Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
GENETIC CONTROL OF GLUCOSINOLATES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0192787
Grant No.
(N/A)
Project No.
CA-D-PLS-7033-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Project Director
Kliebenstein, D.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Sciences
Non Technical Summary
The flavour and nutritional quality of vegetable crops is affected by numerous agricultural practices and environmental stress because the plant responds. This happens because the plant responds to these inputs by altering the production of its chemicals which are the flavor and nutritional components. By developing a complete model and thereby understanding of what inputs a plant uses as signals to control its flavor and nutritional chemistry, it should become possible to develop an almost engineering like precision in the ability to properly handle a plant to maximize the flavor and nutritional profile. This can include altering agricultural practices or changing the breeding of the plant.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012420106010%
2022420106010%
2021440108010%
2042420106010%
2041440108010%
2062420106010%
2061440108010%
2152420106010%
2151440108010%
7011440100010%
Knowledge Area
206 - Basic Plant Biology; 204 - Plant Product Quality and Utility (Preharvest); 215 - Biological Control of Pests Affecting Plants; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms; 701 - Nutrient Composition of Food;

Subject Of Investigation
1440 - Cole crops; 2420 - Noncrop plant research;

Field Of Science
1060 - Biology (whole systems); 1000 - Biochemistry and biophysics; 1080 - Genetics;
Goals / Objectives
The glucosinolate/myrosinase system in the Brassicaceae (Arabidopsis thaliana, Brassica rapa and other cole crops) has long been believed to control a variety of traits including insect and pathogen resistance. Further, this system also has direct impacts on human nutrition such as inducing anti-cancer defenses and causing goiter disease. The biological activity of this system is controlled by the structure of the glucosinolate and by the final activation product formed. This binary control on biological activity is unique among plant secondary metabolites where the compounds structure is the only factor impacting activity. Recent work has dramatically advanced the current knowledge of this system on both the biosynthesis and activation side. However, there are a significant number of holes in our knowledge. The major hole of agricultural relevance is how this system is regulated. Recent work with several Brassica mapping populations has shown us that both glucosinolate biosynthesis and activation differed between plant tissues and developmental age. We proceeded to investigate if this was also true in our model plant, Arabidopsis. We have shown that there is dramatic tissue and developmental control of all glucosinolate related phenotypes and this is under genetic control. We have identified a number of genetic loci that control this variation providing us an excellent avenue within which to begin studying the genetics of these regulatory processes. Once identified, these regulatory genes can then be studied in Brassica crops. We have several major objectives for this research. The first is to identify all of the gene controlling variation in glucosinolate phenotypes. This includes a large number of regulatory factors that transduce a known environmental stimulus. This is allowing us to develop a model of how glucosinolate activation and synthesis and regulation are controlled within the plant in response to a complex environment. We will finally take these new environmental stimuli and test if they impact glucosinolates in isolation or combination with each other. This should allow us to develop a model for how glucosinolates are regulated in a complex environment that could better inform agronomic and breeding decisions in brassica vegetable production.
Project Methods
Most of this work uses either induced mutant alleles or natural genetic variation in genes to identify genes and their functions. Most of the mutations that we study are naturally occurring polymorphisms that are present in wild-populations of Arabidopsis thaliana and ostensibly under any natural selective pressure acting upon these populations. We use natural variation for a number of reasons. The primary reason is that naturally variable loci within Arabidopsis thaliana, are frequently variable within the diverse cruciferous vegetables. As such, the identification of these loci leads to the ability to directly generate genetic markers for marker assisted selection within the cruciferous vegetables allowing for rapid germplasm modification in actual crops. Another basic reason to focus on these loci is that they allow us to understand what selective pressures are acting positively or negatively on these polymorphisms and thereby predict how wild populations will respond to different selective pressures. Another reason for using natural variation is that the lines used typically contain a large number of homozygous lesions. Thereby, we can repeatedly measure a phenotype that may have a low genetic component like insect resistance. A repeated measure analysis on most populations containing induced mutations would be nearly impossible as they typically contain only a few mutations that are heterozygous. Thus, we can study the impact of a large number of lesions on traits with low heritabilitys. In addition to genetics I utilize a broad array of genomics and metabolomics techniques to speed up the analysis. I have developed a series of techniques that allow for 96-well plate extraction and analysis of aqueous soluble metabolites in addition to DNA genotyping. This allows the use of large population sizes for studies of low heritability traits. In addition, I am using metabolite profiling techniques in high-throughput fashion to study entire pathways at a single time. With the glucosinolate pathway, we can use a single extraction and HPLC analysis to study all 35 glucosinolates and all potential glucosinolate activation pathways in Arabidopsis thaliana. This combination of high-throughput metabolite profiling allows for the generation of large data sets that address specific issues like insect resistance with maximal statistical power. The combination of genetics, natural variation and high-throughput metabolite assays is a powerful combination to understand the biological importance of the glucosinolate/myrosinase system and secondary metabolites in general. Finally we utilize genomics with systems biology approaches to develop new understanding of how the entire system functions. This includes combining transcriptomics, metabolomics, metabolite profiling and high-throughput phenotyping to test the link between different genes in controlling a given phenotype as well as unexpected links between different phenotypes. This is critical to do this systems approach to develop a deeper understanding of the connections within the plant as this is fundamental to being able to intentionally manipulate a plant at will.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with researchers from different breeding companies. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. More recently, I have been able to expand the target audience into companies working on using glucosinolates as nutritional supplements by having discussions with Jarrow Formulas, a world leader in nutritional supplement production. In addition consultations were established with Arvegenix, Inc to aid in the establishment of a new glucosinolate containing cover crop for midwestern Soybean production. I have also begun to add VP Genomics to investigate the effect of defense compounds on Botrytis cinerea resistance and how this may be applied to Controlled Environment Agricultural systems. For the Academic community, I gave over 29 seminars during this time frame and published 64 journal articles and book chapters to disseminate our research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The research project in the past year allowed several opportunities for training and professional development. The four graduate students involved in this project over the time frame (Jason Corwin, Rachel Kerwin, Michelle Tang and Nicole Soltis) have attended one or more research meetings wherein they presented their research as posters. These meetings were meetings with less than 80 attendees allowing for extensive interactions allowing all four graduate students to develop their research connections and to practice presenting their work. Drs Corwin and Kerwin graduated with their PhD mid-way through the project and they both have post-doctoral research positions in large research labs and are receiving invitations for job interviews as faculty. The project is also becoming a platform whereby myself, and all of the graduate students worked to develop a novel cohort based undergraduate training regime wherein we hire three undergraduates who are willing to commit to at least two years of research. These students are developing their own research project wherein they are investigating the effect of hybrid vigor on defense metabolism and pathogen resistance. This is occurring within the confines of the larger project an the goal is to train them to be the equivalent of mid-career graduate students by the end of their tenure. This effort is currently in its third year and we are recruiting a new cohort of students. The previous cohort graduated in the past year and they are either working in the biotech industry or pursuing graduate school studies. How have the results been disseminated to communities of interest?To disseminate this work, we have utilized two approaches that differ for the target audiences. For the genomics and breeding industry, I have given several presentations at various company headquarters. But the largest communication effort on this fron is to have numerous small discussions with researchers from different breeding companies. This includes a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. More recently, I have been able to expand the target audience into companies working on using glucosinolates as nutritional supplements by having discussions with Jarrow Formulas, a world leader in nutritional supplement production. In addition consultations were established with Arvegenix, Inc to aid in the establishment of a new glucosinolate containing cover crop for midwestern Soybean production. I have also begun to add VP Genomics to investigate the effect of defense compounds on Botrytis cinerea resistance and how this may be applied to Controlled Environment Agricultural systems. For the Academic community, the communication is almost entirely focused on seminars and publications. I gave over 29 seminars during this time frame and published 64 journal articles and book chapters to disseminate our research. What do you plan to do during the next reporting period to accomplish the goals?We are continuing this project.

Impacts
What was accomplished under these goals? In the past five years, we have had a number of major accomplishments that have helped us to further our goal of understanding the genetic architecture of glucosinolate metabolism. I have briefly described these accomplishments below. 1) We collaborated to show that glucosinolates show circadian oscillations in cabbages and Arabidopsis post-harvest. In our work, we showed that the phytonutrient glucosinolate content of brassica's has a wide range of variation across the day even after harvest and that cold-storage locked the plant into lower levels. This suggests that it may be possible to make vegetables and produce healthier by manipulating their storage environment after harvest. It also suggests that certain vegetables and produce may have optimal times of day in which they should be consumed or processed to maximize their nutritional value. This had been known for some vitamins but this was the first evidence that the broader array of phytonutrients may also be subject to this circadian oscillation both pre- and post-harvest. 2) We showed that attempting to link across different phenotypes using natural genetic variation is fraught with a high level of error particularly false negative error where associations between phenotypes can be missed. Innumerable studies measure the phenotypic variation of several phenotypes in common populations to attempt and link the phenotypes. Often this is looking at traits important for yield and defense or yield and nutritional content. These studies often show no coordination in these traits yet when working to breed for yield there is always a residual link. We looked at glucosinolate and growth variation in a set of Arabidopsis populations in which we knew the existence of a link and the mechanism by which it occurs. Standard genetic analysis indicated that these two phenotypes were unlinked even though we knew the must be coordinated. A deeper analysis showed that we could identify this link but had to move from standard approaches to a more powerful ANCOVA based methodology. Thus, the studies suggesting that there is no linkage across different phenotypes in genetic mapping populations have to be re-evaluated with these new methods to ensure that these observations were not erroneous. 3) We showed that genomic variation within the organellar genomes, mitochondrial and plastidic, is a key component of natural variation in most eukaryotic organisms. In most studies of quantitative genetic variation, the research focuses on variation in the nuclear genome due to technical and biological simplification. However, this left open the question of how genetic variation within the organellar genomes could influence phenotypic variation. Our work showed that the organellar genomic variation had a large consequence upon metabolic and growth variation. However, this was not additive variation and instead it appeared to be caused by epistatic interactions with nuclear loci. These interactions were frequently with pairs of nuclear loci. This suggests that the organellar genomic variation must be included in natural variation studies as well as in breeding programs. By including this variation in breeding programs, it may be possible to increase our ability to conduct predictive breeding because up to this point, the organelle was an ignored aspect of the genetic equation. 4) We collaborated with the lab of Dr. Siobhan Brady here at the University of California, Davis to investigate the scale of the transcriptional regulatory network that influences the accumulation of glucosinolates within Arabidopsis thaliana. This work utilized a Yeast-1-hybrid approach wherein we cloned all of the promoters for the genes encoding enzymes for glucosinolate biosynthesis and systematically tested ~760 transcription factors for the ability to bind these promoters. This analysis showed that there are likely at least 100 different transcription factors that can modulate the expression of genes in the pathway. A validation test with mutants showed that at least 80% of these can alter glucosinolate accumulation within the plant. This work suggests that the linear master regulatory model for the control of metabolites is likely incorrect. Instead, when working to modulate glucosinolates, the breeder will need to consider a more complex intertwined model whereby at least 80 transcription factors can modulate the accumulation of these metabolites. The identity of these transcription factors shows that they function to link defense metabolism with numerous biotic and abiotic inputs generating a model whereby the metabolite accumulation can be finely tuned in response to a highly variable environment. 5) Another major accomplishment focused upon understanding the genetic architecture of glucosinolates and other traits came from a meta-analysis of quantitative trait locus mapping studies. In this work, we investigated all of our previous quantitative trait locus mapping studies to obtain an estimate of how many loci are found per population depending upon the size of the population. Previous work had suggested that the relationship between population size and quantitative trait locus number was log linear with a maximum number of quantitative trait loci found at somewhere between 200 and 400 individuals. Our work however showed that there was no difference in the log-linear and linear models for this range and that populations needed to be at least 1000 individuals for simple populations with only two parents before we could tell which model was most accurate. This work suggests that we may be vastly underestimating the number of loci controlling any given phenotype in nearly every population tested to date. Thus, this work shows that we need larger simple populations to truly measure the genetic architecture of any given trait. 6) We were able to extend this within the lab using a multiple year field trial experiment. This allowed us to show for the first time that specific glucosinolate gene variants do alter fitness in the field using Arabidopsis thaliana. This involved the planting of dozens of specific gene variants in the field and measuring fitness over several years at two different field sites. Critically, this showed that the fitness effects were always significant but that the optimal genotype differed every year in every field site. This provides some of the first empirical evidence of fluctuating selections' effects on specific genotypes. This shows that natural genetic variation can be maintained by selection and that selection does not necessarily favor a single genotype. Thus, in this system, there is no optimal genotype for all conditions and if a breeder was going to apply these lessons to an agronomic system they would have to allow for a system to have a suite of genotypes all inter-planted. 7) In a suite of papers, we showed that a specific glucosinolate, allyl glucosinolate, can directly regulate plant growth and development. Defense metabolites are typically considered to be downstream outputs of the plant with little to no direct consequence to the plant other than by acting as toxins to defeat biotic attackers. Our efforts are beginning to overturn this long term view and we have recently shown that allyl glucosinolate can lead to altered plant growth and appears to function as a direct growth signal. Using genome-wide association mapping, we could show that this functions via an interplay of secondary metabolism and what appears to be a novel branch of the key plant hormone, auxin, signaling pathway. Thus, glucosinolates are not solely defensive toxins but also have the ability to be regulatory compounds in planta. This is modulated by the specific structure of the glucosinolate and future work is required to assess if other glucosinolates can also act as signals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, J.-Z., Li, B., Xiao, Y., Ni, Y., Ke, H., Yang, P., de Souza, A., Bjornson, M., He, X., Shen, Z., Balcke, G.U., Briggs, S.P., Tissier, A., Kliebenstein, D.J. and K. Dehesh. (2017) Induction of ER body formation and indole glucosinolate metabolism is initiated by the plastidial retrograde signaling metabolite, MEcPP. Molecular Plant 10(11) 1400-1416
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhang, W., Corwin, J.A., Eshbaugh, R., Copeland, D., Feusier, J., Chen, F., Atwell, S. and D.J. Kliebenstein (2017) Plastic transcriptomes stabilize immunity to pathogen diversity: The jasmonic acid and salicylic acid networks within the Arabidopsis/Botrytis pathosystem. Plant Cell 29(11)2727-2752
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kerwin, R.E., Feusier, J., Rubin, M., Corwin, J.A., Lin, C., Muok, A., Larson, B., Li, B., Joseph, B., Francisco, M., Copeland, D., Weinig, C. and D.J. Kliebenstein. (2017) Epistatic by Environment Interactions Among Arabidopsis thaliana Glucosinolate Genes Impact Complex Traits and Fitness in the Field. New Phytologist 215(3)1249-1263
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Markelz R.J.C., Covington M.F., Brock M., Devisetty U.K., Kliebenstein D.J., Weinig C. and J.N. Maloof. (2017) Using RNA-seq for genomic scaffold placement, correcting assemblies, and genetic map creation in a common Brassica rapa mapping population. G3 5(7)2259-2270
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wisecaver, J.H., Borowsky, A.T., Tzin, V., Jander, G., Kliebenstein, D.J. and A. Rokas (2017) A global co-expression network approach for connecting genes to specialized metabolic pathways in plants. Plant Cell 29(5)944-959
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Douglas, S.J., Li, B., Kliebenstein, D.J., Nambara, E. and C.D. Riggs. (2017) A novel FILAMENTOUS FLOWER mutant suppresses brevipedicellus developmental defects and modulates glucosinolate and auxin levels. PLoS One 12(5) e0177045
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Copley, T.R., Aliferis, K.A., Kliebenstein, D.J. and S. Jabaji. (2017) An integrated RNAseq-1H NMR Metabolomics approach to understand Soybean primary metabolism regulation in response to rhizoctonia foliar blight disease. BMC Plant Biology 17(84)1-18
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Corwin, J.A. and D.J. Kliebenstein (2017) Quantitative Resistance: More than perceiving a pathogen. Plant Cell 29(4) 655-665


