Mechanisms of insect resistance induced by treatment of Lycopersicon esculentum seeds by jasmonic acid

by magisterartium (student)

Mechanisms of insect resistance induced by treatment of Lycopersicon esculentum seeds by jasmonic acid.

Abstract: Research has suggested that jasmonic acid (JA) treatment of tomato (Lycopersicon esculentum) seeds would cause a constant and higher rate of induced resistance to insect herbivores through increased defence gene expression, leading to increased concentrations of polyphenol oxidases (PPOs). It is already known that methyl jasomnate (MeJA) and jasmonic acid (JA) are vital in the octadecanoid wound-signalling pathway and that the exogenous application of JA to tomato crops has been shown to increase PPO levels. However, during this study, the treatment of seeds did not lead to any statistically significant evidence to support current theories on this, but, did show that JA treatment on tomato seeds to have a slight positive effect on the increase of PPO activity in the wounded and unwounded plants. At the present this study does not give strong enough evidence to support the general release of JA treated seeds for use in agriculture at this in the near future. However, this is not to say that JA application exogenously is not a viable option in natural herbivore resistance. It must be understood, that the results here are not wholely conclusive either way and could be due to erroneous experimental data. The fact that this study has shown there to be no positive effects, this is not to say that future investigation would not show that the treatment of seeds via JA is a viable method of induced herbivore resistance.

I.Introduction

The aim of this study was to examine the effects of the plant signalling hormone jasmonic acid (JA) on the induced resistant mechanisms of Lycopersicon esculentum , or common tomato plants, when the seeds were treated with a 1.5mM solution of JA, most notably the effect on polyphenol oxidase (PPO), a proteinase inhibitor in plants. Polyphenol oxisase, an oxidative enzyme has been found to act as a protease inhibitor in the guts of foraging insect herbivores (Ryan et al., 1990) and have also been found to be commonly used in plants as marker genes to the induced wound response.

In their natural environment, damage is inflicted upon plants by many means such as drought effects, the effects of increased salinity, but most notably by herbivore grazing. To combat the grazing of herbivores, plants have evolved unique mechanisms to reduce the damage inflicted upon them. Not only do plants need to combat the direct effects of herbivore grazing, but also the indirect effects. For example, microbial pathogens such as Agrobacterium tumefaciens which causes crown gall disease, may enter at and attack the wound site, which has resulted in the evolution of a complex system of defence against both the herbivores and pathogens. The responses are almost always induced responses and the response pathways are directly activated as a result of herbivore wounding.

Figure 1a. (Roberts M. R., et al., 2001) Showing a basic scheme for a plant response to wounding. Mechanical wounding, simulating herbivore grazing elicits the expression of local and systemic genes. Shown are the general events, from wounding which causes a calcium ion flux to occur, inducing the expression of genes which from wound healing proteins and the synthesis of signalling hormones, which in turn leads to defence gene expression and then the expression of defence molecules. Also shown is the systemic signalling pathway where the wounding induces signals which cause defence genes to be expressed in unwounded leaves.

Jasmonic acid, the inducement molecule used in this study was first identified as a signaling molecule in induced resistance to wounding when studies lead to the identification of methyl jasmonate (MeJA) as a strong inducer of proteinase inhibitor (Pin) genes in tomato plants. JA has been found to be present in many plant species and is involved in regulating diverse plant functions including plant resistance and senescence (Creelman et al., 1997) Studies have shown that although MeJA is not elicited in sufficient quantities or over sufficient distanced to induce Pin genes in neighbouring plants, JA does act as a systemic signaling molecule, inducing the Pin genes elsewhere in an unwounded area of the plant (Farmer et al., 1992). Jasmonic acid biosynthesis is catalysed by a process called the ocatdecanoid pathway, catalysation occurring via several different enzymes which are regulated by compartmentalization in different sub-cellular vacuoles.

