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Article

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

by
Haseena Gulzar
1,
Muhammad Asif Nawaz
1,
Asad Jan
2,
Farhat Ali Khan
3,
Sumaira Naz
4,
Muhammad Zahoor
4,*,
Dil Naz
5,
Riaz Ullah
6,
Essam A. Ali
7 and
Hidayat Hussain
8
1
Department of Biotechnology, Shaheed Benazir Bhutto University, Sheringal Dir Upper, Peshawar 18000, Khyber Pakhtunkhwa, Pakistan
2
Institute of Biotechnology and Genetic Engineering, Agriculture University Peshawar, Peshawar 18000, Khyber Pakhtunkhwa, Pakistan
3
Department of Pharmacy, Shaheed Benazir Bhutto University, Sheringal Dir Upper, Peshawar 18000, Khyber Pakhtunkhwa, Pakistan
4
Department of Biochemistry, University of Malakand, Chakdara Dir Lower, Malakand 18800, Khyber Pakhtunkhwa, Pakistan
5
Department of Zoology, University of Malakand, Chakdara Dir Lower, Malakand 18800, Khyber Pakhtunkhwa, Pakistan
6
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
7
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
8
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Salle), Germany
*
Author to whom correspondence should be addressed.
Agronomy 2021, 11(10), 1899; https://doi.org/10.3390/agronomy11101899
Submission received: 20 August 2021 / Revised: 7 September 2021 / Accepted: 17 September 2021 / Published: 22 September 2021
(This article belongs to the Special Issue Analysis of the Genetic Diversity of Crops and Associated Microbiota)
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Abstract

:
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.

1. 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 saccharide residues in the cell membranes offering various applications in therapeutics, diagnostics, the food industry, plant breeding, agriculture, and biotechnology. The potential applications of plant lectins in glycobiology, medicines, and agriculture have been studied and reported. Plant lectins are produced commercially for their use in histochemistry, cytochemistry, blotting, affinity chromatography, biosensors, and microarray technologies [1,2,3,4,5,6,7].
Oryza sativa L. is a salt-sensitive plant species with significant genetic variability within the cultivated gene pool that can be exploited to develop salt-resistant varieties. The effectiveness of plant lectins on pathogenic fungi, insects, HIV, and cancer, as observed for numerous legume lectins, also needs to be explored for other plant species [8,9,10,11]. More than a hundred lectin molecules have been isolated from plants, viruses, bacteria, invertebrates, and vertebrates, including mammals. About 18 different types of lectins of plants are reported to have immunomodulatory effect in case of infection or disease such as cancer [12]. The role of lectins in abiotic and biotic stress resistance in rice has been highlighted by many researchers [13,14,15,16,17,18,19,20,21]. Knowing about the genes encoding these lectins and their role in plant defense offers many applications in plant breeding and genetic engineering to improve cultivars. Differences in lectin structure and carbohydrate specificity are related to their different functions. Depending on carbohydrate specificity, major lectins are divided into mannose-binding lectins, galactose, N-acetyl galactosamine binding lectins, N-acetylglucosamine binding lectins, N-acetyl neuraminic acid-binding lectins, and fucose binding lectins. Plant defense is the primary application of plant lectins in response to biotic and abiotic stresses [22,23,24]. The product of a novel rice gene ’RAB21′, induced by abscisic acid and water stress, express a protein ‘Rab 21′ [25,26] and organ-specific expression of another rice gene ’SALT’ in response to salt stress and drought clarifies the activation of specific genes during stress conditions [15,16,17,18,19,20].
Moreover, the regulation of the salt-responsive gene SALT from rice by hormonal and developmental signals showed the differential expression of these genes. The amino acid sequence of rice protein encoded by three genes (a gene SALT (length 145), SALT, and OsJ_01682) showed 100% similarity [16,17,18,19]. Jiang et al. examined the evolutionary history of lectins in rice, Arabidopsis, and soybean and their expression analysis from full-length cDNA, expression sequence tag (EST), microarray, and massively parallel signature sequencing (MPSS) datasets. There are 267 lectin coding genes in rice, of which 254 have been confirmed by expression profiles, which belong to two families, i.e., B- lectin (mannose-binding lectins) and leg-B (legume lectins). Of 267 genes, 91 are related to biotic stresses, 68 to abiotic stresses such as cold, drought, salinity, and 109 genes to biotic and/ abiotic stresses. Lectin expression has also been found to be tissue-specific. Data analyses (MPSS database methods) have shown that after infection by the fungus (Megnaporthe grisea) and bacterium (Xanthomonas oryzae), a total of 58 and 62 lectin genes were differentially expressed, respectively, among which both of the pathogens induced regulation of 29 genes [23]. Various lectin engineering methods are available, by which specificity and stability of a known lectin scaffold could be improved, and non-lectin proteins maybe modified to acquire a sugar-binding function [27]. Hemagglutination (HA) assays are a convenient and quick way of measuring the presence and amount of lectin molecules in a given sample. Lectins of any origin, including multivalent plant lectins, can bind the sialic acid on RBC surfaces and cause blood agglutination. HA is a very convenient method to detect these entities in laboratory settings. Classically, a visual observation method is widely employed in laboratories for routine coagulation assays due to their simplicity. Visual observations in microplates can readily determine hemagglutination titer [28,29,30,31,32,33,34,35,36,37]. This study aimed to assess the presence of seed storage lectins in various rice accessions mainly originated from Pakistan without stress to know the inherent capability of rice genotypes expressing phyto-agglutinin.