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with researchers from different breeding companies. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. More recently, I have been able to expand the target audience into companies working on using glucosinolates as nutritional supplements by having discussions with Jarrow Formulas, a world leader in nutritional supplement production. In addition consultations were established with Arvegenix, Inc to aid in the establishment of a new glucosinolate containing cover crop for midwestern Soybean production. For the Academic community, I gave numerous seminars and published 14 journal articles and book chapters to disseminate our research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The research project in the past year allowed several opportunities for training and professional development. The two graduate students (Michelle Tang and Nicole Soltis) have attended one or more research meetings wherein they presented their research as posters. These meetings were meetings with less than 80 attendees allowing for extensive interactions allowing both Miss Tang and Miss Soltis to develop their research connections and to practice presenting their work. The project is also becoming a platform whereby myself, Nicole Soltis and Michelle Tang are working to develop a novel cohort based undergraduate training regime wherein we hire three undergraduates who are willing to commit to at least two years of research. These students are developing their own research project wherein they are investigating the effect of hybrid vigor on defense metabolism and pathogen resistance. This is occurring within the confines of the larger project an the goal is to train them to be the equivalent of mid-career graduate students by the end of their tenure. This effort is currently in its third year and we are recruiting a new cohort of students. The previous cohort graduated in the past year and they are either working in the biotech industry or pursuing graduate school studies. How have the results been disseminated to communities of interest?The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with researchers from different breeding companies. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. More recently, I have been able to expand the target audience into companies working on using glucosinolates as nutritional supplements by having disccusions with Jarrow Formulas, a world leader in nutritional supplement production. In the past several months, the lab has begun assisting in the efforts to develop a new crop. Consultations were established with Arvegenix, Inc to aid in the establishment of a new glucosinolate containing cover crop for midwestern Soybean production. For the Academic community, I gave numerous seminars and published 14 journal articles and book chapters to disseminate our research. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we are working to clone the genes that control the ability of glucosinolates to regulate defense and development. We are also working to identify common regulatory factors that modulate both primary and secondary metabolism. The identification of these genes will allow us to better understand how this metabolic cross-talk occurs and what fitness benefits it may provide to the plant. We are also working to more formally test how glucosinolates may provide resistance to fungal pathogens in both Brassica and Arabidopsis as well as to extend this analysis beyond Brassica.

Impacts
What was accomplished under these goals? In the past year, we have had three major accomplishments that have helped us to further our goal of understanding the genetic architecture of glucosinolate metabolism. I have briefly described these accomplishments below. First in a suite of papers, we showed that a specific glucosinolate, allyl glucosinolate, can directly regulate plant growth and development. Defense metabolites are typically considered to be downstream outputs of the plant with little to no direct consequence to the plant other than by acting as toxins to defeat biotic attackers. Our efforts are beginning to overturn this long term view and we have recently shown that allyl glucosinolate can lead to altered plant growth and appears to function as a direct growth signal. Using genome-wide association mapping, we could show that this functions via an interplay of secondary metabolism and what appears to be a novel branch of the key plant hormone, auxin, signaling pathway. Thus, glucosinolates are not solely defensive toxins but also have the ability to be regulatory compounds in planta. This is modulated by the specific structure of the glucosinolate and future work is required to assess if other glucosinolates can also act as signals. Secondly, we have begun to identify additional defensive roles genes for glucosinolate metabolism. Modulating glucosinolate metabolism within Brassica napus showed that altering glucosinolate biosynthesis could lead to altered resistance to generalist necrotrophic pathogens including, Sclerotinia sclerotorium and Botrytis cinerea. Extending this work to studying quantitative resistance of Arabidopsis thaliana to Botrytis cinerea identified natural variation in glucosinolate metabolism as a key component of variation in resistance to this generalist pathogen. We are extending this work to assess if this is because of the toxicity of these compounds, their potential signaling roles or perhaps a blend of both functions that leads to this biological role. Finally, we have begun using our knowledge in the glucosinolate pathway to assess how to utilize systems biology and genomics analysis to understand what these studies are telling the researcher. As a key to this effort, we have been mapping the transcriptional networks underpinning both primary and secondary metabolism within Arabidopsis thaliana. This was extended by utilizing network analysis and theory to investigate the correlational structure of metabolite variation within Arabidopsis, Tomato and rice to directly test if the correlational structure really provides biological insight and if this insight requires measuring all of metabolism. This work showed that it was not necessary to measure all of metabolism and that by using the known structure of the metabolic network, it was possible to identify a very small subset of metabolites whose measurement would provide 90% or more of the network information. This work would allow a researcher to greatly streamline data collection and analysis in any effort to utilize metabolomics for biological insight.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cacho, N.I. and D.J. Kliebenstein (2016) Nonlinear selection and a blend of convergent, divergent and parallel evolution shapes natural variation in glucosinolates. Advances in Botanical Research 80:31-55
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Schwahn, K., K�ken, Kliebenstein, D.J., A., Fernie, A.R., and Z. Nikoloski. (2016) Observability of plant metabolic networks is reflected in the correlation of metabolic profiles. Plant Physiology 172(2)1324-33
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Li, B., Zhang, Y., Mohammadi, S.A., Huai, D., Zhou, Y., and D.J. Kliebenstein. (2016) An Integrative Genetic Study of Rice Metabolism, Developmental Growth and Stochastic Variation Reveals Potential C/N Partitioning Loci. Scientific Reports 6:30143
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Francisco, M., Joseph, B., Calligan, H., Li, B., Corwin, J.A., Lin, C., Kerwin, R., Burow, M. and D.J. Kliebenstein. (2016) Genome wide association mapping in Arabidopsis thaliana identifies novel genes involved in linking allyl glucosinolate to altered growth and defense. Frontiers in Plant Science. 7:1010
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kliebenstein, D.J. (2016) False idolatry of the mythical growth versus immunity tradeoff in molecular systems plant pathology. Physiological and Molecular Plant Pathology 95:55-59
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Francisco, M., Joseph, B., Calligan, H., Li, B., Corwin, J.A., Lin, C., Kerwin, R., Burow, M. and D.J. Kliebenstein. (2016) The defense metabolite, Allyl glucosinolate, modulates Arabidopsis thaliana growth dependent upon the endogenous glucosinolate pathway. Frontiers in Plant Science. 7:774
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Lachowiec, J., Queitsch, C. and D.J. Kliebenstein. (2016) Molecular mechanisms governing differential robustness of development and environmental responses in plants. Annals of Botany 117(5)795-809
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Corwin, J.A., Eshbaugh, R., Subedy, A. and D.J. Kliebenstein (2016) Expansive Phenotypic Landscape of Botrytis cinerea shows Differential Contribution of Individual Genetic Diversity and Plasticity. Molecular Plant Microbe Interactions 29(4)287-298
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Bethke, G., Thao, A., Xiong, G., Li, B., Soltis, N.E., Kliebenstein, D.J., Katagiri, F., Pauly, M. and J. Glazebrook. (2016) Pectin biosynthesis is critical for cell wall integrity and immunity in Arabidopsis thaliana. Plant Cell 28(2)537-556
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Corwin, J.A., Copeland, D., Feusier, J., Subedy, A., Eshbaugh, R., Palmer, C., Maloof, J. and D.J. Kliebenstein (2016) The Quantitative Basis of the Arabidopsis Innate Immune System to Endemic Pathogens Depends on Pathogen Genetics. PLoS Genetics 12(2):e1005789
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Gaudinier, A., Tang, M. and D.J. Kliebenstein (2015) Transcriptional networks governing plant metabolism. Current Plant Biology 3-4:56-64
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Soltis, N.E. and D.J. Kliebenstein. (2015) Natural variation of plant metabolism: genetic mechanisms, interpretive caveats, evolutionary and mechanistic insights. Plant Physiology 169(3)1456-68
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang, Y., Huai, D., Yang, Q., Cheng, Y, Ma, M., Kliebenstein, D.J. and Y. Zhou. (2015) Overexpression of three glucosinolate biosynthesis genes in Brassica napus identifies enhanced resistance to Sclerotinia sclerotorium and Botrytis cinerea. PLoS One 10(10)e0140491
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang, W., Kwon, S.-T., Chen, F. and D.J. Kliebenstein (2016) Isolate dependency of Brassica rapa resistance QTLs to Botrytis cinerea. Frontiers in Plant Science 7:161


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with researchers from different breeding companies. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. More recently, I have been able to expand the target audience into companies working on using glucosinolates as nutritional supplements by having disccusions with Jarrow Formulas, a world leader in nutritional supplement production. For the Academic community, I gave numerous seminars and published 14 journal articles and book chapters to disseminate our research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The research project in the past year allowed several opportunities for training and professional development. The senior graduate student on the project, Jason Corwin, gave several seminars at USC, UNC and PSU on the project. This gave him significant experience in developing his presentation style and allowed him to obtain several offers for post-doctoral researcher to apply his toolset developed via this project to other avenues. Additionally, Rachel Kerwin, another senior graduate student on the project gave several seminars at various universities in her search for a post-doctoral position. This was in addition to her presentation at the International Ecology and Evolution meetings. This provided her significant experience in her own presentation style and led to several post-doctoral research offers. Similarly, two junior graduate students (Michelle Tang and Nicole Soltis) have attended one or more international research meetings wherein they presented their research as posters. These meetings were international meetings with less than 80 attendees allowing for extensive interactions allowing both Miss Tang and Miss Soltis to develop their research connections and to practice presenting their work. The project is also becoming a platform whereby myself, Nicole Soltis and Michelle Tang are working to develop a novel cohort based undergraduate training regime wherein we hire three undergraduates who are willing to commit to at least two years of research. These students are developing their own research project wherein they are investigating the effect of hybrid vigor on defense metabolism and pathogen resistance. This is occurring within the confines of the larger project an the goal is to train them to be the equivalent of mid-career graduate students by the end of their tenure. This effort is currently in its second year and has eight undergraduate students. How have the results been disseminated to communities of interest?The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with researchers from different breeding companies. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. More recently, I have been able to expand the target audience into companies working on using glucosinolates as nutritional supplements by having disccusions with Jarrow Formulas, a world leader in nutritional supplement production. In addition to my outreach efforts, the projects outreach efforts were greatly expanded by the presentation of four seminars by the senior graduate student, Dr. Jason Corwin, to researchers at universities during his search for a post-doctoral research position. For the Academic community, I gave numerous seminars and published 14 journal articles and book chapters to disseminate our research. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we are working to clone the genes that control the ability of glucosinolates to regulate defense and development. The identification of these genes will allow us to better understand how this metabolic cross-talk occurs and what fitness benefits it may provide to the plant. We are also working to more formally test how glucosinolates may provide resistance to fungal pathogens in both Brassica and Arabidopsis as well as to extend this analysis beyond Brassica.

