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a Dep. of Biology and Microbiology, South Dakota State Univ., Brookings, SD 57007
b Plant Science Dep., South Dakota State Univ., Brookings, SD 57007
* Corresponding author (Neil_Reese{at}sdstate.edu)
Received for publication November 21, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Roots withdraw substances from the soil, but they also release substances influencing nutrient availability (Brady, 1990; Thibaud et al., 1994). An active transport system pumping H+ ions in or out of the cells in contact with the growing medium maintains the pH gradient central to plant mineral nutrition (Thibaud et al., 1994). The rhizosphere, the first few millimeters of soil in contact with the roots, may have a pH up to 2 units higher or lower than the soil directly around it, which can significantly affect the uptake of most plant nutrients. Plants experiencing an iron deficiency generally increase the H+ output into the rhizosphere to acidify the soil and release organic acids and other compounds that chelate the iron in the surrounding area. The same event is seen in plants experiencing nitrogen deficiency, but to a lesser extent. Net H+ levels increased 2 to 4 µmol H+ g-1 fresh wt. h-1 in response to low nitrogen while they increased to about 28 µmol H+ g-1 fresh wt. h-1 for iron in response to iron deficiencies (Marschner, 1995). Nitrogen-fixing legumes tend to acidify their rhizospere because they take up more cations (ammonium) than anions (nitrate), which is counterbalanced by H+ release (Tang et al., 1997).
Ervio (1991) has suggested that the most important factor for the uptake of cationic heavy metals by plants is the pH of the soil in which they are growing. Elemental sulfur, added to soils contaminated with cadmium and planted with common mustard (Sinapis alba L.), was shown to acidify the soil and make the cadmium more available to the plant. The study also showed that when high levels of sulfur were added the pH dropped significantly and plant growth was inhibited. At pH 5 to 5.5 optimum plant growth was achieved, but was accompanied by significantly increased levels of cadmium uptake (Tichy et al., 1997). Plant uptake of Cd2+ was also measured within control and limed soils. The control plant having the greatest accumulation of cadmium contained 213 µg Cd g-1 dry plant matter, while the highest concentration in plants from the limed soil contained only 67 µg Cd g-1 dry plant weight (Krebs et al., 1998). In the study reported here, the effect of plant-driven changes in rhizosphere pH on cadmium uptake in an agar medium was investigated. A morphological and rhizosphere pH evaluation of available accessions of strawberry clover was also completed to determine its usefulness for reclamation purposes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphological Comparison
Seeds from each accession were grown in a greenhouse in potting soil in 10-cm pots so morphological comparisons between accessions could be made. The plants were watered three times a week and fertilized with time-released fertilizer sticks (Jobes Plant Food Spikes, 6% N, 12% P, and 6% K; Weatherly Consumer Products, Lexington, KY). The plants were given a day–night regime of 16 and 8 h, 25 ± 5°C, and natural light supplemented with high-pressure sodium lamps (230–1000 µmol m-2 s-1). Observations were made after 6 mo of growth.
Media Preparation
A colorimetric pH indicator (bromocresol purple) was added to orchid agar to determine the change in rhizosphere pH induced by the roots of each accession (adapted from Gollany and Schumacher, 1993). An orchid media [containing 6.09 mM Ca(NO3)2, 3.78 mM (NH4)2SO4, 2.08 mM MgSO4, 1.84 mM KH2PO4, 1.65 mM FeSO4, 49.67 µM MnSO4, and 58.43 mM saccharose) in a 1% agar solution was prepared with deionized water. The media was brought to a 15 µM Cd2+ concentration (1.69 mg of Cd2+ was added to each tube). Bromocresol purple (0.9 g dm-3) was added to the media and the pH was adjusted to 7.0 with 1 M NaOH, and the mixture was sterilized in an autoclave. Four milliliters of agar was pipetted into autoclaved 16- x 100-mm culture tubes sealed with sterile 18-mm Bacti Capall lids (Kendall Healthcare Products, Mansfield, MA) to allow sterile air exchange. Six control tubes were poured for each run, three with a pH of 7.6 and three with a pH of 5.0.
