Pea Lectin Research Articles

Density of Compatible Ligands on the Surface of Food Particles Modulates Sorting Efficiency in the Blue Mussel Mytilus edulis

The adhesion between food particles and mucus is a fundamental process in particle sorting in suspension-feeding bivalves that requires specific recognition. Interactions between carbohydrate-binding proteins (lectins) expressed on the feeding organs and carbohydrates present on microbial cell surface can provide this specificity. Microalga cell surface carbohydrates (MCSC) represent unique patterns that can be considered as species-specific fingerprints. In this study, sorting efficiencies in blue mussels Mytilus edulis fed with microalgae having modified MCSC and engineered microspheres coated with target carbohydrates was measured. The nature and quantities of surface carbohydrates required to trigger sorting in mussels was evaluated and the relationship between ligand quantities and sorting efficiency (SE) was determined. Mussels fed with Chlamydomonas which MCSC were blocked with ConA or PEA lectins (affinity to mannose and glucose) led to a significant decrease of the sorting efficiencies, not observed when the lectin UEA (affinity to fucose) was used. The ability of commercial lectins to inhibit sorting was not linear and a threshold was noted between 30 and 45 ug lectins per million algae cells. Further, mussels were fed with microspheres coated with neoglycoproteins. Results showed that glucose-BSA, but not fucose-BSA, has an effect on particle sorting in mussels, and 1.08 x 109 molecules of glucose per microspheres, corresponding to a density of 6.99 x 106 molecules of glucose per µm2, triggers particle selection. These findings support that selection of food particles by mussels rely on the strength of the bond between suspended particle and the mucosal layer that mediate sorting, and that these bonds depend on the quantity of compatible ligands on each particle.

Legume Lectins with Different Specificities as Potential Glycan Probes for Pathogenic Enveloped Viruses.

Pathogenic enveloped viruses are covered with a glycan shield that provides a dual function: the glycan structures contribute to virus protection as well as host cell recognition. The three classical types of N-glycans, in particular complex glycans, high-mannose glycans, and hybrid glycans, together with some O-glycans, participate in the glycan shield of the Ebola virus, influenza virus, human cytomegalovirus, herpes virus, human immunodeficiency virus, Lassa virus, and MERS-CoV, SARS-CoV, and SARS-CoV-2, which are responsible for respiratory syndromes. The glycans are linked to glycoproteins that occur as metastable prefusion glycoproteins on the surface of infectious virions such as gp120 of HIV, hemagglutinin of influenza, or spike proteins of beta-coronaviruses. Plant lectins with different carbohydrate-binding specificities and, especially, mannose-specific lectins from the Vicieae tribe, such as pea lectin and lentil lectin, can be used as glycan probes for targeting the glycan shield because of their specific interaction with the α1,6-fucosylated core Man3GlcNAc2, which predominantly occurs in complex and hybrid glycans. Other plant lectins with Neu5Ac specificity or GalNAc/T/Tn specificity can also serve as potential glycan probes for the often sialylated complex glycans and truncated O-glycans, respectively, which are abundantly distributed in the glycan shield of enveloped viruses. The biomedical and therapeutical potential of plant lectins as antiviral drugs is discussed.

Man-Specific Lectins from Plants, Fungi, Algae and Cyanobacteria, as Potential Blockers for SARS-CoV, MERS-CoV and SARS-CoV-2 (COVID-19) Coronaviruses: Biomedical Perspectives.

Betacoronaviruses, responsible for the “Severe Acute Respiratory Syndrome” (SARS) and the “Middle East Respiratory Syndrome” (MERS), use the spikes protruding from the virion envelope to attach and subsequently infect the host cells. The coronavirus spike (S) proteins contain receptor binding domains (RBD), allowing the specific recognition of either the dipeptidyl peptidase CD23 (MERS-CoV) or the angiotensin-converting enzyme ACE2 (SARS-Cov, SARS-CoV-2) host cell receptors. The heavily glycosylated S protein includes both complex and high-mannose type N-glycans that are well exposed at the surface of the spikes. A detailed analysis of the carbohydrate-binding specificity of mannose-binding lectins from plants, algae, fungi, and bacteria, revealed that, depending on their origin, they preferentially recognize either complex type N-glycans, or high-mannose type N-glycans. Since both complex and high-mannose glycans substantially decorate the S proteins, mannose-specific lectins are potentially useful glycan probes for targeting the SARS-CoV, MERS-CoV, and SARS-CoV-2 virions. Mannose-binding legume lectins, like pea lectin, and monocot mannose-binding lectins, like snowdrop lectin or the algal lectin griffithsin, which specifically recognize complex N-glycans and high-mannose glycans, respectively, are particularly adapted for targeting coronaviruses. The biomedical prospects of targeting coronaviruses with mannose-specific lectins are wide-ranging including detection, immobilization, prevention, and control of coronavirus infection.