Impacts
What was accomplished under these goals? In the past year, we have had three major accomplishments that have helped us to further our goal of understanding the genetic architecture of glucosinolate metabolism. I have briefly described these accomplishments below. First, at the conclusion of a multiple year field trial experiment, we were able to show for the first time that specific glucosinolate gene variants do alter fitness in the field using Arabidopsis thaliana. This involved the planting of dozens of specific gene variants in the field and measuring fitness over several years at two different field sites. Critically, this showed that the fitness effects were always significant but that the optimal genotype differed every year in every field site. This provides some of the first empirical evidence of fluctuating selections' effects on specific genotypes. This shows that natural genetic variation can be maintained by selection and that selection does not necessarily favor a single genotype. Thus, in this system, there is no optimal genotype for all conditions and if a breeder was going to apply these lessons to an agronomic system they would have to allow for a system to have a suite of genotypes all inter-planted. Secondly, in a suite of papers, we showed that the glucosinolate metabolites directly regulate plant growth and development as well as defense signaling. Defense metabolites are typically considered to be downstream outputs of the plant with little to no direct consequence to the plant other than by acting as toxins to defeat biotic attackers. Our efforts are beginning to overturn this long term view and we have recently shown that a specific glucosinolate can regulate jasmonic acid wound signaling. Jasmonic acid signaling is known to regulate glucosinolates and as such, this has established a feedback loop whereby the defense output, glucosinolate metabolite, can be used to regulate the upstream signaling pathway. Similarly, we have shown that glucosinolates can control flowering time in both the lab and the field. Thus, glucosinolates are not solely defensive toxins but also have the ability to be regulatory compounds in planta. Finally, we have begun to identify additional defensive roles and regulatory genes for glucosinolate metabolism. This has shown that new glucosinolate enzyme genes do not require the evolution of new regulatory transcription factors but these new genes can be regulated by conserved transcription factors that are far older then the genes being regulated. These regulatory properties are key to control the expression of the glucosinolate genes to allow them to provide fungal resistance in a number of Brassica plants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cacho, N.I., Kliebenstein, D.J. and S.Y. Strauss. (2015) Macroevolutionary patterns of glucosinolate defense and tests of the Resource Availability and Defense-Escalation hypothesis in Streptanthus (s.l. Brassicaceae). New Phytologist 508(3)915-927
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Atwell, S. Corwin, J.A., Subedy, A., Denby, K.J. and D.J. Kliebenstein (2015) Whole genome resequencing of Botrytis cinerea isolates identifies genetic underpinnings for a broad host lifestyle. Frontiers in Microbiology. 6:996
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Brady, S.M., Burow, M., Busch, W., Carlborg, �, Denby, K.J., Glazebrook, J., Hamilton, E., Harmer, S.L., Haswell, E.S., Maloof, J.N., Springer, N.M. and D.J. Kliebenstein (2015) Reassess the t-test: interact with all your data via ANOVA. Plant Cell 27(8)2088-94
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Joseph, B. Lau, L. and D.J. Kliebenstein. (2015) Quantitative variation in responses to root spatial constraint within Arabidopsis thaliana. Plant Cell 27(8)2227-43
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Jensen, L.M., Halkier, B.A., Kliebenstein, D.J. and M. Burow. (2015) Investigation of the multifunctional gene AOP3 expands the regulatory network controlling glucosinolate production. Frontiers in Plant Science 6:762
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Jensen, L.M., Henriette, S.K.J., Halkier, B.A., Kliebenstein, D.J. and M. Burow. (2015) Natural variation in cross-talk between glucosinolates and onset of flowering in Arabidopsis. Frontiers in Plant Science 6:697
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Burow, M., Atwell, S., Fancisco-Candeiro, M., Kerwin, R.E., Halkier, B.A. and D.J. Kliebenstein (2015) The glucosinolate biosynthetic gene AOP2 mediates feedback regulation of jasmonic acid signaling independent of its known enzymatic function. Molecular Plant 8(8)1201-12
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Nafisi, M., Stranne, M., Fimognari, L., Atwell, S., Martens, H.J. , Pedas, P., Hansen, S.F., Nawrath, C., Scheller, H.V., Kliebenstein, D.J. and Y. Sakuragi. (2015) Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses. Frontiers in Plant Science 6:550
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang, Y., Li, B., Huai, D., Zhou, Y. and D.J. Kliebenstein (2015) The conserved transcription factors, MYB115 and MYB118, control expression of the newly evolved benzoyloxy glucosinolate pathway in Arabidopsis thaliana. Frontiers in Plant Science 6:343
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Liu, J.D., Goodspeed, D., Sheng, Z., Li, B., Yang, Y., Kliebenstein, D.J., J. Braam. (2015) Keep the clock running: light/dark cycles during postharvest storage preserve the tissue integrity and nutritional content of leafy plants. BMC Plant Biology 15(1)92
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Sohrabi, R., Huh, J.-H., Badieyan, S., Harinantenaina, L., Kingston, D.G.I., Kliebenstein, D.J., Sobrado, P. and D. Tholl. (2015) In Planta variation of volatile biosynthesis: An alternative biosynthetic route to the formation of the pathogen-induced volatile homoterpene DMNT via triterpene degradation in Arabidopsis roots. Plant Cell 27(3)874-890
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kerwin, R.E., Feusier, J., Rubin, M., Corwin, J.A., Lin, C., Muok, A., Larson, B., Li, B., Joseph, B., Francisco, M., Copeland, D., Weinig, C. and D.J. Kliebenstein (2015) Field evidence that fluctuating selection can maintain natural genetic variation in Arabidopsis thaliana defense. eLife 4:e05604
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Taylor-Teeples, M., Lin, L., Trabucco, G.M., de Lucas, M., Turco, G., Doherty, C., Toal, T.W., Gaudinier, A., Young, N.F., Xiong, G., Corwin, J., Tsoukalas, N., Pauly, M., Kliebenstein, D.J., Tagkaopoulous, I., Breton, G., Ahnert, S., Kay, S.A., Brady, S.M. and S.P. Hazen. (2015) Environmental, Developmental and genotype-dependent regulation of xylem cell specification and secondary cell wall biosynthesis in Arabidopsis thaliana. Nature 517(7536)571-5
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Joseph, B., Corwin, J.A. and D.J. Kliebenstein. (2015) Genetic variation in the nuclear and organellar genomes control stochastic variation in the metabolome PLoS Genetics 11(1)e1004779


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with researchers from different breeding companies. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. More recently, I have been able to expand the target audience into companies working on using glucosinolates as nutritional supplements by having disccusions with Jarrow Formulas, a world leader in nutritional supplement production. For the Academic community, I gave numerous seminars and published 9 journal articles and book chapters to disseminate our research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The research project in the past year allowed several opportunities for training and professional development. Foremost, one of the post-doctoral researchers working on this project, Dr. Bindu Joseph, gave numerous seminars to industry and was hired as a senior researcher at Bayer Crop Science in North Carolina to work on plant breeding solutions. This is direct evidence that the project is developing individuals in demand in industry. The projects junior graduate student, Michelle Tang, received a NSF pre-doctoral fellowship and was able to attend the International Statistics Course at the University of Washington over the summer to familiarize herself with large dataset analysis. The senior graduate student on the project, Jason Corwin, gave three seminars at USC, UNC and PSU on the project. This gave him significant experience in developing his presentation style and allowed him to obtain several offers for post-doctoral researcher to apply his toolset developed via this project to other avenues. The project is also becoming a platform whereby myself, Dr. Baohua Li and Michelle Tang are working to develop a novel cohort based undergraduate training regime wherein we hire three undergraduates who are willing to commit to at least two years of research. These students will develop their own research within the confines of the larger project an the goal is to train them to be the equivalent of mid-career graduate students by the end of their tenure. This effort started this past year. How have the results been disseminated to communities of interest? The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with researchers from different breeding companies. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. More recently, I have been able to expand the target audience into companies working on using glucosinolates as nutritional supplements by having disccusions with Jarrow Formulas, a world leader in nutritional supplement production. In addition to my outreach efforts, the projects outreach efforts were greatly expanded by the presentation of four seminars by the senior post-doctoral researcher, Dr. Bindu Joseph, to different breeding companies. For the Academic community, I gave numerous seminars and published 9 journal articles and book chapters to disseminate our research. What do you plan to do during the next reporting period to accomplish the goals? In the next year, we are working to understand how glucosinolates themselves can feed-back regulate numerous core physiological processes such as growth and development. We are working to clone genes that control this process to better understand what this metabolic cross-talk is providing to the plant. Interestingly, the plant appears to specifically recognize numerous glucosinolates to regulate different transcription and growth processes. Thus, this feedback regulation is central to how the glucosinolate pathway incorporates into the plant.

Impacts
What was accomplished under these goals? In the past year, we had two key accomplishments that are focused upon our major goal of understanding the genetic architecture of glucosinolates. I have briefly described these accomplishments below. First, we collaborated with the lab of Dr. Siobhan Brady here at the University of California, Davis to investigate the scale of the transcriptional regulatory network that influences the accumulation of glucosinolates within Arabidopsis thaliana. This work utilized a Yeast-1-hybrid approach wherein we cloned all of the promoters for the genes encoding enzymes for glucosinolate biosynthesis and systematically tested ~760 transcription factors for the ability to bind these promoters. This analysis showed that there are likely at least 100 different transcription factors that can modulate the expression of genes in the pathway. A validation test with mutants showed that at least 80% of these can alter glucosinolate accumulation within the plant. This work suggests that the linear master regulatory model for the control of metabolites is likely incorrect. Instead, when working to modulate glucosinolates, the breeder will need to consider a more complex intertwined model whereby at least 80 transcription factors can modulate the accumulation of these metabolites. The identity of these transcription factors shows that they function to link defense metabolism with numerous biotic and abiotic inputs generating a model whereby the metabolite accumulation can be finely tuned in response to a highly variable environment. Our second major accomplishment focused upon understanding the genetic architecture of glucosinolates and other traits came from a meta-analysis of quantitative trait locus mapping studies. In this work, we investigated all of our previous quantitative trait locus mapping studies to obtain an estimate of how many loci are found per population depending upon the size of the population. Previous work had suggested that the relationship between population size and quantitative trait locus number was log linear with a maximum number of quantitative trait loci found at somewhere between 200 and 400 individuals. Our work however showed that there was no difference in the log-linear and linear models for this range and that populations needed to be at least 1000 individuals for simple populations with only two parents before we could tell which model was most accurate. This work suggests that we may be vastly underestimating the number of loci controlling any given phenotype in nearly every population tested to date. Thus, this work shows that we need larger simple populations to truly measure the genetic architecture of any given trait.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Trontin, C., Kiani, S., Corwin, J.A., Hematy, K., Yansouni, J., Kliebenstein, D.J. and O. Loudet. (2014) A tandem of receptor-like kinases is responsible for natural variation in shoot growth response to mannitol treatment in A. thaliana. Plant Journal 78(1)121-33
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: D.J. Kliebenstein (2014) The orchestration of plant defense systems; genes to populations. TIPS 19(4)250-255
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: D.J. Kliebenstein (2014) Interview with Daniel Kliebenstein TIPS 19(4)204-205
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: D.J. Kliebenstein (2014) Synthetic biology of metabolism: Using natural variation to reverse engineer systems Current Opinion in Plant Biology 19(1)20-26
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: D.J. Kliebenstein (2014) Quantitative Genetics and Genomics of Plant Resistance to Insects. Annual Plant Reviews 47(1)235-262
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Li, B and D.J. Kliebenstein (2014) Metabolic Network Modulator 1, an At-hook motif containing gene with pleiotropic effects on natural variation in primary metabolism. Frontiers in Plant Science 5(1)415
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Joseph, B. Atwell, S. and D.J. Kliebenstein (2014) Meta-analysis of Metabolome QTLs in Arabidopsis: Can we estimate the network size controlling genetic variation of the metabolome. Frontiers in Plant Science 5(1)461
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Li, B., Gaudinier, A., Taylor-Teeples, M., Nham, N.T., Ghaffari, C., Benson, D.S., Steinmann, M., Gray, J.A., Brady, S.M. and D.J. Kliebenstein. (2014) Promoter based integration in plant defense regulation Plant Physiology 166(4)1803-20
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kwon, S.-T. and D.J. Kliebenstein (2014) Response of Turnip to Botrytis cinerea infection and their relationship with glucosinolate profiles. Korean J. Plant Res. 27(4): 371-9


