Dean DellaPenna - US grants

The activity of the tomato the tomato fruit pectin-hydrolyzing enzyme polygalacturonase (PG) increases dramatically during ripening due to the presence of three closely related isoforms, PG1, PG2A and PG2B. The Pg2 isoforms are composed of single catalytic PG polypeptides while PG1 is a heterodimer consisting of one PG2 polypeptide in tight association with an ancillary glycoprotein, the PG converter. The involvement of each isoform in pectin degradation and other aspects of fruit ripening remain unclear. The PG isoform levels in planta appear to be determined by the level of converter protein. In order to increase our understanding of the biosynthesis, assembly and biological activity of cell wall enzymes such as PG, the levels of individual PG isoforms will be manipulated in vivo by altering the levels of converter protein in transgenic wild-type plants. To accomplish this the converter protein will be purified, cloned and its structure and expression during wild-type fruit ripening studied. Converter protein levels will be manipulated by expressing a converter cDNA in sense and antisense orientations in transgenic wild-type tomato plants presumably resulting in accumulation of only PG1 or PG2. These studies will increase our knowledge of the effect of individual PG isoforms on polyuronide degradation and other physiological parameters associated with fruit ripening. Several enzymes and structural proteins are secreted from plant cells into the cell wall. The activities of these proteins are essential for normal growth and development as well as specialized functions such as fruit ripening and plant defense. An important question is how the activities of such important proteins is controlled when they are secreted from the cell and not subject to the normal mechanisms associated with the regulation of cytoplasmic enzymes. This proposal describes an interesting approach to studying the processing and function of an extracellular enzyme, polygalacturonase, which degrades polyuronides in the cell wall during fruit ripening. //

This award will support the purchase of necessary pieces of equipment for a multi-user plant growth and tissue culture facility at University of Arizona. NSF and the University will share the cost on a 1:1 basis. Primary users of the facility consist of 17 mostly young faculty members in the Departments of Plant Sciences and Plant Pathology. The facility will be used for research and teaching in the areas of plant growth and development, plant physiology/biochemistry, plant genetics/molecular biology, transformation technology, and host- pathogen interactions. Specific pieces of equipment include growth chambers, light banks, Laminar flow hoods, and shakers, and they will be installed in the new Agricultural Research Laboratory.

9408826 DellaPenna A long-term goal of this research is understanding the functional significance of developmentally regulated, polyuronide modifications in plants. As part of prior NSF funding, the Beta- subunit of polygalacturonase (PG1) was purified; cDNAs encoding the protein were isolated and characterized. Beta-subunit mRNA accumulates to high levels in developing fruit and is to a large degree temporally separated from catalytic PG2 expression. Homologous sequences have been identified in a wide variety of plant species suggesting a broad functional role for the protein in plants. The current working theory is that the Beta-subunit defines a new class of bifunctional plant cell wall proteins that interact both with structural components of the wall and catalytic proteins, to localize and/or regulate metabolic activities within the cell wall. This hypothesis can now be tested in transgenic tomoto fruit in which Beta-subunit expression is inhibited by antisense RNA. The proposed research will further address long- standing questions of how pectin-degrading enzymes are targeted and regulated in plants and what the functional significance of pectin degradation is in various tissues and cell types. ***

A grant has been awarded to Dr. Wayne Loescher and colleagues at Michigan State University to acquire controlled environment chambers for plant biological research. Such chambers are necessary for three major reasons: (1) the potential to achieve consistent, standard, or optimum environmental conditions not found in greenhouses or fields; (2) containment and sanitation to avoid contamination; and (3) the opportunities to improve experimental designs and thus the efficiency of the research.

The work to be done and the methods to be used are the result of recent advances in plant genomics and proteomics, and also in plant development, biochemistry, breeding, genetics, molecular biology, and plant-environment and plant-pathogen/pest interactions. Contemporary work in plant biology and particularly that in genomics often utilizes Arabidopsis thaliana as a model. Although this plant's size and short reproductive cycle make it well suited for growth in controlled-environments, it is nonetheless quite sensitive to environmental cues. Moreover, successfully using large populations for mutant selection and evaluation often depends on abundant, highly-consistent and/or well-regulated environmental conditions. Beyond Arabidopsis, many Michigan State University plant researchers also utilize crop species, and attempting to grow these under defined environmental conditions can present significant challenges, e.g., high light requirements for most crops cannot be met in older growth chambers or under conventional glasshouse conditions in the winter. For molecular genetic work, and especially that with transgenic plants, containment is important, and is often required in working with such systems. Pollen and other propagules must be contained, and, conversely, unique genetic materials must be protected from foreign pollen. Avoiding contamination by pests and diseases is also essential for all plants, and this is more readily achieved in growth chambers than in glasshouses.

