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Published in J. Environ. Qual. 33:54-60 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Bioremediation and Biodegradation

Overexpression of ATP Sulfurylase in Indian Mustard

Effects on Tolerance and Accumulation of Twelve Metals

Ami L. Wangelinea, Jason L. Burkheada, Kerry L. Halea, Stormy D. Lindbloma, Norman Terryb, Marinus Pilona and Elizabeth A. H. Pilon-Smits*,a

a Biology Department, Colorado State University, A/Z Building, Fort Collins, CO 80523
b Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94270

* Corresponding author (epsmits{at}lamar.colostate.edu).

Received for publication October 14, 2002.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Indian mustard [Brassica juncea (L.) Czern.] transgenics overexpressing ATP sulfurylase (APS plants) were shown previously to have higher levels of total thiols, S, and Se. The present study explores the effect of ATP sulfurylase overexpression on tolerance and accumulation of other metals, both oxyanions and cations, reasoning that some anions may react directly with ATP sulfurylase, while other ions may be bound by its thiol end products. The APS transgenics were compared with wild-type plants with respect to tolerance and accumulation of As, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, V, W, and Zn, supplied individually in agar medium (seedlings) or in hydroponics (mature plants). At the seedling stage, APS transgenics were more tolerant than wild type to As(III), As(V), Cd, Cu, Hg, and Zn, but less tolerant to Mo and V. The APS seedlings had up to 2.5-fold higher shoot concentrations of As(III), As(V), Hg, Mo, Pb, and V, and somewhat lower Cr levels. Mature APS plants contained up to 2.5-fold higher shoot concentrations of Cd, Cr, Cu, Mo, V, and W than wild type. They also contained 1.5- to 2-fold higher levels of the essential elements Fe, Mo, and S in most of the treatments. Mature APS plants showed no differences in metal tolerance compared with the wild type. Overexpression of ATP sulfurylase may be a promising approach to create plants with enhanced phytoextraction capacity for mixtures of metals.

Abbreviations: APS, plants overexpressing ATP sulfurylase • WT, wild type


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
TOXIC METALS AND METALLOIDS are increasingly released into the environment by human activities such as industry, mining operations, use of ammunition, traffic, and agriculture, resulting in contamination that threatens natural ecosystems and human well-being (Lantzy and Mackenzie, 1979; Nriagu, 1979; Ross, 1994). Currently, the USA is spending around $3 billion a year on remediation of toxic inorganic elements, comprising 35% of the total U.S. funds spent for environmental cleanup (Glass, 1999, 2000). Some examples of conventional remediation methods for metals include soil washing, excavation and reburial of the soil, and soil stabilization by, for example, concrete capping.

Alternative phytoremediation methods for metals and metalloids make use of the natural ability of plants to acquire minerals from their environment and to stabilize soil and create an upward water flow in the process. Phytostabilization may involve the prevention of leaching through an upward water flow resulting from plant transpiration, reduced runoff owing to aboveground vegetation, and reduced soil erosion through soil stabilization by plant roots (Berti and Cunningham, 2000). In the technology called rhizofiltration, accumulation of metals by roots in a hydroponic setup is followed by harvesting of the plant biomass (Dushenkov and Kapulnik, 2000). Phytoextraction, another promising technology in metal phytoremediation, involves the accumulation of metals in shoot tissue followed by harvesting of the shoot biomass (Blaylock and Huang, 2000). The metal-laden plant material may be used for nonfood purposes or ashed, followed by recycling of the metals or disposal in a landfill (Chaney et al., 2000). These metal phytoremediation technologies are already being used effectively (Salt et al., 1998; Blaylock, 2000), and are gaining acceptance, since phytoremediation is relatively cost-effective and aesthetically pleasing, and may be used in conjunction with more conventional remediation methods.

To further increase the effectiveness of metal phytoremediation, several approaches may be employed, including identification of new suitable plant species via screening studies, optimization of agronomic practices for maximal element uptake, and improvement of selected plant species by classical breeding or genetic engineering.