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with different large breeding companies like BASF. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. For the Academic community, I gave numerous seminars and published 7 journal articles and book chapters to disseminate our research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The research project in the past year allowed several opportunities for training and professional development. The senior graduate student on the project, Jason Corwin, spent several weeks collaborating in Europe where he spent time training other graduate students in genotyping by sequencing approaches. Additionally he attended several international meetings during this time and presented his work at these meetings and at a number of other research labs. This provided him with excellent exposure. Additionally the research project required significant computational avenues. Instead of collaborating to accomplish this, the senior postdoc and senior graduate student in the lab worked to learn computer programming for digital image analysis and computational statistics. This allowed them to extend their skill sets beyond wet lab research into in silico research. How have the results been disseminated to communities of interest? The target audience for this project is large breeding companies and other genomics industries in addition to academic research labs. To facilitate this I gave several presentations and had a number of discussions with different large breeding companies like BASF. In addition I had a number of consultations with smaller focused breeding companies like D'Arrigo brothers on the breeding of glucosinolate crops. For the Academic community, I gave numerous seminars and published 7 journal articles and book chapters to disseminate our research. What do you plan to do during the next reporting period to accomplish the goals? In the next year, we are working to clone additional genes controlling the quantitative variation in glucosinolates. Further, we have extended to doing whole genome Yeast-1-hybrid analysis to identify the complete complement of transcription factors for the entire pathway. This is critical to understand how the pathway is regulated at the whole genome level. We anticipate that both of these approaches will be published in the upcoming year. In coordination with other groups we are also working to identify genes that control how the plant perceives the glucosinolates within the plant. The plant appears to specifically recognize a number of its own chemicals to regulate both transcription and growth by unknown mechanisms. Thus, this feedback regulation is central to how the pathway is regulated.

Impacts
What was accomplished under these goals? In the past year we have had three critical accomplishments geared towards the major goal of understanding the genetic architecture of glucosinolates. I have briefly described each of these accomplishments below. First, we collaborated to show that glucosinolates show circadian oscillations in cabbages and Arabidopsis post-harvest. In our work, we showed that the phytonutrient glucosinolate content of brassica's has a wide range of variation across the day even after harvest and that cold-storage locked the plant into lower levels. This suggests that it may be possible to make vegetables and produce healthier by manipulating their storage environment after harvest. It also suggests that certain vegetables and produce may have optimal times of day in which they should be consumed or processed to maximize their nutritional value. This had been known for some vitamins but this was the first evidence that the broader array of phytonutrients may also be subject to this circadian oscillation both pre- and post-harvest. Secondly, we showed that attempting to link across different phenotypes using natural genetic variation is fraught with a high level of error particularly false negative error where associations between phenotypes can be missed. Innumerable studies measure the phenotypic variation of several phenotypes in common populations to attempt and link the phenotypes. Often this is looking at traits important for yield and defense or yield and nutritional content. These studies often show no coordination in these traits yet when working to breed for yield there is always a residual link. We looked at glucosinolate and growth variation in a set of Arabidopsis populations in which we knew the existence of a link and the mechanism by which it occurs. Standard genetic analysis indicated that these two phenotypes were unlinked even though we knew the must be coordinated. A deeper analysis showed that we could identify this link but had to move from standard approaches to a more powerful ANCOVA based methodology. Thus, the studies suggesting that there is no linkage across different phenotypes in genetic mapping populations have to be re-evaluated with these new methods to ensure that these observations were not erroneous. Finally, we showed that genomic variation within the organellar genomes, mitochondrial and plastidic, is a key component of natural variation in most eukaryotic organisms. In most studies of quantitative genetic variation, the research focuses on variation in the nuclear genome due to technical and biological simplification. However, this left open the question of how genetic variation within the organellar genomes could influence phenotypic variation. Our work showed that the organellar genomic variation had a large consequence upon metabolic and growth variation. However, this was not additive variation and instead it appeared to be caused by epistatic interactions with nuclear loci. These interactions were frequently with pairs of nuclear loci. This suggests that the organellar genomic variation must be included in natural variation studies as well as in breeding programs. By including this variation in breeding programs, it may be possible to increase our ability to conduct predictive breeding because up to this point, the organelle was an ignored aspect of the genetic equation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Joseph, B., Corwin, J.A., Li, B., Atwell, S. and D.J. Klibenstein (2013) Cytosolic genetic variation and extensive cytonuclear interactions influence natural variation in the metabolome. eLife 2:e00776
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Joseph, B., Corwin, J.A., Z�st, T., Li, B., Iravani, M., Schaepman-Strub, G., Turnbull, L.A., and D.J. Kliebenstein. (2013) Hierarchical nuclear and cytoplasmic genetic architectures for plant growth and defense within Arabidopsis. Plant Cell 25(6)1929-45.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Goodspeed, D., Liu, J.D., Chehab, E.W., Sheng, Z., Francisco, M., Kliebenstein, D.J. and J. Braam. (2013) Post-harvest circadian entrainment enhances crop pest resistance and phytochemical cycling. Current Biology 23(13)1-7
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Atwell, S. and D.J. Kliebenstein (2013) Conducting genome wide association mapping of metabolites The Handbook of Plant Metabolomics . 1(1)255-73
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: D.J. Kliebenstein (2013) Regulatory evolution, the veiled world of chemical diversification Journal of Chemical Ecology 39(3)349
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: D.J. Kliebenstein (2013) Making new molecules - evolution of structures for novel metabolites in plants Current Opinion in Plant Biology 16(1)112-117


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: In the past year, we have made significant progress in understanding two major areas of secondary metabolite plant biology. These areas are biological activity and computational analysis of genetic architecture. Plant secondary metabolites have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. However, these phytonutrients also help the plant in defending against various biological pests such as insects or fungi. We were able to use a multi-generational competition experiment to show that different profiles of glucosinolates were beneficial under diverse plant/aphid interactions. Interestingly it was not that a single profile was best but that each profile was good under certain conditions. We were then able to transition this to show how the prevalence of aphids in the natural environment selected for distinct glucosinolate profiles across the European continent. This is one of the first instances where competition between multiple aphid species and a single plant has been shown to determine genetic diversity on a continental scale. In this analysis we also found indications that other loci were being structured by aphid competition and direct competition between plants for resources and we are working to better understand this structuring. Secondary metabolite accumulation is typically controlled by numerous quantitative trait loci (QTLs) that epistatically interact to determine the profile. Identifying each of these individual loci is time-consuming. Thus, we worked to develop a series of computational and systems biological analyses over the past year to test how to best tweeze apart the link of the loci to each other and to the underlying metabolomics. This allowed us to develop approaches to rapidly link genes to causing specific QTLs. Unexpectedly, our computational analysis of metabolic networks showed that all existing approaches were unable to properly generate the genetic network controlling glucosinolate metabolism. All of these approaches are fundamental to the analysis of quantitative variation in most organisms and this was the first comparison of predicted to empirical networks. We are testing this analysis to see if we can improve computational network prediction approaches. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: The target audiences for this research are largely the breeding companies as there are not sufficient funds for individual producers to implement separate breeding projects. Additionally, the specific crop research boards also are not sufficiently funded to implement significant breeding projects with a long term outlook. To transmit the knowledge from this project to these target audiences, I am presenting seminars at numerous companies and having informal discussions with several of them to transmit ideas on glucosinolate variation and how this can be implemented into their breeding design. I am also utilizing the observations about genomics and systems biology of the glucosinolate pathway to expand these discussions to other phenotypes and systems outside of Brassicas and glucosinolates. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Glucosinolates are the major secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc. They impact nutritional quality by acting as anti-cancer agents in human diet and also defend the crop against insect herbivory. In the past year, we have worked to develop new computational approaches that would speed up the rate of identification of genes in any potential plant species. These new systems biological approaches can be utilized for any phenotype but we have largely focused them on plant secondary metabolites as well as primary metabolism. This would help to improve the ability to conduct breeding in numerous plant species simultaneously improve nutritional content and herbivore defense into these plants. This would benefit both the consumer in improved health but also the producer through decreased pesticide input. Finally, the identification of methodologies to more rapidly identify and characterize QTL greatly enhances and facilitates future plant breeding independent of the crop and/or species. As most agronomic improvements are accomplished through plant breeding, these methodology improvements could speed up the development of any crop. We are working to understand how different agronomic factors such as climate interact with these naturally variable neturaceuticals. We have moved one of these genes from the lab to the field to better understand how altering this gene can influence in a mechanistic context different agronomic outcomes. Specifically, this gene, Elong, changes the resistance to multiple different aphids but in a manner where the allele at this locus simply swaps the type of aphid that targets the plant. In some crops, one allele is preferred over another for human consumption so we can provide direct predictions about which aphids will be critical to control for each genotype of the Brassica crops. As these aphids have different seasonalities, we can provide direct information to the grower about when and how to conduct aphid suppression. This allows us to assist the grower in modifying their agronomic practices to increase the flavor and nutritional quality of the crucifer crop and optimize financial benefits to the producer and health/enjoyment benefits to the consumer. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet. To extend our knowledge about the agronomic consequences of plant secondary metabolism, we have recently expanded our systems approaches to begin understanding the systems description of plant/pathogen interactions that are in part controlled by glucosinolate related metabolites. Within this high-detail time course of the interaction of plants with the fungus Botrytis cinerea, we have shown that there is a single decision point in the progression of this disease that is critical for the development of a growing lesion. This decision point in part relies upon the plant chemical defense but is also the result of complex interaction of the plant and pathogen.

