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Enhancing Phytoremediation and Plant Growth in Poplar and Willow
Sharon DotyAssistant Professor
College of Forest Resources
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Why do we need to enhance phytoremediation?
Removal of Pollutants by CYP2E1 Transgenic Poplar
Funding: Superfund Basic Research Program of NIEHS
P.I. Stuart Strand, UWCo-P.I. Sharon Doty, UW
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Trichloroethylene (TCE)
Used for decades as metal degreaser, dry cleaning agent, solvent, and anestheticFollowing use, it was dumped outsideOne of the most widespread contaminants in the environment (60% of SuperFund sites)Toxic to the liver, kidney, CNS, and likely carcinogenicPersistent in the environment
Mammalian Cytochrome P450 2E1 Catalyzes TCE Metabolism
Chloral
Trichloroacetic Acid
Trichloroethanol
Trichloroethylene
2E1
Transformation of Poplar (P. tremula x alba N717-1B4) using A. tumefaciens
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Transgenic plants were assayed for increased metabolism of pollutants
CYP2E1 Transgenic Poplar Had Increased Metabolism of TCE
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0.5
1
1.5
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2.5
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3.5
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μg T
CEO
H/g
m ti
ssue
Vector Control Aspen r2E1 Transgenic Aspen
132X Average Control121X Average Control
Doty, unpublishedS. Doty, et al. (2007) Proc. Natl. Acad. Sci. 104(43):16816-16821.
CYP2E1 transgenic poplar removed TCE at a faster rate
Transgenic Plant % Removal Rate *
No plant control 0.8 +/- 1.1 0.1 +/- 0.1
Vector Control 2.6 +/- 0.3 0.4 +/- 2.8
CYP2E1 #78 86.9 +/- 11.4 20.3 +/- 4.6
S. Doty, et al. (2007) Proc. Natl. Acad. Sci. 104(43):16816-16821.
Rate: ug TCE/day*gm fresh weight
Cytochrome P450 2E1 Has Multiple Substrates
ChloroformCarbon Tetrachloride
Vinyl ChlorideBenzene
Increased Removal of Chloroform from Solution by CYP2E1 Transgenic Poplar
Doty, unpublished
0
100000
200000
300000
400000
500000
0 1 2 3 4 5 6 7
Chl
orof
orm
(GC
pea
k ar
ea)
Time (Days)
Unplanted
Vector Ctrl
r2E1#78
r2E1#20
Increased Removal of Carbon Tetrachloride from Solution By CYP2E1 Transgenic Poplar
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0.5
1
1.5
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0 1 2 3 4 5 6 7Car
bon
Tetr
achl
orid
e (u
g/m
l)
Time (Days)
Unplanted
Vector Ctrl
r2E1#78
r2E1#20
CYP2E1 Transgenic Poplar Removed More Pollutants from Air
TCE
Benzene
Vinyl Chloride
CYP2E1 Poplar Plants Removed TCE from Air At A Faster Rate
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5,000
10,000
15,000
20,000
25,000
0 1 2 3 4 5 6 7
Time (days)
TC
E C
once
ntra
tion
(ug/
m3)
UnplantedVector Controlr2E1#78
Identification of Plant Genes Involved in TCE Metabolism
Proposed Fate of TCE in Plants
Shang, T. Q., et al. (2001) Phytochemistry 58:1055-1065.
Approaches to identify plant genes involved in TCE metabolism
Poplar microarrays (Dr. Fred Farin, UW)Arabidopsis microarrays (Dr. Mary Schuler, UI)
Test candidate genes in yeast
TCE dosed Un-dosed
Phases of detoxification in plants
(Benoit Van Aken, 2008 Trends in Biotechnology 26(5):225-227)
Function Enzyme KH200+TCE #78+TCE
Upregulated 68 472
Phase I TransformationCytochrome P450,Reductases,Dehalogenases
2 55
Phase II ConjugationGlycosyltransferases (UGT),Glutathione-S trasnferases (GST), Acyltransferases 1
811
Phase III
Compartment- ation
ATP binding cassette(ABC transporter)
1 7
PeroxidaseOxidaseOxidoreductase
3 467
Unknown … …
Log FC >2Kang, unpublished
Microarray Results: Vector Control vs CYP2E1Poplar Genes Induced by TCE Exposure
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Current Research
Field testing of transgenic poplar (NIEHS funded)
Analysis of plant genes involved in TCE metabolism (NIEHS funded)
Developing transformation protocols for willow (CPBR funded)
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Summary of Transgenic Phytoremediation
Expression of mammalian P450 2E1 gene in transgenic poplar led to a strong increase in TCE metabolism and removal of TCE (53X), and a strong increase in removal of chloroform (9X), carbon tetrachloride, vinyl chloride (3X) and benzene (10X).
