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

Outputs
Target Audience:The scientific community interested in the nutritional properties of crop plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided opportunity for training of a postdoctoral fellow. How have the results been disseminated to communities of interest?The results been disseminated to communities of interest through publication in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Of the four project areas progress was made only in two, Genetic and Biochemical Dissection of Plant Sulfate Transceptor and the Mechanisms of Electron Transport in Sulfate Assimilation. No progress was made on the other projects because of the lack of funds. In the Dissection of Plant Sulfate Transceptor Since publication of our finding that an Arabidopsis sulfate transporter may function as an S sensor (Plant J 2014) the discoveryhas generated wide interest among members of the plant nutrient sensing community, the yeast nutrient sensing community, and the plant-microbe interaction community. First, investigators in plant nutrient sensing, in particular those interested in nitrogen and phosphate sensing, were keenly interested in the potential of a parallel to the nitrate transporter/sensor NRT1, and the phosphate transporter/sensor. The finding that SULTR1';2 may also be a nutrient sensor suggests that evolution has co-opted of transporters for sensor function. A paper was published showing that the yeast SUL1 and SUL2 sulfate transporters act as S sensors (http://www.jbc.org/content/290/16/10430.long). This represents a remarkable parallel to our finding in Arabidopsis. The authors shared their paper with us prior to publication and discussed the implications of their finding. Our paper was referenced by in the yeast paper. Of course, due the facile and rapid molecular system that is yeast, the biochemistry and structure function studies will outpace investigators working with plants. Our first approach will be to determine whether the yeast and Arabidopsis systems are truly analogous. This is discussed in the Outcomes section of the report. Despite the finding in yeast we still are left with the problem of solving how S sensing is controlled and coordinated in a complex higher eukaryote. Lastly, recent interest has been expressed by researchers in the plant microbe interaction field. A confidential communication from a scientist reported that they have evidence for SULTR1;2 function in controlling mycorrhizal symbiosis. They were interested in a number of the reagents that we have prepared for our project. Therefore, our discovery has already had wide impact on the development of the principal discipline(s) of the project. We have discussed above the impact of our work in Arabidopsis on the yeast field. We chose to describe yeast as within the principal discipline of the project because we define our work as nutrient sensing, regardless of the experimental system being studied. However, yeast is in fact a different species and thus could be considered an "other discipline." Some of the last project period was devoted to investigation of an unanticipated outcome of the project. As described in the Products section, while searching for genes that are controlled by the SULTR1;2 S sensor, a gene was discovered that may play an important role in controlling root architecture in response to S deprivation. The exciting possibility was deemed significant enough to devote a portion of the last years' project period on the controller of root architecture. The goal of the project is to gain a mechanistic understanding of how plants sense their nutritional status with respect to the macronutrient sulfur (S) using Arabidopsis thaliana as a vascular plant model. The sulfate transporter SULTR1;2 was identified that shows properties of being a sensor of sulfur status in that specific alleles show a phenotype of mis-regulating sulfur response genes (genes whose expression is increased or decreased after starving plants for sulfur). The specific project goals are to: identify the specific signaling pathway/signaling molecule that SULTR1;2 interacts. Several different regulatory pathways have been described that control the S-deficiency response. To understand how SULTR1;2 functions as a sensor it is imperative to know which signaling pathway it controls and what molecule it senses. The second goal is to understand how SULTR1;2 interacts with the signaling machinery, will be investigated by assessing the plasma membrane localization of the protein and its membrane topology. To function as a sensor, SULTR1;2 must be positioned in cells in such a way that will allow it to interact with signal transduction proteins. The third objective is characterize the relationship between structure and function of specific domains SULTR1;2 that mark the sensing function of the protein. A series of gene reconstructions & directed mutations are planned to map the sensing domain. Another goal centers on a transformative and unanticipated outcome of the project. One of the sulfur response genes that is controlled by SULTR1;2 encodes a protein of unknown function, but with homology to an E. coli protein named ChaC, also of unknown function. Our transformative results indicate that Arabidopsis ChaC is a type gamma-glutamyl cyclohydrolase involved in glutathione degradation/turnover not previously known. Its likely function is the release and utilization of the reduced sulfur moiety of glutathione during sulfur starvation. During the full term of the project we have made major headway in characterizing a high affinity sulfate transporter that likely also has a function in sensing sulfur. This accomplishment was made using a combination of genetic and biochemical approaches between two laboratories. The project was a collaborative effort between Zhi-Liang Zheng of Lehman College- the City University of New York, and the Tom Leustek of Rutgers The State University of New Jersey. During the most recent past project period the work was divided into two major activities, (1) following up on three objectives that were initiated in the second year project period, and (2) analysis of the role of At5g26220 (GGCT2;1) in possible control of root architecture changes induced by S starvation. This objective was added after an initial discovery during the current project that prompted two requests for REU supplements. The results are transformative and an unanticipated outcome of the project.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Zheng Z-L , Zhang B, Leustek T, (2014) Transceptors at the boundary of nutrient transporters and receptors: a new role for Arabidopsis SULTR1;2 in sulfur sensing. Frontiers in Plant Science 5:710 doi: 10.3389/fpls.2014.00710
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chung J-S, Lee H-N, Leustek T, Knaff DB, Kim C-S (2015) The Arabidopsis thaliana adenosine 5-phosphosulfate reductase 2 (AtAPR2) participates in flowering time and glucose response. Journal of Plant Biology 58: 128-136
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Lee J, Joshi N, Pasini R, Dobson RC, Leustek T (2015) Inhibition of Arabidopsis growth by the allelopathic compound azetadine 2-carboxylate is influenced by differing catalytic properties of prolyl-aminoacyl-tRNA synthetase isoforms. The Plant Journal, resubmission invited September 15, 2015
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Leustek T, Zheng Z-L (2015) SULTR1;2 in S Nutrient-Status Control in Arabidopsis. Molecular Physiology and Ecophysiology of Sulfur, Proceedings of the International Plant Sulfur Workshop, De Kok, L.J., Hawkesford, M., Rennenberg, H., Saito, K., Schnug, E. (Eds.) PP81-91 DOI 10.1007/978-3-319-20137-5_8

