I. Introduction The aim of the project was to study the effects of jasmonic acid, one of the plant hormones involved in induced plant responses, when applied to tomato seeds before they are sown with a view to the effects initiating a positive effect and induced responses being ‘switched’ on constantly.
Over the years, studies have shown that many plants, both wild and agricultural have the ability induce responses to herbivore damage, induced damages being those which are demonstrated after a herbivore attack has taken place (Constable et al., 1996).
In many plants, the responses are regulated in time and space by a highly complex regulatory networks, (M. R. Roberts et al., 2001) which in turn are modulated by interactions with other signalling pathways. The key signalling hormones which will be discussed are jasmonic acid (JA), ethylene, asbscic acid (ABA) and salicyclic acid (SA) along with the roles which ion fluxes, protein phosphorylation cascades and active oxygen species play in the inducement of defence responses against herbivore wounding. The responses both in individual leaves and systemically, over the unwounded areas of the plants will also be discussed.
II. Response Overview When herbivore damage is sustained, the immediate surrounding areas around the wound will consist of different populations of cells. The wound site will consist of damaged cells which are beyond repair but which may also provide a key role in the initiation of an induced response as they will release signalling molecules which can act as elicitors of responses in the neighbouring intact cells or will act as defensive toxins which can cause damage or kill the attacking organism.
The cells surrounding the wound site, although not being directly damaged, will be under considerable stress as they will be acting as a barrier to prevent plant pathogens such as Agrobacterium tumefaciens, which causes crown call disease on the stems of plants and trees, to enter the wound site. The barriers are formed via the process of cell wall strengthening which is partly achieved by the rapid linking of existing cell wall proteins (Bradley et al., 1992), then after the initial response, by the surrounding cells, the wound site is sealed by a calcium-induced callose synthesis process, which blocks the plasmodesmata, preventing the movement of molecules between neighbouring cells.
Herbivore damage also causes the inducement of chemical defences against herbivores, not only in the wounded area of the plant but systematically, in other, unwounded areas of the plant. These induced chemical responses include alkaloids and phenolics. Also included are defensive proteins such as protease inhibitors and PPO which prevent the digestion of plant material in the herbivores gut. Other proteins which deter insect herbivores are amylase inhibitors and lectins.
Figure 1. (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.
III. Discussion In 1990, Farmer and Ryan identified jasmonic acid as a potential wound signalling molecule, after identifying methyl jasmonate (MeJA) as a very strong inducer molecule of proteinase inhibitor (Pin) genes in tomato plants.
In 1996, Constable and Ryan observed the inducement of PPO via wound inducement and via methyl jasmonate. They ran a series of assays in several species of plant to determine which plants had the highest PPO activity when left as a control and when mechanically or chemically induced. Their studies showed that certain families of plants exhibit different PPO levels, the Poaceae, Brassicaceae, and Fabaceae, (with the exception of Glycine max) families all having low PPO activity, even when mechanically induced or MeJA induced. The Solanaceae family, which potato and tomatoes belong to, and the Salicaceae family both exhibited relatively high PPO activity when compared to the other families studied.
Howe and Ryan, 1999 ran a series of systemin suppressor experiments in order to identify the genes involved in the tomato wound response pathway. Systemin, an 18-amino-acid peptide regulates the defence gene response to herbivory or mechanical wounding. They discovered that transgenic plants overexpress the systemin precursor prosystemin and from the transgene 35S::prosys, express wound induced defence proteins including PPO and proteinase inhibitors. They studied the role of prosystemin in the plants wound response pathway by isolating and characterising mutations that suppress 35S::prosys-mediated phenotypes, ie those which express higher levels of PPO and proteinase inhibitors than normal tomato plants under the same conditions.
They identified ten recessive, extragenic supressors (those which were extra to a normal plants genetic makeup), two of which defined new alleles of the previously identified def-1 gene which is a mutation that blocks wound induced and systemic resistance which then leaves the plant without defences and susceptible to herbivore grazing. The remaining eight mutants defined four loci, two of which were insensitive to systemin and lacked systemic wound response. The results confirmed the theory that prosystemin and systemin are both functional in the transduction of systemic induced resistance in the tomato plants and that wound expression is induced in 3 genes, Def-1, Spr-1 and Spr-2 (those insensitive to systemin) by the actions if wounding, systemin and 35S::prosys.
Thaler in 1999 investigated the effects of JA as a natural elicitor to induce resistance to herbivores. Here, she applied jasmonic acid to the foliar regions of the plant and found that the application of JA increased levels of polyphenol oxidase. The plants which were induced received 60% less leaf damage than the control plants. Thaler also observed that although the plants which were treated produced less flowers, the fruit yield from the crop between the treatments. She also was that there was no difference between the yields of the induced plants and of the control plants under both natural control conditions and under reduced herbivore levels. Thaler concluded that the fact that there were no differences in yield was due to the low levels of herbivory experienced by the plants and that JA treatment produces a natural herbivore resistance at no cost to the tomato or its yield and this treatment could be of high value in agriculture, reducing the use of man-made pesticides, providing a cheap, natural alternative.
