Improving the Nutritional Quality and Stress Tolerance of Food Crop Species

The cellular physiology, genetics and molecular biology of mineral and heavy metal ion uptake, translocation, deposition and homeostasis, as well as the metabolism and accumulation of organic constituents of plant foods that impact human nutrition will be studied. An interdisciplinary approach will be taken integrating microelectrode techniques with membrane biochemistry and molecular biology.
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Specifically, these approaches will be used to <ol> <li>Study and maximize the accumulation of nutritionally important mineral elements (Fe, Zn, Ca) in edible portions of crop plants;
<li>Investigate mechanisms of Cd uptake and transport, in order to minimize Cd (and other heavy metal) uptake in edible plant parts;
<li>Elucidate the molecular regulation of biosynthetic and matabolic pathways involved in accumulation of organic constituents of plant foods that could impact their nutritional and health-promoting properties; and
<li>Investigate the physiology and molecular regulation of aluminum tolerance mechanisms in important grain crops (wheat, maize, barley).</ol>
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PROGRESS: 2000/10 TO 2001/09
<ol> <li>What major problem or issue is being resolved and how are you resolving it? The unifying theme for this CRIS project is the interdisciplinary application of experimental approaches from plant genomics, molecular biology, genetics and physiology to study two separate research issues in crop improvement: enhancing the nutritional quality of plant foods and improving the ability of crop plants to grow on marginal soils. This research has 3 major goals: 1) to understand the cellular processes in plants that regulate carotenoid synthesis, as a means of developing strategies by which we can selectively modify carotenoid content and thus improve nutritional quality in crops; 2) to identify the suite of genes conferring Zn hyperaccumulation in the metal hyperaccumulating plant species, Thlaspi caerulescens, in order to develop strategies both to improve Zn content in plant-based foods and to develop plants better suited for the remediation of surface soils contaminated with heavy metals; and 3) to elucidate the mechanisms and the underlying genes crop plants employ to tolerate toxic levels of aluminum (Al) on acid soils. The research on carotenoids uses a model system, the Or mutation in cauliflower, that causes normally carotenoid-deficient tissues to accumulate large amounts of beta-carotene, in much the same fashion we would like to do in staple food crops. The research on improving micronutrient density of plant foods involves the functional genomics of Zn tolerance and transport in the unique Zn hyperaccumulator, Thlaspi caerulescens. Another important outcome of this work is to develop plant species better suited for the phytoremediation of heavy metal contaminated soils. The research on Al tolerance focuses on mapping of Al tolerance genes in populations of wheat and other cereals segregating for Al tolerance with identification and characterization of physiological mechanisms of Al tolerance. We are also using several different molecular approaches aimed at cloning Al tolerance genes, which ultimately will be used to improve this trait in more Al sensitive cereal crops and other food crops.
<li>How serious is the problem? Why does it matter? Improving the nutritional quality of plant foods is a worthy goal, as diet-related diseases in the U.S. such as cardiovascular disease and cancer costs the economy approximately 200 billion dollars annually. More specifically, as vitamin A deficiency is a serious problem in developing countries, it is estimated that providing adequate vitamin A to the world's human population could reduce upwards of 2 million deaths annually. Reducing the incidence of these diseases through increased carotenoid content of plant-based foods will result in significant economic benefits to the U.S. and other countries. Furthermore, deficiencies of the micronutrients Fe and Zn negatively impact the health and productivity of 2 billion people worldwide, as well as significant sections of the U.S. population (premenopausal women and young children). Enhancing the micronutrient density of plant foods can be a sustainable solution to this nutrition problem. Improving the ability of farmers to cultivate crops on marginal soils such as acidic, Al toxic soils, is important as acid soils comprise over 50% of the world's potentially arable lands, as well significant land areas in the United States. While liming of acid soils ameliorates problems associated with soil acidity, this is neither an economic option for poor farmers nor an effective strategy for alleviating subsoil acidity. Marginal soils contaminated with heavy metals is also a serious worldwide problem both for human health and agriculture. Cleanup of hazardous wastes by the currently used engineering-based technologies has been estimated to cost at least $400 billion in the U.S. alone. Recently, there has been considerable interest in the use of terrestrial plants as an alternative, 'green technology' for the phytoremediation of surface soils contaminated with toxic heavy metals. 3. How does it relate to the National Program(s) and National Component(s)? National Program Code 302 (70%)Plant Biological and Molecular Processes National Program Code 202 (30%) Soil Resource Management. The research on understanding the molecular and biochemical basis of carotenoid synthesis and micronutrient accumulation in plants, with the ultimate aim of improving food nutritional quality, addresses a two key components of National Program 302 (Analysis and Modification of Plant Genomes and Biological Processes that Determine Plant Productivity and Quality). The research on plant Al tolerance and heavy metal hyperaccumulation also addresses these same components as well as Component II (Nutrient Management) of National Program 202.
