Intraspecific differences in heavy metal accumulaton, distribution and uptake kinetics in the metallophyte, Deschampsia caespitosa by Richard Stuart Cahoon A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Land Rehabilitation Montana State University © Copyright by Richard Stuart Cahoon (1983) Abstract: The objective of this research is the investigation of intraspecific differences in heavy metal accumulation and distribution in a higher plant species which is known to exhibit heavy metal tolerance. By studying isogenic lines of a higher plant species which differ only in their response to heavy metals, the fundamental nature of metal tolerance and ion transport in a biological system is elucidated. Two races of the grass, Deschampsia caespitosa were compared under a variety of metal-stress (Cu, Cd) conditions. One race, "Tailings", was collected on a metalliferous waste site. It exhibits heavy metal tolerance. The other race, "Agricultural", was grown from commercial seed and appeared to be heavy metal sensitive. Three experimental systems were used to compare races: (1) Sand-solution culture - a defined sand-nutrient solutipn technique was used to determine the influence of (a) race, (b) organ, (c) ion, (d) time and, (e) metal loading rate on tissue metal concentration. (2) Liquid-batch culture - was used to determine the accumulation and biochemical distribution of Cd109 in races of D. caespitosa as a function of time. (3) Macrophyte Reactor - reactor engineering principles were used to develop a novel technique for measuring ion uptake kinetics of tissues of higher plants. Using a continuous stirred tank reactor (CSTR) Cd uptake kinetics of the races were compared. These experiments lead to the conclusion that metal-tolerant "Tailings" plants accumulate less Cu or Cd than "Agricultural" plants. This difference is more significant with Cu than Cd. Acropetal (root to shoot) transport of Cu or Cd is much higher in "Agricultural" than "Tailings". Similar differences in Cd uptake kinetics (i.e., "Agricultural" greater than "Tailings") Were observed. Two hypotheses are posited to explain the phenomenon: "Dying Osmometer" theory - Accumulation and distribution differences can be explained on the basis of the death of "Agricultural" root cells in heavy metal solutions. "Tailings" cells do not die under these conditions. "Altered Carrier" theory - Racial differences in metal accumulation and distribution can be explained by the ion "carrier" model. Conformational alterations in specific ion "carrier" proteins in cell membranes result in ion uptake kinetic differences. Other biological mechanisms which may play a role in the observed intraspecific differences are discussed.  INTRASPECIFIC DIFFERENCES IN HEAVY METAL ACCUMULATION DISTRIBUTION AND UPTAKE KINETICS IN THE METALLOPHYTE, Deschampsia caespitosa by Richard Stuart Gaboon A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Land Rehabilitation • MONTANA STATE UNIVERSITY Bozeman, Montana December 1983. ii main lib. CIIcI Cop'^ APPROVAL of a thesis submitted by Richard Stuart Cahoon This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies. Chairperson, Graduate Committee Approved for the Major Department Date C ^ \ . Head, Major Department Approved for the College of Graduate Studies ZZ ■- Z -f2 Date Graduate Dean ill STATEMENT OF PERMISSION TO USE In presenting this.thesis in partial fulfillment of the requirements for a master's degree at Montana State University, I rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowl edgment of source is made. Permission for extensive quotation from or reproduction of this thesis may be granted by my major professor, or in his/her absence, by the Director of Libraries when, in the opinion of either the proposed use of the material is for scholarly purposes. Any copying or use of the material in this thesis for financial gain shall not be allowed without my written permission. agree that the Library shall make it available to borrowers under Date 1 2- iv Page INTRODUCTION................ .. . ........................... .. ! Research Goals 2 Research Objectives................................... . The Metallophyte Phenomenon .......... .................. Biosorption of Elements by Higher Plants ............ . Higher Plants and Heavy Metal Soil Ions. . . ........... Metal Tolerant Plants and Hyperaccumulation ........ Mechanisms.of Heavy Metal Ion Regulation by Metallophytes .................... 6 Significance of Research into Metal Tolerance and Hyperaccumulation by Metallophytes ............. 11 Biology. . . . . . . . . . . .......................... 11 Agriculture................■........................... 12 Environmental Engineering and Land Reclamation......... 13 Biogeochemical Prospecting ............................ 14 HEAVY METAL IONS AND HIGHER PLANTS..................... 15 Zinc. . ............................................. 15 Copper............................ . . ............ 17 . Lead......................... 18 Nickel.................. 18 Cadmium.................. 19 Aluminum. ........................................... 20 Maganese................................. 20 BIOLOGY OF ION TRANSPORT IN HIGHER PLANTS .................. 22 ^RESEARCH STRATEGY . . . ...................................... 29 General .................................................. 29 Experimental Species Deschampsia caespitosa .............. 31 Selection.......................... 31 Collection of Deschampsia caespitosa "TAILINGS"........ 33 Preparation of D. caespitosa "TAILINGS" and "AGRICULTURAL"..................................... 33 TABLE OF CONTENTS CN CO CO m XJD V TABLE OF CONTENTS--Continued Page Copper.................... 35 Cadmium............................................ '36 EXPERIMENTAL TECHNIQUES ...................................... 37 Sand-Solution Culture - Experiment I ................. 37 Batch Solution-Cd Isotope - Experiment II.............. 38 Macrophyte Reactor - Experiment III.................... 38 The Macrophyte Reactor: Theoretical Basis ............. 40 Statistical Analysis.................. 43 EXPERIMENTATION . '........................................... 45 Experiment I - Sand-Solution Culture..................... 45 Materials and Methods.................................. 45 Results and Discussion.................................... 47 "Tailings" vs "Agricultural" differences............ 54 Copper vs Cadmium differences ...................... 54 Root vs Shoot differences . ........................ 58 Loading rate differences......... 58 Harvest time differences............................ 62 Biochemical fractionation .......................... 64 Conclusions from Experiment I..................... 68 Experiment II - Cd Batch System........................ 69 Materials and Methods......................... 69 Results and Discussion.................... . . . . i . . . 70 Conclusions................................................. 87 Experiment III - "Macrophyte Reactor" - Cd uptake Kinetics. 88 Materials and Methods.......................... . . . 88 Results and Discussion................. 94 Conclusions............................................. 103 SUMMARY AND DISCUSSION............ 105 Summary of Conclusions............................. 105 Experiment I.................................... . . . 105 Experiment I I ...................................... 106 Experiment III...................................... 106 Discussion............................................ 107 "Dying Osmometer" Theory............ H O "Altered carrier" Theory .............................. Ill LITERATURE CITED....................................... 112 APPENDICES................................. 125 Appendix I - Analysis of Variance Table - Experiment I. . . 126 Appendix 2 - ANOVA Table - Experiment II.................. 127 vi Page Table I. Typical Concentrations of Mineral Elements in Foliage of Normal Plants (from Hewitt, 1975)........ 4 Table 2. Elements Hyperaccumulated by Plant Species.......... 7 Table 3. Data from analysis of tailings material collected at Deschampsia caespitosa collection site at Anaconda, MT. All elements in yg ml of a cold- water extraction of saturated paste . .............. 32 Table 4. Concentrations of metals in samples of D. caespitosa collected in October 1981 on the Anaconda Reduction Works tailings ponds....................... 32 Table 5. Modified IX Hoagland,.and Arnon nutrient solution used in heavy metal uptake experiments. .............. 34 Table 6. Concentration of Cd in tissues of races of Deschampsia caespitosa grown in acid-washed sand-nutrient solution culture...................................... 48 Table 7; Concentration of Cu in tissues of races of Deschampsia ■ caespitosa grown in acid-washed sand-nutrient solution culture..................................... 49 Table 8. T-statistic of comparisons of means of metal concentra­ tions in tissues of races of Deschampsia caespitosa . 55 Table 9. Analysis of tissue metal concentrations as a function of metal loading rate-comparison of two races ................................... 59 Table 10. T-statistic of "Tailings" ("T") vs "Agricultural ("A") race comparisons at different loading rates . . 59 Table 11. Average metal concentration of both D. caespitosa races, whole plant; all loading rates,, over time. . . LIST OF TABLES 62 vii 12. Cu and Cd concentrations in "wall" and "cytopolasmic fractions of races of D. caespitosa groxm in sand- solution culture with applied metals............ 65 Table 13. Concentration of Cd in plant tissue of "Tailings" and 1 Agricultural" races of p. cae$pifcosa^grown in aerated, batch culture spiked with Cd ........ 71 Table 14. Comparisons of groups using paired-t test . . . . . . 76 Table 15. Average Cd concentration (yg g-1) in biochemical fractions of D. caespitosa grown in batch culture spiked with Cd"1" .......................... 79 Table 16. Percent distribution of Cd in fractions, over time, of two races of D. caespitosa-grown in aerated, batch culture spiked with Cd1 .................... 85 Table 17. Comparisons of percent Cd distribution in fractions GfigJssuM-of D. caespitosa races grown in batch, Cd solution culture. ............................ 86 Table 18. Comparison of specific Cd uptake rates for whole plant, root, and shoot tissues of two races of D. caespitosa using t-test.......................... 94 Table 19. Net specific Cd accumulation rates (k .) of two races of D. caespitosa grown in continuous-flow solution culture........................................... 95 Table 20. Relative growth rate of root and shoot of Agricul- turla and Tailings races of D. caespitosa grown in continuous stirred tank reactor........... 102 LIST OF TABLES— Continued Page viii Page Figure I. Schematic diagram of higher plant root ion transport system.............................................. 23 Figure 2. Schematic diagram of theoretical continuous stirred tank macrophyte reactor (CSTMR) .................. . 41 Figure 3. Concentration of Cu in ROOTS of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in sand-solution culture at three Cu loading rates . . . 50 Figure 4. Concentration of Cu in SHOOTS of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in sandsolution culture at three Cu loading rates. . 51 Figure 5. Concentration of Cd in ROOTS of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in sandsolution culture at three Cd loading rates . . . 52 Figure 6. Concentration of Cd in SHOOTS of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in sandsolution culture at three Cd loading rates . . . 53 Figure 7. Average concentration of Cu in tissues of "Agri­ cultural" (A) and "Tailings" (T) races of D. caespitosa as a function of Cu loading rate ........ 60 Figure 8. Average concentration of Cd in tissues of "Agri­ cultural" (A) and "Tailings" (T) races of D. caespitosa as a function of Cd loading rate . . . . . 