Effect of Jasmonic Acid on Biomass and Enzyme Activity in Switchgrass and Sorghum Jocelyn Bidlack and Jim Bidlack

1. Effect of Jasmonic Acid on Biomass and Enzyme Activity in Switchgrass and Sorghum Jocelyn Bidlack and Jim Bidlack Department of Biology, University of Central Oklahoma, Edmond, OK 73034 • Experimental design: The growth and treatment of the samples took place on the roof of Howell Hall at the University of Central Oklahoma. The experimental design consisted of four replications, each consisting of two species and four treatments arranged in a randomized block design (Figure 2). Borders, composed of untreated switchgrass and sorghum, were used to reduce the effect of differing environments on the plant growth. On June 01 2010, the pots were seeded with 2.582 g (3000 seeds) switchgrass or 0.668 g (25 seeds) of sorghum. The sorghum germinated on June 03, at which time ~10-15 seedlings emerged. The switchgrass germinated on June 06, at which time ~300 seedlings emerged. The seedlings were thinned on June 12; the switchgrass was thinned to 100 seedlings per pot and the sorghum was thinned to 10 plants per pot. On June 18th, the plants were fertilized with miracle grow 20:20:20 liquid fertilizer, and on June 28 and July 18 a granular 29-0-5 fertilizer was applied for a total N treatment of 238 kg/hectare, a total P2O5 treatment of 73 kg/hectare and a total K2O treatment of 113 kg/hectare. • Figure 2. Examining the experimental design. • Treatment: On July 12 the plants were treated with jasmonic acid (JA) (Figure 4). Neat JA was purchased from Sigma-Aldrich, 50 mg of JA was dissolved in 5 mL of methanol. From the concentrated solution, 0.0 mM, 0.5 mM, 1.5 mM and 5.0 mM treatment solutions were prepared. Triton X-100 was added to the solutions to act as a surfactant enabling absorption of the compound through the cellular membrane. Treatment consisted of 10 mL dosages of JA solutions applied via a spray bottle to the leaves and stems of the plants. Treatment resulted in the application of 0.0 mmol, 0.005 mmol, 0.015 mmol or 0.05 mmol of JA per pot respectively. • Harvest: On consecutive days between July 33 an July 33, the plants were harvested (Figure 3). The plants were cut at pot level and weighed immediately to determine fresh biomass. To obtain dry biomass, the plants were placed in a paper bag and dried at 45° C for 2 days and then weighed. Percent moisture was calculated from these results. • . • Figure 3. Harvest of replications 1 and 2. • Figure 4. JA treatments for replication 1 . Switchgrass: a. 0.0 mM, b. 0.5 mM, c. 1.5 mM, and d. 5.0 mM. Sorghum: e. 0.0 mM, f. 0.5 mM, g. 1.5 mM , and • h. 5.0 mM • Enzyme extraction: The basal 10 cm of plant material was removed from each sample and stored on ice for enzyme extraction. The samples were immediately homogenized in a 50 mM Tris buffer (pH 7.0) containing 0.1 M sucrose, 1% polyvinylpyrrilodone, 4 mM cysteine and 1 mM DTT. The resulting products were strained through 4 layers of cheese cloth and centrifuged (Figure 5) to obtain subcellular isolation of: 1) the microsomes containing hydroxymethyl-glutaryl CoA reductase and 2) cytosol containing the phenylalanine ammonia lyase . • Figure 5. Centrifugation products from Replication 1: a. switchgrass 0.0 mM, • b. sorghum 0.0 mM, c. switchgrass 0.5 mM, d. sorghum 0.5 mM, e. switchgrass 1.5 mM, f. sorghum 1.5 mM , g. switchgrass 5.0 mM, and h. sorghum 5.0 mM. • Enzyme Assay: A spectrophotometric assay (Figure 6) was conducted to determine activity of the PAL enzyme. Phenylalanine ammonia lyase catalyzes production of trans-cinnamic acid from phenylalanine; this reaction is spectrophotmetrically observed as an increase in the absorbance of light at 290 nm. Hydroxymethyl-glutaryl CoA reductase catalyzes oxidation of NADPH and reduction of hydroxymethyl-glutaryl CoA; this reaction is spectrophotometrically observed as a decrease in the absorbance of light at 340 nm. • Figure 6.Spectrophotometric procedure. Table 1. Analysis of variance for biomass and moisture. Sample FW DW % moisture Species ** ** ** Replication ** ** NS Error A NS NS NS JA ** NS NS JA x Species NS NS NS **Significant at p < 0.05 level; NS = not significant. Abstract Optimization of biomass yield in switchgrass (Panicum virgatum) and sorghum (Sorghum bicolor) is essential for efficient and economical production of