JA synthesis begins with the release of lipid precursor molecules from cellular membranes such as the plasma membrane and the chloroplast membranes, but as of yet the membrane types have not been fully identified. Phospholipase D (PLD) has recently been shown to be essential for JA synthesis (Wang et al., 2000) and may produce substrates for phospolipase A2 which activates lipoxygenases or may directly activate them. Lipoxygenases are involved in the next stage of JA synthesis, which probably occirs in the chloroplast. The lipoxygenases convert linolenic acid into lipid hydroperoxides which are then converted into the key intermediate 12-oxo-phytodeienoic acid (OPDA) through the action of allene oxide synthase and allene oxide cyclase (AOS and AOC). In the cytoplasm, OPDA undergoes reduction, and then is subjected to 3 -oxidations in the peroxisome to produce the Jasmonic acid product. In addition to the synthesis of MeJA, Krumm et al., (1995) and Wasternack et al., (1998) have shown that there are other jasmonate-related molecules formed such as bioactive amino acid conjugates.

Figure 1b. A model of the octadecanoid signaling pathway, culminating in the synthesis of MeJA which then induces the plant defences and synthesis of PPOs and other volatiles.

JA signaling in plants is not just limited to the accumulation of high levels in wounded leaves as increases of JA in unwounded leaves have also been observed, although not to the same extent (Bowles, 1998). It is known that JA is a requirement for most systemic wound responses, it is not known if it has specific functions in the systemic leaves or only acts in the wounded leaf to mediate systemic signaling (Roberts et al., 2001, Bowles., 1998). The site of JA synthesis in the plant seems to be instrumental in inducing an appropriate response to herbivore wounding. Farmer and Ryan (1990) and Farmer et al (1992) discovered that the appliance of MeJA and JA exogenously to plants causes the inducement of defence gene expression and can provide resistance to certain insects. Foliar JA application has been shown to increase levels of PPO and induced plants have shown 60% less leaf damage than control plants (Thaler, 1999).

This is a preview of the whole essay

The exogenous application of JA onto foliage acts as a resistance inducer in some crop plants, other studies have shown that treatment of seeds before crop planting can cause induced systemic resistance (ISR) in certain plants. Shailasree et al., (2001) studied the effects of -aminobutyric acid (BABA) on pearl millet and how the treatment of seeds before seeding caused ISR against downy mildew disease in the seeds themselves and plants germinated from those treated seeds.. Plants raised from seeds treated with the inducer showed a disease incidence of 10 and 12% compared to 71 and 76% in the control plants. This suggests that induced resistance in seeds treated with BABA remained operative through the vegetative and reproductive growth of the plants. If ISR can be initiated through seed treatment in these experiments, then it could also be possible in tomato seeds/plants when treated with JA.

II.Materials and Methods

Plant Materials: Lycopersicon esculentum seedlings were treated by soaking in a 1.5 mM solution of jasmonic acid (JA) for 48 hours and were then planted and grown, firstly in a growth box for seven days after germination then repotted and grown for a further 5 weeks under the following controlled conditions; 16 hours light/8 hours dark at 37oc and a light intensity of (LI).

The leaves of the plants were mechanically wounded using a set of laboratory forceps to simulate wounding by pests. The wounds were made across the width of the leaf so that the fibrous midribs were wounded as well as the soft leaf tissue. Any leaves which were wounded and untested were frozen using liquid nitrogen and held in deep freeze (-80oc) for further analysis.

Extraction Buffer 1 and Catechol: For 1L of 0.1M potassium phosphate buffer, 13.2ml K2HPO4 + 86.8ml KH2PO4 + 900ml H2O. For 1/3 (33mM) 4.4ml K2HPO4 + 28.9ml KH2PO4 + 966.7ml H2O. For 500ml 33mM KPO4, 2.2ml K2HPO4 + 14.45ml KH2PO4 + 485ml H2O. The buffer was pH tested using a Denver Instrument Basic pH monitor.

For catechol solution: Catechol has a molecular weight of 110g i.e. 1mole = 110g. Therefore to get a 0.024M solution:

N=CV (volume is set at 500ml – 0.5l)

= 0.024 x 0.5 = 0.012m

N=weight/MR therefore weight= N x MR

=0.012 x 110

=1.32g

For a 50mM solution 2.75g is required (in 500ml) for 50ml catechol solution, 0.255g of catechol in 50ml H2O.

