Journal of Biological Sciences

Year: 2011 | Volume: 11 | Issue: 2 | Page No.: 165-172
DOI: 10.3923/jbs.2011.165.172

Protein Profiling of Brassica juncea (L.) Czern var. Ensabi at Different Developmental Stages

A. F. Toosi, B. Arumugam, B. B. Baki and S. Tayyab

Abstract: Brassica juncea (L.) Czern var. Ensabi is a new local variety of mustard species, found only in Sabah and Sarawak, Malaysia. Electrophoretic characterization of plant proteins during development would be of great interest for comparison to other varieties of Brassica species. This study has been conducted to analyze protein profiles of B. juncea var. Ensabi at different growth stages. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of seed, shoot and root samples of seedling, before-flowering and after-flowering stages of the plant were performed on 10% polyacrylamide gel. Molecular weights of fractionated proteins were determined from a standard plot of log molecular weight versus relative mobility of marker proteins. Five polypeptides of ~66, 54, 39, 34 and 23 kDa, out of 11 protein bands noticed in seed proteins of Ensabi, matched closely with seed storage protein profiles of other varieties of B. juncea. The 29 kDa seed protein, commonly found in B. juncea (L.) Czern was also identified in Ensabi as S8. This protein expressed steadily in shoot samples at all growth stages. Interestingly, two seed proteins, S5 (~54 kDa) and S10 (~23 kDa) were remained expressed in both shoot and root samples throughout the plant development. Both common and unique proteins appeared at different growth stages of the plant. A comparison of the protein profiles at different growth stages suggested steady expression as well as up- and down-regulation of several genes encoding different proteins in B. juncea var. Ensabi.

Keywords: Electrophoresis, Ensabi, growth phase, molecular weight, mustard and protein make-up

INTRODUCTION

Electrophoresis is generally employed for characterization and comparison of germplasm as well as evaluation of protein expression at different developmental stages of plants. Seed storage protein electrophoresis is commonly used as a tool to discover biosystematic relationship and genetic variation among crops for classification and varietal improvement purposes (Turi et al., 2010). Identification of promising genotype(s) for different traits using simple and inexpensive technique such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been shown advantageous in breeding strategies (Kour and Singh, 2004; Sadia et al., 2009).

Brassicaceae or Cruciferae oilseeds such as mustard, canola and rapeseed are crops planted throughout the world mainly for vegetable oil production besides their use as protein-rich food i.e., animal feed and spices. In addition, the plants are also utilized as source of enzymes such as peroxidase to remove phenols from industrial waste (Noshin et al., 2003; Aluko and McIntosh, 2004; Coniglio et al., 2008; Das et al., 2010). Brassicas are best examples of amphidiploids which share molecular, morphological and physiological traits as a result of hybridization of the diploids. In general, mustard seed meal (Brassica juncea L.) contains good amount of protein e.g., 28-36% (Das et al., 2009). Electrophoretic analyses of seed storage proteins of many Brassica species (B. juncea, B. campestris, B. rapa, B. carinata, B. olaracea, B. napus) and their respective varieties have been well documented. These studies revealed genetic diversity i.e., presence of unique species-specific polypeptides as well as common proteins in polypeptide composition of each species. Consequently, this enabled researchers to understand homology and taxonomic relationship among them (Rabbani et al., 2001; Aluko and McIntosh, 2004; Kour and Singh, 2004; Sadia et al., 2009; Turi et al., 2010). For instance, B. Carinata contains polypeptides with molecular weights of 32.6 and 30.7 kDa which are also present in B. rapa and B. napus as species-specific polypeptides, respectively. However, these polypeptides are not present in B. juncea (Sadia et al., 2009). Brassica juncea (L.) Czern have been shown to contain a 29 kDa polypeptide, a common and major protein present in Brassica juncea seed meals (Aluko and McIntosh, 2004). This genetic information on Brassica germplasm is believed to be important to improve edible oil production and nutritional benefits of this crop (Turi et al., 2010).