Genetic engineering is starting to emerge as a relatively rapid and effective way to improve the capacity of plants to tolerate and accumulate metals. Transgenic plants with enhanced metal tolerance and accumulation have been created through several approaches, including overexpression of metal transporter proteins (Samuelsen et al., 1998; Arazi et al., 1999; Van der Zaal et al., 1999; Curie et al., 2000; Hirschi et al., 2000), overproduction of metal-chelating molecules (Evans et al., 1992; de la Fuente et al., 1997; Hasegawa et al., 1997; Goto et al., 1999; Zhu et al., 1999a, 1999b), or even introduction of a bacterial pathway (Rugh et al., 1996; Bizily et al., 1999, 2000). For a review of the development of transgenics for metal phytoremediation, see Krämer and Chardonnens (2001) and Pilon-Smits and Pilon (2002).

Phytochelatins are cysteine-rich metal-chelating peptides involved in heavy metal tolerance and sequestration (Steffens, 1990). The general structure of phytochelatins is ({gamma}-Glu-Cys)n–Gly, where n = 2 to 11 (Rauser, 1995). The cysteine needed for the biosynthesis of phytochelatins is produced by the sulfate assimilation pathway, which is located primarily in the chloroplast in plants (Schwenn, 1994; Leustek, 1996; Leustek and Saito, 1999). After uptake by sulfate permease, sulfate is activated by ATP sulfurylase to form adenosine phosphosulfate, which is subsequently reduced to free sulfite by APS reductase (Setya et al., 1996). Sulfate permease and ATP sulfurylase are induced by sulfur starvation and repressed by feeding sulfate or reduced forms of S (Chen and Leustek, 1995; Logan et al., 1996).

Overexpression of ATP sulfurylase in Indian mustard APS transgenics resulted in twofold higher levels of the organic sulfur compounds glutathione and total nonprotein thiols, as well as higher total sulfur levels (Pilon-Smits et al., 1999). The transgenic APS plants also showed enhanced tolerance to selenium (threefold), increased reduction of selenate to organic forms of selenium, and higher selenium accumulation (Pilon-Smits et al., 1999). These results, obtained using hydroponics, show that ATP sulfurylase is involved in selenate as well as sulfate assimilation, and is probably present in wild-type Indian mustard in amounts that are rate-limiting for the uptake and reduction of sulfur and selenium. The observed enhanced selenium tolerance and accumulation in the APS transgenics suggest they may have enhanced selenium phytoremediation capacity. Indeed, the APS plants showed threefold higher shoot selenium levels compared with wild type when grown on soil naturally rich in selenium (unpublished results). This suggests that hydroponic systems are a useful means to explore plants' phytoremediation capacity and that overexpression of ATP sulfurylase is a promising approach to create transgenic plants with enhanced selenium phytoremediation capacity.

The objective of the present study was to conduct a more comprehensive analysis of metal tolerance and accumulation of the APS transgenics relative to wild-type Indian mustard, with the combined goal to obtain more insight into the involvement of this enzyme in plant metal tolerance and accumulation, and to further explore the phytoremediation potential of the APS transgenics. For instance, it is feasible that the APS transgenics will show higher accumulation of other oxyanions that are similar to sulfate and selenate, such as molybdate, tungstate, chromate, vanadate, and arsenate, if the ATP sulfurylase enzyme can use these ions as a substrate. Also, the higher thiol levels in the APS transgenics may confer enhanced tolerance and accumulation of thiol-bound metals such as cadmium, as was found for other thiol-overproducing transgenics (Zhu et al., 1999a, 1999b). Promising in this respect was the observation that APS transgenics removed more Cd, Cr, Cu, Mn, Pb, and Zn from polluted soil in comparison with wild-type Indian mustard (Bennett et al., 2002). To explore the role of ATP sulfurylase in plant tolerance and accumulation of various metals and metalloids, and the potential of APS plants for phytoremediation of these elements, controlled experiments were done to compare APS and wild-type plants with respect to their accumulation and tolerance of the 12 toxic elements As, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, V, W, and Zn.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Materials
Indian mustard wild-type seeds were from Accession no. 173874, North Central Regional Plant Introduction Station (Ames, IA). Transgenic APS seeds were obtained from plants of this same accession number, genetically engineered to overexpress ATP sulfurylase (APS). The transgenic APS plants overexpress the Arabidopsis thaliana APS1 cDNA including its own chloroplast transit sequence, under the control of the CaMV 35S promoter (Pilon-Smits et al., 1999). The transgenic APS line used for this study was APS8, bred to homozygosity.

