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INDUCTION OF PLANT ALLERGENS BY ENVIRONMENTAL AGENTS
Impact/Purpose:
The overall goal of this proposal is to describe an emerging field of knowledge and show how it may be applied to an environmental problem in a unique way. The problem that will be investigated is the increasing prevalence of allergic disease, including asthma, which has occurred over the last two decades in developed countries. The innovative concept that forms the basis for this project is that changes in environmental conditions can indirectly influence allergic diseases by inducing plants to increase their production of allergens. Thus, the hypothesis that will be tested in this project is that anthropogenic changes in the local environment of plants can enhance their production of some allergenic proteins. These changes in plants may potentiate human sensitization and allergic manifestations. The expression of one of the major allergens, Jun a 3, by mountain cedar trees will be the model system for this investigation. The three specific objectives are to: 1) identify up to three environmental exposures that induce Jun a 3 expression in mountain cedar seedlings and/or pollen in the lab; 2) compare the production of Jun a 3 by trees exposed to higher and lower levels of pollutants in their natural environment; and 3) correlate the production of Jun a 3 in leaves with that in pollen.
Description:
The specific objectives of the project and the progress toward meeting these objectives are listed below:
Identify up to Three Environmental Exposures That Induce the Production of an Allergenic Protein in the Mountain Cedar Tree by Examining the Effects of Exposures on the Expression of the Pathogenesis-Related (PR) Protein Allergen Jun a 3 in the Leaves of Seedlings
Two sets of experiments were performed (Midoro-Horiuti, et al., 1999a; Midoro-Horiuti, et al., 1999b). For the first set, 200 mountain cedar (Juniperus ashei; seedlings were purchased from a small nursery in Mineral Wells, Texas. These were raised from seeds from different trees, so they were not genetically identical. The seedlings were transferred into individual pots and grown in a private greenhouse with individual drip irrigation until they were used for experiments. Some of these plants were used to generate the preliminary data showing the feasibility for this project. Because our original (and only identifiable) supplier of mountain cedar seedlings discontinued this product, all of the subsequent studies were performed on Chinese junipers (Juniperus chinensis, Cupressaceae family) purchased from a large commercial supplier of seedlings for ornamental shrubs and trees. Consistent with the close phylogenetic relationship between J. ashei and J. chinensis, the degree of amino acid identity between these two species was very high (98%) for Jun a 3 and its homologue Jun c 3. In addition, many variants of J. chinensis are used extensively as ornamental shrubs, so this species is a potential cause of allergic sensitization and reactions in many geographic regions.
Chinese junipers are propagated from cuttings and thus are clonal. Despite this genetic similarity, we found that the basal level of expression of Jun c 3 mRNA (Midoro-Horiuti, et al., 2000), in the leaves of the J. chinensis seedlings was several orders of magnitude lower than that of Jun a 3 in mountain cedar seedlings. The difference required a change in the method for quantifying mRNA for Jun c 3 and the house-keeping gene elongation factor alpha (EF1α). Both species of trees were approximately 1 year old at the time of study.
Salicylic Acid Stimulation of Jun c 3 mRNA Expression
The ability of these cedar trees to mount a stress response was demonstrated by exposing them, via their roots, to a 5 mM solution of salicylic acid (Sigma) in water. Salicylic acid is used as a rubber softener and is a potential environmental pollutant. Salicylic acid is also a medicinal karatolyic agent, and small quantities of salicylic acid are used as a food preservative and as an antiseptic in toothpaste. Its only known toxicity is related to ingestion of large quantities. However, salicylic acid has also been recognized to be one of the final intracellular messengers through which environmental stress induces PR-1, 3 and 5 protein expression in plants (Surplus, et al., 1998; Chao, et al., 1999; Hanninen, et al., 1999; Regalado, et al., 2000). Thus, the relatively low direct toxicity of this agent for humans is in interesting contrast with its potential to enhance the expression of PR proteins in plants, some of which (e.g., Jun a 1) may be highly allergenic. Thus its indirect toxicity, through modifying plant gene expression, is potentially much greater than its direct toxic effects on humans or other animals.
