Vacuolar compartmentalization: a second-generation approach to engineering plants for phytoremediation Yi-Ping Tong, Ralf Kneer and Yong-Guan Zhu Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China Engineering plants with greater metal tolerance and accumulation properties is the key to developing phy- toremediators. A recent study by Won-Yong Song et al. has shown that overexpressing the yeast vacuolar transporter YCF1 increases Pb and Cd tolerance and consequently increases the accumulation of these metals in shoots of transgenic Arabidopsis plants even though expression levels of YCF1 were relatively low. This technology can be used to engineer advanced phy- toremediators, increasing their ability to pump heavy metals into a safe compartment while requiring only a small amount of transporters rather than a large amount of chelating peptide material. Excess toxic metals such as Cd, Pb, Cu, As or Ni in soils pose a major environmental and human health problem. Phytoremediation is thought to be a cost effective and environmentally friendly technology to remove toxic metals from soils. An ideal phytoremediator should be fast growing, develop a large biomass, be tolerant to and accumulate high concentrations of toxic metals in the shoot, and be easily cultivated and harvested. Although natural hyperaccumu- lators can tolerate and accumulate high concentrations of toxic metals, they usually have small biomass, grow slowly and cannot be easily cultivated. Now, with the advance- ments of molecular biology, scientists can use genetic engineering to improve the metal accumulation capacity of fast-growing and high-biomass plants. Enhancement of the metal accumulation properties of plants by genetic engineering requires a thorough under- standing of the biological processes involved in metal acquisition from soil to plant roots, metal translocation from roots to shoots, and tolerance to and accumulation of high concentrations of metals. Unfortunately, apart from the mechanisms of tolerance, these biological processes are not well understood, particularly in natural metal hyper- accumulators that have enhanced metal acquisition, translocation, tolerance and accumulation abilities and thus are the most promising source of potential phytor- emediation genes. To our knowledge, no genes from hyperaccumulators have been used in engineering phytor- emediators. Until there is a better understanding of the more complex relations in metal accumulation, the only way to engineer phytoremediators is to increase metal tolerance. Metal tolerance is one of the key prerequisites for phytoremediation. The cellular mechanisms behind metal tolerance in plants, and particularly in bacteria and fungi, are now relatively well understood and several genes conferring metal tolerance have been identified and characterized. Research on microorganisms can provide valuable models for understanding and engineering metal tolerance in higher plants and the number of known potential gene targets in microorganisms is much higher and diverse than in plants [1]. With the exception of mammalian metallothionein [2] and phytochelatin synthase [3], all genes used to date in engineering metal tolerance in plants are of microbial origin. Cellular mechanisms for metal tolerance can be classified into two basic strategies. One strategy is to keep the concentration of toxic metal ions in the cytoplasm low by preventing the metal from being transported across the plasma membrane, either by increased binding of metal ions to the cell wall or by reduced uptake through modified ion channels, or by pumping the metal out of the cell with active efflux pumps, a mechanism that is widespread in metal-tolerant bacteria [1]. The other strategy is to detoxify heavy metal ions entering the cytoplasm through inactivation via chelation or conversion of a toxic ion into a less toxic or easier to handle form and/or compartmentalization. Potential protein targets that have been or might be used for engineering metal tolerance in plant cells are summarized and categorized in Figure 1. Even though there is a variety of different metal tolerance mechanisms, and the reports of transgenic plants with increased metal tolerance and accumulation are many – most, if not all, transgenic plants created to date rely on overexpressing genes involved in the biosynthesis pathways of metal-binding proteins and peptides [2–6], genes that can convert a toxic ion into a less toxic or easier to handle form [7,8], or a combination of both [9]. Increasing cytoplasmic metal binding capacities Inactivation of toxic metal ions through the metal-induced synthesis of phytochelatins and the formation of metal – phytochelatin complexes is the general cytoplasmic mech- anism for trace metal homeostasis in plants and provides tolerance against non-essential heavy metals owing to its relatively unspecific mode of action [10]. Transgenic plants with enhanced chelation capacities that overexpress enzymes involved in the biosynthesis of glutathione (GSH), the precursor for phytochelatin synthesis, exhibit greater heavy metal tolerance and accumulation of heavy metals in the shoot than the controls do [4–6]. However, in some cases, improved chelating capacities do not lead to Corresponding author: Yong-Guan Zhu (ygzhu@mail.rcees.ac.cn). Update TRENDS in Plant Science Vol.9 No.1 January 2004 7 http://plants.trends.com