Figure 6. JA effect on PAL activity Values with the same letter are not significantly different...
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Figure 6. JA effect on PAL activity Values with the same letter are not significantly different Figure 5. JA effect on biomass accumulation Values with the same letter are not significantly different Enzyme extraction: The basal 10 cm of plant material was removed from each sample and put on ice for enzyme extraction. The fresh samples were immediately homogenized in a 50 mM Tris buffer (pH7.0) containing 0.1 M sucrose, 1% polyvinylpyrrilodone, 4 mM cysteine and 1 mM DTT. The resulting products were strained through 4 layers of cheese clothe and centrifuged at 5,000 g for 10 minutes enabling isolation of the microsomal fraction containing HMGR, the supernatant was removed and the pellet discarded. The supernatant was centrifuged at 10,000g for 10 minutes enabling subcellular isolation of the cytosol containing PAL. Figure 3. centrifugation products rep 1 a. switchgrass 0.0 mM microsomal fraction b. Switchgrass 0.0 mM cytosolic fraction c. switchgrass 0.5 mM microsomal fraction d. switchgrass 0.5 mM cytosolic fraction e. switchgrass 1.5 mM microsomal fraction f. switchgrass 1.5 mM cytosolic fraction g. switchgrass 5.0 mM microsomal fraction h. Switchgrass 5.0 mM cytosolic fraction. Effect of Jasmonic Acid on Biomass Accumulation and Enzyme Activity in Switchgrass Jocelyn Bidlack, Paul Olson, Carmen Cowo, Erin Ralstin, Robert Dinger, and Jim Bidlack University of Central Oklahoma, Edmond, OK 73034 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. ‘Alamo’ and ‘Kanlow’ cultivars of switchgrass (Panicum virgatum L.) are potentially efficient and economical crops for the production of biofuel (Cassida et al. 2005). However, under drought conditions, ‘Alamo’ shows a 49% decrease in biomass yield and ‘Kanlow’ shows a 27% decrease in biomass yield (Stroup et al. 2003). Current research conducted by our group is focused primarily on the optimization of bioenergy crop yield and increasing the cultivar’s physiological tolerance to drought thereby enabling expansion of the range of propagation to underutilized, semi-arid lands. To enable economically feasible production a simple and effective method of combating the negative effect of drought on these cultivars is necessary. Under conditions of osmotic stress, plants regulate stomatal closure to conserve moisture by limiting the transpiration flux. Modification of the stomatal opening concurrently limits the uptake of CO 2 , greatly affecting photosynthetic rate and leading to the increased production of reactive oxygen species (ROS) (Sgherri et al. 1993). The ROSs initiate a cascade of reactions, resulting in the destruction of organic macromolecules such as lipids, proteins, and nucleic acids (Shewfelt and Purvis 1995). Jasmonic acid (JA) is a stress-induced hormone that modulates the expression of antioxidative defensive systems. The exogenous application of JA prior to the initiation of osmotic stress has the potential to moderate the effects of drought (Li et al. 1998, Rohwer and Erwin 2008, Wang 1999). Treatment effectively induces biosynthesis and accumulation of the ROS- scavenging antioxidants (Gao et al. 2004), and induces increased activity of the ROS-scavenging enzymes. JA application may alter the production of secondary compounds with antioxidant properties indicated by significantly affecting the activity of hydroxymethyl glutaryl CoA reductase-HMGR (the rate limiting enzyme for the production of terpenoids). In addition, JA application is expected to alter the activity of phenylalanine ammonia lyase (PAL), the rate limiting enzyme for the production of the phenolic compounds such as the cell wall component-lignin. Pretreatment with exogenous JA may provide a mechanism of inducing ROS- scavenging prior to the damaging effect of peroxidation, thereby protecting the photosynthetic mechanism and membranes of the cell and moderating the damaging effects of drought. This preliminary experiment was conducted under standard osmotic conditions with a simplified objective: to determine the concentration of jasmonic acid that most effectively instigates significant affects on biomass accumulation and PAL and HMGR enzyme activity in switchgrass. Data from the experiment were utilized to formulate an appropriate procedure for the application of jasmonic acid in switchgrass. In addition, methods of Literature Cited 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 Sci. 45:682-692. Gao, X., X. Wang, Y. Lu, L. Zhang, Y. Shen, Z. Liang, and D. Zhang. 