Extraction Buffer 2, DOPA and Catalase: For 200ml of 0.1M KPO4 (0.1%SDS) with a pH 7.2, the following were required: 14.34ml 1M K2HPO4 + 5.66ml 1M KH2PO4 + 2ml 10% SDS +178ml H2O. The buffer was pH tested using a Denver Instrument Basic pH monitor.

DOPA was made up as followed then placed in to 1ml aliquots to freeze for later use. For 5mg/ml DOPA in KPO4 pH 7.2: 3.58ml 1M k2HPO4 + 1.42ml 1M KH2PO4 + 250mg DOPA + 45ml H2O

Catalase was made up as follows so that it could be delivered in units of 280/ml: 12mg catalase (from bovine liver, Sigma) + 10ml H2O which was then placed into 1ml aliquots for freezing until required.

PPO Extraction and Assay 1: The tissue employed for the assay was taken from the wounded leaves the leaf tissue being used with the fibrous vascular tissue removed. The tissue was then ground in a hand held mortar and pestle using liquid nitrogen to prevent any reactions occurring between enzymes in the broken down cells. 1g of tissue was then suspended in 5ml of cold buffer (0-4oc) 33mM potassium phosphate pH6.04. The homogenate was then clarified by centrifugation at 1400rpm for 15minutes at 3oC to prevent any enzyme activity. The supernatant was then extracted using a hand pipette and kept on ice to reduce PPO activity in the liquid. This supernatant was then assayed for PPO activity spectrophotometrically at an absorbance level of 540nm every 30 seconds for 5 minutes. The assay solution consisted of 300-500µl of supernatant, 200-500µl catechol solution and the remainder of KPO4 made up to 1ml in a UV transparent cuvette. The assay reaction of PPO oxidation of catechol to orthoquinone was initiated by the addition of the catechol to the buffer and supernatant solution. Before the catechol was added the spectrophotometer was zeroed out on the assay to give a 0 second reaction rate and to give a control of when the rate was being measured from. The assays were repeated 3 times to give a mean value.

PPO Extraction and Assay 2: The tissue employed was taken from 5 week old Lycopersicon esculentum plants, both wounded and unwounded. The leaf tissue was stripped of fibrous vascular tissue and then ground in a hand held mortar and pestle using liquid nitrogen to prevent enzyme activity between the molecules released from broken cell vesicles. 1g of tissue was then suspended in 5ml of cold buffer (0-4oc) 0.1M Na cacodylate buffer, pH 7.2 containing 0.1% SDS. SDS was added to the buffer as a detergent to prevent PPO latency. The homogenate was then clarified by centrifugation at 1400rpm for 15minutes at 3oC to prevent any enzyme activity. The supernatant was then extracted using a hand pipette and kept on ice to reduce PPO activity in the liquid. This supernatant was then assayed for PPO activity spectrophotometrically at an absorbance level of 490nm every 20 seconds for 5 minutes. The assay solution consisted of 50µl of the supernatant, 1ml of DOPA and 1ml of catalase in a UV transparent cuvette. The assay reaction which was followed was that of the conversion of DL-dihydroxyphenolalanine (DOPA) to quinone polymers. In order to prevent the oxidation of the substrate supernatant by peroxidase, 280 units (1ml) of catalase was added. Before the enzyme extraction was added the spectrophotometer was zeroed out on the assay to give a 0 second reaction rate and to give a control of when the rate was being measured from. The assays were repeated 3 times to give a mean value. This assay procedure was a modification of that as described by Sherman et al., (1991).