2. Materials and Methods

The study was carried out in the Institute of Biotechnology and Genetic Engineering (IBGE), Agricultural University Peshawar (AUP), Khyber Pakhtunkhwa, Pakistan.

2.1. Collection of Plant Material

A total of 210 rice accessions (Table 1) were obtained from IABGR/NARC Islamabad and tested for lectins’ presence using HA. Two local varieties, viz., Fakhre Malakand and Boti, and five transgenic rice seeds, making a total of 217 genotypes, were also screened for the existence of lectins for the purpose of comparison with the wild. Gum Arabic (Sigma Aldrich, St. Louis, MO, USA) was used as positive control and simple PBS as a blank. Out of 210 accessions of rice seeds provided by IABGR/NARC Islamabad, only four originated from China, 186 from Pakistan, 12 from IRRI, and the source of 8 accessions was not mentioned in the catalog. Out of 186 accessions originating in Pakistan, 172 belong to Khyber Pakhtunkhwa province, 04 from Punjab, 02 from AJK, 01 from Gilgit Baltistan, and the source provinces of 07 were unknown (Table 1).

2.2. 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 Na2HPO4, and 1.8 mM KH2PO4, 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].

2.3. 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].

2.4. 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].

2.5. 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.

2.6. 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.

2.7. 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].

3. Results

3.1. Rice Lectins Extraction

The results of lectins extraction from rice for the analysis are given in Table 3 and Table 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.

3.2. 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 (Table 3 and Table 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].

3.3. Total Protein Content

Results of the protein content determination in the rice accessions are represented in Table 3 and Table 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].

3.4. 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 D-glucose was used, as large number of samples had to be analyzed.

3.5. 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].

4. 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 [Table 3 and Table 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.

5. 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.

Author Contributions

H.G., M.A.N., A.J. and F.A.K. performed the experiments. S.N. and M.Z. edited the paper initial and revised version. M.Z. and F.A.K. conceived the idea and supervise the process. D.N., R.U., E.A.A. and H.H. did literature survey and are resource persons. All authors have read and agreed to the published version of the manuscript.

Funding

Researchers Supporting Project Number RSP/2021/45, King Saud University Riyadh, Saudi Arabia.

Data Availability Statement

All related data are within the manuscript.

