Much of plant biotechnology R&D is driven by a need to minimize agricultural chemical applications. In addition to the financial advantages of reducing chemical usage, environmental protection groups worldwide and the growing sustainability movement are shunning current agricultural chemical application practices. Use of modern biotechnology techniques has been reported to reduce chemical use in some cases by producing plants better able to resist pests, compete with weeds, and take up soil nutrients.
Phosphorus is difficult for plants to obtain because of its low solubility in soil water. In addition, much of the phosphorus applied to crops is lost through runoff and microbial uptake. Phosphorus binds to the aluminum, iron, and calcium ions present in most agricultural soils, making it insoluble and unavailable to the plants. This is especially true in acid and highly alkaline soils because of higher metal ion solubility.
Luis Herrera-Estrella and his research team estimate that 25% of agricultural land contains alkaline soils that prohibit adequate phosphorus uptake. K. G. Roghothama, of Purdue University, calculates that another 30% of the world's crops are grown in acid soils. Extremely acidic and alkaline soils are typical of developing nations1,2 and, although adjusting the pH levels to between pH 6 and pH 7.5 encourages better phosphorus uptake, the cost is usually prohibitive. The addition of large amounts of phosphate fertilizers is also expensive, as well as environmentally unsound; consequently, decreased crop yields often must be tolerated in developing nations2.
Herrera-Estrella and his team have engineered a tobacco plant that promotes phosphorus uptake in both acid soils and soils high in metals using a technique that could prove economical for developing countries. They exploited the fact that soil organic acids assist roots with phosphorus uptake. Studies conducted by Gardner, Bounty, and others have also indicated that some plants facilitate phosphorus uptake by secreting organic acids into the rhizosphere. Although the secretions are restricted to a zone of soil immediately around the roots, the organic acids may improve phosphorus availability in soil conditions favoring phosphorus loss3.
Alan Richardson and Peter Hocking, of the Commonwealth Scientific and Industrial Research Organization in Australia, believe that organic acids separate bound phosphorus from clay and metals, making it available for plant uptake. In 1988, they reported in a press release that citrate naturally secreted by lupines facilitated phosphorus uptake. (See http://www.pi.csiro.au/Media/MediaReleases/MR19-02-98.htm) Phosphorus uptake was also correlated with the uptake of calcium, potassium, and nitrate3.
Drawing on this information, Herrera-Estrella's team engineered a plant capable of over-secreting citric acid in hopes it would enhance phosphorus uptake. They selected tobacco because it does not normally secrete citrate from the roots, making it simple to quantitate yield improvement from enhanced phosphorus utilization.
The team used Agrobacterium-mediated transformation to introduce a Pseudomonas aeruginosa citrate synthase gene into tobacco cell cultures. Citrate production was under control of a 35S CaMV promoter. They developed two lines of citrate-overproducing tobacco using this system: CSb-4 and CSb-18, which secrete, respectively, two and four times the level of citrate of transgenic control plants lacking the citrate synthase gene.
Citrate synthase is predominantly expressed in the mitochondria. Herrera-Estrella's group introduced and successfully expressed plasmids carrying the citrate synthase gene in the cytoplasm. Cytoplasmic expression of the gene permits its export from the cell and prevents it from being converted into Krebs cycle intermediates in the mitochondria. Their initial experiments entailed growing plants in naturally alkaline soils with low phosphorus levels. Plant life-cycle completion was evaluated by measuring monosodium phosphate assimilation. The CSb-4 and CSb-18 lines completed their life cycles while the control plants failed to achieve anthesis. Phosphorus accumulation in the plant tissues was also evaluated and correlated with life-cycle analyses.
Differences in shoot and fruit dry biomass were then analyzed under low to high phosphorus conditions (22, 44, and 108 ppm). Other soil conditions were kept optimal. No significant differences in shoot biomass were seen between the CSb and control plants in the 22 and 44 ppm phosphorus groups until the plants reached anthesis. Fruit biomass was significantly greater for the CSb-18 plants under the 22 and 44 ppm phosphorus conditions due to an increase in the number of seeds and individual seed size. Plants grown under 108 ppm phosphorus conditions showed no significant differences in shoot and fruit growth.
Mycorrhizal relationships are also known to enhance fertilizer availability in many plants. Herrera-Estrella's group tested phosphorus uptake by the plants grown in the presence of the mycorrhizal fungus Glomus fasciculatum. Again, they showed that the CSb lines were better than the control at capturing phosphorus.
Herrera-Estrella's findings indicate the possibility of reducing supplemental phosphorus applications for crop production. However, further investigations are needed before applying this procedure in the field. Cost effectiveness studies must be evaluated separately on fruit, leaf, and root crops, as well as effects of citrate overproduction on crop taste and appearance. Studies also need to be conducted on the influence of other organic acids on phosphorus uptake. Herrera-Estrella's study does not address phosphorus uptake under acid soil conditions, a significant problem for many agricultural areas.
The group is currently investigating citrate overproduction to improve phosphorus uptake in corn and rice. These two crops have large phosphorus requirements and are responsible for significant phosphorus depletion from farm lands. Perhaps using transgenic citrate-overproducing plants, these crops will one day be better able to use natural phosphorus soil reserves and require smaller applications of fertilizer.
Sources
1. Lopez-Bucio J, Marinez de la Vega O, Guevara-Garcia A, and Herrera-Estrella L. 2000. Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate. Nature Biotechnology18(4):450-453.
2. Roghothama KG. 1999. Phosphate transporters: Molecular tools for enhancing phosphate uptake by plants in acid soils. In Workshop to develop a strategy for collaborative research and dissemination of technology in sustainable crop production in acid savannas and other problem soils of the world, ed. RE Schaffert. Purdue University.
3. Gardner WK and Boundy KA. 1993. The acquisition of phosphorus by Lupinus albus L: IV. The effect of interplanting wheat and white lupine on the growth and mineral composition of two species. Plant and Soil 10:391- 402.
Brian R. Shmaefsky
Department of Biology and Environmental Sciences
Kingwood College
bshmaefs@nhmccd.edu