<li>What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2001 year: An important goal for improving crop aluminum (Al) tolerance is the isolation and characterization of Al tolerance genes. In a project aimed at cloning the major Al tolerance gene in sorghum, several molecular markers closely linked to the Al tolerance locus were identified. A collaboration with Dr. John Mullet of Texas A&M University has been established to determine the physical location of these markers in the sorghum genome and to identify additional closely linked markers, in order to conduct fine-scale mapping and map-based cloning of the sorghum Al tolerance gene. Isolating this gene will allow for the identification of tolerant alleles of similar genes in related grain crops, and for the use these genes to improve the Al tolerance of important grain crops via biotechnological approaches. B. Other significant accomplishment(s), if any: 1. The isolation of the Or gene in orange cauliflower that accumulates high levels of beta-carotene in the curd will provide new insights into the regulation of carotenoid biosynthesis as well as a new molecular tool to increase carotenoid content and thus nutritional quality of important food crops. As a first step towards this goal, molecular mapping of the Or mutation has been conducted and closely linked markers located on either side of the Or gene have been obtained. Thus the stage is set for the identification of more closely linked markers and subsequent map-based cloning of the Or gene. It appears that this gene regulates the flux of carbon through this biosynthetic pathway, which could provide a novel new molecular tool for enhancing beta-carotene content of plant foods. 2. Soils contaminated with heavy metals is a serious worldwide problem both for human health and agriculture, and cleanup of these soils by the currently used engineering-based technologies has been estimated to cost many billions of dollars in the U.S. alone. A better understanding of the molecular basis for heavy metal accumulation and tolerance will enable us to develop transgenic plants suited for the phytoremediation of contaminated soils. This goal is being achieved by elucidating the molecular mechanisms of extreme heavy metal/micronutrient accumulation in the shoots of Thlaspi caerulescens, and it has been found that altered regulation of key micronutrient transport genes plays a role in this trait. To determine the basis for this altered regulation, a Thlaspi transcription factor has been cloned that appears to regulate micronutrient transporter gene expression via changes in plant micronutrient status. It appears that this gene is an important molecular regulator of micronutrient homeostasis, and alterations in this protein appear to confer heavy metal hyperaccumulation and tolerance. This gene may prove to be a useful molecular tool for improving plants for the phytoremediation of heavy metal contaminated soils, which could provide a low-cost alternative to current clean up technologies. 3. Heavy metal tolerance must involve organic ligands that complex and detoxify the heavy metal ions in the plant cell; phytochelatins are the best known type of metal binding ligand and are believed to be involved in plant tolerance to cadmium(Cd). In the zinc/cadmium hyperaccumulator Thlaspi caerulescens, the possible role of phytochelatins (PCs) in the extreme Cd accumulation and tolerance was examined; surprisingly, it was found that PCs do not play a role in Cd accumulation/tolerance. Thus, the possibility that a novel organic ligand that complexes heavy metals in this plant species is now being examined. 4. A major mechanism of aluminum (Al) tolerance in crop plants involves the Al activated exudation of Al binding organic acids from the root apex. In Al tolerant maize that release citric acid in response to Al stress, electrophysiological approaches were used to identify an organic acid channel that when upon binding Al, opens and allows citric acid to flow out of root cells into the soil solution. These findings are significant as they indicate these channels are a key component in Al tolerance and may represent candidates for Al tolerance genes, which can be used to develop improved crops for cultivation on acid soils that comprise approximately half of the world's arable soils. Because of recent findings that anion channels play a key role in crop Al tolerance mechanisms, genes were isolated encoding anion channels from the root tip of a very Al tolerant wheat genotype. Currently, a total of 8 different anion channel genes have been isolated and are being characterized to determine if any of them encode the Al-activated organic acid transporter that mediates Al-induced malic acid release from the root tip of Al tolerant wheat. These studies may facilitate the identification of key molecular determinants of Al tolerance in cereal crops. C. Significant accomplishments/activities that support special target populations: None.