Standardisation Of Protein Assays: To determine the protein concentrations in each of the assays, the Bradford protein assay was carried out. Varied concentrations of BSA (bovine serum albumin) ranging from 10mg/ml to 1.2mg/ml made up to 50ml with the appropriate amount of H2O for each of the protein standards (2000-0). 1.5ml of Coomassie Plus Protein Assay Reagent was added to the BSA solution in 2ml UV transparent cuvettes. The assay was then left to stand for 10 minutes so that the reaction between the BSA and the Coomassie would reach its optimum and for the desired blue colouration to be achieved. The spectrophotometer was blanked on water and then each sample was measured at 595nm. The results were repeated, the 0 reading was then subtracted from the reading taken, as shown by the Coomassie standardisation procedure. This was repeated 3 times so that a mean value could be achieved and then plotted. To take a reading which could be compared to the standards in order to get a standard concentration for the PPO in the assay, 1.5ml of Coomassie Plus Protein Assay Reagent was added to 50µl of the extract. Again, the assay was run for 10 minutes when a reading was taken. The curve plotted was then used to determine the protein concentrations of PPO in the extracted supernatant, which enables the results to be quantified according to the amount of PPO present in the assay.

III.Results

The results used and explained here are those from the PPO extraction and assay 2 as set out in the experimental. This is due to the fact that after the first 3 weeks of plant material and assay material preparation, the PPO extraction and assay 1 did not seem to be giving any significant results although when the enzyme supernatant was added to the assay, a colour change was visible. The reasons for this will be explained in the discussion.

The standardisation procedure was carried out as set out in the materials and methods section. From the standardisation plot and the results of the standardisation assays run it was determined that the JA treated, wounded plants showed a higher concentration of PPO/g of tissue around the wound site at a standard reading of 2000. This was then followed by the wounded control plants which showed a higher concentration than the JA treated unwounded, the protein standard readings as 2000 and 1000 respectively. 1000 was also the reading for the unwounded control plant, although the actual reaction reading was lower than in the wounded control.

FIGURE 2a. Standard plot of the standardisation of protein concentrations. This shows the results of the mean of 3 assays for each concentration of BSA used. From this plot, the concentration of PPO protein in each enzyme assay can be calculated. The assays were spectrophotomically tested at a light absorbance of A595. Error bars show the standard error of the mean +/- 1.0

Figure 2b. This shows the results of the protein standardisation assay as a mean of the 3 values, then when extrapolated from the graph gives a close estimated value of the PPO concentrations in the assays which were run.

JAa – this is the JA treated plant, after 24 hours of induced wounding.

JAb – this is the JA treated plant which was left unwounded.

Cont.c – this is the control plant after 24 hours of induced wounding.

Cont. d – this is the control plant which was left unwounded.

Leaves of control and JA treated plants were mechanically wounded to simulate herbivore grazing and then after an induction period of 24 hours had PPO extraction and assay 2 performed and run on them, along with unwounded control and unwounded JA treated to act as a comparison. All of the plant materials, whether wounded or otherwise showed constitutive PPO activity. In Figure 3, the unwounded, control plant it can be seen that up to 5 minutes the reaction curve is a steady, sigmoidal curve and that the control unwounded reached the highest conversion of DOPA to quinone with a maximum absorbance rate of A490 0.526.

In the wounded control plant, Figure 4 , the rate of reaction and the actual total conversion of DOPA to quinone is less than half that of the unwounded control plant, peaking at A490 0.242

The unwounded, JA treated plant, Figure 5, showed a higher conversion rate and total conversion amount of DOPA to quinone than the wounded control plant, but less than that of the JA treated plant, maximum A490 of 0.339 in the unwounded treated and A490 of 0.381 in the wounded.

The wounded, JA treated plant, Figure 6 which had a standard protein reading of 2000, although showing a erratic rate of reaction on the plot, of DOPA to quinone polymers.

Figure 3. A plot of PPO activity in an unwounded, control plant.

The concentration of PPO in the assay was around 1000 when extrapolated from the data in Figure 2a and 2b . The data used was the mean of 3 assays over 5 minutes. The assays were spectrophotomically tested at a light absorbance of A490. Error bars indicate the SE +/-1.0 The rate of DOPA to quinone polymers was calculated as ∆A/min=0.100 with a correlation coefficient of 0.953

Figure 4. A plot of PPO activity in a wounded, control plant.

The concentration of PPO in the assay was around when extrapolated from the data in Figure 2a and 2b . The data used was the mean of 3 assays over 5 minutes. The assays were spectrophotomically tested at a light absorbance of A490. Error bars indicate the SE +/-1.0 The rate of DOPA to quinone polymers was calculated as ∆A/min=0.051 with a correlation coefficient of 0.963

Figure 5. A plot of PPO activity in an unwounded, JA treated plant.

