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RELATIONS OF HEAVY METAL SEQUESTRATION AND PRODUCTION OF METAL ION LIGANDS IN PLANTS UNDER DIFFERENT ENVIRONMENTAL CONDITIONS Teresa W-M Fan1, Fabienne Baraud2, Andrew N. Lane3, and Richard M. Higashi4 ABSTRACT: Root exudation of metal ion ligands (MIL) is vital to nutritional acquisition of Fe and Zn, and may also be important to mobilization of metal contaminants by plants. We have developed a multidimensional NMR and GC-MS approach for broad profiling of exudate and tissue components including MIL. Amino and organic acids plus mugineic acid (MA) phytosiderophores were identified and quantified. SH-rich peptides were also analyzed using fluorescent probe and SDS-PAGE. The MIL profile differed among plants and genotypes. MA exudation by wheat and barley roots was induced by Fe deficiency, which is consistent with MA’s role in Fe acquisition. Cd treatments of wheat greatly reduced the exudation of MA and other MIL; yet transition metal sequestration into roots increased substantially. This suggests that metal uptake may be mediated by a different mechanism in Cd-contaminated rhizosphere. SH-rich peptides (i.e., phytochelatins) and other MIL accumulated greatly in Cd-treated wheat tissues, possibly related to the intracellular immobilization of Cd and transition metals. Moreover, co-treatment with soil humic substance (HS, an important extant rhizosphere ligand) alleviated Cd-induced loss of wheat biomass and root exudation, while causing a higher sequestration of Cd and transition metals into roots. This is contrary to HS role as a competitive chelator. Vascular plants are known to utilize two different strategies to mobilize Fe(III) from soils (Marschner, 1991). The “Strategy II” plants (graminaceous monocots) release powerful iron chelators into root exudates to complex Fe(III) for uptake. The iron chelators produced are termed phytosiderophores (PS), which consist mainly of mugineic acid and its derivatives, 2’-deoxymugineic acid (2’-DMA) and 3-epi-hydroxymugineic acid (3-epi-OHMA) (Kawai et al., 1988). In addition to Fe(III), PS also complex with Zn(II) and Cu(II) to facilitate their uptake by plants (Römheld, 1991; Hopkins et al., 1998) However, it is unclear whether PS are involved in the acquisition of heavy metal ions (e.g. Cd(II), Pb(II), Sr(II), Cs(II)) commonly found in soils contaminated via a variety of industrial, military and urban activities. Also uncertain is the involvement of exudate components other than PS in metal ion mobilization, primarily for lack of a comprehensive knowledge of root exudate composition. Moreover, once absorbed, it is largely unknown how these metal ions (usually toxic to plants) are translocated to shoots or sequestered inside root cells. Insights into these questions are crucial to the design and implementation of plant-based remediation of metal contamination from soils and sediments. Without prior knowledge, analysis of crude plant root exudates requires a profiling approach, for which we developed a combined nuclear magnetic resonance (NMR) spectroscopy and gas chromatographymass spectrometry (GC-MS) method. We have used this approach for a simultaneous determination of known, unexpected, and even unknown metal ion ligands (MIL), directly from crude exudates (Fan et al., 1997 & in press). With this tool in hand, it is now possible to examine various environmental and genetic factors that influence intra- and extra-cellular MIL profiles, and their role in metal ion acquisition. 