Cross-Reactivity and Sequence Homology Between Alpha-Synuclein and Food Products: A Step Further for Parkinson's Disease Synucleinopathy.

Introduction: Parkinson’s disease is characterized by non-motor/motor dysfunction midbrain neuronal death and α-synuclein deposits. The accepted hypothesis is that unknown environmental factors induce α-synuclein accumulation in the brain via the enteric nervous system. Material and Methods: Monoclonal antibodies made against recombinant α-synuclein protein or α-synuclein epitope 118–123 were applied to the antigens of 180 frequently consumed food products. The specificity of those antibody-antigen reactions was confirmed by serial dilution and inhibition studies. The Basic Local Alignment Search Tool sequence matching program was used for sequence homologies. Results: While the antibody made against recombinant α-synuclein reacted significantly with 86/180 specific food antigens, the antibody made against α-synuclein epitope 118–123 reacted with only 32/180 tested food antigens. The food proteins with the greatest number of peptides that matched with α-synuclein were yeast, soybean, latex hevein, wheat germ agglutinin, potato, peanut, bean agglutinin, pea lectin, shrimp, bromelain, and lentil lectin. Conclusions: The cross-reactivity and sequence homology between α-synuclein and frequently consumed foods, reinforces the autoimmune aspect of Parkinson’s disease. It is hypothesized that luminal food peptides that share cross-reactive epitopes with human α-synuclein and have molecular similarity with brain antigens are involved in the synucleinopathy. The findings deserve further confirmation by extensive research.

Characterization and antimicrobial activity of lectins purified from three Egyptian leguminous seeds

Lectins are carbohydrate-binding proteins that play vital roles in many biological processes. In this study, lectins from three Egyptian cultivars (fava bean, lentil, and pea) were isolated by precipitation with different concentrations of ammonium sulfate. The purification process was performed by affinity chromatography using mannose agarose. The highest concentration of purified lectins (1.48 mg/g) was recorded in pea at 90% saturation. SDS-PAGE of the purified lectins revealed bands of low molecular weights (14 to 18 kDa). The complete amino acid sequences of purified lectins were assessed using mass spectrometry (MS), which indicated the presence of the peptides favin, p54, and psl in fava bean, lentil, and pea, respectively. The lectins showed antimicrobial activity. The highest inhibition zone (35 mm) was measured with lectin purified from lentil against Staphylococcus aureus ATCC 6538, followed by pea lectin (33.4 mm) against Pseudomonas aeruginosa ATCC 10145. To the best of our knowledge, the legume lectins in this study are the first lectins to exhibit antifungal activity against Candida albicans, with the maximum inhibition zone (25.1 mm) observed with purified lectins of fava bean. Additionally, the first scanning electron microscope (SEM) images showing agglutination and clumping of microbial cells exposed to tested lectins are provided. These findings proved that Egyptian legume lectins are distinct from other lectins reported in previous studies and demonstrated their potential as antimicrobial agents against human pathogenic microorganisms.

Pea lectin inhibits cell growth by inducing apoptosis in SW480 and SW48 cell lines

Globally, colorectal cancer is the third most common type of malignant tumor, after lung and breast. Here anticancer property of pea lectin was evaluated against colorectal cancer cell lines SW480 and SW48. The cells were treated with different doses of lectin for 3 days in vitro and the inhibitory effects were found in a dose dependent manner. At the high dose(1.0 mg/ml) 62% and 63% cell growth inhibitions were observed for SW48 and SW480 cell lines, respectively. Cell growth inhibition was further studied by colony formation of the cell lines and the numbers of colonies in SW480+pea lectin and SW48+pea lectin cells were noted significantly lower in comparison to that of control cells. Cell morphological study revealed that pea lectin induced apoptosis both in SW48 and SW480 cell lines, which was further confirmed by caspase inhibitors. Involvement of intrinsic mitochondrial pathway in the apoptosis progression was confirmed by caspase inhibitors and increased of caspase-3 & -9 proteins expressions. Expression levels of p53 and p21 protein were also increased significantly in both cell lines with the decreased of PARP1 protein expression. G2/M and G0/G1 cell cycle arrested was noted in SW48 and SW480 cell lines, respectively, after treatment with pea lectin.

