Progress 02/01/14 to 01/31/19
Outputs
Target Audience:Plant biotechnology industry: Sulfur nutrition is essential for plant biomass production. Genetic and biotechnological engineering of sulfur sensing mechanisms in plants contribute to food and energy production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Yinghua Wang (undergraduate assistant, Department of Biochemistry and Molecular Biology, Michigan State University) obtained hands-on training of laboratory skills under the supervision of the PI Hideki Takahashi, postdoctoral researchers Anne-Sophie Bohrer and Wei Dong, and a graduate student Katerina Lay. The PI Hideki Takahashi has overseen the entire project. The postdoctoral researchers participated in training undergraduate students (including summer REU students) in the laboratory to develop their mentoring and management skills. In the fall semester of 2018, a graduate student Hannah Parks from the MSU BioMolecular Science Gateway was trained for 10 weeks to fulfill the rotational program. She performed research on interaction of sulfur and iron nutritional responses in plants. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?This project has been renewed to the Hatch project MICL02594 with the following project title and research objectives. Project title: Molecular engineering of sulfur assimilation for enhancement of plant stress resilience Objectives: A. Investigating subcellular compartment-specific regulation of sulfur assimilation and signaling A-1. Deciphering transcriptional and posttranscriptional regulatory mechanisms modulating expression of cytosol- and chloroplast-localizing isoforms of ATP sulfurylase The molecular mechanisms for gene expression of ATP sulfurylase (ATPS), the key enzyme in sulfur assimilation, will be investigated at multiple levels. The research project will be focused on the isoenzymes ATPS1 and ATPS2 playing predominant roles in sulfur assimilation in Arabidopsis. Transcriptional regulation of chloroplast-localizing ATPS1 will be studied in relation to mechanisms associated with a putative GATA transcription factor-binding site found in the 1st intron. In contrast, the chloroplast/cytosol dual-localizing ATPS2 will be studied toward a direction to identify the physiological relevance of the alternative translation initiation mechanism that determines the expression levels of the isoforms to be differentially localized in the chloroplast and the cytosol under specific biotic and abiotic stress conditions. A-2. Characterizing enzymatic and non-enzymatic functions of PAPS synthase complex The research project will be focused on investigating sulfur sensing and signaling mechanisms associated with the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase complex formed between ATP sulfurylase (ATPS) and APS kinase (APK) expressed in the cytosol in Arabidopsis. Adenosine 5'-phosphosulfate (APS) is suggested as a signaling molecule in addition to being an intermediary metabolite affecting the PAPS synthase complex formation. The non-enzymatic functionality of the PAPS synthase and the potential signaling effect of APS will be investigated by monitoring the effect of ATPS and/or APK mutations on expression of sulfur-responsive genes in Arabidopsis. B. Elucidating regulatory networks of sulfur signaling pathways involved in plant defense responses B-1. Probing interactions of transcriptional regulators for sulfur response and jasmonate signaling The protein-protein interactions of an EIL-family transcription factor, SULFUR LIMITATION 1 (SLIM1), and JASMONATE ZIM-DOMAIN (JAZ) transcriptional repressors will be tested to verify their relationships in signaling pathways. The effect of mutations in SLIM1 and JAZ on sulfur assimilation, glucosinolate and camalexin biosynthesis, and defense responses will be investigated using genetic crosses of Arabidopsis mutant lines. The sulfur and jasmonate response phenotypes will be characterized by taking advantage of transcriptomics and metabolomics approaches to identify the downstream effect of the signaling pathways on gene expression and metabolism. B-2. Investigating integration of sulfur signaling with metabolic pathways affecting plant defense responses This research project will be focused on studying the protein-protein interaction network of SLIM1, which is known to be associated with pathways modulating specialized metabolism and plant defense responses. Besides glucosinolate and camalexin biosynthesis, pathways for coumarin biosynthesis and exudation are suggested as additional regulatory targets of the SLIM1 network associated with MYB transcription factors. This project will aim at elucidating the SLIM1 network regulating the biosynthesis of this phenolic compound in addition to sulfur-containing specialized metabolites, glucosinolate and camalexin, produced for plant defense. Research will be conducted using Arabidopsis mutant lines to determine how these metabolic pathways are differentially regulated and diverted one another to enhance the plant resilience to biotic factors in the environment.
Impacts
What was accomplished under these goals?
