Phosphorus (P) is an absolutely critical nutrient for plant growth. This has large implications for the agricultural industry; fertilizers must be applied where soils do not contain sufficient P. However, the world’s store of P is finite, and costs of extraction are steadily rising (Gamuyao et al. 2012). Therefore, it is of vital importance to pursue species of crops, such as Kasalath rice, that are well suited for growth in low-P environments. While scientists have known for years that Kasalath rice is capable of producing high yields under the stress of minimal P and other nutrients, they have not before identified the specific gene that contributes this attribute. Gamuyao et al. conducted an investigation to discover what genetic trait Kasalath possess that is absent in other varieties of rice, and what physical effects this trait ultimately conveys. They concluded that a gene dubbed phosphorus-starvation tolerance 1 (PSTOL1) promotes the extended development of roots, allowing plants to absorb higher quantities of all nutrients, including P.—Chad Redman
Gamuyao, R., Chin, J. H., Pariasca-Tanaka, J., Pesaresi, P., Catausan, S., Dalid, C., Slamet-Loedin, I., Tecson-Mendoza, E. M., Wissuwa, M., Heuer, S., 2012. The Protein Kinase Pstoll from Traditional Rice Confers Tolerance of Phosphorus Deficiency. Nature 488, 535–541.
Gamuyao et al. searched for the genetic mechanism behind the P-starvation survival trait of certain types (aus-type) of rice, specifically the variety Kasalath. Kasalath has become vital to the agricultural industry throughout Southeast Asia due to its ability to produce high yields in poor soil. Originating in eastern India, this rice has been implemented for years in a poverty stricken region of the world where rice can be the only source of calories and 60% of farmland is composed of poor and problem soils (Gamuyao et al.). However, it has remained a mystery as to how Kasalath rice could survive in such nutrient-deprived ground; that is the mystery Gamuyao et al.set out to uncover.
While the actual investigative techniques were quite technical, the basic notion of what these researches did can be understood in simpler terms. They began by sequencing the region of Kasalath DNA that has been associated previously with P-starvation tolerance, named Pup1. That is to say, they read what instructions the DNA was giving the rice. From this analysis, Gamuyao et al. were able to identify four specific instructions that they thought were possible contributors to Kasalath’s resistance to P-starvation. From here, the scientists narrowed the possible candidates down from four to just one using a process known as quantitative polymerase chain reaction (qPCR). In a nut shell, using qPCR allowed researchers to decide what piece of Kasalath DNA exists in the Pup1 region that does not exist in other varieties of rice. Ultimately, the gene that was singled out was called phosphorus-starvation tolerance 1 (PSTOL1). After isolating the bit of DNA that they suspected was responsible for Kasalath’s resilience, Gamuyao et al.performed several experiments to test the physical impact of PSTOL1, beginning by artificially inserting the gene into rice varieties that do not naturally have P-starvation tolerance. The real test was to observe the root development of these transgenic specimens. Next, researchers placed these transgenic plants into solutions varying in P concentration (100µM and 10µM), with controls that did not possess the gene in question. Again, root development was the main subject of study.
The following procedures were conducted to confirm the hypothesis of the researchers that their mystery gene, PSTOL1, impacts root development. First, Gamuyao et al. used RNA interference to down-regulate the gene in Kasalath plants; they made it so the rice plant could not use its PSTOL1 gene. Next, the scientists analyzed the expression of PSTOL1 during the root development of a plant by “watching” for the presence of a marker in the roots of a plant that indicate PSTOL1 activity. Additionally, both transgenic plants (plants that have had the P-starvation gene artificially implanted in them and which express this gene at a very high rate) and control plants (Kasalath) were grown in dirt, and root samples were taken from them for an Affymetrix gene-array. This analysis essentially checks for how cells in the plant are using PSTOL1to make the structures that provide the plant with P-starvation resistance. Finally, using hydroponics, Gamuyao et al. performed more qPCR on root samples from plants to detect the presence of more molecules that indicate PSTOL1is active in root development.
Clearly, this procedure was a highly comprehensive one. Similarly, the results yielded were convincing and complete. Original sequencing of the Pup1 region revealed four possible P-starvation tolerance sites, and further qPCR found all but the PSTOL1gene to be shared to some extent between Kasalath and other, non-resistant varieties of rice.
The results of the initial transgenic plant analysis, where rice plants not possessing PSTOL1 were implanted with the gene, were promising; these artificially created varieties also exhibited P-starvation resilience. This was a good indication that PSTOL1 was the missing link from non-resilient varieties of rice. When the transgenic plants were grown in mediums containing varied concentrations of P, they developed longer roots with more surface area than control plants, regardless of their treatment. This result demonstrates that PSTOL1 is an active gene independent of P concentration in the environment.
On to the procedures that were used as confirmation of the Gamuyao et al. hypothesis that PSTOL1 works through affecting root growth, when the researchers inhibited PSTOL1, they found significantly less root mass was produced by the plant. In the marker experiment, while the actual observations were rather complex, the conclusion was that PSTOL1 promotes early root development in Kasalath and the transgenic plants. Moreover, similar results were acquired in the Affymetrix gene-array. Gamuyao et al. found 23 genes within the transgenic plants that were expressed at different rates as compared to the control rice plants regardless of stress situations, namely P-starvation. This data suggests that PSTOL1 is, in effect, regulating the expression of these 23 other genes, all of which are related to stress responses such as low P and flooding. Putting the proverbial cherry on top, Gamuyao et al.conducted one last qPCR analysis on root material from these same transgenic plants, ultimately reinforcing the independence of PSTOL1 and its associated genetic impacts from levels of P in the soil.