Progress 09/15/02 to 09/14/05
Outputs
Glucosinolates are a class of secondary metabolites that mainly occur in cruciferous plants. Upon tissue disruption, glucosinolates are hydrolyzed to unstable intermediates that rearrange to isothiocyanates, thiocyanates or nitriles. Although the primary biological function of glucosinolates in plants is unknown, their breakdown products are proposed to act as allelochemicals and to play a role in plant defenses against herbivores, pests, and pathogens. As components of feed and food, products of glucosinolate hydrolysis have long been of toxicological and pharmocological interest. Depending on glucosinolate composition and on the prevalence of hydrolysis products, consumption of glucosinolates by mammals has been linked with goitrogenic effects or with a reduced risk of developing cancer. In a collaborative study, we obtained corroborating biomedical evidence for the chemo preventive activities of isothiocyanates. The cancer-preventive properties of certain
isothiocyanates have renewed interest in glucosinolate biosynthesis. We have taken a genetic approach to dissect glucosinolate biosynthesis in Arabidopsis thaliana. We developed a bioassay for isothiocyanates in single leaf disks to rapidly monitor glucosinolate content in large populations of insertional mutant collections. The bioassay is based on the ability of isothiocyanates to induce carcinogen-detoxifying enzymes such as quinone reductase (QR) in cultured mouse cells. After screening leaf disks of 16,500 plants for altered QR inducer potency, we identified 15 T-DNA activation-tagged lines that show changes in glucosinolate content and composition (GCC lines). We cloned three genes and focused on the functional characterization of GCC7/IQD1. We used a series of loss- and gain-of-function mutations to demonstrate that IQD1 positively affects glucosinolate accumulation via the expression of multiple pathway genes. Interestingly, using the same set of mutations, we showed that IQD1
not only modulates glucosinolate production but also enhances general plant defense responses. For example, transgenic lines that over express IQD1 show increased resistance against infection by necrotrophic fungi and deterred feeding attacks by insects, whereas loss-of-function mutations caused the opposite effect. IQD1 is a member of a large gene family in Arabidopsis and rice, which share a novel calmodulin-binding domain (IQ67 domain). We demonstrated that recombinant IQD1 interacts with bovine calmodulin in vitro and with all Arabidopsis calmodulins tested. Yeast two-hybrid assays indicated that IQD1 interacts with Arabidopsis calmodulins in vivo. A preliminary deletion analysis demonstrated that the conserved IQ67 domain participates in calmodulin interaction. We also showed that IQD1 has the ability to interact with nucleic acids in vitro, which raises the prospect that IQD1 and other proteins of the 33-member IQD protein family in Arabidopsis directly regulate defense-related
nuclear gene expression, possibly in response to altered calcium. The exciting prospect arises that IQD1 and related proteins are important regulatory molecules that integrate calcium signaling in plant defense response.
Impacts
Isolation of plants with altered glucosinolate metabolism and identification of the mutated genes will provide molecular tools to probe the biological function of glucosinolates in plants. More importantly, genes involved in glucosinolate biosynthesis will allow for metabolic engineering of crops relevant to human nutrition for improved content and composition of ant carcinogenic glucosinolates. The finding that IQD1 is a nuclear regulator of glucosinolate metabolism and general plant defense responses is of high relevance to US agriculture.
Publications
- Levy, M., Q. Wang, C.D. Grubb, S. Abel (2003) GCC7, a nuclear regulator of glucosinolate accumulation in Arabidopsis. 14th International Conference on Arabidopsis Research. Madison, WI, June 20-24. Abstract #224
- Levy, M., Q. Wang, C.D. Grubb, S. Abel (2003) GCC7, a nuclear regulator of glucosinolate accumulation in Arabidopsis thaliana. Annual Meeting of the Phytochemical Society of North America. Peoria, IL, August 9-13. Abstract #P1
- Dingley, K.H., E.A. Sanchez, K.W. Turteltaub, S. Abel, S.E. Ebeler, A.E. Mitchell, S.A. Burns, F.M. Steinberg and A.J. Clifford (2003) Effect of dietary supplements with chemopreventive potential on the bioavailability and adduct formation of a low-dose of the heterocyclic amine 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhiP). Nutr. Cancer. 46:212-221
- Levy, M., Q. Wang, Kaspi, R., Parrella, M.P., and Abel, S. (2005) Arabidopsis IQD1, a novel calmodulin-binding nuclear protein, stimulates glucosinolate accumulation and plant defense. Plant J 43:79-96
- Levy, M., Rachmilevitch, S. and Abel, S. (2005) Transient Agrobacterium-mediated gene expression in the Arabidopsis hydroponics root system for subcellular localization studies. Plant Mol Biol Rep 23:179-184.