The objectives of this research project are to investigate sulfur signaling mechanisms and subcellular compartmentalization of sulfate assimilation pathways in plants. In line with these objectives, we pursued research on sulfur signaling mechanisms with relevance to our working hypothesis proposing signaling effect of protein-protein interactions between plasma membrane-localizing sulfate transporter (SULTR), cytosolic ATP sulfurylase (ATPS2), and cytosolic APS kinase (APK3) in Arabidopsis thaliana. As we reported previously, the protein-protein interaction between ATPS2 and APK3 is modulated by catalytic functions of these enzymes. In particular, the catalytically inactive form of APK3 was unable to interact with ATPS2 in the cytosol, suggesting that accumulation of APS prevents the ATPS2-APK3 enzyme complex formation. In addition, the catalytic mutation in APK3 was found to repress the SULTR1;2 promoter activity in Arabidopsis protoplast. These results implicate that APS itself or signals associated with the ATPS2-APK3 enzyme complex act on pathways controlling transcription of S-responsive genes in plants. We are currently studying the effect of APS and non-catalytic APK3 on promoter activities of other S-responsive genes in Arabidopsis. In addition to S-responsive sulfate transporters, SULTR1;1, SULTR1;2 and SULTR2;1, a putative transcriptional repressor SULFUR DEFICIENCY INDICUED1 (SDI1) controlling glucosinolate biosynthesis has been selected as as a target. In this project, we also pursued research on regulation of chloroplast-localizing form of ATP sulfurylase (ATPS1) in Arabidopsis. ATPS1 is the major flux-controlling enzyme exclusively localized in the chloroplast for sulfur assimilation. Based on literature information suggesting a correlation between gene expression and natural variation of the ATPS1 gene in Arabidopsis, we predicted a putative GATA transcription factor-binding site in the 1st intron is involved in transcriptional regulation of ATPS1. The putative GATA transcription factor-binding site was found in the 1st intron of ATPS1 in Col-0 and Sha accessions, but absent in Bay-0 accession that shows relatively lower ATPS1 transcript accumulation. The ATPS1-luciferase translational fusion gene construct with the Bay-0 type nucleotide substitutions at the putative GATA-binding site in the 1st intron showed low luciferase activity relative to the same construct with the Col-0 haplotype in Arabidopsis protoplasts. These results suggested that the putative GATA transcription factor-binding sequence predicted in the 1st intron is acting as a cis-regulatory element for ATPS1 gene expression.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Takahashi, H. Sulfate transport systems in plants: functional diversity and molecular mechanisms underlying regulatory coordination
Journal of Experimental Botany, https://doi.org/10.1093/jxb/erz132
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Plant biotechnology industry: Sulfur nutrition is essential for plant biomass production. Genetic and biotechnological engineering of sulfur sensing mechanisms in plants contribute to food and energy production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two undergraduate students, Michael Rankin and Yinghua Wang, from the Department of Biochemistry and Molecular Biology at Michigan State University obtained hands-on training of laboratory skills under the supervision of the PI Hideki Takahashi, postdoctoral researchers Anne-Sophie Bohrer and Wei Dong, and a graduate student Katerina Lay. This team organization in the laboratory is aimed at encouraging the postdoctoral researchers to develop their mentoring and management skills. The PI Hideki Takahashi oversees the entire project and is responsible for successful accomplishments. In the fall semester of 2017, a graduate student Ron Cook from the MSU BioMolecular Science Gateway was trained for 10 weeks to fulfill the rotational program and performed research on interaction of sulfur-responsive SSPs and putative receptor-like kinases suggested as possible candidate receptors based on analysis of microarray data. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The protein-protein interaction between sulfate transporter (SULTR), cytosolic ATP sulfurylase (ATPS2), and cytosolic APS kinase (APK3) will be studied in relation to sulfur sensing and signaling mechanisms. Based on the progress made during the previous term, we hypothesize that ATPS2-APK3 protein complex in the cytosol is the key component in sulfur sensing mechanisms controlling gene expression of SULTR1;1 and SULTR1;2. The proposed action mechanisms of sulfur sensing and signaling may involve protein(s) interacting with the ATPS2-APK3 protein complex and the putative repressor function of the intermediary metabolite APS. To this end, we will first plan to model the structure of the ATPS2-APK3 protein complex based on structural information known for a human PAPS synthetase, and then map the amino acid residues that are predicted to be essential at the interface of the protein-protein interaction. The prediction will be followed by experimental validations, introducing point mutations into the amino acid residues of ATPS2 or APK3 and testing their catalytic functions as well as the capability of maintaining the bi-functional ATPS2-APK3 complex. We also aim at identifying proteins associated with the ATPS2-APK3 protein complex through biochemical methods as we planned during the previous term of this research project. In line with this research direction, we have already collected evidence that substitutions of a few essential amino acid residues modulate catalytic properties and affect association and dissociation of the ATPS2-APK3 protein complex. Based on prediction of the protein structure, we additionally expect to obtain insights into amino acid residues or specific domains of the enzymes that are considered to be essential for the ATPS2-APK3 complex formation. Thus, we are now positioned to elucidate the roles of this key enzyme complex in sulfur sensing and metabolism through a rationalized experimental approach taking the advantage of information on the molecular structure. Furthermore, we hypothesize that a transport metabolon consisting of a transporter-enzyme complex may additionally impact the sulfur sensing and signaling mechanisms at the entry step of