Samples were collected before and 1, 2, and 5 days after transiently exposing the roots of the plants to salicylic acid. Total RNA was extracted from the leaves using a hot-borate method (Wilkins and Smart, 1996), and first strand cDNA synthesis was performed with reverse transcriptase. Real-time polymerase chain reactions (PCRs) were performed with primers for Jun c 3 (sense: 5’-CAACTGAGCTGCACAGTCTCC-3’; antisense: 5’-GGGTTGATGGCAAGAGGAATGT-3’) and a housekeeping gene, EF1α, sense: 5’-GGATCTCAAGAGAGGATATGTGGC-3’; antisense: 5’-CTGGTGCATACCCATTTCCAATTT-3’) was analyzed at the same time to normalize for the quantities of mRNA in each assay and the amplification efficiency in each sample. The fluorescence intensity was monitored for Jun c 3 and EF1α. The results (Figure 1) are expressed as the fold changes of the ratio of Jun c 3 to EF1α. Given the very low basal expression of Jun c 3, expression in the leaves increased approximately 2,000 fold by 5 days after salicylic acid stimulation. However, simply transiently submerging the soil of the plants in water (control group) increased the Jun c 3 expression approximately 800 fold after 5 days, relative to the pretreatment values for the same plants.
Figure 1. The Effect of 5 mM Salicylic Acid on the Expression of Jun c 3 mRNA was Analyzed by Real-Time PCR. The mean values of five trees are shown as the mean +/- standard deviation (SD) of the fold change of Jun c 3: EF1α ratio from those from before stimulation.
Despite this large fold increase, the amount of Jun c 3 expression in the salicylic acid-treated plants was not significantly (via multiple measures test) greater than that in the controls. The lack of statistical significance is most likely due to the wide variation in the basal as well as stimulated levels of mRNA for Jun c 3 between individual trees, presumably due to multiple environmental conditions of the plant that we could not identify and thus controlled .
In order to demonstrate that the observed changes in Jun c 3 mRNA can be associated with changes in protein expression, we extracted protein from the leaves of the seedlings (Karenlampi, et al., 1994) that were harvested before and after salicylic acid exposure. The Jun c 3 protein in the extracts was assessed by Western blotting, using anti-MBP-rJun a 3 rabbit IgG (Togawa, et al., 2006) and HRP-anti-rabbit IgG (Zymed). The results are shown in Figure 2 below. Of note is the difference between the Jun c 3 protein responses in two different trees.
Figure 2. Western Blotting Analysis of Jun c 3. Jun c 3 protein after salicylic acid treatment in leaves was analyzed using anti-Jun a 3 polyclonal antibody.
Effects of Ultraviolet C Stimulation on Jun c 3 mRNA
Chinese juniper seedlings were exposed to ultraviolet (UV) light in the C wavelengths (Friedberg, et al., 1995; Ullrich, 1999). Leaves were collected before and after the exposure. In these experiments, the trees were exposed to 250 mW/cm2 of UVC for varying times, up to 10 minutes.
Figure 3 shows the time course of the response in five individual trees to various doses of UVC light (0, 5, and 10 minutes of exposure). Despite the fact that these trees are clonal and were treated simultaneously, the shape of the individual responses still varied extensively. Three of the five trees in the treated group had responses greater than the sham treated trees, which were transported to the exposure facility but not exposed to UVC radiation (UVC 0 min). Figure 4 shows the same data expressed as the mean and standard deviation of the fold increase compared to before irradiation.
Figure 3. Time Course of Jun c 3/EF1α Expression in Individual Trees After UVC Irradiation. J. chinensis seedlings were irradiated at 250 μW/cm2 for 0, 5, and 10 minutes. Leaves were collected from each seedling. mRNA expression was analyzed by real time-PCR.
Figure 4. The Effects of Time Post Exposure to UVC on the Expression of Jun c 3 mRNA in Five Irradiated Seedlings are Summarized. Values are expressed as the fold change in the Jun c 3: EF1α, relative to pre-irradiation values. Mean ± SD values are shown.
These results suggest an increase in the Jun c 3 mRNA expression in three of five plants in each group and not in the other two plants in each group. By 10 days after the irradiation, there was approximately a 10,000 fold increase in the Jun c 3 mRNA in leaves of trees exposed for 5 minutes and a 100,000 fold increase in those exposed for 10 minutes. The sham exposed trees demonstrated up to a 77 fold increase. The housekeeping gene EF1α varied by only about 6 fold between individual trees, indicating that the changes in Jun c 3 mRNA expression is not due to a generalized enhancement in gene expression in these plants. Using a repeated measure statistic, there was no statistical difference in the Jun c 3 mRNA between the groups of plants exposed to different amounts of UVC. Increasing the group sizes might have resulted in statistically significant differences between the groups. However, it is clear that within the treated groups, individual trees could be characterized as “responders” or “non-responders.” When only the responders (three of five trees treated for 5 or 10 minutes) were considered, they expressed a statistically significant increase in Jun c 3 mRNA compared to the sham treated groups.
The third environmental agent we tested is harpin (Dong, et al., 1999). This peptide, produced by Pseudomonas syringae is currently being used as a pesticide on agricultural crops. However, its mode of action is indirect, as it is known to induce some PR proteins in plants and thereby enhance their resistance to microbial pathogens.