The chemical forms and concentrations of the metals and metalloids used are shown in Table 1. These chemical forms were chosen to have high solubility in water and to have minimal effect on the nutrient solution composition. No precipitation was observed when these metal salts were added to agar medium or nutrient solution. The concentrations used were aimed to give approximately 50% reduction in plant growth (on a fresh weight basis) compared with untreated controls, a degree of toxicity expected to optimally reveal any differences in tolerance between the APS transgenics and the wild type (WT).


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  		Table 1. Elements and concentrations used for treatments of seedlings and mature plants.

 
Metal Tolerance and Accumulation Experiments
Seedlings
To compare the metal tolerance of APS and WT Indian mustard plants at the seedling level, WT and APS seedlings were grown axenically from seed on agar medium containing one of the selected metals. For each experiment, a treatment without added metal was run in parallel as a control. The seeds were surface-sterilized as described by Pilon-Smits et al. (1999), and 36 sterilized seeds were sown in a grid pattern on agar medium in Magenta tissue culture boxes (Sigma, St. Louis, MO). The agar medium contained half-strength Murashige and Skoog (MS) salts and vitamins (Murashige and Skoog, 1962), 10 g L–1 sucrose, and 4 g L–1 agargel (Sigma); individual metals were added to the final concentrations shown in Table 1. These concentrations were aimed to give an approximately 50% reduction in wild-type seedling fresh weight, corresponding with an approximately 75% reduction in seedling root length compared with untreated controls. The magenta boxes with the different treatments were randomly arranged and the seedlings were allowed to grow for 7 d in a growth cabinet at 25°C and a 16 h light–8 h dark photoperiod. Individual seedlings were then harvested and washed, and root length was measured as a parameter for metal tolerance (Murphy and Taiz, 1995). To correct for any differences between experiments, metal tolerance was expressed as relative root length, calculated as root length in the presence of a metal divided by root length on control medium.

Mature Plants
To compare metal tolerance at the mature plant level, seeds of WT and APS transgenics were surface-sterilized, sown on agar medium as described above, and grown for 4 d. The seedlings were then transferred to sand, watered with half-strength Hoagland's nutrient solution daily (Hoagland and Arnon, 1938), and grown in a greenhouse at 25°C, 16 h light–8 h dark photoperiod until they were 35 d old. At this point, the plants were transferred to a greenhouse nutrient film technique setup as described by Zhu et al. (1999a). After 7 d of adjustment to the new medium, the fresh weights of the plants were measured and the individual metal treatments were started. One metal was given at a time at the concentrations shown in Table 1; for each experiment a control treatment without metal was run in parallel. These metal concentrations were expected to give an approximately 50% reduction in fresh weight in mature plants compared with untreated controls. Ten replicate plants were used per plant genotype (WT or APS) per treatment. The nutrient solutions were replaced every 3 d for a total of 14 d, after which the plants were harvested, washed, weighed, and dried for elemental analysis.

Elemental Analysis
Seedlings
Seedling shoot tissue was collected and dried for 48 h at 80°C for elemental analysis. Per treatment, five dried shoot samples of six or seven seedlings each were weighed and acid-digested for 6 h at 130°C in concentrated nitric acid according to the method of Zarcinas et al. (1987). After dilution of the acid digests with distilled water the total elemental concentrations in the digests were measured using inductively coupled plasma–atomic emission spectrometry (ICP–AES; Thermo Elemental, Franklin Lakes, NJ) according to the method of Fassel (1978), using appropriate standards and quality controls.

Mature Plants
After harvesting, the plants were dried for 48 h at 80°C. Each individual shoot was ground in a Wiley mill and homogenized, and a sample was taken for acid digestion and analysis by ICP–AES as described above.