The broader significance and scientific importance of this equipment is that modern controlled environments offer much more flexibility in manipulating temperature, light, humidity, and atmospheric composition. Computer controls and monitoring systems now allow for manipulating temperatures, light intensity and duration so as to replicate specific seasonal and daily environmental variations and combinations and monitor experimental conditions for long periods. New generation growth chamber designs minimize vertical temperature gradients, horizontal light gradients, humidity and other atmospheric differences along the borders and walls, thus maximizing the efficiency of chamber space. These factors, along with increased equipment reliability, also reduce the numbers of replicates required for experimentation. Most importantly, new generation equipment allows researchers who want specific conditions (e.g., temperature, light quality, intensity, and duration, or atmospheric manipulations such as CO2 enrichment) much improved capabilities. The new facilities will provide a multi-investigator, interdepartmental resource to support the broad spectrum of plant biology research at MSU. Progress is expected in developing new crops and new and better varieties of traditional crops, in understanding how plants function, how they respond to environmental conditions and resist environmental stresses such as temperature, drought, and salt, and how they might better resist pests and diseases.

Plants synthesize two major groups of carotenoids: carotenes, which are
cyclized or uncyclized hydrocarbons (e.g. lycopene and beta carotene), and
xanthophylls, which are variously oxygenated derivatives of carotenes (e.g.
lutein and zeaxanthin). Carotene hydroxylases (OHases) add hydroxyl groups
to the rings of cyclic carotenes and are essential for the synthesis of all
xanthophylls in plants. This proposal performs a detailed analysis of
carotene hydroxylases in plant tissues by isolating genes defining specific
carotene hydroxylases and modifying the expression of one or more activity
by mutant analysis. The consequences of such modifications on carotenoid
and xanthophyll synthesis and composition in photosynthetic tissues will
provide important insights into the role of each enzyme in the pathway and
the consequences of pathway engineering in food crops. Given the importance
of carotenes as antioxidants in the human diet and the role(s) of
carotenoids in decreasing the incidence and progression of macular
degeneration, various cancers and vitamin A deficiency in certain subgroups
of the global population, a further understanding of the enzymes involved
in carotenoid and xanthophyll synthesis may have beneficial impacts on the
overall health of the population.

During its lifecycle, a plant is exposed to a wide range of abiotic and biotic stresses that can cause production of reactive oxygen species (ROS) in excess of that normally generated by metabolism and for signaling in the absence of stress. Although all subcellular compartments produce ROS, chloroplasts are the major ROS source in photosynthetic tissues. In order to avoid oxidative damage and death, plants have evolved an interconnected, complementary network of compounds (e.g., ascorbate, tocopherols, carotenoids, glutathione), enzymes (e.g., superoxide dismutases, peroxidases, catalases) and biochemical responses (e.g., non-photochemical quenching) that serve to minimize plastid ROS production under stressful conditions or rapidly detoxify excess ROS species or byproducts that are produced.
Tocopherols, more commonly known as Vitamin E, are the major lipid soluble antioxidant in plastids and are proposed to be an essential component of the plastid antioxidant network. Studies in mammals and artificial membranes indicate tocopherols are important for membrane structure, quenching of singlet oxygen and other ROS, scavenging of chain-propagating lipid peroxy radicals and other "non-antioxidant" functions related to signaling and transcriptional regulation. In plants, tocopherols are assumed to function similarly though experimental evidence for this is lacking. During the past five years, the DellaPenna laboratory has cloned all enzymes of the tocopherol biosynthetic pathway in Arabidopsis and developed mutant/transgenic lines whose leaves are completely deficient in tocopherols or accumulate tocopherols at 4-5 times wild type levels. These tocopherol-modified lines will be used:

1. To define the role(s) of tocopherols in scavenging specific ROS species and control of lipid peroxidation.
2. In genetic combinations with mutations that affect other components of the antioxidant network to assess functional compensation and redundancy of antioxidant network components.
3. To study a novel germination phenotype in tocopherol deficient lines.
4. In whole genome expression analysis to assess the response of individual antioxidant network genes and other cellular components to tocopherol modifications.