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

Outputs
Target Audience: The target audience reached was the scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project involved the training of a postdoctoral scientist in carrying out a biochemical/molecular research project in plants. How have the results been disseminated to communities of interest? Yes- as publications for the transceptor project, No for the maize nutrtional improvement project. What do you plan to do during the next reporting period to accomplish the goals? Complete at least two publications that are now being drafted on the maize nutritional value project. We also will continue to perform experiemnts on this same project.

Impacts
What was accomplished under these goals? No work was carried out on the third and fourth project objectives due to a lack of funding for these projects. These projects include Mechanisms of Electron Transport in Sulfate Assimilation; and Lysine Biosynthesis and its Control in Plants. The first projects aims to define the transcriptome and the underlying epigenetic regulation of the primary nitrogen and sulfur responses in maize The second project,Biochemical Dissection of Plant Sulfate Transceptor; was the one on which the most progress was made and that resulted in the listed publication. Significant progress was made on the first project, Epigenetic Regulation of Nutrient Uptake, Assimilation and Utilization in Maize, but no publications have yet resulted, although the manuscripts are in the final stages of preparation. In brief, the results of the research indicate that upregulating the sulfur assimilation pathway in maize, leading to increased biosynthesis ofcysteine and methionine,results in the upregulation of specific seed storage proteins in maize that have a higher proportion of these amino acids. The net result is that the amount of cysteine and methionine increases in the maize kernals. This finding has significant implications for improvement of the nutrional value of maize as animal feed. Maize that is grown as it typically is under field conditions, does not have enough cysteine and methionine to make it a complete nutritional source. Therefore, the production of animal feed from such maize includes supplementation of the feed with the missing amino acids. The procedure developed for this project promises the production of maize that does not need to be supplemented with cysteine and methionine. Indeed, the levels that we have measured in maize kernals developed for this project have the highest levels of cysteine and methionine that have yet been reported.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Xiang X, Pan G, Rong T, Zheng Z-L, Leustek T (2014) A luciferase-based method for assay of 5'-adenylylsulfate reductase. Analytical Biochemistry 460: 22-28

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audiences reached during this reporting period include the scientific community focused on improvement of plant nutrient use efficiency as well as the wider scientific community with interests in plant biochemistry, molecular biology, genetics, plant genome annotation, and crop improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project has provided opportunities for training of undergraduate students, graduate students, and postdoctoral associates. All of the classes of student have worked on the project as scientists carrying out lab research, preparing their data into reports, presenting their data in lab, local and national conferences. In addition, on Project Area 2 we have developed a collaboration with a professor at the City University of New York and this collaboration has provided the opportunity for students working on the project to interact with another research laboratory as part of a team effort. How have the results been disseminated to communities of interest? Some of the results have been published in peer -eviewed journals. In addition, presentations of the work have been presented at local, and national conferences. Lastly, the PI on the project has recently been invited to present research results derived from this project at an international conference in 2014. What do you plan to do during the next reporting period to accomplish the goals? Continue to work toward the project goals and continue to seek extramural funding opportunities to fund the project. Funding from the HATCH project, while critical and essential, provides only a small part of the funding needed to carry out a modern, molecular/biochemcial type research effort. The real importance of HATCH funding is to provide a small portion of the seed money needed to obtain research results that can be used to seek extramural funding. So, although HATCH funding is small, it is very very important and critical.