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Progress 01/01/04 to 12/31/04
Outputs
We have extended the functional characterization of the calmodulin-binding protein, GCC7, as a nuclear regulator of the glucosinolate pathway. We used a series of loss- and gain-of-function mutations in Arabidopsis to demonstrate that GCC7 positively affects glucosinolate accumulation via the expression of multiple glucosinolate pathway genes. Interestingly, using the same set of mutations, we could show that GCC7 not only modulates glucosinolate production but also enhances general plant defense responses. For example, transgenic lines that overexpress GCC7 show increased resistance against infection by necrotrophic fungi (Botrytis) and deterred feeding attacks by insects (cabbage looper, aphids), whereas loss-of-function mutations caused the opposite effect. GCC7 is a member of a relatively large multigene family in Arabidopsis (33 genes) and rice (29 genes), which we named IQD families for their presence of a conserved and plant-specific novel calmodulin-binding
domain, called the IQ66 domain. The exciting prospect arises that GCC7/IQD1 and related proteins are important regulatory molecules that integrate calcium signaling in plant defense response.
Impacts
Isolation of plants with altered glucosinolate metabolism and identification of the mutated genes will provide molecular tools to probe the biological function of glucosinolates in plants. More importantly, genes involved in glucosinolate biosynthesis will allow for metabolic engineering of crops relevant to human nutrition for improved content and composition of anticarcinogenic glucosinolates. The finding that GCC7/IQD1 not only modulates glucosinolate metabolism but also general plant defense responses is of high relevance to US agriculture.
Publications
- Levy, M., Q. Wang, and Abel, S. (2003) A novel nuclear calmodulin-binding proteins modulates glucosinolate accumulation in Arabidopsis. 15th International Conference on Arabidopsis Research. Berlin, Germany, July 11-14. Abstract T07-086.
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Progress 01/01/03 to 12/31/03
Outputs
Glucosinolates are a class of more than 150 secondary metabolites that mainly occur in cruciferous plants. Upon tissue disruption, glucosinolates are rapidly hydrolyzed to unstable intermediates that spontaneously rearrange to isothiocyanates, thiocyanates, or nitriles. Although the primary biological function of glucosinolates in plants is unknown, their breakdown products are proposed to act as allelochemicals and to play a role in plant defenses against herbivores, pests, and pathogens. Furthermore, indolyl glucosinolates can be converted into indole-3-actetic acid and may thus participate in regulating auxin homeostasis in cruciferous plants. As components of feed for livestock and food for humans, products of glucosinolate hydrolysis have long been of toxicological and pharmocological interest. Depending on glucosinolate composition and on the prevalence of hydrolysis products, consumption of glucosinolates by mammals has been linked with goitrogenic effects
(thiocyanates), or with a reduced risk of developing cancer (isothiocyanates). In a collaborative study (Dingwell et al., 2003), we obtained corroborating biomedical evidence for the chemopreventive activities of isothiocyanates. The cancer-preventive properties of certain glucosinolate-derived isothiocyanates have renewed interest in glucosinolate biosynthesis and its regulation. We have taken a molecular genetic approach to dissect glucosinolate biosynthesis in the model plant Arabidopsis thaliana. We previously developed a novel bioassay for glucosinolate-derived isothiocyanates in single leaf disks to rapidly monitor glucosinolate content in large populations of insertional mutant collections. The bioassay is based on the ability of isothiocyanates to induce carcinogen-detoxifying enzymes such as quinone reductase (QR) in cultured murine hepatoma cells. After screening leaf disks of 16,500 plants for altered QR inducer potency, we identified 15 T-DNA activation-tagged lines that
show inheritable changes in glucosinolate content and composition (GCC lines). We cloned three genes by plasmid rescue and focused our studies on gene GCC7 during the past year. Biochemical analyses showed that GCC7 is targeted to the cell nucleus and binds to calmodulin in a calcium-dependent manner. Generation of loss- and gain-of-function mutations convincingly demonstrated its role as a regulatory protein of biosynthetic genes of the glucosinolate pathway. To our knowledge, GCC7 is the first described nuclear regulator of glucosinolate biosynthesis.