1 Dept. of Land, Air & Water Resources, Univ. of California, One Shields Ave., Davis, CA 95616, USA ERPCB, Campus II - Sciences 2, Boulevard du Maréchal Juin, 14032 Caen Cedex, France 3 National Institute for Medical Research, Mill Hill, London NW7 1AA, UK 4 Crocker Nuclear Lab., Univ. of California, One Shields, Ave., Davis, CA 95616, USA 2 1 In this report, we analyzed the root exudation profile of several crop species as shown in Figure 1. It is clear that the exudation profile differed among plant species and genotypes. The barley exudate had 3epi-OHMA and MA as the dominant PS while wheat exudates were rich in 2’-DMA. The relative quantity of glycinebetaine (GB, an osmolyte), amino acids, and organic acids also differed between the two species. Rice exudate was very low in PS, which may account for its lower tolerance to Fe deficiency than barley and wheat. Since many of these exudate components can influence microbial activities, how species or genotypic differences in root exudate composition affect plant-microbe interactions and modulate metal sequestration is under study in our laboratory. Figure 1 – Root exudation profiles of barley, wheat, and rice analyzed by 1H NMR. Two wheat genotypes differing in salt tolerance were surveyed: salt-sensitive Chinese spring, CS and salt-tolerant amphiploid of CS and European saltgrass, AgCS. Tissue metabolite profiles were also analyzed by a combination of 1H NMR, GC-MS, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Example analysis of thiol-rich peptides in both plant and animal tissues is shown in Figure 2, where thiol-rich peptides were derivatized by fluorescent probe bromobimane (BrB) and separated by SDS-PAGE. Fluorescent bands of < 3.5 kDa were prominent in Cd-treated plant tissues (lanes 6, 8) while a fluorescent band of 17.7 kDa was related to a higher Cd content in the clam tissues (lanes 3, 4, 5). kDa 25.6 17.7 11.5 6.5 3.5 Figure 2 – SDS-PAGE of bromobimane-tagged SH-rich peptides and proteins from wheat, rice, and clam tissues. Lanes 1, MW standards; 2, rabbit metallothionein (MT); 35, clam MT extracts; 6, Cd-treated and 7, control wheat root extracts; 8, Cd-treated rice shoots. <3.5 Moreover, we examined the interactive effect of Cd(II) and soil humate (an important extant rhizosphere MIL) on root 8 7 6 5 4 3 2 1 exudation and internal metabolism in relation to metal acquisition by CS wheat. Wheat roots responded to Cd treatment by a large accumulation of Cd as well as decrease in growth (data not shown) and in major exudate components while transition metals accumulated to a higher level than under control conditions (e.g. Fig. 3). Thus, it is clear that these low molecular weight MIL including 2’-DMA were not responsible for the enhanced metal acquisition. This does not rule out the role of macromolecular exudate components (e.g. extracellular polysaccharides) in facilitating metal uptake. Coupling Cd with humic treatment partially alleviated Cd inhibition of root growth (data not shown) and exudation of 2’-DMA, Ala, and acetate while 2 R = OH, MA R = H, 2'-DMA 1 R = R'= OH, 3-epi-OH-MA acetate glycinebetaine COOH- H COO-H H COO1' 1" epi-OHMA MAs MES succinate Ala Gly lactate malate 2'-DMA GAB MA CS Wheat Exudate Barley Exudate C C R' 2 3 C N C 3' NH C 3" OH H C H 2' R H 2" H H H 4 MAs malate 2'-DMA Thr Val GAB malate Rice Exudate Gly glycinebetaine 2'-DMA succinate lactate Ala 2'-DMA 2'-DMA Leu Val Ile Glu AgCS Wheat Exudate MES lactate Unknown polyol acetate Unknown 2'-DMA 2'-DMA 5.0 4.5 4.0 3.5 1H 3.0 2.5 Chemical Shift (ppm) 2.0 1.5 1.0 5.0 2'-DMA 4.5 4.0 3.5 1H 3.0 2.5 Chemical Shift (ppm) 2.0 1.5 