Martin H. Spalding - US grants

By comparison with C3 plants, Chlamydomonas reinhardtii and other unicellular algae exhibit unusual photosynthetic characteristics. It has been proposed that these characteristics result from the operation of a CO2- concentrating system, which increases the intracellular CO2 concentration to a level which suppresses photorespiration by competitive inhibition of RuBP oxygenase. One of the principal components of the proposed system is a protein which catalyzes the active transport of inorganic carbon across either the plasmalemma or the chloroplast envelope. However, direct, physical evidence for the existence of this transporter, or any other component of the CO2-concentrating system other than carbonic anhydrase, is lacking. Activity of the CO2-concentrating system is under environmental control. Cells grown at limiting CO2 concentrations (air- adapted) show activity, but cells grown at elevated CO2 concentrations (CO2-enriched) show no activity of the CO2- concentrating system. We have identified 7 polypeptides which increase in abundance during adaptation to limiting CO2 concentrations or are specific for cells grown under such conditions. We will continue to extend these observations to comparisons between wild-type and mutants of Chlamydomonas deficient in the CO2-concentrating system to help identify which polypeptides might be involved directly in inorganic carbon accumulation. Using antibodies generated against one or more of these "air-specific" polypeptides, we expect to: (1) determine whether appearance of the "air-specific" polypeptides during "induction" of the CO2-concentrating system correlates with the appearance of the capacity for inorganic carbon accumulation, (2) determine the intracellular location of the corresponding antigens, (3) determine whether Chlamydomonas mutants deficient in the capacity for inorganic carbon accumulation contain polypeptides immunologically cross-reactive with these antibodies, (4) investigate the appearance of translatable mRNA for "air-specific" polypeptides during the "induction" of the CO2-concentrating system. Genes differentially expressed during adaptation of CO2-enriched cells to limiting CO2 will be identified by differential screening of a cDNA library and, if time permits, by molecular cloning of the genes corresponding to the "air-induced" polypeptides. Attainment of all objectives will allow us to better assess the potential for transferring the CO2-concentrating system into higher plants. Some algae can take up carbon dioxide dissolved in water and photosynthesize sugars. How they concentrate the dissolved gas is unknown but could be an important trait to introduce into higher plants. This work investigates the mechanism of uptake and its subsequent enhancement of photosynthesis.***//

This award will support a two-year collaborative project between Professor Martin H. Spalding, Department of Botany, Iowa State University, and Dr. Murray R. Badger, Australian National University, Canberra, under the U.S.-Australia Cooperative Science Program. The scientists will study aspects of the photosynthetic process in the unicellular alga Chlamydomonas reinhardtii. This and other unicellular algae are very efficient in their use of external inorganic carbon for photosynthesis, and exhibit additional interesting photosynthetic characteristics as well. The cooperating researchers believe these effects are the result of intracellular carbon dioxide concentration levels in the algae which are significantly greater than in most higher plants. A "carbon dioxide-concentrating mechanism" has been proposed by the investigators which involves energy-requiring transport of inorganic carbon (as carbon dioxide or bicarbonate ion) into and within the algal cells, followed by enzyme-catalyzed conversion of accumulated bicarbonate ion to yield enhanced levels of carbon dioxide. It is the aim of this cooperative project to understand better the transport mechanism and to determine the location of the enzyme (carbonic anhydrase) within the cell. Both the U.S. and Australian groups have been active in this area of research, using complementary approaches and techniques. Professor Spalding and his colleagues have emphasized molecular genetic studies of the CO2-concentrating mechanism in microalgae, while Dr. Badger and his group in Australia have focused their related efforts on cyanobacteria, and have developed special mass spectrometric and centrifugation techniques to aid in the analyses. The cooperative project will build upon the strengths of both groups, and will involve exchanges by the principal investigators as well as by their postdoctoral associates.

Abstract

This research will determine the function of subtilase genes in Arabidopsis. Subtilase genes encode subtilisin-like serine proteases, proteins that play important roles in plant growth and development. In mammalian systems, some subtilases are called "hormone convertases" because they cut other proteins releasing bioactive peptides that serve as hormones and/or function in cell-to-cell signaling. The properties of some plant subtilases suggest they may play similar roles in plant development and physiology.