Statistical Analyses
Statistical analyses for tolerance and accumulation were performed using the software program JMP-IN (SAS Institute, 1999). Statistically significant differences between treatments and plant types (t tests, {alpha} = 0.05) are indicated in the text and by asterisks in the corresponding figure or table.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The ATP sulfurylase overexpressing (APS) Indian mustard transgenics were shown previously to have enhanced rates of sulfate and selenate reduction and to contain higher levels of total thiols, S, and Se (Pilon-Smits et al., 1999). The objective of this study was to explore the effects of ATP sulfurylase overexpression on tolerance and accumulation of other metals, both oxyanions and cations, reasoning that oxyanions may react with the ATP sulfurylase enzyme directly, and cations may be bound by thiols.

Seedling Metal Tolerance and Accumulation
No significant differences in growth were observed between APS and wild-type (WT) seedlings grown on control agar medium without added metals. In the presence of metals there were some significant differences. Judged from root growth inhibition, the APS seedlings were significantly more tolerant than WT to As(III), As(V), Cd, Cu, Hg, and Zn, with the largest difference on Cd where APS relative root growth was more than double that of WT (Fig. 1) . The APS seedlings were less tolerant than WT to Mo and V, with the greatest difference on Mo where relative APS root growth was about one-half that of WT (Fig. 1). No significant differences in tolerance were found between APS and WT plants for Cr, Mn, Ni, Pb, and W.



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  		Fig. 1. Tolerance of wild-type (WT) and APS (i.e., overexpression of ATP sulfurylase) transgenic Indian mustard seedlings to 12 metal(loid)s, supplied at concentrations as indicated in Table 1. Tolerance is expressed as the ratio of root length in the presence of metals relative to root length on control medium. Values shown are the mean and standard error of the mean of 36 seedlings each. Significant differences between APS and WT seedlings ({alpha} = 0.05) are indicated by asterisks.

 
The enhanced tolerance of the APS plants to As(III), As(V), Cd, Cu, Hg, and Zn could be due to their enhanced thiol levels. These elements were all shown to be bound by phytochelatins (Maitani et al., 1996; Cobbett and Goldsbrough, 2000; Pickering et al., 2000; Schmöger et al., 2000). Binding of excess amounts of these metals by the thiol-containing peptides glutathione and phytochelatins may facilitate their vacuolar sequestration and detoxification, leading to enhanced tolerance.

The reduced tolerance of the APS seedlings to Mo and V may be because ATP sulfurylase can react directly with these oxyanions. Wilson and Bandurski (1958) reported that ATP sulfurylase can use molybdate, tungstate, chromate, and selenate as a substrate, but only the reactions with sulfate and selenate resulted in stable end products. If the products of these reactions are unstable, this will lead to a loss of energy due to futile cycling of ATP to ADP and back. Also, competition with other oxyanions may lead to lower levels of the normal end products of sulfate assimilation. Together, this may explain the lower tolerance of the APS seedlings to Mo and V. The APS seedlings did not show reduced tolerance to two other oxyanions tested (Cr and W), however. These differences in oxyanion tolerance may be related to the different tissue concentrations of these elements (see below).

The APS seedlings had significantly higher shoot concentrations than WT of As(III), As(V), Hg, Mo, Pb, and V, with more than double the WT levels for both forms of As and almost threefold higher Hg levels (Fig. 2) . The APS seedlings had somewhat (approximately 10%) lower Cr levels in their shoots than WT, and there were no significant differences in shoot metal concentrations between APS and WT when treated with Cd, Cu, Mn, Ni, W, or Zn (Table 2).



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  		Fig. 2. Shoot metal concentrations in seedlings of wild-type (WT) and APS (i.e., overexpression of ATP sulfurylase) transgenic Indian mustard supplied with six metals, at concentrations as listed in Table 1. Values shown represent the mean and standard error of the mean of five samples consisting of six or seven seedlings each. All metals shown here were present at significantly different concentrations ({alpha} = 0.05) between WT and APS seedlings. Additional results from metals that were accumulated to a similar extent by both plant types are shown in Table 2.