biofuel. Four treatments of jasmonic acid (JA) (0.0 mM, 0.5 mM, 1.5 mM, and 5.0 mM) were applied to assess the effect of species, treatment, and the species x treatment interaction on biomass and the activities of enzymes involved in development of factors affecting pest resistance. Results indicated that fresh weight (FW), dry weight (DW) and percent moisture varied significantly among species. The 5.0 mM concentration of JA resulted in decreased FW and DW in switchgrass, and decreased FW in sorghum. Significant differences, as affected by species and the species x treatment interaction, suggest that species selection and JA treatment are worthwhile considerations when evaluating biomass yield of these species. Interestingly, the high JA treatment decreased production in both species, which may be an important factor concerning treatment concentrations in further studies. The lack of significant effects on biomass in the lower JA treatments provides indication of potential use of this growth regulator for pest resistance without decreased biomass. Additional experiments are being investigated on activities of phenylalanine ammonia lyase and hydroxymethyl-glutaryl CoA reductase to determine how JA may or may not be affecting enzymes affecting pest resistance. a b c d e f g h Introduction Research involving the production of biofuel has been spearheaded in recent years by the impending decline in fossil fuel availability and environmental concerns associated with fossil fuel production and use. The benefits of switchgrass (Panicum virgatum L.) and sorghum (Sorghum bicolor (L.) Moench) as potentially efficient and economical crops for the production of biofuel have been expounded in recent studies (Antonopoulou et al. 2008, Cassida et al. 2005). Optimization of these crops’ yield is essential for the eventual transition of biofuel production from experimental to commercial applications. Crop yield is significantly hindered by disease and pest infestation; however the spraying of fungicides and pesticides is expensive, requiring multiple treatments significantly contributing to the cost of biofuel production. In addition, fungicides and pesticides are corrosive chemical compounds that contribute to the degradation of the environment. As an alternative to these traditional remedies, the application of jasmonic acid has been implemented as an environmentally safe and effective method of endowing pest resistance to many commercial crops including corn, potato, and tomato (Cohen et al. 1993, Schmelz et al. 2003, Thaler 1999). Jasmonic acid is a naturally-occurring compound that acts as an elicitor in metabolic pathways leading to the production of defensive secondary compounds. The exogenous application of low concentrations of jasmonic acid effectively induces the production of these desirable defensive compounds (Mason and Mullet 1990); in the event of infestation, treatment may increase the yield of these valuable alternative crops. Further research is needed to determine the effect of exogenous JA on switchgrass and sorghum. The objectives of this experiment were to determine 1) the effect of species and jasmonic acid treatment on the biomass of switchgrass and sorghum, 2) the effect of species and jasmonic acid treatment on the activity of enzymes involved in the production of defensive secondary compounds and 3) what concentration(s) of jasmonic acid treatments significantly affect biomass within species. Results and Discussion Analysis of variance revealed significant differences in fresh weight (FW), dry weight (DW), and percent moisture as affected by species, and differences in FW as affected by the species x JA interaction (Table 1). The FW and DW of sorghum were significantly higher than FW and DW of switchgrass (Table 2). Differences in biomass were expected between species because of differences in annual and perennial growth habits. Switchgrass, a perennial, is unlikely to devote energy to extensive biomass accumulation for a single year, whereas sorghum, an annual, relies on biomass accumulation for competitive reproduction and survival within a single year. Replication had a significant effect on the FW and DW of the samples (Table 1). These differences were probably due to varying effects of shading by sorghum on bordering and medial replications. Replication 4 was discarded from analysis