Inter-laboratory studies for the validation of two singleplex (tE9 and pea lectin) and one duplex (pat/bar) real-time PCR methods for GMO detection

This paper presents the inter-laboratory studies for the successful validation of qualitative real-time Polymerase Chain Reaction methods for the detection of Genetically Modified Organisms in food and feed. Two singleplex TaqMan methods were developed for the detection of the tE9 terminator present in several genetically modified events and the detection of the endogenous lectin gene from pea; the tE9 terminator donor organism. In addition, a TaqMan duplex method was validated, which allows the simultaneous detection of the pat and bar elements.The paper explains the different acceptance parameters used during the in-house validation, the method transferability study and finally the inter-laboratory validation, how this validation was conducted and the problems encountered. Statistical methods for the validation of the qualitative methods are also presented. This information can be used by laboratories for the verification of these methods during their implementation and for the validation of new methods.

Purification and Characterization of a Lectin from Green Split Peas (Pisum sativum).

Lectins have captured the attention of a large number of researchers on account of their various exploitable activities, including antitumor, immunomodulatory, antifungal, as well as HIV reverse transcriptase inhibitory activities. A mannose/glucose-specific lectin was isolated from green split peas (a variety of Pisum sativum) and characterized. The purification step involved anion-exchange chromatography on a DEAE-cellulose column, cation-exchange chromatography on an SP-Sepharose column, and gel filtration by fast protein liquid chromatography (FPLC) on Superdex 200. The purified lectin had a native molecular mass of around 50kDa as determined by size exclusion chromatography. It appeared as a heterotetramer, composed of two distinct polypeptide bands with a molecular mass of 6 and 19kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of green split pea lectin shows some degree of homology compared to lectins from other legume species. Its hemagglutinating activity was inhibited by glucose, mannose, and sucrose, and attenuated at pH values higher than 12 or lower than 3. Hemagglutinating activity was preserved at temperatures lower than 80°C. The lectin did not show antifungal activity toward fungi including Fusarium oxysporum, Botrytis cinerea, and Mycosphaerella arachidicola. Green split pea lectin showed a mitogenic effect toward murine splenocytes and could inhibit the activity of HIV-1 reverse transcriptase.

Pea lectin receptor-like kinase functions in salinity adaptation without yield penalty, by alleviating osmotic and ionic stresses and upregulating stress-responsive genes.

Lectin receptor-like kinases (LecRLKs) are members of RLK family composed of lectin-like extracellular recognition domain, transmembrane domain and cytoplasmic kinase domain. LecRLKs are plasma membrane proteins believed to be involved in signal transduction. However, most of the members of the protein family even in plants have not been functionally well characterized. Herein, we show that Pisum sativum LecRLK (PsLecRLK) localized in plasma membrane systems and/or other regions of the cell and its transcript upregulated under salinity stress. Overexpression of PsLecRLK in transgenic tobacco plants confers salinity stress tolerance by alleviating both the ionic as well the osmotic component of salinity stress. The transgenic plants show better tissue compartmentalization of Na(+) and higher ROS scavenging activity which probably results in lower membrane damage, improved growth and yield maintenance even under salinity stress. Also, expression of several genes involved in cellular homeostasis is perturbed by PsLecRLK overexpression. Alleviation of osmotic and ionic components of salinity stress along with reduced oxidative damage and upregulation of stress-responsive genes in transgenic plants under salinity stress conditions could be possible mechanism facilitating enhanced stress tolerance. This study presents PsLecRLK as a promising candidate for crop improvement and also opens up new avenue to investigate its signalling pathway.