Acknowledgments

Authors are thankful to Researchers Supporting Project Number RSP/2021/45, King Saud University Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 11 01899 g001a 550Agronomy 11 01899 g001b 550Agronomy 11 01899 g001c 550
Figure 1. HA capability of rice (Oryza sativa L.) lectins (a–g) against 5% Californian breed rabbit RBCs. The + signs are showing HA activity. The HA activities for different rice genotypes are weak (+), high (++/+2), highest (+++/+3) and negative (-), using 5% Californian breed rabbit RBCs.
Figure 1. HA capability of rice (Oryza sativa L.) lectins (a–g) against 5% Californian breed rabbit RBCs. The + signs are showing HA activity. The HA activities for different rice genotypes are weak (+), high (++/+2), highest (+++/+3) and negative (-), using 5% Californian breed rabbit RBCs.
Agronomy 11 01899 g001aAgronomy 11 01899 g001bAgronomy 11 01899 g001c
Agronomy 11 01899 g002 550
Figure 2. Comparison of rice lectins capability of HA is shown against 1% rabbit RBCs (A), 3% B+ human RBCs (B), 5% Californian-breed rabbits RBCs (C), 5% broiler chicken RBCs (D), and 3% local rabbit RBCs (E).
Figure 2. Comparison of rice lectins capability of HA is shown against 1% rabbit RBCs (A), 3% B+ human RBCs (B), 5% Californian-breed rabbits RBCs (C), 5% broiler chicken RBCs (D), and 3% local rabbit RBCs (E).
Agronomy 11 01899 g002
Table
Table 1. Rice (Oryza sativa L.) genotypes used, provided by IABGR/NARC Islamabad.
Table 1. Rice (Oryza sativa L.) genotypes used, provided by IABGR/NARC Islamabad.
S. No Accession No. Country, Year of Release Province/
Donor No/Area 		District
1–47216–7219China, 1983C-12, C-13, C-14, C-15 respectivelyNA
57225Pakistan, 1983PunjabSheikhupura
67234//Khyber PakhtunkhwaUnknown
77235////Malakand
8–117236–7239////Unknown
12–137241–7242////Unknown
147258Pakistan, 1984//Parachinar
157260////Kurrum Agency
16–187261–7263////Pachinar
197268////Parachinar
207269////Unknown
217271////Unknown
227280////Unknown
237281Pakistan, 1987//Mansehra
24–297284–89Pakistan, 1984//Chitral
30–357290–7295////Swat
36–397296–99////Mansehra
407396Pakistan, 1987////
417397////Swat
427411//UnknownUnknown
437413//////
447414//////
457416//////
46–477418–7419//////
487420//Gilgit, BaltistanNA
49–517593–7595//Khyber PakhtunkhwaMalakand
52–557597–7600//////
567601Pakistan, 1989//Dir
57–647603–7610//////
63–847611–7630////Chitral
85–937632–7640////Swat
947641////Unknown
95–1047648–7657////Mansehra
105–1067672–7673//PunjabGujrat
107–1087734–7735//AJKNA
109–1107737–7738//Khyber PakhtunkhwaUnknown
111–1137753–7755////Besham
1147756////Unknown
1157759////Mansehra
1167760////Unknown
1177762//////
1187763//PunjabGujranwala
1197765////Bahawalnagar
1207766////Bahawalpur
121–1477783–7809Pakistan, 1991Khyber PakhtunkhwaChitral
148–1557811–7818//////
156–1597819–7822//////
160–1637824–7827//////
164–1667828–7830////Unknown
167–1697831–7833//////
1707836//////
1717839////Unknown
1727847Pakistan, 1973RGP-ARCNA
1737850//////
1747859//////
1757863//////
176–1777864–7865Pakistan, 1972////
1787869Pakistan, 1973////
1797883//////
1807886//////
1817937Pakistan,1991//Chitral
1828083//////
1838085//////
18417940////Swat
185–18617,941–17,942Pakistan, 2000//Nowshehra
187–18917,943–17,945Pakistan, 1996//Chitral
19017,946Pakistan, 1997//Mansehra
191–20217,947–17,958IRRI, 1999NANA
20318,231Source not mentioned in the catalog
