Jyoti Shah - US grants

Five controlled environment plant growth chambers will facilitate research of a cross-disciplinary group at Kansas State University (KSU) on the response of plants to biotic and abiotic environmental stresses. These chambers will provide controlled growth conditions crucial for understanding the mechanism by which plants perceive and respond to environmental cues.

The acquisitions will facilitate the individual and collaborative research of the group members on understanding the biochemical, physiological and molecular changes in plants exposed to stress, identification of genes involved in stress signaling, development of plant varieties resistant to pathogens and insects, and understanding the molecular basis of cell division and cell death in plants. The growth chambers will enhance the quality and productivity of research at KSU by providing infrastructure for innovative research, through cooperative use of facilities and increased collaboration. Students will benefit from specialty courses providing training on plant stress response. The equipment will further facilitate and enhance the recruitment and training of women and minorities in plant biochemistry, physiology, molecular biology and genetics at KSU.

Plant growth, development and productivity are influenced by environmental cues. The stress of disease, insect infestation, increasing global temperatures, limited water supply and chemical toxicity, limit plant productivity. The proposed work on understanding the perception and response of plants to biotic and abiotic environmental stresses will expedite the development of plant varieties with enhanced resistance to stress, thereby improving plant productivity, food quality, public health and the environment

Membrane lipids comprise diverse molecular species, and their composition differs from membrane to membrane. In addition, membrane lipid composition changes in response to internal and external cues. Furthermore, within a membrane, there may be microdomains with distinct lipid constituents and particular functions. However, it is not understood how these distinct compositions and their dynamics are generated and what their functions are in the cell. The objectives of this project are to use a metabolomic approach to determine cellular membrane lipid composition and to understand the regulation and role of membrane lipid compositional dynamics in plant responses to stresses. A highly sensitive approach based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) will be established. ESI-MS/MS will be employed to profile membrane lipid molecular species and to determine the compositional dynamics in Arabidopsis plants undergoing temperature and drought stresses. To understand how lipid changes are regulated, this project will investigate enzymes involved in generating the membrane lipid compositional dynamics. Arabidopsis lines, abrogated of various isoforms of phospholipase D, the major lipolytic enzyme family, will be instrumental in the analysis. In addition, lipid molecular species of the defense mutant ssi2 that is defective in stearoyl-ACP desaturase and its suppressor lines will be profiled to determine the relationship between lipid composition and alterations in defense responses. The capability to combine full lipid profiling with cellular analysis of the machinery that generates compositional changes should yield new information on how cellular machinery and metabolites interact in a dynamic manner in the cellular response to changing environments. Membrane lipids are vital biological constituents, providing structural backbones for biological membranes and crucial resources for producing second messengers in regulating cellular and organismal functions. Membrane lipids comprise diverse molecular species, and the composition differs from membrane to membrane. The lipid composition changes in response to internal and external cues. However, it is not understood how these distinct compositions and their dynamics are generated and what their functions are in the cell. This project will establish a highly effective approach based on electrospray ionization tandem mass spectrometry and use it to determine cellular membrane lipid composition and the role of membrane lipid compositional changes in plant responses to stresses. Extensive profiling of membrane lipids and their metabolites will yield unprecedented information on how cellular machinery and metabolites interact in a dynamic manner in the cellular response to changing environments.

This project assembles researchers with complementary expertise to develop the metabolomic capacity to quantitatively profile lipid molecular species and their changes in plants. The long-term goal of this project is to understand how cellular lipids and their metabolites interact to produce and maintain complex cell membranes and to regulate cell functions. The goal of this project is two-fold: One is to develop a comprehensive capacity to fully profile cellular lipid species, using a mass spectrometry-based, common platform. The other is to use the profiling capability to determine the metabolic function of enigmatic, patatin-like acyl hydrolases and several putative lipases involved in plant stress responses. The specific objectives of this application are to: (1) profile and quantify regulatory lipids: free fatty acids, selected fatty acid derivatives, lysolipid species, and phosphoinositides; (2) profile and quantify neutral glycerides: diacylglycerol and triacylglycerol; (3) profile and quantify sphingolipid molecular species; (4) discover new lipid species and metabolites; (5) use the profiling capability to determine the metabolic functions of putative lipolytic enzymes: the patatin-like proteins, RL-PLA, AAM10310, AtSABP2, PRLIP1, PAD4, and EDS1. Sensitive and efficient lipid profiling by electrospray ionization tandem mass spectrometry (ESI-MS/MS) has the potential to achieve full characterization of cellular lipids. The development of this capability will contribute greatly to the emerging, comprehensive research strategy, and metabolomics. Lipid profiling will provide a powerful strategy to address biological questions that involve the function of lipids. The functional studies will produce insights into in vivo substrates and products of Arabidopsis lipid-hydrolyzing enzymes, the conditions under which the enzymes are activated, and the spatial distribution of the activity.