Impacts
What was accomplished under these goals? The outcomes of all subprojects have been to move our understanding of the specific system forward. This will result in further opportunities for dissemination of research results through publications and the assembly of a body of preliminary results needed to compete for extramural funding. With regard to the impact of the research the following statements apply. Project Area 1: Epigenetic Regulation of Nutrient Uptake, Assimilation and Utilization in Maize has provided prime opportunity to develop a more nutritious and more efficient maize. Project Area 2: Genetic and Biochemical Dissection of Plant Sulfate Transceptor, our understanding of how plants sense sulfur nutrition status provides insight into the molecular mechanism that plants use to efficiently utilize inorganic nutrients in fertilizers. Project Area 3: Mechanisms of Electron Transport in Sulfate Assimilation, the work provides an understanding of the structure and function relationships at the active site of an important enzyme in sulfur assimilation, leading to synthesis of key amino acids like cysteine and methionine that determine nutritional value of crop plants. Project Area 4: Lysine Biosynthesis and its Control in Plants, the outcome/impact has been to further the understanding of how an aminotransferase functions in promoting resistance to a bacterial pathogen. The project goals are in four individual project areas and the accomplishments for each will be separately described. Project Area 1: Epigenetic Regulation of Nutrient Uptake, Assimilation and Utilization in Maize- several attempts to obtain funding for this project have failed and so no further progress has been made. Project Area 2: Biochemical Dissection of Plant Sulfate Transceptor- significant progress has been made on this project resulting in a major publication at the end of 2013. In summary, genetic evidence has been obtained that plants can sense the level of sulfur available to them. Sulfur is an essential nutrient in the soil. When plants are starved for sulfur they respond with wholesale restructuring of gene expression, metabolism, and morphology. The project being studied sought to identify the sensor of sulfur nutrient by preparing a reporter gene plant under which a sulfur-response gene promoter was linked to a reporter gene. In this way the sulfur starvation response could be readily observed. Mutants were isolated that led to upregulation of the sulfur-response promoter even under sulfur-sufficient conditions. The mutants mapped to a gene encoding a high affinity sulfate transporter. Although the mutant plants are defective in high affinity sulfate transport, the growth conditions can be manipulated such that the plants do not show evidence of sulfur deficiency, yet the reporter gene is misregulated nonetheless. Project Area 3: Several attempts to obtain extramural funding for this project have failed. Nevertheless, a new assay was developed to study a key enzyme of sulfur reduction referred to as 5’-aadenylylsulfaye (APS) reductase using a luciferase-based assay for measurement of the reaction product adenylate (AMP). A luciferase-based assay method was developed for measurement of 5’-adenylylsulfate (APS) reductase (APR), an enzyme of the reductive sulfate assimilation pathway in prokaryotes and plants. 5’-AMP production is measured using a coupling system that generates luminescence with luciferase. The method is shown to provide an accurate measure of APR kinetic properties and can be used both for endpoint and continuous assays. APR activity can be measured from pure enzyme preparations as well as from crude protein tissue extracts. In addition, the assay is ideally suited for high throughput sample analysis of APR in a microtiter dish format. Project Area 4: On this project a we have focused on a key enzyme of lysine biosynthesis referred to as LL-diaminopimelate aminotransferase (DapL), found in plants and selected group of primarily pathogenic bacteria. Following on a previous study that identified possible inhibitors of DapL, we studied the effect of a select group of inhibitors on several different forms of the enzyme. The goal of the project is to identify compounds for possible leads in development of antibiotics or herbicides/algaecides.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhang B, Pasini R, Dan H, Joshi N, Zhao Y, Leustek T, Zheng Z-L (2014) Aberrant gene expression in mutants of the Arabidopsis sulfate transporter SULTR1;2 suggests a possible regulatory role for this sulfate transporter in response to sulfur nutrient status. The Plant Journal 77: 185197
• Type: Journal Articles Status: Published Year Published: 2013 Citation: McKinnie SM, Rodriguez-Lopez EM; Vederas JC, Crowther JM, Suzuki H, Dobson RC, Leustek T, Triassi AJ, Wheatley MS, Hudson AO (2014) Differential response of orthologous L,L-diaminopimelate aminotransferases (DapL) to enzyme inhibitory antibiotic lead compounds. Bioorganic & Medicinal Chemistry 22: 523-30
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Gao H, Subramanian S, Couturier J, Naik S, Kim S-K, Leustek T, Knaff D, Wu H-C, Vignols F, Huynh B, Rouhier N, Johnson M (2013) Arabidopsis thaliana Nfu2 accommodates [2Fe-2S] or [4Fe-4S] clusters and is competent for in vitro maturation of chloroplast [2Fe-2S] and [4Fe-4S] cluster-containing proteins. Biochemistry 52: 6633-6645

