Research Article - Annals of Biological Research ( 2018) Volume 9, Issue 2
The aim of this research was to investigate the effect of one plant activator on peroxidase (EC 1.11.1.7) and protease (EC 4.3.1.1) activity in Solanum lycopersicum Riogrande and H2274 varieties. Plant activator was applied to 10 weeks old seedlings in recommended dose, two and four fold doses (2,4 and 8 ml/L) by spraying to the leaves under in vivo conditions. The leaves of the seedlings were harvested for analysis 24 and 48 h after plant activator applications. All of the experiments were replicated three times. Peroxidase activity changing in S. lycopersicum varieties while compared with control group. 48 h after 8 ml/L activator application in Rio Grande and H2274 varieties POX activity increased as 248% and 247.4% respectively. Protease activity changing in S. lycopersicum varieties was compared with the control group. 48 h after 4 ml/L activator application in RioGrande and H2274 varieties PRO activity increased as 90.6% and 83.7%, respectively. Plant activator application has changed the total peroxidase and total protease levels depending to the time and concentrations in different levels. Thus, the use of chemicals with low doses and activators can prevent different plant related diseases by increasing the efficiency of self defence mechanisms in plants by virtue of which our country can hold an important place in agriculture in future and also foreign sales will contribute to resolve the phyto-pathological problems of tomato
Plant activator, Peroxidase, Protease, S. lycopersicum, Riogrande, H2274
Various enzymes are synthesized in plants which are also involved in the defence mechanism of which the peroxidases are utterly important. Peroxidases (EC 1.11.1.7) are the precursor enzymes that are synthesized in chloroplasts in a large majority of plants and also actively take part in the functioning of the defence mechanism [1]. Peroxidases are involved in defence reactions of plants against pathogens and every kind of stress factors [2]. Peroxidase (POX) cleaves water and oxygen by toxic H2O2 after metabolic events, so the POX reaction is measured by monitoring the formation of oxygen [3]. Proteases are involved in many aspects of plant physiology and development. On the other hand, they are necessary for protein turnover, degradation of damaged, misfolded and potentially harmful proteins and also provide free amino acids required for the synthesis of new proteins. Studies have described proteases are associated with several types of plant PCD, including involved in all aspects of the plant life cycle ranging from the mobilization of storage proteins during seed germination to the initiation of cell death and senescence programs [4-6]. Proteases also take part in oxidative stress, tracheary element development and the selective breakdown of regulatory proteins by the ubiquitin/proteasome pathway by which they are able to control the key aspects of plant growth, development, and defence [7,8]. In recent years, demand of conventional fungicides used against pathogen related diseases are gradually decreasing as producers and consumers becoming more health conscious and biological control and Integrated Pest Management (IPM) programs becoming more popular. Bioactivators used to increase natural disease resistance were one of the basics in biological control. The use of plant activators has increased in recent years with the developments in organic agricultural practices. Use of plant activators to reduce plant diseases is relatively new and very few activators are commercially available. Green Miracle is a long chain fatty acid based new generation stress alleviator for improving the plant health. By virtue its reflective nature Green Miracle alleviates the plant from thermic stress by reducing the rate of transpiration through reflecting greater amount of light that fall on the plant.
Plant materials
Two varieties of S. lycopersicum Mill. Riogrande and H2274 seeds were taken and treated with distilled water for 5 h before the sowing process. Then, seeds were sowed in plastic pots (10 × 30 cm) containing a mixture of 1:2 perlitepeat. Seedlings were grown under plant growth chamber conditions (light/dark of 16/8 h at 25 ± 20°C), relative humidity 60-70%. Ten weeks old seedlings were used for plant activator applications.
Plant activator
In this research Green Miracle plant activator were used for the experiments. This plant activator was taken from Konserve Microbial Agriculture and Animal Product Ltd. Company. Green Miracle is a long chain fatty acid based new generation stress alleviator for improving the plant health. To determine the effects of the plant activator, the criteria which can be defined as the total protease and peroxidase in leaves were studied.
Preparation of leaf extracts
Healthy leaves of Riogrande and H2274 varieties of tomato seedlings were harvested for the applications. For the preparation of crude leaf extract, 0.5 g of fresh leaf were homogenized in 5 ml of cold sodium phosphate buffer (0.05 M, pH 6.5) for 30 s and then centrifuged at 13,000 rpm for 20 min at 4°C.
Enzyme analyses
Peroxidase activity in leaf extracts was assayed spectrophotometrically. 1 ml of assay mixture containing 0.05 M sodium acetate buffer (pH 6.5), 0.2 ml of 0.1 M pyrogallol, 0.1 ml of 90 mM H2O2 and an aliquot of the crude leaf extract containing 10-40 μg proteins were mixed together immediately before the evaluation. The peroxidase enzyme activity was measured at 300 nm according to Kanner and Kinsella [9].