The project will have broader impacts. In addition to training graduate students through research activities, this project will bring current knowledge of metabolic profiling and functional genomics to the classroom. It will also provide an opportunity to broaden participation of underrepresented groups in the plant sciences. The capability and service provided by the Kansas Lipidomics Research Center will be important to many researchers. The project will identify important metabolic and regulatory steps mediating plant growth and stress responses. Manipulation of these steps may lead to production of crop plants with enhanced stress tolerance and increased product quality and/or productivity.

Systemic acquired resistance is an inducible defense mechanism in plants that confers enhanced resistance against a broad-spectrum of pathogens. The activation of SAR requires the translocation of an unknown factor through the phloem. The Arabidopsis thaliana SFD1 gene, which encodes a dihydroxyacetone phosphate (DHAP) reductase, is required for SAR. DHAP reductases catalyze the interconversion of DHAP and glycerol-3-phosphate, which provides the glycerol backbone for glycerolipid biosynthesis. Lipid metabolism is altered in the sfd1 mutant, suggesting that a lipid(s) has an important role in SAR. The long-term objectives of this proposal are to study the role of SFD1 in SAR, thereby testing the above hypothesis. To achieve these objectives, a combination of genetic, molecular and biochemical approaches will be pursued to characterize the biochemical function of SFD1. In addition, the role of a SFD1-derived factor in the phloem during the activation of SAR will be addressed with a novel bioassay in combination with highly sensitive mass-spectrometric approaches. The proposed research will contribute greatly to the understanding of long-distance signaling in SAR. The project will provide an excellent opportunity to integrate state-of-the-art research and education by bringing current knowledge of lipid metabolism and the genetic, molecular and biochemical basis of plant stress responses into the classroom. In addition to providing training to graduate students and post doctoral fellows, this project will engage undergraduates in research in the biological sciences and broaden the participation of underrepresented groups. In particular, an undergraduate student from a non-Ph.D.-granting institution will be recruited to work on the project each summer. Results from this project will have potential benefits to society at large. The lipid(s) identified by the proposed study could provide new targets for enhancing disease resistance in plants, thus limiting the utilization of toxic chemicals to protect plants, thereby improving the quality of our environment and health.

Metabolomic profiling and functions of oxidized membrane lipids in plant stress responses

Ruth Welti and Gary L. Gadbury, Kansas State University
Jyoti Shah, University of North Texas
Xuemin (Sam) Wang, University of Missouri, St. Louis and Danforth Plant Science Center

Increasing evidence indicates that environmental stresses, such as freezing, high salinity, and pathogen infection, lead to oxidative modification of plant membrane lipids to produce "ox-lipids". In contrast to oxylipins, such as jasmonic acid and its derivatives, whose significance in plant growth and defense against stress has been well documented, little is known about the functions of ox-lipids in plants. Ox-lipids may function as mediators signaling stress responses, they may represent damage that could serve as a protective buffer against oxidative damage elsewhere in the cell, or they may be long-term modifications that might function as stress "memory". Thus, ox-lipids have the potential to be essential mediators of plant response to the environment. The goals of the research project are to understand the role of ox-lipids in plant responses to biotic and abiotic stresses and to determine the function of members of two enzyme families, lipoxygenases and acyl hydrolases, which are likely to play important roles in the metabolism of oxidized lipids. The project will test the hypotheses that patterns of ox-lipids are fingerprints of individual stresses and that production and/or removal of specific ox-lipids by lipoxygenases and acyl hydrolases contributes to plant adaptation to stress. Under freezing and high salinity stress (abiotic stress) and infection by a fungal pathogen, Botrytis cinerea, and a bacterial pathogen, Pseudomonas syringae (biotic stresses), the stress-response phenotype and production of ox-lipids by wild-type plants and lipoxygenase- and acyl hydrolase-deficient mutant plants will be documented. The data will shed light on the roles of lipoxygenases and acyl hydrolases in stress responses and in production of specific ox-lipid patterns. Analysis of the stress-phenotype and ox-lipid profiles will lead to identification of ox-lipids that are candidates for mediating plant stress responses. The function of candidate lipid mediators will be tested by lipid analysis and phenotypic analysis of plants overexpressing enzymes that produce the candidate lipids and by supplementing mutant and wild-type plants with the putative mediators. The results have the potential to fill critical gaps in understanding of how lipid metabolic enzymes, cellular lipids, and their metabolites interact to influence plant performance.