The function of nearly 60 subtilase genes in Arabidopsis will be investigated through collaboration with European partners in The Arabidopsis Subtilase Consortium (TASC). A listing of all the genes in the subtilase gene family (and their subfamily identification) is found at http://csbdb.mpimp-golm.mpg.de/csbdb/dbcawp/psdb/main/mgenes.html. This research will focus on the role of ten subtilase genes that are highly regulated during shoot regeneration in tissue culture. Three of these subtilase genes appear to influence shoot development in that the expression of these genes is controlled by a major quantitative trait locus (QTL) that determines shoot regeneration efficiency. Shoot regeneration efficiency varies in Arabidopsis and in some important economic crops where it can impede the successful application of genetic engineering technologies. Another seven subtilase genes are differentially regulated during shoot or root regeneration.

A proteomics approach (the study of a large number of proteins using protein expression profiling) will be used to understand the function of the subtilases. The function of these genes in vivo will be examined by quantitatively analyzing 2D difference gel electrophoresis (DIGE) profiles of T-DNA insertion mutants and transgenics with induced expression of subtilase genes. Subtilase target sites will be determined by analyzing the products from the limited digestion of common substrate proteins by the recombinant enzyme. Optimal subtilase target sites will be determined by designing and testing the hydrolysis of synthetic peptides related to the cleavage sites for common substrates.

Research results will shared with the public and other members of the scientific community through http://csbdb.mpimp-golm.mpg.de/csbdb/dbcawp/psdb.html. Information at this site is edited by the Altmann lab and will be updated on a monthly basis. This research is significant to the goals of the 2010 project in that it contributes to our understanding of a group of important genes that influence plant growth and development. This research makes a unique contribution in that it uses new tools in proteomics to address gene function.

Broader impact

This project has broader impact because it contributes knowledge to the role of proteolysis and peptide signaling in plant development. Little is known about bioactive plant peptides from genomics because peptides are often embedded in other proteins and are not released until cleaved by proteases, such as subtilases.

The project also has broader impact because it provides lab research training for Iowa State University and Des Moines Area Community College undergraduates. Some of the undergraduate trainees will also have the opportunity to conduct research during the summer in international labs of partners associated with TASC. To encourage ISU minority undergrads to participate in this project, we will host a meeting each fall in the Roy J. Carver Co-Laboratory for the local chapter of Minorities in Agriculture, Natural Resources and Related Sciences (MANRRS).

PI: Carolyn Lawrence (Iowa State University; awardee)
coPI: Taner Z. Sen (Iowa State University)


Maize researchers cannot easily leverage all available genetic and genomic data because the online locations of all resources are not easy to find and the sequence-indexed resources generated by individual projects must be searched independently. In addition, it is often the case that when a project?s funding period ends, the generated data are lost because they are not moved to long-term repositories: these once-funded project sites degrade over time and sometimes disappear entirely. This project will overcome these challenges in collaboration with the community of maize researchers by launching POPcorn (PrOject Portal for corn), a needs-driven resource and data pipeline. POPcorn will make available (1) a centralized Web-accessible resource to search and browse ongoing maize genomics projects, (2) a single, stand-alone tool that makes use of Web services and minimal data warehousing to enable researchers to carry out sequence searches at one location that return matches for all participating projects? related resources, (3) a set of tools that enable collaborators to migrate their data to MaizeGDB, the long-term model organism database for maize genetic and genomic information, at their projects? conclusion, and (4) generalized, freely available code that other research communities could use to meet similar needs. The development of the project portal, upload tools for maize data, and tutorials will serve as an outreach activity directly, support basic research, and accelerate the acquisition and utilization of new knowledge that translates directly into this important production crop?s agronomic improvement. Thus, POPcorn will aid in the identification of the molecular-level phenotypes manifesting as traits that plant breeders select for and will lead to improvements in food, fuel, and nutrition. In addition, project personnel will actively participating in the NSF?s Plant Genome Research Outreach to American Indian Undergraduates program at Iowa State University. Thus, these activities will promote research, education, and dissemination of maize data to a broad audience, while developing a new generation of scientists.