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: There are four aspects of the Hatch project: 1. Epigenetic Regulation of Nutrient Uptake, Assimilation and Utilization in Maize 2. Genetic and Biochemical Dissection of Plant Sulfate Transceptor 3. Mechanisms of Electron Transport in Sulfate Assimilation 4. Lysine Biosynthesis and its Control in Plants Report on 1. Epigenetic Regulation of Nutrient Uptake, Assimilation and Utilization in Maize: significant progress has been made in preparing transgenic maize with enhanced nutrient uptake and assimilation. In brief, four different genetic engineering stratgeies were attempted, two of which produced marked accumulation of the high methionine zeins in kernels. The specific strategies that produced the effect were overexpression of an enzyme from Arabidopsis thaliana for sulfur assimilation into cysteine termed serine acetyltransferase. The second, surprising result was that overexpression of a sulfur reduction enzyme from Escherichia coli, 5'phosphoadenosine, 3'-phosphosulfate (PAPS) reductase also produced significant increases in the high Met zeins. The plants are currently in the second transgenic generation where we will evaulate the heritability and effectiveness of the transgenes. Report on 2. Genetic and Biochemical Dissection of Plant Sulfate Transceptor: significant progress has also been made on this project and a manuscript on results is currently being prepared. In brief, we have gathered convincing evidence, based on analysis of Arabidopsis thaliana mutants, that a high affinity sulfate transporter termed SULTR1;2 has a second function in sensing the sulfur nutrition status of Arabidopsis. It also appears that the sulfur sensor is very likely detecting the intracellular concentration of reduced sulfur compounds. Cys and glutathione are likely candidate compounds, rather the the concentration of sulfate. Additional work went into development of test systems that will be used to dissect the domains of the sulfur transceptor that enable it to sense sulfur. Progress on 3. Mechanisms of Electron Transport in Sulfate Assimilation: not much progress has been made on this project. An attempt was made to obtain funding for this project from the National Science Foundation, but the project was not funded. I am currently resubmitting the proposal. In regards to research progress, six different site directed mutants were prepared on the enzyme 5'-adenylylsulfate reductase and it was shown that conservative replacement of an active site residue produces no change in activity, whereas a major change in amino acid eliminates activity. Work is now being performed to determine the substrate binding affinity. Progress on 4. Lysine Biosynthesis and its Control in Plants We have now analyzed the catalytic properties of a homolog of the lysine biosynthesis enzyme L-diaminopimelate aminotransferase from Arabidopsis thaliana and have found that it has broad substrate specificity, preferring either methionine as the amino donor. This result sets the stage for a grant proposal on this project. Work was also completed that resulted in a publication in Functional Plant Biology. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Target audiences are basic researchers interested in basic aspects of plant function. PROJECT MODIFICATIONS: No changes

Impacts
The outcomes for all subprojects has been to move our understanding of the specific system forward. This will result in further opportunities for dissemination of research results through publications and the assembly of a body of preliminary results needed to compete for extramural funding. With regard to the production of a product, one project 1. Epigenetic Regulation of Nutrient Uptake, Assimilation and Utilization in Maize has provided prime opportunity to develop a more nutritious maize. With regard to 2. Genetic and Biochemical Dissection of Plant Sulfate Transceptor, our understanding of how plants sense sulfur nutrition status provides insight into the molecular mechanism that plants use to efficiently utilize inorganic nutrients in fertilizers. With respect to 3. Mechanisms of Electron Transport in Sulfate Assimilation, the work provides an understanding of the structure and function relationships at the active site of an important enzyme in sulfur assimilation leading to synthesis of key amino acids like cysteine and methionine that determine nutritional value of crop plants. Lastly, with respect to 4. Lysine Biosynthesis and its Control in Plants, the outcome/impact has been to further the understanding of how an aminotransferase functions in promoting resistance to a bacterial pathogen.

Publications

• Jones-Held S, Ambrozevicius AP, Campbell M, Drumheller B, Harrington E, Leustek T (2012) Two Arabidopsis thaliana dihydrodipicolinate synthases, DHDPS1 and DHDPS2, are unequally redundant. Functional Plant Biology 39: 1058-1067

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

Outputs
OUTPUTS: A genetic complementation system was developed that will allow us to dissect the structure and function of a dual function high affinity sulfate transporter and sulfur sensor called SULTR1;2. SULTR1;2 is expressed in cortical and epidermal cells of roots in Arabidopsis. Genetic evidence suggests this protein functions as a sulfur sensor in addition to its well-known function as a sulfate transporter. In past work three different point mutations were characterized, one of which, sel1-17, appears to disrupt sensing but not transport function. The complementation system is intended to map the regions of SULTR1;2 responsible for sensing. Given its restricted expression pattern complementation system was developed using the SULTR1;2 promoter. The promoter vector was constructed so that mutant versions of SULTR1;2 can be cloned into it. The complementation system was also designed so SULTR1;2 mutant forms can be tested for transport function, so the vector was designed with a mechanism to move the SULTR1;2 mutant into a yeast plasmid for testing in a yeast sulfate transport defective strain. To test complementation in both Arabidopsis and yeast we placed an epitope tag (FLAG) at the carboxyl terminus of SULTR1;2. The epitope tag will allow us to measure the expression level and to control for expression. Lastly, in order to test for complementation in plants a specific Arabidopsis tester strain was constructed. The strain carries a TDNA insertion in the SULTR1;2 gene. This allele has been named sel1-18 and is a complete knockout of the SULTR1;2 expression. The sel1-18 strain was transformed with a reporter gene construct consisting of a sulfur-responsive promoter from the At2g44460 gene linked to LUC. Currently, we have constructed three variants in the complementation vector, the wild type SULTR1;2 (SEL1) as well as two point mutant alleles sel1-17 (defective in sensing but not transport) and sel1-15 (defective in sensing and transport). These have been successfully tested for transport function in yeast.The constructs have also been transformed into the Arabidopsis tester strain. So far we have shown that SEL1 complements both the sensing and transport functions of the tester strain. We have also demonstrated that the FLAG-tagged protein can be detected when expressed in yeast and in Arabidopsis. We are now growing the initial plants transformed with sel1-15 and sel1-17. Our initial assessment is that neither of these alleles complements the sensing defect of the tester Arabidopsis strain. In a second aspect of our project, we are attempting to engineer improved nutritional value in maize by transgenic expression of genes necessary for sulfate assimilation. There were deleterious consequences to the expression. The plants were stunted and sickly, and emitted enormous amounts of toxic reduced sulfur intermediates. We are attempting to enhance Met kernel accumulation while mitigating the negative effects. PARTICIPANTS: Project Participants Thomas Leustek, Project Director, supervision of the project Rita Pasini, Technician, maintence of laboratory operation and research on plant sulfur sensing. Christopher Trischetta, Research Intern, plant sulfur sensing project. These individuals were paid to work on the project. Unpaid persons working on the project are not listed. Partner Organizations Waksman Institute, City University of New York, Texas tech University, the 2011-2014 National Science Foundation, Title: "Collaborative Research: Genetic and Biochemical Dissection of Plant Sulfate Transceptor," IOS-1121521, $340,001 Collaborators on the project include Jo Messing, Yongrui Wu, and Jose Joplanta of the Waksman Institute. With them we are collaborating of transgenic approaches to increasing nutritional value of maize kernels by enhancing the accumulation of methionine. Collaborators at outside universities include Zhi-Liang Zheng, from the Department of Biological Sciences, Lehman College, City University of New York with whom we are collaborating on the sensing of sulfur nutrient in Arabidopsis. We also collaborate with Dr. David Knaff, Texas Tech University on the structure and function of an enzyme of the plant sulfate reduction pathway. Training or professional development Opportunities of undergraduate students abound. During the 2011 project period 5 students were trained. A graduate exchange student from the Sixuan University School of Agriculture arrived for a 1 year internship in Septemebr of 2011. Xiaoli Xiang has been transforming maize plants with constructs aimed at increasing the nutritional value of maize kernels. TARGET AUDIENCES: Target audiences The plant basic knowledge community including researchers at universities, institutes, and non-profit organizations as well as applied researchers and R&D researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The sensing defect of sel1-15, sel1-17, and sel1-18 can be genetically complemented by SEL1. A system is now in place to map the domains of SULTR1;2 that control sensing and transport. Maize kernel methionine content can be controlled by the sulfate assimilation rate. There are deleterious consequences for increasing sulfate assimilation stemming from accumulation of toxic intermediates of the assimilation pathway. It may be possible to mitigate the deleterious effects by controlled expression of sulfate assimilation genes.