Broader Impacts: Carrying out the proposed work will provide training for multiple students and postdoctoral trainees at four institutions and bring current knowledge of metabolic profiling, functional genomics, and stress biology to the classroom. It will broaden the participation of underrepresented groups in research through the McNair Program at the University of North Texas, the Summer Undergraduate Research Opportunity Program at Kansas State University, the Des Lee Collaborative Scholarships at the University of Missouri, and the Danforth Plant Science Center NSF REU-Site program, which has achieved over 30% participation by underrepresented minority groups in the past several years. It will involve high school students in the research through the Texas Academy of Mathematics and Science at University of North Texas and through the Students and Teachers as Research Scientists (STARS) program in St. Louis. Organization of mass spectral data on plant lipids, and particularly on stress-induced lipids, into a web-accessible database will provide a foundation for further investigation of the structure and function of lipids, and particularly novel lipids, and will facilitate integration of lipidomics data with other metabolomics and functional genomics data. Analytical capabilities developed in this work will become enabling technologies available to researchers worldwide via the Kansas Lipidomics Research Center. This work also will provide insight into the identity of metabolic steps with potential to enhance stress tolerance in plants and improve agricultural productivity and quality.

Aphids are a large group of phloem-feeding insects that cause extensive damage to plants and vector important viruses. However, our understanding of plant defense against aphids is limited. The interaction between Arabidopsis thaliana and Myzus persicae, commonly known as green peach aphid (GPA), has uncovered a role for the Arabidopsis TPS11 gene, which exhibits homology to trehalose metabolism genes, in plant defense against aphids. The goal of this project is to characterize the involvement of TPS11 in trehalose metabolism, and the role of trehalose metabolism in plant defense against aphids. A combination of biochemical, molecular, genetic and genomic tools combined with insect bioassays and behavioral studies will be utilized to accomplish these goals. Since trehalose and its metabolites are present at low levels in plants, it is anticipated that this study will uncover a regulatory function of trehalose metabolism in plants. The proposed activity will enhance understanding of plant defense against GPA, which is a pest of >50 plant families and vectors several important viruses. In addition, it will fill important gaps in our knowledge of trehalose metabolism and its involvement in plant stress response.

Broader Impacts: This project will benefit a graduate student and a post-doctoral fellow who will receive training in biochemistry, molecular biology and genetics. In addition, through the PI's instructional activities this project will bring current knowledge of plant signaling, carbohydrate metabolism and stress response into the classroom. This project will also provide an opportunity to train undergraduates and high school students and broaden the participation of underrepresented groups in research. Results from this project will also have potential benefits to society at large, because they will identify steps in plant metabolism that mediate defense against insects. In the future, manipulation of these steps may augment aphid resistance and enhance quality and productivity of plants.