<li>Describe the major accomplishments over the life of the project including their predicted or actual impact. 1. To date, a significant body of biochemical, molecular, and cytological information in relation to the mode of action of the cauliflower Or gene has been obtained. Results strongly suggest that this gene controls the development of plastids in plant cells (structures in plant cells such as chloroplasts, the site of photosynthesis) and that the plastid is the site of beta-carotene accumulation in plants carrying this gene. The predicted impact of these findings is that it forms a foundation upon which attempts to clone this gene will be conducted. 2. Research into the mechanism by which the Or gene induces beta-carotene accumulation in cauliflower demonstrated unambiguously that the beta-carotene accumulates in large crystalline sheets within plastids, and that the Or gene appears to induce the formation of these sheets by directing plastids to become chromoplasts. This is significant because it suggests that if the Or gene can be cloned, it may direct this same phenomenon within plastids of the endosperm of grains such as wheat and rice. 3. Additional research was undertaken to gain insight into the action of the Or gene. Callus cultures were developed from Or plants that can be used for experimentation in lieu of continual growth of whole cauliflower plants. The cultures were successfully established and found to produce heightened levels of beta-carotene. These will provide an alternative and readily available source of material for biochemical and molecular experiments in the future. 4. Al-induced organic acid release was identified as the first significant Al tolerance mechanism in crop plants. Previously, most of the research on Al tolerance mechanisms had been speculative, and had not identified any actual tolerance mechanisms. 5. Research on Al tolerance mechanisms has identified a membrane transport protein in the outer cell membrane of cells of Al tolerant roots, which is a channel that binds Al and then opens, allowing the detoxifying organic acid to flow out into the soil. This was a key finding in understanding the cellular mechanism of Al tolerance, and has directed research towards the cloning of the gene that encodes this transport protein. 6. Research on the genetic and physiological complexity of Al tolerance in wheat showed that Al tolerance in wheat involves the functioning of several genes that all act in concert to enhance a single Al tolerance mechanism based on Al-induced release of Al-detoxifying malic acid from the root tip. These findings have directed subsequent research that is focused on this particular tolerance mechanism for efforts to clone Al tolerance genes in wheat. 7. Research was conducted into Al tolerance mechanisms in related cereal species (wheat, barley, maize, sorghum, rice), in order to see if they employ different tolerance mechanisms. It was found that these species employ a similar mechanism based on organic acid release from the root tip, which differed only in specific details such as the type of organic acid released (malate or citrate), the root region releasing the organic acid, and whether the response was constitutive or induced by Al exposure. These findings are important, for they suggest that similar or related genes regulate Al tolerance in each of these cereal crop species. 8. Because there is a controversy in the literature as to whether roots of Al tolerant genotypes release enough organic acids to bind and detoxify most of the Al in the soil at the root surface, we used a microanalytical technique to directly sample very small solution volumes (nanoliters) at the root surface and measure organic acid concentrations in this media. It was found that roots of Al tolerant wheat plants release more than enough malic acid to chelate all of the soluble Al in the soil at the root surface. 9. Research on heavy metal and micronutrient accumulation in the zinc and cadmium hyperaccumulator Thlaspi caerulescens showed that a number of micronutrient transport sites were stimulated in the roots and leaves of this plant. This led to the cloning one of the first micronutrient transport genes in any plant species, and characterization of this gene led to the determination that metal hyperaccumulation involves increased expression of micronutrient transport genes. 10. Research on the molecular mechanism(s) of micronutrient/heavy metal hyperaccumulation in Thlaspi caerulescens showed that the regulation of genes involved in micronutrient nutrition by plant micronutrient status was altered in this plant, causing the up regulation of a number of micronutrient transporters and genes involved in metal tolerance. This finding has led to the search for key regulatory gene(s) whose alteration may identify the molecular switch needed to transform normal plants into micronutrient and heavy metal hyperaccumulator. 11. Research on the use of plants to rededicate soils contaminated with the toxic radio nuclide cesium-137 (a product of nuclear reactor operation) identified a plant species, Amaranthus retroflex us, that was much more effective in extracting this radioisotope from contaminated soils than other plant species. Field studies showed that this plant was very promising for phytoremediation of radio cesium contaminated sites.
<li>What do you expect to accomplish, year by year, over the next 3 years? Not applicable, as this CRIS project has terminated.
<li>What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? During the past year, reprints of 8 different scientific publications were sent out to over 80 scientists in the U.S. and 10 foreign countries. The research was presented in invited talks at 9 international symposia, 4 invited seminars, and 10 presentations at National meetings (American Society of Plant Biologists meeting in Providence, RI, and Agronomy Society of America meetings in Minneapolis, MN). We also maintain several web pages - Web addresses: http://www.css.cornell.edu/research/USPSNL/uspsnl.html; http://www.scas.cornell.edu/faculty/kochian/index.htm; http://www.plantbio.cornell.edu/faculty.php?fid=42; http://132.236.156.26/bti2/bti2 page.taf?page=giovannoni lab which describe our research programs, as well as list recent publications, the members of the laboratory, and links to other ARS web pages.
<li>List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below) Improving aluminum tolerance in small grains. Agricultural Research Magazine. 2000. v. 48. p. 23.