6.1 Figure 9. Percent distribution of total Cu and Cd in tissues of "Agricultural" (A) and "Tailings" (T) plants grown in sand-solution culture................................67 Figure 10. Average concentration of Cd in root and shoot "WALL" fractions over time of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in aerated, liquid batch culture spiked with Cd-1 .............. 72 LIST OF FIGURES ix LIST OF FIGURES— Continued Page Figure 11. Average concentration of Cd in root and shoot "CYTOPLASMIC" fractions over timfe of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa Sr5$i in aerated» liquid batch culture spiked with Figure 12. Average concentration of Cd in root and shoot "MEMBRANE" fractions over time of "Agricultural" (A) and "Tailings) (T) races of D. caespitosa grown in aerated, liquid batch culture spiked with Cd109 Figure 13. Average per cent distribution of total ROOT Cd in cellular fractions of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa (n=2) liquid batch culture spiked with Cd1 ............ Figure 14. Average per cent distribution of total SHOOT Cd in cellular fractions of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in- aerated, liquid batch culture spiked with Cd1 . . Figure 15. Average ROOT/SHOOT Cd distribution (as per cent of total Cd accumulated) in "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in aerated, liquid batch culture spiked with Cd^ . . Figure 16. Average ROOT/SHOOT Cd distribution (Root Cd con­ centration/ shoot Cd concentration) over time in "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown integrated, liquid batch culture spiked with Cd ................ Figure 17. Diagram of continuous stirred tank macrophyte reactor (CSTMR) used to evaluate Cd uptake kinetics of D. caespitosa . . . . . . . . . . . . Figure 18. Diagram of one of six flasks in the continuous stirred tank macrophyte reactor (CSTMR) .......... Figure 19. Theoretical and experimental dilution curve of continuous stirred tank reactor (CSTMR) for a conserved substance pulsed into system............ Figure 20. Daily pH measurements of effluent stream from continuous stirred tank.macrophyte reactor (CSTMR) containing either "Agricultural" (A) or "TAILINGS" (T) races of D. caespitosa. . . . . . . . . . . . 73 . 74 81 . 82 83 84 89 90 92 97 X LIST OF FIGURES— Continued Page Figure 21. Daily net specific Cd uptake rate for ROOT tissues (k ^ ROOT [=] Ug Cd g tissue min ) of "Agricultural" ana "Tailings" races of D. caespitosa grown separately in continuous stirred tank macrophyte reactor (SCTMR) . .......................................... 98 Figure 22. Volume displacement of ROOTS, SHOOTS, and WHOLE PLANT of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in continuous stirred tank macrophyte reactor (CSTMR) . . . . . . . . . . . 100 « ' I Figure 23. Relative growth rate ( — . ) of ROOTS and SHOOTS of "Agricultural" (A) and "Tailings (T) races of D. caespitosa grown in continuous culture (CSTMR) contining 0.25 X Arnon^and Hoagland*s solution spiked with 1.0 US ml Cd as CdNO^ ................ 101 xi Abstract ■ The objective of this research is the investigation of intraspecific differences in heavy metal accumulation and distribution in a higher pl^nt species which is known to exhibit heavy metal tolerance. By studying isogenic lines of a higher plant species which differ only in their response to heavy metals, the fundamental nature of metal tol­ erance and ion transport in a biological system is elucidated. Two races, of the grass, Deschampsia caespitosa were compared under a variety of metal-stress (Cu, Cd) conditions. One race, "Tailings", was collected on a metalliferous waste site. It exhibits heavy metal tolerance. The other race, "Agricultural", was grown from commercial seed and appeared to be heavy metal sensitive. Three experimental systems were used to compare races: (1) Sand-solution culture - a defined sand-nutrient, solutipn technique was used to determine the influence of (a) race, (b) organ, (c) ion, (d) time and, (e) metal loading rate on tissue metal concentration. (2) Liquid-batch culture - was used to-determine the accumulation and biochemical distribution of Cd1 y in races of D. caespitosa as a function of time. (3) Macrophyte Reactor - reactor engineering principles were used to develop a novel technique for measuring ion uptake kinetics of tissues of higher plants. Using a continuous - stirred tank reactor (CSTR) Cd uptake kinetics of the races were compared. These experiments lead to the conclusion that metal-tolerant "Tailings" plants accumulate less Cu or Cd than "Agricultural" plants, this difference is more significant with Cu than Cd. Acropetal (root to shoot) transport of Cu or Cd is much higher in "Agricultural" than "Tailings". Similar differences in Cd uptake kinetics (i.e. , "Agri­ cultural" greater than "Tailings") Were observed. Two hypotheses are posited to explain the phenomenon: "Dying Osmometer" theory - Accumulation and distribution differences can be explained on the basis of the death of "Agricultural" root cells in heavy metal solutions. "Tailings" cells do not die under these conditions. "Altered Carrier" theory - Racial differences in metal accumulation and distribution can be explained by the ion "carrier" model. Conforma­ tional alterations in specific ion "carrier" proteins in cell membranes result in ion uptake kinetic differences. Other biological mechanism^ which may play a role in the observed intraspecific differences are discussed. I INTRODUCTION Increasingly sophisticated methods of detection as well .as heightened environmental awareness have led to the realization that heavy metal ions pose a serious environmental problem. These heavy metal ions, toxic to most biologic systems in relatively small concentrations, appear to be an ubiquitous by-product of a tech­ nological society. Industrial process effluents, atmospheric emissions, urban runoff, municipal sewage and wastewaters, mining and large scale construction projects are all associated with heavy metal contamination of soil, air and water (Bradshaw & Chadwick, 1981; Buchauer, 1973; Burkitt et al., 1972; Lagerwerff & Specht, 1970). Heavy metal contamination by anthropogenic activity has given rise to an intriguing phenomenon: resilient biological systems which have adapted to these contaminants. The existence of metallophytes, higher plants which exhibit the capacity to tolerate/accumulate normally toxic heavy metal concentrations in the environment, provides an opportunity to investigate the nature of the mechanisms of ion uptake in plants and the nature of tolerance to soil ionic stresses. Tolerant species often "hyperaccumulate" essential and nonessential elements providing more research questions of biologic, environmental, and 2 agricultural importance. This study seeks to characterize the nature of the metallophyte phenomenon via physiological and bio- ■ chemical comparisons of metal tolerant and metal sensitive ecotypes of a single species under metal stress conditions. This research also develops several techniques for the analysis of ion accumula­ tion by higher plants. These techniques include a novel reactor design capable of yielding information on ion uptake kinetics by higher plants. Research Goals The goals of this research are several fold: i (1) To determine whether intraspecific differences in heavy metal ion accumulation and transport exist in a known higher metallophyte species. (2) To characterize those differences if they exist. (3) To establish a data base for further research into the nature and possible technological application of metallophytism in higher plants. (4) To determine the applicability of chemical engineering principles to the evaluation and exploitation of higher plant physiological phenomena. To attain these goals, the following research objectives have been established: Research Objectives (1) To evaluate intraspecific differences in heavy metal accumulation and transport with traditional experimental techniques of sand-solution culture. (2) To evaluate possible intraspecific biochemical dif­ ference in metal accumulation and transport using radioisotope techniques. (3) To develop a technique to measure metal ion uptake kinetics in a higher plant and to use the method to evaluate possible differences between physiological races of the selected experimental species. 3 The Metallophyte Phenomenon Biosorption of Elements by Higher Plants The concept of plant uptake of nutrients from the soil by roots is just over three hundred years old.. Prior to the published work of Glauber (1656), in which he stated that saltpetre was taken from manure by plants, water and air were considered the source of plant constituents. In subsequent experiments de Saussure (1804) unequivocally demonstrated that plants take up a portion of their constituents from the soil. For the next fifty years, workers like Boussingault and Liebig continued to develop the science of plant nutrient uptake. During this period, confusion arose over the molecular nature of the nutrients taken up from the soil by the plant. The development of nutrient solution culture techniques in the 1860's confirmed that, with rare exception, (HBO3, MoO^, HMoO^), elements were taken up by plants in ionic form. This technical development also lead to the search for essential micronutrients and to the distinction between biologically active vs. passive ion uptake. In the 1950 s, American workers developed purification techniques that allowed them to demonstrate that chlorine is an essential plant nutrient. In so doing, they completed a list of ions which are considered as essential for higher plant life. The list of 4 ions which are essential to all higher plants is comprised of C, 0, H, P, K, Ns S, Ca, Fe, Mg, Mn, Cu, Mo, Zn, B, Cl (Steward, 1963). Elements which are not required by all plants but which are essential to particular species: Co, I, V, Na, Se, Al, Si, Cr, F, (Mortvedt et al., 1972). Higher plants tend to absorb all elements present in root zone -O' (rhizosphere) solution but exhibit uptake rate selectivity (Hewitt, 1975) (Table I). Table I. Typical Concentrations of Mineral Elements in Foliage of Normal Plants (from Hewitt, 1975) Element PPM Dry Matter Total mM in Cells Nutrient Solution (mM) N ■ 15,000-35,000 150-350 15.0P 1,500-3,000 7-14 1.0S 1,000-3,000 4.5-140 1.5Ca 10,000-50,000 35-175 5.0Mg 2,500-10,000 15-60 1.5K 15,000-50,000 55-180 5.0Na 200-2,000 1-12 1.0Fe 50-300 0.15-0.75 0.1Mn 25-250 0.06-0.6 0.01Cu 5-15 0.01-0.03 0.001Zn 15-75 0.03-0.15 0.002Co 0.2-29 0.005-0.05 0.0002B 15-100 0.2-1.3 0.05Mo 0.5-5 0.004-0.075 0.0005Cl 100-1,000 0.4-4 0.1 As research data gathers on nutrient sorption, the evidence increasingly points towards wide species variation in selectivity of quantity and type of ion taken up (Rorison et al., 1968). Furthermore, workers have shown ion uptake variations within species. Earley (1943) demonstrated varietal differences in rate of Zn 5 uptake by soybean cultivars. This intraspecific variation suggests a genetic basis of ion uptake selectivity. This subject is reviewed by Epstein and Jeffries (1964) and is thoroughly discussed in Epstein's pioneering work on the biochemical genetics of nutrient uptake by higher plants (Epstein, 1972). The intraspecific variation in mineral nutrition of plants from different habitats shown by Goodman (1968) with Lolium perenne may have its basis in mechanisms similar to those elucidated by Woolhouse (1968), Jeffries et al. (1968) and Brown and Jones (1979). These workers describe the alteration of molecular ion transport mechanisms by ecotypes in order to adapt to particular ionic regimes in the rhizosphere. Higher Plants and Heavy Metal Soil Ions Higher plant species characteristic of metal contaminated soils have been recognized for several centuries. Thalius, in 1588, noted Minuartia verna as a metal indicator (Ernst, ]965)i Others (Williams, 1830; Henwood, 1857; Baumann, 1885) mention species found consistently on metal contaminated soils. Studies of "metallophytes" (plants which tolerate/accumulate inimical metal ions) remained descriptive until the work of Prat (1934), in which he demonstrated the physiological differences between metalliferous mine colonizing and non-mine colonizing populations of