//
//
//
20423,718
205–20623,727–23,728
207–20823,731–23,732
20923,736//
21023,744
211BotiPakistanKhyber, PakhtunkhwaSwat
212Fakhre Malakand////Malakand
213–217Five Transgenic seedsNANANA
Table
Table 2. Method of two-fold serial dilution, corresponding titer values, HA units/mL, and specific activity.
Table 2. Method of two-fold serial dilution, corresponding titer values, HA units/mL, and specific activity.
Dilution Number Ratio and %Age Concentration of the Diluted Sample Dilution Factor HA Titer Value (HU/mL) Specific Activity = Titre/mg of Protein
Original sample1:1 (100%)1/2011e.g., Protein Concentration = 6.2 mg/mL and titre = 16 Therefore, Specific activity of the plant/animal lectins showing visible HA will be 16/6.2 = 2.58
11:2 (50%)1/211/22
21:4 (25%)1/221/44
31:8 (12.5%)1/231/88
41:16 (0.063%)1/241/1616
51:32 (0.0313%)1/251/3232
61:64 (0.01563%)1/261/6464
71:128 (0.007813%)1/271/128128
81:256 (0.0039063%)1/281/256256
91:512 (0.00195313%)1/291/512512
101:1024 (0.0009766%)1/2101/10241024
111:2048 (0.0004883%)1/2111/20482048
121:4096 (0.000244141%)1/2121/40964096
The percentage concentration of the diluted sample is calculated as a ratio multiplied by 100. Reciprocal of highest dilution showing visible agglutination.
Table
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.
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.
S. NoAccession No.HAProtein Conc. mg/mLS. NoAccession No.HAProtein Conc. mg/mLS. NoAccession No.HAProtein Conc. mg/mLS. NoAccession No.HAProtein Conc. mg/mL
17216+5.815547599+5.381077734+34.2691607822+36.425
27217+5.18557600+8.031087735+24.8081617824+4.608
37218+5.865567601+6.331097736+6.2451627825+26.545
47219+5.77577603+5.671107737+24.9311637826+6.785
57225-7.3587604+4.9941117738+4.9981647827+24.956
67234+4.621597605+6.651127753+3.0561657828+24.897
77235+34.996607606+5.9651137754+4.7031667829+24.672
87236+3.87617607+24.8781147755+25.421677830+5.125
97237+24.731627608+25.21157756+35.2851687831+35.585
107238+24.344637609+6.251167759+37.841697832+4.28
117239-4.293647610+6.941177760+34.7701707833+6.335
127241+4.624657611+36.1151187762+35.3751717836+35.685
137242+24.871667612+35.9651197763+24.1671727839+24.354
147258+35.735677613+26.5751207765+4.4631737847+24.912
157260+36.26687614+36.9951217766+5.1551747850+4.504
167261+24.98697615+6.6651227783+6.041757859+5.79
177262+34.326707616+36.821237784+4.8841767863+5.045
187263+3.372717617+5.161247785+36.8451777864+4.197
197268+34.365727618+36.2351257786+36.6751787865+4.398
207269+24.706737619+26.4651267787+6.8251797869+4.515
217271+34.326747620+35.31277788+27.131807883+4.327
227280+35.105757621+35.8151287789+26.141817886+35.665
237281+5767622+36.3051297790+36.8151827937+5.94
247284+6.165777623+4.9151307791+37.5851838083+36.215
257285+35.445787624+34.4211317792+26.621848085+36.78
267286+36.95797625+25.7551327793+26.818517940+26.765
277287+36.565807626+6.7851337794+25.7618617941+5.51
287288+36.13817627+6.981347795+36.18518717942+5.54
297289+36.995827628+35.3751357796+7.2818817943+6.055
307290+5.07837629+5.61367797+27.5618917944+4.716
317291+4.418847630+6.8551377798+37.52519017945-5.395
327292+24.037857632+6.431387799+37.5119117946+26.915
337293+23.761867633+5.11397800+35.76519217947+5.375
347294+3.929877634+25.3151407801+3.54519317948-5.795
357295+4.376887635+5.251417802+33.35219417949+5.515
367296+7.245897636+35.221427803+4.46519517951+5.27
