Xuemin Wang - US grants

9511623 Wang Lipid-based signaling via the octadecanoid pathway, which leads to the formation of regulators such as jasmonic acid, plays an important role in plant physiological processes. Jasmonate and related oxygenated lipids have been shown to act as endogenous signals activating defense gene expression and regulating growth and development. Alph-linolenic acid has been shown to be the precursor for the synthesis of these regulators. However, very little is known about lipolytic release of alpha-linolenic acid in the octadecanoid signaling pathway. Which lipid hydrolyzing enzyme is activated in the signaling process? Which glycerolipids are hydrolyzed? How is the hydrolysis regulated? These questions are important because making available the fatty acid precursor is an early step in the sequence of events in signal transduction and may control the signaling pathway. The goal of this research is, therefore, to investigate the involvement, nature, and regulation of deacylation by lipolytic enzymes in the octadecanoid signaling pathway. Stimulus-induced lipid turnover and release of alpha-linolenic acid will be investigated in castor bean leaves in response to wounding. Wounding is chosen because it is a well characterized stimulus that induces the jasmonate signaling system. Completion of the proposed work will fill a critical gap in our knowledge of the cellular roles of membrane lipid-hydrolyzing enzymes in plant growth and environmental responsiveness.

Regulation and Function of Membrane Lipid Hydrolysis in Lipid-Mediated Signaling in Plants
Grant proposal number: IOS-0818740
PI: Xuemin Wang


PROJECT SUMMARY

Cell membranes are rich sources for producing bioactive compounds that affect plant growth, development, and stress responses. This project investigates the molecular mechanism by which one class membrane lipid-hydrolyzing enzyme phospholipase D (PLD) and its product phosphatidic acid (PA) mediate plant survival, growth, and biomass accumulation. The central hypothesis is that specific PLDs and PAs mediate plant growth by interacting with proteins in carbon metabolism and translation regulation. This project will characterize the interaction of PLD/PA with one group of enzymes in carbon metabolism and another in translation regulation. The PA/PLD-effector interactions will be characterized using different approaches, including quantitative biophysical analysis and open-ended profiling of proteins and lipids in the regulatory complexes. The intellectual merits of the research are to discover direct connections of membrane-based signaling with energy metabolism and growth control and to identify novel regulatory processes in plants with potential to improve plant production.

The proposed activity will have broader impacts on science, education, and society. The proposed work has the potential to transform current knowledge on the molecular and biochemical mechanism by which cells integrate stress and growth cues for optimizing plant production. It will provide opportunities to train students and researchers in emerging, under-explored disciplines in plant biology. It will serve as a platform to broaden participation of underrepresented groups in research. The information will be disseminated to enhance science and education through lectures and seminars, classroom teaching, scientific meetings, and publications. The knowledge gained may be applicable to developing crop plants with enhanced stress tolerance and productivity.

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

This award provides support for purchase of a phosphor-imager to be placed in the University's Plant Biotechnology Center. There are already several items of genomics-related equipment in the Center, but there is no shared-use imager, a workhorse instrument in modern molecular biology. Among the uses planned are research on the structure and function of expressed genes, functional and comparative genomics, gene mapping, plant cell-cycle studies, investigation of non-host disease resistance mechanisms, and regulation and function of lipid-based signaling. In addition to advancing research, the instrument will broaden the training of students, post-doctoral fellows, and visiting scientists by providing greater access to the latest and most up-to-date equipment. Imagers of this type replace the use of film in many research procedures that require localization of radioactively-labelled proteins and nucleic acids after polyacrylamide gel electrophoresis. Use of the imager speeds up detection and measurement, as well as avoiding the need for chemicals and equipment used in film processing.

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.

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.

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.