because of discrepancies in fertilization rates and differing environmental influences throughout the experiment. The JA treatment had a significant effect on fresh weight (FW) in both species according to the ANOVA (Table 1). Further analysis using LSD suggested that in switchgrass, the 5.0 mM treatment of JA resulted in significantly lower FW compared to the control and 0.5 mM treatments, and significantly lower DW compared to the control treatment (Table 3). The LSD analysis also suggested that in sorghum, the 5.0 mM JA treatment resulted in significantly lower FW compared to the control, 0.5 mM and 1.5 mM treatments (Table 3). The inhibitory effect of the high JA treatment is a valuable observation providing guidance concerning treatment concentrations in future studies. The 5.0 mM treatment consisted of the application of 5 mmol of JA to each pot regardless of growth habit, thus the moles per unit cm of plant material was much higher for the switchgrass compared to sorghum, causing the effect of the treatment to be exaggerated in the switchgrass . The enzyme activities of phenylalanine ammonia lyase and hydroxymethyl-glutarayl CoA reductase are currently being analyzed for relative differences in species and species x JA treatment. The experiment yielded important preliminary data for future investigation of JA’s effect on yield and pest resistance in these valuable crops. Table 2. Biomass and percent moisture of species. Species FW (g) DW (g) % moisture Switchgrass 296.3 b† 62.50 b 79.67 a Sorghum 1454 a 419.2 a 71.11 b †Means followed by the same letter within a column are not significantly different. Table 3. Biomass and moisture as affected by JA. [JA] FW DW % moisture --------------- Switchgrass ---------------- 0.0 mM 391.7 a† 90.00 a 81.97 a 0.5 mM 355.0 a 70.00 ab 79.87 a 1.5 mM 270.0 ab 56.67 ab 79.27 a 5.0 mM 168.3 b 33.33 b 77.56 a ---------------- Sorghum ------------------- 0.0 mM 1516 a 445.0 a 73.19 a 0.5 mM 1508 a 416.7 a 72.51 a 1.5 mM 1472 a 415.0 a 69.67 a 5.0 mM 1322 b 400.0 a 68.89 a †Means for each species within a column with the same letter are not significantly different. Acknowledgements We thank Charles MacKown of the USDA-ARS Grazinglands Research Laboratory for providing switchgrass seeds and for advice throughout the experiment. Funding was provided by the Experimental Program to Stimulate Competitive Research (EPSCoR) – Research Experience for Undergraduates (REU) program. • Materials and Methods • On 17 May 2010, switchgrass seeds were obtained from the USDA-ARS Grazinglands Research Laboratory in El Reno, Oklahoma. Sorghum seeds were purchased from Ross Seed Company in El Reno on the same day. • Germination study: To determine the germination rates of the two species, a preliminary germination study was conducted (Figure 1). Two Petri dishes were lined with moistened filter paper and 10 seeds of a given species were deposited on the dish and covered for three days. Four repetitions of the study were completed and the average germination rate for each species recorded for use in the seeding rate calculation. The average germination rates for switchgrass and sorghum were 10% and 80%, respectively. • Figure 1. Germination study. a b c d Literature Cited Antonopoulou, G., H.N. Gavala, I.V. Skiadas, K. Angelopoulos, and G. Lyberatos. 2008. Biofuels Generation from Sweet Sorghum: Fermentative Hydrogen Production and Anaerobic Digestion of the Remaining Biomass. Bioresource Technology 99: 110-119 Cassida K.A., J.D. Muir, M.A. Hussey, J.C. Read, B.C. Venuto, and W.R. Ocumpaugh. 2005. Biofuel Component Concentrations and Yields of Switchgrass in South Central U.S. Environments. Crop Science 45: 682-692 Cohen Y., U. Gisi, and T. Niderman. 1993. Local and Systematic Protection Against Phytophthora infestans Induced in Potato and Tomato Plants by Jasmonic Acid and Jasmonic Methyl Ester. Phytopathology 83: 1054-1062 Mason H.S., J. E. Mullet. 1990. Expression of two soybean vegetative storage protein genes during development and in response to water deficit wounding and Jasmonic acid. Plant Cell 6:569-579. Schmelz E.A., H.T. Alborn, and J.H. Tumlinson. 2003. Synergistic Interactions between Volicitin, Jasmonic Acid and Ethylene mediate Insect-induced Volatile Emission in Zea mays. Physiologia Plantarum 117: 403-412 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 e f g h