Tocopherols, more commonly known as Vitamin E, are the major lipid soluble antioxidant in chloroplasts of all plants and are proposed to be an essential component of the plastid antioxidant network. The research being undertaken will provide a comprehensive understanding of tocopherol function(s) in photosynthetic tissues of Arabidopsis. This work will also define and characterize the integration and complementation of tocopherols with and by other components of the plastid antioxidant network (Vitamim C, carotenoids and enzymes that scavenge reactive oxygen). Because of the evolutionary conservation of the antioxidant network in chloroplasts, the data obtained will be of direct relevance to a wide range of plant species and crops.

As the site of photosynthesis, the chloroplast is the defining organelle of green plants and may be thought of as the world's life-support system. It is an attractive target for functional genomics because it participates in a wide array of biosynthetic processes yet has a similar number of genes and metabolic processes as a bacterial cell. The aim of this project is to determine functions for the ~4,400 nuclear genes predicted to encode plastid-targeted proteins in Arabidopsis. Computer methods are being used to create structural models for many plastid proteins and sequenced bacterial genomes are being analyzed to link genes of unknown function with those of known function. Together, these informatics approaches will generate hypotheses for the roles of genes of unknown function. Knockout lines will be analyzed for chloroplast, plant, and seed morphology, as well as photosynthetic parameters and selected chloroplast-synthesized metabolites. In-depth characterization of mutants with morphological or metabolic phenotypes will be performed, including analyzing flux through the central metabolic network in seeds. The informatic and phenotypic data will be made available to the research community through a project website (http://plastid.msu.edu ).

Broader Impacts: By enriching the annotation of nuclear genes encoding plastid-targeted proteins, the research will lay the foundation for a comprehensive understanding of plastid function throughout the life cycle of plants. The project will contribute to the objectives of the 2010 Project by attempting to improve the annotation of approximately 15% of the protein-coding genes of Arabidopsis. Because a number of the traits that will be studied are important targets in crops, these results should inform approaches to transgenic crop improvement and molecular breeding of economically important plants.

The project will involve interdisciplinary training of students and postdoctoral researchers. Each trainee will be exposed to multiple areas of science, ranging from informatic approaches, through high-throughput phenotypic analysis and flux measurements, to data analysis methods, preparing them for 21st century biology careers. In addition to cross-training of students, this project will integrate research and education at three levels: 1.Summer research experiences for undergraduate students and secondary school teachers; 2.Summer research opportunities for faculty from primarily undergraduate schools; 3.Training of participants in cutting edge computer-based curriculum development tools using the Lon-CAPA web-based course management system (www.lon-capa.org).

DESCRIPTION (provided by applicant): Medicinal plants produce a wealth of pharmaceutical compounds such as taxol, vincristine, and morphine. Unfortunately, the specialized secondary metabolic pathways leading to such compounds remain poorly understood and progress in elucidating and manipulating these taxonomically restricted metabolic pathways has been correspondingly slow. This has been exacerbated by the limited development of "omics"-level resources for medicinal plants, which has meant that as a group, research in medicinal species have not benefited to the same extent from the genomics revolution, as have research in model plants and agronomic crop species. This proposal describes the combined use of state-of-the-art sequencing technologies, metabolomics capabilities, and bioinformatics to develop an unrestricted, public resource to address this growing gap in our knowledge base of species-specific plant metabolism and accelerate the identification and functional analysis of genes involved in natural product biosynthesis in 20 widely used medicinal plant species. This resource will provide the research community with user-friendly access to the DNA sequences and expression profiles of each plant's transcriptome and associated metabolome, which we anticipate will have a translational effect on drug development. To achieve this goal, we will utilize next generation sequencing approaches to determine the near-complete set of mRNAs encoded by each medicinal plant species. Transcriptome profiling of up to 20 chemically diverse tissues/treatments per species using the RNA-Seq method from Illumina will be performed and correlated with metabolite profiles generated through LC-TOF and GC-MS for these same samples. All sequence and gene expression data will be deposited into NCBI and made available, along with metabolite profiling data at medicinalplantgenomics.msu.edu, a custom website developed by the research consortium. Thus, this NIH Grand Opportunities Grant will provide searchable and downloadable databases for medicinal plant gene sequences, expression profiles and metabolites that can be accessed and utilized by the research community to facilitate discovery of the pathways and genes responsible for biosynthesis of key pharmaceuticals. High throughput sequencing of genomes and transcriptomes has revolutionized and accelerated the pace and progress of research across the life sciences and this proposal will for the first time extend these advances into the medicinal plant arena on a broad scale. PUBLIC HEALTH RELEVANCE: This proposal describes the combined use of state-of-the-art DNA sequencing technologies, metabolomics capabilities, and bioinformatics to develop an unrestricted, public resource to advance our knowledge base of species-specific plant metabolism and accelerate the identification and functional analysis of genes involved in natural product biosynthesis in 20 widely used medicinal plant species. This resource will provide the research community with user-friendly access to the DNA sequences and expression profiles of each plant's transcriptome and associated metabolome, which we anticipate will have a translational effect on drug development.

