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The relationship of isoenzymes of peroxidase and esterase with rice allelopathy

Liuqing Yu 1, Di Li 1,2, Zhengbo Zhang1, Yongjun Zhou 1 and Jianping Zhang 1

1 China National Rice Research Institute, State Laboratory of Rice Biology, Hangzhou 310006, China.

Editorial Office of Molecular Plant Breeding, Beijing 100080, China. Email


Rice leaves were collected from rice at the stage of 3 to 4 leaves. The homogenates of the rice leaves were prepared in a mortar with the sample of two grams plus two millilitre extracting reagent under ice-bath condition. Samples of supernatant were prepared with extracting and centrifuging with an equal quantity of 40 percent sucrose solution. Electrophoresis of peroxidase (POD) isoenzyme and esterase (EST) isoenzyme were conducted to show the relationship between the isoenzymes and rice allelopathy, and the activities of the two isoenzyemes investigated. The results indicated that the activity of POD from the sample of rice leaf was higher than that from rice root. A potential allelopathic variety I-Kung-Pao showed 0.20 OD470/g FW/min activity of POD in the leaf, but significantly reduced 0.12 OD470/g FW/min in the root. Among the five varieties of rice, the potential allelopathic rice cultivars, I-Kung-Pao and PI312777, gave more strong activities of POD than non-allelopathic varieties D-gu, No-Iku1716 and Xiushui 63 in their leaves. The bands of EST isoenzyme on gel after electrophoresis were varied among the five varieties of rice, American allelopathic rice variety PI312777 had seven bands of A, B, C, D, E and F. Chinese allelopathic rice variety I-Kung-Pao had five bands of A, B, C, D and F. The EST of C was the characteristic brand of both allelopathic rice cultivars. However, the non-allelopathic rice cultivars had much less concentration of EST isoenzyme, even it was absent in Xiushui 63, the non-allelopathic rice cultivar.

Media summary

Potential allelopathic rice had greater activity of peroxidase isoenzyme and more spectrum bands of esterase isoenzyme than non-allelopathic rice varieties.

Key Words

Peroxidase isoenzyme, esterase isoenzyme, allelopathy, rice


In the germplasm of rice there were only a very few cultivars (lines) with allelopathic potential, and the difference in allelopathic potentials among cultivars is determined by the genotype. Allelopathy is quantitative trait in genetics (Ebana et al. 2001, Jensen, Olofsdotter and Courtois 2001, Jensen, Courtois and Shin 2001, Zeng et al. 2003).

Isoenzymes technique is an effective tool to study the life phenomena at the molecular level, and which is generally applied in the study of genetics, heterosis, and physiology (Zhu et al. 2004, Zhu et al. 1985). However, the reports about the study of rice allelopathy with this method are very few.

The objective of this study was to study the relationship of the allelopathy to the isoenzymes by analyzing the isoenzymes of peroxidase and esterase of the allelopathic rice cultivars and the non-allelopathic rice cultivars that were determined by bioassay.

Materials and Methods

The rice varieties with allelopathic potential were I-Kung-Pao and PI312777, and the non-allelopathic rice varieties were Xiushui 63, D-gu and No-Ikul716. The allelopathic rice variety PI312777 was kindly provided by Dale Bumpers National Rice Research Centre, and the other rice cultivars were provided by the Chinese National Rice Research Institute.

Agents were applied with Acrylamide, N N-Methylenebisacrylamide, TEMED, Coomassie Brilliant R- 250 and β-Mercaptoethanol from Sigma; Tris, agarose from Gibaco company, the other reagents from the company in China.

The rice seeds were sterilized with 1% NaClO (v/v) solution for 30 min, and then rinsed 3 to 4 times with sterilized water, soaked in water for 48h, then cultivated at 28. The pre-germinated seeds were cultivated in the greenhouse, and the seeds of barnyardgrass were inoculated in the rice pot. The rice leaves were picked at the stage of tillering for analyzing peroxidase and esterase. The activities of peroxidase (POD) in the rice leaves was determined using a modification of the methods of Zhang (Zhang XZ 1992). The 0.5g fresh rice leaves and roots were ground into homogenates with 50 ml phosphate buffer (pH 7.0) under ice-bath conditions. Crude peroxidase solution samples were obtained after centrifuging (10,000 rpm; at 4) for 15 min with the 10 ml homogenates.