The concentration of PPO in the assay was around 1000 when extrapolated from the data in Figures 2a and 2b . The data used was the mean of 3 assays over 5 minutes. The assays were spectrophotomically tested at a light absorbance of A490 Error bars indicate the SE +/-1.0 The rate of DOPA to quinone polymers was calculated as ∆A/min=0.064 with a correlation coefficient of 0.938

Figure 6. A plot of PPO activity in a wounded, JA treated plant after 24 hours. The concentration of PPO in the assay was around 1500 when extrapolated from the data in Figures 2a and 2b. The data used was the mean of 3 assays over 5 minutes. The assays were spectrophotomically tested at a light absorbance of A490 Error bars indicate the SE +/-1.0 The rate of DOPA to quinone polymers was calculated as ∆A/min=0.82 with a correlation coefficient of 0.952

IV. Discussion

A selection of tomato seeds were treated with JA and the plants which germinated from these seeds were tested for induction of foliar PPO and their relative concentrations to control plants by mechanical wounding. All of the plants which were assayed showed PPO activity, including the unwounded, control plants. PPOs found in plants have broad substrate specificities and therefore the reaction rates in some assays would understandably vary depending on what substrate the rate of reaction was being measured using.

In the results section, it was mentioned that the first series of assays which were carried out did not yield results which were statistically viable for any plots or comparisons. As said before, different plant PPOs have different substrates which the react best with. As the first assay which was used was based on that of the conversion of catechol to orthoquinone via PPO as a catalyst. This assay was modified from an assay which was first used on potato tubers. There is a strong possibility that the reason the first assays which were run gave no results which any conclusion could be drawn from because of the different plant materials that were used. The potato tuber cells which were used would from the tomato leaf material used, such as containing chloroplasts which are required for JA biosynthesis. Although, tomato and potato plant defences have been shown to be similar, the PPOs which are present in the tubers and those which are present in tomato foliage would have different substrates and therefore the assay which was designed initially for potato tuber PPO levels to be measured would not work with the tomato substrate assay. This conclusion is partially backed up by the research of Ryan and Constable (1998) which shows a forty fold increase in PPO levels between wounded potato tubers and those of tomatoes when run under a similar assay as was run to gain results which could be used for this study.

When analyzing the results, one of the major anomalies was the fact that the unwounded control plant had a higher PPO reaction rate, a high protein content and also had the highest total conversion of DOPA to quinone recorded. Once this issue was resolved and a theory had been established to explain why the values were so high then it was easier to explain the results of the JA treated plants. In plants, an inducement of a wound response does not necessarily mean that the plant has been wounded. In recent years it has been discovered that MeJA and JA are not only released under wounding situations, but that plants respond to many environmental stresses in the same way. Under drought stress for example, a plant will release JA and also abscisic (ABA), which is another plant signaling hormone. ABA is released in order to induce the closure of stomatal pores to reduce water loss via transpiration. It has also been put forward that JA is released under drought conditions (Thaler and Bostock, 2004), in order to have a stronger defence against any insect wounding that may occur whilst the plant is in its weakened state. Under the growing conditions in the greenshouses, some of the plants became dehydrated, but mostly during the early assays these were not used. However, if a dehydrated unwounded plant had been selected then this could account for both the high protein level recorded and the high PPO reaction rate. If ABA and JA were both present in the samples as a drought response, then the protein standardasition assay would have shown a higher protein level than in the other assays, and as JA is released under drought, then PPO levels would be higher, therefore explaining the huge increase in DOPA conversion compared to wounded and treated plants.

With the exception of the anolomous result for the unwounded control, the three other plants gave positive results. Although no direct comparison could be made between the wounded and unwounded controls, the fact that the JA treated wounded and the JA treated unwounded plants both gave higher rates of DOPA conversion, ∆A/min=0.64 and ∆A/min0.82 respectively, compared to ∆A/min=0.51 in the wounded control, gives some weight to the theory that induced resistance was passed on through the vegetative and reproductive stages of the plants life, with the JA treatment activating the induced resistance in the seedlings. Although there isn’t any major significant difference in the wounded and unwounded JA treated rates, there is a difference, which could be expanded on.