2004. Jasmonic acid is involved in the water- stressed-induced betaine accumulation in pear leaves. Plant Cell Environ. 27:497–507. Sgherri, C., C. Pinzino, and F. Navari-Izzo. 1993. Chemical changes and O 2 · − production in thylakoid membranes under water stress. Physiol. Plant. 87:211–216. Shewfelt, R., and A. Purvis. 1995. Toward a comprehensive model for lipid peroxidation in plant tissue disorders. Hort. Science 30:213-218. Stroup, J., M. Sanderson, J. Muir, M. McFarland, and R. Reed. 2003. Comparison of growth and performance in upland and lowland switchgrass types to water and nitrogen stress. Bioresource Technol. 86:65-72. Wang, C. 1999. Methyl jasmonate reduces water stress in strawberry. J. Plant Growth Regul. 18:127–134. Li, L., J. Van Staden, and A. Jager. 1998. Effects of plant growth regulators on the antioxidant system in seedlings of two maize cultivars subjected to water stress. Plant Growth Treatment: On July 12 the plants were treated with Jasmonic acid. Neat Jasmonic acid 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 was added to the solutions to act as a superfactant enabling absorption of the compound into the intracellular space. Treatment consisted of 10 mL dosages of treatment 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. Figure 1: Treatment application Harvest: On consecutive days between July 33 an July 33, the plants were harvested. 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 weighed. Figure 2. Switchgrass JA treatments replication 1 a. 0.0 mM b. 0.5 mM c. 1.5 mM d. 5.0 mM Materials and Methods Experimental design: Growth and treatment of the experiment took place on the roof of Howell Hall at the University of Central Oklahoma. The experimental design consisted of four repetitions, each of which was arranged in a randomized block design. On June 01 2010, the pots were seeded with 2.582 g switchgrass. The switchgrass germinated on June 06 and the plants were thinned to 100 seedlings per pot on June 12. On June 28 and July 18 a granular fertilizer was applied for a total N treatment of 150kg/hectare, a total P treatment of 60 kg/hectare and a total K treatment of 30 kg/hectare. a b c d Figure 7. JA effect on HMGR activity Values with the same letter are not significantly different Abstract ‘Alamo’ and ‘Kanlow’ switchgrass (Panicum virgatum L.) cultivars are high-yielding perennials with substantial lignocellulosic content applicable for biofuel production. However, these cultivars are indigenous to moist, lowland areas and possess negligible tolerance to drought conditions. A physiochemical effect of drought in plants is the generation of “reactive oxygen species” (ROS), which damage membranes, proteins, and nucleic acids of the cell. The oxylipin hormone, jasmonic acid (JA) may be exogenously applied as potentially effective treatment to enhance drought tolerance through elicitation of ROS-scavenging enzyme activity. An initial study was conducted to determine effective JA concentrations for elicitation of significant changes in biomass yield and enzyme activity in switchgrass. Four concentrations of JA (0.0 mM, 0.5 mM, 1.5 mM and 5.0 mM) were evaluated by assessing biomass yield and the activity of enzymes essential for production of phenolics (phenylalanine ammonia lyase; PAL) and terpenoids (hydroxylmethyl glutaryl CoA reductase; HMGR). Results indicated a significant effect on biomass accumulation in some JA treatments. Results also showed that PAL activity decreased and HMG-CoA activity increased as a result of JA application. Data from this experiment will be utilized to formulate a reliable method of JA treatment applicable for increasing switchgrass tolerance to drought conditions. Enzyme Assay: A spectrophotometric assay was conducted to determine the activity of the PAL enzyme. Phenylalanine ammonia lyase enables efficient production of trans-cinnamic acid from phenylalanine, this reaction is spectrophotmetrically observed as an increase in the absorbance of light at 290 nm. A spectrophotometric assay was conducted to determine the activity of the HMGR enzyme. Hydroxymethyl glutaryl CoA reductase oxidizes NADPH and reduces 3-hydrox-3- methylglutaryl-CoA to produce NADP + and Mevalonic acid . This reaction is spectrophotometrically observed as a decrease in the absorbance of light at 280 nm indicative of a decrease in the concentration of the conjugated HMG-CoA substrate and as a decrease in the absorbance of light at 340 nm indicative of the oxidation of NADPH to NADP + . Results and Discussion Analysis of the results indicated that the highest concentration of JA (5.0 mM) significantly affected biomass