ARTIFICIAL ASSOCIATIVE SYMBIOSES BEtWEEN TOMATO PLANTS AND FUNGISTATIC Rhizobium

Biomethods in plant protection against pests and diseases considered the most prospective alternative to chemicals which pollute soil and water causing concern about public health. The possibility of creating an artificial association of nodule bacteria with plants to protect them from the adverse effects of pathogenic fungi can be realized using one of the specific mechanism of nodule bacteria attachment to the roots of leguminous plants by plant lectins, able to recognize and specifically bind with different carbohydrates, particularly polysaccharides of rhizobia cell wall. In our study we used the composite plants of tomato (Lycopersicon esculentum) Dubok variety and bacterial strains associated with the roots of wild legumes from the collection of Institute of Biochemistry and Genetics (IBG USC RAS). «Hairy rooted» tomato plants were obtained by treatment with Agrobacterium rhizogenes ATCC 15834, containing vehicle gene construction pCambia 1305.1 with inserted pea lectin gene psl under cauliflower mosaic virus 35S promoter. The antagonistic activity of bacteria towards pathogens was tested by dual culture study. The ability of microorganisms to produce siderophores and cyanide was analyzed. Few isolates were identified by sequencing of the 16S rRNA gene fragments. By screening of the collection of isolates from nodules of wild legume from tribe Viceae the candidate strains were detected, particularly Rhizobium leguminosarum, Pseudomonas sp. and Stenotrophomonas rhizophila, with fungistatic activity against Fusarium solani, F. oxysporum, Fusarium sp. and F. oxysporum f. sp. lycopersici. Prodiction of siderophores was detected in two members of Pseudomonas genus, S. rhizophila and R. leguminosarum. Two Pseudomonas strains, the 14M and 103, and S. rhizophila were shown to produce cyanide. It was also found that treatment of roots transgenic by psl gene with R. leguminosarum 116 strain reduced the amount of hyphae of the pathogen F. oxysporum f. sp. lycopersici in the rhizosphere of tomato plant that could potentially contribute to plant defense against pathogenic fungi. Thus, the use of lectins as transgenes in roots allows us to obtain artificial association with rhizobia in non-symbiotic plants such as tomato, which in combination with the use of microorganisms possessing fungistatic activity can more effectively protect the plant root system o

ИСКУССТВЕННЫЕ АССОЦИАТИВНЫЕ СИМБИОЗЫ МЕЖДУ ТОМАТОМ И РИЗОБИЯМИ, ОБЛАДАЮЩИМИ ФУНГИСТАТИЧЕСКОЙ АКТИВНОСТЬЮ

Biomethods in plant protection against pests and diseases considered the most prospective alternative to chemicals which pollute soil and water causing concern about public health. The possibility of creating an artificial association of nodule bacteria with plants to protect them from the adverse effects of pathogenic fungi can be realized using one of the specific mechanism of nodule bacteria attachment to the roots of leguminous plants by plant lectins, able to recognize and specifically bind with different carbohydrates, particularly polysaccharides of rhizobia cell wall. In our study we used the composite plants of tomato ( Lycopersicon esculentum ) Dubok variety and bacterial strains associated with the roots of wild legumes from the collection of Institute of Biochemistry and Genetics (IBG USC RAS). «Hairy rooted» tomato plants were obtained by treatment with Agrobacterium rhizogenes ATCC 15834, containing vehicle gene construction pCambia 1305.1 with inserted pea lectin gene psl under cauliflower mosaic virus 35S promoter. The antagonistic activity of bacteria towards pathogens was tested by dual culture study. The ability of microorganisms to produce siderophores and cyanide was analyzed. Few isolates were identified by sequencing of the 16S rRNA gene fragments. By screening of the collection of isolates from nodules of wild legume from tribe Viceae the candidate strains were detected, particularly Rhizobium leguminosarum, Pseudomonas sp. and Stenotrophomonas rhizophila, with fungistatic activity against Fusarium solani, F. oxysporum, Fusarium sp. and F. oxysporum f. sp. lycopersici. Prodiction of siderophores was detected in two members of Pseudomonas genus, S. rhizophila and R. leguminosarum. Two Pseudomonas strains, the 14M and 103, and S. rhizophila were shown to produce cyanide. It was also found that treatment of roots transgenic by psl gene with R. leguminosarum 116 strain reduced the amount of hyphae of the pathogen F. oxysporum f. sp. lycopersici in the rhizosphere of tomato plant that could potentially contribute to plant defense against pathogenic fungi. Thus, the use of lectins as transgenes in roots allows to obtain artificial association with rhizobia in non-symbiotic plants such as tomato, which in combination with the use of microorganisms possessing fungistatic activity can more effectively protect the plant root system o against pathogens.

