table 1; Fig. 3; spot 7 in Fig. 5c) and 1-aminocyclopropane1-carboxylate synthase (ACC synthase) (Table 1; Fig. 3; spot 8 in Fig. 5c). The identity of the remaining protein spots could not be determined using the present available peptide mass values and databases.

4. Discussion The present study demonstrated the optimized protein extraction protocol for H. brasiliensis seed and subsequently, the proteomes of two different phases of the rubber seed, i.e. the mature dry seed and the germinated seed. The study was undertaken as an early effort to establish a platform to identify characteristic proteins involved in the biochemical and molecular processes underlying rubber seed germination. As sample preparation for 2D-PAGE is critical, rigorous extraction procedure is crucial as plant tissues in general are low in protein content. Rubber seed for instance, is estimated to contain only approximately 17.6 g of proteins per 100 g of rubber seed, with the remaining consisting of fat (48.5 g) and carbohydrate (22.9 g), water (8.5 g), ash (2.5 g), calcium (120 mg) and phosphorus (430 mg) [6]. In addition, plants are also rich in IEF interfering compounds such as salt, polysaccharides, phenolics, organic acids, pigments, terpenes and inhibitory ions [7]. Several methods for plant protein extraction have been described [8–10]. Amongst the widely used methods included precipitation by tricholoroacetic acid (TCA), acetone or TCA and acetone; direct solubilization of ground material using standard urea containing lysis buffer with CHAPS, carrier ampholytes and DTT and extraction of proteins in aqueous phase using Tris–HCl, with each method having both advantages and disadvantages. In this report, we

Fig. 5. Rubber seed proteins identified by peptide mass fingerprinting: (a) shows representative proteins decreased in abundance, (b) shows proteins unique to the mature dry rubber seed proteome and (c) shows conserved proteins.

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utilized phenol/chloroform method to extract rubber seed proteins and SDS to solubilize the proteins as the direct solubilization method commonly used by others yielded low protein recovery in addition to incomplete IEF caused perhaps by the presence of high concentration of interfering compounds [11]. The phenol/chloroform protein extraction method in combination with solubilization with SDS resulted in H. brasiliensis seed protein fractions suitable for IEF. The sequential extraction steps in the phenol/chloroform extraction protocol had probably removed much of the interfering polysaccharides, RNA and DNA contaminants and the SDS solubilization step has been reported to improve extraction and separation of basic proteins [12]. The proteomes of H. brasiliensis seeds established using the method showed the presence of at least 261 protein spots. These spots represented perhaps the most abundant H. brasiliensis seeds proteins. Other studies on the detection of seed proteins of plants such as barley [13] and Arabidopsis thaliana [14] by 2D-PAGE reported as high as 1000 seed proteins and as low as 100 proteins for wheat [15] and rice [16]. This suggest that the detection of proteins in a given extract is very much dependent on the samples, methods of protein extraction, separation and detection. This is further illustrated in the present study as no attempts were made to include the water-soluble protein extracts, resulting in the lower number of protein spots present. Lower recovery of protein from H. brasiliensis may be also due to the loss of the very hydrophobic proteins such as the membrane proteins and the high molecular weight proteins. Of the proteins detected in the H. brasiliensis proteome by PDQUEST software, two protein spots unique to the mature dry seed gels and three protein spots that decreased in abundance in the germinated seed gels were identified by mass spectrometry. The two unique proteins were acidic lectin and gibberellin 20-oxidase. The three proteins that decreased in abundance were a starch branching enzyme, a putative beta-glucosidase and MutT/nudix family protein. Both beta-glucosidase and MutT/nudix family protein possess plant cell wall hydrolytic activity associated with mobilization of the endosperm in germinating grain or during the growth of vegetative tissues [17]. These proteins were absent or decreased in abundance perhaps because the seeds had already germinated and therefore were no longer required for subsequent growth processes. Gibberellin 20oxidase for example, is an enzyme that catalyzes the last three steps in the synthesis of active gibberellins [18], a hormone required in seeds germination, was perhaps also no longer required following the germination. A negative feedback control by gibberellins has been observed in maize seedlings [19], thus suggesting that a similar feedback mechanism also occurred during rubber seed germination. The starch branching enzyme is known to exist in multiple isoforms, in which, their expressions may differ at different developmental stages of the storage organ [20]. The decreased of the starch branching enzyme IIb abundance

may reflect the differential expression of its isoforms at different stages, as seen in pea embryo [21]. Though there were significant changes in the levels of at least 24 proteins (9 proteins that were increased in abundance and 15 proteins that were decreased in abundance) in the germinated seed proteomes, the bulk (>60%) of seed proteins remained consistent to that of the non-germinating dormant seed proteome. These included three that were identified by peptide mass fingerprinting, namely ribulose bisphosphate carboxylase small chain (RuBisCO small subunit), tyrosine decarboxylase and 1-aminocyclopropane-1-carboxylate synthase (ACC synthase). RuBisCO enzyme is a common enzyme involved in plant photosynthesis [22]. Tyrosine decarboxylase is a common plant enzyme involved in the biosynthesis of numerous secondary metabolites, including hydroxycinnamic acid amides proposed to form a physical barrier against pathogens [23]. ACC synthase gene, on the other hand, is the key regulatory enzyme in the ethylene biosynthetic pathway in plants, vital for emergence of germinating seedlings [24]. Significantly more seed proteins have been identified for a number of other plant seeds especially Arabidopsis. Gallardo et al. [14] in their study, identified proteins associated with triacylgycerol catabolism, cell cycle activity, radicle emergence and defense mechanism and photosynthesis during the germination sensu stricto and radicle emergence of Arabidopsis. Several other studies have also identified other proteins or mRNAs associated with dormancy and seed germination [25–27]. However, unlike these earlier studies, no genome sequence of H. brasiliensis or closely related plant species is presently available for the complete identification of the protein spots. The present study nonetheless demonstrated that 2D-PAGE and mass spectrometry could be successfully applied to the global study of protein expressions in H. brasiliensis. The established proteomes were well resolved for protein expression studies and identification of H. brasiliensis proteins, even with limited amount of protein materials. Further examination of proteins present only in the germinating H. brasiliensis may shed some light into their roles in the resumption of metabolic activity during germination.

Acknowledgement The authors thank Dr. Chee Hui-Yee for providing the rubber seeds used in the study.

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