377297+4.712907637+34.6951437804+37.2919617952+4.99
387298+24.560917638+24.3821447805+35.3119717953+5.755
397299+3.718927639+7.161457806+35.50519817954+4.9185
407396+37.35937640+6.811467807+33.84519917955+6.26
417397+5.265947641+25.111477808+24.81520017956+5.875
427411+36.26957648+4.9131487809+36.08120117957+5.38
437413+35.1967649+5.1751497811+34.5620217958-5.59
447414+35.65977650+4.6171507812+35.6720318231+4.0485
457416+34.985987651+24.1051517813+36.1220423718+5.68
467418+35.4997652+24.5651527814+34.8420523727+5.575
477419+361007653+25.781537815+34.49520623728+25.66
487420+35.631017654+6.7851547816+24.31520723731+23.577
497593+4.1681027655+5.631557817+36.3120823732+33.8585
507594+7.51037656+7.3351567818+4.3920923736+36.22
517595+4.5221047657+6.231577819+37.0721023744+3.6395
527597+4.5791057672+24.1141587820+35.64211Boti+2……
537598+5.291067673+4.9581597821+26.56212F.M+22.79
213–217 = Five transgenic seeds of rice, where only two showed visible HA (+2) with 3.54 and 3.79 protein content.
The hemagglutination (HA) results of 217rice (Oryza sativa L.) genotypes; accessions (210), local varieties (02) and transgenic seeds (05) showing weak (+), high (++/+2), highest (+++/+3) and negative (-), using 5% Californian breed rabbit RBCs provided by IABGR/ NARC Islamabad. Above values of protein concentration are mg/100mg of rice flour. Protein contents were measured via microplate reader at 595 nm using Bradford reagent and the difference of (±0.4) was noted between the values of protein concentration. (……) represent (missing value); Boti & F.M (Fakhre Malakand) are local varieties.
Table
Table 4. HA, protein content (mg/mL), titer values, and specific activity in the seeds of 106 rice (Oryza sativa L.) accessions.
Table 4. HA, protein content (mg/mL), titer values, and specific activity in the seeds of 106 rice (Oryza sativa L.) accessions.
S. NoAccession No.HAProtein Conc. mg/mLTitre ValueSpecific activity HU/mgS. NoAccession No.HAProtein Conc. mg/mLTiter ValueSpecific activity HU/mgS. NoAccession No.HA Protein Conc. mg/mLTitre ValueSpecific activity HU/mg
17235+34.99627 = 12825.621377619+26.46523 = 81.237737800+35.76522 = 40.693
27236+23.8725 = 328.268387620+35.325 = 326.037747802+33.35223 = 82.386
37237+24.73127 = 12827.055397622+36.30523 = 81.268757804+37.2923 = 81.097
47238+24.3427 = 12829.49407624+34.420524 = 163.6195767805+35.3122 = 40.753
57242+24.87127 = 12826.28417625+25.75522 = 40.695777806+35.50523 = 81.453
67258+35.73528 = 25644.63427627+6.9821 = 20.286787807+33.84523 = 82.081
77260+36.2622 = 40.638437628+35.37529 = 51295.25797808+24.81522 = 40.8307
87261+24.9822 = 40.803447634+25.31522 = 40.752807809+36.0821 = 20.3289
97262+32.16322 = 41.849457636+35.2224=163.065817811+34.5623 = 81.754
107268+34.36523 =81.8328467637+34.69524 = 163.407827812+35.6723 = 81.411
117269+24.70622 = 40.8499477641+25.1124 = 163.131837813+36.1222 = 40.654
127271+34.326210 =1024236.70487651+24.10522 = 40.974847814+34.8423 = 81.653
137280+35.10523 = 81.5670497652+24.56522 = 40.876857815+34.49525 = 327.119
147285+35.44523 = 81.469507655+5.6328 = 25645.47867816+24.31520 = 10.231
157286+36.9523 = 81.151517672+24.11422 = 40.972877819+37.0723 = 81.1315
167287+36.56523 = 81.2185527735+24.808526 = 6413.309887820+35.6424 = 162.8368
177288+36.1323 = 81.3050537737+24.931527 = 12825.955897821+26.5622 = 40.609
