Semi-Quantiﬁcation of Lectins in Rice ( Oryza sativa L.) Genotypes via Hemagglutination

: Lectins are unique glycoproteins that react with speciﬁc sugar residues on cell surfaces resulting in agglutination. They offer enormous applications in therapeutics, diagnostics, medicine, and agriculture. Rice lectins are naturally expressed during biotic and abiotic stresses suggesting their importance in stress resistance physiology. The objective of this study was to determine the presence and relative concentration of lectins in different accessions of rice obtained from IABGR/NARC Islamabad mainly originated from Pakistan. About 210 rice accessions including 02 local varieties and 05 transgenic seeds were screened for seed lectins using a hemagglutination (HA) assay with 5% Californian bred rabbits’ erythrocytes. A protein concentration of 3–8 mg/100 mg of seed ﬂour was measured for all the rice accessions; the highest was 8.03 mg for accession 7600, while the lowest noted was 3.05 mg for accession 7753. Out of 210 accessions, 106 showed the highest HA activity. These 106 genotypes were further screened for titer analysis and speciﬁc activity. The highest titer and speciﬁc activity were observed for accession 7271 as 1024 and 236 hemagglutination unit (HAU), respectively. The selected accessions’ relative afﬁnity and HA capability were evaluated using blood from four different sources: human, broiler chicken, local rabbit, and Californian-breed rabbit. The highest HA activity was observed with Californian-breed rabbit RBCs. The lectin assay was stable for about 1–2 h. After the required investigations, the accessions with higher lectin concentration and HA capability could be used as a readily available source of lectins for further characterization and utilization in crop improvement programs.


Introduction
Lectins are carbohydrate-binding proteins present in every form of life. These are non-immune in origin and can recognize and bind unique sugars present on cell surfaces, resulting in cell agglutination. These are major glycoproteins that react with specific

Extraction of Lectins from Rice
The dry, mature seeds were finely grounded in a mortar and pestle. Seed meal was defatted with two volumes of hexane twice by centrifugation, and then dried overnight at room temperature [38]. About 0.2 g of defatted seed flour was added to 1 mL of 1 X phosphate buffer saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 , pH 7.4), shaken for 4 h on an orbital shaker before centrifugation. The suspension was then cleared by centrifugation at 10,000× g for 10 min at 4 • C, and the extract was stored at −20 • C [21].

Preparation of RBCs
About 2 mL of Californian breed rabbit blood was obtained in ethylene diamine tetraacetic acid (EDTA) tubes from animal husbandry of Agriculture University Peshawar Khyber Pakhtunkhwa, Pakistan.It was immediately centrifuged at 2000 rpm for 10 min. The serum was discarded, and the pellet was washed twice with 1 X PBS. Finally, the washed erythrocytes were suspended in 1 X PBS at a concentration of 5% in different flasks and used for HA [33].

HA Assay
The assay was carried out in a standard U-bottomed microplate. The accessions showing the highest agglutination were serially diluted 2-fold as Bing et al. described [34]. A 50 µL of the clarified lectin extract was mixed with half volume (25 µL) of 5% Californian breed rabbit erythrocytes and incubated for 30 min at room temperature. HA was observed with the naked eye, measured titer, representing reciprocal of highest dilution upon which full agglutination was achieved. The specific activity of HA is defined as the titer of HA per mg of protein [21,39].

Total Protein Estimation
A standard but miniaturized photometric Bradford method was used to estimate the protein content in the seed extracts having BSA as standard using a 96 welled U-bottomed microplate via reader at 595nm [40,41]. The detailed method is given in Table 2.

Hemagglutination Inhibition Assay (HIA)
Hemagglutination inhibition assay (HAI or HI) using D-Glucose was performed as described by Adenike and Ereton, (2004) [42]. Serial dilutions of the carbohydrate solution (D-Glucose; initial concentration 200mM) were incubated at room temperature with equal volume of the lectin extract for 30 min. After that 50 µL of 5% rabbit erythrocytes were added, mixed, and incubated for further 30 min.

Screening Blood Affinity for Rice Lectins
The affinity of different blood types with rice lectins was screened using various percentages (3-5%) of erythrocytes obtained from various sources, including human (blood group B+), broiler chicken, and two strains of rabbit, i.e., local and Californian breed rabbit [20,42].

Rice Lectins Extraction
The results of lectins extraction from rice for the analysis are given in Tables 3 and 4. The procedure of lectin assay was miniaturized as a large number of samples (i.e., 217) of rice (Oryza sativa L.) had to be analyzed; also, the plant material available was less in quantity (5-10 seeds). Thus 96-welled microplate was used for HA. Table 3. Hemagglutination activity (HA) with Californian breed rabbit RBCs and protein content (mg/mL) in the seeds of rice (Oryza sativa L.) accessions using Bradford method.

