Progress 07/01/08 to 06/30/13
Outputs
Target Audience: Breeders, Scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The most significant outputs of the project include four peer-reviewed research articles published from data generated by my research group. I established collaborations with six Virginia Tech professors, four international researchers (China, Germany), one international company (New Zealand) with U.S. distribution, and one regional collaborator (IALR, Danville, VA). The project was a significant component of the research of four graduate students leading to one Master’s and two Ph.D. degrees. These students presented their research in poster and oral formats at research conferences on plant biology with regional, national and international attendance. Four undergraduates were trained in research methods. How have the results been disseminated to communities of interest? Peer reviewed publications targeted to the scientific research community. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The project focused on the glucosinolate-myrosinase system, the major bioactivated chemical defense against generalist pathogens and herbivores found in crucifers. This chemical defense provides a broad plant resistance and therefore has a strong positive effect on crucifer crop productivity. Crucifers produce two different major classes of glucosinolate structures and have large gene families encoding myrosinase, however the biological significance of this biochemical diversity was largely unexplored. The goal of the five-year project was to change our knowledge of the relationship between the biochemical diversity within the glucosinolate-myrosinase system and plant resistance against specific crop pests. We found that one class of glucosinolates was more effective against pests that cause extensive cellular breakage and death such as necrotrophic microbes or chewing insects whereas, the other glucosinolate class was more effective against pests that infect plants but allow the plant to survive such as biotrophic microbes and aphids. We found that the two myrosinases detected in arabidopsis leaves are collectively active under a broad range of temperature (10 to 65 degrees Celsius) and pH values (5-11) normally not found in the plant suggesting they are adapted to activity outside of the plant. These results will allow breeders to enhance the natural pesticide activity of crucifer crops by targeting changes in glucosinolate abundance cognizant of the feeding strategy of specific pests and generating combinations of myrosinase isoenzymes in a plant cultivar that extend the temperature and pH range of myrosinase activity. By exploiting the natural pesticides of crucifers, we can reduce our dependence on fossil fuel-derived chemical pesticides and minimize our chemical contamination of the agricultural environment.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Zhang, J.X., Sun, X.M., Zhang, Z.P., Ni, Y.W., Zhang, Q., Liang, X.M., Xiao, H.B., Chen, J.P., and Tokuhisa, J.G. (2011) Metabolite profiling of Arabidopsis seedlings in response to exogenous sinalbin and sulfur deficiency. Phytochemistry 72, 1767-1778. DOI: 10.1016/j.phytochem.2011.06.002
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: In the past year the lab has provided expertise and conducted collaborative research on how changes in the glucosinolate-myrosinase chemical defense system affect insects feeding on crucifer crops. Insects feeding on plants reduce crop yields because the insects transmit plant pathogens and reduce plant vigor, making plants more susceptible to disease. 1) I have had published a manuscript on the biochemical properties of two myrosinases purified from Arabidopsis that bioactivate the chemical defense system. 2) In collaboration with Prof. Tom Kuhar and graduate student Anna Wallingford, I have measured glucosinolates in broccoli plants over a period of crop development coincident with feeding by the Harlequin bug, Murgantia histrionica to monitor changes in glucosinolate abundance and individual compounds. 3) In collaboration with Prof. Barry Flinn, I am conducting baseline measurements of glucosinolate content in pennycress (Thlaspi arvense) seed. 4) Studies are continuing on the gene expression and metabolite reallocation by plants in response to feeding by root herbivores. PARTICIPANTS: I have collaborations with Profs. David Bevan, Asim Esen, Barry Flinn, Tom Kuhar, Dorothea Tholl, and members of their research groups. Individuals working on the projects include the postdoctoral researcher Changhe Zhou, graduate students Anna Wallingford and Jingyu Zhang, and undergraduate Kristen Clermont. All of the students have received advanced training in research methods, strategies and ethics. TARGET AUDIENCES: The pennycress project is based in the Institute for Advanced Learning and Research, Danville Virginia. The Institute was founded in 2002 to develop and attract emerging technology and talent for Southern Virginia's economic prosperity to broaden the agricultural and manufacturing base by enabling economic and community transformation in the region. The Harlequin bug research began as a trap crop project at the Eastern Shore AREC to address pest problems in high value vegetable crops. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The chemical defenses that plants have can be beneficial or detrimental to agricultural productivity. On the one hand, these defenses minimize crop damage by insects. 