Protease activity in the crude leaf extracts was assayed spectrophotometrically. 0.5 ml enzyme solution (supernatant) was added to 2.5 ml casein solution and kept under 30°C without shaking in a water bath for incubation for 20 min. Again, 2.5 ml TCA (Tri-Carboxyl-Acid) solution was added to each test tube and kept under 30°C without shaking in a water bath for incubation for 30 min. After that, suspension filtered through a coarse filter paper tube with a clean glasstube. 0.5 ml of filtrate was taken to a clean glass tube and 2.5 ml 0.5 M Na2CO3 was added to each tube. Twofold diluted 0.5 ml Folin-Ciocalteu reagent was added to each tube. The tubes were stored at room temperature without shaking for 30 min. The protease enzyme activity was measured at 660 nm in spectrophotometer [10].
Plant activators have been used extensively in recent years in the fight against harmful organisms. Using the plant activator to pre-stimulation of plant defence systems provides protection against pests and disease by which it is possible to improve the efficiency. Thus, the plant activators can used to combat pests which can be antibacterial, fungicidal, insecticidal, etc. [11]. According to our research results, peroxidase activity changing in RioGrande and H2274 varieties were determined after plant activator application, while compared with the control group. Most effective application for POX changing was the 8 ml/L application after 48 h in RioGrande and H2274 varieties where the POX activity increased 248% and 247.4% respectively. Most effective total protease activity changing was 4 ml/L application after 48 hours in RioGrande and H2274 varieties as 90.6% and 83.7% respectively. Results of these assays and statistical data of SPSS represented in Tables 1 and 2.
| Group | POX activity (mg/mL/min) | St. Dev. | Compared Groups | Protease activity (U/ml/min) | St. Dev. | Time (h) | |
|---|---|---|---|---|---|---|---|
| Control | 54.000 | 85.440 | 1.4** | 0.1177 | 0.00153 | 1.2** | 24 |
| 2 ml/L | 54.290 | 49.771 | 2.4** | 0.1577 | 0.01450 | 1.3** | 24 |
| 4 ml/L | 58.236 | 664.816 | 3.4** | 0.1947 | 0.02050 | 2.3** | 24 |
| 8 ml/L | 112.900 | 618.304 | 0.1320 | 0.01500 | 3.4** | 24 | |
| Group | POX activity (mg/mL/min) | St. Dev. | Compared Groups | Protease activity (U/ml/min) | St. Dev. | Time (h) | |
| Control | 533.333 | 76.376 | 1.4** | 0.1180 | 0.00300 | 1.4** | 48 |
| 2 ml/L | 536.166 | 59.448 | 2.3** | 0.1783 | 0.00751 | 2.3** | 48 |
| 4 ml/L | 617.466 | 33.871 | 2.4** | 0.2253 | 0.01450 | 2.4** | 48 |
| 8 ml/L | 187.94 | 53.935 | 3.4** | 0.1553 | 0.00651 | 3.4** | 48 |
** Statistically meaningful groups gave (p<0.05).
Table 1: Statistical relationships between the groups after 24 and 48 h plant activator applications in S. lycopersicum RioGrande seedlings.
| Groups | POX activity (mg/mL/min) | St. Dev. | Compared Groups | Protease activity (U/ml/min) | St. Dev. | Time (h) | |
|---|---|---|---|---|---|---|---|
| Control | 547.467 | 338.711 | 0.1350 | 0.1808 | 1.2** | 24 | |
| 2 ml/L | 510.467 | 256.642 | 0.1967 | 0.01300 | 1.3** | 24 | |
| 4 ml/L | 568.667 | 382.797 | 3.4** | 0.2240 | 0.01650 | 2.3** | 24 |
| 8 ml/L | 1.203.333 | 450.925 | 0.1677 | 0.00900 | 1.4** 3.4** | 24 | |
| Groups | POX activity (mg/mL/min) | St. Dev. | Compared Groups | Protease activity (U/ml/min) | St. Dev. | Time (h) | |
Control |
547.467 | 338.711 | 1.3** | 0.1353 | 0.01550 | 1.2** 1.3** | 48 |
| 2 ml/L | 615.267 | 373.258 | 2.4** | 0.2047 | 0.01050 | 2.3** | 48 |
| 4 ml/L | 627.167 | 312.903 | 3.4** | 0.2477 | 0.01550 | 2.4** 3.4** | 48 |
| 8 ml/L | 1.902.433 | 487.828 | 1.4** | 0.1747 | 0.01450 | 1.4** | 48 |
If the value is less than 0.05, the difference between the examined data or groups is statistically significant (95% confidence). Thus, there is a statistically significant difference shown in Tables 3-6. There is no difference by 0.053 in Table 5 but if the confidence level is taken as 90%, there will be a difference.
