Phytoremediation of Persistent Organic Pollutants: Mechanistic studies and Field

Application

Jason C. WhiteHead, Department of Analytical Chemistry

The Connecticut Agricultural Experiment Station

Persistent Organic Pollutants (POPs) in Soil• They persist for decades• Global distribution• Likely mutagenic, estrogenic, carcinogenic effects• Bioaccumulation, biomagnification• Other remediation strategies are ineffective due to

high degree of sequestration; plants should not facilitate their remediation Aldrin Chlordane DDT/DDE

Dieldrin Dioxins Endrin

Furans Heptachlor Lindane

Mirex PCBs Toxaphene

EPA Dirty Dozen

Uptake and translocation of weathered p,p’-DDE by

selected plant species

0

500

1000

1500

2000

2500

3000 MustardCloverRyeS. squashW. squashZucchiniPumpkin

Roo

ts

Roo

ts

Roo

ts

Roo

ts

Ste

ms

Ste

ms

Ste

ms

Ste

ms

Leaf

Leaf

Leaf

Leaf

Frui

t

Frui

t

Frui

t

Frui

t

p,p'

-DD

E (n

g/g)

Soi

l

Soi

l

Soi

l

Soi

l

Soi

l

Soi

l

Soi

lR

oots

Roo

ts

Roo

tsS

hoot

s

Sho

ots

Sho

ots

Cucurbita pepo ssp ovifera

Cucurbita pepo ssp pepo

Lupi

n

Cano

la

Clov

er

W. S

quas

h

Mus

tard

Cucu

mbe

r

Pean

ut

Pige

onpe

a

Vet

ch

p,p'-DDE in root system

Rye

% P

hyto

extr

acte

d

0.00

0.75

1.50

2.25

Pum

pkin

52 15 370.65

1.8

910.88

32

9.7 40

9.0

p,p'-DDE in shoot system

Phytoextraction (% removal) of weathered p,p’-DDE by plant species. Numbers in green indicate mass ratio

of root to soil (values are 10-4)

Chl

orda

ne (n

g/g)

0

2500

5000

7500

21000

24000

Soil Roots LeavesStems Fruit

SoilZucchiniCucumberTomatoSpinachCorn

Uptake and translocation Uptake and translocation of chlordane from soil of chlordane from soil by selected plant speciesby selected plant species

DDX in the xylem sap of 3 cucurbitsZucchiniSquashCucumber

DDE DDT

DDD

Xyle

m s

ap C

once

ntra

tion

(ng/

mL)

0.0

0.5

1.0

1.5

5.0

10.0

15.0

20.0

Root mass (g, dry weight)0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

DD

E Fl

ow (n

g/h)

0

5

10

15

20

25

30

35Zucchini

SquashCucumber

CAES Research on the C. pepossp pepo system

• Focused on two separate lines of investigation:1. Optimizing remediation- Field/pot studies in an

effort to further enhance contaminant uptake (cropping strategy, surfactants, organic chelators or TPAs, fungal inocula, intercropping)

2. Mechanistic studies- hydroponic, rhizotron, grafting, breeding, and molecular studies to elucidate governing processes.

The C. pepo ssp pepo system• Mechanistically, you need to consider two areas• Ex planta- Release and uptake of a highly sequestered

residue from soil to roots- Plant exudates are released to facilitate nutrient acquisition from soil. Species vary widely in exudation. Greater amounts of low molecular weight organic acids result in soil matrix deconstruction

- This disruption in soil structure inadvertently increases the bioavailability of weathered POPs

• In planta- Translocation of a hydrophobic residue from roots to shoots.

- Hydrophobic contaminants must move with water through root tissue and cross into the vascular cylinder. Should be minimal but some plants are clearly more effective than others.

ZucchiniSquashCucumber

DDE DDT

DDD

Xyle

m s

ap C

once

ntra

tion

(ng/

mL)

0.0

0.5

1.0

1.5

5.0

10.0

15.0

20.0

Tota

l Org

anic

Aci

d (p

icom

oles

)

0

2000

4000

6000

8000

Zucchini Squash Cucumber Control

* Organic acids summed are citric, malic, succinic, tartaric, malonic

Zucchini exude more organic acids, whether you look directly at the root surface (left) or in the rhizosphere pore water (right)

Tota

l Org

anic

Aci

d (p

icom

oles

)

0

2000

4000

6000

8000

10000

12000

Zucchini Squash Cucumber Control

Accumulation of chlordane components from soil by shoots of intact or grafted zucchini and cucumber plants