Figure 2. This figure, taken from Thaler, (1999) shows the amount of damage received to control and jasmonic acid treated plants. This shows that the treated plants had a greater resistance than the control plants.
In 2000, Bauke et al., studied the selective inducement of responses in Arabidopsis. They suggest that in Arabidopsis, a specialist insect herbivore causes the inducement of certain wound response pathways and this was shown by the fact that when certain insects grazed on the test plants, depending on the insect, various genes were activated. For example, JA was induced by the feeding of the diamondback moth and regulated the -glucosidase (BGL 1) gene, where as ethylene was induced by the Egyptian cotton worm, which regulated the control of a purative calcium-binding elongation hand protein, used in wound responses (Figure 4.).
The study also showed that the insect herbivores are selective in which induced resistance affects them. When the plants were treated with JA, they showed induced resistance to the diamondback moth, with the amount of leaf material lost reduced after treatment. When a different set of plants were treated with ethepton, a molecule close to ethylene, the grazing of the Egyptian cotton worm was reduced significantly as shown in Figure 3, however, that of the diamondback moth was unaffected.
Figure 3. (Bauke A. et al., 2000) This study showed the effect of ethylene treatment on Arabidopsis. The plants are numbered 0-6, representing the leaf area lost to herbivores. The arrows indicate the leaf areas grazed upon. Those treated with ethepton showed a greater resistance to the egyptain cotton worm but not to the diamondback moth.
This shows the inducement of defensive genes in Arabidopsis when treated with ethephon. And when treated with methyl jasmonate. Jasmonic acid is shown to induce a wider range of defensive genes than ethephon.
The inducement of systemic resistance through seed treatment with an inducer was suggested by Shailasree et al., in 2001. In this paper, they go on to describe the effects of the treatment of pearl millet seeds with -aminobutyric acid (BABA) in order to induce resistance to downy mildew disease which is caused by the oomycetous fungas Sclerospora graminicola. Their treatments of seeds undergoing 6 hour soakings in a 50mM BABA solution resulted in a 23% disease incidence in the treated seeds, whereas in the untreated, control seeds the disease incidence was 98%, showing that treatment of seeds gave 75% protection against disease.
Following this, Shailasree et al., allowed the seeds to germinate and gave a secondary does of downy mildew disease to see if induced resistance has been stimulated in the new seedlings. The response of the plants was that they showed a disease incidence of only 10 and 12% respectively, depending on where the disease had been introduced, either at tillers or inflorescence axes. Compared to the 71 and 76% disease incidence in the set of control plants, suggested that the resistance which was induced in the seeds by BABA treatment remained operative during the vegetative and reproductive growth cycles.
Figure 5. (Shailasree et al., 2001) Shows the incidence of downy mildew disease in pearl millet plants after seed treatment with BABA as an inducer of systemic resistance.
The studies of Mathieu et al., 1991, Felix and Boller, 1995 and Thain et al., 1990 have shown that one of the most rapid responses following wounding of a plant or the application of elicitors which induce responses are a series of ion fluxes in the plasma membrane and data suggests that these fluxes play a part in the activation of plant defence genes. The studies have shown that elicitors and wounding cause a rapid depolarisation in the plasma membranes electrical potential, driven by an efflux of K+, an influx of protons and the alkalinisation of the extracellular medium surrounding the wound site. When chemical agents which disrupt the fluxes are applied to the cells, a disruption of defence gene expression can be seen.
An example by Messiaen in 1999 showed that when fusicoccin, a fungal toxin was applied, H+-ATPase was activated in the plasma membrane and the membrane becomes hyperpolarised, inhibiting the expression of the gene which expresses systemic wound inducement and glycan synthesis. In 1999 it was also shown by Schaller and Oecking that some wound response genes were activated with the application of, H+-ATPase inhibitors when there is no other wound/elicitor induced stimulus present. Calcium ions also play a casual role in the wound signalling pathway. Knight et al., 1993 gave evidence to show that following wounding there is a rapid increase in cytoplasmic Ca2+ concentrations, and when calcium channel blockers are applied to a wound site, and molecules which cause the mobilisation of intracellular Ca2+ stores, elicitor induced gene expression has been seen (Leon et al., 1998)
The induced defence mechanisms of a plant are complex, with many signalling pathways and molecules involved in the inducement of defence genes, including various ions, plant hormones and even mutant genes. But most notably the effects of jasmonic acid when applied both externally or induced internally must be taken into account, as this, most studies show is the mainstay of induced plant defence mechanisms.