Melandrium silvestre. This study was unique until the 1950's when workers in the United Kingdom and Germany began studying higher plants found growing on areas contaminated with heavy metals due to mining disturbance. Bradshaw (1952) described lead and zinc tolerant 6 populations of Agrostis tenuis. Later, Wilkins (1957, 1960) used rooting techniques to show tolerance of mine populations of Festucs ovina to lead. Workers in Germany (Schwanitz and Hahn, 1954; Repp, 1963; Baumeister, 1954; Baumeister & Brughardt, 1956; Wachsmann, Broker, 1963) added to the list of metal tolerant species. Met.al Tolerant Plants and Hyperaccumulation. A large body of literature exists which describes the use of metallophytes as tools of geologic prospecting (Wild, 1970; Cole, 1965; Cannon, 1957, 1960). These "geobotanical indicator" species, as well as other metallophytes, exhibit an interesting biological phenomenon; they appear to extract large amounts of certain elements from the soil (Table 2). The kind and quantity of ion accumulated depends upon plant species and soil conditions. These species accumulate tissue concentrations of ions many times greater than those found in the soil solution and often in excess of levels considered adequate for biological function. Table 2, though not exhaustive, has been broken down into two categories: (I) hyperaccumulation of essential elements (2) hyperaccumulation of non-essential elements and.compounds The hyperaccumulation of essential elements by biological systems is, perhaps, to be expected. However, this selective accumulation of nonessential elements and compounds poses intriguing questions. Mechanisms of Heavy Metal Ion Regulation by Metallophytes Critical concentrations of heavy metal ions may interfere with biological systems via several actions: (I) competition with essential elements for uptake or for O 7 t Table 2. Elements Hyperaccumulated by Plant Species ION PLANT S P E C I E S ________ REFERENCE Zn * Agrostis tenuis Silene inflate Thlaspi alpestre Turner & Marshall, 1972 Baumeister, 1967 Ernst, 1968 Cu * Agrostis alba Melandrium sglvestre Minuartia verna Agrostis tenuis Aeolanthus biformifolius Prat & Komarek, 1934 Prat & Komarek, 1934 Ernst, 1968 . Bradshaw et al., 1965 Malaisse et al.; 1978 Mn * Caultheria hispidula Vaccinium myrtilloides Triticum aestivum Glycine max Lactuca sativa Gerloff et al., 1966 Gerloff et al., 1966 ’ Foy et al., 1973 Heenan & Carter, 1976 Sonneveldt & Voogt, 1975 Mo * Fabaceae sp. Lycopersicon esculentum Fabaceae sp. Dye & O'Hara, 1959 Johnson, 1966 Barshad, 1948 B * various species Oertli & Kohl, 1961 Cook 1916 Co * Haumaniastrum robertii Pimelea suteri Nyssa'sylvatica Morrison et al., 1979 Lyon et al., 1968 Beeson et al., 1955 Cl * Agrostis tenuis Atriplex pelycarpa Cordukes & Parups, 1971 ■ Chatterton et al., 1970 Cd Pinus taeda Liriodendron tulipifera Lactuca sativa Raphanus sativus Apim graveolens Hordeum vulgare Kelly et al., 1979 Kelly et al., 1979 Haghiri, 1973 Haghiri, 1973 Haghiri, 1973 Cutler & Rains, 1974 Se Astragalus bisulcatus Astragalus sp. Shrift, 1969 Robinson & Edgington, 1945 Si Equisitaceae sp. Robinson & Edgington, 1945 F Lycopersicon sp. Rosa sp. Festuca rubra Gurirtsmon et al., 1957 Gurirtsmon et al., 1957 Johnson et al., 1976 Cr Pimelea suteri Lyon et al.., 1965 8 Table 2. (cont.) ION PLANT SPECIES REFERENCE Sr Ulmus sp. Atriplex sp. Vaneslow5 1966 Wallace et al., 19.72 Pb Lolium perenne Pinus taeda Hordeum vulgare Raphanus sativus Jarvis et al., 1977 Roffe, 1973 Dowdy & Larson, 1975 Alloway5 1968 Ni Alyssum serpyllifolium Alyssum bertolonii Pearsonia metallifera Brooks et al.5 1981 Lee et al., 1978 Lee et al., 1978 As Agrostis tenuis Porter & Peterson5 1977 Al Camelia sp. Sporobolus caperesis Pinus sp. Matsumoto et al., 1976 Moomaw et al., 1959 Suchting5 1948 Ba Brazil nut Wagner, 1936 I Spinacea oleracea Brassica sp. Beeson5 1941 Beeson5 1941 V Various species Beath5 1943 Ag Eriogonum ovalifolium Henwood, 1857 Hg Alsine setaceae Linstow5 1929 Sn Trientalis europeae Linstow5 1929 Ra Bertholetia excelsa Hewitt5 1975 Li Thalictrum sp. Hewitt5 1975 Br Cucurbitaceae sp. Hewitt5 1975 U Sarcobatus sp. Hewitt5 1975 Au Equisetum palustre Hewitt, 1975 Rare Earths Castanea sp. Carya sp. Milton et al., 1944 Robinson & Edgington5 1945 Polychloro- biphenyls Spartina alternivolia IMrozek et al., 1982 essential elements 9 functional Sites5 competitive inhibition (2) inactivation of enzymes due to irreversible binding or denaturation non-competitive inhibition (3) alteration of nucleic acid organization (4) . alteration of membrane structure (5) alteration of various macromolecular structures (e.g., spindle apparatus) (6) alteration of cytoplasmic water structure (7) alteration of cytoplasmic colloid structure Those species which exhibit hyperaccumulation of particular ions (Table 2) must, obviously, avoid these deleterious effects. Higher plants have several possible tactics at their disposal with which to deal with elevated metal concentrations. These tactics can be divided into two types: external and internal. External - mechanisms which affect the adsorption and absorption of ions onto the root surface. These mechanisms involve alteration of the chemistry of the rhizosphere in order to make ions more or less available. Foy et al. (1978) have demonstrated that crop species alter the rhizosphere pH, rendering Al ions insoluble and thus unavailable for uptake by the plant. Levitt (1980) terms this, "metal ion stress avoidance". Ernst (1976) states that there is no evidence that higher plants can exclude ions in the soil solution, though exclusion has been demonstrated in unicellular Chlprella (Foster, 1977). Internal -mechanisms which effectively reduce toxic cellular levels of heavy metals or otherwise mitigate their toxic effects upon 10 cellular components. A list of these theoretically possible mechanisms and the experimental evidence, or lack thereof, which supports or fails to support their validity follows: (1) Differential uptake of ion - demonstrated in fungi (Okamoto and Euwa, 1974), algae (Foster, 1977) and higher plants (Ernst, 1972) (2) Removal of ion via deposition — in cell walls; demonstrated for Zn, Cu, Pb, Al (Ernst, 1972; Peterson, 1969; Turner & Marshall, 1972; Malone et al., 1974). In vacuoles; not demonstrated for heavy metals, however evidence for depo­ sition in vacuole by halophytic species (Greenway & Munns, 1980). (3) Removal of ion via extrusion - no evidence for this mech ism in metallophytes; demonstrated in halophytes (Greenway & Munns, 1980). (4) Chelation or complexation of ion by organic compound - demon­ strated in higher plants for Cu (Rauser & Curvetto, 1980), Ni (Thompson & . Tiffin, 1974), Zn (Rauser, 1981), Cd (Wagner & Trotter, 1982). (5) Selective translocation to non-vital areas of the plant - demon­ strated in higher plants for Al (Sivasabramaniam ,&" Talibudeen, 1972), Ni (Gambi, 1967), Pb (Roffe, 1973), Cu (Reilly & ' Reilly, 1973; Bradshaw et al., 1965), Zn (Turner & ' Marshall, 1972). (6) Alternate metabolic pathways which by-pass inhibited enzymes - not experimentally verified in higher plants. (7) Increased production of inhibited enzyme(s) - not experi­ mentally verified. . (8) Production of organic antagonist - not experimentally verified,. however, Mathys (1975) has shown a correlation between metal ion tolerance and production of glycosides in Cruciferae. (9) Decreased requirements for products of inhibited system - not experimentally verified. (10) Production of altered enzyme such that enzyme is rendered insensitive to metal ion effects - in-vivo activity of some enzymes of Cu tolerant ecotypes is less inhibited by elevated solution copper than enzymes of Cu-sensitive (Mathys, 1975); stages of the TCA (Krebs) cycle of Cu tolerant plants are not inhibited to the same extent as in Cu sensitive (Ernst, O 11 1976); root acid phosphatases of Cu-tolerant ecotypes are less sensitive to Cu.ions than non-tolerant (Woolhouse, 1970). (11) Increased uptake of H„0t increased growth, dilution of ion - not experimentally verified. Significance of Research into Metal Tolerance and Hyperaccumulation by Metallophytes . Investigations into the nature of the metallophyte phenomenon are an important and fertile area df: research. These studies have implications for various fields: Biology The bulk of evidence for the elucidation of the process of organic evolution is based upon phylogenetic studies of fossil records. However,some biological systems have exhibited, evolutionary development over relatively short time periods. A classic example of this process is the development of "industrial melanism" in lepidopterous species near factories in the United Kingdom. The unique significance of soil chemical composition as a factor in the selective complex of plant evolution is based upon the fact that there is, at the membrane surfaces of root cells bounding the apoplast, a direct confrontation between the environment (i.e., soil ions) and the structural and catalytic components of the biological system. Upon radicle extension into the soil, there exists an interface of protein-mediated activity at the plasmalemma with the external environment. As a result, the study of biochemical and physiological adaptation to excess metal concentrations in the soil and the plant offers a unique opportunity to investigate the process of plant evolution. 12 Given the genetic basis for the phenomenon of heavy metal tolerance and accumulation in higher plants, it seems logical to explore the mechanisms by which "metallophytes" exhibit these characters. The characterization of the ecological, physiological, and biochemical basis of the phenomenon presents the possibility of integrating those factors involved in genome-environment interaction. Investigations into heavy metal uptake by metallophytes can provide insight into the basic biological processes of ion uptake. These processes can be characterized via the physiological and biochemical comparison of metal sensitive and metal tolerant, as well as metal accumulating and non-accumulating populations of the same species. This approach can provide data to elucidate the biochemical and genetic nuance of ion uptake, translocation, and assimilation. Agriculture Studies of metallophytic mechanisms provide a basis for a more thorough understanding of ion uptake. Such studies will lead to a more thorough understanding of the relationship of plants and soil chemistry. Such an understanding of the nature of ion-related physiology may lead to a refinement of current agricultural technology. Describing the molecular nature of metallophytic mechanisms will lead to the development of plant species which are suited to less favorable soil environments. This concept runs counter to the traditional approach of manipulating the environment via irrigation, fertilization, amendments, etc. to suit the plant. By investigating the nature of 13 the mechanisms by which higher plants tolerate and accumulate potentially inimical ions in the rhizosphere, the foundation will be laid for the use of crop species which ameliorate their own soil environment rather than relying upon traditional methods of soil manipulation. This concept, discussed in some recent literature (Christiansen and Lewis, 1982; Wright, 1976) provides much opportunity for research and could revolutionize agricultural science, leading to developments in the manner in which we use technology in agricultural systems. Environmental Engineering and Land Reclamation The prospect of developing a technology wherein selected higher plant metallophytes are utilized to ameliorate environmental contamination problems is a distinct possibility. Metallophyte species have already proven useful in the reclamation of metalliferous waste areas (Gadgil, 1968; Johnson et al., 1977; Smith and Bradshaw, 1970). The use of higher plants in biosorptive water treatment systems (Bouwer and Chaney, 1974) suggests that metallophytes could be utilized to ameliorate metal contamination of mine drainage, urban runoff, and industrial effluents, etc. The demonstrated ability of higher plants to accumulate organic contaminants from soils (Mrozek et al., 1982) and to metabolize these compounds into benign substances (Harborne, 1977), suggests the possibility of utilizing selected species to reclaim contaminated land and to cleanup some industrial effluents and toxic soil residues remaining after hazardous material spills. Biogeochemlcal Prospecting The use of metallophytic species as indicators of ore bodies is not a novel practice. Agricola in his "Be re Metallica" of 1556, described the anomalous growth and coloration of plants associated with veins and outcrops of metal ores. This concept, though not thoroughly developed in this country (Cannon, 1957, 1960), has been utilized extensively in Australia, New Zealand, South Africa, and the Soviet Union (Brooks, 1972). Recent advances in the field of biogeochemical prospecting utilize aerial photography and other remote sensing techniques to identify areas of metal accumulation. These techniques often rely upon the unique spectral reflectance of metallophytes under conditions of metal hyperaccumulation (Homer et al., 1980). 