Soil and Biota of Serpentine: A World View
2009 Northeastern Naturalist 16(Special Issue 5):155–162
Manganese Hyperaccumulation in Phytolacca americana L.
from the Southeastern United States
A. Joseph Pollard1,*, Heather L. Stewart1, and Caroljane B. Roberson1
Abstract - Most plants that hyperaccumulate metals are restricted to soils with
elevated concentrations of those metals. Recent reports have suggested that some
Phytolacca species in China can hyperaccumulate manganese (Mn). Phytolacca
americana L. (Pokeweed) is a ubiquitous weed of roadsides and waste areas in its
native range in the southeastern United States, and has no known association with
high-Mn soils. We investigated whether Mn hyperaccumulation also occurs among
such plants. Field-collected samples contained approximately 2000 μg Mn g-1 dry
weight, whereas other species from the same site ranged from 50 to 450 μg g-1.
Seedlings of P. americana were transplanted to the laboratory and grown in nutrient
solutions ranging up to 8 mM Mn. After three weeks, Mn concentration in leaves
exceeded 32,000 μg/g or 3.2%. This result suggests that P. americana possesses a
latent physiological ability to hyperaccumulate Mn, even if this trait is rarely, if ever,
expressed within its native range.
Introduction
Hyperaccumulation of metals or metalloids by plants is a relatively rare
phenomenon, documented in fewer than 500 species (Baker and Brooks
1989, Baker et al. 2000, Reeves and Baker 2000). Hyperaccumulation is
the uptake and storage of elements in above-ground portions of plants, to
concentrations far higher than in the soil or in other plant species in the
community. There has been considerable interest in hyperaccumulation because
of its potential applications in phytoremediation (Lasat 2000, McGrath
and Zhao 2003, Salt et al. 1998) and phytomining (Brooks et al. 1998), and
because it provides a model system for studying basic questions in plant
physiology, ecology, and evolution (Boyd 2004, Milner and Kochian 2008,
Pollard 2000).
Over 300 species hyperaccumulate nickel (Ni) from serpentine soils,
representing about 75% of the known examples of hyperaccumulation. The
generally recognized criterion for Ni hyperaccumulation is 1000 μg Ni per
gram of dry plant matter, or 0.1% (Baker et al. 2000, Reeves and Baker
2000). At least seven other elements are reported to be hyperaccumulated.
Mn hyperaccumulation has been described in approximately 10 species
(Baker and Brooks 1989, Fernando et al. 2008, Min et al. 2007). The criterion
for Mn hyperaccumulation is 10,000 μg g-1 or 1.0% on a dry-weight
basis, reflecting the general abundance of this element in soils and biological
materials (Baker and Brooks 1989). The majority of Mn hyperaccumulators
1Department of Biology, Furman University, Greenville, SC 29605, USA.
*Corresponding author - joe.pollard@furman.edu.
156 Northeastern Naturalist Vol. 16, Special Issue 5
are also from serpentine soils, especially in New Caledonia (Fernando et al.
2008), but some have been reported from industrially polluted soils.
The Chinese species Phytolacca acinosa Roxb. (Phytolaccaceae) has
been reported to hyperaccumulate Mn in an area of Hunan Province contaminated
by mine tailings (Xue et al. 2004), and further studies have shown
that the physiological ability to tolerate and hyperaccumulate Mn also occurs
in populations of this species from uncontaminated soils (Xue et al.
2005). More recently, it has been found that the related species Phytolacca
americana L. (Pokeweed) also hyperaccumulates Mn from contaminated
soils in China (Min et al. 2007, Peng et al. 2008). Although this species has
been introduced into China and other areas worldwide, it is native to North
America, especially the southeastern United States, where it is one of the
most abundant and ubiquitous broadleaf ruderal species on disturbed sites
and roadsides. It has no recorded association with serpentine or any other
high-Mn soils within its native range.
The goal of this study was to determine whether P. americana from its
native area in North America possesses the ability to hyperaccumulate Mn.
In order to characterize the physiological response of P. americana to high-
Mn substatrates, tolerance and uptake of Mn were studied in hydroponic
solutions amended with Mn at various concentrations. Because the species
is common on uncontaminated soil, we also examined the uptake of Mn from
typical soils in comparison to other species in the same community.