Analysis of upregulated genes in response to TCE in poplar revealed many genes known to be involved in detoxification of pollutants. Analysis of these genes may lead to improved phytoremediation.
Enhancing Plant Growth using Nitrogen-Fixing Endophytes of
Poplar and Willow
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Contaminated areas often have poor soil
Abandoned, polluted commercial propertiesPhytoremediation depends on good plant growth
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Nitrogen fixation in root nodules
Chemical fertilizers are petroleum-based; getting costlierSome microbes convert N2 gas to ammonia
Nitrogen fixation occurs within root nodules of only some plant species
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EndophytesMicroorganisms that live within plants without causing diseaseIncreased nutrient acquisition, pathogen resistance, stress tolerance
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Nitrogen-fixing endophytes of poplar and willow
Doty, et al. (2005) Symbiosis 39: 27-35
Doty, et al. (2009) Symbiosis 47:23-33
In wild settings, poplar and willow grow under low-nitrogen conditions
Isolated endophytes; identified using 16S rRNA gene sequencing
Some poplar and willow endophytes grow well in medium without ammonium or nitrate
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0.05
0.1
0.15
0.2
0.25
0.3
0 5 11 23 36 50 71 76.5 96 103
Time (hours)
OD
600
AzotobacterC58WPBWW5WW9WW11
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Plant Growth Promotion by Endophytes of Poplar
NFM Rope
Sterile sand
Kentucky bluegrass
Grass growth promotion assay
Room temp, 16/8 hr light/darkness
After 50 days, cleaned and dried
Weighed for shoot and root respectively
Determined for N content using a CHN analyzer
Gang Xin, Genyun Zhang, and Sharon Doty (manuscript in preparation)
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1
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N content total N N content total N total N
N c
onte
nt, %
or t
otal
N, m
g w/ WPBControl
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
1 2 3Ave
rage
dry
wei
ght p
er c
ham
ber,
g
w/ WPBControl
Shoot Root Whole plant
Shoot RootWhole plant
42% biomass increase with WPB inoculation
37% total N increase with WPB inoculation
Gang Xin
WPB enhances the growth of rice under nitrogen limitation
Expt. Performed in Dr. Rodriguez’s lab
Summary of Endophyte Characterization
Initially focused on Burkholderia strain, WPB.WPB shares high nifHDK gene similarity with known B. vietnamiensis strains.High nitrogenase activity was determined using both the 15N2 incorporation assay and the Acetylene Reduction Assay.WPB can significantly promote growth of Kentucky bluegrass, crabgrass, and rice. Similar experiments with poplar and corn are underway.
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Current Research
Propagation of internally-sterile poplar
Growth effectsMicroarrays
Analysis of willow endophytes
Acknowledgements for Endophyte Research
Gang Xin: WPB studies
Helped with acetylene reduction assays or strain identification: Z. Khan, A. Vajzovic, G. Singleton, and J. W. Kang
Gengyun (George) Zhang: Turfgrass studies
Rice studies:
Dr. Regina Redman and Dr. Rusty Rodriguez
Acknowledgements for Phytoremediation
ResearchProfessor Stuart Strand (Principal Investigator)
FUNDING: DOE and NIEHS
Allison Moore
Azra Vajzovic
Zareen Khan
Andy James
Glenda Singleton
Jun Won Kang
Keum Young Lee
George Zhang