accumulation (Figure 5). The 0.5 mM concentration and the 1.5 mM concentration conveyed the general trend of decreased biomass with the addition of JA application however, the results were not significant (Figure 5). The JA treatment had a significant effect on PAL activity; the 0.5 mM concentration and the 5.0 mM concentration elicited a significant decrease in PAL activity. The 1.5 mM concentration induced a reduction in activity however, the effect was not significant. The JA treatment had a significant effect on HMGR activity; the 5.0 mM concentration elicited a significant increase in activity while the 0.5 mM and 1.5 mM concentrations induced significantly decreased activity compared to the control. Jasmonic acid is endogenously produced as a stress hormone, the application of exogenous jasmonate induces a simulated, stressed condition resulting in decreased primary productivity and decreased activity of PAL, an enzyme essential for lignin production. However, high levels of JA increase the activity of enzymes for the production of defensive compounds. The average endogenous concentration of JA in a drought-stressed plant is ~0.5 mM, the exogenous application of 0.5 mM, 1.5 mM, and 5.0 mM evaluated the effect of JA at 1X, 3X and 10X the natural concentration. Results suggest that application of high concentrations of jasmonic acid diverts resource allocation from biomass accumulation to the production of secondary protective compounds in a concentration dependent manner. Under conditions of drought, application of 5.0 mM JA may increase biomass accumulation over untreated samples due to the increased activity of HMGR for the production of antioxidative terpenoids which serve to alleviate the deleterious effects of ROS production in drought-stressed switchgrass. Further study in necessary to determine whether high concentrations of JA effectively increases drought tolerance in switchgrass. Figure 8. spectrophotometric procedure a b c d e f g h Acknowledgements Funding for this project was provided by a Research, Creative, and Scholarly Activities (RCSA) grant from the Office of Research and Grants at the University of Central Oklahoma. Seed for the project was generously donated by Johnston Seed Company.
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Transcript of Figure 6. JA effect on PAL activity Values with the same letter are not significantly different...
Figure 6. JA effect on PAL activityValues with the same letter are not significantly different
Figure 5. JA effect on biomass accumulation Values with the same letter are not significantly different
Enzyme extraction: The basal 10 cm of plant material was removed from each sample and put on ice for enzyme extraction. The fresh samples were immediately homogenized in a 50 mM Tris buffer (pH7.0) containing 0.1 M sucrose, 1% polyvinylpyrrilodone, 4 mM cysteine and 1 mM DTT. The resulting products were strained through 4 layers of cheese clothe and centrifuged at 5,000 g for 10 minutes enabling isolation of the microsomal fraction containing HMGR, the supernatant was removed and the pellet discarded. The supernatant was centrifuged at 10,000g for 10 minutes enabling subcellular isolation of the cytosol containing PAL.
Figure 3. centrifugation products rep 1 a. switchgrass 0.0 mM microsomal fraction b. Switchgrass 0.0 mM cytosolic fraction c. switchgrass 0.5 mM microsomal fraction d. switchgrass 0.5 mM cytosolic fraction e. switchgrass 1.5 mM microsomal fraction f. switchgrass 1.5 mM cytosolic fraction g. switchgrass 5.0 mM microsomal fraction h. Switchgrass 5.0 mM cytosolic fraction.
Effect of Jasmonic Acid on Biomass Accumulation and Enzyme Activity in Switchgrass Jocelyn Bidlack, Paul Olson, Carmen Cowo, Erin Ralstin, Robert Dinger, and Jim Bidlack
University of Central Oklahoma, Edmond, OK 73034
IntroductionResearch 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. ‘Alamo’ and ‘Kanlow’ cultivars of switchgrass (Panicum virgatum L.) are potentially efficient and economical crops for the production of biofuel (Cassida et al. 2005). However, under drought conditions, ‘Alamo’ shows a 49% decrease in biomass yield and ‘Kanlow’ shows a 27% decrease in biomass yield (Stroup et al. 2003). Current research conducted by our group is focused primarily on the optimization of bioenergy crop yield and increasing the cultivar’s physiological tolerance to drought thereby enabling expansion of the range of propagation to underutilized, semi-arid lands. To enable economically feasible production a simple and effective method of combating the negative effect of drought on these cultivars is necessary.