Brassica juncea (L.) Czern var. Ensabi is a new local variety of mustard species found only in Sabah and Sarawak, Malaysia. A handful number of researches have been initiated to characterize allelopathic activity, physical and chemical properties of B. juncea var. Ensabi (Tossi and Bakar, 2007) in an attempt to explore the use of its extract as natural herbicide. Electrophoretic characterization of different polypeptides of B. juncea var. Ensabi would be of great interest for comparison to other varieties of Brassica species. Here, we present our data on protein profiling of this plant at different growth stages such as seeds, seedling, before-flowering and after-flowering stages, using SDS-PAGE and their comparison within each stage or different stages of development.

MATERIALS AND METHODS

All these experiments were conducted at RIMBA ILMU and Biochemistry Laboratory of the Institute of Biological Sciences, University of Malaya, Malaysia (July-December, 2009).

Chemicals: Bio-Rad (2000) prestained SDS-PAGE standards, broad range (Catalog No. 161-0318, Control 310004830) and reagents for SDS-PAGE were purchased from Sigma Chemical Co., USA. All other reagents used were of analytical grade purity. All the experiments were performed at 28°C unless otherwise stated.

Plant materials: Mature seeds of Brassica juncea (L.) Czern var. Ensabi were collected from Ensabi plants grown in the University of Malaya campus and stored with silica gel in darkness at 4°C before use. Fifty fresh seeds were placed in each petri dish, previously lined with 9 cm diameter Whatman No. 1 filter paper. The filter paper was moistened with 6 mL of water. The petri dishes were placed in growth chambers with a temperature regime of 25°C and exposed to fluorescent light with an intensity of 630 Em^-2sec^-1. All petri dishes were augmented with 6 mL of deionized water at 3 day intervals in order to maintain moisture conditions. Germinated seeds (complete seedlings) were removed from the petri dishes 14 days after sowing and used in subsequent experiments.

A part of seeds of Ensabi were sown in wooden boxes previously filled with garden soil of Malacca series in an insect-proof house. After germination, the plants were subjected to 12 h of natural sunlight outdoor (mean midday radiation of 1812 μmole photon m^-2sec^-1), 384 μmole photon m^-2sec^-1 inside the insect-proof house, mean ambient temperatures of 33±2°C (day) and 25±2°C (night) at Rimba Ilmu, University of Malaya, Kuala Lumpur, Malaysia. Plants were harvested at two different growth stages i.e., before- and after- (physiological maturity) flowering stages. These plants received a dose of fertilizer as 300 kg of NPK (15-15-15) ha^-1 plus 150 kg of nitrogen ha^-1 in split dose i.e., full NPK with half nitrogen at sowing and remaining half of nitrogen fertilizer 25 days after planting. Full dose of NPK was applied in the row below the seeds in the form of ammonium nitrate (34%), triple super phosphate and muriate of potash as the source of N (nitrogen), P (phosphorous) and K (potassium), respectively. These plants (before-and after-flowering stages, collected 52 and 86 days after germination, respectively) were immediately washed with double distilled water to remove soil or other adhered materials and used in other experiments.

Sample preparation: Roots and shoots of seedlings as well as each harvest (before-and after-flowering stages) were separated. Five grams of each material (seeds, shoots and roots obtained at seedling, before-and after-flowering stages, respectively) were soaked separately in 0.06 M sodium phosphate buffer, pH 6.8 overnight. These samples were grounded using mortar and pestle with 10 mL of the same buffer and filtered through four layers of cheese cloth to remove fiber debris. The filtrate of each sample was centrifuged at 5500 rpm for 30 min at 5°C. The supernatant was then vacuum filtered again with Whatman No. 42 filter paper. Finally, the last filtrate of each sample was stored at 4°C.

Protein estimation: Protein concentration in the plant samples was determined by Bradford (1976) method using BSA as the standard.

