S Shahab, N Ahmed
S Shahab, N Ahmed. Growth Promotion Of Mung Beans By Bacterial Pyrroloquinoline. The Internet Journal of Genomics and Proteomics. 2003 Volume 1 Number 1.
Pyrroloquinoline (PQQ) is an important cofactor of bacterial dehydrogenases, linking the oxidation of many different compounds to the respiratory chain. There have been very few studies of the functional role of PQQ in plant growth. CMG 860 is a native bacterial isolate having multidimensional biofertilizing abilities. CMG 860 has been found to have
Pyrroloquinoline quinone [4, 5-dihydro-4, 5-dioxo-1H-pyrrolo-[2, 3-f] quinoline-2, 7, 9-tricarboxylic acid is an aromatic, tricyclic ortho quinone that serves as the redox cofactor for several bacterial dehydrogenases. PQQ was the first of the class of quinone cofactors that have been discovered in the last 18 years and make up the prosthetic group of quinoproteins[3,6,7.] PQQ is water soluble, heat stable, and has the ability to carry out redox cycles [15,17]. It has been reported that PQQ acts as a reactive oxygen species (ROS) scavenger by directly neutralizing reactive species in
Although plants and animals do not produce PQQ themselves, PQQ has invoked considerable interest because of its presence in human milk and its remarkable antioxidant properties. PQQ was the vitamin identified after 50 years in 2003 [3, ,2]. Although biological functions of PQQ are not fully understood  PQQ has attracted considerable interest because of its presence in a wide variety of foods and its remarkable antioxidant properties [8,9,14].
There have been few studies of the functional roles of PQQ in plants. It is known that PQQ stimulates pollen germination in vitro in the plant species Lilium, Tulipa, and Camellia [16,14]but the mechanisms are unclear. PQQ is at least 100 times more efficient than ascorbic acid, isoflavonoids, and poly phenolic compounds in assays assessing redox cycling potentials [20, 15] In addition to scavenging superoxide, PQQ could also scavenge other toxic free radicals, as do vitamin E, b-carotene and carotenoids, vitaminC, flavonoids, conjugated linoleic acid, and phenolic compounds PQQ is found in plant and animal tissues in the nanogram to gram range even though plants and animals do not produce PQQ themselves[10,11]
Genes involved in PQQ synthesis have been cloned from
Materials and Methods
Reaction mixture , Cycling condition Amplification and Sequencing
PCR amplification of
Mutation in pqq Gene
A stock solution of acridine orange (5mg/25ml) as a mutagenic agent was prepared. An aliquot of 10 µl overnight grown culture was inoculated in test tubes containing variable concentrations of acridine orange (10µl, 50µl, 100 µl, 200 µl, and 400 µl). These test tubes were incubated at 37°C for 24 hours at 100 rpm. A100µl culture from 10-5, 10-6 and 10-7 dilutions were spread over nutrient agar plates and incubated for 24 hrs at 37°C to get isolated colonies. About 100 colonies were randomly selected and tooth picked on to a control (nutrient agar plate) and test plate (tris minimal agar plate having 5mM zinc phosphate). The colonies which lost phosphate solubilization activity were initially selected and designated as CMG860 mutant colonies CMG860 M2 and CMG860 M4.
Auxin Production Activity
Auxin production activity of the wild type CMG860 and mutants CMG860M2 and CMG860M4 were carried out by using salkovski reagent method 
Isolation of pqq Mutants
Mutant strains M2 and M4 were studied for the growth promotion of the mung beans seedlings. Briefly, the surface sterilized seeds were incubated with late log phase cultures of wild type CMG860 and mutant strains CMG860M2 and CMG860M4 by incubating them with 10ml of respective bacterial suspension (10-7cfu per seed) in a sterile 15ml test tube. Non inoculated control seeds were incubated with 15 ml of nutrient broth. The mutant strains CMG860M2 and CMG860M4 were then finally selected on the basis of their demolished plant growth promotion activities.
Revival of Plant Growth Promotion activities of Mutants in Pots
Mutant strains CMG860M2 and CMG860M4 were analyzed by the method of Choi
Set A contained: O/N grown culture of CMG860 wild type
Set B contained: O/N grown culture of CMG860M2/CMG860M4
Set C contained: synthetic PQQ +O/N grown culture of CMG860M2 /CMG860M4
Two days old mung beans seedlings were immersed in 10 ml O/N culture of wild type CMG860 (Set A) , mutants CMG860M2 / CMG860M4 (Set B) and in 10 ml of 100nM of synthetic PQQ (Set C) for 1 hr, and then transferred into the plastic pots contained 200gm autoclave sand, and the surplus synthetic PQQ solution that remained after treatment was poured into the pot of Set C. Pots were placed in a green house and set to a 16-h light/8-h-dark cycle with a relative humidity of 60%. Shoot length and root length of the plants were recorded after 10 days.