Publications

• Hudson AO, Klartag A, Gilvarg C, Dobson RC, Garbelini-Marques F, Leustek T (2011) Dual diaminopimelate biosynthesis pathways in Bacteroides fragilis and Clostridium thermocellum. Biochemica Biophysica Acta-Proteins and Proteomics 1814:1162-8.

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

Outputs
OUTPUTS: We have continued our investigation of how plants sense the macronutrient sulfur (S). S is an inorganic macronutrient required by plants for the synthesis of essential S-containing compounds like cysteine and methionine. S is available to plants in the form of the sulfate ion present in the rhizosphere, which is taken up by roots and then is translocated to shoots. Plants are able to sense their S status. The information is used to make adaptive changes to metabolism, growth, and development . The project is based on mutants of Arabidopsis thaliana that misregulate a gene that is normally expressed when plants are starved for S. The mutants express the response gene even when they are grown with adequate S. Three mutant alleles were isolated in a gene encoding a high affinity sulfate transporter. This finding leads to the hypothesis that the sulfate transporter may have a second function in sensing S status. All of the work in the past year has focused on testing the hypothesis. In this way we hope to define how plants sense their S status and adapt to varying level of S in their environment. The second project on which significant progress was made focuses on understanding the control of amino acid biosynthesis. The aspartate-derived family of amino acids, lysine, threonine, and methionine are formed via a branched metabolic pathway. Their synthesis is controlled by a complex interplay of allosteric enzyme regulation and control of gene expression. One Arabidopsis mutant that accumulates enormous quantities of threonine carries a mutation in the DHDPS2 gene, one of two homologous genes encoding dihydrodipicolinate synthase, the first enzyme for lysine biosynthesis. The mutants are viable, meaning that the two DHDPS genes have redundant functions. Our aim of this project is to understand how a mutation in a redundant lysine biosynthesis gene affects the biosynthesis of a different amino acid, threonine. We hypothesize that DHDPS2 has a regulation function that DHDPS1 does not have. The information on both projects are in the process of being disseminated to the intended audience through the submission of manuscripts for publication in leading peer reviewed journals. Additional projects are also ongoing, but less research progress has been made. These include a characterization of the Epigenetic Regulation of Nutrient Uptake, Assimilation and Utilization in Maize and Mechanisms of Electron Transport in Sulfate Assimilation both of which are either in the planning or research stages, for which proposals have been submitted in 2010 to Federal Granting Agencies. PARTICIPANTS: Principle Investigator: Thomas Leustek, conceived and planned the experiments, wrote grant proposals and manuscripts, prepared data figures and carried out general laboratory management. Technician: Rita Pasini, carried out the research, experiments, and data collection. She reported the results to Thomas Leustek. Rita also implemented general laboratory management. TARGET AUDIENCES: The target audiences included the plant biology research community including basic scientists interested in regulation of biochemical processes and applied scientists interested in application of the technology to crop plants. By way of example, it may be possible to improve the nutritional properties of crop plants like maize by modifying the control of S-sensing and/or lysine biosynthesis. PROJECT MODIFICATIONS: No modifications have been made.