1) A better understanding of the basic biochemistry of the glucosinolate-myrosinase system can lead to targeted enhancement of this chemical defense. Myrosinase is the critical bioactivator of this defense system. In a manuscript published early this year, my colleagues Profs. David Bevan and Asim Esen, postdoctoral researcher Changhe Zhou and I demonstrated how robust this enzyme is to extreme temperature and pH conditions and proposed that the plant takes its chemical defense deep into the gut of herbivorous insects. 2) We know that the glucosinolate-myrosinase system affects herbivore feeding behavior. The harlequin bug changes its feeding targets as a pest of broccoli as the crop flowers and matures. We have extracted and measured broccoli glucosinolates to see if insect behavior correlates with changes in glucosinolate content or profiles. Glucosinolates of particular structure and at high concentration can be detrimental to humans and livestock. 3) An emphasis on novel biofuel production has led to the cultivation of pennycress for its seed oil. The residual seedmeal has a high glucosinolate content that is toxic to most animals thus precluding its use as a feedstock. Prof. Barry Flinn leads a program at a regional research institute, the IALR in Danville VA, to enhance the biofuel potential and reduce the glucosinolate content of pennycress through a mutagenesis-screening program. I am establishing a baseline profile of glucosinolates in pennycress and will screen novel pennycress plant lines for altered glucosinolate profiles.
Publications
- Zhou, C.H., Tokuhisa, J.G., Bevan, D.R., and Esen, A. (2012) Properties of beta-thioglucoside hydrolases (TGG1 and TGG2) from leaves of Arabidopsis thaliana. Plant Sci. 191:82-92.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: I have led a team to develop and further optimize a novel method for aeroponic plant cultivation and investigate the chemical defense system in roots of crucifer plants. The aeroponic cultivation method uses calcined clay as a soilless potting substrate. The media is porous with low cation exchange capacity, which gives it high water retention capability, sufficient access for gas exchange, and the ability to rapidly manipulate nutrient levels. The root mass is easily and rapidly harvested with minimal tissue damage. In collaboration with Drs. Martha Vaughan and Dorothea Tholl, we have used the system to investigate root herbivory by Bradysia second instar larvae on wild type and mutant Arabidopsis plant lines. A graduate student in Biological Sciences, Jingyu Zhang, has been optimizing aeroponic plant cultivation and fungus gnat cultures to screen more mutant plant lines for chemical defense factors that affect plant defense potential. In collaboration with Prof. Asim Esen, we have investigated the molecular properties of the glucosinolate-myrosinase defense system in crucifer plants to identify plant adaptations that increase the efficacy of the plant defense response. For both investigations, our findings have been disseminated through publication. Dr. Martha Vaughan currently works at the USDA. PARTICIPANTS: I have collaborated with Profs. Dorothea Tholl, Sue Tolin, and Asim Esen, and members of their research groups. Individuals working on the projects include the postdoctoral researcher Changhe Zhou, graduate students Jung-hyun Huh, Sabrina Majumder, Martha Vaughan, Anna Wallingford, and Jingyu Zhang, and undergraduate student Valerie Siira. All of the students have received advanced training in research methods and strategies. TARGET AUDIENCES: Virginia Tech had a University Open House on November 12, 2011. The public was invited to visit research buildings. I discussed my research programs with various members of the public. Scientific community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Investigations of plant chemical defenses have revealed fundamental insights into plant chemical defense that can be exploited to strengthen sustainable agriculture in the United States. Development of the aeroponic system has allowed us to examine details of the root herbivory by Bradysia and the plant defense system. During the current reporting period, we have optimized the ability to germinate seeds and grow seedlings in the calcined clay media rather than rather transplant older seedlings into the media and further develop the cultivation of the Bradysia insect colony. We showed that the two myrosinase isoenzymes are extremely robust; they are glycosylated and function in a broad pH and temperature range, and in the presence of hydrolytic enzymes. These properties indicate the capability to function not only in damaged plant tissue at the site of herbivory but also in the insect midgut. This hypothesis will be further explored using site-directed mutagenesis of the genes coding for TGG1 and TGG2 and reintroducing the altered genes into a TGG-null background of Arabidopsis. The characterization of the enzyme component of the glucosinolate-myrosinase chemical defense system of Arabidopsis, the myrosinases TGG1 and TGG2, was completed and submitted for publication to Plant Science and is accepted pending revisions. These studies show that the defense is multifaceted. The discovery of how natural plant insecticides function in the plant increases our opportunities to generate new natural insecticides and approaches to insect pest management. The article describing the aeroponic cultivation has been accessed 2770 times so far and ranks 38th for most viewed in the last year of Plant Methods publications.