Seed Culture
The seeds were sterilized for 10 min in a 10% commercial bleach solution and shaken at 144 rpm on an orbital shaker (VWR Scientific, West Chester, PA) in sterile water for 6 h. One seed was placed in each media-filled agar tube and the tubes were inserted into 1-inch (25.4-mm) black foam racks to protect the roots from direct exposure to light. The seedlings were kept on a day–night regime of 16 and 8 h, 22 ± 3°C, and a photon flux density of 130 µmol m-2 s-1. The tubes were randomized weekly during the growing period.
Two studies were conducted, the first using 24 µM Cd for 5 wk and the second using 15 µM Cd for 2 and 5 wk. Twenty replications from each of four accessions were grown for each experiment. At the end of each study, rhizosphere pH and seedling cadmium were measured.
Colorimetric Analysis of Media
A set of standards was made from the colorimetric agar. One liter of agar was prepared and divided into nine parts. Each part was adjusted to one of the desired pH values (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) prior to autoclaving and eight culture tubes for each pH were filled with 4 mL. Tubes of the same brand, size, and glass composition were used throughout the study to avoid variation in optical density and concentration.
The agar filled tubes were scanned two at a time into a graphics program (Adobe Systems, 1996) at 300 dpi on a flatbed scanner (S1200; Umax Data Systems, Hsinchu, Taiwan) with a transparency top light. The tubes were laid in a foam template to exclude outside light during the scanning process. A 75- x 100-pixel rectangular area was selected from the upper portion of the agar on each tube scan and a histogram was made of the selected area. The mean value given on the red channel was used for the analysis.
The red channel luminosity values and their corresponding pH values were entered into a spreadsheet program (Microsoft Excel; Microsoft, 1996). The values were graphed and a polynomial trend line was plotted (Fig. 1) . The resulting equation was used to determine the pH values of the treatment tubes, scanned, and analyzed in the same manner.
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Hydroponic Plant Culture
A hydroponic system containing four shallow tanks was constructed with 0.6- x 2.4-m sides and a plywood top and bottom, with the tanks lined with roofing rubber. An additional polyvinyl sheet was used to line each of the tanks. Each tank held 90 L when filled to a depth of 15.2 cm and was aerated by two 30.4-cm air stones connected to aquarium pumps. There were holes in the top allowing for 21 plants to grow in each tank, but only 15 were used to prevent variation in the areas directly above the air stones. Black plastic mesh was fitted into custom-drilled saucers to support the plants during growth. Seeds were started in vermiculite contained in small cylinders suspended upon the plastic mesh. A cotton string wick was fastened to the mesh and placed in the growing solution to keep the vermiculite hydrated. Once the seedlings were large enough, the wick was removed and the vermiculite was washed through the mesh.
The plants were grown in half-strength modified Hoagland's solution (Hoagland and Arnon, 1950), with two times the prescribed iron provided as NaFeEDTA. The pH was adjusted to approximately 5.5 and maintained with concentrated HCl. Natural daylight was supplemented with two 400-W high-pressure sodium lamps (Superlite series; Ruud Lighting, Racine, WI) to provide a day–night regime of 16 and 8 h, 21 to 30°C, and photon flux density of 200 to 600 µmol m-2 s-1.
Cadmium Treatments for Hydroponic Culture
Strawberry clover accessions were grown hydroponically to study their ability to accumulate and tolerate high concentrations of Cd2+. The accessions for the first experiment were selected based on morphological characteristics such as height and leaflet size. The second experiment consisted of plants chosen based on rhizosphere pH. Fifteen accessions were grown in each of two experiments for a total of 30 accessions. All plants were allowed to grow for 60 d without cadmium prior to treatment. Treatments were as follows: (i) control, (ii) +5 µM cadmium, (iii) +25 µM cadmium, and (iv) +50 µM cadmium.