Publications

  • D.J. Kliebenstein 2012. "Model misinterpretation within biology; phenotypes, statistics, networks and inference". Frontiers in Plant Science 3(13)1-5.
  • Windram, O., Madhou, P. , McHattie, S., Hill, C., Hickman, R., Cooke, E., Jenkins, D., Penfold, C., Baxter, L., Breeze, E., Kiddle, S., Rhodes, J., Atwell, S., Kliebenstein, D.J., Kim, Y.-S., Stegle, O., Zhang, C., Tabrett, A., Legaie, R., Moore, J., Finkenstadt, B., Wild, D.L., Mead, A., Rand, D., Benyon, J., Ott, S., Buchanan-Wollaston, V. and K.J. Denby 2012. "Arabidopsis defence against Botrytis cinerea: chronology and regulation deciphered by high-resolution temporal transcriptomic analysis". Plant Cell 24(9)3530-57.
  • Zust, T., Heichinger, C., Grossniklaus, U., Harrington, Kliebenstein, D.J. and L.A. Turnbull 2012. "Natural enemies drive geographic variation in plant defence genes". Science 338(6103)116-119.
  • D.J. Kliebenstein 2012. "Exploring the shallow end; estimating information content in transcriptomics studies". Frontiers in Plant Science 3:213.
  • D.J. Kliebenstein 2012. "Plant defense compounds: Systems approaches to metabolic analysis". Annual Reviews of Phytopathology 50;155-173.
  • Osbourn, AE and D.J. Kliebenstein 2012. "Making new molecules, evolution of pathways for novel metabolites in plants". Current Opinion in Plant Biology 15(4)415-23.
  • Xiao, Y., Savchenko, T., Baidoo, E.E.K., Chehab, W.E., Hayden, D.M., Tolstikov, V., Corwin, J.A., Kliebenstein, D.J., Keasling, J.D. and K. Dehesh 2012. "Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes". Cell 149(7)1525-38.
  • Hageman Blair, R. Kliebenstein, D.J. and G.A. Churchill 2012. "What can causal networks tell us about metabolic pathways". PLoS Computational Biology 8(4)e1002458.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: In the past year, we have made significant progress in understanding three major areas of secondary metabolite plant biology. These areas are biological activity, genetic architecture of metabolic variation and regulation. Plant secondary metabolites have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. Phytonutrients also aid the plant in defending against various biological pests such as insects or fungi. We are currently using a model plant/pest interaction to begin understanding how genetic variation in the pest is able to overcome specific defenses within the plant and how broad this genetic variation may be. We have also shown that the plant is not able to differentiate between closely related insect herbivores. Secondary metabolite accumulation is typically controlled by quantitative trait loci (QTLs), each of which is time-consuming to identify. We are analyzing the genetic architecture controlling metabolism within wild plant accessions to test the ability of genome wide association (GWA) mapping to rapidly clone QTLs. This has shown that there are 100s to thousands of genes controlling natural variation in plant primary and secondary metabolism. We are applied new approaches for to use network analysis to identify over 20 novel genes involved in glucosinolate regulation within Brassicaceous plants. We have also extended our understanding of genetics to show that the stochastic variation in agronomically important traits is genetically controlled and as such it will be impossible to breed for an absolute phenotypic value. The glucosinolates chemical structure regulates its potential biological activity. For example, if the plant accumulates 4-methylthioalkyl glucosinolate instead of the anti-cancer 4-methylsulfinylalkyl glucosinolate, a difference of only an oxygen, the vegetable does not provide the same anti-cancer activity. We have identified a number of regulatory genes that control both the amount and specific structure of the glucosinolate and we are re-introducing them into the plant to test the impact on glucosinolate function. This has shown that the system is highly connected with at least 20 regulatory factors controlling the chemicals accumulation. As a new observation we have shown in the past year that the regulation is birectional with the metabolites controlling the activity of the transcription factors as well as the circadian clock. Thus, there is an intimate connection between glucosinolate content and plant growth suggesting a need to modify how companies plan to alter crucifer vegetables phytonutrient profiles either through traditional breeding or transgenic technologies. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: The target audiences for this research are largely the breeding companies as there are not sufficient funds for individual producers to implement separate breeding projects. Additionally, the specific crop research boards also are not sufficiently funded to implement significant breeding projects with a long term outlook. To transmit the knowledge from this project to these target audiences, I am presenting seminars at numerous companies and having informal discussions with several of them to transmit ideas on glucosinolate variation and how this can be implemented into their breeding design. I am also utilizing the observations about genomics and systems biology of the glucosinolate pathway to expand these discussions to other phenotypes and systems outside of Brassicas and glucosinolates. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Glucosinolates are the major secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc. They impact these vegetables nutritional quality by acting as anti-cancer agents in human diet and also defend the crop against insect herbivory. In the past year, we have identified at least 20 new genes controlling this process that makes it possible to simultaneously breed improved nutritional content and herbivore defense into these plants. This would benefit both the consumer in improved health but also the producer through decreased pesticide input. We are also working towards understanding how different agronomic factors such as seasonality and planting density alter the production of these phytonutrients. The genes we identified in the past year have allowed us to begin explaining in a mechanistic context the different agronomic inputs that can be expected to alter glucosinolate accumulation. We have also extended this to show how altering the glucosinolates is directly influencing the agronomic behavior of the plant. This is allowing us to assist the grower in modifying their agronomic practices to increase the flavor and nutritional quality of the crucifer crop thus optimizing financial benefits to the producer and health and enjoyment benefits to the consumer. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet. Finally, the identification of methodologies to more rapidly identify and characterize QTL greatly enhances and facilitates future plant breeding independent of the crop and/or species. We have recently expanded this to develop novel genome wide association approaches that will greatly benefit the plant breeding community. Finally, we have shown that it will be impossible to have absolute precision in glucosinolate breeding as there are genetic sources of variance within the chemicals accumulation.This is allowing us to show that individual technologies will not be the most successful and instead combinatorial systems approaches are required. As most agronomic improvements are accomplished through plant breeding, these methodology improvements could speed up the development of any crop.

Publications

  • Chan, E.K.F., Rowe, H.C., Corwin, J.A. Joseph, B. and D.J. Kliebenstein. (2011) Combining genome wide association mapping and transcriptional networks to identify novel genes controlling glucosinolates in Arabidopsis thaliana. PLoS Biology 9(8)e1001125.
  • Zust, T., Shimizu, K., Joseph, B., Kliebenstein, D.J. and L.A. Turnbull. (2011). Using knockout mutants to reveal the costs of defensive plant secondary metabolites. Proceedings of the Royal Society B: Biological Sciences 278(1718)2598-2603.
  • Bidart-Bouzat, M.G. and D.J. Kliebenstein. (2011) An ecological genomic approach challenging the paradigm of differential plant responses to specialist versus generalist insect herbivores. Oceologia 167(3)677-89.
  • Jimenez-Gomez, J.M., Corwin, J.A., Joseph, B., Maloof, J.N. and Kliebenstein, D.J. (2011) Genomic analysis of QTLs and genes altering natural variation in stochastic noise PLoS Genetics 7(9)e1002295.
  • Kliebenstein, D.J. (2011) The quantitative genetics of phenotypic error or uniformity Frontiers in Genetics 2(59)1-2.
  • Kliebenstein, D.J. (2011) Combining genomics platforms to find the genes and systems controlling adaptation in ecology and evolution. in Ecology in the Post Genomic Era / Omics reveal the life of plants. Eds. A.J. Nagano and S. I. Morinaga. Bun-ichi Sogo Shuppan Co., Tokyo, Japan, volume 34 pp 181-206
  • Chaplin Kramer, R. Kliebenstein, D.J., Chiem, A., Mills, N. and Kremen, C. (2011) Chemically mediated tritrophic interactions: opposing effects of glucosinolates on a specialist herbivore and its predators. Journal of Applied Ecology 48(4)880-887.
  • Chehab, E.W., Kim, S., Savchenko, T., Kliebenstein, D.J., Dehesh, K. and J. Braam. (2011) Intronic T DNA insertion renders Arabidopsis opr3 a conditional JA producing mutant. Plant Physiology 156(2)770-8.
  • Kerwin, R.E. Jimenez Gomez, J.M., Fulop, D. Harmer, S.L., Maloof, J.N. and Kliebenstein, D.J. (2011) Network quantitative trait loci mapping of circadian clock outputs identifies metabolic pathway to clock linkages in Arabidopsis. Plant Cell 23(2);471-85.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: In the past year, we have made significant progress in understanding three major areas of secondary metabolite plant biology. These areas are biological activity, genetic architecture of metabolic variation, glucosinolate hydrolysis control and glucosinolate biosynthesis . Plant secondary metabolites have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. Phytonutrients also aid the plant in defending against various biological pests such as insects or fungi. We are currently using a model plant/pest interaction to begin understanding how genetic variation in the pest is able to overcome specific defenses within the plant and how broad this genetic variation may be. Secondary metabolite accumulation is typically controlled by quantitative trait loci (QTLs), each of which is time-consuming to identify. We are analyzing the genetic architecture controlling metabolism within wild plant accessions to test the ability of genome wide association (GWA) mapping to rapidly clone QTLs. This has shown that GWA of plant metabolomics is highly susceptible to environmental interactions and natural selection. We are developing and testing new approaches for to use network analysis to interrogate GWA data to identify novel genes involved in glucosinolate regulation within Brassicaceous plants. The cruciferous phytonutrients, glucosinolates, are relatively inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We have extended our previous work to show how gene families homologous to previously identified genes are interacting to quantitatively control this important phenotype. This is illuminating a role for chaperones to aid the hydrolysis enzyme, myrosinase, to pass through the endoplasmic reticulum. The glucosinolates chemical structure regulates its potential biological activity. For example, if the plant accumulates 4-methylthioalkyl glucosinolate instead of the anti-cancer 4-methylsulfinylalkyl glucosinolate, a difference of only an oxygen, the vegetable does not provide the same anti-cancer activity. We have identified a number of regulatory genes that control both the amount and specific structure of the glucosinolate and we are re-introducing them into the plant to test the impact on glucosinolate function. Interestingly in the past year, we have used the identified genes to show that there is an intimate connection between glucosinolate content and plant growth suggesting a need to modify how companies plan to alter crucifer vegetables phytonutrient profiles either through traditional breeding or transgenic technologies. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The target audiences for this research are largely the breeding companies as there are not sufficient funds for individual producers to implement separate breeding projects. Additionally, the specific crop research boards also are not sufficiently funded to implement significant breeding projects with a long term outlook. To transmit the knowledge from this project to these target audiences, I am presenting seminars at numerous companies and having informal discussions with several of them to transmit ideas on glucosinolate variation and how this can be implemented into their breeding design. I am also utilizing the observations about genomics and systems biology of the glucosinolate pathway to expand these discussions to other phenotypes and systems outside of Brassicas and glucosinolates. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Glucosinolates are the major secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc. They impact these vegetables nutritional quality by acting as anti-cancer agents in human diet and also defend the crop against insect herbivory. We have identified numerous genes controlling this process that makes it possible to simultaneously breed improved nutritional content and herbivore defense into these plants. This would benefit both the consumer in improved health but also the producer through decreased pesticide input. We are also working towards understanding how different agronomic factors such as seasonality and planting density alter the production of these phytonutrients. This is being extended to testing how the time of day influences the content of these factors. This is allowing us to assist the grower in modifying their agronomic practices to increase the flavor and nutritional quality of the crucifer crop thus optimizing financial benefits to the producer and health and enjoyment benefits to the consumer. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet. Finally, the identification of methodologies to more rapidly identify and characterize QTL greatly enhances and facilitates future plant breeding independent of the crop and/or species. We have recently expanded this to develop novel genome wide association approaches that will greatly benefit the plant breeding community. This is allowing us to show that individual technologies will not be the most successful and instead combinatorial systems approaches are required. As most agronomic improvements are accomplished through plant breeding, these methodology improvements could speed up the development of any crop.