Publications
- Vaughan,M.M., Tholl,D., and Tokuhisa,J.G. (2011). An aeroponic culture system for the study of root herbivory on Arabidopsis thaliana. Plant Methods 7, 5.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: I have used a novel method for aeroponic plant cultivation to investigate the chemical defense system in roots of crucifer plants and to characterize the plant chemical defense response to macronutrient deficiency. In collaboration with Dr. Dorothea Tholl, we developed an aeroponic cultivation method using calcined clay as a soilless potting substrate. The media is porous, which gives it high water retention capability yet allows for its facile removal from the root mass. The low cation exchange capacity of the media permits rapid manipulation of nutrient levels. We have prepared a manuscript on the aeroponic culture system for submission to Plant Methods. An undergraduate Biological Sciences major, Valerie Siira, has researched the relationship of plant nutrition and plant chemical defense levels. Martha Vaughan, a graduate student, has demonstrated the utility of the culture system for the investigation of plant responses to root herbivory by fungus gnat larvae (Bradysia). Graduate student Junghyun Huh has used the aeroponic system to investigate the plant infection process caused by the causal agent for root rot (Pythium irregulare). She has developed staining methods to distinguish the attachment and hyphal penetration stages of Pythium infection and has begun to screen plant lines lacking defense compounds for increased susceptibility. In collaboration with Asim Esen, Changhe Zhou and David Bevan, we are publishing a manuscript on the characterization of the myrosinase enzyme of the glucosinolate-myrosinase plant defense system. PARTICIPANTS: One undergraduate student Valerie Siira has participated in this project. Two graduate students, Martha Vaughan and Junghyun Huh, have contributed. Collaborators include Drs. David Bevan (Biochemistry), Asim Esen (Biological Sciences), and Dorothea Tholl (Biological Sciences). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Research results during the reporting period indicate that plants do not exhibit a marked increase in chemical defenses against herbivory under optimal versus suboptimal macronutrient conditions. Indeed, our results indicate a reduction in the glucosinolate-myrosinase defense system under optimal nutrition. These results support the Plant Vigor Hypothesis which postulates that higher plant growth rates favor insect herbivory. These results suggest an opportunity in sustainable agriculture to use levels of nitrogen fertilizer that may be suboptimal for maximum growth but optimal for plant defense responses. The cost savings and reduced runoff pollution associated with reduced nitrogen application and the enhanced crop yield due to increased plant resistance may compensate for the reduced crop yield anticipated by suboptimal growth. Larval feeding is increased by the absence of indole glucosinolates and the plant defense response is mediated by the plant defense hormone jasmonate. We have characterized the enzyme component of the glucosinolate-myrosinase chemical defense system of crucifer plants. We showed that the enzyme is extremely robust; it works in at a pH range from 4 to 11, at temperatures from 10-70 degrees C, and in the presence of hydrolytic enzymes. These properties indicate the capability to function not only in damaged plant tissue at the site of insect feeding but also in the insect midgut. These results indicate a potential insecticide target in the insect midgut that could be activated by robustly-engineered enzymes. We have prepared a manuscript on the characterization of myrosinase for submission to Planta. Research in the areas of the plant defense response in roots and the relationship between plant nutrition and plant defense capability has identified some fundamental biology that can be exploited to develop sustainable agriculture in the United States. To use less nitrogen fertilizers yet have a more strongly defended crop plant may have broad application in agriculture. The discovery of new targets for natural plant insecticides increases our opportunities to generate new insecticides using these novel targets.