SDS-polyacrylamide gel electrophoresis: SDS-PAGE was performed to study the protein profiles of B. juncea var. Ensabi at different developmental stages according to the method of Laemmli (1970) on 10% (w/v) polyacrylamide gel in tris-glycine buffer, pH 8.3 containing 0.1% SDS. Sample incubation buffer also contained 5% (v/v) β-mercaptoethanol. About 10 μL of sample containing 5-20 μg of protein was loaded in each well and electrophoresis was carried out for about 2 h. The gel was stained with 0.2% (w/v) coomassie brilliant blue R-250 and destained in 5% methanol, 7% acetic acid solution. Different marker proteins along with their molecular weights (adapted from technical bulletin of Bio-Rad (2000) prestained SDS-PAGE standards for Tris-HCl gel) given in parentheses: myosin (198, 510 Da), β-galactosidase (116, 254 Da), BSA (84, 796 Da), ovalbumin (53, 896 Da), carbonic anhydrase (37, 418 Da), soybean trypsin inhibitor (29, 051 Da), lysozyme (19, 809 Da) and aprotinin (6, 845 Da) were used for calibration. It is important to note that molecular weight values of some of these preparations are not matched with the reported values due to the formation of dye-protein complex as reported by the manufacturer (Bio-Rad, 2000).

Statistical analysis: Distances travelled by the protein band and bromophenol blue front were measured. Relative mobility (R_m) values of different protein bands were calculated using the following formula:

Data of log molecular weight (log MW) of different marker proteins were plotted against their R_m values. Regression analysis of these data yielded the following straight-line equation:

The above equation was used to determine the molecular weight of various fractionated proteins at different growth stages of B. juncea var. Ensabi.

RESULTS AND DISCUSSION

Electrophoretic patterns of protein samples obtained at different growth stages of Brassica juncea (L.) Czern var. Ensabi, namely, seed (S), shoot of the seedling (SH_S), root of the seedling (R_S), shoot of the plant before-flowering (SH_BF), root of the plant before-flowering (R_BF), shoot of the plant after-flowering (SH_AF) and root of the plant after-flowering (R_AF) showed presence of a number of fractionated proteins ranging from low to high molecular weights (Fig. 1a-c). The molecular weights of these proteins were determined using a standard curve between log molecular weight and relative mobility (R_m) of different marker proteins as described in ‘MATERIALS AND METHODS’ and listed in Table 1-4. A slight variation in the R_m values of certain marker proteins used in this study (Lane 1 of Fig. 1a-c) compared to those shown in the technical bulletin supplied by the manufacturer can be ascribed to the use of a low percentage and non-gradient polyacrylamide gel against a gradient gel employed by the manufacturer.

Consequently, both high molecular weight (myosin) and low molecular weight (aprotinin) protein bands were omitted in the construction of the standard curve. Use of the remaining six marker proteins in plotting the standard curve showed a strong correlation coefficient of 0.995. Furthermore, straight-line equation yielded similar molecular weights of marker proteins by using their R_m values.

A total of 11 major protein bands were noticed in seed (S) sample (Lane 2 of Fig. 1a-c) ranging in molecular weight from 140, 610 to 19, 327 Da (Table 1). Out of these fractionated proteins, seven proteins marked as S3, S4, S6, S7, S9, S10 and S11 were present in relatively higher amounts (Lane 2 of Fig. 1a-c). These proteins represent the storage, structural and biologically active proteins including enzymes associated with the hydrolysis of stored food (amylase, maltase, protease, carbohydrase and lipase), germination, aerobic/anaerobic respiration, lectins and enzyme inhibitors (Fukushima, 1991;Mandal and Mandal, 2000).