15 HEAVY METAL IONS AND HIGHER PLANTS The heavy metals as defined by Passow et alt (1961), comprise . thirty-eight elements. All these elements share one thing in common, they are all toxic to biological systems at relatively low concentrations. Fortunately not all of the thirty-eight elements exist in concentrations that pose environmental health hazards. In fact, increasingly sophisticated analytical techniques have revealed that of the thirty-eight heavy metals, relatively few exist at critical levels in soil or water; the elements of concern, and those emphasized in this report are Cu, Zn, Pb, Ni, Cd, Al, Mn. Zinc - This element, an essential nutrient, is required in small quantities. Its presence is required in several enzyme systems (Clarkson & Hanson, 1980). Because of its small size, Zn tends I to form tetrahedral complexes. For unknown reasons, the plant cell has need for stable metalloenzyme complexes in.which the coordination is tetrahedral, thus, Zn.best fits this need. Zinc appears to play the role of enzymatic cofactor and is absolutely essential for various metabolic pathways, i.e., the synthesis of tryptophan. All plants contain some level of Zn. The occurence of high Zn concentrations in plant tissues grown on Zn-contaminated soils was first established in the 1800's. More 16 recent work on the phenomenon of Zn accumulation has revealed the following: (1) Different species, growing in the same Zn-contaminated soil, differ in the degree to which they take up Zn • (Ernst, 1965). (2) Different plant organs accumulate different quantities of Zn. Generally, roots accumulate most of the Zn. In some species, however, the inflorescence seems to be the prime accumulating organ. While most Zn-accumulators retain the largest portion of Zn in root tissues, the pattern of distribution depends upon the species (Nicolls et al., 1965; Cole et al., 1968). (3) The quantity of Zn in plant tissues varies with change in season and often shows an increase as season progresses, Zn uptake being correlated with growth (Ernst, 1965). (4) Levels of Zn in tissues vary within species, implicating genetic control.of uptake (White, 1976;. (5) No experimental evidence exists for an active Zn-carrier. (6) Uptake of Zn appears to be independent of metabolic activity as it is unaffected by metabolic inhibitors and temperature; dead tissues take up more Zn than living tissue (Rathore et al., 1970); However, subsequent trans­ location does seem to be metabolically dependent (Chandel & Saxena, 1980). (7) Zinc is involved in cell wall synthesis (Hewitt et al., 1954); Zn accumulating species appear to incorporate Zn into the cejLjL wall matrix' (Turner & Marshall, 1971, 1972). The amount of Zn incorporated in the cell wall is corre­ lated with tolerance of high Zn levels in the soil by the plant (Petersong ,-1969) ! Zn tolerant Agrostis tenuis accumulate more cell wall Zn than Zn sensitive A. tenuis. Furthermore, the amount of Zn in the shoot/amount of Zn in the root (shoot/root ratio) is highest in Zn tolerant species. The greatest proportion of Zn taken up is incorporated in the cell walls of the root and at stem nodes (Peterson, 1969). (8) Low CEC (cation exchange capacity) of roots of tolerant species appears to significantly reduce Zn absorption rate (Ernst, 1972a). 17 (9) Tplerance to high soil Zn can be conferred by way of reciprocal grafts of root and shoot tissue (White, 1976). Similar results have been found with uptake of B (Brown & Jones, 1971). I _ • Copper - This element, an essential nutrient, is a bound moiety in many redox enzymes. The role of Cu as a strong competitor for ligands makes it vital for much enzymatic activity and toxic in any but low concentrations. Investigations into the aspects of Cu accumulation have not received the attention that has accompanied Zn uptake. Workers have reported the following findings on Cu accumulation: (1) Species grown on identical soil Cu concentrations, differ in the quantity of copper which they accumulate in their tissues (Bateman & Wells, 1917). (2) Copper uptake mechanisms are apparently different from Zn uptake mechanisms (Peterson, 1969). (3) Copper levels in aerial parts remain constant with increasing soil copper concentration until a "threshold" concentration is reached at which time the Cu content in above-ground parts rises dramatically. Species differ in the level at which this "threshold" uptake occurs (Nicolls et al., 1965). (4) Roots consistently contain more Cu than aerial parts (Ernst, 1968). (5) In solution culture, tolerant and non-tolerant races of Agrostis tenuis exhibit very slight increases in shoot tissue Cu with increases in soil Cu concentration. Roots, however, show highly significant increases , (Bradshaw et al., 1965). (6) Species show a remarkable ability to "hyperaccumulate" tissue Cu from low soil Cu concentrations (Bradshaw et al., 1965). (7) Cu appears to be translocated via a specific chelatot (Thompson & Tiffin, 1974). 18 (8) Copper is selectively translocated from sensitive roots . to tolerant aerial parts in Becium homblei (Reilly & Reilly, 1973). (9) High copper concentrations (16 micromoles CuSO,) in nutrient solution apparently triggers the production of copper- thioneins (low molecular weight proteins that bind metals in mercaptide complexes (Rauser & Curvetto, 1980) in copper tolerant Agrostis gigantea (see also Morrison et al., 1979; Malaisse & Gregoire, 1978). Lfad - This element exhibits no known function in plant cells. Not being an essential element, its pattern of uptake should differ sub­ stantially from the previously discussed metals. Although Pb contamination is an environmental problem, little work has been done to characterize its uptake by higher plants. A review of this literature reveals: (1) The pattern of Pb uptake resembles that of Cu, initially static tissue levels in aerial parts with increasing soil concentration and subsequent rapid uptake at a threshold level (Nicolls et al., 1963); (2) Uptake of Pb is not influenced by metabolic inhibitors or low temperatures but is dependent upon soil pH (in Phaseolus, Zea, Glycine) Arvik & Zimdahl, 1974); (3) Plant species show variability in degree of Pb uptake as well as in localization of deposition (Rolfe, 1973). (4) Pb which is taken into the cell is concentrated in the dictyosomes (of corn root cells); lead appears to be subsequently incorporated into the cell wall matrix (Malone et al., 1974); (5) Roots typically accumulate more Pb than shoots (in Lolium perenne) (Jarvis et al., 1977). Nickel - A review of the literature reveals scant details on the nature of Ni uptake. Wild (1970) has demonstrated that the pattern of uptake of Ni resembles that of Cu. Similarly, some reports indicate that Ni "hyperaccumulators" exist (Antpnovics & Bradshaw, 1971). The following details of Ni uptake have been reported: (1) Ni appears to accumulate in the epidermis of leaves and in the schlerenchyma between vascular bundles (in Alyssum bertoloni, a Ni accumulator) (Gambi, 1967). (2) Ni is translocated via a specific chelator (Thompson & Tiffin, 1974). Cadmium - This element has received attention because of its toxic effects in low concentrations and accumulation in the food chain. Cd has no known role in metabolic function. However, the accumulation of Cd by higher plants has been observed by several workers. Some crop species appear to be such "hyperaccumulators" of Cd that health concerns have been voiced. This is particularly evident in the use of municipal sewage sludges as fertilizer (Hinesly et al., 1978; Turner, 1973). The following details of Cd accumulation have been published: (1) The pattern of Cd uptake is similar to Pb. Initial uptake is via diffusion and exchange adsorption; subsequent translocation and sequestering involves metabolic activity (Cutler & Rains, 1974); (2) There exists no experimental evidence for an active carrier of Cd; (3) Cd concentrations (in soybean cultivars) is in the order stems > leaves > pods > beans (Haghiri, 1973); (4) Cd is accumulated against a concentration gradient (in Barley) and is retained primarily in roots and the lower stem. Cd appears to be "irreversibly sequestered" once taken up by the cell (Cutler & Rains, 1974). Dead root tissues accumulate more Cd than living tissue (Jarvis et al., 1977); (5) Cd accumulation is under genetic control (Hinesly et al., 1978); (6) Species differ in the degree to. which they take up Cd (Haghiri, 1973; Miles & Parker, 1979). 20 (7) All species tested exhibit increased tissue Cd levels with increasing soil Cd concentrations; uptake rate varying between species (Kelly et al., 1979). Aluminum - This element is not an essential plant nutrient. Aluminum toxicity is rare in temperate climates but common in tropical soils. The work by Foy et al. (1978) is tangentially related to metal uptake in that it involves plant mechanisms which inhibit Al uptake. These avoidance mechanisms exhibit a unique survival tactic, manipulation of the chemistry of the rhizosphere by the plant root. Olsen et al. (1981) have investigated similar tactics which involve decreasing the soil pH in order to render Fe more available. Work on the accumulation of Al has shown that: (1) Al tolerance (in barley) appears to be controlled by one major, dominant gene (Reid, 1971); (2) Al tolerance may be due to a chelating mechanism (Grine & Hodgson, 1969); (3) Al-sensitive wheat and barley varieties accumulate higher Al concentrations in aerial parts than Al-tolerant (Foy & Fleming, 1978); (4) Tea plant (Camellia spp.) selectively translocates Al to non-sensitive areas (older leaves), avoiding more sensitive areas (new leaves and buds) (Sivasabramaniam & Talibudeen, 1972); (5) Al is located in leaf epidermis cells which exhibit distinctly thickened walls (Matsumoto, 1976); (6) Some Al-tolerant species have the ability to increase the rhizosphere pH, rendering Al less available (Chamura & Koike, 1960; Adams & Pearson, 1970; Clark & Brown, 1974), Manganese - Ionic Mn is an essential nutrient. As such, one can expect Mn uptake mechanisms to exist in all species. Excessive uptake 21 ' - | - | occurs when the divalent form Mn is present in high soil concentrations. This situation typically arises.under reducing conditions. The scant reports on Mn accumulation have demonstrated the following: (1) Plant species and cultivars differ greatly in their ability to tolerate excess soluble Mn (Foy, 1973); (2) Mn tolerance is associated with greater retention in the root (Andrew & Hegarty, 1969); I | .(3) Mn-tolerant rice plants apparently oxidize Mn (available) to Mn (less available) (Engler & Patrick, 1975). 