Methods
Manganese uptake from non-metalliferous soils
The study site was an occasionally mowed clearing in secondary oakpine
woodland on the campus of Furman University, Greenville, SC, USA
(34.9218°N, 82.4362°W). The soil in this area, a Cecil clay loam, was not
expected to be enriched in Mn either geologically or anthropogenically. Three
soil samples were collected from the upper 15 cm of the soil profile at locations
distributed across the study area. Soil samples were dried and weighed,
digested in concentrated HNO3, boiled to dryness, redigested in hot HCl, filtered,
and diluted with deionized water. Extracts were analyzed using flame
atomic absorption spectrophotrometry (Model 551, Instrumentation Laboratory,
Wilmington, MA, USA).
Leaves were collected from P. americana and six other common broadleaf
herbaceous species. The other plants analyzed were also common ruderal
plants, including both native and introduced species, and both perennials and
annuals (Table 1). Five plants were sampled from each species, on the dates
indicated in Table 1. One fully expanded leaf was collected from each plant.
Leaf samples were dried, weighed, ashed at 500 °C in a muffle furnace, dissolved
in concentrated HCl, and then diluted for analysis by flame atomic
absorption spectrophotometry as above.
2009 A.J. Pollard, H.L. Stewart, and C.B. Roberson 157
Manganese uptake from hydroponic solutions
Young seedlings of P. americana at the 2–3 leaf stage were carefully
removed from the soil in August 2006, at the study site described earlier.
Any remaining soil was rinsed from the roots using deionized water, and
the plants were transferred to rafts constructed of polystyrene foam covered
with polyethylene sheeting. Each raft containing five seedlings was floated
in a 350-mL container of nutrient solution. The nutrient solution consisted of
modified 0.25-strength Hoagland solution macronutrients (Hoagland and Arnon
1950) containing 1.25 mM KNO3, 0.25 mM KH2PO4, 1.0 mM Ca(NO3)2,
and 0.5 mM MgSO4, together with modified Long Ashton micronutrients
(Hewitt and Smith 1975) containing 25 μM Fe-EDDHA, 25 μM H3BO3,
5 μM MnSO4, 0.5 μM CuSO4, 0.5 μM ZnSO4, and 0.25 μM NaMoO4.
Plants were grown in an environmental chamber (Model E7/2, Conviron,
Winnipeg, MB, Canada) programmed for 16 hr days at 26 °C, and 8 hr nights
at 22 °C. Solutions were continuously aerated and changed twice per week.
After 10 days growth, the Hoagland’s solution was amended with MnCl2
to provide manganese at 5 concentrations: 5μM (the control micronutrient
concentration), 1 mM, 2 mM, 5 mM, and 8 mM. After 15 days cultivation,
plants were separated into roots and shoots, rinsed in deionized water, and
analyzed for Mn content as described for the field-collected samples.
All statistical analyses were conducted using JMP 7.0 (SAS Institute Inc.,
Cary, NC, USA).
Results
Manganese uptake from non-metalliferous soils
The mean concentration of Mn in the soil ranged from 78 to 630 μg g-1,
with a mean of 376.8 μg g-1. Mean concentrations of Mn in leaf tissues from
the seven plant species studied are shown in Figure 1. Phytolacca americana
averaged >2000 μg Mn g-1, which was over 4X higher than in any other species
sampled. Analysis of variance indicated that the differences between
species were statistically significant (F = 6.96; df = 6, 28; P < 0.0001).
Means comparisons using the Tukey-Kramer multiple comparison procedure
Table 1. Species studied in comparative analyses of manganese content under field conditions.
N = native, I = introduced, A = annual, and P = perennial.
Species Family Common name Origin Duration
Acalypha rhomboidea Raf.1 Euphorbiaceae Three-seed Mercury N A
Boehmeria cylindrica (L.) Sw.2 Urticaceae False Nettle N P
Chenopodium album L.1 Chenopodiaceae Lambsquarters I A
Erechtites hieraciifolia (L.) Raf. Asteraceae Burnweed N A
ex DC2
Phytolacca americana L.2 Phytolaccaceae Pokeweed N P
Rumex crispus L.2 Polygonaceae Curly Dock I P
Solanum carolinense L.2 Solanaceae Carolina Horsenettle N P
1Samples collected in September 2007
2Samples collected in August 2006
158 Northeastern Naturalist Vol. 16, Special Issue 5
with α = 0.05 indicated that P. americana was significantly different from all
other species, but that there were no significant differences in Mn accumulation
among the other six species.