ASSOCIATIVE SYMBIOSIS BETWEEN RHIZOBIA AND TRANSGENIC TOMATOES INCREASES PLANT RESISTANCE TO Fusarium oxysporum f. sp. lycopersici

It has been shown that a tomato cultivar Gruntovy Gribovsky 1180 transformed with pea lectin gene (psl ) has been shown to form a stable associative symbiosis with a Rhizobium leguminosarum bacteria that have fungistatic activity. This provides the protection of the plant root system from the phytopathogen Fusarinm oxysporum f. sp. lycopersici. The survival of the plants transgenic for the psl gene and incubated with rhizobia made up 81.5% after the fungal contamination. The analogous value for the nontransgenic plants was equal to 47.6%.

Pea lectin inhibits growth of Ehrlich ascites carcinoma cells by inducing apoptosis and G2/M cell cycle arrest in vivo in mice

Pea (Pisum sativum L.) lectin is known to have interesting pharmacological activities and of great interest on biomedical research. In the current research pea lectin was purified followed by ion exchange chromatography on DEAE column and affinity chromatography on glucose-sepharose column. The lectin shown 11.7–84% inhibitory effect against Ehrlich ascites carcinoma (EAC) cells at the concentration range of 8–120μg/ml in RPMI 1640 medium as determined by MTT assay. Pea lectin was also shown 63% and 44% growth inhibition against EAC cells in vivo in mice when administered 2.8mg/kg/day and 1.4mg/kg/day (i.p.) respectively for five consequent days. When Pea lectin injected into the EAC bearing mice for 10 days its significantly increased the hemoglobin and RBC with the decreased of WBC levels toward the normal. Apoptotic cell morphological change of the treated EAC cells of mice was determined by fluorescence and optical microscope. Interestingly, cell growth inhibition of the lectin was significantly reduced in the presence of caspase inhibitors. Treatment with the lectin caused the cell cycle arrest at G2/M phase of EAC cells which was determined by flow cytometry. The expression of apoptosis-related genes, Bcl-2, Bcl-X and Bax was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Intensive increase of Bax gene expression and totally despaired of Bcl-2 and Bcl-X gene expression were observed in the cells treated with Pea lectin for five consecutive days.

Genetically modified α-amylase inhibitor peas are not specifically allergenic in mice.

Weevils can devastate food legumes in developing countries, but genetically modified peas (Pisum sativum), chickpeas and cowpeas expressing the gene for alpha-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. αAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system. This modification was thought to be responsible for the reported allergenicity in mice of the transgenic pea but not the bean. Here, we observed that transgenic αAI peas, chickpeas and cowpeas as well as non-transgenic beans were all allergenic in BALB/c mice. Even consuming non-transgenic peas lacking αAI led to an anti-αAI response due to a cross-reactive response to pea lectin. Our data demonstrate that αAI transgenic peas are not more allergenic than beans or non-transgenic peas in mice. This study illustrates the importance of repeat experiments in independent laboratories and the potential for unexpected cross-reactive allergic responses upon consumption of plant products in mice.

Comparative analysis of the antibacterial activity of some phytolectins

The aim of this work was to analyse the comparative effects of the antibacterial properties of partially purified lectins from the seeds of Artocarpus heterophyllus (jack fruit), Canavalia ensiformis (jack bean), Lens culinaris (lentil) and Pisum sativum (pea) against the bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The lectins were isolated by partial purification using ammonium sulphate precipitation and dialysis. The antimicrobial activity was studied using agar well diffusion method. The results showed that the Jack fruit lectin had a potent anti-bacterial activity against S. aureus, B. subtilis, E. coli and P. aeruginosa whereas Pea and jack bean lectin were found to be effective bacteriostatic agents which reduced the growth of bacteria and lentil lectin showed the least antibacterial activity. A comparison of the antibacterial activity of phytolectins with conventional antibiotics namely ampicillin and tetracycline was also carried out. Studies revealed that the antibacterial activities of the conventional antibiotics are higher than that of the plant extracts at the same concentration in accordance to literature.DOI: http://dx.doi.org/10.3329/icpj.v2i2.13192 International Current Pharmaceutical Journal 2013, 2(2): 18-22