PI: Dean DellaPenna (Michigan State University)

CoPIs: C. Robin Buell (Michigan State University), Edward S. Buckler (Cornell University/USDA-ARS), and Torbert R. Rocheford (Purdue University)

Key Collaborators: Michael A. Gore (University of Arizona/USDA-ARS) and Jianbing Yan (CIMMYT, Mexico)

Senior Personnel: Dick Johnson (University of Illinois at Urbana-Champaign) and Theresa M. Fulton (Cornell University)

Carotenoids are a group of several hundred distinct chemical compounds that are essential for vision and immune system function and as natural colorants in fresh and processed foods. Some carotenoids (betacarotene, alpha-carotene and beta-cryptoxanthin) are the major provitamin A compounds in the human diet while others such as lutein and lycopene play a role in decreasing the incidence and severity of macular degeneration or prostate cancer, respectively. The goal of this project is to identify the genes and their most useful variants (alleles) in maize that determine the levels and compositions of carotenoids and vitamin E in maize seed. Using information obtained using the model plant Arabidopsis, maize genes corresponding to those responsible for the biosynthesis of carotenoids and vitamin E will be identified. Knowledge of the natural variation in the content and composition of these compounds in maize seed will be combined with the recently published genome sequence of maize and detailed analysis of gene expression during maize seed development to determine which specific alleles of these genes contribute the most to beneficial levels of these compounds in maize seed. The alleles identified can then be used in maize breeding programs to increase the levels of these compounds to enhance the quality of food and feed derived from maize. This same approach will also enable the identification of new and novel genes that play significant roles in determining the levels of carotenoids and vitamin E in maize. The combined information obtained will provide a road map for generating similar changes for these and other vitamins in other agricultural crops that serve as major food and feed sources for humanity.

Understanding the molecular genetic basis of biochemical traits in agricultural seed crops that are essential for nutrition in humans and animals is a key component of meeting future global food and feed needs. The research will provide a comprehensive genetic assessment of natural variation in two such biochemical pathways and will serve as a model for genome scale integrative analysis of other areas of plant metabolism in maize and other agricultural plant species. The large body of publically available gene expression data generated in this study will provide an unparalleled resource that will greatly impact the maize research community. The research will provide a unique environment for educating the next generation of scientists through engagement of high school, undergraduate, and graduate students as well as postdoctoral associates in research at interfaces of plant genetics, genomics, quantitative genetics and plant biochemistry. The researchers will engage under-represented groups in plant scientific research through targeted recruitment of Hispanic undergraduates to the research programs at all three universities. This outreach will include educating preschool children, K-12 students, and undergraduates in the concepts of genetics, plant breeding, biochemistry, nutrition, food sources and their relevance to diet and health. As vitamin deficiencies are a pervasive global health issue, a practical outcome of this research will be to provide the basis for more expedient and cost effective marker-assisted selection programs in maize for enhanced dietary levels of carotenoids and vitamin E in the US and developing countries. Toward this end, the project have established a network with researchers at CIMMYT (Mexico) and IITA (Nigeria) to facilitate seamless transfer of relevant results into active international breeding programs targeting developing countries. All data generated through this project will be available at the project website (http://www.maizegenomics.plantbiology.msu.edu) and through long-term data repositories that include the NCBI GEO (http://www.ncbi.nlm.nih.gov/geo/) and Short Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi), MaizeGDB (http://www.maizegdb.org/) and TAIR (http://www.arabidopsis.org/).