the sulfate assimilation pathway directly or through modulating the ATPS2-APK3 interaction. While we have collected evidence for SULTR-ATPS2 interaction in Arabidopsis protoplasts in vivo, which supports the idea of a transport metabolon for sulfate assimilation, we consider that additional interacting protein partners may be involved as modulators for signaling. One of these components can be the cytosolic O-acetylserine(thiol)lyase (OASTL-A), which is known to interact with the C-terminal STAS domain of SULTR1;2. OASTL-A also interacts with Ser acetyltransferase (SAT5) to form a cysteine synthase complex (CSS) when sulfide is available in excess under sulfur-replete conditions, but freed when OAS accumulates under sulfur deficiency. Therefore, the cellular sulfur status or the concentrations of substrates for Cys biosynthesis in the cytosol necessarily influence the SULTR-OASTL-A interaction, and in turn, the activity of the SULTR-ATPS2 sulfate transport metabolon at the plasma membrane. The research on putative sulfate transport metabolon will be conducted, considering these potential metabolic interplays with the ATPS2-APK3 and CSC. In addition to providing the interfaces for interactions with the metabolic enzymes, SULTRs are suggested to be functional as dimers. The results of our preliminary experiments indicate homo- and heteromeric interactions of SULTRs in yeast and plant cells. The homology of plant SULTR with bacterial and animal SLC26 anion transporters also supports the dimer configuration. Thus, this portion of the research project will be targeted toward the analysis of protein-protein interactions of multiple components. Similar to the experimental approaches planned for studying the functions of the ATPS2-APK3 complex, we will fully take the advantage of structural information to investigate the putative sulfur sensing functions of the protein complexes formed between the SULTR dimers or those interacting with ATPS2 and OASTL-A. The gene expression levels of SULTR1;1 and SULTR1;2 will be monitored as readout of signals associated with these putative sensing mechanisms in Arabidopsis mutant lines (sultr1;1, sultr1;2, atps2, apk3, oastl-a, and sat5) and in protoplasts expressing the mutant forms of SULTRs, ATPS2, APK3, OASTL-A and SAT5 to be verified to have defects in protein complex formation. In addition to studying the sulfur sensing mechanisms, we will continue to investigate the effect of sulfur nutrition and sulfur-responsive SSPs on root development and sulfur metabolism. We hypothesize that sulfur-responsive SSPs, such as CAPE4 and CLE2 identified from transcriptome analysis, may carry information specifically associated with sulfur-derived signals and act on regulatory pathways controlling development of root systems in response to changes in sulfate availability in the environment. The physiological roles of these SSPs and their putative receptor kinases in the primary root, lateral root, and root hair development will be studied using Arabidopsis mutants defective for either the SSP or the receptor functions. Following these phenotypic characterizations, we will conduct transcriptome analysis of Arabidopsis mutants to identify downstream components of the sulfur-responsive SSP signaling modules. We also plan to characterize the homologs of these sulfur-responsive SSPs and putative receptors in crop species to extend our knowledge toward development of root traits relevant to improvement of sulfur use efficiency and stress mitigation.
Impacts
What was accomplished under these goals?
The objectives of this research project are to investigate sulfur signaling mechanisms and subcellular compartmentalization of sulfate assimilation pathways in plants. In line with these objectives, we pursued research on sulfur signaling mechanisms with relevance to our working hypothesis proposing signaling effect of protein-protein interactions between plasma membrane-localizing sulfate transporter (SULTR), cytosolic ATP sulfurylase (ATPS2), and cytosolic APS kinase (APK3) in Arabidopsis thaliana. To investigate the effect of ATPS2 and APK3 on sulfur signaling, the wild-type and atps2 and apk3 mutant lines were grown under sulfate-replete conditions followed by a sulfate-deficiency treatment and further to receive resupply of sulfate for observation of changes in gene expression profiles of sulfur-responsive genes in relation to sulfur starvation and recovery responses. The results obtained from a series these sulfate withdrawal and re-supply experiments suggested that transcript levels of SULTR1;1, SULTR1;2 and SULTR2;1 increase in both the atps2 and apk3 mutant lines relative to the wild type under sulfate-starved conditions. In contrast, those transcripts were seen differently in two mutant lines in response to sulfate re-supply, as they were strongly repressed in apk3 mutant whereas moderately in atps2 relative to the wild type. These gene expression profile data provided an implication that both the presence of the ATPS2-APK3 protein complex and accumulation of APS as an intermediate of the two-step metabolic pathway for PAPS biosynthesis could have negative impact on gene expression of SULTRs. To further investigate the relevance of the PAPS biosynthetic pathway in the cytosol in sulfur signaling, point mutations were introduced into ATPS2 and APK3 to modify their catalytic functions and ability to form the bi-functional protein complex. Our preliminary results indicated that a mutation in ATPS2 that favors the enzyme catalysis for the reverse reaction (APS hydrolysis) strengthens the interaction with APK3 and represses a luciferase reporter gene expression controlled under the SULTR1;2 promoter. Furthermore, mutagenesis of APK3 into a catalytically inactive form led to dissociate the ATPS2-APK3 complex and repress the SULTR1;2 promoter-luciferase expression, indicative of a role of the intermediary metabolite APS involved in regulation of sulfur-responsive gene expression. The PI Hideki Takahashi gave a research talk on this topic at the 11th International Plant Sulfur Workshop. In addition to studying ATPS2-APK3 interactions, we pursued research on sulfur-responsive small signaling peptides (SSPs) to extend our research target toward characterizing signaling pathways that modulate root development in response to changes in sulfur availability. Our study in the aspect