22 BIOLOGY OF ION TRANSPORT IN HIGHER PLANTS ■ The transport of ions from aqueous solutions into root and shoot cells of higher plants is vital to life on earth. The difficulty in analyzing the processes which are involved in ion ■ accumulation and assimilation in higher plants is reflected in the general lack of basic data in this area. A review of the pertinent literature (Liittge & Pitman, 1976; Pitman, 1977; Nissan, 1974; Anderson, 1972; Clarkson & Hanson, 1980; Briggs et al., 1961; Wardlow & Passioura, 1976; LUttge & Higinbotham, 1979; Nye & Tinker, 1977) reveals the complexity of the various interactive processes involved .in higher plant-ion transport phenomena. A synthesis of this literature suggests the following general mechanisms of ion transport into the xylary stream. Once ions are at. the root surface they may move through the root tissues via either of two pathways, (I) the apoplasm and (2) the symplasm (Figure I). The apoplast is that zone which includes all extracellular wall material and intercellular spaces. Ions may be transported in the apoplast up to a layer of suberized material (casparian strip) which separates root cortical cells from vascular tissue. A combination of exchange sorption and diffusion allows ions to move in the apoplast. The symplasm is that zone which is 23 EXTERNAL SOLUTION Figure I. P leem odesm ita CYTO/SYMFLASM VACUOLE m plasmelemma SYMPLASMl C PATHWAY N A PO PLASMIC PATHWAY CASPARIAN STRIP Schematic diagram of higher plant root ion transport system. N = diffusive transport processes. F = convec­ tive transport processes. (Adapted from Pitman [1971]). bounded by the external surface of the cell membrane (plasmalemma). Considerable evidence (Gunning & Robards, 1976) indicates that the cytoplasm in the root is connected by channels (plasmodesmata) makinS the symplasm a contiguous system. Movement by ions through the symplasm is not understood though plasmodesmata and cytoplasmic streaming have been implicated. The existence of the casparian strip necessitates ion transport across the plasmalemma prior to transfer to the xylary stream. Here, at the plasmalemma/apoplast interface, lies the crucial transport mechanism debate. Is ion transport through the membrane a purely physico-chemical process or is it mediated by metabolic activity? Though individuals ascribe various levels of importance to each mechanism, the available evidence (i.e., ion selectivity, transport against diffusion and electrical potential differences) suggests a large role for biological structures in ion transport. Of the various models which have been put forth to describe the empirical results, the one which is widely accepted by physiologists and which elicits much debate is termed the "carrier" hyppthesis (Epstein, 1972; LaUchli, 1976). This hypothesis is based upon the concept of enzymatic saturation. (Nissen, 1974) which implicates protein "carriers" as the mode of ion transport across the plasmalemma. Like enzymes, these carriers may be more or less specific for particular substrates, and will exhibit saturation kinetics described by Michaelis-Menten type equations. Using this approach, Epstein (1972) stated that there are two distinct mechanisms of potassium 25 uptake by barley roots. Nlssen (1983) alleges that there are several distinct saturation kinetics phases in ion uptake as a function of external ion activity. The following evidence corroborates the existence of protein "carriers" which mediate ion transport: (1) Sulfate and phosphate binding protein can be isolated from a variety of cell membrane systems (Rothfield, 1971) (2) Potassium transport in barley and sunflower is regulated by allosteric control of K -carrier activity by cytoplasmic potassium concentration (Glass, 1977; Nye & Tinker, 1969; Pettersson, & Jensen, 1978) (3) Existence of a constitutive phosphate uptake mechanism in Neurospora (Lowendorff et al., 1974) The review articles and texts on biological transport mentioned above generally agree that the "carrier” hypothesis at least partially explains a wide variety of ion transport phenomena. A corollary to the "carrier" hypothesis is that differences in uptake characters, especially within species (Lauchli, 1976) may be explained on the basis of protein conformational alterations. In fact, nutritional ecotypes do exhibit enzymatic differences (Lauchli, 1976). Cox and Thurman (1978) demonstrated that zinc-tolerant Anthoxanthum forms less stable metal-enzyme complexes (lower Km) than zinc-sensitive clones. Wainwright and Wpolhouse (1975) demon­ strated similar enzyme differences between Cu-tolerant and Cu- sensitive races of Agrostis tenuis. The in-vivo activity of several enzymes of Cu-tolerant ecotypes is less inhibited by elevated solution copper than enzymes of Cu-sensitive plants (Mathys, 1975). 26 Lauchli (1976) has stated that ions are "carried" across the plasmalerrana from the apoplasm, transported to xylary parenchyma by unknown mechanisms and then actively secreted into the xylem stream by "carriers" located at the plasmalemma of the cells lining the xylem. Pitman (1977) concludes that while the apoplast to symplast and symplast to xylem ion transfer processes are similarly mediated by "carriers", they are distinctly different systems based on metabolic inhibitor studies. The implication of the carrier hypothesis is that ion uptake characteristics are genetically controlled. This fits with the few studies which attempt to correlate ion uptake parameters with genetics. In 1934, Weiss stated that iron "utilization" in soybean was under monogenic control. Similarly, Pope and Munger (1953a, 1953b) demonstrated that boron and magnesium uptake and/or trans­ location in a celery variety is controlled by a single allele. Boron deficiency in tomato has been linked to a single, recessive gene (Wall & Andrus, 1962). Bernard and Howell (1964) showed that phosphorus accumulation in a soybean is also governed by a single gene. Epstein (1972) discusses much of this type of work. Other workers (Brown & Devine, 1980; Brown et al., 1971; Jones, 1974; Clark, 1975; Wright, 1976; Clarkson & Hanson, 1980) have shown significant ion transport differences between genotypes, suggesting genetic control at one or several loci. Most of the above reports discuss the nature of the ion "carrier". However, other processes are involved in ion uptake phenomena. For example, removal of an 27 ion from the symplasm directly affects uptake rate and may be accomplished by extracellular secretion, or complexation in the cytoplasm or cell wall. Several investigators have suggested that cell wall composition (i.e. CEC) (Ernst, 1972) may play an im­ portant role in uptake processes. Complexation of metal within the wall matrix has been demonstrated for Zn, Cu, Pb, and Al (Peterson, 1967; Ernst, 1972; Turner & Marshall, 1972; Malone et al., 1974). Complexation of ions in cytoplasmic chelates has been demonstrated in several plant-ion systems (Rauser & Corvetto, 1980; Wagner & Trotter, 1982). These intracellular chelates are relatively low molecular weight proteins that have the peculiar characteristics of low aromatic amino acid moieties and high cysteihyl residue content. In mammalian systems, metals can induce the transcription of genes directing the synthesis of proteinaceous chelates (Hamer & Wallfng, 1982). Deposition in the vacuole also represents another process by which the intracellular ion concentration and ion uptake may be affected. Depositional processes have been implicated in biological mechanisms of tolerance to Na+ and Cl' accumulation (Greenway & Munns, 1980). The various transport systems in the higher plant root are. diagrammed in Figure I. Control of the uptake process can take place at any of the sites (N) shown. Genetic control of these various processes is implicit even though the rate limiting step per se may be a strictly physical process (i.e., passive adsorption to wall matrix). In other words, the rate-limiting step in ion transport in a particular species for a particular ion may be under 28 monogenic (i.e., cytoplasmic chelates) or polygenic (i.e., altered membrane and wall structure) control. 29 RESEARCH STRATEGY General The focus of this research is a characterization of the bio­ logical mechanisms of heavy metal accumulation in a higher plant which exhibits unusual metal-tolerance capacity. The primary tool of this investigation is the comparison of a known metal-tolerant species with a metal-sensitive race of the same species under various metal-stress conditions. Assuming that the races are essentially identical, differences in physiological response to rhizosphere heavy metals allow for an investigation of the basic mechanisms which differentiate these races. Indeed, the study of physiological "mutants" led to Garrod's (1909) "one gene- one enzyme" hypothesis of biochemical genetics. The use of intra­ specific, isogenic comparisons of physiologic response to heavy metals is a first step towards an understanding of the fundamental biological mechanism underlying ion transport. The primary means of comparing these races is by determining the accumulation and distribution of heavy metals in plant tissues under different culture conditions of metal stress. From differences in metal accumulation and distribution under identical conditions one may infer differences in transport characteristics. Distribution of metals in organs (root vs. shoot) and biochemical fractions 30 allows the investigator to draw conclusions concerning metal accu­ mulation (and thus transport) mechanisms. Some consideration is also given to a comparison of growth rates in the experimental races. A review of the literature shows that ion accumulation characteristics vary widely between ions. It seems useful, therefore to compare accumulation patterns of two dissimilar heavy metals in these two physiological races. Finally, this research develops a technique for precise control over rhizosphere conditions which permits accurate determination of ion uptake kinetics and other physiological responses under tightly controlled rhizosphere conditions. Using these approaches, the following questions have been posed: (1) Are there heavy metal accumulation differences between metal-tolerant and metal-sensitive races of the same species? (2) Are there differences in heavy metal distri­ bution in organs (root/shoot) between races? (3) Are there differences in heavy metal dis­ tribution in biochemical fractions of the two races? (4) Are there differences in heavy metal accumula­ tion/ distribution relative to the species of ion? (5) Are there differences in heavy metal accumula­ tion/distribution relative to species of ion and genotype? (6) What are the general magnitudes of accumulation/ distribution differences, if they exist? 31 (7) Can established methods of reactor design engineering be applied to a higher plant root-ion system? (8) Can a "macrophyte reactor" be designed to yield reliable ion uptake kinetic data for the heavy metal-metallophyte system? (9) Are heavy metal accumulation differences (if they exist) reflected in differences in ion uptake kinetics? (10) What are the general magnitudes of ion uptake kinetics differences, if they exist? (11) How- do differences in heavy metal accumulation, distribution, and uptake kinetics relate to established and purported biological mechanisms? (12) Do the patterns of differences in heavy metal accumulation, distribution, and kinetics suggest fundamental mechanisms of ion transport in biological systems? (13) Do these differences imply biological mechanisms of heavy metal tolerance and accumulation in higher plant systems? Experimental Species Deschampsia. caespitosa Selection A suspected metal tolerant grass species was collected on the Anaconda Reduction Works tailings ponds in the fall of 1981. Analysis of the tailings material (Table 3) showed elevated levels of several metals and low pH. Determination of metal concentration in tissues of this grass (Table 4) indicated accumulation of several metals well above that considered adequate or normal (Mortvedt, 1972) The collected species was taxonomically determined as Deschampsia caespitosa (L.) Beauv. commonly referred as "tufted hairgrass". 