SDS-PAGE Analyses and Electrophoretic Band Characterization of Three Cultivars of Grass Pea based on Seed Storage Proteins

Grass pea (Lathyrus sativus L.) is a member of the Vicieae tribe and family Fabaceae, consists of about 160 annual and perennial grain legume crop plants and many of them are economically important. This species is represented by several sub-species and important cultivars. Grass pea (khesari/Teora/Lakh/Lakhadi in India) is prized for its high dietary protein content (26-32%), adaptability to harsh environments, disease tolerance and low input requirements for cultivation (Urga et al., 2005). Despite its drought resistance, grass pea is unaffected by excessive rainfall and can be grown in flood-prone areas, including those with poor soils and thick clay (Sinha, 1980). Grass-pea can easily be grown on marginal land under normal as well as adverse environmental conditions. Because of its ease of cultivation, grass pea is considered a traditional common crop by more than 100 million people in drought-prone areas of Asia and Africa and cheapest food legume for low-income families and rich source of protein and good qualities of essential amino acids. Grass pea also contains an undesirable and toxic non-protein amino acid called ß-N-oxalyl-L-α, ß-di-aminopropionic acid (ODAP) which can lead to‘neuro-lathyrism’ a disease causing paralysis of lower limbs if consumed in high quantity (300-400 g/day) as a staple food for a long period of time (for continuous 2-4 months) (Singh and Rao, 2013; Mondal and Puteh, 2014; Basaran et al., 2016; Ozbek Yazici et al., 2020). The ODAP content of different grass pea genotypes ranged from 1.55 to 20.8 mg/g seeds (Arslan et al., 2017). Its seeds also contain a high amount of free L-homoarginine (1.8±0.06 g/100 g of seed flour), which may act as a precursor of lysine (1.76±0.11 g/100g of seed flour, C6H14N2O2) (Mariame Cisse et al., 2013). Lysine is an essential amino acid and the building blocks of protein.Lysine helps to produce collagen in the body and it is essential for the development of carnitine, a nutrient that aids in the conversion of fatty acids into energy and lowers cholesterol. It also appears to have antiviral properties. As a fodder crop it is both palatable and extremely nutritious and for sheep its high lysine content promotes improved wool quality (Tubby, 2009).

Grass pea seed proteins are synthesized at a particular stage during seed development to be utilized during germination. These proteins are therefore called storage proteins. Seed storage proteins are highly physiologically stable, being unaffected by environmental conditions and easily to handle. The major storage seed proteins are albumins (water soluble), globulins (salt soluble), glutelins (alcohol soluble) and prolamins (acid or alkali soluble) etc. of seeds and these proteins display a number of characteristic features (Osborne, 1924; Katsube et al., 1999). Thus, it was reported that biochemical and molecular analysis, particularly of electrophoretic analysis of seed proteins as revealed by electrophoretic band profiles of seed proteins of SDS-PAGE (Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis) have provided valid evidence for detecting and assessing relationships even at the level of cultivars (Ladizinsky and Hymowitz, 1979; Kamel et al., 2003; Javaid et al., 2004; Celebi et al., 2009; Hameed et al., 2009; Jukanti et al., 2017). So, the objective of present study was to determine the differences among three important cultivars of Lathyrus sativus based on seed storage protein by using SDS-PAGE and to obtain information useful for a breeding program.

Plant material
 
The seeds of 3 different cultivars of grass pea (Fig 1 A-C) namely B-1(Nirmal), flower initiation time 58 days), Prateek (flower initiation time 67 days) and BIO L-212 (Ratan), flower initiation time 52 days) were officially procured from Pulses and Oilseeds Research Station, Berhampore, West Bengal, India. These cultivars were chosen and selected as experimental materials only on the basis of their flowering time (an agronomic character). 100 seed weights of three cultivars were 32.4 gm (Prateek), 35.5gm (Nirmal) and 42.5 gm (Ratan) respectively.


 
Extraction of seed proteins
 
From each cultivar’s 0.2 g dry seed was homogenised in chilled 2 ml of 0.2 M phosphate buffer in a pre-chilled pestle and mortar (pH-8.2). The extracts were centrifuged at 4°C for 15 minutes at 10,000 rpm. The crude proteins were extracted and the supernatant was used for protein profiling. By using a dye-binding assay, the protein concentration of extracts was determined immediately and directly from the supernatant using the method of Bradford (1976). A standard curve of 595 nm absorbance versus 10-80 µ g of BSA was also drawn and the amount of protein in the sample was computed using this curve. Repetition of same experiment was done 3 times in order to check the reproducibility of the method.

Seed protein sub-fractions (albumins, globulins, glutelins and prolamins) from Lathyrus seeds were extracted and quantified as described by Vaz et al., (2004).
 
SDS-PAGE
 
To denature the protein, the supernatant was mixed (1:1) with 2x sample buffer (Laemmli, 1970) and heated in a 1.5 ml eppendorf tube in a water bath at 85°C for 3 minutes just before starting the electrophoresis process. Then the protein samples were then run through a one-dimensional SDS-PAGE in a 1 mm thick gel slab (4% stacking gel= 2.5 cm height and 10% resolving gel= 5.5 cm height). The gel was 8 x 7.3 cm2 in total.

Electrophoresis was performed in a vertical electrophoresis apparatus (Bio-Tech India Pvt. Ltd) using a discontinuous buffer system as per the method of Laemmli (1970). 20 µl protein samples were loaded into each well of the gel using a micro-pipette. Protein molecular weight marker (molecular weight range= 14-97 kDa) from Chrommas Biotech, India, was used in one well of the same gel. To see the movement of protein in the gel, 0.02 per cent bromophenol blue (BPB) was added to the protein sample as a tracking dye. The gel was run in continuous current mode at 10 mA. The gel was then stained with 0.025 % Coomassie brilliant blue (CBB) R-250 for an overnight staining.
 
