Multidisciplinary research at the organismal, cellular, and molecular levels must continue to improve agronomic, horticultural and forestry plant systems if we are to provide adequate food, feed, and fiber for a growing world population. In this endeavour, the improvement of plants to exploit marginal lands in suboptimal environments, and to utilize inputs more efficiently and sustainably, must remain high research priorities. Suboptimal environments are recognized as the most important factors contributing to the failure to achieve yield potential in crop plants around the globe.

Rapid progress is being made in mapping and sequencing the genomes of several important crop species. These maps and gene banks help researchers identify target genes that can be manipulated to increase natural resistance to pests and pathogens, to enhance their nutritional value, and to confer greater adaptability to environmental extremes. While the cloning of genes that condition plant environmental stress resistance is in its infancy, technical procedures for Agrobacterium-mediated and particle bombardment transformation and regeneration of fertile transgenic plants are becoming routine. We are now at a cross-roads in agricultural history where plant biotechnology provides opportunities to create plants with altered stress resistance. Biotechnology has made it possible to cross the 'species barrier' and offers an additional tool for crop improvement. In fact several commercial products (insect and herbicide resistant crops) are now available to farmers. Recent successes include the development of plants which are genetically engineered to withstand oxidative stresses associated with chilling and high light intensities, and plants engineered for the accumulation of osmoprotectants conferring increased drought and salt tolerance.

As part of this international effort to improve crop productivity by reducing environmentally mediated yield losses, the Center for Plant Environmental Stress Physiology was established at Purdue University in 1987. This interdisciplinary Center is comprised of a core of faculty in the Horticulture Department with common research and teaching interests in plant-environment interactions and biotechnology. Its mission is to increase understanding of plant susceptibility to adverse environmental conditions which limit achievement of crop yield potential. The Center’s primary objectives are to elucidate mechanisms of plant resistance to these environmental factors, and to train future scientists to meet the challenge of feeding a growing world population on a rapidly diminishing acreage of arable land in the face of global climate change.

The Center for Plant Environmental Stress Physiology has developed a curriculum to train doctoral students in the biology of plant response to the environment. We propose a comprehensive course of study for a group of doctoral students who will receive unique training in plant responses to the environment, metabolic and biophysical plant physiology, and modern genetic and molecular biological techniques. This will facilitate the development of new crop plants with altered resistance to adverse environmental conditions, including drought, salinity, low temperatures, heavy metals and nutrient deficiencies. Our multidisciplinary approach will give students an appreciation for the complexity of plant sensing of environmental signals, an understanding of how these signals are transduced to alter plant metabolism and growth, and the knowledge required to manipulate, by biotechnology, plant yield for the benefit of mankind.

Three separate interdisciplinary programs (the Plant Physiology Program, the Genetics Program, and the Biochemistry and Molecular Biology Program) comprising faculty from both the School of Agriculture and School of Science encompass a majority of our current graduate students in plant biology and biotechnology. Representatives from all these programs have recently established a unified "Plant Biology Program" to consolidate and more efficiently handle many of the organizational and recruiting functions of these interdisciplinary efforts. The USDA Fellows would be admitted to the Graduate School through this Plant Biology Program. The graduate students entering the Plant Biology Program are expected to have satisfied a rigorous scientific-based undergraduate curriculum, including advanced calculus, organic chemistry, physics, and cell, developmental and genetic biology, before admission to the Graduate School. Each incoming student is required to complete three rotations through participating laboratories during the first semester in residence. Upon completion of rotations, it is expected that they will select a laboratory and thesis advisor to complete their studies. In consultation with the major advisor, the student selects an advisory committee consisting of three additional members, with two from the Center for Plant Environmental Stress Physiology and one from another department or discipline.

The curriculum is designed to be flexible and allow each student to develop a plan of study consistent with their past training and career goals. The required coursework "core" includes General Biochemistry I and II, and Plant Anatomy, and two additional courses selected from Plant Growth and Development, Plant Molecular Biology (or Eukaryotic Genetics) and Evolution, all taken within the first year of study. Each USDA Fellow will also complete lecture and seminar courses in Plant Responses to the Environment during the first year. In addition to the multidisciplinary core curriculum, students will be required to complete at least 6 credits of courses in their area of specialization selected from the approved list, and 2 credits of Advanced Topics for the given area of specialization (Plant Physiology Option or Plant Biochemistry and Molecular Biology Option). Students may also elect special modular-based laboratory courses to gain special experience in biochemical and molecular biology technologies. A written preliminary exam, covering the first year's coursework, is given at the end of the second semester. Successful completion of this exam is required to maintain USDA fellowship status.