32 Table 3. Data from analysis of tailings material collected near Deschampsia caespitosa^collection.site at Anaconda, MT. All elements in Hg ml of a cold-water extraction of ___ ______saturated paste. SARA 1.68 PH 3.37 EC (mmhos) 6.10 Ca 411.0 Hg ml ! Mg 350.0 Hg ml : Na Fe 191.2 Hg ml 3.32 Hg ml 1 Zn 340.0 Hg ml ! Cu 364.0 Hg ml Mn 1000.0 Hg ml 0.10 Hg ml ^Pb Cd 4.10 Hg ml Al 155.0 Hg ml *Sodium adsorption ratio Table 4. Concentrations of metals in samples of D. caespitosa collected in October 1981 on the Anaconda Reduction Works tailings ponds. Samples were decomposed by perchloric acid digestion. The residue was analyzed for metals by atomic absorption flame spectrophotometer. Deschampsia caespitosa samples Metals in Pg g-I ', dry tissue Cu Cd Zn Mn Mg Fe Pb Ni ' Standing Dead Litter 238 19.8 419 682 930 1838 59.0 — Living Roots & Shoots 1000 25.5 550 670 1150 9750 231 3.57 33 This species is catholic in distribution in the northern hemisphere. It is a "bunch" grass which reproduces yegetatively by tillering. Metal tolerance in this species has been previously reported (Cox & Hutchinson, 1979; Surrbrug, 1982). The D. caespitosa collected at the metal tailings site is referred to throughout this report as the "TAILINGS" race or popu­ lation. Seed of Deschampsia caespitosa from agricultural-field grown plants was obtained from the Oregon State University Seed Laboratory. This population is referred to as the "AGRICULTURAL" race or population. Collection of Deschampsia caespitosa "TAILINGS" Fifteen to twenty individual clumps of D. caespitosa were collected randomly across a several acre grassed site on the tailings ponds. Individual clumps were selected at least five meters apart. The plants were dormant when collected in October and were transported in tailings to the greenhouse. Preparation of D. Caespitosa "TAILINGS" and "AGRICULTURAL" Two months after fielcj collection (December, 1981) "Tailings", plants were washed numerous times with double distilled water to remove adhering tailings from the root system. One hundred and fifty smaller clumps were removed from the 15 larger clumps, rinsed with distilled HgO and were placed in 125 ml plastic beakers which contained acid-washed (0.1N HNO3) sand which was saturated with Arnon and Hoagland*s (Hewitt, 1975) (IX) solution (Table 5). 34. Plants were watered daily with nutrient solution and double distilled water only, until the initiation of: EXPERIMENT I, five months later; EXPERIMENT 2, fifteen months later; EXPERIMENT 3, seventeen months later. Table 5. Modified IX Hoagland and Arnon nutrient solution used in heavy metal uptake experiments. . SALT CONCENTRATION KNO3 1.03 g £ 1 Ca(NO3)2 • 4H20 0.708 g A™1 ■INH4H2PO4 0.231 g a Mg SO4 * 7H20 0.466 g JT1 H3BO3 4H20 2.86 mg JT1 ' -IMn Cl2 • 1.80 mg & -ICu SO4 • 5H 0 0.077 mg &-IZn SO. • 4 7H2° 0.218 mg & I H2MoO4 • H2O 0.078 mg &IFe Na EDTA 0.164 g & Seeds of the "AGRICULTURAL" race were germinated in December 1981 in identical acid-washed sand, nutrient solution culture. These plants were watered identically to the.tailings population until the onset of experimentation. All specimens were kept in the greenhouse during the preparation phase. Experimental Heavy Metals Two heavy metal ions were chosen for experimentation because of their similarities and differences. Copper and Cd were - 35 selected because of their importance as environmental contaminants. Both induce toxicity in living systems at relatively low concen- ■ !rations. These metals are essentially different in their functional relationships to biological systems. Each exhibits different coordina­ tion chemistry. Copper is a required element for biological function whereas Cd assumes no known role in metabolic processes. This essential difference could result in different patterns of accumulation in the two races of metallophyte. ' Copper. Like all members of the transition metals, this element has an incompletely filled d-orbital. Copper is commonly found in the II oxidation state though I and III oxidation states are also relatively stable. . Like other transition metals, Cu forms numerous inorganic and organic complexes. The chemistry of Cu is complex and a source of intense interest and debate (Leckie & Davis, 1979). The physiological role of Cu is also a complex problem. It provides organisms with a metal which in its reduced state readily binds and reduces . In its oxidized form, Cu is readily reduced. In protein complexes Cu has a high redox potential. These properties have been exploited by use in enzymes that hydroxylate monophenols (oxidizing them to create complex polymers), terminate electron transport chains, detoxify superoxides, oxidize amines, and act generally as cytoplasmic oxidases. Only Cu fills this vital biochemical niche. Although the primary biological role of Cu lies in its relationship with proteins in metal-enzyme complexes. 36 it can act in other ways (Peisach, 1966): (1) in trigger and control mechanisms; (2) in structural forms (i.e., elastin, collagen) (3) as a Lewis acid (i.e., Cu"̂ " accepts electron pairs)J (4) as redox catalyst. The copper requirement of plants is quite low relative to other nutrients. It is essential in amounts near 3-10 ppm dry weight but toxic in amounts over 50 ppm (Clarkson & Hanson, 1980; Nriagu, 1979). Cadmium. This metal belongs to the IIb group of elements qnd is almost always found in the II oxidation state. Although Cd exhibits no known vital role in biological processes, it is accumulated by higher plants. (Nriagu, 1979). Growth reduction due to Cd has been shown in both soil and solution studies (Jastrow and Koeppe, 1980). Cadmium is known to complex with certain proteins, to substitute for other metals in metalloenzymes. Ernst (1980) has reviewed the data on cadmium effects on plants and has stated the following: (1) Cd inhibits in vitro activity of several enzymes; ■ (2) Cd has a high affinity for sulfhydryl groups; (3) Nitrate reductase is very sensitive to low : concentration of Cd; (4) Cd reduces the amount of chlorophyll in leaf mesophyll cells; (5) Cd inhibits photosynthesis; (6) Cd induces chromosomal aberrations. EXPERIMENTAL TECHNIQUES Sand-Solution Culture - Experiment I Acid-washed silica oxide wetted with a defined nutrient solution has been widely applied in plant-ion investigations (Hewitt & Smith, 1975). It permits a certain level of control to the investigator but does not allow direct evaluation of uptake kinetics. The sand-solution system was used in Experiment I to determine if any heavy metal accumulation or distribution differences exist between the experimental races. The system was also used to evaluate differences arising due to different mepal ions (Cu, Cd). Finally, Experiment I describes accumulation and distribution differences due to metal loading rate and in time (plant age). This experiment was carried out in the greenhouse which adds environmental variables to the system. However, if one assumes that each race responds iden­ tically to environmental factors other than rhizosphere metals, the system may be used to make, comparisons between races. Response variable in this experiment is metal concentration in harvested plant tissue as determined by atomic absorption flame spectro­ photometry . 38 Batch Solution-Cd Isotope-Experiment II Batch solution culture is a common technique to investigate plant-ion relations (Nye & Tinker, 1969). Determination of ion uptake kinetics in batch systems is very difficult, if not impossible, owing to the changing concentrations of ionic species in the reactor vessel over time. Batch culture was used in Experiment II for one purpose: to determine the biochemical distribution of Cd ions in the two experimental races of D . caespito$a. Experimental use of radio isotopes has, perhaps.more than any other technique, lead to a greater understanding of the nature of ion transport in plants. Its utility lies in the investigator's ability to detect minute ion concentration differences over short time periods. Cadmium was selected for experimentation because of a commercially available source of Cd"*"®̂ (half life = 456 + 10 days) and the fact that isotopes of Cu have half lives which preclude feasible use. Response variable in this experiment was concentration 109of Cd based upon measurements of gamma radiation from Cd . . Though gamma emitters pose safety problems, they are experimentally convenient due to the fact that sample preparation is very simple (i.e. quenching is not a factor). Macrophyte Reactor - Experiment III Nye and Tinker (1979) have pointed out the conceptual failure in the use of batch (solid and liquid media) culture systems to obtain ion uptake data. They state that continuous-flow systems are required for accurate evaluation of the kinetic characteristics 39 of ion accumulation by higher plants. With this motivation and the existing body of literature on the modeling of environmental processes (Waite & Freeman, 1977) and reactor kinetics (Smith, 1970), Experi­ ment III was designed to determine the feasibility of constructing a "macrophyte reactor" suitable for obtaining ion uptake kinetic data. The objectives of this experiment are: (1) To construct a reactor which would provide control of a continuous-flow nutrient solution stream around the roots of D. caespitosa,. (2) To obtain ion uptake kinetics data from the Deschampsia-heavy metal system. (3) To compare heavy metal accumulation kinetics between the two experimental races of D. caespitosa. Several investigators have used flowing solutions to determine xion uptake in plants (Clement et al., 1974; Asher et al., 1965; Edwards & Asher, 1974; Van de Dijk, 1981; Veen, 1977; Wild et al., 1974) and more recent systems have employed elaborate control of particular ions in the nutrient solution (Breeze et al., 1982). All of these systems are batch or semi-batch reactors which means■ that ambient nutrient concentrations around roots are changing, over time. Accurate kinetic data requires constant nutrient composition at the root surface. Bloom and Chapin (1981) have presented the only system which actually employs a continuous-flow reactor. None of the reports in the literature uses reactor kinetics theory to develop a means of describing the biological processes involved in ion accumulation in higher plants. 40 The development of a "macrophyte reactor" represents a novel scientific activity. It is important, therefore, to present a theoretical treatment of the system. The Macrophyte Reactor: Theoretical Basis Development of the macrophyte realtor is based upon the law of conservation of mass. Simply stated, this law means that mass influx into a system minus accumulation must equal mass efflux. Figure 2'.- depicts the basic flow of material in the macrophyte . reactor system. The reactor system contains a finite volume, V 3which has the units of cubic length (L .). Volumetric fluid flow 3 -Ithrough the system, F has units of volume per time (L t ). Mass concentration of a component in the fluid entering thq reactor is -3termed and has the units of mass per volume (ML ). Concentration of C leaving the reactor also has units of mass per volume (ML ;. The macrophyte system is modeled as a continuous stirred tank reactor (CSTR) and,, as such, complete mixing of the reactor fluid is assumed. This means that the concentration of C in the reactor effluent is identical to the concentration in the reactor. Input of radiant energy to the reactor is denoted as Mass balances in a CSTR are described by the following: ACCUMULATION = TRANSPORT + TRANSFORMATION Mathematically, this equation is: ac ■V^r = FC. - FC + RV (I)dt i 41 M ACROPHYTE REA CTO R N E T ACCUMULATION = NET TRANSPORT + TRANSFORMATION v dC = FCi - F C + RV d t Figure 2. Schematic diagram of theoretical continuous stirred tank macrophyte reactor (CSTMR) of volume V containing constituent C with throughput flow F. Radiant energy input is denoted as Qn. Mass balance equation for C across the reactor is shown where R is a composite process rate term. 42 where the left hand side of the equation is a differential expressing the rate of change or rate of accumulation of C in the reactor. The right hand side of the equation is comprised of a rate of change component due to mass transport [F(C^-C)] and a process rate (R) due to transformation. The rate term R has the units of mass 3 “1per volume per time (ML- ^ ). R may take on positive or negative values depending upon the reaction taking place within the reactor. The CSTR establishes a steady-state condition which eliminates the left hand side of equation (2) : dC dt KC1 - C) + R Thus, in a CSTR, mass balance equations can be reduced to an algebraic expression: I(C1-C) = R (3) The term — is important as it gives the dilution rate (D) of reactor contents: g q V 1) V(L3) D (t 3O The reciprocal of dilution rate, .