All plants were maintained for 30 d in the culture medium, and the cadmium concentrations were checked weekly using a flame atomic absorption spectrophotometer (PerkinElmer Model 503). At the end of the treatment period, the plants were harvested. The roots were soaked in a 100 mM CaCl2 solution to remove any cell wall associated and free space cadmium (Wagner and Yeargan, 1986). The roots and shoots were separated and frozen for further use. The cadmium in the tissues was measured in the same manner as listed above for the seedlings.
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RESULTS |
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Under the long-day conditions 72 accessions flowered. The flowers ranged in color from white to bright pink, most plants had 10 or fewer flowers, but a few formed as many as 145 (Accession 237925). Of these flowering plants, 28 set seed. The seed pods turned brown and dried out when the seeds were ripe (Table 1).
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The dry weights of all seedlings increased as the rhizosphere pH decreased in the 2-wk experiment (see Fig. 2A) . Both of these increases in dry weight were statistically significant. In the 5-wk experiment with rhizosphere pH values from the 5-wk point (see Fig. 2B), this relationship had diminished and was no longer statistically significant. This suggests that either H+ ions had saturated the pH-indicating agar to the point where the majority of the nutrients and cadmium were in solution, or that the limits of the pH indicator had been reached. When all seedlings are examined together, there is a positive correlation between dry weight and total cadmium that is statistically significant for both experiments (see Fig. 2C,D).
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Hydroponic Cadmium Uptake
A study of hydroponically grown plants was conducted to obtain an overall view of the effects of different levels of cadmium on strawberry clover as well as its distribution within the plant. The accessions for the first experiment were picked based on morphological characteristics and the accessions for the second experiment were picked for their acidic or basic rhizosphere pH characteristics based on results from the initial rhizosphere pH study.
Cadmium concentrations in the roots and shoots increased as the treatment level of cadmium increased (Fig. 3) . Most of the cadmium was deposited in the roots of the plants.
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Plant size decreased when compared with the controls as the levels of cadmium to which they were exposed increased. There was also a decrease in the number of plants that reached flowering and in total stolon production. As soon as 3 d after initial cadmium exposure, the leaves began to yellow, increasing in severity at the higher cadmium levels. After further exposure, new leaf size decreased, older leaves became spotty, and some leaves began to die in the medium- and high-level cadmium treatments. By the end of the treatment period the roots took on a grey color and their growth was stunted as in the shoots. There was also little or no root nodulation seen in the cadmium treatments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The morphological examination of strawberry clover makes clear the variability of characteristics among the accessions (Jauert, 1999). Accessions with desirable characteristics for use in either phytoextraction or phytostabilization of cadmium-contaminated soils are available. Some accessions contained tall plants with dense foliage useful in the phytoextraction process. They could be harvested easily with plenty of biomass aboveground for cadmium translocation. Plants of this nature that do not naturally move large amounts of metal into their shoots may, alternatively, be candidates for chelator-induced phytoextraction. Many accessions were low growing and very quick to spread. Phytostabilization calls for this type of plant. It will not crowd out the desired crop plant and it could cover and stabilize a large area.
With more than 1.5 pH units between its high and low mean rhizosphere pH (see Table 2), strawberry clover holds great possibility for use in phytoremediation. Those accessions with a substantial ability to acidify the rhizosphere could prove useful in treating alkaline soils, which, along with their other drawbacks, may be more susceptible to flooding.
The continuous phytoextraction process requires plants with certain characteristics. Plants must be able to tolerate high levels of metal in their shoots and roots, translocate the metal from its roots into its shoots for harvest, and take up the metal quickly. The hyperaccumulators that have been examined for use in phytoextraction of cadmium are able to take up more than 5000 µg Cd g-1 into their shoots from a 5 µg Cd mL-1 solution (Baker et al., 1994). The drawback to hyperaccumulators is their low biomass and slow growth. In the hydroponic cadmium uptake study, strawberry clover shoots were shown to take up as much as 52 µg Cd g-1 from a 3 µM Cd2+ growing solution and as much as 701 µg Cd g-1 from a 50 µM Cd2+ solution (see Fig. 3). Despite lower shoot concentrations, the strawberry clover grown in low levels of cadmium achieved high biomass, grew quickly, and produced long stolons. Their affinity for accumulation is clear from the root levels seen in the control tanks that had only low levels of Cd2+, which may have come from the plastic liner. Some plants were able to live in the higher concentrations, although their growth was stunted. Research into chelator-induced phytoextraction may reveal a greater use for this species in reclamation.