PROJECT SUMMARY Misalignments and disruption of the circadian clock lead to metabolic and physiological dysfunctions. The clock regulates metabolism whereas metabolic activities feedback to influence circadian rhythms, and this interplay between the clock and metabolism coordinates physiology. However, one major knowledge gap is the limited understanding of the mechanism by which metabolism affects clock function. The goal of the proposed research is to elucidate the molecular mechanism by which the circadian clock and lipid metabolism are interconnected through the interaction and reciprocal regulation between lipid mediators and major clock regulators using the model organism Arabidopsis thaliana. The feasibility of the proposed research is supported by recent findings that the central glycerolipid metabolic intermediate, phosphatidic acid (PA), directly binds to the clock transcription factor LHY (LATE ELONGATED HYPOCOTYL), manipulations of PA-metabolizing activities alter clock outputs, and disruptions of the clock perturb lipid accumulation in Arabidopsis. The hypothesis is that the PA-LHY interaction functions as a cellular conduit to integrate the circadian clock with lipid metabolism and mediate lipid production and organismal responses to changing environments. To test the hypothesis, Aim 1 will characterize PA interaction with the clock regulators by determining the lipid binding specificity to LHY, the amino acid residues involved in PA binding, and the intracellular location of the PA-LHY interaction using subcellular-specific PA biosensors and mass spectrometry. Aim 2 will address how altered PA metabolism entrains the circadian clock and mediates stress responses by identifying genes/enzymes responsible for producing PA species that alter clock function. Through quantifying the effect of cellular PA changes on the expression of genes involved in clock regulation, these data will be used to model how cellular PA changes lead to alterations in circadian rhythms and clock outputs. Aim 3 will determine how the circadian clock affects lipid metabolism by using clock mutants to assess how misalignments between internal circadian rhythms and the external environment affect lipid metabolism and accumulation. In addition, clock-targeted genes in lipid metabolism will be identified and tested for roles in the circadian regulation of lipid accumulation. The proposed studies will reveal new regulatory mechanisms for both the circadian clock and lipid metabolism and will advance the current understanding of the interplay between these two pathways. The results are relevant to human health because PA is a lipid mediator involved in mammalian clock regulation and various pathological processes, and the basic molecular mechanism of the clock is conserved between plants and humans. Therefore, the impact of the proposed work is to advance foundational knowledge for the molecular interconnection between lipid metabolism and the clock in eukaryotes, and the information has the potential for future strategies for understanding and mitigating metabolic and physiological dysfunctions associated with clock disruptions.

Sexual reproduction mixes the genetic materials from two parents, which is the driving force for natural selections and a basis for plant breeding improvements. However, the cellular and molecular mechanisms that ensure the transfer of the genetic information from each parent to the next generation are largely unknown. This project addresses how specific enzymes that cleave phospholipids, which are building blocks of cell membranes, such as those of sperm and egg cells, affect the sperm genome transfer in maize seed production. Genetic defects in those enzymes lead to some embryos without the sperm genome, producing maternal haploids with genetic information only from the egg. This study will investigate how those enzymes affect membrane lipid changes and how the lipid changes lead to haploid formation. Thus, findings from this project will advance the fundamental, mechanistic understanding of sperm genome stability and haploid seed production with potential applications to crop improvement, such as haploid induction technology to enhance crop breeding and production for food, feed, fuel, and industrial feedstocks. This project will have additional broader impacts on science, education, and society by providing opportunities for training of students and postdoctoral researchers and serving as a platform to broaden participation of underrepresented groups in research. The research materials, including various maize lines, will be distributed to community and the results and large datasets will be disseminated to enhance science and education through lectures and seminars, in classroom teaching, at national and international meetings, and through timely publications in peer-reviewed journals.<br/><br/>The goal of this project is to elucidate how two pollen-specific phospholipases, pPLAIIφ and PLD3, are involved in sperm genome transmission and haploid seed production in cereal crops. The research tests the hypotheses that loss of the pPLAIIφ and PLD3 activities perturbs lipid homeostasis, membrane structures, and sperm-egg membrane fusion during fertilization, leading to haploid induction and decreased seed production. The supporting objectives are to: 1) determine the role of pPLAIIφ and PLD3 catalytic activities in haploid and seed production, 2) characterize the effect of the phospholipases on pollen and sperm membrane lipid homeostasis, and 3) measure lipid dynamics and effects on sperm-egg fusion. The results will advance the understanding of sperm genome stability and sexual reproduction and have the potential to identify chemical and molecular modifiers applicable to enhance haploid induction for crop improvement.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.