Impacts
Isolation of plants with altered glucosinolate metabolism and identification of the mutated genes will provide molecular tools to probe the biological function of glucosinolates in plants. More importantly, genes involved in glucosinolate biosynthesis will allow for metabolic engineering of crops relevant to human nutrition for improved content and composition of anticarcinogenic glucosinolates.
Publications
- Dingley, K.H., E.A. Sanchez, K.W. Turteltaub, S. Abel, S.E. Ebeler, A.E. Mitchell, S.A. Burns, F.M. Steinberg and A.J. Clifford (2003) Effect of dietary supplements with chemopreventive potential on the bioavailability and adduct formation of a low-dose of the heterocyclic amine 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhiP). Nutr. Cancer. 46:212-221
- Levy, M., Q. Wang, C.D. Grubb, S. Abel (2003) GCC7, a nuclear regulator of glucosinolate accumulation in Arabidopsis. 14th International Conference on Arabidopsis Research. Madison, WI, June 20-24. Abstract #224
- Levy, M., Q. Wang, C.D. Grubb, S. Abel (2003) GCC7, a nuclear regulator of glucosinolate accumulation in Arabidopsis thaliana. Annual Meeting of the Phytochemical Society of North America. Peoria, IL, August 9-13. Abstract #P1
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Progress 01/01/02 to 12/31/02
Outputs
Glucosinolates are a class of more than 150 secondary metabolites that mainly occur in cruciferous plants. Upon tissue disruption, glucosinolates are rapidly hydrolyzed to unstable intermediates that spontaneously rearrange to isothiocyanates, thiocyanates, or nitriles. Although the primary biological function of glucosinolates in plants is unknown, their breakdown products are proposed to act as allelochemicals and to play a role in plant defenses against herbivores, pests, and pathogens. Furthermore, indolyl glucosinolates can be converted into indole-3-actetic acid and may thus participate in regulating auxin homeostasis in cruciferous plants. As components of feed for livestock and food for humans, products of glucosinolate hydrolysis have long been of toxicological and pharmocological interest. Depending on glucosinolate composition and on the prevalence of hydrolysis products, consumption of glucosinolates by mammals has been linked with goitrogenic effects
(thiocyanates), or with a reduced risk of developing cancer (isothiocyanates). The cancer-preventive properties of certain glucosinolate-derived isothiocyanates have renewed interest in glucosinolate biosynthesis and its regulation. We have taken a molecular genetic approach to dissect glucosinolate biosynthesis in the model plant Arabidopsis thaliana. We have developed a novel bioassay for glucosinolate-derived isothiocyanates in single leaf disks to rapidly monitor glucosinolate content in large populations of insertional mutant collections (Wang et al., 2002). The bioassay is based on the ability of isothiocyanates to induce carcinogen-detoxifying enzymes such as quinone reductase (QR) in cultured murine hepatoma cells. To date we have assayed duplicate leaf disks of 16,500 plants that represent 5,500 T-DNA activation-tagged lines for altered QR inducer potency. HPLC analysis verified that we have isolated 15 lines that show inheritable changes in glucosinolate content and
composition. These lines are currently further characterized and subjected to gene cloning by plasmid rescue. To date, we have cloned three genes that are predicted to encode a putative transporter, a putative nuclear protein, and a putative transcription factor. Studies to verify and to elucidate the functions of these genes in glucosinolate biosynthesis are underway.
Impacts
Isolation of plants with altered glucosinolate metabolism and identification of the mutated genes will provide molecular tools to probe the biological function of glucosinolates in plants. More importantly, genes involved in glucosinolate biosynthesis will allow for metabolic engineering of crops relevant to human nutrition for improved content and composition of anticarcinogenic glucosinolates.
Publications
- WANG, Q., GRUBB, C.D., ABEL, S. (2002) Direct analysis of single leaf disks for chemopreventive glucosinolates. Phytochem. Anal. 13:152-157.
- LEVY, M., WANG, Q., GRUBB, C.D. and S. ABEL (2002) Mutational analysis of glucosinolate biosynthesis in Arabidopsis. Plant Biology 2002, Denver, CO, August 3-7, Poster #720
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