(-) is detention time "0" which gives the average time spent by a particle in the reactor. It has the units of t. It is important to note that all terras on the left hand side of equation (3) can be easily measured or controlled. The utility of this approach in ion uptake studies is apparent. By constructing a reactor of known volume in which the fluid contents 43 are close to Ideally mixed and by measuring the influent and effluent concentrations of an ion, one may describe an inferred process rate for ion accumulation by root tissues. It is important to "keep in mind that the process rate R may be composed of several other process rates. That is, the process rate R is probably composed of at least two distinct processes: (I) ion influx into tissues and (2) ion efflux from tissues. R is, therefore, a net process term. Statistical Analysis The major objective in this research is the determination of differences, if they exist, in heavy metal accumulation, distribution and kinetics between the two races of D. caespitosa. Comparison of sample means using t-and paired t-tests is the primary statistical tool for making these determinations. In addition, Analysis of Variance is used in EXPERIMENT I to evaluate the significance of MAIN effects (e.g. type of metal ion, organ, loading rate, etc.) and factor interactions. Regression analysis is used in EXPERIMENT III to determine differences in slopes of uptake rate over time between the two races. The null hypothesis in all comparisons of sample means is: Ho : yI = y2 . That is, there are no differences in metal accumulation, distribution, or uptake rates between means of "TAILINGS" and 44 of "AGRICULTURAL" races. The alternative hypothesis is 2 . •ha : yI * y 45 EXPERIMENTATION Experiment I - Sand-Solution Culture Materials and Methods Plants of the two races ("TAILINGS" and "AGRICULTURAL") of Deschampsxa caespitosa were removed from acid-washed sand medium, rinsed with double distilled water and transplanted into 125 ml, plastic beakers with drainage holes and filled with 100 g of clean acid-washed sand. Five individual clumps of plant material were placed in each beaker, watered with IX Arnon & Hoagland's (A & H). solution and placed in a IO0C chamber for 3-days. The.beakers were subsequently transferred to a greenhouse an4 were watered daily . with 5 ml/beaker of IX A & H solution. Four weeks later plants were arranged in a completely randomized factorial design. Three heavy metal loading rates were selected on the basis of the literature. Reports (Mortvedt et al., 1975) indicate that the selected ranges should yield non-toxic, moderately toxic, and highly toxic responses to Cu and Cd. Cadmium loading rates were lower than Cu (at the HIGH loading rate) because of reports of toxic responses in plants at quite low concentrations of Cd. Copper was applied to the plastic beakers daily, in 46 the following amounts: ■ LOADING ' Cu Mass of Cu RATE- ml/beaker/day CONCENTRATION Applied/beaker/day low - 3.0 0.019 pg ml 0.06 pg/beaker/day med 3.0 • 10.0 Pg ml ^ 30.0 pg/beaker/day. high . 3.0 20.0 pg ml 60.0 pg/beaker/day Cadmium as aqueous Cd(NO^^ solution was applied to beakers in the following amounts: LOADING RATE ml/beaker/day Cd Mass, of Cd CONCENTRATION Applied/beake.r/dt Z low 3.0 <0.001 pg ml <0.003 pg/beaker/day med 3.0 5.0 pg ml ̂ 15.0 pg/beaker/day high 3.0 10.0 pg ml ̂ 30 pg/beaker/day Plants were watered every morning with unmeasured amounts of double-distilled water and every evening with (I) IX nutrient solution (controls) or (2) nutrient solution with metal spike at 3.0 ml per day. In order to determine total metal in roots and shoots three of four replicate beakers per treatment were selected randomly at each sampling period. Plants were harvested at 0,3,6, and 9 weeks after initiation of metal application. They were removed from sand, rinsed thoroughly with double distilled water to remove all sand, rinsed with 0.1 N HNO^, rinsed again with double distilled water, blotted dry and oven dried at 70°C. Samples were then ground with a stainless steel Wiley Mill to pass a 40 mesh screen. The samples were weighed, then digested in a mixture of nitric and perchloric acids and the metal concentration determined by atomic 47 absorption spectrometry (Munshower & Neuman, 1978; In order to obtain preliminary data on the gross biochemical location of accumulated copper or cadmium, three beakers of each treatment (.15 plants) were harvested as above and were fractionated as follows: Plant samples were separated into root and shoot and were frozen at -7Q°C after the HNOg rinse. Samples were cut up with stainless steel scissors and were homogenized in 25 mM KHgPO^/KgHPO^ buffer (pH 7.4) with a chilled Sorvall tissue homogenizer for 10 minutes at high speed. Volume of the homogenate was determined and an aliquot removed for dry weight analysis. The homogenate was centrifuged at 10,00.0 g for 20 minutes, the supernatant solution collected and labeled as "cytoplasmic" fraction. The pellet debris was resuspended in 0.1 N HNOg and recentrifuged at 10,000 g for 20 minutes. The. . supernatant was collected and added to the "cytoplasmic" fraction. The pellet was collected and labeled "wall" fraction. Prepared "wall" samples were digested as above and metal concentration measured by atomic absorption spectrometry. Liquid samples were analyzed directly by atomic absorption spectrometry. Results and Discussion The mean concentration of metals in the two races as a function of metal ion, loading rate and harvest time are tabulated in Tables 6 and 7. The accumulation of Gd and Cu over time in the two races is shown in Figures 3,4 and 5,6. One-way analysis of variance CTi Table 6. Concentration of Cd in tissues of races of Deschampsia caespitosa grown in acid-washed sand-nutrient solution culture. Data are in pgCd/g: tissue dry weight as determined by acid digestion and atomic absorption spectrophotometry. Data are for four sample times (0,3,6,9 weeks), three Cd loading rates (low = < 0.001 yg/day; med = 15 yg/day; high = 30 yg/day Data are average of three independent samples + standard error of the mean. TAILINGS RACE_________ _________ -________________ AGRICULTURAL RACE Loading Week Rate Root low 0.20 + 0.0 0 med 0.20 + 0.0 high 0.20 + 010 low 0.20 + 0.0 3 med 18.5 + 0.20 high . 34.9 + 2.6 low 0.30 + 0.10 6 med 32.0 + 4.6 high 62.3 + 1.5 low 0.20 + 0.0 9 med 28.2 + 6.2 high 67.3 + 3.2 Loading Shoot Rate 0.20 + 0.0 low 0.20 + 0.0 med 0.20 + 0.0 high 0.20 + 0.0 . . low 20.4 + 8.5 med 38.6 + 8.3 high 0.18 + 0.02 ' low 13.1 + 2.3 med 26.7 + 3.3 high 0.30 + 0.05 low 15.1 + 2.4 med 30.3 + 5.2 high Root Shoot 0.20 + 0.0 0.20 + 0.0 0.20 + 0.0 0.20 + 0.0 0.20 + 0.0 0.20 + 0.0 0.20 + 0.0 0.20 + 0.0 19.2 + 0.81 24.1 + 2.6 34.8 + 0.26 53.4 + 4.7 0.20 + 0.0 0.20 + 0.0 29.7 + 0.38 41.8 + 4.7 66.6 + 2.5 65.3 + 5.8 1.4 + 0.12 0.20 + 0.0 34.5 + 2.1 22.0 + 0.64 158.7 + 18.8 58.3 + 8.3 ■>os Table 7. Concentration of Cu in tissues of races of Deschampsia caespitosa grown in acid-washed sand-nutrient solution culture. Data are in yg Cu/g tissue dry weight as determined by atomic absorption spectrophotometry. Data are for four sample times (0,3,6,9 weeks), three Cu loading rates (low = < 0.06 yg/day; med = 30 yg/day; high = 60 yg/day). Data are average of three independent samples + standard error of the mean. "TAILINGS" "AGRICULTURAL" Week Loading Rate Root Shoot Loading Rate Root Shoot low 4.7 + 0.94 8.8 + 1.9 low 16.2 + 1.9 12.7 + 2.6 0 med 4.7 + 0.94 8.8 + 1.9 med 16.2 + 1.9 12.7 + 2.6 high 4.7 + 0.94 8.8 + 1.9 high 16.2 + 1.9 12.7 + 2.6 low 7.6 + 0.47 8.8 + 0.12 low 12.7 + 0.69 13.1 + 2.1 .3 med 37.5 + 4.5 20.4 + 0.57 med 74.4 + 3.0 30.7 + 3.0 high 91.4 + 4.7 34.4 + 1.7 high 114.7 + 8.1 85.2 + 1.2 low 7.4 + 1.7 6.4 + 0.37 low 17.3 + 2.8 5.7 + 1.2 6 med 69.4 + 1.4 19.3 + 1.8 med 96.7 + 3.8 26.4 + 1.0 high 125.7 + 16.4 35.8 + 4.8 high 196.7 + 2.0 79.3 + • 1.5 low 5.7 + 0.55 5.8 + 0.09 low 3.7 + 0.27 7.5 + 0.24 9 . med 28.1 + 1.2 21.9 + 2.4 med 93.2 + 4.7 20.5 + 2.5 high 82.4 + 1.3 42.5 + 4.6 high 202.5 + 34 109.3 + 11.7 50 A high "5 A med ' I T med WEEKS Figure 3. Concentration of Cu in ROOTS of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in sand- solution culturg at tljiree Cu loading ratesflow = 0.06 Mg beaker da^^ ; mej = 30 Mg beaker day ; high = 60 Mg beaker day ). Data are means of three independent determinations. Cu measured by atomic absorption flame spectrophotometry. 5i A high WEEKS Figure 4. Concentration of Cu in SHOOTS of "Agricultural" (A) and "Tailings" (T) races of D . caespitosa grown in sand- solution C^lture1St three Cu loading rates (low = 0.06 Mg beaker djy ;_ijied = 30 Mg beaker 1 day-1; high = 60 Mg beaker day ). Data are means of three inde­ pendent determinations. Cu measured by atomic absorption flame spectrophotometry. 52 CD 50 W E E K S Figure 5. Concentration of Cd in ROOTS of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa grown in sand- solution culturegt thrje Cd loading rates (low = <0.001 ug beaker d̂ iy ;_med = 15 Mg beaker-1 day-1; high = 30 Mg beaker day ). Data are means of three independent determinations. Cd measured by atomic absorption flame spectrophotometry. y* t 9 C d / g ti ss u e 53 150 - __^T high "D A med - * T med A.T low WEEKS ' PpSISiiEindependent determinations. Cd measured by atomic absorption flame spectrophotometry. 54 indicates that replicates of samples are homogeneous (F = 0.01), eliminating replicate to replicate variability as a factor. Figures 3,4 and 5,6 show that under identical conditions of metal loading rate and harvest time the "Agricultural" race accumulates more metal than the "Tailings" race. The difference is exacerbated with time and appears to be more significant with Cu.than with Cd. Analysis of variance indicates that race, ("Agricultural" vs "Tailings"), organ (root, shoot), metal (Cu, Cd), loading rate (low, med, high), and harvest time (0,3,6,9 weeks) all elicit significant (P = 0.001) effects of metal concentration in tissues (Appendix I). Analysis of the data using, a t-statistic reveals several significant differences (Table 8). "Tailings" vs. "Agricultural" differences. Averaged over all treatments, the "Agricultural"race accumulates almost twice as much heavy metal as the "Tailings" race. This basic difference is more pronounced with Cu than Cd and with shoots more than roots. Visual assessment of the races indicated that Cu had a more dramatic effect on the "Agricultural" race than did cadmium. The "Tailings" race did not appear to be adversely affected by either metal treatment. Copper vs. Cadmium differences. Averaged over all treatments, at identical loading rates there is a significantly higher (p =0.001) amount of Cu in plant tissues than Cd (1.94 times more). Differences in metal concentration between races and organs are less significant Table 8. T-statistic of comparisons of means of metal concentrations in tissues of races of Deschampsia caespitosa ("Agricultural" ("A"), "Tailings" ("T")). Data from Experiment I (sand-solution culture). yg metal/g dry tissue______Statistical Comparison calculated tabled level of TREATMENT AVERAGED OVER. n X min max t-value t-value significance "A" Race Roots & shoots; Cu .