of root development has been focused on two SSPs in Arabidopsis, CAPE4 and CLE2, which have been identified through transcriptome analysis of the sulfur responses. During this reporting period, we made a progress in characterizing the sulfur-responsive phenotypes of Arabidopsis roots, in particular with a focus on functions of CLE peptides and its receptor CLV1. Based on analysis of root phenotypes of the clv1 mutants, we found that CLE-CLV1 signaling pathway inhibits development of lateral roots under sulfur-deficiency, although such an effect appears to be abolished by re-supply of sulfate. Our results also suggested that putative regulatory components that may function downstream of the CLE-CLV1 pathway are either active under sulfur deficiency to inhibit lateral root development or inactive upon sulfur replenishment for recovery of root development. Additionally, gene expression analysis suggested that sulfate supply attenuates the effect of pathways feedback controlling CLE2 and CLE3 gene expression. We also worked on methods development to determine auto-phosphorylation of putative receptor kinases for the sulfur-responsive SSPs, aiming at understanding the physical interactions of SSPs and receptor kinases.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Takahashi, H. "Molecular networks of sulfate transport, signaling and regulation." 11th International Plant Sulfur Workshop, Conegliano, Italy. September 16-20, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Bohrer, A.-S. and Takahashi, H. "Control of sulfur metabolic pathway in Arabidopsis thaliana through enzyme complex formation in the cytosol." 1st Annual North American Mass Spectrometry Summer School. Madison, WI. August 6-9, 2018. (Poster Presentation)
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Plant biotechnology industry: Sulfur nutrition is essential for plant biomass production. Genetic and biotechnological engineering of sulfur sensing mechanisms in plants contribute to food and energy production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Undergraduate students from the Department of Biochemistry & Molecular Biology at Michigan State University (Michael Rankin and Yinghua Wang) obtained hands-on training of laboratory skills under the supervision of the PI (Hideki Takahashi), postdoctoral researchers (Anne-Sophie Bohrer and Wei Dong) and a graduate student (Katerina Lay). This team organization in the laboratory is aimed at encouraging the postdoctoral researchers to develop their mentoring and management skills. The PI (Hideki Takahashi) oversees the entire project and is responsible for guiding them to successful accomplishments. In Summer 2017, Katherine Martinez from State University of New York at Oneonta participated in this project for 10 weeks as an REU student. Katherine presented a poster at the Mid-Michigan Symposium for Undergraduate Research Experiences based on results of her research conducted in my laboratory under this project. In Fall 2016 and Spring 2017, Genevieve Hoopes from the MSU BioMolecular Science Gateway was trained for 10 weeks as a rotational graduate student to perform research with an emphasis on characterization of sulfur-responsive SSPs. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The protein-protein interaction between sulfate transporter (SULTR), cytosolic ATP sulfurylase (ATPS2), and cytosolic APS kinase (APK3) will be studied in relation to sulfur sensing mechanisms. The effect of SULTR-ATPS2 and ATPS2-APK3 interactions on sulfur signaling pathways and expression of sulfur-responsive genes will be examined in Arabidopsis. Our preliminary data indicates transcript repression of SULTR1;1 and SULTR1;2, which normally occurs in wild-type Arabidopsis plants under sulfur-replete conditions, can be eliminated in atps2 mutant lines. This could implicate direct roles of the SULTR-ATPS2 or ATPS2-APK3 interactions associated with transcript repression of SULTR1;1 and SULTR1;2. To test this hypothesis regarding the potential sulfate sensing and signaling roles of the SULTR-ATPS2 and ATPS2-APK3 protein complexes, SULTR1;1 and SULTR1;2 gene expression levels will be determined in Arabidopsis cells expressing the catalytic point mutants of ATPS2 and APK3 modulating these transporter-enzyme or enzyme-enzyme interactions. Furthermore, binding of cytosolic O-acetylserine(thiol)lyase (OASTL) to SULTR in the presence or absence of ATPS2 and APK3 catalytic point mutants and the subsequent impact on SULTR1;1 and SULTR1;2 gene expression will be tested. Since OASTL is a subunit of cysteine synthase complex (CSS) that dissociates when OAS accumulates under sulfur deficiency and re-assembles when sulfide is available under sulfur-replete conditions, the sulfate sensing and signaling mechanisms in action through the function of SULTR-ATPS2 and SULTR-OASTL may be additionally controlled by the cellular metabolic status affecting Cys biosynthesis. Transgenic Arabidopsis lines that individually overexpress plastidic ATPS1, plastidic ATPS2 and cytosolic ATPS2 constructed in my laboratory will be utilized to answer these questions regarding the effect of sulfur metabolism and assembly of CSS on SULTR-ATPS2 and SULTR-OASTL interactions. The work on sulfur-responsive SSPs will be pursued with an emphasis on characterizing their functions to control the root growth, sulfur-responsive gene expression, and sulfur metabolism. In addition to CAPE4, we have identified CLE2 as another sulfur-responsive SSP but showing an opposite expression profile. Unlike CAPE4 being induced by sulfur starvation, CLE2 expression was significantly repressed when Arabidopsis seedlings were transferred from sulfur-replete to sulfur-deficient conditions. The effect of the CLE2-CLV1 peptide-receptor signaling module on expression of sulfur-responsive genes, including SULTR1;1, SULTR1;2, SHM7 and SDI1, will be investigated using the promoter-luciferase reporter system and Arabidopsis protoplasts on the same platform as we conducted relevant experiments for CAPE4. Furthermore, the physiological relevance of this signaling module will be evaluated in relation to sulfur response phenotypes by comparing the root growth and the transcript expression profile of sulfur-responsive marker genes in the wild type, CLE2-overexpressing lines, cle2 and clv1 mutants under sulfur-deficient and sulfur-replete conditions.