Impacts
With regard to the sulfate sensing project, outcomes included the findings that the sulfate transporter allelels, sel1-15, sel1-16, and sel1-17 all are defective in sensing S status. sel1-15, sel1-16 are also defective in sulfate transport, but sel1-17 is as able to transport as is the wild type. In addition, sel1-17 plants show the same sulfate content as wild type over a wide range of sulfate concentrations, indicating that the S-sensing defect is not related to an inability to take up or assimilate sulfate. It also appears that the sensing defect of sel1-15 and sel1-16 is not due solely to an inability to take up or assimilate sulfate because when the mutant plants are grown at ultra high external sulfate the transport defect is partly overcome, yet S-sensing remains defective. Finally, it was demonstrated that the S-sensing defect is wide-ranging. The mutants were isolated based on the misregulation of a single S-response gene. We have now analyzed a range of S-response genes and found that many of them (although, importantly, not all) are misregulated in sel1-15, sel1-16, and sel1-17. These gene expression results support the hypothesis that the high affinity sulfate transporter is also an S-sensor. It functions in regulation of a subset of S-response genes, but there are some that are regulated independently of the high affinity sulfate transporter. The work from the prior year has added significant new data in support of the S-sensor hypothesis. Based on the past years work a manuscript has been written and submitted for publication to Proceeding of the National Academy of Sciences and we are currently awaiting the outcome of the review. The funding from HATCH was critical to performing the research and the outcomes reported here. With regard to the regulation of lysine biosynthesis, a DHDPS1 mutant was analyzed and found to have normal threonine content, unlike the DHDPS2 phenotype. Alleles were combined and segregants identified with various combinations and copy numbers of wild type DHDPS. The results indicated that the DHDPS2 gene is the source of about 60% of the DHDPS activity in Arabidopsis leaves, and DHDPS1 is the source of the remaining 40% of the activity. Plants with only a single copy of the wild type DHDPS2 gene, as the only functional DHDPS, did not accumulate threonine. The DHDPS activity was slightly in these plants than in plants lacking wild type DHDPS2, with two functional copies of DHDPS1 (these plants do not accumulate threonine). The results indicate that threonine accumulation is not a function of reduced DHDPS activity in the mutants, rather it is related directly to a functional copy of DHDPS2 gene. These results imply that DHDPS2 has a regulatory function that is not substituted or replaced by DHDPS1. A manuscript of the above-described results is now in preparation, and a manuscript of related work has been submitted to Biochemica Biophysica et Acta Proteins and Proteomics. We are now awaiting the outcome of the review.

Publications

• No publications reported this period

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

Outputs
OUTPUTS: Activities: Research efforts focused on several projects related to amino acid biosynthesis and improvement of crop nutrition. (1) Regulation of the aspartate family of amino acids, threonine, lysine, and methionine, all derived from aspartate. (2) Regulation of 5'-adenylylsulfate reductase, a key enzyme for the synthesis of cysteine and methionione. (3) Function of prolyl aminoacyltRNA synthetase, a central enzyme in the utilization of proline. (4) Function of LL-diaminopimelate aminotransferase, a central enzyme needed for the synthesis of lysine. (5) Global control of amino acid biosynthesis in plants. (6) Identification of tyrosine O-sulfation of proteins in plants and bacteria. (7) Sensory perception of sulfur nutrient by plants, a key step in the control of sulfur metabolites including cysteine and methionine. Events: The PlantBiology conference hosted by the America Society of Plant Biologists was attended in July of 2009 where results were presented and discussed. Ph.D. degree obtained by Jiyeon Lee, Plant Biology Graduate program, dissertation title: "Analysis of the enzymological properties of prolyl-tRNA synthetases in plants focusing on the mis-activation of the proline analog azetidine-2-carboxylic acid" M.Phil. degree obtained by Varinnia Gomes, Food Science Graduate program, thesis title: "Structure and Function Analysis of 5'-Adenylylsulfate Reductase (APR) in Plants" Services: No consulting, counseling or tutoring was conducted Products: None to report PARTICIPANTS: Project Participants PD-Thomas Leustek Rita Pasini technician Jiyeon Lee Graduate assistant Varinnia Gomes graduate assistant Melinda Martin- Research Assistant Professor TARGET AUDIENCES: The scientific community PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
(1) The ongoing project focuses on explaining the threonine accumulation phenotype of an Arabidopsis mutant in one of two genes for dihydrodipicolonate synthase (DHDPS2) encoding the first enzyme of lysine biosynthesis. We discovered that the accumulation of threonine is not repressible by treating plants with lysine and methionine, treatments that would be expected to block threonine synthesis, nor is the growth-sensitivity of the mutant to combined treatment with threonine, lysine and methionine greater than wild type. These results suggest that threonine accumulation is not directly due to biosynthesis, or that the mutant is less sensitive to feedback inhibition. If the later hypothesis is correct it would suggest that DHDPS may interact with aspartate kinase, the first enzyme of the pathway, perhaps in the role of a regulatory subunit. We are currently attempting to detect such an association. (2) We are investigating the role played by the cysteine residues in the function of 5'-adenylylsulfate reductase and have produced mutant forms of the enzyme that are now being used to examine the function of specific cysteine residues. (3) The project centered on the misutilization of a chemical analog of proline called azetidine-2-carboxylic acid. It was found that the form of prolyl tRNA synthetase localized within chloroplast/mitochondria is much better able to distinguish beteen proline and A2C than is the form of the enzyme localized in the cytosol. This result suggests a mechanism for evolution of resistance to A2C. (4) Bacterial mutant of the LL-dimainipimelate aminotransferase was created and found to be lethal unless the mutant is provided with diaminopimelate in the growth medium. This is the first demonstration in any organism/species that LL-dimainipimelate aminotransferase is an essential gene and points the way to identifying inhibitors of this enzyme that might be developed in to herbicides and antibiotics. (5) The phenotype of histidine biosynthesis mutants were analyzed. This is a cataloging effort to determine which will be the best to use in analysis of a gene regulation response whereby all amino acid biosynthesis and metabolism pathways are coordinately regulated. (6) A number of different proteins have been identified that appear to be modified after translation by sulfation of tyrosine residues in both plants (Arabidopsis) and bacteria (E. coli). Individual proteins were identified and we are now in a position to begin to analyze the function of this protein modification. (7) Mutants of Arabidopsis that are unable to sense the presence of sulfur as an inorganic nutrient were analyzed. Two alleles were found to be defective in sulfate transport, while the third allele was found to be fully capable of sulfate transport. This work sets the stage for identifying the mechanism that plants use to sense the sulfur in their environment.

