Progress 09/15/05 to 03/14/10

Outputs
OUTPUTS: We identified IQD1, which enhances glucosinolate accumulation and general-induced plant defense responses against herbivory and Botrytis infection when overexpressed in Arabidopsis thaliana. We demonstrated that the encoded protein is localized to the cell nucleus, binds to calmodulin (CaM) in a calcium-dependent manner, and modulates glucosinolate pathway gene expression. IQD1 defines a new class of plant-specific CaM-binding proteins of unknown biochemical functions and biological roles. We focused our research on structure-function studies of IQD1 and demonstrated that the protein binds in vitro to several CaMs and CaM-like polypeptides (CML) of Arabidopsis, which we confirmed in vivo for CaM1 and CaM2 by using yeast two-hybrid assays. Using radioactively labeled CaM2, we determined the apparent dissociation constant of the IQD1:CaM2 interaction (Kd=600 nM), which indicates a moderate interaction and suggests that IQD1 interacts in vivo with CaM-like or other target proteins. We generated cDNA libraries from 2-week-old Arabidopsis seedlings as well as from flowers to screen for IQD1-interacting proteins in yeast. A set of candidate proteins that strongly interact with IQD1 in yeast was identified. Deletion analysis of IQD1 confirmed that the highly conserved IQ67 domain, which is the defining feature of IQD1 and related proteins, mediates the interaction between IQD1 and CaM/CMLs. Interestingly, we observed that IQD1 displays affinity to single-stranded nucleic acids such as ssDNA, poly(A) and poly(G), which suggests that IQD1 plays a role in regulating gene expression at the posttranscriptional level. Our preliminary data suggest that binding of CaM to IQD1 in vitro alters the nucleic acid-binding properties of IQD1. A genome-wide comparative analysis of IQD1-related genes and their encoded proteins revealed the existence of relatively large families of similar proteins in Arabidopsis (33 members) and rice (29 members) that are specific to plants and arose early during the evolution of land plants. A survey of available iqd knockout lines suggests that IQD proteins have important roles during plant growth and development. Interestingly, knockout mutations in an additional gene, IQD33, also lead to reduced glucosinolate accumulation. Our findings were disseminated in publications, poster abstracts at scientific meetings, and oral presentations. PARTICIPANTS: Steffen Abel, PI: Supervised and coordinated research, analyzed data and wrote manuscripts Maggie Levy, post-doctoral researcher: Identified IQD1 and conducted research to characterize Arabidopsis lines with altered IQD1 expression levels Tatyana Savchenko, post-doctoral researcher: Conducted research to characterize the biochemical properties of IQD1) Aaron Adamson, postdoctoral researcher: Performed yeast two-hybrid screens to identify IQD1-interacting proteins Jessie Selby, MSc student: Characterized knock-out lines for IQD and CaM genes Brandon Zip, undergraduate student: Analyzed plants for glucosinolate content by HPLC TARGET AUDIENCES: The nature of this project was largely fundamental research. Therefore, the community of plant biologist was the target audience, which was informed by publications and oral presentations at scientific meetings. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues, such as drought, salinity, cold and heat stress, wounding, oxidative stress, herbivory, or pathogen response. A thorough understanding calcium signaling circuits at the cellular and systemic levels will be necessary for the long-term improvement of plant species of high societal value in agriculture and forestry. IQD1 is the founding member of a novel, plant-specific, and large family of putative CaM target proteins containing about 30 members in Arabidopsis and rice. We conclude from our research that IQD1 and related proteins function as nucleic-acid binding proteins in the cell nucleus, which regulate a set of genes with important roles in plant defense against herbivory and necrotrophic fungi. In addition, the potential importance of the IQD gene family for the development of crop traits has recently been uncovered in tomato. Elevated expression of SUN/IQD12, caused by retrotransposon-mediated gene duplication, dramatically alters tomato fruit shape, resulting in extremely elongated and often seedless fruits (Science 319:1527-1530, 2008). Furthermore, work by others recently showed that other members of the Arabidopsis IQD gene family mediate responses to the growth hormone gibberellic acid. We anticipate that IQD genes and proteins are important signaling components in diverse processes during plant growth and development and thus feasible molecular targets for crop improvement.