HA Assay
Approximately all the accessions showed the presence of lectins via HA out of 217 (210 wild, 2 cultivated and five transgenic) accessions, 109 accessions (106 wild, 1, cultivated, 2 transgenic) gave strong agglutination results (Tables 3 and 4; Figure 1a-g). The accessions (109) exhibiting high HA activity were further serially diluted for titer analysis and specific activity as shown in Table 4. The respective titer values observed for 7816 and 7271 were 4-1024, making a range of 4.315 (lowest)-1024 (highest) HA unit (HAU) and their specific activity as 0.231-236. This micro assay requires only 25 to 100 µL of extract, whereas the conventional glass slide method requires at least 1000 µL of plant extract mixed with the same amount of RBC suspension for a single assay [32,33].
HA is used to detect lectins in the crude or clarified extracts of plants. Purified rice seed extracts were serially diluted by a two-fold dilution method till well#10. The last wells (11 and 12) were reserved for positive (gum arabic) and blank (PBS). Figure 1a-g shows most of the accessions' HA assay for lectins using 5 % Californian breed rabbit erythrocytes. The microplate selected for the HA assay was a U-bottomed with 196 wells distributed in 12 columns (from 1-12) and 08 rows (from A-H). Numbering rows as A, B and C etc. in the figures was for convenience to correctly insert the data in the tables below.
Usually, HA is carried out at room temperature, and an equal volume of lectin extract and RBCs suspension is used [21,32,39]. However, this ratio may vary as described in other studies (Zhang et al., 2000) [9]. In this microassay, precise agglutination results were obtained with 25µL of rabbit RBCs mixed with 50 µL of rice extract [ Figure 1a-g]. For accurate and rapid results during cold weather, the microplate can be kept in an incubator instead of room temperature. A visible lattice of RBCs at the bottom of the well was taken as positive and button formation was recorded as negative. The interpretation of HA on a glass slide is different [32]. One of the rules of selection is that the agglutination should settle fast and non-agglutination should settle slowly. Strong positive, weak positive, and intermediate positive were differentiable with the naked eye. In a U-bottom microplate, strong positive samples showed a lattice or network spread throughout the bottom. Diffused button at the bottom showed partial agglutination and sharp button shows negative agglutination results [32,33]. However, these observations and interpretations can vary from lab to lab, expert to expert, type and concentration of RBCs, incubation time, nature of the sample agglutinin, and method of hemagglutination assay [31]. The lectin assay was stable for about 1-2 h, also reported by Zhang et al. [9].

Total Protein Content
Results of the protein content determination in the rice accessions are represented in Tables 3 and 4. For all (i.e., 217) of the tested genotypes, a protein concentration of 3-8 mg/100mg of seed flour was observed, the highest among which was 8.03 mg for accession 7600, while the lowest was 3.05mg for accession 7753 [40]. Sun et al. observed 5-13 g/100g of proteins in rice [41].

HAI
The rice lectins from all the genotypes were not inhibited by simple monosaccharide (D-glucose) moieties [9]. Other carbohydrates specificity, like mannose and complex polysaccharides needs to be sorted out for these rice accessions. In this study only Dglucose was used, as large number of samples had to be analyzed.

Affinity Comparison of Various Types of Blood for Rice Lectins
HA capability of the rice lectins against different types of blood RBCs was compared with each other as represented in Figure 1. The blood obtained from humans (B+), rabbits (local and Californian breed), and broiler chickens were screened for comparative agglutination results with rice lectins. Californian breed rabbit erythrocytes showed more precise results than human and broiler erythrocytes. However, we have recognized broiler chickens as a cheap and readily available source for blood agglutination assays of plant lectins on a large scale ( Figure 2). Heard (1955) identified the different blood groups in rabbits and found that some rabbits' red cells are agglutinated more strongly than the red cells of others and to a higher titer. In this study, Californian breed rabbit erythrocytes showed more affinity for rice lectins than local rabbits [33,35].

