2005 Progress Report: Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins

EPA Grant Number: R829479C021
Subproject: this is subproject number 021 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
Investigators: Cheng, Zong-Ming
Institution: University of Tennessee - Knoxville
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 2004 through September 30, 2007 (Extended to December 31, 2007)
Project Period Covered by this Report: October 1, 2004 through September 30, 2005
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

The objectives of this research project are to: (1) construct the transformation vectors with the already cloned metallohistin cDNA agNt84 under control by constitutive promoter CaMV35S; (2) transfer the genes into a woody plant, a Populus hybrid, and annual plants, Brassica napus and B. juncea, and confirm transformation by polymerase chain reaction (PCR) and Southern blot; (3) determine levels of RNA and protein expression in different tissues; (4) characterize the transgenic plants for their metal accumulation capacity and phytoremediation potential; and (5) determine the short- and long-term effects of the overexpression of metallohistin on growth and development of the transgenic plants.

Progress Summary:

Objective 1: To Construct the Transformation Vectors With the Already Cloned Metallohistin cDNA agNt84 Under Control By Constitutive Promoter CaMV35S

AgNt84 was PCR amplified from the initial cDNA clone using forward 5’CAGCGGATCCCATTGTCTCCAATCCTCTTCA3’ and reverse primer 5’TGACGAGCTCAGACGTACGTACAACTGATAGCAT3’, which introduced BamHI and SacI sites, respectively. The BamHI-SacI fragment was then ligated into pBI121, in front of a CaMV35S promoter as shown in Figure 1. The vector was introduced into Agrobacterium tumefaciens strain GV3850.

Figure 1. pBINAgNt84: Binary Vector Used for Transformation

Objective 2: To Transfer the Genes Into a Woody Plant, a Populus Hybrid, and Annual Plants, Brassica juncea, and To Confirm Transformation by Polymerase Chain Reaction and Southern Blot

Because Populus and Brassica transformation is slower, we proceeded to transform tobacco and Arabidopsis to evaluate the construct. Arabidopsis thaliana ecotype Col1 plants were transformed by the floral dip method [1] and Nicotiana tabacum cv Xanthi were transformed by the leaf disc transformation method.

Objective 3: To Determine Levels of RNA and Protein Expression in Different Tissues

Expression of AgNt84 was confirmed by northern blot (Figure 2). The protein, however, was in an unextractable form, tightly bound to the cell wall. Immunostaining of cell wall pellets showed a strong fluorescent signal from transgenic plants compared to controls (Figure 3).

Figure 2. Northern Blot of Transgenic Arabidopsis Lines. Ten μg total RNA was run and probed with the AgNt84 cDNA clone. Bottom panel is 18S RNA shown for equal loading.

Figure 3. Immunostaining of Pellets From Cell Wall Protein Extraction Show That AgNt84 is Still Tightly Bound Within the Cell Wall. Immunostaining was with rabbit preimmune and anti-AgNt84 immune serum, followed by incubation with FITC-linked goat anti-rabbit IgG as the secondary antibody.

Objective 4: To Characterize the Transgenic Plants for Their Metal Accumulation Capacity and Phytoremediation Potential

Preliminary experiments were conducted to assess the metal binding ability of transgenic plants by a dithiazone staining method. Tobacco plants hydroponically grown in 5 mM MES buffer without Cd showed no staining characteristic of Cd accumulation. When grown on buffer containing 200 mM Cd(NO3)2, staining was observed starting from day 1 in root tissue (Figures 4 and 5 and Table 1).


rubber band disk

tube buffer band

Figure 4. Hydroponically Grown Tobacco Plants. The first two figures are diagrams of the setup of the hydroponic containers, and the remaining pictures are of the plants at various steps in the metal uptake experiment. A. A diagram of the disk in the container. The view is through the opening of the petri dish container. The disk can easily be moved in and out of the container, which facilitates rinsing the plants. Each plant is placed in a hole in the disk. B. A diagram of the setup of the hydroponic container. The disk is above the buffer or media so that the roots are in contact. In addition, the microfuge tubes hold up the disk to allow the disk to remain above the buffer or media. C. Picture of tobacco plants hydroponically grown in MS. D. Tobacco plants in the process of being rinsed with water in the laminar flow hood. E. Tobacco plants in MES buffer containing 200 μM Cd(NO3)2 4H2O. A bigger lid was used to accommodate the size of the plants. The picture was taken after the plants were rinsed with water.

Figure 5. Metal Binding in Transgenic Tobacco: Dithizone Staining of Tobacco WT and Transgenic Lines T12 and T17 Hydroponically Grown in the Presence or Absence of 200 mM Cd (NO3)2. Arrows indicate staining resulting from the formation of Cd dithizonate. The color of dithizonate ranges from an orange-red to red-violet color in order of increasing Cd concentration in the tissue. Intense staining is observed with transgenic plants compared to WT.

Table 1. Cadmium Uptake of T10, T12, T17, and Wild-Type Tobacco on 200 mm Cd. Blank areas indicate where sample was lost during processing, and NC indicates where no calculation was done because the amount of cadmium was below detection levels. A. Blank samples. B. Day1 samples. C. Day 3 samples.

A. Blanks – No Incubation With Cadmium.

Shoot

Root

Total dry mass (g)

Tobacco Line

g

mg/L Cd

mg Cd/g dry wt.

Tobacco Line

g

mg/L Cd

mg Cd/g dry wt.

WT

0.41

0.017

4.09

WT

0.07

0.0015

2.14

0.48

WT

0.43

<0.001

NC

WT

0.07

<0.001

NC

0.50

WT

0.32

0.001

0.31

WT

0.07

<0.001

NC

0.39

10

0.26

<0.001

NC

10

0.05

<0.001

NC

0.31

10

0.06

<0.001

NC

10

0.06

<0.001

NC

0.06

10

024

0.014

5.71

10

0.05

0.0046

9.2

0.29

12

0.13

<0.001

NC

12

0.04

<0.001

NC

0.17

12

0.30

<0.001

NC

12

0.05

<0.001

NC

0.35

12

0.22

<0.001

NC

12

0.04

0.0046

11.5

0.26

17

0.30

<0.001

NC

17

0.07

<0.001

NC

0.37

17

0.23

<0.001

NC

17

0.03

<0.001

NC

0.26

B. Day 1 – Incubation With Cadmium for 1 Day.

Shoot

Root

Total dry mass (g)

Tobacco Line

g

mg/L Cd

mg Cd/g dry wt.

Tobacco Line

g

mg/L Cd

mg Cd/g dry wt.

WT

0.30

4.879

1,626

WT

0.06

4.880

8,133

0.36

WT

0.19

0.988

520

WT

0.05

3.669

7,338

0.24

WT

0.23

0.333

145

WT

0.08

3.334

4,168

0.31

WT

0.16

0.311

194

WT

0.04

2.181

5,453

0.20

WT

0.21

1.226

584

WT

0.05

6.070

12,140

0.26

10

0.33

1.686

511

10

0.07

6.000

8,571

0.40

10

0.16

10

0.03

5.860

19,533

0.19

10

0.22

2.576

1,171

10

0.05

8.280

16,560

0.27

12

0.16

1.309

818

12

0.04

4.786

11,965

0.20

12

0.16

1.275

797

12

0.02

4.126

20,630

0.18

12

0.22

3.170

1,441

12

0.05

7.290

14,580

0.27

17

0.22

1.850

841

17

0.07

7.540

10,771

0.29

17

0.29

3.421

1,180

17

0.07

6.460

9,229

0.36

C. Day 3 – Incubation With Cadmium for 3 Days.

Shoot

Root

Total dry mass (g)

Tobacco Line

g

mg/L Cd

mg Cd/g dry wt.

Tobacco Line

g

mg/L Cd

mg Cd/g dry wt.

WT

0.07

1.973

2,819

WT

0.10

5.490

5,490

0.17

WT

0.41

2.505

611

WT

0.13

7.110

5,469

0.54

WT

0.24

WT

0.11

5.370

4,881

0.35

WT

0.23

WT

0.04

3.280

8,200

0.27

WT

0.34

1.094

322

WT

0.15

6.910

4,607

0.49

WT

0.31

2.834

914

WT

0.08

9.360

11,700

0.39

WT

0.18

1.618

899

WT

0.05

5.980

11,960

0.23

10

0.77

7.460

969

10

0.16

14.360

8,975

0.93

10

0.18

3.384

1,880

10

0.04

8.140

20,350

0.22

10

0.35

5.610

1,603

10

0.08

12.110

15,138

0.43

12

0.21

12

0.07

10.230

14,614

0.28

12

0.17

0.976

574

12

0.02

4.792

23,960

0.19

12

0.29

4.202

1,450

12

0.11

11.400

10,364

0.40

12

0.28

2.005

716

12

0.07

7.760

11,086

0.35

12

0.22

10.440

4,745

12

0.06

7.430

12,383

0.28

17

0.20

2.646

1,323

17

0.06

6.580

10,967

0.26

17

0.21

3.691

1,758

17

0.26

7.970

3,065

0.47

17

0.43

2.635

618

17

0.13

16.080

12,369

0.56

Objective 5: To Determine the Short- and Long-Term Effects of the Overexpression of Metallohistin on Growth and Development of the Transgenic Plants

Overall, transgenic plants appeared normal, indicating that overexpression of a metallohistin gene did not interfere with normal plant cell metabolism.

Future Activities:

After we demonstrated the feasibility in plants (tobacco and Arabidopsis), we are in the process of transforming Populus and Brassica juncea. After transformation is confirmed, we will characterize metal binding and accumulations.

Journal Articles:

No journal articles submitted with this report: View all 1 publications for this subproject

Supplemental Keywords:

sustainable industry, waste, agricultural engineering, bioremediation, environmental engineering, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology, bioremediation, carcinogen, contamination, phytodegradation, pollutant, toxicity, heavy metal, environmental cleanup,, Scientific Discipline, Waste, TREATMENT/CONTROL, POLLUTANTS/TOXICS, Treatment Technologies, Technology, Chemicals, Biochemistry, Bioremediation, Molecular Biology/Genetics, Biology, plant-based remediation, metallohistins, transgenic plants, plant uptake studies, biotechnology, plant biotechnology, phytoremediation

Relevant Websites:

http://www.cpbr.org Exit

Progress and Final Reports:

Original Abstract
  • 2006 Progress Report
  • 2007
  • Final

  • Main Center Abstract and Reports:

    R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
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    R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
    R829479C005 Development of Novel Plastics From Agricultural Oils
    R829479C006 Conversion of Paper Sludge to Ethanol
    R829479C007 Enhanced Production of Biodegradable Plastics in Plants
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    R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
    R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
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    R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
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    R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
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    R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
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