The proposal for this study is available in the appendices and is noticeably different to the experiments carried out. Although the study has stuck to the basic outline of the proposal, changes had to be made due to unforeseen difficulties. Firstly, the actual growing of the plants took at first, a minimum waiting time of two-three weeks. When the plants were ready to be wounded and then have the assays run, it was noticed that the method being used was not compatible with the tomato plants and so a new experimental had to be drawn up, leading to the writing of PPO extraction and Assay 2. At this point, it was realized that there was not enough time left in the laboratory schedule to allow the re-growing and then repeating of all the experiments, and so it was decided to just examine the main effects of the treatment, to see if it increased PPO levels in wounded and unwounded plants. To improve the study, assays to determine the concentrations of PPO expressed at different times after wounding in same age plants, ie after 0, 2, 4, 6 hours would be run as would assays to determine the concentration of PPO expressed at same time on different age plants, eg 4 hours after wounding on a 1, 2, 3 week old plant up to 5 weeks and finally a set of assays to determine the concentration of PPO expressed on different age plants at different times after wounding. Also, by increasing the dosage of JA, this might show an increase in induced resistance.

In conclusion to this study, it would be recommended that more time is given to researching the possibility of JA seed treatment in crop plants, as although the findings of this study were not statistically significant, they do show some evidence that JA has a positive effect of PPO levels and induced wound resistance and along with other studies such as those by Thaler (1997) and Shailasree (2001), gives weight to the use of naturally induced resistance via seeds rather thatn the use of chemical pesticides.

V. Acknowledgements

I would like to thank Dr Mike Roberts for his time and patience in the laboratory and Dr Nigel Paul for the loan of equipment.

V!. References

Bowles, D. (1998) Signal transduction in the wound response of tomato plants. Phil. Trans. R. Soc. Lond. B 353:1495-1510

Creelman, R. A. and Mullet J. E. (1997) Biosynthesis and action of jasmonates in plants. Annual Review of Plant Molecular Biology 48:355-381

de Bruxelles, G. L., and Roberts, M. R. (2001) Signals Regulating Multiple Responses To Wounding and Herbivores. Critical Reviews in Plant Science 20(5):487-521

Devoto, A. and Turner J., G. (2003) Regulation of jasmonate-mediated plant responses in Arabidopsis. Annals of Botany 92:329-337

Howe, G. A. and Ryan C.A. (1999) Suppressors of systemin signaling identify genes in the tomato wound response pathway. Genetics Society Of America. 153:1411-1421

Krumm, T., Bandemer, K. and Boland, W. (1995) Induction of volatile biosynthesis in the Lima bean (Phaseolus lunatus) by leucine and isoleucine conjugates of 1-oxo and 1-hydroxyindan-4-carboxylic acid: Evidence for amino acid conjugates of jasmonic acid as intermediates in the ocadecanoid signaling pathway. FEBS Lett. 377:523-529

Ryan, C. A. (1990) Proteinase inhibitors in plants: genes for improving defences against insects and pathogens. Annual Review of Phytopathology

28:425-449

Shailasree, S., Sarosh, B. R., Vasanthi, N. S. and Shetty, H. S. (2001) Seed treatment with -aminobutyric acid protects Pennisetum glaucum systemically from Sclerospora graminicola Pest Management Science 57:721-728

Sherman, T. D., Vaught, K. C. and Duke, S. O., (1991) Polyphenol Oxidase Phylogeny. Phytochemistry 30:2499-2505

Thaler, J. S. (1999) Induced resistance in agricultural crops: Effects of Jasmonic acid on herbivory and yield in tomato plants. Environmental Entomology 28:30-37

Thaler, J. S. and Bostock, R. M. (2004) Interactions between abscisic-acid-mediated responses and plant resistance to pathogens and insects. Ecology 85:48-58