Discussion
Identification of stable and resistant genotypes through evaluation of proteins involved in defense is integral for varietal development and release. Plant defense is the primary application of plant lectins in response to biotic and abiotic stresses [10,11]. The study shows that seed storage lectins are present in nearly all 217 rice genotypes (Table 3). A significant highest lectin activity was observed in 109 (i.e., 106 wild) genotypes out of 217 (210 wild), which were further investigated for titer values and specific activity. Twenty-two accessions showed high titer values (ranges 64-1024) and specific activities (ranges 13-170 HU/mg).
The 106 wild accessions of rice (Oryza sativa L.) that exhibited highest agglutination as compared to the rest (210) showed their inherent capability of expressing higher concentration of phytoagglutinins (Table 4). One of the few parameters at molecular level that can easily be used to find the resistant genotypes to biotic and abiotic stresses [16][17][18][19][20][21][22]. For semi-quantification of seed lectins in rice a method described in Table 2 was used. The detail of which are given in Table 3. The same method is used for the finding of viral titer [30].
Two local varieties (Fakhre Malakand, Boti) and five transgenic rice varieties were used for the purpose of comparison with the wild relatives. We have found a considerable higher protein concentration, titer values and specific activity in wild accessions of rice as compared to the cultivated and transgenic ones, depicting greater genetic diversity of seed storage lectins [Tables 3 and 4].
HA experiments were carried out at room temperature, i.e., 25 • C. This lectin assay of rice (Oryza sativa L.) was miniaturized as only a limited seed sample of each accession was available, and a large number of samples (i.e., 217) had to be analyzed. Thus, 96-welled microplate was used for HA. The ratio of seed flour (g) to protein extraction buffer (mL) was kept the same as used by Bhagyawant et al. [33]. However, this ratio may vary in different studies of plant lectins [2,23].
The U-bottomed microplate was used for HA and the results were recorded through visual observation. In U-shaped wells of microplate strong positive samples appeared as a completely spread lattice while the partially agglutinated showed a diffused button and sharp button reflected negative agglutination results. This interpretation may vary from lab to lab or according to the type of microplate (i.e., U-bottomed or V-bottomed) or simple glass slide used [32]. Visual reading of the plate also depends on the concentration of erythrocytes used. Usually, granular appearance of the button and resistance to flow was considered positive.
There is a difference between the extent of HA of different blood types with rice lectins, viz. blood of human (B+) and two rabbits (local and Californian breed) and chicken broiler as observed in the study. Californian breed rabbits' 5% red cells agglutinated with rice lectins more strongly than the red cells of the local strain and had higher titer (Figure 1a-g. Heard (1955) identified the same results with different rabbit blood groups [38]. Levine and Landsteiner [36] and Kellner and Hedal [37] encountered the same variation in the agglutinability of rabbit RBCs. The 5% red cells of Californian bred rabbit agglutinated with rice lectins more strongly than the red cells of the local strain, and to a higher titer. Human blood group B+ (3%) and 1% rabbit RBCs did not give precise agglutination results, while 5% broiler chicken RBCs and 3% local rabbit RBCs showed clear and satisfactory results ( Figure 2). The titer values and specific activities observed for rice kernel lectins may increase with trypsinized rabbit RBCs. However, we have not used trypsin-treated blood for HA [2,16].
The values observed by Peumans et al. 1998 for untreated and trypsin-treated rabbit red blood cells (0.3 and 0.6 µg/mL, respectively) indicate that the treatment has little effect on HA. Trypsin-treated human erythrocytes agglutinated only at higher concentrations of Calsepa (5 µg/mL), whereas no clear agglutination was observed with untreated human red blood cells [2]. Hirano et al. [16] and Zhang et al. [20] used 1% trypsinized rabbit RBCs to detect lectins in rice extracts, while Bhagyawant et al. [33] used 3% trypsin treated rabbit erythrocytes for the detection of legume lectins and Gulzar et al. [32] used 2% human RBCs for various plants.
Hemagglutination inhibition assay (HAI) was carried for carbohydrate specificity of rice lectins using D-glucose. No apparent inhibition was noticed in the HAI. Therefore, our samples did not show specificity for glucose. Most of the stress-related lectins found in rice are mannose-specific, although glucose specific lectins have also been found in rice [17][18][19][20][21][22][23][24].
According to the analysis of Zhang et al., [20] mannose-binding rice lectins (MRL) have a specific role in plant defense, as they appear exclusively as a response to salt stress. This may also suggest the role of protein-carbohydrate interaction in plant defense against stress. In the same year 2000, Hirano and his coworkers discovered jacalin-related MRL as a mixture of iso-lectins related to salt inducible 'SALT' gene product with constitutive expression as lectin activity was detected in all parts of the plant at all the stages of growth. Furthermore, they argue that some unintentional stress factors during growth, such as water stress, may have induced the jacalin-related MBL expression in rice [16,20].
In short, lectins are a complex group of proteins with structural diversity and an affinity for several carbohydrates; they are involved in a global response, and respond to biotic and abiotic stresses.

Conclusions
A selected range of indigenous (192) and few exotic (25) rice accessions were investigated for lectin concentrations in this study. They exhibited high HA, reflecting a high lectin concentration in the seeds of wild rice. The findings suggest inclusion of the species in plant breeding programs for lectin alleviation in the cultivated varieties. HA variation for investigated lectins can be utilized for mapping lectin encoding genes governing biotic and abiotic stress resistance in rice as lectins are present in every part of the non-stressed rice plant. Thus, lectins as part of the seed storage proteins can be used as a passive-defense mechanism which might reflect their inherent resistance to biotic and abiotic stresses. Further experiments are needed in this connection to validate the present results in present and other cultivars of rice.