| ANOVA | |||||
|---|---|---|---|---|---|
| Mean value | |||||
| Sum of Squares | df | Mean Square | F | Sig. | |
| Between Groups | 0.009 | 3 | 0.003 | 13.414 | 0.015 |
| Within Groups | 0.001 | 4 | 0.000 | ||
| Total | 0.010 | 7 | |||
Table 3: ANOVA test for protease activity in S. lycopersicum RioGrande (24 and 48 h)
| ANOVA | |||||
|---|---|---|---|---|---|
| Mean value | |||||
| Sum of Squares | df | Mean Square | F | Sig. | |
| Between Groups | 0.011 | 3 | 0.004 | 420.854 | 0.002 |
| Within Groups | 0.000 | 4 | 0.000 | ||
| Total | 0.011 | 7 | |||
Table 4: ANOVA test for protease activity in S. lycopersicum H2274 (24 and 48 h)
| ANOVA | |||||
|---|---|---|---|---|---|
| Mean value | |||||
| Sum of Squares | df | Mean Square | F | Sig. | |
| Between Groups | 13460171 | 3 | 4486724 | 6.359 | 0.053 |
| Within Groups | 2822110 | 4 | 705,527 | ||
| Total | 16282280 | 7 | |||
Table 5: ANOVA test for POX activty in S. lycopersicum Rio Grande (24 and 48 h)
| ANOVA | |||||
|---|---|---|---|---|---|
| Mean value | |||||
| Sum of Squares | df | Mean Square | F | Sig. | |
| Between Groups | 14532873 | 3 | 4844291 | 7.702 | 0.039 |
| Within Groups | 2515731 | 4 | 628,933 | ||
| Total | 17048603 | 7 | |||
Table 6: ANOVA test for POX activty in S. lycopersicum H2274 (24 and 48 h)
In our research, L. esculentum Mill Rio Grande and H2274 varieties POX activity increased 248% and 247.4% respectively. A similar study was carried out by Kiprak [12] on the change of POX activity in Capsicum annuum. Peroxidase activity changing in Capsicum annuum L. var. grossum seedlings was determined in different levels, after plant activators application, while compared with control group. 24 h after 1.2 ml/L dose of Crop-Set application to the in vivo growth seedlings of Capsicum annuum L. var longum, POX activity increased 180% and after 48 h 2.4 ml/L dose of Crop-Set application. Both of the plant activators significantly increased POX activity in both plant varieties according to the exposure time. In our work in vivo conditions related to POX enzyme production; the highest enzyme activity was observed at 48 h in the plant groups to which the plant activator was applied. In S. lycopersicum H2274 and S. lycopersicum RioGrande species, the most effective dose was found to be four times the dose of the recommended dose.
There are a variety of studies on the use of casein as substrate for proteolytic activities in different plants. Mahajan et al. [13] used casein as a substrate to screen twenty plants of the Euphorbiaceae family for proteolytic activities in leaves. Among these selected plants; Pedilanthus tithymaloides has the highest caseinolytic activity, followed by Euphorbia tirucalli, Euphorbia nivulia and Euphorbia nerifolia 9.0, 6.42, 4.56 and 4.38 U/mg proteins, respectively.
Our study has found that, the protease activity of tomatoes RioGrande and H2274 was found to be increased. In another research conducted by Veerasamy et al. [14] zeatin application also showed increased in protease activity. In our research; total protease activity increasing in RioGrande and H2274 varieties after 48 h with 4 ml/L plant activator applications was as 90.93% and 83.07%, respectively.
As a result, exports of agricultural products due to pesticide residues to minimize the role of the terms of the negative effects, plant activators is necessary to traditional chemical control of the manufacturer as a more preferred alternative methods. Plant activators, to per unit area more and high-quality product that delivers, drugs commonly used in agricultural production today is inevitable.
Future research in this field will be; an active role in the functioning of the defence mechanism and protease, a key enzyme in the metabolism of the protein, interact each other. Various inhibitors will be used to interaction between these two enzymes.
This research is a part of Mr. Ertugrul Osman Bursalioglu’s PhD thesis which is successfully completed in July 2013 in Canakkale Onsekiz Mart University, Graduate School of Natural and Applied Sciences, Biological Science.