Wasternack, C., Ortel, B., Miersch, O., Kramell, R., Beale, M., Greulich, F., Feussner, L., Hause, B., Krumm, T., Boland, W., and Parthier, B. (1998) Diversity in octadecanoid-induced gene expression of tomato. Journal of Plant Physiology. 152:345-352

VI Appendices

Plant Raw Data:

Control, Unwounded Plant Material

1 g of plant material per 5ml of cold buffer (0-4oc) 0.1M Na cacodylate buffer, pH 7.2 containing 0.1% SDS

Control, Wounded (24 Hours) Plant Material

1 g of plant material per 5ml of cold buffer (0-4oc) 0.1M Na cacodylate buffer, pH 7.2 containing 0.1% SDS

JA Treated, Unwounded Plant Material

1 g of plant material per 5ml of cold buffer (0-4oc) 0.1M Na cacodylate buffer, pH 7.2 containing 0.1% SDS

JA Treated, Wounded (24 Hours) Plant Material

1 g of plant material per 5ml of cold buffer (0-4oc) 0.1M Na cacodylate buffer, pH 7.2 containing 0.1% SDS

Octadecanoid Pathway Enzyme Abbreviations

Allene oxide cyclase (AOC) Allene oxide synthase (AOS) Phospholipase (PLD) Jasmonic acid carboxyl methyltransferase (JMT) Lipoxygenase (LOX) OPDA reductase3 (OPR3)

(iii) Initial Project Proposal

Aims and Objectives

The aim of the project is to determine whether the treatment of tomato seeds by jasmonic acid (JA) induces a greater and/or earlier response of polyphenol oxidase (PPO) when the plant is wounded than would be seen in an untreated wild type plant, and how the treatment of the tomato seeds alters the mechanisms of this response. Polyphenol oxidase is one of the major anti-herbivore proteins induced in the tomato plant and is induced by methyl jasmonate (MeJA) (Constable C.P. and Ryan C.A. (1996) A Survey Of Wound- And Methyl Jasmonate-Induced Leaf Polyphenol Oxidase In Crop Plants Phytochemistry 47: 507-511) and previous studies have shown that by spraying mature crop tomato plants with JA then a greater resistance to insect grazing is shown and that by treating the seeds the same effect is produced (Thaller J.S. (1999) Induced Resistance in Agricultural Crops: Effects of Jasmonic

Herbivory and Yield in Tomato Plants Environ. Entomol. 28(1): 30-37).

This study is to determine the degree of resistance increase if it actually occurs.

Methodology

The experiments to be undertaken are as follows:

Concentrations of PPO expressed at different times after wounding in same age plants, ie after 0, 2, 4, 6 hours.
Concentrations of PPO expressed at same time on different age plants, eg 4 hours after wounding on a 1, 2, 3 week old plant up to 5 weeks.
Concentrations of PPO expressed on different age plants at different times after wounding.
Also, how the concentrations of PPO expresses varies between treated and untreated plants of same age, same times after wounding.

The reaction to be quantified is that of catechol orthoquinone, catalysed by PPO.

To gain the PPO extract, the wound response will be induced on the tomato plants leaves.
The leaf will be removed after a set time of 0, 2, 4, 6 hours.
The leaf will be ground down using a mortar and pestle and liquid nitrogen in order to for a powder which contains intact proteins.
The leaf power will be mixed with 33mM, pH6.0 potassium phosphate buffer up to 5ml for every 1g of leaf used in order to be centrifuged at 4oC for 15 minutes to produce a supernatant containing the PPO enzyme.
This extract is to be kept on ice at all times.

To determine the concentration of PPO the following procedure will be used:

6 cuvettes to have 100μl of catechol added to them.
Each cuvette to have 650,700,750,800,850,900μl of potassium phosphate buffer added to each respectively,

Each solution is to be made up to 1ml, therefore 250,200,150,100 and 50μl are to again be added respectively to the cuvettes, the remaining cuvette to be the control of buffer and catechol.
The absorbance of each cuvette will be measured at 540nm every 30 secs for 5 minutes.

The experiment will be repeated for each of the treatments and at different ages and times after wounding.