Because the School of Agriculture will provide matching funds, we propose a total of six fellowships (four from the USDA National Needs Fellowship Program, and two from the School of Agriculture) in the following research areas. The suggested research areas are designed to correspond to the strengths and experience of the participating faculty:

1. Isolation of plant salt tolerance determinants by molecular genetic complementation

Paul M. Hasegawa and co-workers have determined that constitutively active yeast calcineurin mediates salt tolerance of transgenic tobacco plants. Calcineurin is a Ca²⁺- and calmodulin-dependent protein phosphatase that is an integral component of a salt stress signal cascade that regulates ion homeostasis in yeast. Further, mRNA of a NaCl responsive Ca²⁺-ATPase gene is differentially regulated in plants expressing calcineurin. These results, and isolation of plant cDNAs that suppress yeast calcineurin null mutations, indicate that calcineurin is involved in a stress signaling pathway required for salt adaptation of plants. A USDA Fellow will utilize salt-sensitive yeast mutants to isolate plant determinants of salt tolerance. These will be identified as plant supressors of salt-sensitive yeast mutants, or homologs of yeast genes that complement these mutants. These genes might mediate ion homeostasis function in the stress signal cascade that coordinately controls processes which facilitate adaptation. This project will combine techniques of molecular genetics and plant physiology to dissect salt adaptation of plants into precise mechanistic entities.

2. Identification of genes associated with winter survival

The goal of Ed Ashworth's laboratory is to identify characteristics and genes that facilitate winter survival of woody plants. Investigations are being conducted in Cornus sericea, as this species is one of the most freeze-tolerant of all plants. Cold acclimated twigs from this species can survive in liquid nitrogen. This level of freeze-tolerance is remarkable, and genes that contribute to winter survival in this species would be good candidates for transfer to other less hardy species. Ed Ashworth’s group has identified a cell wall protein that accumulates in the fall as tissues cold acclimate, is present at maximum levels in winter, decreases in abundance in spring as tissues deacclimate and lose cold hardiness, and is absent in summer. A cDNA encoding this protein has recently been isolated and partially sequenced. The gene apparently codes for a dehydrin-like protein. A USDA Fellow will identify additional genes whose expression is increased in C. sericea during cold acclimation. Techniques for protein and RNA isolation from wood tissue have already been developed in this laboratory. The student will use differential display to identify transcripts that are unique to cold acclimating tissues. These transcripts will be amplified, radiolabeled, and used to probe an existing cDNA library made from cold acclimating wood tissue. It is anticipated that this approach will lead to the isolation of genes that are unique to extremely freeze-tolerant taxa.

3. Molecular genetics of heavy metal tolerance

Peter Goldsbrough and co-workers have shown that phytochelatins (PCs) and metallothioneins (MTs) are two classes of metal ligands produced by plants. Analysis of PC-deficient mutants of Arabidopsis confirm that PCs are required for tolerance to Cd, Hg and Pb. Other experiments suggest that MTs may be involved in tolerance to Cu, Zn and possibly other metals. These observations indicate that PCs and MTs likely play a significant role in the sequestration and distribution of metal ions in plants. Manipulating the expression of these ligands may alter the distribution of metal ions among plant organs. A USDA Fellow will examine the effects of manipulating the expression of PCs and MTs on metal ion distribution in Arabidopsis. The open reading frame of each Arabidopsis MT will be used to construct chimeric genes driven by different promoters so that MT expression will be increased in leaves, stems or roots. Transgenic plants with ectopic expression of MTs will be analyzed for tolerance to metals (Cu, Zn, Cd and others) and accumulation of these metals in different organs. Manipulation of PC synthesis will be accomplished by organ specific expression of gamma-GluCys synthetase in the GSH-deficient cad2 mutant. The USDA Fellow will test the hypothesis that increased expression of plant MTs or PCs in specific tissues will result in a corresponding increase in the accumulation of metals in these tissues. These chimeric MT genes will also be transferred into a PC-deficient mutant to examine any interaction between PCs and MTs in metal accumulation.

4. Characterization of signal transduction during phosphorus stress

The goal of K.G. Raghothama's research program is to understand the molecular mechanisms of plant response to phosphate (Pi) deficiency. Plants sense and respond to Pi deficiency by altering their morphology, physiology and biochemistry. The signal transduction pathway involved in the Pi starvation response mechanism is not clear. Recently K.G. Raghothama has cloned and characterized the Pi transporter genes from plants. Expression of these genes is enhanced during Pi starvation. The 5’ regions of the Pi transporter genes are being analyzed for the presence of conserved elements found in Pi starvation-induced genes of yeast and other microorganisms. A USDA Fellow will dissect the signal transduction pathway in plants by generating transgenic Arabidopsis thaliana plants expressing the reporter gene luciferase under the control of a Pi transporter gene promoter. Seeds of the transgenic plants will be chemically mutagenized, and seedlings will be screened for a lack of the reporter gene activity. Further characterization of the seedlings will lead to identification of mutations in genes that are required for activation of the Pi starvation response. The mutants obtained in this study will serve as valuable genetic tools to clone and characterize the genes involved in the Pi starvation signal transduction pathway in plants.

