Submitted to: The Plant Cell
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/17/2001
Publication Date: 9/18/2001
Citation: Interpretive Summary: We wanted to identify the part in the structure of a transporter that enables it to do the work of transporting calcium in a plant. We would liken this transporter to an engine for our purposes here, in order to illustrate the point of this complicated and ambitious genetic experiment. We examined yeast as our plant model. In doing so, we were able to identify yand define the "engine" part that enables the nutrient transport, and we also showed that this part can do the same thing when it is put on other engines. In other words, by better understanding the genetic mechanism that moves nutrients into and through plants, we become better equipped to manipulate plants in order to increase the amount of nutrients they can absorb. Our ultimate goal is to improve the nutrient content of plant-based foods. Overall, this study demonstrates that yeast is an excellent model for researchers to use in order to study how plant transporters work at the egenetic level. The ability to genetically alter the transport function in yeast, and to express these altered genes in plants, offers the potential to manipulate nutrient storage and signaling functions, and to surpass agricultural limitations currently mandated by environmental conditions.
Technical Abstract: Ca2+ levels in plants are controlled in part by H+/Ca2+ exchangers. The Arabidopsis H+/Cation exchangers, CAX1 and CAX2 were identified by their ability to suppress yeast mutants defective in vacuolar Ca2+ transport. CAX1 has a much high capacity for Ca2+ transport than CAX2, and CAX1 appears to help regulate plant cytosolic Ca2+ levels; however, the amino acid residues involved in CAX-mediated Ca2+ binding and translocation have not been identified. An Arabidopsis thaliana homolog of CAX1, CAX3, is 77% identical (93% similar) and when expressed in yeast localized to the vacuole but does not suppress yeast mutants defective in vacuolar Ca2+ transport. Chimeric constructs and site directed mutagenesis showed that CAX3 can suppress yeast vacuolar Ca2+ transport mutants if a nine amino aci region of CAX1 is inserted into CAX3. A single leucine to isoleucine change within this region caused CAX3 to weakly suppress the yeast Ca2+ sensitivity. Biochemical analysis in yeast showed that these alterations caused increased vacuolar H+/Ca2+ exchange. This nine-amino acid region is highly variable among the plant CAX-like genes and exchanging the nine- amino acid region of CAX1 into CAX2 greatly increase the vacuolar Ca2+ transport properties of this chimeric protein. This study suggests that this region is involved in CAX-mediated Ca2+ transport.