Impacts
What was accomplished under these goals?
The objectives of this research project are to investigate sulfur signaling mechanisms and subcellular compartmentalization of sulfate assimilation pathways in plants. In line with these objectives, we pursued research to determine protein-protein interactions between plasma membrane-localizing sulfate transporter (SULTR), cytosolic ATP sulfurylase (ATPS2), and cytosolic APS kinase (APK3) in Arabidopsis. According to results obtained during the previous reporting period, APK3 was suggested to be an essential component that competitively inhibits SULTR-ATPS2 protein complex formation, which led us to present a model that implicates regulation of sulfate influx across the plasma membrane being dependent on two interrelated protein-protein interaction events. The model hypothesizes binding of ATPS2 with SULTR at the entry step to modulate the sulfate influx due to ATPS catalysis in the reverse reaction being thermodynamically favored for hydrolysis of adenosine 5'-phosphosulfate (APS) to generate sulfate. The SULTR-ATPS2 complex, however, can be forced to dissociate by ATPS2-APK3 complex formation in the presence of APK3. To test this hypothesis, point mutations were introduced into ATPS2 and APK3 to modify their catalytic functions and to examine how these mutations affect the formations of the SULTR-ATPS2 and ATPS2-APK3 protein complexes. The fusion gene constructs with or without the catalytic point mutations in ATPS2 and APK3 were made to test the protein-protein interactions in bimolecular fluorescence complementation assays, luciferase complementation imaging, and epitope-tag pull-down assays. Our preliminary results suggest a key regulatory role of a metabolic intermediate APS in modulating the ATPS2-APK3 interaction in the cytosol. We will continue the research in this aspect with an aim to elucidate the nature of sulfate transporter-sulfur assimilatory enzyme complex formation. The research project has also made a progress toward a direction to identify small signaling peptides (SSPs) expressed in Arabidopsis roots in response to changes in sulfur nutritional conditions. Based on a survey of transcriptome datasets, we identified CAPE4 as an SSP-coding gene induced under sulfur-starved conditions in Arabidopsis. The transcriptome dataset from our previous study on sulfur limitation 1 (slim1) mutant suggested that CAPE4 gene expression is under control of SLIM1. Hierarchical clustering analysis of sulfur-responsive genes provided a cluster of genes co-regulated with CAPE4 in Arabidopsis. Sulfur limitation inducible genes, such as high-affinity sulfate transporters (SULTR1;1 and SULTR1;2), Ser hydroxylmethyltransferase (SHM7) and sulfur deficiency induced protein (SDI1) were present in the cluster. The receptors for CAPE4 are yet unknown. However, several candidates for receptor-like kinases were identified through predictions based on transcriptome analyses, and at least one of them was shown to induce the promoter activity of SULTR1;2 with the luciferase reporter system in Arabidopsis protoplasts when it was co-expressed with CAPE4. These findings provide new perspectives on interactions of SSP and sulfur signaling mechanisms involved in regulation of sulfur-responsive gene expression in plants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Maruyama-Nakashita, A., Suyama, A., Takahashi, H. (2017). 5?-non-transcribed flanking region and 5?-untranslated region play distinctive roles in sulfur deficiency induced expression of SULFATE TRANSPORTER 1;2 in Arabidopsis roots. Plant Biotechnol. 34, 5155. doi: 10.5511/plantbiotechnology.16.1226a
- Type:
Other
Status:
Published
Year Published:
2017
Citation:
Takahashi, H. Molecular basis of sulfur nutritional response in plants. Agriculture and Agri-Food Canada, London, ON, Canada. Aug 29, 2017. (Invited Seminar)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Martinez, K., Bohrer, A.-S., and Takahashi, H. Small peptide signaling pathways involved in plant sulfur responses. Mid-Michigan Symposium for Undergraduate Research Experiences (Mid-SURE). Jul 26, 2017. East Lansing, MI, USA.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Plant biotechnology industry: Sulfur nutrition is essential for plant biomass production. Genetic and biotechnological engineering of sulfur sensing mechanisms in plants contribute to food and energy production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Undergraduate students from the Department of Biochemistry & Molecular Biology at Michigan State University (Michael Rankin, Sarah Hodge, and Yinghua Wang) obtained hands-on training of laboratory skills under the supervision of the PI (Hideki Takahashi) and postdoctoral researchers (Anne-Sophie Bohrer and Wei Dong). This team organization in the laboratory is aimed at encouraging the postdoctoral researchers to develop their mentoring and management skills. The PI (Hideki Takahashi) oversees the entire project and is responsible for navigating them for accomplishments. Michael Rankin participated in REU program during the summer semester (Plant Genomics@MSU) and presented a poster at the Mid-Michigan Symposium for Undergraduate Research Experiences based on results of his research conducted in my laboratory under this project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The research project will be focused on studying biological relevance of protein-protein interaction of sulfate transporter (SULTR) and cytosolic ATP sulfurylase (ATPS2) in relation to sulfur sensing mechanisms. Sulfate concentration in the cytosol can be modulated by sulfate influx across the plasma membrane and metabolic conversion of sulfate to APS requiring catalytic activity of ATPS2 in the cytosol. Thus, the SULTR-ATP2 