The reaction included 1.9 ml acetate buffer (pH 5.0), 1 ml 0.1% guaicol, 0.01ml crude peroxidase solution and 1 ml 0.08% H2O2. The OD470 values were investigated after 2 min reaction. Distilled water was used to substitute 0.08% H2O2 to rectify the spectrophotometer. The activities of POD were measured in the form of OD470g-1FWmin-1.

The activity of the esterase (EST) in rice leaves was investigated using a modification of the method of Asperen (1962), using α-naphthyl acetate esterase substrate with Fast Blue as staining solution. Solutions of α-naphthyl acetate esterase 200mg solute in 10 ml 50% acetone, and blue RR salt 200mg solute in 90 ml 0.2 mol/L phosphate buffer (pH 7.0), were mixed together and filtered for evaluating the esterase activities. 1 ml esterase crude solution mixed with 5 ml staining solution for 1 h at 37. The activities of esterase were measured in the form of OD600g-1FWmin-1.


Analysis of the activities of peroxidase (POD) in rice

The activities of the POD in the different rice cultivars were significantly different, the activities of I-Kung-Pao was the highest, and that of PI312777 was secondary, and that of three non-allelopathic rice cultivars were very low (Figure 1).

Analysis of the peroxidase (POD) in the rice leaves

The numbers of bands of the POD in the rice from different cultivars on gel after electrophoresis were different, the allelopathic variety PI312777 had five bands(A,B,C,D,E), and the Chinese allelopathic rice variety I-Kung-Pao had three bands(A,C,D). Compared to the non-allelopathic cultivars Xiushui 63, D-gu and No-Iku1716, the band A was the characteristic band of the allelopathic cultivars which both of the I-Kung-pao and PI312777 had (Figure 2).

Figure1. Peroxidase activity in rice leaves from different varieties

Figure 2. Peroxidase in the rice leaves from different cultivars
Note: 1. Xiushui 63 2. I-Kung-Pao 3.D-gu 4. PI312777 5. No-Iku1716

Analysis of the activities of esterase(EST) in rice

In the rice leaves, the activities of EST was the highest in cv I-Kung-Bao, and PI312777. The activities of EST of these two allelopathic rice cultivars were higher than that of the non-allelopathic rice cultivars XiuShui 63, D-gu and No-Iku1716 (data not shown).

Analysis of the esterase (EST) in rice leaves

The American allelopathic rice cultivars PI312777 had six bands (A,B,C,D,E,F) after electrophoresis, and the Chinese allelopathic rice cultivar I-Kung-Pao had five bands (A,B,C,D,F). Compared to the non-allelopathic rice cultuvars, Xiushui 63, D-gu and No-Iku1716, the EST of C was the characteristic band of the allelopathic rice cultivars, which were much reduced or absent in the non-allelopathic rice cultivars (Figure 3).

Figure 3. Comparison of esterase isoenzyme in rice from different varieties Note: 1. Xiushui63 2. No-Iku1716 3. I-Kung-Pao 4.D-gu 5. PI312777


Li et al. (2004), evaluated the allelopathy of over 470 rice cultivars from Chinese rice germplasm with the method of the root-length of barnyardgrass and the root-length of lettuce, and concluded that Chinese variety I-Kung-Pao, a Taiwan native variety, was a rice variety with allelopathic potential. Zhang et al. (2005) identified I-Kung-Pao as a rice variety with allelopathic potential using the method of allelopathic index (AI) with the specific secondary metabolites as HELP markers.

Compared to the potentially allelopathic American rice PI312777, the Chinese allelopathic potential rice variety I-Kung-Pao had higher activities of POD and EST. Both of them had the POD A and EST C that did not exist in the non-allelopathic rice cultivars, indicating that the POD A and EST C represented the characteristics of the allelopathic potential rice cultivars. However, further research is needed to verify these results because the number of the rice cultivars in this experiment was limited.


This work was financially supported by the National Natural Foundation of China (301706200 and 30370942).


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