 

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  		Table 2. Shoot metal concentrations in wild type (WT) and APS (i.e., overexpression of ATP sulfurylase) seedlings after 7 d of growth at the concentrations shown in Table 1.{dagger}

 
The higher shoot levels of As and Hg in the APS seedlings may be the result of their higher tolerance to these elements, leading to better overall health, transpiration, and root–shoot translocation. No differences in shoot concentrations of other thiol-bound metals (Cd, Cu, and Zn) were found, however; perhaps these metals are retained more in the root.

The higher levels of Mo and V could be due to upregulation of the sulfate uptake system, if these oxyanions can make use of sulfate permease transporter proteins, as suggested by certain studies (Leggett and Epstein, 1956; Marschner, 1995; Leustek, 1996). Upregulation of sulfate permease may be a reaction to overexpression of ATP sulfurylase, and may also explain the enhanced levels of S and Se observed earlier in APS plants (Pilon-Smits et al., 1999). The higher levels of Mo and V inside the APS plants offer an additional explanation for their lower tolerance to these elements. The APS seedlings did not show enhanced accumulation of the other oxyanions Cr and W, possibly explaining why they showed the same tolerance as wild type to these elements at the seedling level.

Mature Plant Metal Tolerance and Accumulation
There were no significant differences in metal tolerance between APS and WT mature plants, as judged from fresh weight gain when healthy mature plants were exposed for 14 d to the 12 individual metals in a nutrient film setup (results not shown).

The APS transgenics, however, did show enhanced accumulation of a range of metals, both cations and anions. The APS mature plants contained significantly higher shoot concentrations than WT for Cd, Cr, Cu, Mo, V, and W, with the Cd and V concentrations being almost double and triple those of WT, respectively (Fig. 3) . The APS and WT plants showed no differences in shoot metal concentration when supplied with As(III), Hg, Mn, Ni, Pb, and Zn (Table 3).



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  		Fig. 3. Shoot metal concentrations in wild-type (WT) and APS (i.e., overexpression of ATP sulfurylase) transgenic Indian mustard plants supplied with six metals at the concentrations listed in Table 1. Values shown represent the mean and standard error of the mean of 10 plants, each sampled once. All metals shown here were present at significantly different concentrations ({alpha} = 0.05) between WT and APS plants. Additional results from metals that were accumulated to a similar extent by both plant types are shown in Table 3.

 

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  		Table 3. Shoot metal concentrations in mature plants after 14 d of treatment at the concentrations shown in Table 1.{dagger}

 
Of the metals or metalloids accumulated to higher levels in shoots of APS transgenics compared with WT, four were supplied as oxyanions (Cr, Mo, V, and W) and their enhanced uptake may be due to upregulation of sulfate transporter systems, as hypothesized above. The other two, Cd and Cu, can be complexed by thiols; perhaps more efficient binding by thiols can somehow stimulate their uptake. The same effect was not observed, however, for other thiol-bound elements such as As and Hg.

To investigate the effect of ATP sulfurylase overexpression on plant levels of other, essential macro- and micronutrient shoot concentrations of Cu, Fe, Mg, Mn, Mo, S, and Zn were determined by ICP–AES for all mature plant treatments. Interestingly, the APS plants had higher levels of the essential elements Fe, Mo, and S in the majority of treatments, with on average 1.5-fold higher Fe levels and more than 2-fold higher Mo and S levels (Table 4). The higher Mo and S levels may be the result of upregulation of sulfate transporters as a result of ATP sulfurylase overexpression, if molybdate can be taken up by sulfate permease. The reason for the higher Fe levels is not clear. Some plant Fe is complexed with S in iron–sulfur clusters, which obtain their S from cysteine (Amrani et al., 2000; Mihara et al., 2002). Higher availability of S for Fe–S clusters may stimulate enhanced Fe uptake. The S for molybdenum cofactor (MoCo) is also derived from cysteine (Amrani et al., 2000), and enhanced availability of S may also stimulate Mo uptake.


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  		Table 4. Shoot concentrations of the essential elements Fe, Mo, and S in mature plants after 14 d of treatment with various metal(loid)s at the concentrations shown in Table 1.