187289+36.99523 = 81.1436547755+25.4225 = 325.9040907822+36.42523 = 81.245
197292+24.037527 = 12831.702557756+35.28522 = 40.7568917825+26.54524 = 162.444
207293+23.76122 = 41.0635567759+35.226623 = 81.2374927827+24.95625 = 326.456
217298+24.560527 = 12828.067577760+33.180325 = 3210.07937828+24.89725 = 326.5346
227396+37.3522 = 40.5442587762+33.583321 = 20.558947829+24.67226 = 6413.698
237411+36.26210 = 1024163.57597763+32.778322 = 41.4397957831+35.585210 = 1024183.34
247413+35.123 = 81.5686607785+34.563324 = 163.506967836+35.68522 = 40.7036
257414+35.6523 = 81.4159617786+34.4523 = 81.797977839+24.35422 = 40.9188
267416+34.98523 = 81.6048627788+24.753328 = 25653.857987886+35.66523 = 81.412
277418+35.424 = 162.9629637789+24.093325 = 327.8176998083+36.21523 = 81.287
287419+36210 = 1024170.66647790+34.543326 = 6414.0861008085+36.7829 = 51275.516
297607+24.878522 = 40.8199657791+35.056623 = 81.58210117940+26.76522 = 40.591
307608+25.222 = 40.7692667792+24.413322 = 40.90610217946+26.91521 = 20.289
317611+36.11523 = 81.3082677793+24.533322 = 40.88210323728+25.6629 = 51290.45
327612+35.96523 = 81.3411687794+23.8422 = 41.041610423731+23.57726 = 6417.89
337613+26.57523 = 81.2167697795+34.123322 = 40.970010523732+33.85926 = 6416.586
347614+36.999524 = 162.2858707797+25.0420 = 10.19810623736+36.2223 = 81.286
357616+36.8223 = 81.1730717798+35.016623 = 81.594107F.M+22.7921 = 20.71
367618+36.23523 = 81.2830727799+35.006623 = 81.597108109T.S+23.54,3.7921 = 20.28, 0.26
The 109 rice (Oryza sativa L.) genotypes; accessions (106), local varieties (02), transgenic seeds (02), showing the presence of lectins (+) high (+2/++) or highest (+3 /+++) (HA) results using Californian breed 5% Rabbit RBCs, were screened further for titer values and specific activity (titer/mg of protein) F.M; Fakhre Malakand, local variety. TS.; transgenic seeds. The above values of protein concentration are mg/100mg of rice flour. Protein contents were measured via a microplate reader at 595 nm using the Bradford reagent, and the difference of (±0.4) was noted between the values of protein concentration when multiple readings were taken. Titer values are the reciprocal of the highest two-fold dilution (i.e., 1/2n = 2n, where n = 0, 1, 2, 3) showing visible agglutination. Specific activity was measured by dividing titer values over the protein content (titer/mg of protein).
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Gulzar, H.; Nawaz, M.A.; Jan, A.; Khan, F.A.; Naz, S.; Zahoor, M.; Naz, D.; Ullah, R.; Ali, E.A.; Hussain, H. Semi-Quantification of Lectins in Rice (Oryza sativa L.) Genotypes via Hemagglutination. Agronomy 2021, 11, 1899. https://doi.org/10.3390/agronomy11101899

AMA Style

Gulzar H, Nawaz MA, Jan A, Khan FA, Naz S, Zahoor M, Naz D, Ullah R, Ali EA, Hussain H. Semi-Quantification of Lectins in Rice (Oryza sativa L.) Genotypes via Hemagglutination. Agronomy. 2021; 11(10):1899. https://doi.org/10.3390/agronomy11101899

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Gulzar, Haseena, Muhammad Asif Nawaz, Asad Jan, Farhat Ali Khan, Sumaira Naz, Muhammad Zahoor, Dil Naz, Riaz Ullah, Essam A. Ali, and Hidayat Hussain. 2021. "Semi-Quantification of Lectins in Rice (Oryza sativa L.) Genotypes via Hemagglutination" Agronomy 11, no. 10: 1899. https://doi.org/10.3390/agronomy11101899

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