complex may function as a key component controlling the flux of sulfate transport and subsequent metabolism. SULTR can also bind O-acetylserine(thiol)lyase (OASTL-A1), a subunit of cysteine synthase complex (CSS) suggested to be associated with sensing of cellular sulfur status as it dissociates when O-acetylserine accumulates under sulfur deficiency while reassembles when sulfide is available under sulfur-replete conditions. In this project, the combinatorial effect of these protein-protein interactions on sulfate uptake will be determined using Arabidopsis atps2 and oastl-a1 mutants. Given these implications suggesting potential roles of these plasma membrane-bound protein complexes in sulfur sensing, we will investigate the effect of SULTR-ATPS2 and SULTR-OASTL-A1 interactions on sulfur signaling pathways modulating expression of sulfur-responsive genes in Arabidopsis. The atps2 and oastl-a1 mutants will be used for transcript profiling of sulfur-responsive genes that are responsible for sulfate uptake, sulfate reduction and glucosinolate biosynthesis or genes involved in their transcriptional regulation. In addition, transient promoter-reporter assay systems will be employed to determine the effect of SULTR-ATPS2 and SULTR-OASTL-A1 interactions on transcriptional regulations of sulfur-responsive genes in Arabidopsis protoplasts in vitro. The research project will be further extended toward investigating how sulfate metabolism in plastids and cytosol can differentially affect SULTR-ATPS2 and SULTR-OASTL-A1 interactions and sulfur signaling pathways in plants. Transgenic Arabidopsis lines overexpressing plastidic ATPS1, plastidic ATPS2 or cytosolic ATPS2 have been constructed in my laboratory to address these questions and to elucidate the relationships between the plastid-cytosol sulfur metabolic flux partitioning and sulfur signaling.
Impacts
What was accomplished under these goals?
The project with an aim to investigate the subcellular compartment-specific functions of sulfate assimilation pathways has progressed in a direction to identify protein-protein interaction between plasma membrane-localizing sulfate transporter (SULTR) and cytosolic ATP sulfurylase (ATPS2) in Arabidopsis. We tested potential interfering effects of cytosolic APS kinase (APK3) and O-acetylserine(thiol)lyase (OASTL-A1) on SULTR-ATPS2 protein-protein interaction, and found that presence of either of these alternative interacting partners of ATPS2 and SULTR could lead to dissociate the SULTR-ATPS2 protein complex. These results provide new insights into flux control mechanisms of sulfate transport and APS/PAPS biosynthesis involving protein-protein interaction of plasma membrane-localizing sulfate transporter and cytosolic sulfur metabolic enzymes. We expect to publish these results in the next reporting period. During this reporting period, the work on sulfur-deficiency-induced repressor proteins controlling glucosinolate biosynthesis in Arabidopsis was accepted for publication in Science Advances. The results presented in this paper demonstrate the role of a novel transcriptional repressor protein in sulfur metabolic partitioning between the primary and secondary pathways. The work was done in collaboration with groups in Germany (Max Planck Institute of Molecular Plant Physiology) and Japan (RIKEN and Kyushu University). The PI (Hideki Takahashi) contributed to this paper as a senior co-author.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Aarabi, F., Kusajima, M., Tohge, T., Konishi, T., Gigolashvili, T., Takamune, M., Sasazaki, Y., Watanabe, M., Nakashita, H., Fernie, A.R., Saito, K., Takahashi, H., Hubberten, H.-M., Hoefgen, R., Maruyama-Nakashita, A. (2016). Sulfur-deficiency-induced repressor proteins optimize glucosinolate biosynthesis in plants. Sci. Adv. in press.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Yoshimoto, N., Kataoka, T., Maruyama-Nakashita, A., Takahashi, H. (2016). Measurement of uptake and root-to-shoot distribution of sulfate in Arabidopsis seedlings. Bio-Protocol 6(1): e1700. http://www.bio-protocol.org/e1700
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Bohrer, A.-S., Takahashi, H. (2016). Compartmentalization and regulation of sulfate assimilation pathways in plants. Int. Rev. Cell Mol. Biol. 326:1-31. doi:10.1016/bs.ircmb.2016.03.001
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Rankin, M., Bohrer, A.-S., and Takahashi, H. Protein-protein interactions of sulfur metabolic enzymes and sulfate transporters in plants. Mid-Michigan Symposium for Undergraduate Research Experiences (Mid-SURE). Jul 27, 2016. East Lansing, MI, USA. (Poster Presentation)
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Plant biotechnology industry: Sulfur nutrition is essential for plant biomass production. Genetic and biotechnological engineering of sulfur sensing mechanisms in plants contribute to food and energy production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Undergraduate students (Nicholas Rykulski and Jocelyn Olvera) obtained hands-on training of laboratory skills under the supervision of the PI (Hideki Takahashi) and postdoctoral researchers (Anne-Sophie Bohrer and Wei Dong). Nicholas Rykulski coauthored a research paper. This team organization is aimed to encourage postdoctoral researchers in the laboratory to develop mentoring and management skills. The PI (Hideki Takahashi) oversees the entire project and is responsible for navigating them for accomplishments. How have the results been disseminated to communities of interest?The long-term goal of this project is to clarify the sulfur sensing mechanism and its control over sulfate uptake, metabolism and plant biomass production. During this reporting period, the progress and perspective of this