Manganese uptake from hydroponic solutions
The concentration of Mn in the leaves and roots of P. americana plants
grown in Mn-amended hydroponic solutions is shown in the upper panel of
Figure 2. Increasing levels of Mn in the hydroponic solution led to increases
in tissue concentrations of Mn in both the root and shoot. Leaf Mn concentrations
reached 8000 μg g-1 in the lowest Mn-amended solution (1 mM),
and exceeded 30,000 μg g-1 in the 8 mM solution. Root concentrations were
always less than leaf concentrations. An ANCOVA indicated that tissue
Mn concentrations differed significantly depending on hydroponic solution
concentration (F = 165.40; df = 1, 46; P < 0.0001) and the plant part being
tested, i.e., leaf vs. root (F = 12.66; df = 1, 46; P = 0.0009). However, the interaction
between solution concentration and plant part was not statistically
significant (F = 2.59; df = 1, 46; P = 0.1143), suggesting that the proportionality
between leaf and root accumulation was relatively constant at all
solution concentrations.
Total mass of both root and shoot systems declined with increasing Mn
concentration (Fig. 2, lower panel). The decline in mass due to solution
concentration was statistically significant (ANCOVA: F = 10.03; df = 1, 46;
P = 0.0027). Once again, the interaction term was not significant (F = 2.57;
Figure 1. Manganese concentration in leaves of seven species growing on an uncontaminated
clay loam soil in Greenville, SC, USA. Each bar on the graph represents
the mean of five plants; error bars indicate standard error of the mean. Species codes:
Ar = Acalyphya rhomboidea, Bc = Boehmeria cylindrica, Ca = Chenopodium album,
Eh = Erechtites hieraciifolia, Pa = Phytolacca americana, Rc = Rumex crispus, and
Sc = Solanum carolinense.
2009 A.J. Pollard, H.L. Stewart, and C.B. Roberson 159
df = 1, 46; P = 0.1160), suggesting that Mn toxicity affected shoots and roots
similarly. The declines in mass were associated with visible symptoms of
toxicity such as necrotic lesions on the leaves at intermediate concentrations
and mortality in the highest concentration.
Figure 2. Responses of Phytolacca americana (Pokeweed) seedlings to 15 days of
cultivation in Hoagland’s solution amended with varying concentrations of MnCl2.
Upper panel: Mn concentration in leaves and roots; lower panel: mass of shoot system
and root system. Each data point represents the mean of five plants; error bars
indicate standard error of the mean.
160 Northeastern Naturalist Vol. 16, Special Issue 5
Discussion
The total Mn concentration in soils at the study site is consistent with typical
values reported elsewhere for strong acid extracts of uncontaminated soils
(Gilkes and McKenzie 1988). The Mn concentrations found in the leaves of
six species other than Phytolacca americana fall in the “normal” range for
plants growing on uncontaminated soils (Clarkson 1988, Reeves and Baker
2000). The concentration of Mn in P. americana was significantly higher than
that in all other species growing in the same area, exceeding 2000 μg g-1. It has
previously been reported that some plants on normal soil may have Mn concentrations
exceeding 1000 μg g-1 (Denaeyer-De Smet 1966, Reeves and Baker
2000). The behavior of P. americana is similar to that reported for P. acinosa in
China (Xue et al. 2005), which accumulated approximately 1850 μg Mn g-1 on
uncontaminated soil having an Mn concentration of about 600 μg g-1.
In hydroponic culture, P. americana approached the hyperaccumulation
criterion of 10,000 μg Mn g-1 (1%) in solutions of 1 and 2 mM Mn, and exceeded
it in 5 and 8 mM solutions. Again this is comparable to Chinese populations of
both P. acinosa (Xue et al. 2004, 2005) and P. americana (Min et al. 2007, Peng
et al. 2008). A further point of agreement is that in all cases, Mn concentrations
in shoots exceeded those in roots, a pattern that is typical of metal hyperaccumulators
but not found in the majority of plants (Reeves and Baker 2000).
Xue et al. (2004) considered P. acinosa to be remarkably Mn-tolerant,
because it was capable of surviving, flowering, and fruiting in 10 mM Mn solution,
experiencing mortality only at 15 mM. In comparison, P. americana may
be somewhat less tolerant, with nearly complete mortality in 8 mM solutions.
On the other hand, P. americana plants grew successfully with internal leaf
concentrations of approximately 10,000 μg Mn g-1, whereas the tissue toxicity
threshold for most plants is typically much lower (Horst 1988), suggesting that
the species does possess at least some degree of inherent Mn tolerance. But fundamentally,
metal tolerance is a relative, not absolute, character (Pollard et al.