Chloroplasts are green organelles of plant cells and are the metabolic factories of the cell. Chloroplasts convert light into chemical energy, which is then used by the cell to synthesize hundreds of thousands of compounds upon which life on the planet depends. Like any factory, chloroplasts do not operate in isolation but rather collaborate with other organelles to synthesize most plant compounds. Collaborative synthesis is most common for non-polar (fat-soluble) compounds such as plant oils, pigments and volatile flavor compounds, but how non-polar biosynthetic intermediates move between plant organelles has remained unknown. The project builds on the discovery of a novel mechanism for this movement of compounds between organelles; to extend the factory analogy, we have found that rather than using carts to transport intermediates between organelles, as has long been assumed, the chloroplast and other organelles build a shared wall (membrane) which creates a common pool of non-polar intermediates to be accessed by enzymes in either organelle. The investigators will take biochemical, genetic and molecular approaches to test the range of compounds accessible at this interface and define its limits and operating principles. The resulting data will have broad implications and impacts in the engineering of plant metabolism to produce non-polar metabolites for food, feed, fuel and industrial applications. Training of undergraduates, especially for student's who are the first to attend college in their family, will be an integral component of the research. Data from these studies will be distributed to the public through peer-reviewed manuscripts or through seminars or talks and poster presentations at meetings. Once published, all transgenic or mutant lines, constructs, and other molecular tools will be either donated to the Arabidopsis stock center or will be provided upon request to interested parties in keeping with standard research practices.

This collaborative research project is directed at identifying a subset of the ~40,000 genes in the corn genome that work together to determine the levels of five essential and limiting dietary vitamins in kernels: vitamin E and the four B vitamins, B1 (thiamin), B2 (riboflavin), B3 (niacin) and B6 (pyridoxine). By combining approaches similar to those used in the Human Genome project, the researchers will identify alleles, special variations in these "vitamin" genes, and learn how to put them together to generate high amounts of vitamins in corn kernels. An important outcome of this research will be the knowledge by which to enhance these micronutrient levels in corn kernels such that diets in which maize is a major component provide a balanced nutritional content. Such direct translation of these findings will be the eventual incorporation and fixation of identified alleles in maize breeding programs that are favorable for the increased levels of vitamins E and B to enhance the food and feed supply chain. In addition, this research will provide guiding principles for parallel efforts in other agricultural crops and thus enable predictive breeding and metabolic engineering of more nutritious crops worldwide. Finally, integration of research with education within the project will permit training of the next generation of plant scientists with knowledge of plant genetics, breeding, genomics, biochemistry, and bioinformatics.

This project seeks to leverage the tremendous genetic and genomic tool sets developed in maize the past decade to advance and accelerate our fundamental understanding of the genes, alleles and genetic mechanisms controlling synthesis and accumulation of vitamins that are limiting in maize grain and hence result in vitamin deficiencies in maize-based diets: four B vitamins (B1, thiamine; B2, riboflavin; B3, niacin; B6, pyridoxine) and vitamin E. This project brings together a team of scientists with divergent but complementary knowledge and skills that together will allow the genes, alleles and underlying mechanisms controlling these nutritional traits to be elucidated and the knowledge deployed on a global scale. Specific objectives are to (i) perform genome-wide association studies with the maize Ames inbred line panel (n~2,000) to identify and resolve quantitative trait loci (QTL) controlling accumulation of these micronutrients; (ii) assess the role of rare alleles by constructing and analyzing segregating F2 populations derived from Ames lines that are extreme outliers for traits; (iii) determine the contribution of expression QTL and presence-absence variants (PAVs) to vitamin composition using whole transcriptome sequencing data obtained from grain 24 days after pollination in 500 inbred lines that represents the phenotypic variation of the Ames panel; and, (iv) perform genomic prediction with the Ames panel to accelerate the efficiency of breeding improved grain micronutrient composition in developing countries. The broader impacts of this project to the broader scientific community and public will be ensured through a set of coordinated activities that engage students, postdoctoral associates, scientists and the public. Data and biological resources generated in this project will be made accessible to the community. Data will be disseminated through publications, project websites and long-term repositories such as the NCBI's SRA and MaizeGDB.