Publications

• Levy M, Wang Q, Kaspi R, Parrella PP, Abel S (2005) Arabidopsis IQD1, a novel calmodulin-binding nuclear protein stimulates glucosinolate accumulation and plant defense. Plant J 43:79-96 (NRI Cover Story).
• Levy M, Rachmilevitch S, 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.
• Abel S, Savchenko T, Levy M (2005) Genome-wide comparative analysis of the IQD gene families in Arabidopsis thaliana and Oryza sativa. BMC Evolutionary Biology 5:72 (1-25).
• Grubb CD, Abel S (2006) Glucosinolate metabolism and its control. Trends Plant Sci 11:89-100.

Progress 09/15/07 to 09/14/08

Outputs
OUTPUTS: During the previous year, we followed up on our observation that IQD1 interacts in vitro with representative members of the major clades of Arabidopsis calmodulins (CaM) and calmodulin-like proteins (CML), CaM2, CaM4, CML8 and CML9. Using radioactively labeled CaM2 generated by in vitro transcription/translation in the presence of [35S]Met followed by affinity chromatography purification and proteolytic removal of the affinity tag, we determined by Scatchard plot analysis the apparent dissociation constant of the IQD1:CaM2 interaction. The obtained value of Kd=600 nM indicates only a moderate affinity of IQD1 for CaM2 and suggests that IQD1 may interact in vivo with so far unknown CaM/CML or other target proteins. To identify such IQD1-interacting proteins, we constructed two cDNA libraries from 2-week-old Arabidopsis seedlings and from Arabidopsis flowers, which we interrogated in a yeast two-hybrid screen using IQD1 as well as IQD20, the smallest member of the IQD family, as bait proteins. We conducted multiple screens for each IQD protein and cDNA library and identified a total of 11 proteins that reproducible interact with the bait protein in the yeast assay. However, none of the identified putative interacting proteins belongs to the CaM/CML family. We are currently verifying the interaction of IQD1 and IQD20 with the putative proteins using alternative approaches, such as in vitro pulldown assays. In addition, we have started a systematic analysis of available T-DNA insertion lines of other members of the Arabidopsis IQD family. Our initial results demonstrate based on morphological phenotypes that IQD proteins play important roles in plant growth and development. PARTICIPANTS: Dr. Tanya Savchenko, a post-doctoral fellow, conducted the in vitro interaction studies between IQD1 and CaM/CML proteins from Arabidopsis. After a vacancy that lasted several months, her position was filled by Dr. Aaron Adamson who conducted the yeast two-hybrid screens. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Because of the turnover in essential personnel and the difficulty to recruit a qualified post-doctoral researcher, I requested a one-year no-cost extension.

Impacts
Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues, such as drought, salinity, cold and heat stress, wounding, oxidative stress, herbivory, or pathogen response. A thorough understanding calcium signaling circuits at the cellular and systemic levels will be necessary for the long-term improvement of plant species of high societal value in agriculture and forestry. Our structure-function studies of IQD1, which is a member of a novel family of plant-specific CaM-interacting proteins of unknown functions, will pave the way to understand the biochemical and biological roles of this emerging class of proteins that are likely to link calcium signaling pathways to the regulation of nuclear gene expression in plant development and in response to various environmental challenges. This year, the group of Esther van der Knaap (Ohio State University) published the identification of IQD12 in tomato, which affects the shape of tomato fruits. Her report was chosen as the cover story of Science (319:1527-1530). The finding that IQD12 determines tomato shape and our preliminary data on IQD gene knockout mutants of Arabidopsis highlight the importance of IQD proteins in plant and crop biology.