research project were highlighted in the AgBioResearch 2014 Annual Report. In addition, the PI (Hideki Takahashi) was invited to the 10th International Workshop on Sulfur Metabolism in Plants and gave an oral presentation on this research project. What do you plan to do during the next reporting period to accomplish the goals?Research will be focused on investigating the biological relevance of subcellular compartmentalization and protein complex formation of ATP sulfurylase (ATPS) and APS kinase (APK) in plants. Protein-protein interaction between ATPS and APK has been successfully detected in Arabidopsis protoplasts by YFP-based bifluorescence complementation assays. Luciferase complementation imaging methods are also being pursued to probe compartment-specific formation of the ATPS-APK complexes. In addition to studying the protein-protein interactions through fluorescent or luminescent imaging approaches, epitope-tagged ATPS and APK isoforms from Arabidopsis will be co-expressed with sulfotransferase and desulfo-glucosinolates for reconstitution of glucosinolate biosynthesis in tobacco leaves to biochemically validate compartment-specific metabolic fluxes of sulfate assimilation associated with ATPS-APK complex formation. Furthermore, the impact of the overexpression of plastidic ATPS1 or cytosolic ATPS2 on sulfur signaling will be investigated by analyzing transcriptome profiles and expression levels of sulfur-responsive genes. During the next reporting period, we also aim to identify ATPS-APK-interacting proteins through proteomic screening of protein candidates that may bind to epitope-tagged ATPS-APK complexes. The interacting proteins are expected to be the modifiers altering the metabolic fluxes or the protein components potentially affecting the S-signaling mechanisms associated with the ATPS-APK protein complexes either in plastids or cytosol. Furthermore, we will pursue a direction towards investigating functional interplay between sulfate transporters and ATPS-APK protein complexes to gain new insights into sulfate sensing and flux control mechanisms.
Impacts
What was accomplished under these goals?
This project is aimed to elucidate the molecular mechanisms of sulfur assimilation in plants. During this reporting period, the work on plastid-cytosol dual-localization of ATP sulfurylase, ATPS2, resulted in two publications in a journal Frontiers in Plant Science (ISI impact factor = 4). Our findings of subcellular localizations of Arabidopsis ATPS2 documented the genetic origin of cytosolic ATP sulfurylase, and answered a long-standing unresolved question regarding subcellular partitioning of sulfur assimilation in plants. Furthermore, the study on sulfur deficiency response of a low-affinity sulfate transporter, SULTR2;1, demonstrated the roles of sulfur-responsive cis-elements located in the 3'-nontranscribed intergenic region. The sulfur-responsive cis-elements were identified to be essential for the induction of SULTR2;1 that mediates root-to-shoot transport of sulfate under sulfur deficiency. The results were published in a high profile journal Plant Cell (ISI impact factor = 10).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Maruyama-Nakashita, A., Watanabe-Takahashi, A., Inoue, E., Yamaya, T., Saito, K., and Takahashi, H. (2015). Sulfur-responsive elements in the 3-nontranscribed intergenic region are essential for the induction of SULFATE TRANSPORTER 2;1 gene expression in Arabidopsis roots under sulfur deficiency. Plant Cell 27: 12791296.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Bohrer, A.-S., Yoshimoto, N., Sekiguchi, A., Rykulski, N., Saito, K., and Takahashi, H. (2015). Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana. Front. Plant Sci. 5: 750. doi: 10.3389/fpls.2014.00750
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Bohrer, A.-S., Kopriva, S., and Takahashi, H. (2015). Plastid-cytosol partitioning and integration of metabolic pathways for APS/PAPS biosynthesis in Arabidopsis thaliana. Front. Plant Sci. 5: 751. doi: 10.3389/fpls.2014.00751
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Takahashi, H. Whole plant regulation of sulfate transport systems in Arabidopsis. 10th International Workshop on Plant Sulfur Metabolism. Sep 1-3, 2015. Goslar, Germany. (Invited Oral Presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Bohrer, A.-S., Takahashi, H. Plastid-cytosol partitioning of metabolic sulfur metabolism in Arabidopsis thaliana. Jul 19-24, 2015. Gordon Research Conference on Plant Metabolic Engineering. Waterville Valley, NH, USA. (Poster Presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Bohrer, A.-S., Dong, W., Yoshimoto, N., Olvera, J., Rykulski, N., Chen, J., Takahashi, H. Protein-protein interaction underlies functional interplay of high-affinity sulfate transporters facilitating sulfate uptake in plant roots. Jul 19-24, 2015. Gordon Research Conference on Plant Metabolic Engineering. Waterville Valley, NH, USA. (Poster Presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Dong, W., Yoshimoto, N., Kopriva, S., Takahashi, H. Regulation of sulfur assimilation by chloroplast-localizing ATP sulfurylase in Arabidopsis thaliana. Jul 19-24, 2015. Gordon Research Conference on Plant Metabolic Engineering. Waterville Valley, NH, USA. (Poster Presentation)
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Progress 02/01/14 to 09/30/14
Outputs
