The Scientific World Journal

Volume 2014 (2014), Article ID 421249, 6 pages

http://dx.doi.org/10.1155/2014/421249

Research Article

Tissue Fractions of Cadmium in Two Hyperaccumulating Jerusalem Artichoke Genotypes

Xiaohua Long,¹ Ni Ni,1,2 Zhaopu Liu,¹ Zed Rengel,³ Xin Jiang,² and Hongbo Shao1,4

¹Jiangsu Provincial Key Laboratory of Marine Biology, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China

²State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China

³School of Earth and Environment, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

⁴Key Laboratory of Coastal Biology & Bio-Resources Utilization, Yantai Institute of Coastal Zone Research (YIC), Chinese Academy of Sciences (CAS), Yantai 264003, China

Received 15 March 2014; Accepted 26 March 2014; Published 14 April 2014

Academic Editor: Xu Gang

Copyright © 2014 Xiaohua Long et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

In order to investigate the mechanisms in two Jerusalem artichoke (Helianthus tuberosus L.) genotypes that hyperaccumulate Cd, a sand-culture experiment was carried out to characterize fractionation of Cd in tissue of Cd-hyperaccumulating genotypes NY₂ and NY₅. The sequential extractants were: 80% v/v ethanol (), deionized water (), 1 M NaCl (), 2% v/v acetic acid (), and 0.6 M HCl (). After 20 days of treatments, NY₅ had greater plant biomass and greater Cd accumulation in tissues than NY₂. In both genotypes the fraction was the highest in roots and stems, whereas the and fractions were the highest in leaves. With an increase in Cd concentration in the culture solution, the content of every Cd fraction also increased. The and ratios in roots were lower in NY₅ than in NY₂, while the amount of other Cd forms was higher. It implied that, in high accumulator, namely, NY₅, the complex of insoluble phosphate tends to be shaped more easily which was much better for Cd accumulation. Besides, translocation from plasma to vacuole after combination with protein may be one of the main mechanisms in Cd-accumulator Jerusalem artichoke genotypes.

1. Introduction

Cd is one of biotoxic metal elements, which has strong chemical activity and long-term toxicity and is relatively mobile in plants [1, 2]. It is also one of the major environmental pollutants. Moderate Cd contamination of arable soils can result in considerable Cd accumulation in edible parts of crops [3–5]. Cd can be present in plant tissues in concentrations that are nontoxic to crops but can contribute to substantial Cd dietary intake by humans [6].

Existing methods of cleaning up Cd-contaminated soils are expensive, such as mechanical removal and chemical engineering [7]. Comparatively, phytoextraction has a great potential in ameliorating Cd-contaminated soils because it is a cost-effective, environmentally friendly approach applicable to large areas [8, 9].

Plant resistance to metal toxicity stress includes avoidance and tolerance [10]. Avoidance frequently results in exclusion, whereas accumulation in plant tissues must be linked with internal tolerance mechanisms. These tolerance mechanisms might rely on metal being retained mainly in roots, with transport to photosynthetically active above-ground tissues impeded. In addition, tolerance may be underpinned by metals existing in nonactive (nontoxic) forms in plant tissues. Such nontoxic forms may, for example, include binding of metals in the cell wall or complexation with organic acid and proteins mostly in the vacuole [11].

We have previously reported that two Jerusalem artichoke genotypes, NY₂ and NY₅, when grown in Cd contaminated soils did not suffer from Cd toxicity, even though Cd concentration not only in roots but also in leaves and stems exceeded 100 mg kg⁻¹ dry weight [12], which is the main feature of hyperaccumulators. Hence, NY₂ and NY₅ genotypes showed a potential to be used in phytoremediation of Cd-contaminated soils via phytoextraction. However, the research so far has mainly concentrated on Cd accumulation and plant physiological properties rather than on Cd chemical forms in Jerusalem artichoke genotypes. The Cd chemical forms in plant tissues are expected to be linked to Cd tolerance via detoxication mechanisms. An understanding of Cd-tolerance mechanisms in Jerusalem artichoke genotypes is a prerequisite for quick screening ofgermplasm for improved Cd accumulation and tolerance. This study was aimed at characterizing the distribution of Cd chemical forms in Jerusalem artichoke genotypes that hyperaccumulate Cd.

2. Materials and Methods

2.1. Plants

Two Jerusalem artichoke (Helianthus tuberosus L.) genotypes, NY₂ and NY₅, were selected from the Nanjing Agricultural University Experimental Station (“863 Program”) at Laizhou County in Shandong Province, China. Previous work [12] indicated that these two genotypes have the capacity to hyperaccumulate Cd.

2.2. Experimental Setup

The tests were carried out in a greenhouse at Nanjing Agricultural University (N32° 2′ 6.25′′, E18° 50′ 23.47′′), Nanjing, China. The average temperature throughout the test period was between 26.6 ± 4.4°C (daytime) and 22.0 ± 2.4°C (night), and the relative humidity was 61.5 ± 1.3% (daytime) and 68.0 ± 1.9% (night). Tuber slices with buds were germinated on sand moistened with 1/2 Hoagland nutrient solution in an incubator. The nutrient solution was replaced every second day. At trefoil stage, young plants were transplanted into porcelain pots. About one week later, Cd treatments were imposed (0, 2.5, 5.0, or 10 mg L⁻¹ as CdCl₂2.5 H₂O). Each Cd treatment was replicated in three pots, and two uniform plants were allowed to grow in each pot at a uniform spacing. Sampling was carried out after 3-week treatment duration.

2.3. Plant Sampling and Analysis

Roots were washed in deionized water, and then shoots and roots were separated, weighed, and used for sequential extraction to determine chemical forms of Cd [13, 14]. Briefly, 1 gram fresh leaf, stem, or root material was cut into pieces of 1-2 mm², transferred into a beaker with 10 mL of extractant (Table 1), and kept at 25°C overnight (20–24 h) on a shaker [15]. The following day the solutes were saved, and the residues were extracted again overnight with the next extractant. In total, there were five sequential extractions.

Table 1: Extractants and relevant extracted chemical forms of Cd.

Extract ion reagent
Code
Predominant forms of extracted Cd

80% v/v ethanol
FE
Cd-nitrate, Cd-chloride, Cd-amino acid complexes

Deionized water
FW
Soluble Cd-organic acid complexes, Cd(H₂PO₄)₂

1 M NaCl
FNaCl
Cd-pectates, Cd-polypeptide, or Cd-protein complexes

2% v/v acetic acid
FAcet
Sparingly soluble CdHPO₄, Cd₃(PO)₂, and/or other Cd-phosphate complexes

0. 6 M HCl
FHCl
Cd-oxalate

The extracts were digested with a concentrated acid mixture of HNO₃–HClO₄ (3 : 1 v/v) and heated at 160°C for 5 h. After cooling, the extracts were diluted, filtered, and made up to 25 mL with 5% v/v HNO₃. The Cd concentration in the extract was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, IRIS Intrepid II XSP; Thermo Electron Company, USA). The analyses were carried out in triplicate.

2.4. Symbol Meanings

FE, FW, FNaCl, FAcet, and FHCl show the amounts of the Cd-containing fractions extracted by ethanol, water, NaCl, acetic acid, and HCl, respectively.

2.5. Statistical Analysis

All statistical tests were performed using SPSS 13.0. Two-way ANOVA was used to determine the significance of genotype and Cd treatment effects on Cd forms. Mean treatment differences were separated by the least significant difference (LSD0.05) test if  -tests were significant (; Fisher’s protected test).

3. Results

3.1. Effects of Cd Treatments on the Biomass and Its Components of Two H. tuberosus Genotypes

Even though the two Jerusalem artichoke genotypes showed good tolerance to Cd toxicity, NY₂ showed some wilting in the high-Cd treatments. Compared to control, the Cd treatment (2.5 and 10 mg kg⁻¹) decreased leaf, stem, root, and total biomass for both NY₂ and NY₅ (Table 2). In contrast, the 5 mg kg⁻¹ Cd treatment increased the biomass and its components for NY₂. In every Cd treatment, the biomass and its components of NY₅ were lower than in the 0 mg kg⁻¹ Cd supply, but there was no significant difference.

Table 2: Effects of different cadmium treatments on the fresh biomass of two H. tuberosus genotypes.

Genotype
Cd supply (mg L⁻¹)
Leaves (g plant⁻¹)
Stem (g plant⁻¹)
Root (g plant⁻¹)
Whole plant (g)

NY₂
0
24.3a
10.7ab
23.3a
58.2a

2.5
24.6a
11.1ab
21.1a
56.8a

5
28.5a
13.0a
28.4a
69.9a

10
15.4b
8.3b
11.1b
34.9b

NY₅
0
29.8a
13.0a
25.8a
68.5a

2.5
29.1a
12.8a
17.1b
58.9a

5
30.1a
13.9a
20.9ab
64.9a

10
27.5a
12.7a
16.9b
57.1a

Different letters within a column indicate the significant differences among the treatments ().

3.2. Chemical Forms of Cd in Plants

3.2.1. Effects of Cd Treatments on Cd Chemical Forms of Roots in Two Jerusalem Artichoke Genotypes

As can be seen from Table 3, the distribution ratios raised with increased Cd supply in both NY₂ and NY₅. In control group, the difference between the five forms was not remarkable. In treatment group, the Fw ratio increased and was higher than the FR ratio in NY₂ and NY₅. FNACl occupied the most proportion in both two Jerusalem artichoke genotypes; secondly, FAcet and FE were the least.

Table 3: Fractionation of Cd in roots of two Jerusalem artichoke genotypes.

Genotype
Cd supply (mg L⁻¹)
FE (g g⁻¹)
FW (g g⁻¹)
FNaCl (g g⁻¹)
FAcet (g g⁻¹)
FHCl (g g⁻¹)

NY₂
0
11a
8.4a
11a
12a
8.3a

2.5
41c
48c
223a
127b
35c

5
38b
103b
655a
127b
54b

10
62b
150b
1675a
329b
91b

NY₅
0
3.7c
4.9bc
13a
7.9b
7.8b

2.5
51c
36c
304a
113b
33c

5
55b
83b
469a
259ab
63b

10
80b
117b
1280a
387b
245b

Different letters within a row indicate significant differences among the fractions ().

There were some differences of the five main chemical forms in roots between NY₂ and NY₅. The Fw ratio was higher in NY₂ than that in NY₅. Water extracts the soluble Cd organic acid complex, Cd (H₂PO₄)₂, which is poisonous and tends to cause harm to plants. The FAcet ratio of high accumulator was higher than low accumulator, while the Fw ratio was lower. Ethylic acid extracts unsoluble CdHPO₄, Cd₃(PO)₂, or Cd-phosphate complexes, which may be better for Cd accumulation in high accumulator.

3.2.2. Effects of Cd Treatments on Cd Chemical Forms of Stems in Two Jerusalem Artichoke Genotypes

We can see from Table 4 that the distribution ratios were raised with increased Cd supply in both NY₂ and NY₅. Compared to the roots, every proportion of the five chemical forms in stems decreased greatly. Similarly, FNaCl occupied the most proportion in both two Jerusalem artichoke genotypes; secondly FAcet and FR were the least. The Fw ratio was higher in NY₂ than that in NY₅. The FAcet ratio of high accumulator was higher than low accumulator, while the Fw ratio was lower.

Table 4: Cd chemical forms of stems in two Jerusalem artichoke genotypes.

Genotype
Cd supply (mg L⁻¹)
FR (g g⁻¹)
FW (g g⁻¹)
FNaCl (g g⁻¹)
FAcet (g g⁻¹)
FHCl (g g⁻¹)

NY₂
0
7.0a
5.8a
6.6a
8.7a
6.4a

2.5
17c
14c
126a
66b
35b

5
18b
28b
278a
48b
34b

10
22b
43b
315a
91b
57b

NY₅
0
2.9c
5.3b
7.4a
6.5ab
6.4ab

2.5
13c
13c
99a
54b
31bc

5
16a
17a
195a
92a
47a

10
32c
49bc
393a
135b
131b

Different letters within a row indicate the significant differences among the forms ().

3.2.3. Effects of Cd Treatments on Cd Chemical Forms of Leaves in Two Jerusalem Artichoke Genotypes

The distribution ratios were raised with increased Cd supply in both NY₂ and NY₅ (Table 5). Compared to stems, the five forms were further reduced in different degrees, especially FNaCl. FAcet covered the most part, 38% and 41%, respectively, in NY₂ and NY₅.

Table 5: Cd chemical forms of leaves in two Jerusalem artichoke genotypes.

Genotype
Cd supply (mg L⁻¹)
FR (g g⁻¹)
FW (g g⁻¹)
FNaCl (g g⁻¹)
FAcet (g g⁻¹)
FHCl (g g⁻¹)

NY₂
0
8.7a
5.2a
5.9a
9.4a
8.9a

2.5
13c
6.9c
20c
70a
47b

5
8.3b
12b
17b
44a
42a

10
15bc
9c
20b
56a
45a

NY₅
0
1.4c
5.3b
7.3a
6.5ab
7.5a

2.5
7.4b
6.6b
14b
45a
47a

5
10b
8.9b
14b
61a
78a

10
15b
15b
41b
129a
112a

Different letters within a row indicate the significant differences among the forms ().

The FW ratio was the lowest in leaf instead of the FR ratio in NY₂. Ethanol extracts Cd-nitrate, Cd-chloride, and Cd-amino acid. The lowest FNaCl ratio might be beneficial for protecting leaves because NaCl extracts mainly Cd-pectates, Cd-polypeptide, or Cd-protein.

4. Discussion

4.1. Cd Distribution in Cells of Roots

Cd concentration showed the same order (root > stem > leaf > tuber) and Cd accumulation in plant components showed the order of stem > leaves > roots for both NY₂ and NY₅ in previous work [12]. Cd chemical forms in plants were linked with Cd transporting activity, among which FR and FW were the strongest; secondly, FNaCl, FAcet, and FHCl were the weakest [16]. Most of Cd enriched in roots after absorbing by plants and the amount transported up to shoots was usually a little [17]. Cd concentrated in roots which might be related to Cd that formed stable large molecule complex with protein, polysaccharide, ribose, and nuclein in roots, deposited [18], and then lightened poisoning to organs in shoots. Cd accumulation in roots is usually accredited to cell wall [19]. Cell wall is the first protective screen protecting cell protoplast from being harming by heavy metals. Cellulose, hemicelluloses, xylogen, and pectic substance which consist of cell wall have abundant active perssads, such as carboxyl, oxhydryl, and aldehyde group. A part of external Cd being passed through will combine with these perssads. It prevents large amount of Cd from going into plasma and reduces toxicity [16]. Especially in the condition of short time and low concentration, this tolerance system of the combination of Cd and cell wall is most important [20]. However, some scholars consider oppositely that the amount of Cd combined with root cell wall is much less than that in the cell [19, 21, 22]. Vacuole is a Cd accumulating place in higher plants, but not the main point, only in the condition of high Cd concentration [19, 23]. As can be seen from Table 3, the absolute advantage laid with the FNaCl ratio, showing that Cd mainly adheres to protein. This is because that Cd has very strong affinity with protein or hydrosulphonyil in other organic compounds [24]. On the one hand, the combination of Cd and protein in plants can decrease the amount of free Cd, reducing its availability and mobility and avoiding harm to plants. Cd also may be combined with enzymes and functional proteins, disturbing their regular function and disordering physiological and biochemical metabolism [25]. Because of the higher Fw ratio and the lower FAcet ratio, the amount of Cd is more poisonous and is transported more quickly in NY₂ than that in NY₅, with NY₂ showing fewer biomass of roots in some degree and NY₅ showing normal for the growth of 20 days.

4.2. Cd Transporting from Roots to Shoots

The transporting of Cd from roots to shoots and accumulated in shoots is a very complicated process. There are many studies on it [26]. It is usually considered that Cd absorbed by roots is transported to other components in plants through the xylem. Root metal ions go into root vascular bundle through endoderm and the inner casparian strip. Passing through casparian strip is difficult, so this translocation is mainly carried out in young roots in which casparian strip has not been formed completely [27]. Then mental ions may be transported up to shoots by transpiration. There are many reports on long-distance translocation and the system of Cd long-distance translocation in plants is controversial. The Fw and FNaCl ratios were reduced substantially in both NY₂ and NY₅, so compared to control, stems did not suffer toxicity in NY₅ while except when at the Cd concentration of 10 mg L⁻¹ and the biomass of stem in NY₂ decreased by small degrees.

4.3. Cd Accumulation and Distribution in Leaves

Cd in leaf cells mainly comes from the water translocation from vascular bundle to leaf tissue which indicates that transpiration plays an important role in heavy mental accumulation [19]. Similar to root cells, the combination of leaf cell wall and Cd decreases Cd concentration in cell sap, lightening toxicity to leaf cells. However, the interception of cell wall plays a secondary role and the main detoxication mechanism is in the vacuole [19]. There are rich small molecule substances, such as GSH, oxalic acid, histidine, citrate, and phosphoric acid in vacuole. Cd avoids contacting with organelle to realize Cd detoxication through chelation or laydown with those small molecule substances [28, 29]. Compared to other chemical forms, the FAcet ratios have absolute advantage in both two Jerusalem artichoke genotypes showing that a considerable part of Cd tends to form insoluble phosphate in leaves; accordingly, the amount of free Cd which is poisonous becomes low (Table 5). Therefore, from the appearance, there is almost no remarkable difference between the biomass of treatment group and that of control group in NY₂ and NY₅ leaves. Previous studies have documented that Cd exists and transports in ion form in some certain plants [30, 31]. But in some Cd-accumulators, Cd existence is mostly in organic combination [31].

5. Conclusions

In summary, Cd toxicity and tolerance mechanism are most complex. Different plants, even different strains of the same plant or different ecological types, may show diverse Cd tolerance ability and mechanism. According to the previous study, compared with NY₂, genotype NY₅ may be a better candidate for phytoremediation of and biofuel production on Cd-contaminated soils. The present study implied that in high accumulator, namely, NY₅, the complex of insoluble phosphate tends to be shaped more easily which is much better for Cd accumulation. Besides, translocation from plasma to vacuole after combination with protein may be one of the main mechanisms in Cd-accumulator Jerusalem artichoke genotypes.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Xiaohua Long and Ni Ni contributed to the paper equally.

Acknowledgments

The authors are grateful for the financial support of National Natural Science Foundation of China (no. 31201692), the National Key Projects of Scientific and Technical Support Programs funded by the Ministry of Science and Technology of China (no. 2011BAD13B09), the Project of a Special Fund for Public Welfare Industrial (Agriculture) Research of China (no. 200903001-5), the Ministry of Science and Technology of Jiangsu Province (no. BE2011368), and Fundamental Research Funds for Central Universities (no. Y0201100249).

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