Stephen H. Howell
Proposal number IOS-0919707
ER stress transducing transcription factors in plants

Plant stress tolerance is a highly valued trait given the demands for producing crops for food and fuel under all kinds of environmental conditions, particularly those exacerbated by climate change. The goal of this project is to better understand the mechanism by which plants perceive adverse environmental conditions and how they protect themselves from the stress caused by these conditions. This project will focus on two factors called transcription factors that "turn on" the expression of stress response genes. These transcription factors are unusual in that they are "dormant" and attached to membranes in the cytoplasm of plant cells under normal conditions, but are activated and released from the membranes and enter the nucleus under stress conditions. The two transcription factors are activated by different stresses -- one by heat and the other by salt stress -- and each turns on a different set of genes. The investigators will determine how the factors are activated, how they distinguish different stresses and how they are relocated from one compartment of the cell to another. The factors turn on different sets of genes, and so the investigators will also examine how the factors selectively activate different target genes. This system represents a newly discovered stress response pathway in plants and has prospects for manipulating plants to achieve greater stress tolerance. The project will also provide the opportunity to train George Washington Carver interns in the area of plant stress research.

Intellectual Merit:

Algae play a major role in two areas of global concern, climate change and renewable biofuels, are emerging as prime topics on the world stage. Algae of all types account for approximately one-half of carbon dioxide (CO2) recycled from the atmosphere and 'fixed' via photosynthesis into sugars, proteins, and organic substances needed by all living organisms on earth, including humans. Thus, algae are critical to maintaining low levels of atmospheric CO2, a potent greenhouse gas. The fact that many fast growing, easy to culture algae also are oil-rich has recently drawn the attention of scientists and engineers around the globe to the possibility of using algae as an abundant, potentially inexpensive, source of renewable and sustainable biofuels that will lessen the needs for highly polluting, expensive and environmentally nonfriendly fossil fuels. Research to be conducted collaboratively between the laboratories of Dr. Don Weeks at the University of Nebraska-Lincoln and Dr. Martin Spalding at Iowa State University is aimed at elucidating the mechanisms underlying the ability of algae to serve as ?super sponges? of CO2 from the environment. The Spalding/Weeks laboratories recently discovered two proteins, HLA3 and LCIA, which provide the algal cell, Chlamydomonas reinhardtii, with the ability to scavenge very low levels of inorganic carbon (CO2 and bicarbonate) from their aquatic environment. Ongoing research sponsored by NSF will focus on the molecular mechanisms by which these inorganic carbon transporters work, where in the cell they are located and how these molecules interact with other components of the cell to allow efficient CO2 uptake and utilization for photosynthesis. In addition, the use of the newly discovered inorganic carbon transporters to augment CO2 uptake and photosynthetic efficiency in algal cells involved in biofuel production will be explored.

Broader Impacts:

This research will contribute significantly to the training of undergraduate and graduate students and postdoctoral associates participating in the project at both Iowa State University and at the University of Nebraska. It also will contribute to broadening the education of high school and undergraduate students and of high school biology teachers that will participate during summer internships and camps. Because members of underrepresented groups (e.g., African Americans, Hispanics, and Native Americans) are specifically recruited for the high school and undergraduate internships, this research also will provide opportunities for broadening educational experiences for these groups. Postdoctoral associates and students trained in our projects will find ample opportunities in academic and industrial positions focused on algal biology and biotechnology and its application to critical societal needs.

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Abstract for Award no. 1517256 (MCB-SSB)

This project will develop and test predictive mathematical models of the chemical reactions (metabolism) by which maize (corn) transforms the sugars produced during photosynthesis into compounds such as starch that are stored in the seeds. The goal is to understand starch metabolism better and provide the means for enhancing food production. This study will reveal fundamental principles of metabolic function and dynamics, and provide interdisciplinary training (in mathematical modeling, genetic engineering and metabolic biochemistry) to students engaged in this project.

Starchy endosperm (SE) is the major site of carbohydrate and protein storage in cereals, but atypical constraints complicate detailed resolution of the system. These include a steady state oxygen level of essentially zero, coincident with energy-expensive breakdown and re-synthesis of building blocks prior to storage polymer formation. A major question in SE metabolism is how ATP is generated to support starch and protein synthesis, and how the photo-assimilate is divided between catabolic and anabolic pathways. This project will expand upon existing flux models to account for bio-energetic parameters, and probe the system by directed genetic modifications that impair specific metabolic nodes. Metabolite levels and flux maps will be compared between SE from unaltered and mutant lines. These data will be applied to refine the mathematical models and test hypotheses of how the system diverts chemical resources for storage compound accumulation.