(units are raw area counts)

+ HEPX

Transnonachlor+ Cis-chlordane

+ Trans-chlordane+ MC5a/MC5b

0

40

80

120

0

20

40

60

Zucchini

Zucchini homograft

0

2

4

6

ND ND

Zucchini shoot on cucumber root

0

4

8

12

16Cucumber

NDND

0

4

8

12

NDND

Cucumber homograft

0

5

10

15

20

25Cucumber shoot on zucchini root

Chordanes *

*

*

*

*

*

Mattina et al., 2007

Differential uptake of weathered p,p’-DDE by 2 subspecies of Cucurbita pepo

(ssp pepo and ssp ovifera)

BC

F

0

2

4

6

8

10

12 Roots

BC

F

0

2

4

6

8

10Stems

Gur

neys

prid

e

Hyb

rid c

resc

ent

Yello

w C

rook

Bla

ck m

agic

Blac

k zu

keBl

ack

beau

tyB

utte

r sca

l.C

osta

taE

arly

Pro

Gol

drus

hH

ybrid

but

ter

Hyb

rid ja

ckM

agda

Pat

ty p

anR

aven

Rev

enue

Sene

caS

tars

hip

Sun

burs

tS

unra

y

Zeph

yr

Gur

neys

prid

e

Hyb

rid c

resc

ent

Yello

w C

rook

Bla

ck m

agic

Bla

ck z

uke

Blac

k be

auty

But

ter s

cal.

Cos

tata

Early

Pro

Gol

drus

hH

ybrid

but

ter

Hyb

rid ja

ckM

agda

Pat

ty p

anR

aven

Rev

enue

Sen

eca

Sta

rshi

pS

unbu

rst

Sun

ray

Zeph

yr

pepo- 5.4ovifera- 0.54

pepo- 7.2ovifera- 2.4

Average BCF Average BCF

Stem DDE bioconcentration factors and % DDE phytoextracted for 3 cultivars each of two C.

pepo subspecies

Stem

DD

E B

CF

0

2

4

6

8

10Zucchini(C. pepo ssp pepo)

Non-zucchini summer squash (C. pepo ssp ovifera)

Cost

ata

Gol

drus

h

Rave

n

Zeph

yr

Patty

Y. C

rook

% D

DE

phyt

oext

ract

ed

0.0

0.2

0.4

0.6

0.8

1.0Zucchini(C. pepo ssp pepo)

Non-zucchini summer squash (C. pepo ssp ovifera)

Cost

ata

Gol

drus

h

Rave

n

Zeph

yr

Patty

Y. C

rook

Traditional Breeding Studies- DDE-accumulating zucchini and non-accumulating squash are in the same subspecies. What will be the abilities of the hybrids?

Stem bioconcentration

factors (BCFs) and % DDE

phytoextracted by parental and

F1 hybrid zucchini and

squash

Stem

DD

E B

CF

0

4

8

12

16

Parents F1 Hybrids

A

B

CC

% P

hyto

extr

acte

d

0.0

0.4

0.8

1.2

1.6

Zucchini (C. pepo ssp pepo)Squash ( C. pepo ssp ovifera)Hybrid Zucchini (ssp pepo x ssp ovifera)Hybrid Squash (ssp ovifera x ssp pepo)

Parents F1 Hybrids

A

B

D

C

% DDE Phytoextracted and

Stem BCFs by parental, F1 hybrid, and F1 Backcrossed zucchini and squash

(preliminary data)

% P

hyto

extr

acte

d

0.0

0.5

1.0

1.5Pa

rent

al

F1 h

ybrid

F1 B

ackc

ross

Pare

ntal

F1 h

ybrid

F1 B

ackc

ross

Zucchini (C. pepo ssp pepo)

Squash(C. pepo ssp ovifera)

B

AA

A

BB

Stem

BC

F

0

5

10

15

Pare

ntal

F1 h

ybrid

F1 B

ackc

ross

Pare

ntal

F1 h

ybrid

F1 B

ackc

ross

Zucchini (C. pepo ssp pepo)

Squash(C. pepo ssp ovifera)

A

B

C

A

B B

Uptake of DDE by zucchini and squash exposed to 120 ng/mL DDE under batch hydroponic conditions for 96 hours

Stem

DD

E co

nten

t (ng

/g)

0

200

400

600

800

1000

Zucchini Squash Zucchini Squash

Exposed to DDE

Non-exposed Controls

A

BB

B

A. B..

Cyt P450

ZucchiniDDE

Squash DDE

- + - + - + - +

SHOOT ROOT

PP1

Actin Actin

ZucchiniDDE

Squash DDE

Zucchini and squash that were or were not exposed to DDE were subjected to differential transcript analysis and subtractive hybridization techniques.