2002). Thus, it would be instructive to compare the degree of Mn tolerance in
Phytolacca species with that of other plants in the same community, a test that
has not to our knowledge been conducted. It would also be interesting to test
whether populations from manganiferous soils, such as the Mn-contaminated
region in China, are more tolerant than conspecific populations from uncontaminated
soils. As mentioned earlier, there is no record of P. americana occurring
on manganiferous soils within its native range in North America.
In some cases, high metal concentrations in shoot tissues may occur because
severe metal toxicity leads to nonspecific increases in membrane permeability,
resulting in “breakthrough” of metals into the shoot (Baker 1981, Köhl et al.
1997). In contrast, genuine hyperaccumulation typically involves few if any
detectable symptoms of stress. It is possible that some of the highest tissue concentrations
that have been reported for Phytolacca species, both in the present
study and in the literature, do represent breakthrough accumulation, particularly
in extreme treatments such as 25 and 50 mM hydroponic solutions (Min et al.
2007). On the other hand, high foliar Mn levels achieved in more moderate substrate
concentrations confirm that this species behaves as a hyperaccumulator.
2009 A.J. Pollard, H.L. Stewart, and C.B. Roberson 161
The strict definition of hyperaccumulation proposed by Reeves (1992)
refers to foliar metal concentrations of a plant “growing in its natural habitat.”
Thus, a plant should not be recognized as a hyperaccumulator solely on the basis
of responses to metal-enriched nutrient solutions (Reeves and Baker 2000).
Because the populations of P. americana that we studied in the southeastern
United States grow on non-manganiferous soils, it is not surprising that the Mn
concentrations of plants in their native habitats fall short of the hyperaccumulation
threshold. In this case, perhaps the species should be described as a “latent
hyperaccumulator,” because it possesses the physiology to hyperaccumulate a
metal that it does not typically encounter in elevated concentrations. Outside
the genus Phytolacca, the closest parallel may be the case of North American
Thlaspi montanum L. (Alpine Pennycress), which is reported to have a constitutive,
or species-wide, physiological ability to hyperaccumulate nickel, even
though the great majority of populations do not occur on soils with elevated
nickel (Boyd and Martens 1998). Phytolacca americana represents an even
more extreme case, in which there is no known association with metalliferous
soils in its native range. In this case, it makes little sense to seek direct adaptive
explanations for hyperaccumulation, because it is rarely if ever expressed in the
field. Among the proposed hypotheses which have been advanced for hyperaccumulation
(Boyd and Martens 1992), it may be most appropriate to propose
explanations based on inadvertent uptake, in which the tendency to accumulate
high Mn concentrations occurs as a indirect side-effect of some other physiological
characteristic in the species.
Literature Cited
Baker, A.J.M. 1981. Accumulators and excluders: Strategies in the response of plants to
heavy metals. Journal of Plant Nutrition 3:643–644.
Baker, A.J.M., and R.R. Brooks. 1989. Terrestrial higher plants which hyperaccumulate
metallic elements : A review of their distribution, ecology, and phytochemistry.
Biorecovery 1:81–126.
Baker, A.J.M., S.P. McGrath, R.D. Reeves, and J.A.C. Smith. 2000. Metal hyperaccumulator
plants: A review of the ecology and physiology of a biological resource
for phytoremediation of metal-polluted soils. Pp. 85–107, In N. Terry and G.S. Bañuelos
(Eds.). Phytoremediation of Contaminated Soil and Water. CRC Press, Boca
Raton, fl, USA. 389 pp.
Boyd, R.S. 2004. Ecology of metal hyperaccumulation. New Phytologist 162:563–567.
Boyd, R.S., and S.N. Martens. 1992. The raison d’être for metal hyperaccumulation by
plants. Pp. 279–289, In A.J.M. Baker, J. Proctor, and R.D. Reeves (Eds.). The Vegetation
of Ultramafic (Serpentine) Soils. Intercept Ltd., Andover, UK. 510 pp.
Boyd, R.S., and S.N. Martens. 1998. Nickel hyperaccumulation in Thlaspi montanum
var. montanum (Brassicaceae): A constitutive trait. American Journal of Botany
85:259–265.
Brooks, R.R., M.F. Chambers, and L.J. Nicks. 1998. Phytomining. Trends in Plant Science
3:359–362.
Clarkson, D.T. 1988. The uptake and translocation of manganese by plant roots. Pp.
101–111, In R.D. Graham, R.J. Hannam, and N.C. Uren (Eds.). Manganese in Soils
and Plants. Kluwer Academic Publishers, Dordrecht, The Netherlands. 344 pp.
Denaeyer-DeSmet, S. 1966. Note sur un accumulateur de manganèse, Vaccinium myrtillus.