Publications

• Chung, J.-S., Noguera-Mazon, V., Lancelin, J.-M., Kim, S.-K., Hirasawa, M., Hologne, M., Leustek, T., Knaff, D.B. (2009). The interaction domain on thioredoxin for Pseudomonas aeruginosa 5'-adenylylsulfate reductase, Journal of Biological Chemistry 284: 31181-31189

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

Outputs
OUTPUTS: In 2008 transgenic Arabidopsis plants expressing bacterial diaminopimelate dehydrogenase were prepared and were initially characterized. The aim of this experiment is to deregulate lysine biosynthesis, leading potentially to a method to improve lysine content of crops. Lysine is the major nutrient that limits the nutritional value of plant crops. Analysis of the transgenic plants is ongoing. One article was written and published this year. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Target audience are scientists and educators working on crop nutrients. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The research we conducted has enabled us to define better how lysine is produced. We anticipate that this information will be useful in improving nutritional content in many food plants. We are continuing to build on the knowledge generated by further evaluating the lysine synthesis pathway.

Publications

• Publication related to this project published in 2008: Hudson AO, Gilvarg C, Leustek T (2008) Biochemical and phylogenetic characterization of a novel diaminopimelate biosynthesis pathway in prokaryotes identifies a diverged form of LL-diaminopimelate aminotransferase. J Bacteriol 190: 3256-3263.

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

Outputs
OUTPUTS: In the research period we investigated the diaminopimelate biosynthetic pathway in Geobacter sulfurreducens. This pathway was critical to elucidate to develop an understanding for potential modification of the sulfur-containing amino acids to increase nutrient content of crop plants. We also made progress in understanding the pathways for glutathion and its hydrolysis. This also is important for future enhancement of crop nutrients. We also investigated the genetic regulation of histidine biosynthesis. PARTICIPANTS: Andre Hudson is a post-doc in Dr. Leustek's lab. He is also a minority (Afroamerican). Melinda Martin is an adjunct faculty member involved in this research. TARGET AUDIENCES: Scientists involved in research on nutrient enhancements in crop plants. Plant breeders and crop biotechnologists.

Impacts
Our research on the Geobacter biosynthetic pathways contributes to an understanding of overall nutrient synthesis. This information provides clues as to how amino acid synthesis occurs in food crops. The work on glutathion synthesis and hydrolysis will enable investigators to better modify plant genomes to enhance health nutrient content. The work on genetics of histidine biosynthesis will also be useful to scientists working in the area of gene regulation and expression. Histidine is an important component of chromosomes and plays an integral role in gene expression and regulation.

Publications

• Hudson AO, Gilvarg C, Leustek T (2007) LL-diaminopimelate aminotransferase is essential for diaminopimelate biosynthesis in Geobacter sulfurreducens. In preparation for Journal of Bacteriology
• Hudson AO, Gilvarg C, Leustek T (2007) Biochemical and phylogenetic characterization of a novel diaminopimelate biosynthesis pathway in prokaryotes identifies a diverged form of LL diaminopimelate aminotransferase. J Bacteriology (provisionally accepted December 2007)
• Martin MN, Saladores PH, Lambert, E, Hudson AO, Leustek T (2007) Localization of members of the glutamyl transpeptidase family identifies sites of glutathione and glutathione Conjugate hydrolysis. 144: 1715-1732
• Muralla R, Sweeney C, Stepansky A, Leustek T, Meinke D (2007) Genetic dissection of histidine biosynthesis in Arabidopsis. Plant Physiology 144: 890-903
• Kim SK, Gomes V, Gao Y, Chandramouli K, Johnson MK, Knaff DB, Leustek T (2007) The two domain structure of 5 adenylylsulfate reductase from Enteromorpha intestinalis is a requirement for efficient APS reductase activity. Biochemistry 46: 591-601

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

Outputs
No progress reported this period

Impacts
Work was completed on elucidation of the gene structure for Adenylsulfate (APS) reductase. This is important because it will allow for manipulation of sulfur metabolism in diverse organisms. Sulfur containing amino acids are important nutrients in plants and animal systems. We made significant progress in elucidating the features of lysine and histidine biosynthesis in several plants, algae and microbes. We filed a patent for a new pathway for lysine biosynthesis. It is expected that this work will aid agricultural scientists in developing crop plants that are high in these and other health-related nutrients.