Analysis of gel documentation
 
Finally, the Gel-DocTM XR+ system from Bio-Rad in the United States was used to photograph and scan the gels. Image LabTM software (version 5.0) was used to perform a detailed analysis of protein band patterns in terms of band number, mobility of protein bands, staining intensity, band percentage, lane percentage and molecular weight determination for each band.
 
Quantitative assay of ODAP
 
This study was conducted according to the protocol described by Karadag et al., (2010).

The total seed protein of three grass pea varieties was analysed using one-dimensional denaturing SDS–PAGE, revealing qualitative and quantitative inter-varietal differences in terms of total number of protein bands, location, thickness, staining intensity (Fig 2), relative mobility of bands and molecular weight. The Gel Doc data/electrophorogram (Fig 3 A-C) of total protein profiles showed the number of peaks of different heights obtained on the basis of the staining intensity of protein bands against their respective relative mobility (Rf value) of bands of the studied cultivars.

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During protein profiling of total proteins of experimental cultivars, B-1(Nirmal) and Prateek showed 26 protein bands while 27 protein bands were found in case of BIO L-212 (Ratan). The result shows the relative mobility (Rf value) and molecular weight of protein bands varied from 0.002 to 0.979 kDa and 14.3 to 97.4 kDa (Table 1-3). Quantitative variation of total seed protein and major storage seed protein among different varieties of grass pea studied in the present experiment was also found and is shown in Table 4.



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The quantities of total seed proteins in the present study (Table 4). Prateek cultivar (78.9 μg/ml) has highest total protein content whereas B1 (Nirmal) (65.4μg/ml) has lowest total protein content. Present study also reports intermediate quantity of total seed proteins (73.2 μg/ml) in BIO L-212 (Ratan) cultivar. Present results showed that B1 (Nirmal) and BIO L-212 (Ratan) have the highest albumin content (3.6 μg/ml and 4.5 μg/ml, respectively). Nirmal (7.2 μg/ml) and Ratan (8.0 μ g/ml) had higher globulin content than the other studied variety. Furthermore, it was determined that Ratan has the highest glutelin content (3.4 μg/ml) and the highest prolamin amounts (0.41 μg/ml) among the varieties studied. However, prolamin amounts of all studied taxa were quite low.

In this endeavour, ODAP content estimated in three grass pea cultivars was found very low (Table 4) ranging from 0.06 to 0.08%. These low-toxin (<0.1%) varieties are highly desirable for cultivation.

The SDS-PAGE for water-soluble storage seed protein electrophoresis was used to investigate primarily the diversity of seed storage proteins and inter-varietal genetic differences (Emre et al., 2015) among three grass pea cultivars. The band patterns show variations in the number of bands, band position and band molecular weight, among other things. Each cultivar’s protein profiles exhibit their own electrophoresis pattern, with subunits of varying molecular weight. The differences between varietiesare also observed in both presence and absence of a particular protein band and all three taxa are clearly identifiable from the protein banding patterns. Based on SDS-PAGE results, it can be said that electrophoretic analysis of seed storage proteins demonstrated the similarity between Nirmal and Prateek in terms of same number of protein bands (26) whereas Ratan variety differs by its 27 protein bands. All three cultivars are clearly identifiable from their protein banding patterns in view of both band mobility and relative intensity of the bands.

Furthermore, our present results show that the early flowering characteristics in BIO L-212 (Ratan) cultivar are generally correlated with higher mean levels of total seed protein and glutelin. High seed protein content and early flowering are very important for grass pea breeding strategies, agronomy and improvement (Girma et al., 2012).

Protein content and seed weight are important traits in grass pea. The approximate ranges in protein content and seed weight of the grass pea collection analysed are 26%-32% and 32.4–42.5 g (100 seeds weight), respectively. Interesting, for values of protein content and 100 seeds weight seems to be the valid evidence of same geographical origin, which could be utilized in breeding programmes after further evaluation and characterization. As a result, genotypes with high protein content and/or 100 seed weight may be useful parents for breeding improved genotypes (Bisignano et al., 2003). In other words, strategies for recognize high protein individuals within higher-seeded entries should be suggested.

Based on SDS-PAGE results, it can be concluded that electrophoretic analysis of seed storage proteins can be used as powerful tools for grass pea breeding strategies, agronomy and improvement. In this endeavour, ODAP content among three grass pea varieties was found very low, so these varieties are nutritionally safe for food and feed as well as highly desirable for cultivation. Storage protein profiles of each variety showed its own electrophoresis pattern by which they can also be distinguished biochemically particularly on the basis of number of protein bands, band mobility, relative intensity and molecular weight of individual protein band. However, protein content, seed weight, early flowering characteristics and glutelin content could be used in the selection of superior parental genotypes for breeding programmes.

The authors gratefully acknowledge the assistance and support of the Pulses and Oilseeds Research Station, Berhampore, West Bengal, India for providing the experimental materials. We also gratefully appreciate the cooperation of the Post Graduate Department of Botany, Hooghly Mohsin College (Estd. 1836) in conducting this research work.
 

All authors contributed equally to this paper. Animesh Mondal performed the seed protein extraction, quantitative assay of protein and gel electrophoresis experiments the Gel Doc analysis, interpretations, seed collection and characterizations; Kalyan Kumar De contributed in design, analyses of the results and writing the paper.

The authors declare no conflict of interest.