Under conditions of osmotic stress, plants regulate stomatal closure to conserve moisture by limiting the transpiration flux. Modification of the stomatal opening concurrently limits the uptake of CO2, greatly affecting photosynthetic rate and leading to the increased production of reactive oxygen species (ROS) (Sgherri et al. 1993). The ROSs initiate a cascade of reactions, resulting in the destruction of organic macromolecules such as lipids, proteins, and nucleic acids (Shewfelt and Purvis 1995). Jasmonic acid (JA) is a stress-induced hormone that modulates the expression of antioxidative defensive systems. The exogenous application of JA prior to the initiation of osmotic stress has the potential to moderate the effects of drought (Li et al. 1998, Rohwer and Erwin 2008, Wang 1999). Treatment effectively induces biosynthesis and accumulation of the ROS-scavenging antioxidants (Gao et al. 2004), and induces increased activity of the ROS-scavenging enzymes.
JA application may alter the production of secondary compounds with antioxidant properties indicated by significantly affecting the activity of hydroxymethyl glutaryl CoA reductase-HMGR (the rate limiting enzyme for the production of terpenoids). In addition, JA application is expected to alter the activity of phenylalanine ammonia lyase (PAL), the rate limiting enzyme for the production of the phenolic compounds such as the cell wall component-lignin. Pretreatment with exogenous JA may provide a mechanism of inducing ROS-scavenging prior to the damaging effect of peroxidation, thereby protecting the photosynthetic mechanism and membranes of the cell and moderating the damaging effects of drought.
This preliminary experiment was conducted under standard osmotic conditions with a simplified objective: to determine the concentration of jasmonic acid that most effectively instigates significant affects on biomass accumulation and PAL and HMGR enzyme activity in switchgrass. Data from the experiment were utilized to formulate an appropriate procedure for the application of jasmonic acid in switchgrass. In addition, methods of enzyme extraction and analysis were evaluated and refined in anticipation of future research in simulated drought conditions.
Literature CitedCassida, 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 Sci. 45:682-692.Gao, X., X. Wang, Y. Lu, L. Zhang, Y. Shen, Z. Liang, and D. Zhang. 2004. Jasmonic acid is involved in the water-stressed-induced betaine accumulation in pear leaves. Plant Cell Environ. 27:497–507.Sgherri, C., C. Pinzino, and F. Navari-Izzo. 1993. Chemical changes and O2·− production in thylakoid membranes under water stress. Physiol. Plant. 87:211–216.Shewfelt, R., and A. Purvis. 1995. Toward a comprehensive model for lipid peroxidation in plant tissue disorders. Hort. Science 30:213-218.Stroup, J., M. Sanderson, J. Muir, M. McFarland, and R. Reed. 2003. Comparison of growth and performance in upland and lowland switchgrass types to water and nitrogen stress. Bioresource Technol. 86:65-72.Wang, C. 1999. Methyl jasmonate reduces water stress in strawberry. J. Plant Growth Regul. 18:127–134.Li, L., J. Van Staden, and A. Jager. 1998. Effects of plant growth regulators on the antioxidant system in seedlings of two maize cultivars subjected to water stress. Plant Growth Regul. 25:81–87.
Treatment: On July 12 the plants were treated with Jasmonic acid. Neat Jasmonic acid 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 was added to the solutions to act as a superfactant enabling absorption of the compound into the intracellular space. Treatment consisted of 10 mL dosages of treatment 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.
Figure 1: Treatment application
Harvest: On consecutive days between July 33 an July 33, the plants were harvested. 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 weighed.
Figure 2. Switchgrass JA treatments replication 1 a. 0.0 mM b. 0.5 mM c. 1.5 mM d. 5.0 mM
Materials and MethodsExperimental design: Growth and treatment of the experiment took place on the roof of Howell Hall at the University of Central Oklahoma. The experimental design consisted of four repetitions, each of which was arranged in a randomized block design. On June 01 2010, the pots were seeded with 2.582 g switchgrass. The switchgrass germinated on June 06 and the plants were thinned to 100 seedlings per pot on June 12. On June 28 and July 18 a granular fertilizer was applied for a total N treatment of 150kg/hectare, a total P treatment of 60 kg/hectare and a total K treatment of 30 kg/hectare.