Publications

• Mccoy A, Adams N, Hudson A, Gilvarg C, Leustek T, Maurelli A. 2006. Diaminopimelate aminotransferase, a trans-kingdom enzyme shared by Chlamydia and plants for synthesis of lysine. Proc. Nat. Acad. Sci. 103:17909-17914.
• Hudson A, Singh A, Leustek T, Gilvarg C. 2006. An L,L-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants. Plant Physiology 140: 292-301.
• Stepansky A, Leustek T. 2006. Histidine biosynthesis in plants. Amino Acids 30: 127-142.

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

Outputs
The lysine level in crops is one of the key determinants of nutritional quality. Knowledge about the biosynthesis pathway, which differs from the pathway that had been expected based on comparative biochemistry with bacteria, can lead to strategies for improvement of nutritional quality. Two papers were published that characterize the limitations in the biosynthesis of methionine in plants. Methionine is another key amino acid limiting the nutritional quality of crops. The first paper (Lee et al., 2005) defines that homoserine production provides the major limitation for biosynthesis of methionine. The second paper (Bagga et al, 2006), a work carried out in collaboration with researchers at New Mexico State University, reported that increasing methionine synthesis in alfalfa resulted in increased accumulation of a high-methionine content storage protein. This is a potentially important finding, suggesting a feed-forward regulation of high-methionine-content storage proteins that when free methionine is abundant alfalfa is able to fix it into a protein storage form. Three research papers were published that explored the contribution of sulfate assimilation as a major limitation for cysteine and methionine synthesis. The first paper (Martin et al., 2005), carried out in collaboration with Pioneer Hi-Bred International Inc., found that increasing the activity of a single enzyme in maize necessary for sulfite formation causes large-scale increase in cysteine synthesis. The second paper (Kim et al., 2005) carried out in collaboration with researchers at Texas Tech University, explored the properties of the enzyme for sulfite formation. Although the subject of the paper was the sulfite forming enzyme from a bacteria Pseudomonas aeruginosa, there was a strong connection to plant biology in that the Pseudomonas enzyme shares many biochemical properties with the plant homologous enzyme and previous work (Tsakraklides et al., 2002, Plant J., 32: 879-889) had shown that expression of the Pseudomonas enzyme in transgenic plants was very effective in increase cysteine biosynthesis. The third paper (Kim et al., 2006) also carried out in collaboration with researchers at Texas Tech University, explored the biochemical properties of the sulfite forming enzyme from plants. Finally, a review paper was accepted for publication on the subject of histidine biosynthesis in plants (Stepansky et al., 2006). This paper is a prelude to a major paper that will be published in 2006 on a comprehensive analysis of histidine biosynthesis mutants of the model plant Arabidposis thaliana. Analysis of amino acid biosynthesis in a model plant is directly applicable to crop plants, so it is anticipated that this study will provide key understanding that might be used for crop plant improvement.

Impacts
As a result of this research we have doscovered significant information regarding the pathway for lysine, methionine, and cysteine synthesis. This information will enable to evaluate ways to enhance nutrient levels in food crops. This could result in a significant enhancement of human and animal nutrition.

Publications

• Hudson AO, Singh BK, Leustek T, Gilvarg C (2006) An L,L-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants. Plant Physiology140: 292-302
• Kim S-K, Rahman A, Conover RC, Johnson MK, Mason JT, Moore ML, Leustek T, Knaff DB (2006) Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5 adenylyl sulfate reductase from Enteromorpha intestinalis. Biochemistry (accepted for publication October 28, 2005)
• Bagga S, Potenza C, Ross J, Martin MN, Leustek T, and Champa Sengupta-Gopalan C (2005) Co-expression of cystathionine -synthase and -zein in alfalfa leads to increase of -zein accumulation. In Vitro Cellular and Developmental Biology- Plant (accepted for publication Aug 29, 2005)
• Kim S-K, Rahman A, Mason JT, Hirasawa M, Miginiac-Maslow M, Keryer E, Knaff DB, Leustek T (2005) The interaction of 5 adenylylsulfate reductase from Pseudomonas aeruginosa with thioredoxin. Biochemica Biophysica Acta 1710: 103-112
• Sors TG, Ellis DR, Na GN, Lahner B, Lee S, Leustek T, Pickering IJ, Salt DE (2005) Role of sulfur assimilating enzymes in selenate reduction, tolerance and accumulation in Astragalus. Plant J 42: 785-797
• Lee M-S, Martin MN, Hudson AO, Lee J, Muhitch MJ, Leustek T (2005) Methionine and threonine synthesis are limited by homoserine availability and not the activity of homoserine kinase in Arabidopsis thaliana. Plant J 41: 685-696

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

Outputs
It is too early to report on this project.

Impacts
It is too early to report on this project.

Publications

• No publications reported this period