Improving the Use of Phosphate in Alfalfa

Phosphate (P) has always been one of the most limiting factors in agricultural soil, and will become more of a constraint as world population grows and environmental concerns gain precedence. It is therefore imperative to improve how efficiently and carefully P fertilizers are applied, as well as how efficiently crop plants use those fertilizers. To explore this, Ma et al. (2012) caused alfalfa plants to express genes that are found in a model legume organism, Medicago truncatula, in the hopes that these genes would improve the use of organic phosphate (Po) by alfalfa plants. Po is generally derived from animal manure, and takes the form of phytate in soil, which is difficult for plants to use. The genes MtPHY1and MtPAP1 produce enzymes that can assist in the digestion of Po, and therefore were selected to express in the roots of transgenic alfalfa. Ultimately, it was concluded that transgenic lines of alfalfa demonstrated significantly better use of Po than control lines when grown in either lab conditions or active farm soil.—Chad Redman
                  Ma, X.F., Tudor, S., Butler, T., Ge, Y., Xi, Y., Bouton, J., Harrison, M., Wang, Z.Y. 2012. Transgenic Expression of Phytase and Acid Phosphatase Genes in Alfalfa (Medicago sativa) Leads to Improved Phosphate Uptake in Natural Soils. Mol Breeding 377, 377–391.
                  Ma et al. inserted genes from Medicago truncatula into the roots of alfalfa

plants in order to promote the use of Po. They chose to use genes that had demonstrated significant ability to increase P uptake in other plants previously, and focused on the use of transgenic plants in the real-world conditions of active farm soil. Specifically, the authors selected phytase (MtPHY1) and purple acid phosphatase (MtPAP1) genes, which both produce phytases, or enzymes that break down phytate into usable P. They also identified two different promoters, or regions of DNA that initiate the process of expressing genes, which could be used for both MtPAP1 and MtPHY1 and tested them independently for each of the genes. These promoters are identified as CaMV35S and MtPT1. Thus, Ma et al.created four different transgenic alfalfa lines, combining one gene with one promoter in all four combinations.

                  Researchers began with two different lab tests, the first growing plants in a medium without any P and the second in a medium supplied with Po. In the first condition, each of the transgenic plant lines along with a control line were grown in sand and fertilized with a solution that did not contain any form of P. After two weeks of growth under P-stressed conditions, the plants were harvested for their roots in order to isolate RNA and measure enzyme activity.
                  The second growth condition also utilized sand, but in this case plants were grown in the presence of fertilizer. The fertilizer composition was standard, except that it contained no inorganic phosphate (Pi), only Po. After six weeks of growth plants were harvested and all above-ground parts of the plant were dehydrated using an oven over the course of one week. The dried biomass of each plant group was recorded to compare use of Po.
                  After these lab-generated medium tests, plants were grown in pots containing soils from active farm ground. Two different soils were tested, one from Texas (soil 1) and the other from Oklahoma (soil 2). Soil 1 was generally less nutrient rich, and also more acidic. Soil 2 had a significantly higher concentration of usable P and a fairly neutral pH. In each soil, all four transgenic plant lines, along with control plants, were grown for three weeks without adding any nutrients to the soil. After three weeks, fertilizer without any form of P was applied in order to isolate the effects of P-stress on the plants. In order to determine biomass, two cuttings of the alfalfa plants were performed, the first after eight weeks and the second after another four weeks. These cuttings were dried and weighed. Additionally, the second cuttings of these plants were used to measure total P contained in the plants.
                  Ma et al. present some intriguing findings. From the RNA and enzyme analysis of plants grown in sand without any added P fertilizer, they find that transgenic plants on the whole produce far greater levels of APase, an enzyme that breaks down forms of phosphorous that plants cannot use into forms that may be digested. This observed difference was highly statistically significant. Moreover, there was an observed significant difference between the enzyme activities induced by the two different promoters. MtPT1 controlled genes had higher levels of APase than CaMV35S controlled genes. However, there was no observed difference between the two genes themselves. These results indicate that the transgenic alfalfa is, in fact, superior to the wild type alfalfa. Furthermore, the MtPT1 promoter is more effective in promoting efficient P use than the CaMV35S promoter.
                  Examination of plant roots revealed that transgenic plants showed higher phytase activity than control plants. Phytase is another enzyme that breaks down P into forms that may be easily used by plants, but phytase is specific to braking down phytate. Phytate is the most abundant source of P in soils, so phytase production is essential to effective use of P fertilizers. Interestingly, MtPHY1 plants exhibited higher phytate activity than MtPAP1 plants, which suggests that MtPHY1 transgene alfalfa is preferable.
                  When it comes to growth performance, transgenic plants consistently outperformed wild type alfalfa. As the above enzyme analyses would suggest, the plants with the MtPT1-MtPHY1 construct had the most vigorous growth and the highest dry biomass when grown in sand with Po readily available. Also, there was a strong correlation between enzyme activity and biomass, with transgenic lines clearly using the Po much more efficiently than the non-transgenic line.
                  In natural soils, those taken from active farm ground, similar patterns were observed. Especially in soil 1, transgenic alfalfa grew as expected, much larger and healthier than the control plants. In soil 2, the transgenes MtPHY1 and MtPAP1 were less effective because the pH of soil 2 was close to neutral, which is too basic for the enzymes to work at optimal levels. Moreover, because soil 2 contained a higher concentration of P to begin with, there was less P stress on wild type plants.
                  What Ma et al. have shown is that transgenic alfalfa can be used to alleviate the economic and environmental costs of applying large amounts of P fertilizer. With more efficient P use, particularly in alfalfa expressing the MtPT1-MtPHY1construct, pasture grounds will require less application of P, resulting in fewer weed control issues and also less runoff into aquatic ecosystems.

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