Simultaneous Hyperaccumulation of Nickel, Manganese, and Calcium in Alyssum Leaf Trichomes C. LEIGH BROADHURST,* ,† RUFUS L. CHANEY, † J. SCOTT ANGLE, ‡ TIMOTHY K. MAUGEL, § ERIC F. ERBE, | AND CHARLES A. MURPHY | Animal Manure and Byproducts Laboratory, Animal and Natural Resources Institute, U.S. Department of Agriculture, Henry A. Wallace Agricultural Research Center, Beltsville, Maryland 20705, Agricultural Experim ent Station, University of Maryland, College Park, Maryland 20742, Laboratory for Biological Ultrastructure, Department of Biology, University of Maryland, College Park, Maryland 20742, and Electron Microscopy Unit, Soybean Genom ics and Im provem ent Laboratory, Plant Sciences Institute, U.S. Departm ent of Agriculture, Beltsville, Maryland 20705 We have developed commercially viable phytoremedia- tion/phytomining technologies employing Alyssum Ni- hyperaccumulator species to quantitatively extract Ni from soils. The majority of Ni is stored either in Alyssum leaf epidermal cell vacuoles or in the basal portions only of the numerous stellate trichomes. Here, we report simultaneous and region-specific localization of high levels of Ni, Mn, and Ca within Alyssum trichomes as determined by scanning electron microscopy/energy-dispersive X-ray analysis (SEM/EDX). Plants were grown in high Ni soil, achieving up to 48 400 μgg -1 Ni in total leaf concentration; however, Ca and M n were not enriched in the experimental soils. The region-specific localization of hyperaccumulated Ca, Mn, and Ni occurred in three soil types, five Alyssum species/ecotypes, and over a wide range of soil Ni concentrations. The metal concentration in the trichome basal compartment was ∼15-20% dry weight, the highest ever reported for healthy vascular plant tissue. Introduction More than 400 plant species are known to naturally ac- cumulate high levels of metals such as Cd, Cu, Co, Mn, Ni, and Zn. Hyperaccumulation is defined as accumulation of >1000 μgg -1 in plant dry material for Cd, Ni, and Co, and >10 000 μgg -1 for essential trace elements such as Mn and Zn which are required in larger amounts to support normal metabolism (1). The genus Alyssum (Brassicaceae) contains the greatest number ofreported Ni hyperaccumulators (48), many ofwhich can achieve 3% dry weight Ni in leafbiomass (2). Our research consortium has developed commercially feasible phytoremediation and phytomining technologies that can potentially clean up Ni-contaminated soils (3-5). The technology employs the Ni-hyperaccumulating species Alyssum m urale and A. corsicum to quantitatively extract Ni from a range of soil types. These species are endemic to serpentine (ultramafic-derived) soils throughout Mediter- ranean Europe,butunlikemanyserpentine-endemicspecies, they grow prolifically and hyperaccumulate Ni in other soil types such as limestone soils, organic soils, and loam (4-6). Successfuldevelopment ofhyperaccumulatorspeciesfor large-scale phytoremediation/phytomining requires knowl- edge of Ni localization patterns for each genus/species/ ecotype of interest, and for a variety of realistic growth conditions. The Ni localization patterns have been deter- mined for 10 Alyssum Nihyperaccumulatorspecies/ecotypes (6-11).Nickelis mainlystored in the leaves and is particularly concentrated in epidermal cell vacuoles. There is a positive correlation ofsulfur (S) and Ni, indicating that SO4 2- maybe a counterion for Ni 2+ within the vacuoles (6, 9). The upper and lower leaf surfaces of Alyssum sp. are covered with an overlapping network of branchlike, stalked bifurcate or stellate trichomes (10, 12, 13). The 10 Alyssum Ni hyperaccumulator species/ecotypes mentioned above have stellate trichomes with 8-14 elongate rays, some of which bifurcate.The upper sides ofthe rays are covered with hemisphericalnodules, but the underside is smooth (Figure 1). Trichomes are attached to the epidermis with an ∼20 μm smooth cylindrical pedicle that has a broad fan-shaped compartment at its base. We found previously for A. m urale thatthetrichomebasalcompartment,trichomepedicle,and the epidermal cells adjacent to the trichome basal compart- ment strongly concentrate Ni, but there was no appreciable Ni in the rays or nodules (6). Here, we report simultaneous and region-specific localization ofhigh levels ofNi, Mn, and Ca within Alyssum trichomes. Materials and Methods Horticulture. Alyssum m urale “Kotodesh”, A. m urale “AJ9”, A. corsicum , A. fallcinium , and A. pterocarpum were grown from seed in a greenhouse at USDABeltsville. Twenty-one days later, “Kotodesh” seedlings were transplanted to 250 g pots containingPromixpottingsoilwith an increasingseries of NiSO4‚6H2O addition (0, 5, 10, 20, 40, 80 mmol Ni kg -1 ). NiSO4 is a standard salt for Ni soil enrichment. Carbonates (halfCaCO3/halfMgCO3)were added to each pot at amounts equimolar with NiSO4. Alyssum m urale “Kotodesh”, A.murale “AJ9”, A. corsicum , A. fallcinium , and A. pterocarpum were also transplanted into calcareousmineralsoilassociated with nickel mining tailings from Inco Ltd. operations at Port Colborne, Ontario (Welland soil, Typic Epiaquoll; Canadian classification, Terric Mesisol with 30 g kg -1 CaCO3 added). The Inco soil typically yields 1% Ni in Alyssum whole shoots and 1.7%in leaves and would be phytotoxic to normalplants (14). For control plants, all five Alyssum species were transplanted into Promix only. Initial results showed a significant correlation of Ni and S,especiallyin epidermalcellvacuoles.Because the possibility existed that additional S was provided by NiSO 4 or MgSO4, we started a second group of plants in soils with no added S. Alyssum m urale “Kotodesh” was grown from seed and transplanted after 21 days into 250 g pots containing Promix with an increasing series of NiC4H6O4‚5H2O addition (0, 5, 10, 30, 60, 90 m m ol Ni kg -1 ), and equimolar carbonates as above. Alyssum m urale “Kotodesh”, A. m urale “AJ9”, and A. pterocarpum seedlings were also transplanted into natural Brockman variant serpentine soilfrom Josephine Co.,Oregon (Typic Xerochrepts), with 10 wt % Promix added to improve drainage. *Correspondingauthor phone: (301)504-6550;fax: (301)504-5031; e-mail: broadhul@ba.ars.usda.gov. † Animal and Natural Resources Institute, USDA. ‡ Agricultural Experiment Station, University of Maryland. § Department of Biology, University of Maryland. | Plant Sciences Institute, USDA. Environ. Sci. Technol. 2004, 38, 5797-5802 10.1021/es0493796 Not subject to U.S. Copyright. Publ. 2004 Am. Chem. Soc. VOL. 38, NO. 21, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 5797 Published on Web 09/29/2004