Subtracted cDNAs were cloned into a TA cloning vector and expression in E. coli via colony array analysis was employed (Fig. 1).

Figure 1.

Figure 2.Blast search on isolated

differentially expressed cDNAsincludes sequence homology match to “phloem protein I”

(PP1) and a cytochrome P450. Semi-quantitative RT PCR

confirmed differential regulation (Fig. 2).

Gene Description Accession Number

E value Max % Identity

Functions

2-Oxoisovalerate dehydrogenase (2 OVD)

NM_203027 3e-73 79% oxidative stress

Aspartate aminotransferase (AAT) XM_002515409 5e-108 85% Amino acid metabolism

Brassinosteroid Insensitive 1 associated receptor kinase (Brassino. Inh)

XM_002532844 1e-104 80% defense

Chlorophyll a/b -binding protein -1 DQ471302 1e-76 84% photosynthes is

chlorophyll a/b -binding protein -2 XM_002284820 4e-97 83% photosynthes is

Cystatin (Cystatin like) ABF94587 3e-07 53% cystein proteinase inhibitor

Cysteine Proteinase (Cys Pro) AJ415386 3e-59 82% signaling and abiotic stresses

DNA binding protein with bHLH domain (bHLH)

XP_002521010 3e-32 45% unknown

Glucosylceramidase ( Gluc. Cera.) NM_124368 1e-09 81% Lipid metabolism

Protein phsophatase -2a XM_002513094 2e-60 84% Involved in phosphorylatio n

50S ribosomal protein L5 (L5 Ribo 50S) XM_002511867 7e-51 79% Unknown

Ribulose activase EF506513 1e-122 82% photosynthes is

Putative senescence-associated protein BAB33421 4e-44 79% Stress tolerance

Unknown protein 1 (UK 1) XP_002527893 0.004 48% Unknown

Unknown protein 2 (UK 2) BT009458 8e-27 100% Unknown

Unknown protein 3 (UK 3) AY942801 4e-33 77% Unknown

Unknown protein 4 (UK 4) XM_002532526 3e-48 80% Unknown

Unknown protein (predicted protein mahagunin like) (UK 5)

XM_002519497 1e-52 82% Unknown

Unknown protein (predicted protein with chlorophyll a/b -binding) (UK 6)

U01964 1e-23 83% Unknown

Unknown protein (Predicted protein with DNA binding domain (UK 7)

XP_002531567 4e-09 49% Unknown

Unkown predicted protein (UK 8) XM_002309035 1e-31 78% Unknown

Zeaxanthin epoxidase (Ze. Epo.) DQ641126 2e-98 90% abiotic stress tolerance

List of DDE induced subtracted cDNA clones from zucchini

bHLH

AAT

Rub. Activase

Ze Epo.

Chl a/b 1

Phosphatase 2A

UK 1

UK 3

Cystatin like

UK 4

UK 5

L5 Ribo 50S

2 OVD

Cys Pro.

Chl a/b 2

Brassino. Insensitive

ACT2 control

DDE+

Confirmation of upregulation of zucchini subtracted cDNAs by RT-PCR analysis

–

Conclusions• C. pepo ssp pepo uniquely accumulates significant quantities of

weathered POPs from soil• POP accumulation is dependent on both contaminant

properties and plant genotype• Soil amendments such as organic acids, surfactants, and

fungal inocula can increase POP extraction but effects are cultivar- and treatment-specific

• Although C. pepo ssp pepo has a unique organic acid exudation profile (as a nutrient acquisition strategy), the differences do not explain the observed POP accumulation

• Based on hydroponic, grafting, and molecular data, it appears that a unique transport protein in C. pepo ssp pepo tissue permits POP movement into the vascular cylinder

• Investigations elucidating the nature of this transport system are underway

Acknowledgements• Dr. Om Parkash (UMass), Dr. MaryJane

Mattina, Dr. Brian Eitzer, Dr. Martin Gent, Dr. Neil Schultes

• Dr. Zakia Parrish, Dr. Mehmet Isleyen, Dr. Xiaoping Wang, Dr. Wen-Yee Lee

• Lydia Wagner, Bill Iannucci-Berger, Craig Musante, Terri Arsenault

• Funding from US EPA STAR, USDA CSREES