Analysis of PQQ
To analyze PQQ production of wild type and mutant strains a method of Choi
Extraction of protein from mung bean plants
A 5 day old leaves of mung beans were plucked and immediately placed in vials containing liquid nitrogen. These samples were further dried for 24h in freeze dry system using freezone 6liter benchtop model 77520.
SDS-polyacrylamide gels (12 % w/v) were prepared according to Laemmli (1 1). Samples were diluted in Laemmli sample buffer ( 11) and boiled for 3 min before loading. gels were run for 90 min and then run at room temperature at 100 V . Gels were stained with Coomassie blue staining method. An electrophoresis cell (model:DYCZ_24E) was used. Prestained mol wt markers were purchased from Sigma
Sequence Homology of Amplified PCR Product (pqqA-D)and pqq E of CMG860
The PCR product was purified using quick PCR purification kit. The ABI373 DNA sequencing system was used for sequence pqqA –D operon. A total of 2212 bases were determined which showed 95% homology with pqqBC gene of
Isolation of pqq Mutants
Following random mutagenesis of
Auxin Production Activity
The auxin production activity of wild type and mutants was performed by using salkovski assay reagent method. Both mutant strains (CMG860M2 and CMG860M4) and Wild type CMG860 showed auxin production activity (Figure 2).
Growth Promotion of Mung Been Seedling
Effect of Wild type and mutant strains were checked on the growth of mung beans seedlings. Heights of the plants were measured every 3 days up to 10 days after the treatments. Mutant strains CMG860M2 and CMG860M4 were failed to promote growth of mung beans seedlings while revival of plant growth promotion activities was observed (Figure3 and Figure4) when synthetic pqq was added in O/N culture of mutant strains (CMG860M2 and CMG860M4). The height of mung beans plants treated with wild type CMG860 was increased by approximately 25% at 10 days after treatment (Figure5 and Figure6).
Identification and Quantification of PQQ via RP-HPLC in CMG860 wild type and mutants
Production of PQQ from wild type CMG860 and mutant stains CMG860M2 and CMG860M4 were confirmed via RP- HPLC. The retention time of standard PQQ is 1.8. Quantification of PQQ in wild type CMG 860 was also performed via HPLC by the integration of peak heights. CMG60 wild type produced 2.44 mg/ml of PQQ (Figure7 and 8). No PQQ production was detected in mutant strains CMG860M2 and CMG860M4 (Figure 7 and 8).
The total extracted protein was analyzed via SDS-PAGE Electrophoresis. A band of 37 kb was isolated which was absent from control sample. The presences of 37kb band clearly indicate the difference at protein level.
A=Growth of M2on nutrient agar plate
B= Growth of M4on nutrient agar plate
C= Growth of M2on Tris agar plate
D= Growth of M4on Tris agar plate
Keys:A = Wild Type 860B = Mutant M4
C = Mutant Supplemented with Synthetic pqq
Keys A = Wild Type 860B = Mutant M4 C = Mutant Supplemented with Synthetic pqq
Gene encoding PQQ cofactor was detected in CMG860 by PCR (Figure1). The purified PCR product of CMG860pqqA-D (EU72017) was sequenced. A Total of 2212 bases were determined which showed 95% homology with pqqBC gene of
There have been few studies of the functional roles of PQQ in plants. It is known that PQQ stimulates pollen germination in vitro in the plant species Lilium, Tulipa, and Camellia [16,17]but the mechanisms are unclear. To investigate the role of PQQ as a main plant growth promoting factor in CMG860, mutagenesis experiments were performed. Results of mutagenesis clearly indicated that wild type CMG860 showed better plant growth as compared to mutants (Figure3-4). Wild type CMG860 markedly increased the shoot length of mung beans seedlings up to 114 % and 87 % respectively over mutants M2 and M4 while wild typeCMG860 increased the root length of mung beans seedlings up to 200% over mutants. Cultural analysis of the mutant and wild type CMG860 via HPLC clearly indicated the production of PQQ as main agent responsible for plant growth promotion activities (Figure 3 and 4). The revival of plant growth in presence of synthetic PQQ clearly indicated that PQQ is a plant growth promoter. Therefore it was assumed that the biochemical basis of plant growth promotion mediated by PQQ is more or less similar to that of its growth promotion in mammals . It was likely to propose here that many PGPR produce PQQ, which would illuminate previously unknown plant growth promotion mechanisms. As in mammals, PQQ has great potential to be used as a growth promoting factor in plants. Plant growth promotion by PGPR has received attention for academic and practical reasons because beneficial interactions between PGPR and plants offer tremendous potential for field applications. In order to study the mechanism of plant growth promotion at protein level, The total protein extraction from young leaves of mung bean plants was carried out as the RNA analysis was difficult because of highly degrading potential of RNA hence protein extraction from the mung beans leaves were performed. Total protein was extracted from treated and untreated control samples via Plant protein extraction kit. A band of approximately 37kb was found in test samples which was absent in control samples. These results were in good harmony with those obtained by Adam (1999) and Abdel monem