This MRI award funds the acquisition of a LSM710 laser scanning confocal microscope to
support research and training in plant signaling at the University of North Texas by providing
imaging capabilities that are required for molecular and biochemical studies at the cellular and
sub-cellular levels. In addition, the confocal microscope will enhance research and training in
developmental physiology, eco-toxicology, neurobiology and microbiology. The LSM710 will
come equipped with a 34-channel spectral detector that will facilitate studying the in situ
expression pattern of genes, intracellular protein localization and protein-protein interactions,
changes in Ca2+, reactive oxygen species and sugar fluxes, and macromolecular dynamics.
Broader Impacts: The LSM710 will benefit undergraduate and graduate students and post-docs
by providing them training in the application of imaging technology for research and
development. The confocal microscope will also provide opportunities to encourage and
broaden the participation and training in the Life Sciences of underrepresented minorities, high
school-aged students and future secondary teachers through the McNair program, the Teach
North Texas program, the Texas Academy of Mathematics and Science, the Honors College, the
NSF S-STEM supported (#0807128) Fostering Outstanding Cohorts in Undergraduate Science
(FOCUS) Scholarships program, and the Howard Hughes Medical Institute Undergraduate
Science Education program at the University of North Texas. The LSM710 will support
research by these students. The instrument will positively impact the environment and society
by supporting research on the discovery, function and application of signaling mechanisms in
plants, which will augment sustainable agriculture, industry and renewable natural resources.

The ability to induce defense systemically after a local infection allows plants to control disease spread to additional organs and ward off new infections. Dehydroabietinal, an abietane diterpenoid that is produced by a variety of plants, is a potent activator of systemic disease resistance, which is an inducible defense mechanism activated in the foliage in response to a local infection. The goal of this project is to characterize the involvement of dehydroabietinal as a long-distance signaling molecule in plant defense. A combination of genetic, biochemical, and molecular approaches will be utilized to accomplish this goal. The proposed activity will enhance our understanding of long-distance communication in plant defense and provide important insights into the function of abietane diterpenoids in plants.

Broader Impacts: This project will support the research and training of three graduate and two undergraduate students, and a postdoctoral fellow. They will receive training in plant biochemistry, molecular biology, genetics, organic chemistry, and mass spectrometry. In addition, through the instructional activities of the PI and Co-PIs, this project will bring current knowledge of plant signaling, secondary metabolism, and stress response, in addition to organic synthesis and mass-spectrometry techniques, into the classroom. The project will also provide opportunities to train high school students and future science teachers, and broaden the participation of underrepresented groups in research through several programs that are in place at the University of North Texas. This project will potentially benefit society at large by identifying steps in plant metabolism/signaling that mediate defense against pathogens. In the future these steps could be targeted for augmenting disease resistance in plants and thus enhance the quality and productivity of plants, and by way of limiting the need for toxic chemicals to protect plants, also benefit the environment and public health.

In the cells of a plant, lipids form membranes that separate the cell from its environment and compartments within the cell from one another. These lipids also have crucial metabolic and signaling functions that are only now being established. As plants develop and are exposed to environmental signals, membrane lipids are extensively chemically modified. Recent advances in lipid analysis have revealed that addition of a fatty acid to a membrane lipid, called polar lipid head-group acylation, is a major modification process, but relatively little is known about this process or its physiological significance. This project will address the hypothesis that head-group acylation of lipids functions to improve plant adaptation to environmental stress. To identify the role of head-group acylation when plants are under stress, genes encoding enzymes responsible for head-group-acylated lipid metabolism will be identified. By examining plants that are missing these genes and enzymes, in comparison to plants that contain them, the functions of membrane lipid head-group acylation in plant stress responses will be determined. These activities will identify metabolic steps with potential to enhance stress tolerance in plants and improve agricultural productivity and quality. Relevant data will be integrated into a plant functional genomic knowledge base. Further, the work will provide interdisciplinary training in biostatistics, chemistry, and biology to postdoctoral trainees, and it will broaden the participation of underrepresented groups in research through collaboration with a faculty mentor and undergraduate student at historically black Langston University.

This project aims to improve current understanding of the role of membrane lipid modification in producing and maintaining complex cell membranes, and influencing organismal performance. In the context of whole glycerolipidomes, new mass spectrometry-based approaches will be used to identify and characterize changes in lipid head-group acylation in response to a biotic stress, Pseudomonas syringae infection, and an abiotic stress, phosphate deficiency. To identify the gene product(s) responsible for head-group acylation, the lipid profiles of knockout mutants of candidate genes will be obtained and compared to the lipid profiles of wild-type plants. Data from functional analysis of the knockout mutants under stress will be correlated with lipid levels, providing additional information about the roles of head-group acylation in plant function. The effects of application of acylated head groups or head-group acylated lipids to plants also will be tested. Lipid profiling and functional data will be integrated into a novel metabolic database to expand knowledge of metabolic networks.