The leaves of the cedar seedling were sprayed with a solution of the commercial product, supplied by the manufacturer (Eden Bioscience, Bothell, WA). In order to develop a dose response curve, we used several dilutions of the harpin-containing solution, with the manufacturer recommended dose being indicated by 1x. The results are shown in Figure 5 below.
Figure 5. Jun c 3 Expression After Harpin Treatment. J. chinensis seedlings were treated with 0, 0.5x, 1x, or 5x harpin. Leaf samples were collected 0, 4, and 7 days after treatment. Jun c 3 mRNA expression was analyzed by RT-PCR. The results are expressed as mean ± SD of the fold increase after treatment.
The results of harpin exposure indicate that there was not a significant change in Jun c 3 mRNA after the cedar seedlings were exposed to harpin. This finding is consistent with the previous reports that harpin is an inducer of proteins of the PR-1, 2, and 10 groups (Tampakaki and Panopoulos 2000; Kariola, et al., 2003). We know of no previous evidence concerning its effects on PR-5 protein expression.
Taken together, the results from the three different exposures suggest that the expression of Jun c 3 mRNA in Chinese juniper seedlings has a very low basal rate, but is exquisitely sensitive to certain (e.g., UVC) environmental conditions. Some of these conditions were controlled in our experiments, while other, unidentified conditions were confounding variables in our experiments. These findings may be significant in several possible realms. The expression of plant proteins, such as Jun c 3, which are potentially allergenic, may be modified by the environmental conditions to which plants are commonly exposed. More specifically, since male Chinese junipers are used extensively in landscaping, they grow in close proximity to human populations. Thus, allergen expression in their pollen or other plant parts may lead to sensitization, and on subsequent exposure, allergic symptoms. Another possible ramification of recognizing the exquisite sensitivity of the PR protein responses is that these may provide the scientific underpinning of systems that can be used to continuously monitor our environment for conditions that will have more direct adverse effects on humans or other animals.
Examining the Effects of Exposing Pollen Grains to Gaseous Pollutants and UV Radiation on Their Expression of Jun a 3
Based on our preliminary results of exposing trees to the gaseous pollutant ozone, we learned that gene expression in plant materials, particularly leaves, was susceptible to the high air velocity in our environmental exposure chambers (desiccation). Thus, we discontinue our attempts to use these units, designed for animal exposure, for tree exposure. Future studies with gaseous pollutants will require the design and construction of special exposure chambers for plants.
Compare the Production of Jun a 3 by Trees Exposed to Higher and Lower Levels of Criteria Pollutants in Their Natural Environments
Samples of leaves, cones, and pollen were harvested from mountain cedar trees growing along the corridor between Austin and San Antonio, Texas, during the pollination season. Two groups of trees were selected based on their proximity to the major interstate highway (I-35), with the proximate trees within 50 m of the heavy traffic and the distant ones lying at least 1000 m from the throughway, and in most cases, upwind from the major mobile source of pollutants. mRNA prepared from these samples was subjected to RT-PCR and the cDNA separated on agarose gels. The resulting bands were stained with SYBR green and quantified by densitometry. The results are shown in Figure 6.
The number of samples analyzed to date has not allowed a meaningful assessment of this relationship. Other dual samples (i.e., leaves and pollen) were collected, but have not been analyzed to date.
Figure 6. Jun a 3 Expression from the Field. Leaves or corns of J. ashei trees were collected near or far from I-35. mRNA expression of Jun a 3/EF1α were analyzed by RT-PCR. Means ± SD are shown.
Correlate the Production of Jun a 3 in Leaves with That in Pollen, Under Field Conditions
The small number of samples for which analysis is complete precludes statistical analysis.
During the tenure of this grant, additional studies have been performed which are complementary to the studies described here. In these studies, we have expanded the geographic significance of our hypothesis concerning the role of environmental exposure of pollen-producing plants on human health by cloning and expressing additional PR-5 pollen proteins (Jun a 1 homologues) in cedar trees from both Japan and Southern Europe. Of particular interest, we found that a PR-5 protein of the Italian cypress trees (Cupressus sempervirens), while expressed in very low levels in pollen from a number of sources, is quite sensitizing. Up to 50 percent of the IgE antibodies produced against Italian cypress pollen were found to be directed against the PR-5 allergen Cup s 3 (Togawa, et al., 2006). This suggests that there is a wide variation in the level of expression of Cup s 3 (or its close homologue, Cup a 3, from the Arizona cypress), which is probably environmentally controlled. Following the finding of the Jun a 3 homologue, Cry j 3, in Japanese cedar pollen, our colleagues in Japan have begun to examine the role of environmental conditions on the expression of this allergen.