Besides, proteins required for seed protection i.e., defense against micro-organisms and insects may also constitute the total protein content of the seed (Kelly et al., 1998). The number of protein bands appeared in the electrophoretogram (Lane 2 of Fig. 1a-c) shown in this study is relatively low compared to ~26 bands displayed in previous reports for seed protein profile of B. juncea (Sadia et al., 2009; Turi et al., 2010).

This can be attributed to the relatively lower resolving power of non-gradient 10% polyacrylamide gel used in this study. The seed protein profile shown in this report was compared with the spectrum of seed proteins obtained from 13 B. juncea cultivars (Sadia et al., 2009). Five major peptides with molecular weight values of ~66, 54, 39, 34 and 23 kDa matched closely with S3, S5, S6, S7 and S10 proteins present in B. juncea (L.) Czern var. Ensabi (Fig. 1, Table 1). Appearance of S8, a protein of ~29 kDa in B. juncea var. Ensabi seed sample has been reported to be a commonly occurring polypeptide in B. juncea (L.) Czern (i.e., type: AC Vulcan and Commercial Brown) seed meals (Aluko and McIntosh, 2004). Dhawan and Nainawatee (1994) also showed presence of a 28 kDa water soluble polypeptide as the major protein in B. juncea seed meals besides 12 and 13 kDa polypeptides. Indeed, the protein (S8) remained expressed in shoot samples of B. juncea var Ensabi at all growth stages in the form of SH_S11, SH_BF7 and SH_AF8 (Fig. 1). Polypeptides with molecular weight of 66 and 34 kDa are two common proteins observed between B. juncea and B. napis as well as B. campestris and B. juncea, respectively (Dhawan and Nainawatee, 1994). These two polypeptides were also present in B. juncea var. Ensabi in the form of S3 and S7, respectively (Fig. 1, Table 1).

B. juncea seed meal is reported to contain calcium-soluble protein fraction (Aluko et al., 2004). The composition of this fraction from yellow mustard (Sinapis alba) seed meal has shown the presence of major polypeptides with different molecular weights i.e., 5, 15, 22, 28, 35, 50 and 55 kDa (Aluko et al., 2004). In view of it, four seed proteins, namely, S5 (~54 kDa), S7 (~34 kDa), S8 (~29 kDa) and S10 (~23 kDa) shown in this study (Table 1) seem to belong the same group of calcium-soluble protein fraction. Storage proteins in Brassicaceae oilseeds (mustard) are classified into two major types, labelled as cruciferin or legumin type globulin (11S or 12S/300-360 kDa) and napin or napin-like albumins (1.7S or 2S/15-18 kDa) (Schwenke et al., 1981; Sjodahl et al., 1991; Lonnerdal and Janson, 1972; Crouch et al., 1983). Cruciferin is a hexameric protein where each subunit contains acidic/α (30 kDa) and basic/β (20 kDa) polypeptides linked by a disulfide bond (Dalgalarrondo et al., 1986; Delseny and Raynal, 1999). On the other hand, napin, an allergenic protein in Brassicaceae (i.e., Bra j 1 in B. juncea) (Monsalve et al., 2004) is composed of a large or heavy (10-12 kDa) and a small or light (4-6 kDa) polypeptides linked by four disulfide bonds (Gehrig and Biemann, 1996; Rask et al., 1998). Peptide sequences of 22, 28 and 35 kDa proteins of S. alba were shown to be composed of cruciferin peptides through MALDI-TOF analysis (Aluko et al., 2004). Due to the similarity of B. juncea var. Ensabi seed proteins S7, S8 and S10 (Table 1) with those of S. alba proteins, it can be suggested that these proteins may represent cruciferin peptides. Less resolving power of our non-gradient 10% polyacrylamide gel might be responsible for the lack of characterization of napin-like albumins in B. juncea var. Ensabi.