Bulletin de la Société Royale de Botanique de Belgique 99:331–343.
162 Northeastern Naturalist Vol. 16, Special Issue 5
Fernando, D.R., I.E. Woodrow, T. Jaffré, V. Dumontet, A.T. Marshall, and A.J.M.
Baker. 2008. Foliar manganese accumulation by Maytenus founieri (Celastraceae)
in its native New Caledonian habitats: Populational variation and localization
by X-ray microanalysis. New Phytologist 177:178–185.
Gilkes, R.J., and R.M. McKenzie. 1988. Geochemistry and mineralogy of manganese in
soils. Pp. 23–35, In R.D. Graham, R.J. Hannam, and N.C. Uren (Eds.). Manganese in
Soils and Plants. Kluwer Academic Publishers, Dordrecht, The Netherlands. 344 pp.
Hewitt E.J., and T.A. Smith. 1975. Plant Mineral Nutrition. The English Universities
Press, London, UK. 160 pp.
Hoagland D.R., and D.I. Arnon. 1950. The water-culture method for growing plants
without soil. California Agricultural Experiment Station Circular 347:1–32.
Horst, W.J. 1988. The physiology of manganese toxicity. Pp. 175–188, In R.D. Graham,
R.J. Hannam, and N.C. Uren (Eds.). Manganese in Soils and Plants. Kluwer
Academic Publishers, Dordrecht, The Netherlands. 344 pp.
Köhl, K.I., F.A. Harper, A.J.M. Baker, and J.A.C. Smith. 1997. Defining a metal
hyperaccumulator plant: The relationship between metal uptake, allocation, and
tolerance. Plant Physiology 114(Supplement):562.
Lasat, M.M. 2000. Phytoextraction of metals from contaminated soil: A review of
plant/soil/metal interaction and assessment of pertinent agronomic issues. Journal
of Hazardous Substance Research 2(5):1–25.
McGrath, S.P., and F.J. Zhao. 2003. Phytoextraction of metals and metalloids from
contaminated soils. Current Opinion in Biotechnology 14:277–282.
Milner, M.J., and L.V. Kochian. 2008. Investigating heavy-metal hyperaccumulation
using Thlaspi caerulescens as a model system. Annals of Botany 102:3–13.
Min, Y., T. Boqing, T. Meizhen, and I. Aoyama. 2007. Accumulation and uptake of
manganese in a hyperaccumulator Phytolacca americana. Minerals Engineering
20:188–190.
Peng, K.J., C.L. Luo, W.X. You, C.L. Lian, X.D. Li, and Z.G. Shen. 2008. Manganese
uptake and interactions with cadmium in the hyperaccumulator Phytolacca
americana L. Journal of Hazardous Materials 154:674–681.
Pollard, A.J. 2000. Metal hyperaccumulation: A model system for coevolutionary
studies. New Phytololgist 146:179–181.
Pollard, A.J., K.D. Powell, F.A. Harper, and J.A.C. Smith. 2002. The genetic basis
of metal hyperaccumulation in plants. Critical Reviews in Plant Sciences
21(6):539–566.
Reeves, R.D. 1992. Hyperaccumulation of nickel by serpentine plants. Pp. 253–257,
In A.J.M. Baker, J. Proctor, and R.D. Reeves (Eds.). The Vegetation of Ultramafic
(Serpentine) Soils. Intercept Ltd., Andover, UK. 510 pp.
Reeves, R.D., and A.J.M. Baker. 2000. Metal-accumulating plants. Pp. 193–229, In
I. Raskin and B.D. Ensley (Eds.). Phytoremediation of Toxic Metals. John Wiley
and Sons, New York, NY, USA. 304 pp.
Salt, D.E., R.D. Smith, and I. Raskin. 1998. Phytoremediation. Annual Review of
Plant Physiology and Plant Molecular Biology 49:643–668.
Xue, S.G., Y.X. Chen, R.D. Reeves, A.J.M. Baker, Q. Lin, and D.R. Fernando. 2004.
Manganese uptake and accumulation by the hyperaccumulator plant Phytolacca
acinosa Roxb. (Phytolaccaceae). Environmental Pollution 131:393–399.
Xue, S.G., Y.X. Chen, A.J.M. Baker, R.D. Reeves, X.H. Xu, and Q. Lin. 2005. Manganese
uptake and accumulation by two populations of Phytolacca acinosa Roxb.
(Phytolaccaceae). Water, Air, and Soil Pollution 160:3–14.