a b c d
Figure 7. JA effect on HMGR activity Values with the same letter are not significantly different
Abstract‘Alamo’ and ‘Kanlow’ switchgrass (Panicum virgatum L.) cultivars are high-yielding perennials with substantial lignocellulosic content applicable for biofuel production. However, these cultivars are indigenous to moist, lowland areas and possess negligible tolerance to drought conditions. A physiochemical effect of drought in plants is the generation of “reactive oxygen species” (ROS), which damage membranes, proteins, and nucleic acids of the cell. The oxylipin hormone, jasmonic acid (JA) may be exogenously applied as potentially effective treatment to enhance drought tolerance through elicitation of ROS-scavenging enzyme activity. An initial study was conducted to determine effective JA concentrations for elicitation of significant changes in biomass yield and enzyme activity in switchgrass. Four concentrations of JA (0.0 mM, 0.5 mM, 1.5 mM and 5.0 mM) were evaluated by assessing biomass yield and the activity of enzymes essential for production of phenolics (phenylalanine ammonia lyase; PAL) and terpenoids (hydroxylmethyl glutaryl CoA reductase; HMGR). Results indicated a significant effect on biomass accumulation in some JA treatments. Results also showed that PAL activity decreased and HMG-CoA activity increased as a result of JA application. Data from this experiment will be utilized to formulate a reliable method of JA treatment applicable for increasing switchgrass tolerance to drought conditions.
Enzyme Assay: A spectrophotometric assay was conducted to determine the activity of the PAL enzyme. Phenylalanine ammonia lyase enables efficient production of trans-cinnamic acid from phenylalanine, this reaction is spectrophotmetrically observed as an increase in the absorbance of light at 290 nm.
A spectrophotometric assay was conducted to determine the activity of the HMGR enzyme. Hydroxymethyl glutaryl CoA reductase oxidizes NADPH and reduces 3-hydrox-3-methylglutaryl-CoA to produce NADP+ and Mevalonic acid . This reaction is spectrophotometrically observed as a decrease in the absorbance of light at 280 nm indicative of a decrease in the concentration of the conjugated HMG-CoA substrate and as a decrease in the absorbance of light at 340 nm indicative of the oxidation of NADPH to NADP+.
Results and DiscussionAnalysis of the results indicated that the highest concentration of JA (5.0 mM) significantly affected biomass accumulation (Figure 5). The 0.5 mM concentration and the 1.5 mM concentration conveyed the general trend of decreased biomass with the addition of JA application however, the results were not significant (Figure 5).
The JA treatment had a significant effect on PAL activity; the 0.5 mM concentration and the 5.0 mM concentration elicited a significant decrease in PAL activity. The 1.5 mM concentration induced a reduction in activity however, the effect was not significant. The JA treatment had a significant effect on HMGR activity; the 5.0 mM concentration elicited a significant increase in activity while the 0.5 mM and 1.5 mM concentrations induced significantly decreased activity compared to the control.
Jasmonic acid is endogenously produced as a stress hormone, the application of exogenous jasmonate induces a simulated, stressed condition resulting in decreased primary productivity and decreased activity of PAL, an enzyme essential for lignin production. However, high levels of JA increase the activity of enzymes for the production of defensive compounds. The average endogenous concentration of JA in a drought-stressed plant is ~0.5 mM, the exogenous application of 0.5 mM, 1.5 mM, and 5.0 mM evaluated the effect of JA at 1X, 3X and 10X the natural concentration. Results suggest that application of high concentrations of jasmonic acid diverts resource allocation from biomass accumulation to the production of secondary protective compounds in a concentration dependent manner. Under conditions of drought, application of 5.0 mM JA may increase biomass accumulation over untreated samples due to the increased activity of HMGR for the production of antioxidative terpenoids which serve to alleviate the deleterious effects of ROS production in drought-stressed switchgrass. Further study in necessary to determine whether high concentrations of JA effectively increases drought tolerance in switchgrass.
Figure 8. spectrophotometric procedure
a b c d e f g h
AcknowledgementsFunding for this project was provided by a Research, Creative, and Scholarly Activities (RCSA) grant from the Office of Research and Grants at the University of Central Oklahoma. Seed for the project was generously donated by Johnston Seed Company.