Publications

• No publications reported this period

Progress 09/15/06 to 09/14/07

Outputs
During the previous year, we developed calmodulin pull-down and calmodulin overlay assays to demonstrate that IQD1 interacts in vitro with representative members of the major clades of Arabidopsis calmodulins (CaM) and calmodulin-like proteins (CML), CaM2, CaM4, CML8 and CML9. We created a series of deletion constructs and showed that the highly conserved IQ67 domain, which is the hallmark of the 33 IQD family members in Arabidopsis and of related proteins in other plant species, mediates calcium-dependent interaction with Arabidopsis CaM2. These studies were confirmed by IQD20, the smallest IQD family member in Arabidopsis, which mainly consists of the IZ67 domain. To test whether IQD1 interacts with CaM or CML polpeptides in vivo, we performed yeast two-hybrid assays with an extended set of CAMs and CMLs (13 members). These experiments revealed specific interactions for IQD1 with CaM2, CaM4 and CaM6 as well as for IQD20 with CML13. We further demonstrated that IQD1 interacts with single stranded nucleic acids in vitro (ssDNA and homopolyribonucleotides). Together with the nuclear localization of IQD1 and its highly basic isoelectric point, our data suggest that IQD1 regulates gene expression via calcium-regulated calmodulin interaction in the nucleus. We hypothesize that this mechanism is important for the regulation of plant defense responses as previously reported for IQD1 loss-and gain-of function alleles. Toward a systematic analysis of the biological roles of IQD1 we have created overexpression and knockout lines in the same ecotypes, Columbia and Wassilewskija. These lines will be used for probing the defense response pathways that are targeted by IQD1 and related proteins.

Impacts
Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues, such as drought, salinity, cold and heat stress, wounding, oxidative stress, herbivory, or pathogen response. A thorough understanding calcium signaling circuits at the cellular and systemic levels will be necessary for the long-term improvement of plant species of high societal value in agriculture and forestry. Our structure-function studies of IQD1, which is a member of a novel family of plant-specific CaM-interacting proteins of unknown functions, will pave the way to understand the biochemical and biological roles of this emerging class of proteins that are likely to link calcium signaling pathways to the regulation of nuclear gene expression in plant development and in response to various environmental challenges.

Publications

• No publications reported this period

Progress 09/15/05 to 09/15/06

Outputs
During the previous year, we developed calmodulin pull-down and calmodulin overlay assays to demonstrate that IQD1 interacts in vitro with representative members of the major clades of Arabidopsis calmodulins (CaM) and calmodulin-like proteins (CML), CaM2, CaM4, CML8 and CML9. We created a series of deletion constructs and showed that the highly conserved IQ67 domain, which is the hallmark of the 33 IQD family members in Arabidopsis and of related proteins in other plant species, mediates calcium-dependent interaction with Arabidopsis CaM2. These studies were confirmed by IQD20, the smallest IQD family member in Arabidopsis, which mainly consists of the IQ67 domain. To test whether IQD1 interacts with CaM or CML polypeptides in vivo, we performed yeast two-hybrid assays with an extended set of CaMs and CMLs (13 members). These experiments revealed specific interactions for IQD1 with CaM2, CaM4 and CaM6 as well as for IQD20 with CML13. We further demonstrated that IQD1 interacts with single stranded nucleic acids in vitro (ssDNA and homopolyribonucleotides). Together with the nuclear localization of IQD1 and its highly basic isoelectric point, our data suggest that IQD1 regulates gene expression via calcium-regulated calmodulin interaction in the nucleus. We hypothesize that this mechanism is important for the regulation of plant defense responses as previously reported for IQD1 loss- and gain-of-function alleles. Toward a systematic analysis of the biological roles of IQD1 we have created overexpression and knockout lines in the same ecotypes, Columbia and Wassilewskija. These lines will be used for probing the defense response pathways that are targeted by IQD1 and related proteins.

Impacts
Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues, such as drought, salinity, cold and heat stress, wounding, oxidative stress, herbivory, or pathogen response. A thorough understanding calcium signaling circuits at the cellular and systemic levels will be necessary for the long-term improvement of plant species of high societal value in agriculture and forestry. Our structure-function studies of IQD1, which is a member of a novel family of plant-specific CaM-interacting proteins of unknown functions, will pave the way to understand the biochemical and biological roles of this emerging class of proteins that are likely to link calcium signaling pathways to the regulation of nuclear gene expression in plant development and in response to various environmental challenges.

Publications

• Grubb, C.D. and S. Abel. 2006. Glucosinolate metabolism and its control. Trends Plant Sci. 11:89-100
• Abel, S., Savchenko, T. and M. Levy. 2005 Genome-wide comparative analysis of the IQD gene families in Arabidopsis thaliana and Oryza sativa. BMC Evol. Biology 5:72 (1-25)