Monitoring growth, survival and enzyme system of melon fruit fly Bactrocera cucurbitae (Coquillett) under the influence of affinity purified pea lectin

AbstractPisum sativum agglutinin has been shown to act as a feeding inhibitor for various insect pests belonging to different orders: Lepidoptera, Coleoptera and Hemiptera. In the present study, its insecticidal activity was assessed through monitoring the growth and development of a dipteran pest Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Pea lectin, P. sativum agglutinin (PSA) was purified by single step affinity chromatography on a Sephadex G‐100 and the purification was monitored through hemagglutination activity and SDS‐PAGE. Insect feeding assays were conducted to determine the effect of pea lectin against first and second instar larvae of melon fruit fly B. cucurbitae. Lectin was incorporated in an artificial diet at a varied range of concentrations, 12.5, 25, 50, 100, 200 and 400 μg/mL. The lectin showed highly significant antimetabolic effects in both first and second instars. Time taken for pupation and development as well as percentage pupation and percentage adult emergence were adversely affected. The activity of three hydrolase enzymes (esterases, acid and alkaline phosphatases), five oxidoreductases (superoxide dismutase, catalase, ascorbate peroxidase, peroxidase, O‐demethylase) and one group transfer enzyme (glutathione‐S‐transferases) was also assessed in second instar larvae fed on lectin treated diet at 100 μg/mL concentration. The P. sativum lectin significantly and deleteriously influenced the activity of all these enzymes at all exposure intervals.

Development and verification of PCR based assay to dectect and quantify garden pea lec gene

Genetically modified organisms (GMOs) entering the food chain have became its part, which is necessary to monitor. GMO analyses are used as a control mechanism according to valid acquis communautaire for traceability and labeling of GMOs. Generally, approved PCR based protocols are used and they require stepwise procedures that use amplification of species specific gene as initial point. This study aims to develop and verify PCR based assay for amplification of garden pea lectin gene (Pisum sativum L.) as reference one. Lectin gene was analysed in silico, selected region was amplified and sequenced and new set of species specific primers for identification of garden pea was designed. Conditions of conventional PCR as well as real-time PCR were optimised and specificity of new primer set on DNA extracted from garden pea cultivars as well as DNA extracted from other selected species from Fabaceae family was tested. Quantification of garden pea lectin gene using real-time PCR based on SYBR Green I was optimised and performance characteristics recorded. The characteristics fit to method acceptance criteria range. Plasmid with garden pea lectin sequence was developed and plasmid is available as a positive control.   

Artificial colonization of non-symbiotic plants roots with the use of lectins

Rhizobia have the ability to increase growth of non-legume plants due to the production of phytohormones and protection of plant from diseases and pathogens. However, the practical use of these beneficial bacteria sometimes fails because of their inability to effectively colonize rhizoplane and rhizosphere of inoculated plants. We chose the legume lectins as a factor that allows plants to form associative symbiosis with rhizobia. To test the fact that transgenic tobacco, tomato and rape roots with pea lectin gene may affect specific interaction with rhizobia, transgenic roots have been artificially inoculated by fluorescently-labeled pea rhizobia R. leguminosarum and east galega rhizobia Rhizobium galega. Microscopic and microbiological tests have shown that the number of adhered R. leguminosarum onto tobacco, rape and tomato roots which transformed with pea lectin gene is higher in comparison with the control, but no such effect through inoculation of these plants with R. galegae has been found. This confirms the interaction of R. leguminosarum with pea lectin at the surface of transformed roots. Undoubtedly, the improvement of recognition and attachment processes by using lectins can lead to the achievement of a stable associative relationship between non-symbiotic plants and rhizobia.

Bioengineering of symbiotic systems: Creation of new associative symbiosis with the use of lectins on the example of tobacco and oil seed rape

"Barbate roots" in tobacco and colza transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression oflectin gene on colonization oftransgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and colza. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.

Pea lectin unfolding reveals a unique molten globule fragment chain

Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains -a long β-fragment and a short α-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a β-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ∼ 20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule β-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular.