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III. Cadmium in the Tobacco Plant

III. Cadmium in the Tobacco Plant

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Compared to other crops, tobacco (Nicotiana tabacum) can accumulate

relatively high levels of Cd as shown by the results of a pot experiment using soil

containing 69 ppm Cd: tobacco leaf concentrations were three times higher than

the highest value reported among all other crops tested (Davis, 1984). However,

at relatively low Cd exposure, tobacco might not accumulate more Cd than leafy

crops, such as spinach, Swiss chard, or lettuce (Wagner, 1993).

Various studies have been performed under controlled conditions to study Cd

uptake, phytotoxicity, and tolerance in tobacco. However, the Cd exposures used

in many of the reported studies were much higher than those found in a field

situation, and therefore the results may not be directly applicable for

understanding the field response of tobacco to Cd. Tobacco grown hydroponically with 1.5 mg/l cadmium chloride (CdCl2) developed normally and

generally without visible symptoms of toxicity, despite the accumulation of up to

226 ppm Cd in the lower leaves of the plant. In a separate experiment, necrotic

spots were observed on lower leaves, which contained a mean Cd concentration

of 560 ppm (Phu-Lich et al., 1990). Nicotiana rustica (cv. Pavonii) seedlings

grown hydroponically with 5 –40 mM Cd did not show signs of Cd toxicity, and

reduced root growth was only observed with 40 mM Cd (Voăgeli-Lange and

Wagner, 1996). We have obtained comparable results in our experimentation

(Fig. 1). Exposure to hydroponic levels up to 100 mM CdCl2 was phytotoxic to

varying degrees, depending on the Nicotiana species; and exposure to levels

closer to 10 mM yielded leaf-lamina Cd concentrations around 200 ppb.

Tobaccos (cv. Samsun NN and cv. Samsun) grown in a solution containing

2 mg/l CdCl2 contained up to 255 and 352 ppm Cd in their leaves, respectively,

without showing visible symptoms of toxicity, although the yield of cv. Samsun

NN was significantly reduced by 18.2% in dry weight (Harkov and Brennan,

1981; see also Clarke and Brennan, 1983). Similarly, Clarke and Brennan (1989)

did not observe visible symptoms of toxicity in tobacco that contained up to

383 ppm Cd in its leaves, but the overall plant growth was affected. Interestingly,

after a 30-day treatment with 0.25 ppm Cd, shoot height significantly increased

by 10 –40%, and internode length, leaf number and generally fresh weight also

increased, while leaf and root dry weight generally decreased, compared with

control plants (Clarke and Brennan, 1989). Others have noted an apparent

stimulatory effect of low Cd on the growth of plants and algae (Wagner, 1993).

A stimulatory effect was also described for tobacco callus tissue and tobacco cell

suspension cultures at low to moderate Cd concentrations (Maroti and Bognar,

1985; Hirt et al., 1989, 1990) while high Cd inhibited growth.

Hence, while relatively low Cd levels can stimulate some growth parameters

in tobacco, such as shoot height, very high Cd concentrations are toxic to the

plant. In tobacco fields, exposure to Cd is not expected to cause visible symptoms



Figure 1 Demonstration of phytotoxic effects of CdCl2 during hydroponic greenhouse growth of

Nicotiana species. N. tabacum (A, B, C), N. rustica (D, E, F), and Nicotiana sylvestris (G, H, I)

were grown hydroponically and exposed to 0 mM (A, D, G), 10 mM (B, E, H), and 100 mM (C, F, I)

of CdCl2. The plants were flooded and then drained with growth solution (Hoagland’s solution plus

Cd) three times daily (i.e., the plants were not grown in a continuous state of flooding). The reservoirs

feeding the hydroponic fields were replaced weekly or as needed. The photographs show that

these three species have different sensitivities to Cd exposure. N. sylvestris was nearly killed by the

100 mM dose, and N. tabacum was clearly stunted under the same condition, while N. rustica appeared

to thrive.



of Cd toxicity, since field Cd levels are typically low and also because of

tobacco’s relative tolerance to Cd.

Various effects of high to very high Cd exposure on tobacco physiology have

been reported; they include retarded growth, yellowing of leaves, decreased

biomass, decreased photosynthetic rate, decreased leaf chlorophyll a and b

content, decreased chlorophyll a fluorescence, inhibition of photochemical

activity of photosystem II (but not that of photosystem I), rotting of roots, leaf

chlorosis, and small rust-like circular spots on leaves (Jiang and Li, 1989; Li et al.,

1990, 1992; Phu-Lich et al., 1990; Min et al., 1997; Guadagnini, 2000; Choi et al.,

2001). Increasing the Cd concentrations up to 150 ppm in the growth medium led

to an increase in the protein content of mid-stalk position leaves (cv. Dajinyuan)

(Li et al., 1990, 1992).

It is noted that Cd may also affect tobacco – virus interactions (Citovsky et al.,

1998; Ueki and Citovsky, 2002). For example, Harkov and Brennan (1981) found

that increasing Cd levels from 0 to 2 mg/l in the growth medium increased the

sensitivity of tobacco (cv. Samsun NN) to tobacco mosaic virus (TMV). Unlike

this TMV-hypersensitive cultivar, no evidence could be found for increased

sensitivity to TMV in the systemic host cv. Samsun.

Ex vivo studies (tobacco hairy root, cell and tissue cultures) also reflect upon

Cd phytotoxicity, tolerance and accumulation patterns in tobaccos (Reese and

Roberts, 1984; Piqueras et al., 1999; Fojtova and Kovarik, 2000; Guadagnini,

2000; Nedelkoska and Doran, 2000b; Fojtova et al., 2002). Senescence in BY-2

(tobacco bright yellow) cultured cells required a 10-fold higher Cd concentration,

compared with that inducing animal cell death, suggesting that there are

mechanisms by which tobacco is able to cope with limited exposure to high levels

of this metal (Fojtova and Kovarik, 2000). Different Cd concentrations in the

culture medium can have quite different effects on cell morphology, viability, and

DNA integrity of BY-2 suspension cells (Fojtova and Kovarik, 2000). However,

tolerance to Cd can be increased by progressively elevating Cd levels in the

culture medium (Huang and Goldsbrough, 1988; Domazlicka and Opatrny,

1989). Differences in Cd uptake and tolerance reported among studies may be

due to different initial Cd concentrations in the medium, different exposure time,

different species, or other variables.


Cadmium primarily enters the tobacco plant through the roots. Once in the

roots, it can either be stored or exported to the shoot. In N. tabacum, the

proportion of Cd translocated from root to shoot is higher than in N. rustica,

which efficiently retains more Cd in its roots (Fig. 2; Wagner and Yeargan, 1986).

Cadmium translocation to the shoot appears to be rapid. In N. rustica exposed to



Figure 2 Cd distribution in hydroponic, greenhouse-grown Nicotiana species. Six species of

Nicotiana were grown hydroponically in the greenhouse and exposed to 10 mM CdCl2. The plants

were flooded with growth solution (Hoagland’s solution plus Cd) and drained three times daily. The

plants were grown to flowering and then harvested. The different plant organs were separated. The

respective organs from three plants were pooled together to create one sample. All samples were

lyophilized, digested via microwave in concentrated nitric acid, and analyzed for Cd by inductively

coupled plasma-mass spectrometry (ICP-MS). The data showed that N. rustica distinguished itself by

having higher Cd concentration in the roots and lower concentration in the leaf lamina, compared with

the other five species.

40 mM Cd, the Cd was detected in the leaves within 3 h (Voăgeli-Lange and

Wagner, 1996).

Grafting experiments suggest that N. tabacum roots control the amount of Cd

translocated to the shoots and, therefore, the amount of metal accumulated in the

leaf (Wagner et al., 1988). Indeed, Cd treatment, added in the nutrient solution or

in the soil of stem grafts of N. rustica (cv. Pavonii) shoots on N. tabacum

(cv. KY14) roots, resulted in leaf Cd accumulation typical of N. tabacum, and

vice versa.

Transport to the shoot primarily takes place through the xylem. Xylem loading

is likely facilitated by yet-unidentified membrane-transport processes. Cadmium

can certainly reach the xylem via symplastic transport, but probably also through

the extracellular spaces (apoplastic transport), at least under high-level exposure.

Apoplastic transport has been suggested for zinc (Zn) in T. caerulescens grown

with high external Zn concentrations (White et al., 2002). In the xylem,

transpiration-driven mass flow may play an important role in the movement of

Cd, probably in the form of non-cationic, organic acid complexes, such as Cdcitrate (Senden et al., 1995). Theoretical studies predict that the majority of

the iron (Fe) and Zn (the latter being chemically similar to Cd) in xylem

sap should be chelated by citrate. In contrast, phytochelatins (PCs) and other



thiol-containing ligands do not seem to play a direct role in Cd transport in the

xylem (Salt et al., 1998), although results obtained by Larsson et al. (2002)

suggest that, on prior exposure to high Cd, the presence of PCs in Arabidopsis

thaliana may increase Cd uptake and translocation from roots to shoot. Recently,

Gong et al. (2003) demonstrated that PCs can be transported from roots to shoots

and are indeed involved in Cd root-to-shoot transport in Cd-exposed (20 mM

CdCl2) A. thaliana (see Section V.A.3 for a discussion of PCs).

Once in the shoot, it is possible that some Cd may be redistributed in the

plant via the phloem, as suggested by results of studies on Zn in wheat (Herren

and Feller, 1994) and by data obtained with leaf-applied 109Cd (Cakmak et al.,

2000). The transport mechanisms of Cd from root to shoot remains to

be elucidated in tobacco and other plants, and it likely differs with Cd

exposure level.


The pattern of Cd accumulation may differ substantially within the genus

Nicotiana (Fig. 2; Wagner and Yeargan, 1986). For example, N. tabacum

appears as a leaf and root Cd accumulator, whereas N. rustica is primarily a

root accumulator.

Cd concentration in tobacco, which increases with increasing amounts of Cd

in the growth medium, is usually higher in the leaves than in the stem (MacLean,

1976; Ruick and Schmidt, 1981; Clarke, 1983; Clarke and Brennan, 1989; Mench

et al., 1989). Clarke and Brennan (1989) reported mean leaf and stem Cd

concentrations of 0.7 and 0.3 ppm, respectively, in tobacco grown under

controlled conditions, without exogenous Cd supply. Moreover, N. tabacum can

accumulate higher Cd concentrations in its leaves than in its roots (MacLean,

1976; Clarke and Brennan, 1983, 1989; Logan and Chaney, 1983; Wagner and

Yeargan, 1986; Mench et al., 1989; Keller et al., 2003). By estimating the rootto-leaf Cd ratio from average root and leaf Cd concentrations of 16 cultivars

grown under greenhouse conditions without added Cd (Clarke and Brennan,

1989), ratios of less than 1.0 are obtained (from 0 to 0.75; Fig. 3). Ratios greater

than 1.0 can also be found for tobaccos exposed to low exogenous Cd. The

Brazilian tobacco cultivars, T28, T10, T63, T44, and T48 (Salviano et al., 2001),

and “Taiwan tobacco #5” (Wen, 1983) were grown hydroponically, and higher

Cd concentrations were reported in the roots than in the shoot/leaves (Fig. 3;

Wen, 1983; Salviano et al., 2001). However, these authors found moderate Cd

concentrations in the plants that were grown with no added Cd in the growth

solution. This suggests Cd contamination either during plant growth or during

sample preparation for metal analyses.



Figure 3 Cd concentrations in leaves (shoot for cv. T28, T10, T63, T44, and T48) and roots

of hydroponically or pot-grown tobacco not exposed to exogenous additions of Cd, with root-toleaf (or root-to-shoot) ratios estimated from these average Cd concentrations. Data are from

MacLean (1976), Clarke and Brennan (1983, 1989), Wen (1983), Mench et al. (1989), and

Salviano et al. (2001).


Several studies have reported Cd concentrations in field-grown tobacco

leaves. The Cd concentration is variable, depending on agro-climate, soil

parameters (see Section IV), and cultivar (see Section III.I). Usually, the Cd

concentration in field-grown tobacco leaves (including midribs and veins)

ranges from less than 0.5 to 5 ppm (Westcott and Spincer, 1974; Frank et al.,

1977, 1980, 1987, 1991; Nwankwo et al., 1977; Schmidt et al., 1985; Murty

et al., 1986; Oto and Duru, 1991; Saldivar et al., 1991; Tamboue et al., 1991;

Bell et al., 1992; Yue, 1992; Cai et al., 1995; Gondola and Kadar, 1995; Shariat

and Eddin, 1999; Matsi et al., 2002; Tsotsolis et al., 2002). However, the reports

of these studies typically do not define the leaf stalk position, the tobacco

maturity stage, the tobacco variety or the exact part of the leaves analyzed (e.g.,

with or without midribs). These factors, which are discussed in detail below,

may all, to different extents, influence the reported Cd concentration of tobacco

leaves. Furthermore, the reported Cd concentration measured in tobacco leaves

also depends on sample preparation and the accuracy of the analytical methods

used. Older studies may be more likely to have methodological errors, so care

should be taken when comparing values across studies (Scherer and

Barkemeyer, 1983).



Variation in Cd concentration in tobacco leaves is found both at the micro- and

macrogeographic scales. For example, in Turkey, Oto and Duru (1991) reported

lower concentrations in tobacco from the Aegean region than that from four other

regions investigated. Bell et al. (1992) studied metals in mid-stalk, air-cured

leaves of Maryland tobacco over several years. Of the 402 samples analyzed, 5%

had Cd levels in the range of 0.4– 0.8 ppm, 22.5% in the range of 0.8– 1.5 ppm,

48% in the range of 1.5 –3.2 ppm, 19% in the range of 3.2– 5.7 ppm, and 5.5%

had values between 5.7 and 15.6 ppm. The wide range in Cd levels of the last

group was attributed to growth in the presence of municipal sludge (can be

contaminated with Cd) or inadequate liming (see Section IV.B).

Tobacco grown in Cd-contaminated areas can accumulate high levels of Cd

in the leaves. Grabuloski et al. (1985) found 35.8– 47.7 ppm Cd in tobacco

grown in the vicinity of a Zn and Pb smelter in Macedonia, while values ranged

0.75– 1.82 ppm Cd in the usual tobacco-producing regions of Macedonia. In

Cd-polluted areas of Dayu county in the Jiangxi province of China, high

average Cd concentrations, ranging from 8.6 (Yue, 1992) to 17.4 ppm (Cai et al.,

1995), have been reported in tobacco leaves. In comparison, tobacco leaves

from non-polluted areas of the same province had average Cd levels nearly 10

times lower (1.86 ppm; Cai et al., 1995). Yue (1992) also reported a lower

average Cd value (1.48 ppm) for tobacco from five main tobacco-producing

areas of China, but there were differences among these areas. It has been

noted that field trials using metal-contaminated sludge indicate that Cd in

tobacco can reach exceptionally high values (67.4 ppm in the leaf lamina,

Gutenmann et al., 1982).


More Cd accumulates in lower leaves than in the medium and upper leaves,

suggesting a permanent Cd accumulation mechanism (Westcott and Spincer,

1974; Frank et al., 1977, 1980, 1987, 1991; Wagner and Yeargan, 1986; King,

1988; King and Hajjar, 1990; Phu-Lich et al., 1990; Miele et al., 2002). Studies

performed on cured tobacco leaves from Canadian farms located in the prime

growing regions of the watersheds of Lake Erie and Lake Ontario indicate that

cured sand (lower) leaves have a higher average Cd concentration than upper

leaves (Fig. 4; Frank et al., 1977, 1980, 1987, 1991). In contrast, Ruick and

Schmidt (1981) found higher Cd concentrations in the laminae of the top leaves

of tobacco (cv. “S53”) grown in fields in Germany (soil Cd background level:

0.101 ppm), yet the same cultivar grown in the laboratory in normal garden soil

(Cd background level: 0.77 ppm) tended to have somewhat higher Cd

concentrations in the lower leaves.



Figure 4 Tobacco leaf Cd concentration according to stalk position. (A) Tobacco grown

hydroponically with or without addition of Cd to the growth medium (data from Phu-Lich et al.,

1990). (B) Field-grown tobacco from Lake Erie and Lake Ontario watersheds in Canada, sampled

from 1974 to 1976, and from Ontario sales warehouses (1976p) (data from Frank et al., 1977, 1980).

(C) Same as B, but sampled in 1981– 1989 (data from Frank et al., 1987, 1991).





Leaf Cd accumulation in N. tabacum increases with leaf age (Ruick and

Schmidt, 1981; Wagner and Yeargan, 1986). The Cd contents of a mature and an

immature leaf of mature N. tabacum grown with 30 mM cadmium sulfate

(CdSO4) ranged 300 – 750 and 180 – 350 ppm, respectively (Wagner and

Yeargan, 1986). Wagner and Yeargan (1986) reported that Cd was primarily

found as soluble Cd in seedling roots of N. tabacum and N. rustica

(soluble:insoluble ratio 9:1). In contrast, the ratio was 1:1 in mature roots. The

soluble Cd found in roots (both mature and immature) is largely in the form of

anionic, low-molecular-weight Cd-binding peptides (phytochelatins), due to the

relatively high Cd exposure. In mature roots, the nature of the insoluble fraction

is unknown.


Wagner and Yeargan (1986) reported that Cd was almost uniformly distributed

in 38-cm-long mature leaves and 22-cm-long immature leaves of a mature

tobacco, with some enrichment in the center lower portion and the center upper

portion of mature and immature leaves, respectively. Bache et al. (1985) observed

that the leaf lamina contained higher Cd concentrations than did the midribs.

Tobaccos (cv. Virginia 115) grown in pots (soil: 0.98 ppm Cd) contained 1.87 and

1.20 ppm Cd in the laminae and ribs, respectively. When 1% of a sludge containing

87.2 ppm Cd was added to the soil, the values were 5.33 and 4.90 ppm Cd,

respectively. Similar results were obtained for experimentally field-grown

tobacco: 3.18 and 2.24 ppm Cd in the leaf laminae and ribs, respectively; and

67.4 and 36.3 ppm Cd, respectively, when 224 metric tons/ha of a Cdcontaminated sludge (84 ppm Cd) was applied to the soil (Gutenmann et al.,

1982). Under high Cd exposure, we have also observed that the leaf lamina of six

hydroponically grown Nicotiana species contained higher Cd concentrations than

did the midribs (Fig. 2). As the Cd concentration is usually higher in the lower

leaves than in the upper leaves (see Section III.E), the Cd concentrations in the

lamina and in the midribs will also differ according to stalk position. An example

for field-grown tobacco is provided by Ruick and Schmidt (1981), although

these authors did not find the highest Cd concentrations in the lower leaves

(see Section III.E). They reported 1.57 – 2.57 and 1.47– 1.75 ppm Cd in lower leaf

laminae and lower leaf midribs, respectively, while in the mid-leaves, the corresponding values were 1.79– 1.96 and 1.01– 1.35 ppm (Ruick and Schmidt, 1981).

The leaf epidermis of tobacco contains long and short trichomes that may act

as a sink during metal detoxification processes (Choi et al., 2001). Compared

with untreated seedlings, tobacco (cv. Xanthi) that was exposed to 200 mM Cd



for several weeks had up to twofold more trichomes, which secreted more than

10-fold larger crystals (ca. 150 mm long), with an estimated 2.2% Cd content

(Choi et al., 2001). These results suggest that, under extremely high Cd exposure,

trichomes play a role in a Cd storage and disposal mechanism in tobacco.

However, the Cd content of trichomes has not been studied in tobacco grown

under field Cd concentrations.


Within the plant cell, Cd can be found in various cell components (e.g.,

cell wall, cytoplasm, chloroplast, nucleus, vacuoles; Ramos et al., 2002).

Distribution may vary according to the species, organ, and tissue. In addition,

differences among studies also depend on the Cd concentrations used.

Cd can accumulate in the cell vacuoles of higher plants, which may serve

to protect the cell from the toxic effects of Cd (Chardonnens et al., 1998).

When grown hydroponically with 20 mM Cd for 1 week, N. rustica (cv.

Pavonii) predominantly sequesters Cd within the vacuole sap, but not the

tonoplast (Voăgeli-Lange and Wagner, 1990). However, at eld-like Cd concentrations, the accumulation pattern may be different.

Although most Cd is found in the cell wall fraction in lettuce leaves (Ramos

et al., 2002), it is not yet clear to what extent Cd can bind to the cell wall in fieldgrown tobacco. Based on data obtained with tobacco hairy roots cultures exposed

to 20 ppm Cd, it appears that most Cd is not associated with the root cell walls,

although the very high concentrations used in the study do not allow extrapolation

to field-grown tobacco (Nedelkoska and Doran, 2000a,b). Using Cd concentrations up to 7.6 mM, Wagner and Yeargan (1986) reported significant amounts of

Cd in the insoluble fractions containing the tobacco cell walls. In their study, the

soluble:insoluble Cd ratio was usually greater than 1. However, some insoluble Cd

may be due to binding after homogenization. It is still not clear how Cd is

partitioned at the sub-cellular level in conventionally field-grown N. tabacum.


Crop species have been divided into those capable of low, moderate, or high

cadmium accumulation (Arthur et al., 2000). Moreover, Cd concentration can

vary among cultivars/varieties, as was found for various plant species

(McLaughlin et al., 1996). For example, the durum wheat lines, Nile, Biodur,

and Hercules, consistently exhibit low Cd concentration (, 100 ppb) whereas the

cultivar, Kyle, consistently exhibits high (greater than 200 ppb) Cd concentration

(Penner et al., 1995).



Hydroponic studies show that the response of different N. tabacum cultivars to

Cd stress may differ and depend, for example, on Cd supply. Salviano et al.

(2001) found differences among five Brazilian tobacco cultivars grown

hydroponically for 30 days. When no Cd was added to the growth solution,

shoot Cd concentrations of cv. T28, T10, T63, T44, and T48 were 1.8, 1.5, 2.2,

2.5, and 1.9 ppm, respectively, while corresponding root concentrations were 2.1,

3.0, 2.8, 4.8, and 5.2 ppm, respectively. These data suggest different root-to-shoot

transport between these cultivars. In a greenhouse experiment, Clarke and

Brennan (1989) investigated the differences in Cd concentration in 16

commercial cultivars of tobacco exposed for 30 days to one of the three Cd

treatments: 0, 0.25, and 1 ppm Cd as CdCl2. For the 0 ppm Cd treatment, leaf and

root Cd concentrations did not significantly differ among the 16 cultivars; only

the Cd concentration in the stem of cv. G28 was significantly higher (0.7 ppm)

than that of cv. NC-232 or McNair-944 (0.1 ppm each). At higher Cd exposures,

various responses were reported. For example, the leaves of cv. NC-232 reached

a significantly higher Cd concentration (382.6 ppm) than those of cv. Coker-48

(243.4 ppm) at the 1 ppm Cd treatment, but at the 0.25 ppm Cd treatment, the

opposite occurred.

These results show that Cd concentration of a cultivar may vary according

to Cd exposure, and hence, those results from high Cd exposures may not

mimic real-field conditions. This is further illustrated by results obtained by

Wagner and Yeargan (1986). When grown hydroponically with 40 mM Cd, the

leaves of cv. Beinhart 1-1000 contained 35% of the Cd concentration found in

other cultivars (cv. KY14, KY151, KY L4-L8, NC 95, and Burley 21).

However, at lower Cd concentrations, the difference was less, as cv. Beinhart

1-1000 contained 80% of the amount of Cd, compared with other N. tabacum

cultivars. Moreover, the response of different tobacco cultivars to Cd exposure

may also depend on other factors. For example, when infected with TMV, cv.

Samsun (a systemic host) appears to be a better leaf Cd accumulator than cv.

Samsun NN (hypersensitive host) at Cd treatments from 0 to 2 mg/l

(Harkov and Brennan, 1981). When uninfected with this virus, cv. Samsun

also accumulated more Cd than cv. Samsun NN at the 0 ppm Cd treatment,

but a reverse pattern was observed at the 2 ppm Cd treatment (Clarke and

Brennan, 1983).

Published field data also suggest that some differences exist between tobacco

cultivars, although all or part of the differences observed may be due to other

factors, such as differences in soil characteristics, or soil Cd heterogeneity. For

instance, tobacco varieties cultivated in Germany accumulated Cd at different

concentrations when grown in the same, highly Cd-contaminated soil conditions

(Isermann et al., 1983). Schmidt et al. (1985) found higher amounts of Cd in

a flue-cured tobacco than in a Burley variety.

Available published data indicate that differences in Cd uptake and root-toshoot translocation abilities can exist among tobacco cultivars, although most

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III. Cadmium in the Tobacco Plant

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