Publications

  • Chan, E.K.F., Rowe, H.C., Hansen, B.G, and Kliebenstein, D.J. (2010) 'The complex genetic architecture of the metabolome'. PLoS Genetics 6(11)e1001198.
  • Paul-Victor, C., Zust, T. Rees, M., Kliebenstein, D.J. and L.A. Turnbull (2010). 'A new method for measuring RGR can uncover the costs of defensive compounds in Arabidopsis thaliana.' New Phytologist 187(4)1102-11.
  • Chan, E.K.F., Rowe, H.C. and Kliebenstein, D.J. (2010) 'Understanding the evolution of defense metabolites in Arabidopsis thaliana using genome-wide association mapping' Genetics 185(3)991-1007.
  • Burow, M, Halkier, B.A. and D.J. Kliebenstein (2010) 'Regulatory networks of glucosinolates shape Arabidopsis thaliana fitness.' Current Opinion of Plant Biology 13(3)348-53.
  • Sonderby, I.E., Burow, M., Rowe, H.C., Kliebenstein, D.J. and B.A. Halkier (2010) A complex interplay of three R2R3 MYB transcription factors determines the profile of aliphatic glucosinolates in Arabidopsis. Plant Physiology 153(1)348-63.
  • Rowe, H.C. Walley, J.W., Corwin, J.A., Chan, E.K.F., Corwin, J.A., Dehesh, K. and Kliebenstein, D.J. (2010) 'Deficiencies in jasmonate-mediated plant defense reveal quantitative genetic variation in Botrytis cinerea pathogenesis' PLoS Pathogens 6(4):e1000861.
  • Rowe, H.C. and Kliebenstein, D.J. (2010) 'All mold is not alike: the importance of intraspecific diversity in necrotrophic plant pathogens' PLoS Pathogens 6(3):e1000759.
  • Kliebenstein, D.J. (2010) 'Systems biology uncovers the foundation of natural genetic diversity' Plant Physiology 152(2)487-499.
  • Agee, A.E., Surpin, M. Sohn, E.J., Girke, T., Rosado, A., Kram, B.W., Carter, C., Wentzell, A.M., Kliebenstein, D.J., Jin, H.C., Park, O.K., Jin, H., Hicks, G.R., and N. Raikhel. (2010) 'MODIFIED VACUOLE PHENOTYPE1 is an Arabidopsis myrosinase-associated protein involved in endomembrane protein trafficking.' Plant Physiology 152(1)120-132.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: In the past year, we have made significant progress in understanding three major areas of secondary metabolite plant biology. These areas are biological activity, genetic architecture of metabolic variation, glucosinolate hydrolysis control and glucosinolate biosynthesis . Plant secondary metabolites have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. Phytonutrients also aid the plant in defending against various biological pests such as insects or fungi. We are currently using a model plant/pest interaction to begin understanding how genetic variation in the pest is able to overcome specific defenses within the plant and how broad this genetic variation may be. Secondary metabolite accumulation is typically controlled by quantitative trait loci (QTLs), each of which is time-consuming to identify. We are analyzing the genetic architecture controlling metabolism and transcriptional variation within Arabidopsis and rice to identify methodologies to allow for enhanced QTL cloning efficiency. This includes utilizing in-house developed novel approaches for developing biochemical pathways solely from highly-parallel genetic/phenotyping platforms. This was first implemented to recreate the glucosinolate pathway and is now being expanded to other pathways. We are also developing and testing new approaches for genome wide association mapping using the glucosinolate pathway as a test case. The cruciferous phytonutrients, glucosinolates, are relatively inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We have extended our previous work to show how gene families homologous to previously identified genes are interacting to quantitatively control this important phenotype. The glucosinolates chemical structure regulates its potential biological activity. For example, if the plant accumulates 4-methylthioalkyl glucosinolate instead of the anti-cancer 4-methylsulfinylalkyl glucosinolate, a difference of only an oxygen, the vegetable does not provide the same anti-cancer activity. We have identified a number of regulatory genes that control both the amount and specific structure of the glucosinolate and we are re-introducing them into the plant to test the impact on glucosinolate function. This includes the identification of a gene family of MYB transcription factors that are now being used by a number of companies to alter crucifer vegetables phytonutrient profile either through traditional breeding or transgenic technologies. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: The target audiences for this research are largely the breeding companies as there are not sufficient funds for individual producers to implement separate breeding projects. Additionally, the specific crop research boards also are not sufficiently funded to implement significant breeding projects with a long term outlook. To transmit the knowledge from this project to these target audiences, I am presenting seminars at numerous companies and having informal discussions with several of them to transmit ideas on glucosinolate variation and how this can be implemented into their breeding design. I am also utilizing the observations about genomics and systems biology of the glucosinolate pathway to expand these discussions to other phenotypes and systems outside of Brassicas and glucosinolates. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Glucosinolates are the major secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc. They impact these vegetables nutritional quality by acting as anti-cancer agents in human diet and also defend the crop against insect herbivory. We have identified numerous genes controlling this process that makes it possible to simultaneously breed improved nutritional content and herbivore defense into these plants. This would benefit both the consumer in improved health but also the producer through decreased pesticide input. We are also working towards understanding how different agronomic factors such as seasonality and planting density alter the production of these phytonutrients. This analysis is allowing us to assist the grower in modifying their agronomic practices to increase the flavor and nutritional quality of the crucifer crop thus optimizing financial benefits to the producer and health and enjoyment benefits to the consumer. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet. Finally, the identification of methodologies to more rapidly identify and characterize QTL greatly enhances and facilitates future plant breeding independent of the crop and/or species. We have recently expanded this to develop novel genome wide association approaches that will greatly benefit the plant breeding community. As most agronomic improvements are accomplished through plant breeding, these methodology improvements could speed up the development of any crop.

Publications

  • Burow, M, Halkier, B.A. and D.J. Kliebenstein (In Press - 2010) Regulatory networks of glucosinolates shape Arabidopsis thaliana fitness. Current Opinion of Plant Biology v(i)pp-pp.
  • Sonderby, I.E., Burow, M., Rowe, H.C., Kliebenstein, D.J. and B.A. Halkier (In Press - 2010) A complex interplay of three R2R3 MYB transcription factors determines the profile of aliphatic glucosinolates in Arabidopsis. Plant Physiology
  • Rowe, H.C. and Kliebenstein, D.J. (In Press - 2010) All mold is not alike: the importance of intraspecific diversity in necrotrophic plant pathogens. PLoS Pathogens
  • Chan, E.K.F., Rowe, H.C. and Kliebenstein, D.J. (In Press - 2010) Understanding the evolution of defense metabolites in Arabidopsis thaliana using genome-wide association mapping. Genetics v(i)pp-pp.
  • Kliebenstein, D.J. (In Press- 2010) Systems biology uncovers the foundation of natural genetic diversity. Plant Physiology v(i)pp-pp.
  • Agee, A.E., Surpin, M. Sohn, E.J., Girke, T., Rosado, A., Kram, B.W., Carter, C., Wentzell, A.M., Kliebenstein, D.J., Jin, H.C., Park, O.K., Jin, H., Hicks, G.R., and N. Raikhel. (2010) MODIFIED VACUOLE PHENOTYPE1 is an Arabidopsis myrosinase-associated protein involved in endomembrane protein trafficking. Plant Physiology 152(1)120-132.
  • Kliebenstein, D.J. (2009) Advancing genetic theory and application by metabolic QTL analysis. Plant Cell 21(6)1637-46.
  • Kliebenstein, D.J. (2009) Quantification of variation in expression networks. Methods Mol Biol 553:227-45.
  • Kliebenstein, D.J. (2009) Use of Secondary Metabolite Variation in Crop Improvement, in Plant-derived Natural Products: Synthesis, Function, and Application. Eds A.E. Osbourn and V. Lanzotti. Springer Science + Business Media, New York, pp 83-95.
  • Kliebenstein, D.J. and H.C. Rowe. (2009) Anti-rust Antitrust. Science 323(8):1301-2.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: In the past year, we have made significant progress in understanding three major areas of secondary metabolite plant biology. These areas are biological activity, genetic architecture of metabolic variation, glucosinolate hydrolysis control and glucosinolate biosynthesis (1-19). Plant secondary metabolites have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. Phytonutrients also aid the plant in defending against various biological pests such as insects or fungi. We are currently using a model plant/pest interaction to investigate the how phytonutrients such as glucosinolates and pest genetic variation interact to determine how the plant interact with its environment (1,4,14). Secondary metabolite accumulation is typically controlled by quantitative trait loci (QTLs), each of which is time-consuming to identify. We are analyzing the genetic architecture controlling metabolism within Arabidopsis and rice to identify methodologies to allow for enhanced QTL cloning efficiency (2,3,5,6,8,9,11,13,17-19). This includes utilizing in-house developed novel approaches for studying networks controlling metabolism (11,13,17). As QTL are a major target in plant breeding, these approach will allow more efficient germplasm development including the direct introduction of epistasis and genotype x environment into the breeding equation (13,15). The cruciferous phytonutrients, glucosinolates, are relatively inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We have extended our previous work to show how tissue development and agronomic factors influence the physiology and genetics of this phenotype (2,11,15,19). This work has also begun to increase our understanding of the number of genes controlling this important phenotype. The glucosinolates chemical structure regulates its potential biological activity. For example, if the plant accumulates 4-methylthioalkyl glucosinolate instead of the anti-cancer 4-methylsulfinylalkyl glucosinolate, a difference of only an oxygen, the vegetable does not provide the same anti-cancer activity. We have identified a number of biosynthetic genes and are re-introducing them into the plant to test the glucosinolates function (3,9,17). This includes the identification of a gene family for the enzyme required to produce anticancer glucosinolates (9) and the enzyme involved in producing the goiter causing, 2-hydroxy-but-3-enyl glucosinolate (3). We have also identified the source of the stereochemistry important for the anti-cancer glucosinolates activity (10). These genes are currently being utilized by several groups to alter crucifer vegetables phytonutrient profile either through traditional breeding or transgenic PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Glucosinolates are the major secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc. They impact these vegetables nutritional quality by acting as anti-cancer agents in human diet and also defend the crop against insect herbivory. We have identified numerous genes controlling this process that makes it possible to simultaneously breed improved nutritional content and herbivore defense into these plants. This would benefit both the consumer in improved health but also the producer through decreased pesticide input. We are also working towards understanding how different agronomic factors such as seasonality and planting density alter the production of these phytonutrients. This analysis is allowing us to assist the grower in modifying their agronomic practices to increase the flavor and nutritional quality of the crucifer crop thus optimizing financial benefits to the producer and health and enjoyment benefits to the consumer. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet. Finally, the identification of methodologies to more rapidly identify and characterize QTL greatly enhances and facilitates future plant breeding independent of the crop and/or species. As most agronomic improvements are accomplished through plant breeding, these methodology improvements could speed up the development of any crop.

Publications

  • Vergara, F., Wenzler, M., Hansen, B.G., Kliebenstein, D.J., Halkier, B.A., Gershenzon, J. and B. Schneider. (2008) Determination of the absolute configuration of the glucosinolate methylsulfoxide group reveals a stereospecific biosynthesis of the side chain. Phytochemistry 69(15)2737-42.
  • Wentzell, A.M., Boeye, I., Zhang, Z.-Y. and D.J. Kliebenstein. (2008) Genetic networks controlling structural outcome of glucosinolate activation across development. PLoS Genetics 4(10)e1000234
  • Bidart-Bouzat, M.G. and D.J. Kliebenstein. (2008) Differential levels of insect herbivory in the field associated with genotypic variation in glucosinolates in Arabidopsis thaliana. Journal of Chemical Ecology 34(8)1026-1037.
  • H.C. Rowe, Hansen, B.G., Halkier, B.A. and D.J. Kliebenstein. (2008) Biochemical networks and epistasis shape the Arabidopsis metabolome. The Plant Cell 20(5)1199-1216.
  • Kliebenstein, D.J. and H.C. Rowe (2008) Ecological costs of biotrophic versus necrotrophic pathogen resistance, the hypersensitive response and signal transduction. Plant Science 174(6)551-6.
  • Wentzell, A.M. and D.J. Kliebenstein. (2008) Genotype, age, tissue and environment regulate the structural outcome of glucosinolate activation. Plant Physiology 147(5)415-28.
  • Chehab, E.W., Kaspi, R., Savchenko, T., Rowe, H., Negre-Zakharov, F., Kliebenstein, D.J., and K. Dehesh. (2008) Distinct roles of jasmonates and aldehydes in plant-defense responses. PLoS ONE 3(4)e1904.
  • Kliebenstein, D.J. (2008) A role for gene duplication and natural variation of gene expression in the evolution of metabolism. PLoS ONE 3(3)e1838.
  • Hansen, B.G., Halker, B.A. and D.J. Kliebenstein. (2008) Identifying the molecular basis of QTLs: eQTLs add a new dimension. Trends in Plant Sciences 13(2) 72-77.
  • Lankau, R.A. and D.J. Kliebenstein. (2009) Competition and herbivory interact to determine the accumulation and fitness consequences of a secondary compound. Journal of Ecology 97(1):78-88.
  • Kliebenstein, D.J. (2009) A quantitative genetics and ecological model system: Understanding the aliphatic glucosinolate biosynthetic network via QTLs Phytochemistry Reviews 8(1)243-54.
  • Li, J., Hansen, B.G., Ober, J.A., Kliebenstein, D.J. and B.A. Halkier. (2008) A crucifer-specific radiation of flavin-monoxygenases involved in aliphatic glucosinolate biosynthesis. Plant Physiology 148(3)1721-33.
  • Burow, M., Zhang, Z.-Y., Ober, J.A., Lambrix, V.M., Wittstock, U., Gershenzon, J. and D.J. Kliebenstein. (2008) ESP and ESM1 mediate Indol-3-acetonitrile production from Indol-3-ylmethyl glucosinolate in Arabidopsis. Phytochemistry 69(3)663-671.
  • Walley, J.W., Rowe, H.C., Xiao, Y., Chehab, E.W., Kliebenstein, D.J., Wagner, D. and K. Dehesh. (2008) The chromatin remodeler SPLAYED regulates specific stress signaling pathways. PLoS Pathogens 4(12):e1000237.
  • Burow, M., Losansky, A., Muller, R., Plock, A., Kliebenstein, D.J., and U. Wittstock. (2009) The genetic basis of constitutive and herbivore-induced ESP-independent nitrile formation in Arabidopsis thaliana. Plant Physiology 149(1):561-574
  • Hansen, B.G., Kerwin, R.E., Ober, J.A., Lambrix, V.M., Mitchell-Olds, T., Gershenzon, J., Halkier, B.A. and D.J. Kliebenstein. (2008) A novel 2-oxoacid dependent dioxygenases involved in the formation of the goiterogenic 2-hydroxybut-3-enyl glucosinolate and generalist insect resistance in Arabidopsis thaliana. Plant Physiology 148(4):2096-2108.
  • Rowe, H.C. and D.J. Kliebenstein. (2008) Complex genetics control natural variation in Arabidopsis thaliana resistance to Botrytis cinerea. Genetics 180(4):2237-2250.
  • Kliebenstein, D.J. (2009) Quantification of variation in expression networks in Plant Systems Biology, edited by D. Belostotsky. Humana Press.
  • Kliebenstein, D.J. (2009) Quantitative Genomics; analyzing intra-specific variation using global gene expression polymorphisms or eQTLs. Annual Reviews Plant Biology