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: I mentored an undergraduate Biological Sciences major (Valerie Siira) who is conduct research on manipulating chemical defense levels through nutrition. She established an experimental system using arugula cultivated hydroponically or in soilless potting mix and has optimized fertilizer application to alter the natural chemical defense levels in the plant. She has assessed the glucosinolate content of white radish cultivars that are used as crucifer cover crops (collaboration with Brinkley Benson). I mentored a Ph.D. candidate in Biological Sciences (Jung-hyun Huh, collaboration with Dr. Dorothea Tholl) regarding the development of a plant-pathogen system to test chemical defense capabilities in plant roots using Arabidopsis-Pythium irregulare as a pathosystem. I supervised Earl Petzold, a technician, on the preparation of plants with altered levels of chemical defense compounds. I convened a workshop involving four laboratories (McDowell, Tholl, Tokuhisa and Vinatzer) to discuss a major paradigm shift in plant pathogen chemical defense described in two publications. PARTICIPANTS: Valerie Siira, a junior in Biological Sciences, has mastered major techniques associated with the research and has begun to design and implement experimentation on the effect of nutrition on plant chemical defense potential. Sabrina Majumder, Horticulture graduate student at the Master's Level designed and implemented research on the role of glucosinolates in plant resistance against viral infection. Jung-hyun Huh, a Ph.D. candidate in Biological Sciences, has implemented research on the role of glucosinolates in plant resistance against oomycete infection. Earl Petzold, a technician in Horticulture, has prepared a number of transgenic plants altered in glucosinolate levels. Collaborators include Drs. Sue Tolin and Boris Vinatzer (Plant Pathology, Physiology and Weed Science, Virginia Tech), Brinkley Benson (Horticulture) and Drs. Asim Esen and DOrothea Tholl (Biological Sciences, Virginia Tech). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Project modifications based on recent publications in the field are under consideration.
Impacts
Research results during the reporting period indicate that plants grown under sulfate deficiency have significantly lower glucosinolate content without exhibiting classic symptoms of the nutrient deficiency. These results indicate that the chemical defense potential of crucifer plants is reduced before visible nutrient deficiency symptoms are apparent. This system will be used to test the pathogen defense potential of nutrient deficient plants. The 100 umole/g dry weight levels of aliphatic glucosinolates in the roots of white radish indicate a strong biofumigation potential for this cover crop. We will explore novel cultural practices to enhance the biofumigation potential. The Arabidopsis-Pythium pathosystem is very sensitive to changes in indole secondary metabolism but not to aliphatic glucosinolate formation.
Publications
- Uddin, M. M., Ulrichs, C., Tokuhisa, J. G., and Mewis, I. (2009) Impact of glucosinolate structure on the performance of the crucifer pest Phaedon cochleariae (F.), Journal of Applied Botany and Food Quality-Angewandte Botanik, 82(2): 108-113.
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Progress 07/01/08 to 09/30/08
Outputs
OUTPUTS: I have mentored a master's level student of Horticulture on the development of a inoculation method to look at turnip mosaic virus infection of Arabidopsis in collaboration with Dr. Sue Tolin. I have trained the student to identify data available through the web on the expression of genes involved with the production and activation of glucosinolates, which are major plant chemical defense compounds found in crucifer crops. I have trained an undergraduate agricultural sciences major in the protocols necessary to isolate and quantify glucosinolates. We have conducted glucosinolate analyses of crucifer cover crops for ongoing projects with Brinkley Benson and Dr. Ron Morse and of turnip mosaic virus-infected plants with Dr. Tolin. I have consulted with Dr. Asim Esen on the data describing the activation of glucosinolates as defense compounds. Results from the Arabidopsis infected with turnip mosaic virus were presented in poster format to the 25th Mid-Atlantic Plant Molecular Biology Society Conference in Savage, MD (August 21-22, 2008). PARTICIPANTS: Collaborators: The project has resulted in collaborations with Drs. Sue Tolin and Boris Vinatzer (Plant Pathology, Physiology and Weed Science, Virginia Tech), and Dr. Asim Esen (Biological Sciences, Virginia Tech). Training: I have provided training of one undergraduate, one graduate student and one technician. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Research results during the reporting period indicate that plants with lower glucosinolate content have less resistance to turnip mosaic virus. These results are preliminary and need to be replicated with more plant lines and viral strains. Discussions with Dr. Esen on the activation of glucosinolates has led to a reassessment of the critical characteristics for the isoforms of the activating enzymes.
Publications
- No publications reported this period
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