Interestingly, two seed proteins , namely, S5 and S10 of B. juncea var. Ensabi remained expressed in both shoot and root samples at all developmental stages in the form of SH_S7, SH_BF5, SH_AF6; R_S8, R_BF5, R_AF6 and SH_S12, SH_BF8, SH_AF9; R_S11, R_BF9, R_AF9, respectively (Fig. 1,Table 1-4). These seed proteins may possibly represent vital proteins or enzymes required for the growth, biochemical reactions and defense mechanism from the very early stage. Seed protein S11 of B. juncea var. Ensabi (Table 1) seems to represent trypsin inhibitor due to similarity in molecular weight with a trypsin inhibitor, BjTI (MW ~20 kDa) observed earlier in B. juncea (Indian mustard). This protein is supposed to be involved in the regulation of endogenous plant proteinases and protection of seed from diseases and pests besides its storage function (Mandal et al., 2002). Absence of this protein (S11) in shoot and root samples of different developmental stages further strengthens our prediction as BjTI was present only in seeds and not in other plant parts (Mandal et al., 2002). On the other hand, presence of seed protein S2 (MW ~134 kDa) in B. juncea var. Ensabi (Fig. 1, Table 1) contradicted an earlier report suggesting the absence of 135 kDa polypeptide in Brassica seeds (Aluko and McIntosh, 2004). In view of the very low R_m values of both S1 and S2 proteins and being outside the range of R_m values of different marker proteins used in this study, computed values of molecular weights of these proteins remain questionable. Further studies are needed to clarify the presence of high molecular weight proteins in B. juncea var. Ensabi.

When the seeds (embryos) germinated as seedlings, several new proteins, particularly in the molecular weight range ~74-127 kDa appeared both in shoots (SH_S) and roots (R_S) of seedlings (Lanes 3 and 4 of Fig. 1a) due to up-regulation of genes. In general, 12 and 11 protein bands appeared in shoot and root samples, respectively of seedlings of B. juncea var. Ensabi. Out of these bands, SH_S1, 2, 3, 4, 5, 6, 8, 9 and R_S1, 2, 3, 4, 5, 6, 9 represent newly expressed proteins in shoot and root of B. juncea var. Ensabi’s seedlings, respectively (Lanes 3 and 4 of Fig. 1a, Table 2). The newly expressed proteins may account for cell division, enlargement and differentiation during growth and development of seedlings. Mustard plants in Brassicaceae family are known as hyper accumulators due to their ability to concentrate heavy metals in their different plant parts (Garg and Kataria, 2010). Therefore, the newly expressed polypeptides seem to have been evolved for heavy metal ion homeostasis and detoxification (Zhou and Goldsbrough, 1994; Cobbett, 2000). Three additional seed proteins, S4, S6 and S8 were remained expressed in seedlings as R_S7, SH_S10/R_S10 and SH_S11 (Fig. 1a, Table 1, 2), respectively besides S5 and S10 as described above and may serve similar functions. On the other hand, genes for remaining six seed proteins seem to be down-regulated once the seed transformed into seedling stage (Fig. 1a, Table 1, 2). This is possibly due to proteins/enzymes needed for catabolic reactions in seeds are no longer required as the dry mass of food storage declines at the end of seed dormancy for germination and seedling development (Mandal and Mandal, 2000).

A comparison of protein profiles of shoot and root samples of seedlings suggested several similarities as well as presence of unique proteins in each part. Proteins with molecular weight values of ~98, 94, 90, 73, 55, 46, 37 and 23 kDa were common in both shoot and root samples of seedlings in the form of SH_S3, 4, 5, 6, 7, 9, 10, 12 and R_S3, 4, 5, 6, 8, 9, 10, 11, respectively (Lanes 3 and 4 of Fig. 1a, Table 2), suggesting that these proteins share similar functional properties. Distribution of similar proteins (e.g., calcineurin or Ca²⁺/calmodulin-dependent protein phosphatase, classical type of protein kinase c, stress related protein and phytochelatin synthase each of 55 kDa, glyoxalase I of ~58 kDa, an endogenous protein of 85 kDa) in different parts of seedlings has also been shown in different varieties of B. juncea (Sharma and Deswal, 2004; Deswal et al., 2004; Pareek et al., 1998; Deswal and Sopory, 1998; Gasic and Korban, 2005). Proteins such as SH_S1, 2 and 8 as well as R_S1 and 2 were the distinctive polypeptides present in shoot and root of seedlings, respectively (Lanes 3 and 4 of Fig. 1a, Table 2). These proteins may represent the soluble enzymes or polypeptides available in leaves, particularly in chloroplast or stroma (SH_S1, 2 and 8) and in root (R_S1 and 2), responsible for photosynthesis and detoxification of heavy metal absorbed from soil, respectively.