5. Oxidative stress resistance

The focus of Randy Woodson's laboratory is on oxidative stress in plants as related to the development of chilling tolerance and injury. Tomato is being used as a model system to study the role of oxidative stress defense enzymes such as catalase and superoxide dismutase in chilling injury. Tomato exhibits a diurnal variation in chilling tolerance that correlates with changes in the activity of catalase. Randy Woodson and co-workers have shown that treatment of tomato seedlings with hydrogen peroxide elicits resistance to chilling injury and this is associated with the induction of catalase gene expression and increased enzyme activity. More recently, expression of antisense catalase RNA in transgenic plants has resulted in significant reduction in catalase activity. These plants exhibit increased sensitivity to chilling, high light conditions, and other forms of oxidative stress. A USDA Fellow will study the molecular mechanisms involved in the regulation of oxidative defense genes in tomato, including a complete characterization of the catalase gene family and the differential regulation of these genes.

6. Metabolic engineering of osmoprotectant overproduction

The research programs of Ray Bressan, David Rhodes and Bob Joly are primarily focused on the isolation, characterization and manipulation of plant genes that enhance tolerance to osmotic stresses (drought and salinity), including genes that encode enzymes of the biosynthesis of osmoprotectants such as proline, glycinebetaine, 3-dimethylsulfoniopropionate and related onium compounds. In collaboration with Laszlo Csonka (Biological Sciences, Purdue University), Ray Bressan and co-workers have cloned the tomato PRO2 gene encoding the first two enzymes of proline biosynthesis. The gamma-glutamyl kinase encoded by this gene is sensitive to allosteric feedback inhibition by proline. This limits the potential of this gene in conferring proline over-production in transgenic plants. A USDA Fellow will select for mutations of the PRO2 gene (expressed in Escherichia coli) which confer proline over-production and resistance to the toxic proline analog L-azetidine-2-carboxylate as a result of altered sensitivity to feedback inhibition. The USDA Fellow will introduce and express the mutated (feedback insensitive) genes in transgenic plants to determine how they influence flux through the proline biosynthetic pathway as a function of osmotic stress, and whether they confer increased osmotic adjustment and drought and salt tolerance. Bressan, Rhodes and Joly will provide guidance in molecular biology, enzymology and whole plant water relations components of this project, respectively.

1. Grade expectations for course requirements

All graduate students are required to take the core requirements and electives to total 30 credit hours in the first two years of their program of study. All students must maintain a B average, attaining a cumulative index of 3.0/4.0 during all semesters, consistent with University-wide requirements. If their cumulative index falls below 3.0 for two consecutive semesters, students will be removed from the graduate program.

2. Qualifying and preliminary examinations

The written qualifying examination comprises review questions from courses taken in the first two semesters of the program. Upon successful completion of this qualifying examination, a written research proposal, on a subject outside the student's specific area of research but within the scope of plant responses to the environment, and an oral defense of the proposal must be prepared and given within the second year. A two-page preproposal will be submitted for approval by the student's advisory committee selected by the end of the first year of study. The committee comprises a major professor and at least three additional faculty, with two from the Center for Plant Environmental Stress Physiology cooperators and one from another department or discipline. Once the preproposal has been approved, a full proposal is written according to the guidelines of USDA-NRICGP for competitive research proposals. The written proposal is presented and defended orally before the student's advisory committee, and the student is examined orally on the content of the proposal as well as topics related to plant physiology, biochemistry, genetics and molecular biology. The latter examination constitutes the preliminary examination.

3. Teaching requirement

All doctoral students will be required to serve as a teaching assistant for a minimum of one semester in an undergraduate plant biology laboratory.

4. Dissertation defense

A final examination and oral defense of the dissertation will be made before the advisory committee. At least two semesters must elapse between completion of the preliminary oral examination and the scheduling of the final defense. The student will present a special departmental seminar on the dissertation research. The seminar is open to all faculty and students and is followed immediately by the oral defense before the advisory committee.

It is anticipated that the USDA Fellows will require a minimum of 3 years to complete the doctoral program. For students making satisfactory progress, additional time needed will be funded by the department, through departmental research and teaching assistant funds, or by funds generated by the individual investigators.

Students can bypass the Master's program and work directly on the doctoral degree. The Master's bypass requires successful completion of the first two years of coursework, approval of the advisory committee, and submission of a paper to a peer-reviewed journal.