Progress 01/01/07 to 12/31/07

Outputs
In the past year, we have made significant progress in understanding three major areas of secondary metabolite plant biology. These areas are biological activity, genetic architecture, glucosinolate hydrolysis control and glucosinolate biosynthesis (1-10). Plant secondary metabolites have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. Phytonutrients also aid the plant in defending against various biological pests such as insects or fungi. We are currently using a model plant/pest interaction to investigate the how phytonutrients such as glucosinolates and pest genetic variation interact to determine how the plant interact with its environment (1,6). Secondary metabolite accumulation is typically controlled by quantitative trait loci (QTLs), each of which is time-consuming to identify. We are currently analyzing the genetic architecture controlling secondary metabolites within Arabidopsis and closely related species to identify methodologies to allow for enhanced cloning rates (1,2,3,5,8,9 and 10). This includes utilizing in-house developed novel approaches for studying networks controlling metabolism (2,3). As QTL are a major target in plant breeding, this would increase the number of loci that could be used to breed for increased plant production. This also includes an aspect to attempt and develop high-throughput genotyping systems to better enable QTL identification and cloning (2,8). The cruciferous phytonutrients, glucosinolates, are relatively inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We have cloned a locus from Arabidopsis that stimulates isothiocyanate production. Further, we showed that the same loci controlling glucosinolate hydrolysis also determine resistance to herbivory by a major crucifer crop pest, Trichoplusia ni. This locus has helped us identify numerous other potential loci controlling glucosinolate activation through variable gene expression (4). The glucosinolates chemical structure regulates its potential biological activity. For example, if the plant accumulates 4-methylthioalkyl glucosinolate instead of the anti-cancer 4-methylsulfinylalkyl glucosinolate, a difference of only an oxygen, the vegetable does not provide the same anti-cancer activity. We have identified a number of biosynthetic genes and are re-introducing them into the plant to test the glucosinolates function (7,8). This includes both the identification of the enzyme required to produce anticancer glucosinolates and another enzyme involved in producing the goiter causing, 2-hydroxy-but-3-enyl glucosinolate (8). These loci are currently being utilized by several groups on campus to alter crucifer vegetables phytonutrient profile either through traditional breeding or transgenic technologies.

Impacts
Glucosinolates are the major group of secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc. They impact the nutritional quality of these vegetables by acting as anti-cancer agents in human diet and defend the crop against insect herbivory. We have identified numerous loci controlling this process that will make it possible to simultaneously breed improved nutritional content and herbivore defense into these plants. This would benefit both the consumer in improved health but also the producer through decreased pesticide input. We are also working towards understanding how different agronomic factors such as seasonality and planting density alter the production of these phytonutrients. Hopefully, this analysis will allow us to guide the producer to modify their farming practices to increase the flavor and nutritional quality of the crucifer crop to optimize financial benefits to the producer and health and enjoyment benefits to the consumer. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet. Finally, the identification of methodologies to more rapidly identify and characterize QTL will greatly enhance and facilitate future plant breeding independent of the crop and/or species. As most agronomic improvements are accomplished through plant breeding, these methodology improvements could speed up the development of any crop.

Publications

  • Sonderby, I.E., Hansen, B.G., Bjarnholt, N., Ticconi, C., Halkier, B.A. and D.J. Kliebenstein. (2007) A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates in Arabidopsis. PLoS ONE.
  • Wentzell, A.M., Rowe, H.C, Hansen, B.G., Ticconi, C., Halkier, B.A., and D.J. Kliebenstein. (2007) Linking metabolic QTL with network and cis eQTL controlling biosynthetic pathways. PLOS Genetics. 3(9)e162.
  • Burow, M., Zhang, Z.-Y., Ober, J.A., Lambrix, V.M., Wittstock, U., Gershenzon, J. and D.J. Kliebenstein. (2007) ESP and ESM1 mediate Indol 3 acetonitrile production from Indol 3 ylmethyl glucosinolate in Arabidopsis. Phytochemistry.
  • Van Leeuwen, H. Kliebenstein, D.J., West, M.A.L., Kim, K., van Poecke, R., Katagiri, F., Michelmore, R.W., Doerge, R.W. and D.A. St. Clair. (2007) Natural variation among Arabidopsis thaliana accessions for transcriptome response to exogenous salicylic acid. The Plant Cell 19(7)2099-110.
  • Rowe, H.C and D.J. Kliebenstein. (2007) Elevated genetic variation within virulence associated Botrytis cinerea polygalacturonase loci. Molecular Plant Microbe Interactions 20(9)1126-37.
  • Kliebenstein, D.J. , D Auria, J.C., Behere, A.S., Kim, J.H., Gunderson, K.L., Breen, J.N., Lee, G., Gershenzon, J., Last, R.L., Jander, G. (2007) Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana. The Plant Journal 51(6):1062-76.
  • Hansen, B.G., Kliebenstein, D.J. and B.A. Halkier (2007) Identification of an Arabidopsis flavin monooxygenase as the S oxygenating enzyme in aliphatic glucosinolate biosynthesis. The Plant Journal 50(5):902-10.
  • West, M.A.L., Kim, K., Kliebenstein, D.J., van Leeuwen, H., Michelmore, R.W., Doerge, R.W., and D.A. St. Clair. (2007). Global eQTL mapping reveals the complex genetic architecture of transcript level variation in Arabidopsis. Genetics 175(3):1441-50.
  • Kliebenstein, D.J. (2007) Metabolomics and plant quantitative trait locus analysis The optimum genetical genomics platform? In Concepts in Plant Metabolomics, B.J. Nikolau and E.S. Wurtele, eds (Dordrect, The Netherlands: Springer), pp.29-45.


Progress 01/01/06 to 12/31/06

Outputs
In the past year, we have made significant progress in understanding three major areas of secondary metabolite plant biology. These areas are biological activity, genetic architecture, glucosinolate hydrolysis control and glucosinolate biosynthesis (1-7). Plant secondary metabolites have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. Phytonutrients also aid the plant in defending against various biological pests such as insects or fungi. We are currently using several model plants to investigate the how various phytonutrients such as flavonols and glucosinolates help the plant interact with its environment (1,3). Secondary metabolite accumulation is typically controlled by quantitative trait loci (QTLs), each of which is time-consuming to identify. We are currently analyzing the genetic architecture controlling secondary metabolites within Arabidopsis and closely related species to identify methodologies to allow for enhanced cloning rates (4,7). This includes developing novel approaches for studying networks controlling metabolism (2,4). As QTL are a major target in plant breeding, this would increase the number of loci that could be used to breed for increased plant production. This also includes an aspect to attempt and develop high-throughput genotyping systems to better enable QTL identification and cloning (3). The cruciferous phytonutrients, glucosinolates, are relatively inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We have cloned a locus from Arabidopsis that stimulates isothiocyanate production. Further, we showed that the same loci controlling glucosinolate hydrolysis also determine resistance to herbivory by a major crucifer crop pest, Trichoplusia ni. This locus has helped us identify numerous other potential loci controlling glucosinolate activation through variable gene expression (4,7). The glucosinolates chemical structure regulates its potential biological activity. For example, if the plant accumulates 4-methylthioalkyl glucosinolate instead of the anti-cancer 4-methylsulfinylalkyl glucosinolate, a difference of only an oxygen, the vegetable does not provide the same anti-cancer activity. We have identified a number of biosynthetic genes and are re-introducing them into the plant to test the glucosinolates function. Included in this are genes involved in producing the goiter causing, 2-hydroxy-but-3-enyl glucosinolate. These loci are currently being utilized by several groups on campus to alter crucifer vegetables phytonutrient profile either through traditional breeding or transgenic technologies.

Impacts
Glucosinolates are the major group of secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc. They impact the nutritional quality of these vegetables by acting as anti-cancer agents in human diet and defend the crop against insect herbivory. We have identified numerous loci controlling this process that will make it possible to simultaneously breed improved nutritional content and herbivore defense into these plants. This would benefit both the consumer in improved health but also the producer through decreased pesticide input. We also work on improving hydroxycinnamates and flavonols within tomato, the compounds believed to lead to improved health under the high-fat western diet. By understanding how these two major phytonutrients are controlled, we should be able to enable breeding of crops for greater human health benefits. This could lead to direct health benefits to the consumer as well as indirect benefits to society of decreased health care costs. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet. Finally, the identification of methodologies to more rapidly identify and characterize QTL will greatly enhance and facilitate future plant breeding independent of the crop and/or species. As most agronomic improvements are accomplished through plant breeding, these methodology improvements could speed up the development of any crop.

Publications

  • West, M.A.L, van Leeuwen, H., Kozik, A., Kliebenstein, D.J., Doerge, R.W., St Clair, D.A. and R.W. Michelmore. 2006 High-density haplotyping with microarray-based expression and single feature polymorphism markers in Arabidopsis. Genome Research 16(6):787-95.
  • Kliebenstein, D.J., West, M.A.L., van Leeuwen, H., Kim, K., Doerge, R.W., Michelmore, R.W. and D.A. St.Clair. (2006) Genomic survey of gene expression diversity in Arabidopsis thaliana. Genetics 172(2):1179-7789.
  • Whittall, J.B., Voelckel, C., Kliebenstein, D.J. and S.A. Hodges. 2006 Convergence, constraints and the role of regulatory genes during adaptive radiation; Floral anthocyanins in Aquilegia. Molecular Ecology 15(14):4645-8
  • Kliebenstein, D.J. 2006 Metabolomics and plant quantitative trait locus analysis - the optimum genetical genomics platform. Proceedings of the annual plant metabolomics conference. In press
  • Oscar, O.R., Kliebenstein, D.J., Arbizu, C., Ortega, R. and C.F. Quiros. 2006 Glucosinolate survey in cultivated and feral mashua (Tropaeolum tuberosum Ruiz & Pavon). Journal of Economic Botany 60(3):254-64.
  • Kliebenstein, D.J., West, M.A.L, van Leeuwen, H., Kim, K., Doerge, R.W., and D.A. St. Clair. 2006 Identification of QTL Controlling Gene Expression Networks Defined A Prioiri. BMC Bioinformatics 7(1):308.
  • Zhang, Z., Ober, J.A. and D.J. Kliebenstein. 2006 The Gene Controlling the Quantitative Trait Locus EPITHIOSPECIFIER MODIFIER1 Alters Glucosinolate Hydorlysis and Insect Resistance in Arabidopsis. Plant Cell 18(6): 1524-36.


Progress 01/01/05 to 12/31/05

Outputs
In the past year, we have made significant progress in understanding three major areas of secondary metabolite plant biology. These areas are biological activity, genetic architecture, glucosinolate hydrolysis control and glucosinolate biosynthesis (1-7). Plant secondary metabolites have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. Phytonutrients also aid the plant in defending against various biological pests such as insects or fungi. We are currently using a model plant to investigate the how various phytonutrients such as flavonols and glucosinolates protect the plant against biological pests. This has shown that phytonutrients provide protection against fungi like Botrytis cinerea, insects like Trichoplusia ni (Cabbage looper) and ultraviolet radiation (3,4,7). Secondary metabolite accumulation is typically controlled by quantitative trait loci (QTLs), each of which is time-consuming to identify. We are currently analyzing the genetic architecture controlling secondary metabolites within Arabidopsis and closely related species to identify methodologies to allow for enhanced cloning rates (1,5). As QTL are a major target in plant breeding, this would increase the number of loci that could be used to bred for increased plant production. The cruciferous phytonutrients, glucosinolates, are relatively inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We have cloned a locus from Arabidopsis that stimulates isothiocyanate production. Further, we showed that the same loci controlling glucosinolate hydrolysis also determine resistance to herbivory by a major crucifer crop pest, Trichoplusia ni. We have cloned this locus from Brassica vegetables and have shown that it controls differential glucosinolate activation between Brassicaceous vegetables. Thus, by identifying this gene, we will be able to better understand how crucifers defend against insects and help humans defend against cancer. The glucosinolates chemical structure regulates its potential biological activity. For example, if the plant accumulates 4-methylthioalkyl glucosinolate instead of the anti-cancer 4-methylsulfinylalkyl glucosinolate, a difference of only an oxygen, the vegetable does not provide the same anti-cancer activity. We have identified a number of biosynthetic genes and are re-introducing them into the plant to test the glucosinolates function ( 5,6 ). Included in this are genes involved in producing the goiter causing, 2-hydroxy-but-3-enyl glucosinolate. These loci are currently being utilized by several groups on campus to alter crucifer vegetables phytonutrient profile either through traditional breeding or transgenic technologies.