1.0 accumulation of Zn, Ni and Cd in roots was slightly enhanced (Fig. 3). These results suggest that soil humate can modulate both root exudation and internal metabolism. Exudate Ala vs Fe 1200 Exudate GAB vs Ni 150 60 100 800 Fe Ala 80 40 400 40 0 Ctl HS Cd 20 0 0 Ctl Cd+HS Exudate Acetate vs Zn HS 5000 Zn acetate 120 250 200 80 Cd Cd+HS Exudate 2'-DMA vs Cd 160 350 300 50 Cd 2'-DMA 200 4000 150 3000 150 100 2000 100 250 40 50 1000 50 0 0 Ctl HS Cd Cd+HS Exudate Malate vs Cu 0 0 Ctl HS Cd Cd+HS µmole MIL/g root exudate dry wt 0 Figure 3 – Comparison of exudate MIL concentration versus metal content in CS wheat roots under a combination of soil humate (HS, 5 ppm) and Cd (5 ppm) treatments. All MIL declined drastically in exudates while transition metals accumulated more in roots with Cd and Cd+HS treatments. The presence of 5 ppm Cd in growth media resulted in a nearly 1000-fold Cd accumulation in wheat roots. To understand the relation of humic-Cd treatment to intracellular MIL, we analyzed wheat tissue extracts for a number of metabolites including phytochelatins. Due to space limitation, only the result for phytochelatins is shown here in Fig. 4. It is interesting to note that the combined humic and Cd treatment led to a further increase in PC production in roots than Cd treatment alone. This enhancement should help immobolize bioavailable Cd to alleviate its inhibitory effect on growth, as observed in this study. 180 200 160 120 120 Cu malate 80 60 40 0 0 Ctl HS Cd Cd+HS 6000 Wheat Root 14 5000 12 4000 mg MT eq/g µg Cd/g root 10 8 3000 6 2000 µg Cd/g root mg MT equivalent/g root 16 4 1000 2 0 0 Ctl 0.5 Cd HS HS+Cd 180 Wheat Shoot 160 0.4 140 mg MT eq/g µg Cd/g shoot 0.3 120 100 80 0.2 60 40 0.1 20 µg Cd/g shoot mg MT equivalent/g shoot µg Metal/g root dry wt 80 Ni GAB 120 Figure 4 – Increase in Phytochelatin (PC) production in CS wheat tissues in response to humic and Cd treatments. PC was analyzed as in Fig. 2 and calibrated against rabbit metallothionein. Triplicate samples were analyzed per treatment. PC accumulated dramatically in wheat roots in response to Cd, which was accompanied by a large accumulation of Cd. Soil humic treatment further enhanced this PC production and, to a less extent, Cd accumulation in wheat roots. Both increases were proportionally much less in Cd-treated shoot, while humic-Cd treatment did not lead to further increase in shoot Cd. Thus, Cd was mainly sequestered in wheat roots, mostly likely by interacting with PC, and soil humate may enhance this process. 0 0.0 Ctl Cd HS HS+Cd In conclusion, root exudation varied with plant species, genotypes, and treatment conditions. Exudation of PS or common organic or amino acids did not appear to be related to the acquisition of Cd or Cd-induced acquisition of transition metals. Soil humate modulates both root exudation and internal root metabolism to help immobolize available Cd, thereby alleviating its inhibitory effect on growth. REFERENCES Marschner, H. in Plant roots : the hidden half; Waisel, Y.; Eshel, A.; Kafkafi, U., Eds; M. Dekker: New York, 1991, pp 503-526. Kawai, S.; Takagi, S.; Sato, Y. J. Plant Nutr. 1988, 11, 633-642. Römheld, V. Plant and Soil 1991, 130, 127-134. Hopkins, B. G.; Whitney, D. A.; Lamond, R. E.; Jolley, V. D. J. Plant Nutr. 1998, 21, 2623-2637. Fan, T. W. M.; Lane, A. N.; Pedler, J.; Crowley, D.; Higashi, R. M. Analyt. Biochem. 1997, 251, 57-68. Fan, T. W. M.; Lane, A.N.; Shenker, M., Bartley, J.P.; Crowley, D.; and Higashi, R.M. Phytochem., in press. 3 I II H+ reductant ? PS Soil Fe3+–PS ? Humic Fe2+ ? FeR Fe2+ Fe3+ ?