One of the main objectives of this study was to determine if a correlation exists between a plant's ability to acidify the rhizosphere and the amount of cadmium that it accumulates. In screening processes, this relationship would have many uses. Our hypothesis was based in part on the knowledge that nutrient uptake in plants is controlled by the pH of the soil. Many nutrients are charged cations bound to the soil in differing degrees. Plants increase their net excretion of H+ ions to free up minerals as they need them. In legumes, like strawberry clover, plants will even alter the rhizosphere pH based on the type of nitrogen provided. If ammonium cations are in the growing media, the plants acidify it, while nitrate anions provoke the opposite response (Marschner et al., 1986).
When the dry weights are compared against the rhizosphere pH results, a clear trend is evident (Fig. 2A). The seedlings with the lowest rhizosphere pH have the highest dry weights, possibly because they were able to free more nutrients from the agar matrix to be taken up by their roots. In a future research project, an elemental analysis could be performed to further investigate this hypothesis. The 2-wk study was highly significant for this correlation (see Fig. 2A). The lower significance in the 5-wk study for rhizosphere pH vs. dry weight suggests that the limits of the small volume of orchid agar media had been surpassed (see Fig. 2B).
It is known that soil pH has a great influence on the amount of cadmium available to plants. Many studies have been done in soil with different chemicals added to alter the pH. Ryegrass (Lolium perenne L.) was grown in cadmium-contaminated soils amended with CaCl2 to make the soil more alkaline. The concentration of cadmium in the plants dropped from 20 mg kg-1 to 5 mg kg-1 when the soil pH was adjusted from 4 to 7 (Eriksson, 1989). A study on common mustard grown in soil acidified with elemental sulfur suggests the optimum pH for cadmium uptake is between 5 and 5.5. Higher pH values keep the cadmium bound to the soil matrix and lower pH values are detrimental to the plant's health (Tichy et al., 1997). In the pH + Cd agar culture study above, the agar media pH was adjusted to 7.0 and any changes in pH after that were made by the seedlings' roots. As hypothesized, there was a correlation between rhizosphere pH and total cadmium uptake. The plants in the 2-wk study with the lowest rhizosphere pH values contained the most cadmium while those with higher pH values had significantly less cadmium (see Fig. 2G). The regression was highly significant with Prob > F = <0.0001. The relationship in the 5-wk study was not as clearly demonstrated, probably due to the constraints of the media in each tube. After 5 wk most of the tubes were some shade of yellow, indicating that even those plants with a lower rate of acidification had begun to saturate the media–pH indicator with H+ ions. There was also almost as much variability within each accession as there was between accessions, suggesting that it may not be necessary to screen all available accessions when looking for candidates for remediation (see Table 2). Accessions 254916 and 237925 are tall with high rhizosphere pH values and might be useful in phytoextraction. Much of the cadmium was found to remain in their roots. Removal of the metals through this method may still be difficult. Remediation methods demanding shorter plants that spread quickly could employ Accessions 223821 and 236484. It is also likely that within local cultivars there may be strains with suitable abilities to change the rhizosphere pH.
The R2 value for the regression line in Fig. 2G was 0.66, suggesting that all of the cadmium uptake may not be explained by rhizosphere pH. Also, the lack of a clear correlation between the mean rhizosphere pH values of the accessions and their mean cadmium uptake provides us with questions for the future. It is known that phytochelatins and homophytochelatins are the peptides responsible for safe cadmium storage throughout the plant (Gekeler et al., 1989), and have been studied in strawberry clover. A plant's efficiency at producing these peptides may affect uptake. It is also possible that the location of the final resting place of the cadmium in the plant may play a role. A plant depositing cadmium only in its roots may be unable to take up as much as one transporting a high percentage into its shoots. All of these points require further research before any definitive conclusions can be made.
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ACKNOWLEDGMENTS |
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