 
Not only were shoot S levels approximately 1.5-fold higher under control conditions in APS plants, but various metal treatments further enhanced this difference between APS and WT. Treatment with As, Pb, and V, for instance, resulted in a modest increase in WT S levels, but in a two- to threefold increase in APS S levels. Treatment with Cr and Ni even resulted in a decrease in WT S levels but a twofold increase in APS S concentrations. Enzymes involved in sulfate uptake and assimilation are known to be upregulated in response to metal stress to provide cysteine for the production of metal-binding thiols (Chen and Leustek, 1995; Logan et al., 1996). The presence of extra ATP sulfurylase activity appears to somehow stimulate this effect, perhaps by further enhancing sulfate permease levels.

Similar but less pronounced differences were observed for Mo and Fe (Table 4). The shoot Mg levels were also somewhat enhanced in APS plants relative to WT (+20% on average; p < 0.05 for nine treatments); there were no differences in Cu, Mn, or Zn levels (results not shown).

Overall Effect of ATP Sulfurylase Overexpression on Metal Tolerance and Accumulation
There were similarities but also many differences between seedlings and mature plants with respect to metal tolerance and accumulation. The differences in results obtained with seedlings and mature plants may in part be explained by development-related differences in plant metabolism (e.g., differences in endogenous ATP sulfurylase activity). Also, the differences observed between seedlings and mature plants may in part be due to the experimental setup: the seedlings were exposed to the metals continuously from seed, while the mature plants were only exposed during the last 2 wk of their 8-wk life. In any case, the different results obtained from seedlings and mature plants illustrate that one should be careful when drawing conclusions about mature plant metal tolerance and accumulation from experiments with seedlings, and that tolerance by mature plants to acute metal stress may involve different mechanisms than continuous metal tolerance starting from germination.

In spite of the differences in results obtained from seedlings and mature plants, some general trends emerge with respect to the effect of ATP sulfurylase overexpression on plant tolerance and accumulation of oxyanions and cations. The APS plants showed a tendency to accumulate enhanced shoot levels of oxyanions. Shoot Mo and V levels were enhanced in both APS seedlings and mature plants, Cr and W were higher in mature APS plants, and As levels were enhanced in APS seedlings. Tolerance to oxyanions tended to be reduced or unaffected, with the exception of the thiol-bound oxyanion As, to which APS seedlings had higher tolerance. The APS plants contained enhanced shoot levels of certain cations as well, especially thiol-bound cations: Hg and Pb in seedlings and Cd and Cu in mature plants. Seedling tolerance to cations tended to be enhanced, especially for known thiol-bound cations. Possible explanations for these phenomena have already been discussed above.

Does Overexpression of ATP Sulfurylase Show Promise for Metal Phytoremediation?
The APS transgenics tended to accumulate metals to higher shoot concentrations than wild type: of the 12 elements examined, the APS plants contained up to 2.5-fold higher levels of As, Cd, Cr, Cu, Hg, Mo, Pb, V, and W in at least one growth stage. A higher shoot metal concentration is a favorable property for phytoextraction. On the other hand, the APS plants tended to be less tolerant to oxyanions. Whether decreased tolerance would be a factor in a field situation remains to be seen. Promising in this respect, in a study where plants were grown from seed on metal-contaminated environmental soil, APS transgenics removed more Cd, Cr, Cu, Mn, Pb, and Zn from the soil in comparison with wild-type Indian mustard without any differences in biomass production (Bennett et al., 2002). Thus, at less extreme metal concentrations, metal tolerance does not appear to negatively affect the use of APS plants for metal extraction. The finding that APS transgenics show enhanced accumulation of a variety of metals and metalloids suggests that overexpression of ATP sulfurylase is a promising strategy for the creation of plants with enhanced metal phytoremediation capacity, especially in view of the fact that most metal-polluted sites contain mixtures of metals. Of course any field use of transgenic plants for phytoremediation should be preceded and accompanied by a thorough risk assessment study.


   ACKNOWLEDGMENTS
 
This work was supported by USEPA Research Grant G8A11586 awarded to E.A.H. Pilon-Smits and N. Terry, and U.S. National Science Foundation Grant MCB-9982432 awarded to E.A.H. Pilon-Smits, including a Research Experience for Undergraduates supplement for S.D. Lindblom. M. Pilon is supported by NSF Grant MCB-0091163.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





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