Identification of Genes Involved in Metal Transport in Plants

Plants obtain mineral nutrients from the soil. If they are growing in soil with high levels of metals, they will take up an excess of what is needed for growth. Depending on the species, this can be detrimental to growth—or lethal—and can greatly limit the growth range of plants and the productivity of agricultural species. However, some plants have adapted to living in soil containing excess metals. A portion of these species will even hyperaccumulate the metals, leading to exceedingly high levels of metals in the plant tissues. The metal most commonly accumulated is nickel (Ni). How and why certain plants are able to accumulate—and tolerate—high levels of potentially toxic compounds has spawned diverse areas of research, including an article by Talke et al., “Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri,” which appeared in the September 2006 issue of Plant Physiology. Interestingly, most of Ni and zinc (Zn) hyperaccumulators belongs to one family, the Brassicaceae, which includes the well-studied Thlaspi caerulescens and Arabidopsis halleri. Hyperaccumulators such as these typically accumulate the metals in the aboveground biomass through bulk flow of the metals in the xylem from root to shoot. Prior to this, the metals must first be translocated from the root symplast into the xylem apoplast, and in most instances the transporter proteins involved in this process have not been identified. This is a saturable process limited not only by the number of transport proteins present, but also by the variation in the transporters with respect to transport rate, substrate affinity, and substrate specificity (for review, see Pilon-Smits, 2005). Another transport step occurs from the xylem into the leaf cells. At the tissue level, metal may be accumulated in the epidermis and trichomes, while at the cellular level, these excess metals are typically accumulated in the vacuole or cell wall. The metals are often bound by chelators, which are believed to play a role in detoxification of the metals (for review, see Peuke and Rennenberg, 2005; Haydon and Cobbett, 2007). The ability of plants to remove organic contaminants such as metals and accumulate them in aboveground biomass has been taken advantage of in the remediation of contaminated soils. However, not all metal-hyperaccumulating plants have high biomass, a “requirement” for successful use of a plant for phytoremediation. Thus, an important question is what confers the ability to tolerate (elevated) levels of a metal that would be lethal or seriously inhibit the growth of a closely related species, or, more specifically, what makes a plant a hyperaccumulator? A. halleri is a member of the sister clade to Arabidopsis thaliana, but unlike A. thaliana it is a metal hyperaccumulator of Zn and cadmium (Cd). As is typical for hyperaccumulators, the Zn hyperaccumulation in A. halleri is partly due to increased partitioning of the metal from roots to shoots. Using cross-species transcript profiling, Talke et al. (2006) identified a set of candidate genes that are more highly expressed in A. halleri than in A. thaliana when grown under elevated levels of Zn and a set in A. halleri that is transcriptionally regulated by Zn. The change in transcript level of a subset of these candidate genes was further analyzed using real-time PCR and 29 of these were found to encode putative metal homeostasis proteins. Although 11 of these 29 were confirmations of previous findings, the other 18 had never been connected to metal hyperaccumulation. Four of the genes with the highest transcript level in A. halleri, HMA4, ZIP9, ZIP6, and ZIP3, might exist as multiple genomic copies. The genes ZIP9, ZIP6, and ZIP3 are members of the ZIP family of metal transporters and candidates for cytoplasmic metal influx in roots (Guerinot, 2000), while HMA4 (At2g19110) has been characterized as a P1B-type heavy metal ATPase in A. thaliana and is suggested to have a role in root-to-shoot Zn transport (Hussain et al., 2004; Verret et al., 2004; for review, see Colangelo and Guerinot, 2006). The multiplication of these genes within the A. halleri genome could be an explanation for the high levels of expression, as has been recently shown for HMA4 (Hanikenne et al., 2008). All four of these genes are believed to have functions directly related to metal transport; thus, it would be fitting that they would be up-regulated in a hyperaccumulator such as A. halleri. An efficient root uptake system, as well as root-to-shoot translocation of metals, is among the more important characteristics of a metal hyperaccumulator. Studies such as the one by Talke et al. (2006) have identified candidate genes up-regulated in the hyperaccumulator A. halleri relative to A. thaliana, including those involved in root-to-shoot translocation such as HMA4. A. halleri, a known Zn hyperaccumulator, also will hyperaccumulate Cd in aboveground biomass, most likely using components of the Zn (or iron [Fe]) transport. This double use of the transport system can be problematic in biofortification efforts of food crops in which the accumulation of toxic compounds such as Cd is detrimental (Palmgren et al., 2008). Thus, it is important to know and understand the details of the uptake process. The translocation of Cd from root to xylem was studied by Ueno et al. (2008) and their findings confirm the use of components of the Zn transport system for Cd transport. The root-to-shoot translocation was abolished by the metabolic inhibitor carbonyl cyanide 3-chlorophenylhydrazone, strongly suggesting an active transport system. HMA4, a P1B-type heavy metal ATPase involved in Zn root-to-shoot transport, possibly plays a role in this process. Their study also found that Cd is translocated in the xylem predominantly as aqueous free ions rather than being complexed with citrate, which is believed to play an important role in the loading of aluminum and Fe. The flow of the metals through the plant was the focus of a study by Waters and Grusak (2008), with the goal of identifying tissues that served as metal sinks for transport to seed to aid in the biofortification of crop plants by ultimately increasing seed metal content. Waters and Grusak (2008) performed whole-plant partitioning to determine the mineral content of tissues during growth stages in three wild-type A. thaliana ecotypes (Columbia-0, Landsberg erecta, and Cape Verde Islands) and one mutant (Columbia-0∷ysl1ysl3) that has low copper, Fe, and Zn levels in seeds most likely due to lesions in genes proposed to be involved in metal transport from the vascular tissue (Waters et al., 2006). Although ecotype-specific differences were seen in metal partitioning at the tissue level, an increase in shoot metal content over the course of the plants' life cycle was common in all three. They were also able to determine that in A. thaliana, silique hulls are a mineral source for seeds. In the ysl1ysl3 double mutant, there is an increased leaf-to-stem ratio of Cu and Zn, suggesting that metals are not partitioned properly in the plant. The authors propose that this might be due to a decrease in the mobilization of these metals from or translocation through the fruit hulls in the double mutant. Together, this suggests that, in the case of Cu and Zn, these metals need to go through a source tissue (leaves) before being translocated to the seed sinks. YSL1 and YSL3, both of which Talke et al. (2006) found to be more highly expressed in A. halleri, need to be functional for efficient mobilization and are potential targets for the increase of minerals into seeds. The identification of the proteins involved in metal hyperaccumulation, such as in the study by Talke et al. (2006), is an important step in fully understanding the process of tolerance of high metal content in plants. This information can, and has been, used in other studies on metal accumulation and to further research on biofortification of plants.