Impacts
Glucosinolates are the major group of secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc.. They impact the nutritional quality of these vegetables by acting as anti-cancer agents in human diet and defend the crop against insect herbivory. Thus, by understanding glucosinolate production and control it may be possible to breed improved nutritional content and/or herbivore defense into these plants. We also work on the polyphenol phytonutrients and understanding the variation of these compounds. This includes the hydroxycinnamates and flavonols that are believed to be the source of the health benefits of red wine underlying the French Paradox. By understanding how these two major phytonutrients are controlled, we should be able to enable breeding of crops for greater human health benefits. This could lead to direct health benefits to the consumer as well as indirect benefits to society of decreased health care costs. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet. Finally, the identification of methodologies to more rapidly identify and characterize QTL will greatly enhance and facilitate future plant breeding independent of the crop and/or species.

Publications

  • Kliebenstein, D.J., West, M.A.L., van Leeuwen, H., Kim, K., Doerge, R.W., Michelmore, R.W. and D.A. St.Clair. (2005) Genomic survey of gene expression diversity in Arabidopsis thaliana. (In Press) Genetics
  • Kliebenstein, D.J. (2005) Metabolomics and plant quantitative trait locus analysis. The optimum genetical genomics platform. Proceedings of the annual plant metabolomics conference (In Press)
  • Brown, B.A., Cloix, C., Jiang, G.H., Kaiserli, E., Herzyk, P., Kliebenstein, D.J. and G.I. Jenkins. (2005) A UV-B-specific signaling component orchestrates plant UV-protection. Proc. Natl. Acad. Sci. USA 102(50):18225-18230.
  • Kliebenstein, D.J., Rowe, H.C. and K.J. Denby. (2005) Secondary metabolites influence Arabidopsis/Botrytis interactions: variation in host production and pathogen susceptibility. Plant Journal 44(1):25-36.
  • Windsor, A.J., Reichelt, M., Figuth, A., Svatos, A., Kroymann, J., Kliebenstein, D.J., Gershenzon, J. and T. Mitchell-Olds. (2005) Geographic and evolutionary diversification of glucosinolates among near relatives of Arabidopsis thaliana (Brassicaceae). Phytochemistry 66(11):1321-1333.
  • Kliebenstein, D.J., Kroymann, J., and T. Mitchell-Olds. (2005) The glucosinolate-myrosinase system in an ecological and evolutionary context. Current Opinion in Plant Biology 8(3):264-271.
  • Murray, S.L., Adams, N., Kliebenstein, D.J., Loake, G.J. and K.J. Denby. (2005) A constitutive PR-1::Luciferase expression screen identifies Arabidopsis mutants with differential disease resistance to biotrophic and necrotrophic pathogens. Molecular Plant Pathology 6(1):31-41.


Progress 01/01/04 to 12/31/04

Outputs
In the past year, we have made significant progress in understanding three major areas of secondary metabolite plant biology. These areas are biological activity, glucosinolate hydrolysis control and glucosinolate biosynthesis (Kliebenstein 2004). Phytonutrients are a large class of compounds commonly known as plant secondary metabolites. These compounds have beneficial activities when incorporated in human diets by consumption of fruits or vegetables. In addition, phytonutrients also aid the plant in defending against various biological pests such as insects or fungi. We are currently using a model plant to investigate the role of various phytonutrients such as flavonols and glucosinolates in helping to protect the plant against biological pests. This has shown that phytonutrients are able to provide protection against fungi like Botrytis cinerea and insects like Trichoplusia ni (Cabbage looper). This involves the use of plants genetically manipulated, either via transgenic or traditional breeding technologies, to only vary in the production of a specific phytonutrient. Using this system, we can rapidly bioassay specific phytonutrients for biological activity (Denby, et al. 2004). The cruciferous phytonutrients, glucosinolates, are relatively inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be degraded/activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We identified Arabidopsis genetic variation that controlled isothiocyanate versus nitrile production. We have identified a small genomic region that contains this locus and are close to cloning out the underlying gene. Further, we showed that the same loci controlling glucosinolate hydrolysis also determine resistance to herbivory by a major crucifer crop pest, Trichoplusia ni. This locus also appears to control glucosinolate activation in Brassicaceous vegetables. Thus, by identifying this gene, we will be able to better understand how crucifers defend against insects and help humans defend against cancer. The actual chemical structure of the glucosinolate regulates its potential biological activity. For example, if the plant accumulates 4-methylthioalkyl glucosinolate instead of the anti-cancer 4-methylsulfinylalkyl glucosinolate, a difference of only an oxygen, the vegetable does not provide the same anti-cancer activity. Thus, we are currently identifying a number of genes that regulate the glucosinolates biosynthetic structure. Included in this are genes involved in producing the goiter causing, 2-hydroxy-but-3-enyl glucosinolate. By identifying these loci, we should be able to provide tools to alter crucifer vegetables phytonutrient profile either through traditional breeding or transgenic technologies

Impacts
Glucosinolates are the major group of secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc.. They impact the nutritional quality of these vegetables by acting as anti-cancer agents in human diet and defend the crop against insect herbivory. Thus, by understanding glucosinolate production and control it may be possible to breed improved nutritional content and/or herbivore defense into these plants. Further, by understanding a phytonutrients biological activity against various pests and in humans, it will be possible to predict the agricultural ramifications following the alteration of the phytonutrient profile for improved human diet.

Publications

  • Kliebenstein, D.J. (2004) Secondary metabolites and plant/environment interactions: a view through Arabidopsis thaliana tinged glasses. Plant Cell and Environment 27(6):675-684.
  • Denby, KJ, Kumar, P. and Kliebenstein, D.J. (2004) Identification of Botrytis cinerea susceptibility loci in Arabidopsis thaliana. Plant Journal 38(3):473-486.


Progress 01/01/03 to 12/31/03

Outputs
In the past year, we have made significant progress in understanding three major areas of glucosinolate/myrosinase system biology. These areas are phenotypic plasticity, hydrolysis control and insect defense against the glucosinolate/myrosinase system (Wittstock, et al. 2003). Phenotypic plasticity is the observation that a plant will display different phenotypes when grown in different environments. This is similar to the nature versus nurture argument where nature is the plants genotype and nurture is the environment within which it grows. Thus, one could modify a plants glucosinolate content by altering the environment that it grows in. However, most phytonutrients are linked and altering one may have negative impacts on another. We are currently analyzing how altering glucosinolate metabolism impacts the major flavonoids and anthocyanins classes of phytonutrients. This work is currently in progress but is showing that most phytonutrients are intricately linked and alterations geared towards one will dramatically change the levels of another.This is of significant importance to plant breeders and growers who want to alter increase specific phytonutrient levels in response to market demand. By understanding how the different phytonutrients are linked, it may be possible to identify methods or genetic backgrounds whereby one class can be increased without altering other important phytonutrient levels. Glucosinolates are inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be degraded/activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We identified Arabidopsis genetic variation that controlled isothiocyanate versus nitrile production. Further, we showed that the same loci controlling glucosinolate hydrolysis also determine resistance to herbivory by a major crucifer crop pest, Trichoplusia ni. In contrast, this same resistance mechanism had no impact on the other major crucifer pest, Plutella xylostella (Diamondback moth (DBM)). This indicates the glucosinolate/myrosinase system can provide specific herbivore resistance in cruciferous vegetables. We are currently identifying a number of the genes underlying this activation process and studying their biochemistry. It appears that most of these genes act as a large protein complex that is co-localized within the plant cell. To test this we are initiating a number of experiments geared towards testing this including biochemical complex purification as well as GFP/GUS co-promoter localization assays. The hope of this project is to understand how this activation complex is controlled and regulated so as to be able to alter its function and predictably modify the glucosinolate activation state.

Impacts
Glucosinolates are a major group of secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc.. They impact the nutritional quality of these vegetables by acting as anti-cancer agents in human diet and defend the crop against insect herbivory. Thus, by understanding glucosinolate production and control it may be possible to breed improved nutritional content and/or herbivore defense into these plants. Field 43: Publications Wittstock, U., Kliebenstein, D.J., Lambrix, V., Reichelt, M. and J. Gershenzon. (2003) Glucosinolate hydrolysis and its impact on generalist and specialist insect herbivores. Rec Adv Phytochem 37: 101-125 (eds. Romeo JT, Dixon RA, Vol. 37: Integrative Phytochemistry: From Ethnobotany to Molecular Ecology. Pergamon, Amsterdam)

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
In the past year, we have made significant progress in understanding three major areas of glucosinolate/myrosinase system biology. These areas are phenotypic plasticity, hydrolysis control and insect defense against the glucosinolate/myrosinase system. Phenotypic plasticity is the observation that a plant will display different phenotypes when grown in different environments. This is similar to the nature versus nurture argument where nature is the plants genotype and nurture is the environment within which it grows. We showed that Arabidopsis glucosinolates accumulate to different levels depending upon which environment they grow in and that this difference was dependent upon the Arabidopsis variety utilized. This allowed us to identify genetic loci that control how a plant and its environment determine glucosinolate accumulation (Kliebenstein, et al. 2002a). This is of significant importance to plant breeders who want to alter glucosinolate content irrespective of the environment.Glucosinolates are inactive. Glucosinolate activation requires hydrolysis by the enzyme myrosinase into various compounds, thiocyanates, isothiocyanates and nitriles. The specific activity requires that the glucosinolate be degraded/activated into the proper form. For example, if the 4-methylsulfinylalkyl glucosinolate is hydrolyzed into the isothiocyanate, sulforophane, it is a potent anti-cancer agent. If instead it is hydrolyzed into the nitrile, all anti-cancer activity is lost. We identified Arabidopsis genetic variation that controlled isothiocyanate versus nitrile production. Further, we showed that the same loci controlling glucosinolate hydrolysis also determine resistance to herbivory by a major crucifer crop pest, Trichoplusia ni. In contrast, this same resistance mechanism had no impact on the other major crucifer pest, Plutella xylostella (Diamondback moth (DBM)). This indicates the glucosinolate/myrosinase system can provide specific herbivore resistance in cruciferous vegetables (Kliebenstein et al. 2002b). DBM is a worldwide crucifer pest that is not impacted by the crucifers natural anti-herbivory compounds, glucosinolates. Understanding how DBM defends against glucosinolates could provide a strategic target for a species specific pesticide. Simple analysis of DBM feces showed that the plants glucosinolates pass through the insect without being absorbed or degraded. This was surprising as the plant hydrolyzes the glucosinolates upon tissue disruption via the insects chewing. This hydrolysis activates the glucosinolates. However, our analysis of the feces indicates that defends was preventing this activation. Biochemical analysis showed that the DBM gut contains a enzyme, sulfatase, that removes a simple sulfate from the intact glucosinolate. The sulfate removal has dramatic implications for both plant and insect as conceals the glucosinolate from the hydrolytic machinery. Thus, the glucosinolate is not activated and passes harmlessly through the insect (Ratzka, et al. 2002). This sulfatase appears to be DBMs entire defense mechanism and is not present in other related insect species. Thus, it is an ideal target for a species specific pesticide.

Impacts
Glucosinolates are a major group of secondary metabolites/neutraceuticals in cruciferous vegetables including broccoli, cabbage, cauliflower, etc.. They impact the nutritional quality of these vegetables by acting as anti-cancer agents in human diet and defend the crop against insect herbivory. Thus, by understanding glucosinolate production and control it may be possible to breed improved nutritional content and/or herbivore defense into these plants.

Publications

  • REICHELT, M., BROWN, P.D., SCHNEIDER, B., OLDHAM, N.J., STAUBER, E., TOKUHISA, J., KLIEBENSTEIN, D.J., MITCHELL-OLDS, T., AND J. GERSHENZON. 2002 Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana. Phytochemistry 59(6):663-671.
  • KLIEBENSTEIN, D.J., FIGUTH, A., AND T. MITCHELL-OLDS. 2002a Genetic Architecture of Plastic Methyl Jasmonate Responses in Arabidopsis thaliana. Genetics 161(4):1685-1696
  • KLIEBENSTEIN, D.J., PEDERSEN, D. AND T. MITCHELL-OLDS. 2002b Comparative Analysis of Insect Resistance QTL and QTL Controlling the Myrosinase/Glucosinolate System in Arabidopsis thaliana Genetics 161(1):325-332.
  • RATZKA, A., VOGEL, H., KLIEBENSTEIN, D.J., MITCHELL-OLDS, T. AND J. KROYMANN. 2002. Disarming the mustard oil bomb. Proceedings of the National Academy of Sciences (USA) 99(17):11223-11228.