& 144 45.75 0.20 270.6 3.52 3.291 (df=286) 0.001"T" Race Cd; all sample times, loading rates 144 26.33 0.12 158.5 Cu "A" & "T" races; roots 48 36.3 3.2 270.6 o 7 c 3.402 0.001Cd & shoots all sample 48 18.7 0.12 156.3 J • / D times med loading rate only VkQX JHj Roots "A" & "T" races; Cu & 144 40.0 0.20 270.6 O /, O 3.291 (df=286) 0.001Shoots Cd; all sample times, loading rates 144 21.8 0.12 129.2 U • H O Roots Cu "A" & "T" races; all 60 64.4 3.2 270.6 O QO 3.291 (df=118) 0.001Shoots sample times, loading rates 60 29.7 3.6 129.2 Roots ^ "A" & "T" races; all 60 29.5 0.20 192.4 I 57 1.282 (df=118) 0.20Shoots sample times, loading rates 60 20.5 0.12 74.9 "A" Race-Cu "T" Race-Cu Roots & shoots; all sample times, loading rates 60 60 60.9 33.2 3.2 3.3 270.6 158.5 3.07 2.86 (df=118) 0.005 Table 8. (cent) TREATMENT "A" - Cd "T" - Cd "A" roots "T" roots "A" shoots "T" shoots "A" Root-Cu "T" Root-Cu "A" Root-Cd "T" Root-Cd "A" Shoot-Cu "T" Shoot-Cu "A" Shoot-Cd "T" Shoot-Cd "A" Shoot-Cd. "A" Shoot-Cd nT" Root-Cd "T" Shoot-Cd yg metal/g dry tissue______Statistical Comparison________ calculated tabled level of AVERAGED OVER_____ n_____x min max t-value t-value significance Roots & shoots'; all sample timess loading rates .60 30.5 60 19.4 0.20 0.12 192.4 73.7 1.96 1.658 (df=118) 0.10 Cu & Cd; all sample times, loading rates . 72 50.2 72 29.7 0.20 0.20 270.6 158.5 2.41 2.24 (df=142) 0.025 Cu Si Cd; all sample times, loading rates 72 28.4 72 15.3 0.20 0.12 129.2 52.7 3.26 2.807 (df=142) 0.005 All sample times, loading‘rates 36 73.8 36 39.1 3.2 3.3 270.6" 158.5 2.38 2.29 . (df=70) 0.025 All sample times, loading rates 36 28.8 36 20.3 0.20 0.20 192.4 73.7 0.98 0.847 (df=70) 0.40 All sample times, loading rates 36 34.0 36 18.4 3.6 5.7 129.2 . 51.4 2.60 2.29 (df=70) 0.025 All sample times, loading rates 36 22.2 36 12.1 0.20 0.12 24.9 52.7 2.05 1.994 (df=70) 0.050 All sample times, loading rates 36 28.8 36 22.2 0.20 0.20 192.4 74.9 0.80 0.674 (df=70) 0.50 All sample times, loading times 36 20.3 36 12.1 0.20 0.12 73.7 52.7 1.86 1.667 (df=70) .. 0.10 Table 8. (cent) yg metal/g dry tissue_______Statistical Comparison TREATMENT AVERAGED OVER n X min max calculated t-value tabled t-value level of significance "A" Root-Cu "A" Shoot-Cu All sample times, loading rates 36 36 73.8 34.0 3.2 3.6 270.6 129,2 2.94 2.899 (df=70) 0.005 "T" Root-Cu "T" Shoot-Cu All sample times loading rates 36 . 36 39.1 18.4 3.3 5.7 158.5 51.4 3.13 2.899 (df=70) 0.005 58 with Cd whereas these differences are very significant with Cu. Root vs. Shoot differences. Averaged over all treatments, the differences between metal concentration in roots and shoots is highly significant (p = 0.001). This difference is high for Cu (p = 0.001) but diminishes with Cd (p - 0.20). In all cases, there is higher metal concentration in roots than shoots. This is not surprising given the difficulty in removing particles attached to root surfaces and the high ion adsorptive capacity of root cell wall material indicated in the literature (Nye & Tinker, 1977). Loading rate differences. Table 9 shows differences in average concentration of metals in tissues of both races. Comparison using a t-statistic (Table 10) shows significant differences between races at the low and medium loading rate of copper in roots and significant differences with copper at high loading rate for shoots. Cadmium does not exhibit significant differences at any loading rate and organ except for the high rate in shoots. Figures 7 and 8 show the relationship between average metal concentration in roots and shoots in the two races and loading rate. It can be seen that generally, metal concentration in tissues of "Tailings" is lower than metal concentration in "Agricultural" tissues at most loading rates. The differences are more pronounced as loading rate increases. Figure 7, showing copper effects, demonstrates a significant point about these races. The differences between root and shoot concentration are an indication of the level 59 Table 9. Analysis of tissue metal concentrations as a function of metal loading rate-comparison of two races (i.e. "Tailings", "Agricu ltural") . (Data are yg metal/g dry wt ). n = 1-2 Data from Experiment I (sand-solution*culture). Phyiological races of Deschampsia caespitosa ■"TAILINGS" COPPER "AGRICULTURAL" Loading Rate X Loading Rate X low 6.4 low 12.8 ROOT med 34.9 med 70.1 high 76.0 high 132.5 low 7.4 low 9.8 SHOOT med 17.6 med 22.6 high 30.4 CADMIUM high 124.3 low 0.23 low 0.50 ROOT med 19.7 med 20.9 high 41.0 high 65.0 low 0.22 low 0.29 SHOOT med 12.0 ■ med 22.0 high 24.0 high 44.0 Table 10. COMPARISON T-statistic of "Tailings" ("T") vs. "Agricultural" ("A") race comparisons at different loading rates (low, med, high) for ' Cd and Cu, roots and shoots (n=12; df for comparisons=22)„ METAL CALCULATED TABLED LEVEL OF METAL ORGAN LOADING RATE t-value t-value SIGNIFIANCE "T" vs "A" Cu Root low 2.20 3.119 0.005 "T" vs "A" Cu Root med 2.91 2.819 0.01 "T" vs "A" Cu Root high 2.04 1.717 0.10 "T" vs "A" Cu Shoot low 1.71 1.717 0.10 "T" vs "A" Cu Shoot med 1.73 1.717 0.10 "T" vs "A" Cu Shoot high 3.47 3.119 0.005 "T" vs "A" Cd Root low 1.59 1.321 0.20 "T" vs "A" Cd Root med 0.13 0.686 ns "T" vs "A" Cd Root high 1.20 0.858 0.40 "T" vs "A" Cd Shoot low 1.12 0.858 0.40 "T" vs "A" Cd Shoot , med 1.82 1.717 0.10 "T" vs "A" Cd Shoot high 2.16 2.074 0.05 60 low med high Copper loading ra te Figure 7. Average concentration of Cu in tissues of "Agricultural" (A) and "Tailings" (T) races of D . caespi^osa a^ a function of Cu loading^rate p ow = 0.06 tig beaker ^day j med = 30 ug beaker day ; high = 60 Mg beaker day ). Data are means of 12 independent samples averaged over all times, replications. Cu concentration measured by atomic absorption spectrophotometry. 61 SHOQT HOOT SHOOT C a d m i u m l o a d i n g r a t e Figure 8. Average concentration of Cd in tissues of "Agricultural" (A) and "Tailings" (T) races of D. caespitosa as a function of Cd loading rate_^low = <0.003 Mg beaker”1 day”1; med = 15 Mg beaker day”1; high = 30 Mg beaker”1 day”1). Data are means of 12 independent samples averaged over all times, replications. Cd concentration measured by atomic absorption spectrophotometry. 62 of control exercised over transport. In the "Tailings" race, shoot copper concentration, increases gradually as loading rate increases and the difference between root and shoot gradually.increases. In the "Agricultural" race the copper concentration in the shoot increases greatly as loading rate increases such that there is little difference between root and shoot concentration at the high loading rate. The difference between races with Cd at various loading rates is not as significant though Figure 8 does reflect the general trend of higher tissue concentration of Cd in the "Agricultural" race at any particular loading rate. Harvest time differences. Figures.3,4,5,6 show the trend.of metal accumulation over time. A striking.feature is the general leveling off or decline in metal concentration as time progresses. This response is shown in Table 11 which tabulates the average metal concentration (Cu and Cd) in all tissues of both races over time. TABLE 11 TIME______n_____x (yig metal/g dry tissue) 0 weeks 72 5.4 3 weeks 72 32.3 6 weeks 72 42.7 9 weeks 72 43.3 Table 11. Average metal concentration of both D. caespitosai races, whole plant; all loading rates, over time. 63 Figures 3, 4, 5, and 6 show that the decline in accumulation of metal is most significant for the "Tailings" race and Cu treatments. The fact that both races exhibit significant differences in their accumulation of copper relative to cadmium may be explained by the fact that copper is a nutrient ion and cadmium is not. One could expect an organism to readily accumulate a nutrient species while excluding a non-nutrient. This may fit with a selective protein "ion carrier" hypothesis. The significant differences between the races relative to metal accumulation suggest that the "Tailings" population is capable of avoiding or excluding excess metal ions. This is contrary to Ernst's (1975) statement that metal ion exclusion is not possible in higher plant systems. The difference in metal accumulation between races could be explained on the basis of loss of membrane integrity and lysis of "Agricultural" cells. . Jarvis et al. (1976) have shown that freshly killed plant tissue accumulates Cd at a higher rate than living material until saturation of adsorption sites on the cell wall matrix. One would expect that if gradual death of root cells was the cause of increased metal accumulation in the "Agricultural" race, transport to the shoots would decline. However, the data show that metal concentrations in shoots of the "Agricultural" race continued to increase over time. Tills may indicate .a passive leakage of ions into the xylem stream by slowly dying root xylem parenchyma, cells. The data indicates that the "Tailings" race is capable of excluding metals from the roots but, more significantly, it apparently restricts metal ion transport to the shoot. This may implicate an Ct 64 altered transport mechanism at the xylem parenchyma plasmalemma. Overall differences in root vs. shoot can be easily explained by the fact that the root apoplast represents an ion exchange matrix capable of adsorbing large amounts of ions. It is assumed that transport to the shoots is an active biological process and the ratio of root/shoot concentration differences reflect a mechanism of control of metal transport. These data would suggest that the "Tailings" population has the capacity to restrict ion entry into root tissues and transport to the xylary stream while the "Agricultural" race does not possess such a capacity. Variation in metal concentration in tissues over time implicates growth processes as well as transport processes. This is because metals are not known to leach out of tissues in significant amounts once translocated. Growth rate differences may explain the decrease in metal concentration over time. Significantly lower tissue metal concentration exhibited by the "Tailings" race suggests that this genotype can tolerate high intracellular metal levels as well as restrict transport. By the game reasoning, the "Agricultural" race may exhibit both reduced growth rate and lack of metal transport control. Biochemical fractionation. Table 12 lists the concentration of Cu and Cd in "cytoplamic" and "wall" fractions of the "Tailings" and "Agricultural" races. Figure 9 shows the gross biochemical distribution of Cd and Cu. Small sample size precludes any comparisons between genotypes; however, one may conclude that heavy metals Table 12. Cu and Cd concentrations in "wall" and "cytoplasmic" fractions of races of D. caespitosa grown in sand-solution culture with applied metals. Analysis by acid digestion-atomic absorption spectrophotometry. Data are from single independent determinations. RACE ORGAN METAL FRACTION Concentration of Cu in sample (yg Cd/g dry tissue) concentration of Cd in sample (pg Cd/g dry tissue) % of total metal in tissue IIiJiIt . Root Cu "wall" . 18.0 — 86.5 "T" Root Cu "cytoplasm" 2.8 — 13.5 "A" Root Cu "wall" 44.1 - 89.5 "A" Root Cu "cytoplasm" 5.2 — 10.5 • llTn Shoot Cu "wall" 5.0 - 70.4 Ilrjtl Shoot Cu "cytoplasm" 2.1 - 29.6 "A" Shoot Cu "wall" 8.5 - 90.3 "A" Shoot Cu '"wall" 0.9 - 9.7 IIiJitI Root Cd "wall" . - 5.6 34.2 Ilrjll Root Cd "cytoplasm" 5.9 36.0 "A" Root Cd "wall" - 22.3 92.8 "A" Root Cd "cytoplasm" - ’ 1.7 7.2 uTu Shoot Cd "wall" - 1.2 81.8 IliJiU Shoot Cd "cytoplasm" — ■ 0.27 18.2 "A" Shoot Cd "wall" - 2.4 71.7 "A" Shoot Cd "cytoplasm" ■ - 0.96 28.3 Ilipll Root control "wall" 4.1 <1.00 *96.0 ** - Ilipll Root control "cytoplasm" 0.17 <0.10 4.0 "A" Root control "wall" 6.8 <0.01 83.0 "A" Root control "cytoplasm" 1.4 <0.01 17.0 Table 12. (cont) RACE ORGAN METAL FRACTION Concentration of Cu in sample (yg Cd/g dry tissue) concentration of Cd in sample (yg Cd/g dry tissue) % of total metal in tissue Ilipll Shoot control "wall" 1.5 1.47 96.0 97.4 Ilipll Shoot control "cytoplasm" 0.6 0.04 4.0 2.6 "A" Shoot control "wall" 3,2 <0.01 89.0 "A" Shoot control "cytoplasm" 0.4 <0.01 11.0 ft = % Cu in tissue fraction; ** = % Cd in tissue fraction o\c\ is su e 67 .E e I 50 0 19« Cu Cd W A L L Z Z Z Z Z Cu Cd Cu Cd Z W A L L Z / Z W A L L Z Z Z z / W A L L Z z Z ROOT R ( A ) I C Y T O C V T O W A L L Z Z W A L L Z Z Z • . Z Z Z Z Cu Cd Z ZZ ZZz Z ZZ z Z Z Z Z SHOOT A SHOOT Figure 9. Percent distribution of total Cu and Cd in tissues of "Agricultural" (A) and "Tailings" (T) plants grown in sand-solu