Target Audience: Plant biotechnology industry: Sulfur nutrition is essential for plant biomass production. Genetic and biotechnological engineering of sulfur sensing mechanisms in plants contribute to food and energy production. Changes/Problems: Dr. Stanislav Kopriva, an outside collaborator of this research project, has moved to the University of Cologne (Germany) as Professor and Chair of Plant Biochemistry. Collaboration will not change because the experiments on sulfur metabolic flux analysis can be done in his new lab at Cologne. Dr. Kopriva will continue to lead the EU-funded project on plant nutrition to support this collaboration. What opportunities for training and professional development has the project provided? Undergraduate students (Nicholas Rykulski and Jessica Noll) obtained hands-on training of laboratory skills under the supervision of the PI (Hideki Takahashi), postdoctoral researcher (Anne-Sophie Bohrer) and research technician (Steven Lundback). This organization is aimed to encourage postdoctoral researcher and staff in the laboratory to practically develop mentoring and management skills. The PI (Hideki Takahashi) oversees the entire project and is responsible for navigating them for accomplishments. How have the results been disseminated to communities of interest? During this reporting period, the PI (Hideki Takahashi) was invited to organize The 9th International Workshop on Sulfur Metabolism in Plants and gave a presentation on this research project. The long-term goal of this project is to clarify the sulfur sensing mechanism and its control over sulfate uptake, metabolism and plant biomass production. A special issue on this research topic (sulfur metabolism) is organized in an international journal Frontiers in Plant Science. The PI (Hideki Takahashi) is one of the associate editors for this special issue and will also contribute an original research article. What do you plan to do during the next reporting period to accomplish the goals? This project aims to demonstrate how the flux balance between the cytosolic and plastidic PAPS biosynthetic pathways may impact the overall sulfur metabolism and sulfur-responsive transcriptional regulatory networks in plants. We will take a transgenic approach to pursue this specific objective. Transgenic Arabidopsis plants overproducing ATPS2 in cytosol or plastids will be made for conducting comparative sulfur metabolic flux analyses. Chloroplasts and cytosol will be fractionated from the wild-type, atps2 mutant, and ATPS2-overexpressing lines to determine the enzymatic function of ATPS2 isoforms in the individual subcellular compartment. The contribution of PAPS biosynthesis in cytosol and chloroplasts will be estimated based on quantitative metabolic flux data. The impact of ATPS2 overexpression on sulfur signaling will be investigated by analyzing transcriptome profiles and expression levels of sulfur-responsive genes. We will also pursue the objective of elucidating the biological relevance of ATPS-APK complex formation in plants. Assembly and disassembly of ATPS-APK complex will be tested by bimolecular fluorescence complementation and yeast two-hybrid assays. This part should progress during the next reporting period. We expect to identify in vivo interactions between ATPS and APK hypothesized to be controlled by changes in sulfur status of cells. Furthermore, we will continue to work on yeast two-hybrid screening to identify novel ATPS-APK-interacting proteins. During the next reporting period, we expect to obtain a few candidate proteins and analyze their putative roles in controlling ATPS-APK complex assembly and PAPS biosynthesis in yeast and Arabidopsis.
Impacts
What was accomplished under these goals?
This project is aimed to elucidate the molecular mechanisms of sulfur metabolic flux partitioning between plastid and cytosol in plants. The study was focused in particular on the subcellular localization of the key entry enzyme ATP sulfurylase (ATPS) and its interaction with APS kinase (APK), which has been hypothesized to be implicated in sulfur metabolic flux control and sulfur sensing in plants. During this reporting period, we obtained molecular evidence underlying plastid-cytosol dual-localization of ATPS2 in Arabidopsis. Differential localizations of ATPS2 isoforms in plastids and cytosol were due to alternative translational initiations. In addition, ATPS2 promoter activity was found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots. This study unraveled the genetic identity of cytosolic ATPS in Arabidopsis and demonstrated the mechanism controlling its subcellular localization. Furthermore, we conducted yeast two-hybrid experiments to search for potential interactions among ATPS and APK isoforms from Arabidopsis, and obtained evidence for dimerization of cytosolic ATPS2. These results provide new insights into molecular evolution of sulfur metabolic enzymes and their control mechanisms.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Anne-Sophie Bohrer and Hideki Takahashi
Molecular evidence for dual localization of ATP sulfurylase 2 in Arabidopsis thaliana 9th International Workshop on Sulfur Metabolism in Plants, April 14-17, 2014, Freiburg, Germany (Poster Presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Hideki Takahashi
Membrane transport of sulfate and sulfur-containing metabolites in plants
9th International Workshop on Sulfur Metabolism in Plants, April 14-17, 2014, Freiburg, Germany (Invited Oral Presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Anne-Sophie Bohrer and Hideki Takahashi
Chloroplast-cytosol dual localization of ATP sulfurylase in Arabidopsis thaliana. Plant Biology 2014, July 12-16, 2014, Portland, OR (Poster Presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Anne-Sophie Bohrer and Hideki Takahashi
Alternative translation of ATP sulfurylase 2 allows dual localization of sulfate assimilation in chloroplasts and cytosol in Arabidopsis thaliana. 25th International Conference on Arabidopsis Research, July 28-August 1, 2014, Vancouver, Canada (Poster Presentation)
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