Figure 1b (Lanes 3 and 4) shows SDS-PAGE pattern of B. juncea var. Ensabi at before-flowering stage, bearing a total of 8 and 9 protein bands in shoot (SH_BF) and root (R_BF) samples, respectively. Proteins, SH_S9 and R_S2, 4, 5, 6, 9, 10 in shoot and root samples of seedlings remained nearly akin in B. juncea var. Ensabi at before-flowering stage as SH_BF6 and R_BF1, 2, 3, 4, 6, 7, respectively (Table 2, 3). These are the proteins which possibly support continuous elongation of cells, growth and development of the plant during the primary growth stage of plant body, photosynthesis, transpiration, translocation and senescence. Proteins SH_BF1, 2, 3, 4 and R_BF8 are the new proteins appeared in shoot and root, respectively at before-flowering stage compared to seedling stage of B. juncea var. Ensabi (Lanes 3 and 4 of Fig. 1b, Table 3). The newly expressed proteins in shoot may include proteins responsible for initial growth of lateral buds and reproductive organs (flowers) of the plant. Apart from proteins SH_BF5, 8 and R_BF5, 9, all other proteins of shoot and root samples at before-flowering stage were totally distinct (Lanes 3 and 4 of Fig. 1b, Table 3). It seems probable that some of the expected common polypeptides of shoot and root samples were present in very low amount and hence were not visible on the electrophoretogram.

Protein profiles of shoot and root samples at after-flowering stage of B. juncea var. Ensabi are shown in lanes 3 and 4 of Fig. 1c. Poor visibility/low intensity of protein bands in both shoot and root samples may be attributed to either the low content of these proteins present in these tissues naturally or low recovery of proteins from these plant parts during extraction procedures. In spite of the above limitation, a total of 9 protein bands were visible in both shoot and root samples at this stage. Proteins which emerged at seedling stage of B. juncea var. Ensabi, particularly in root samples such as R_S2, 4, 6, 9 and 10 were remained throughout the plant growth. These proteins were represented as R_BF1, 2, 4, 6, 7 and R_AF1, 2, 3, 7, 8 at the before- and after- flowering stages, respectively (Table 2-4). Proteins S4/R_S7 and S6/SH_S10/R_S10 which were visible at both seed and seedling stages were seen in the form of R_AF5 and SH_AF7, respectively at the after-flowering stage (Fig. 1c, Table 1, 4). Shoot proteins SH_AF2, 3, 4, 6 and 9 were found to be similar to root proteins R_AF1, 2, 3, 6 and 9, respectively during this stage, whereas, proteins SH_AF1, 5 and R_AF4 were the unique proteins present in shoot and root, respectively at the after-flowering stage (Lanes 3 and 4 of Fig. 1c, Table 4).

ACKNOWLEDGMENTS

This study was financially supported by the University of Malaya grant in the form of IPPP grant (Grant No.: PS245/2008C) sanctioned to A.F. Toosi. A.F. Toosi also acknowledges the financial assistance (UM Fellowship) received from the University of Malaya. We are indebted to the Head, Institute of Biological Sciences and the Dean, Faculty of Science, University of Malaya for providing necessary facilities. S. Tayyab is a member of CRYSTAL research group.

REFERENCES