accelerated biodegradation of 2,4-d in legume rhizospheres ... · pdf file1 accelerated...

1

Accelerated biodegradation of 2,4-D in legume rhizospheres: elucidation of the

mechanisms responsible

Liz Shaw

The rhizosphere

Distinctive soil-plant root environmentEnriched in organic carbon due to rhizodepositionAs much as 40% of carbon that is photosynthetically fixed is translocatedbelow ground

endo

rhiz

osph

ere

ecto

rhiz

osph

ere

rhiz

opla

ne

2

Rhizodeposit composition

O

CH2OH

H

HO

H

OH

H OH

HO

O

OH

O

NH2 C

CH3

H

COOH

OH

OH

N N OH O

HO

O

NH

OHO

OCH2OH

H

HO

O

OH

H OH

OCH2OH

H O

OH

H OH

OCH2OH

H H

OH

H OH

mucilage

sloughed border cells

‘Rhizosphere effect’

Time (d)0 10 20 30

Min

eral

isat

ion

(% in

itia

l)

0

20

40

60

80

non-plantedLoliumTrifolium

Enhanced biodegradation of 2,4-dichloro-phenoxyacetic acid in rhizosphere soil of red clover

Shaw & Burns (2005) Soil Biol. Biochem. 37, 995

O

ClCl

2,4-D

COOH

3

Summary of research reporting a ‘rhizosphereeffect’ on the biodegradation of pesticides

SoybeanDiphenolic ether: Acifluorfen

Zablotowicz et al., 1997

MaizeChloroacetanilide:Metolachlor and alachlor

Hoagland et al. 1997

Summer cypress, maize, pennisetum

Triazine:Atrazine and simazine

Kruger et al., 1997Marchand et al., 2002Pervovitch et al., 1996Piutti et al., 2002Singh et al., 2004

Red clover, daisy fleabane, barnyard grass, fall panicum, early goldenrod, chicory, timothy grass, green foxtail, sunflower

Phenoxyacetic acid:2,4-D and 2,4,5-T

Boyle and Shann, 1995Boyle and Shann, 1998

Plant speciesPesticideReference

research is descriptive, not mechanistic

Overall aim: to investigate mechanisms of Trifolium rhizosphere enhanced 2,4-D biodegradation

Time (d)0 10 20 30

Min

eral

isat

ion

(% in

itia

l)

0

20

40

60

80


?

4

Overall aim: to investigate mechanisms of Trifolium rhizosphere enhanced 2,4-D biodegradation

Specific objectives:1. To investigate the impact of plant and soil properties on the

enhanced biodegradation2. To compare the number and diversity of 2,4-D degradative

microorganisms in Trifolium, Lolium and non-planted systems

Time (d)0 10 20 30

Min

eral

isat

ion

(% in

itia

l)

0

20

40

60

80


Functional engineering of rhizosphere for benefit (i.e. rhizoremediation)

Time (d)0 10 20 30

Min

eral

isat

ion

(% in

itial

)

0

20

40

60

80


Treatment of mineralization data

t1 = t0[(b-1)/(b+1)]1/b = time at maximum rateMaximum rate = (-a.b)/(4t1)

a = asymptote

Y = a[1+(t/t0)b]-1

time

at m

axim

um ra

te, t

1 (d

)

0

15

20

25

30

non-p

lanted

Loliu

m

Trifoliu

m

max

imum

rate

(d-1

)

0

2

4

6

8

10

a a

a a

b

b

5

The rhizosphere effect has legume specificity

non-p

lanted

Loliu

mHolc

us

Festuc

a

Helian

thus

Stellar

ia

Mysoti

s

Trifoliu

m

Medica

goLo

tus

Max

imum

rate

(d-1

)

0

5

10

15

20

25

non-p

lanted

Loliu

mHolc

us

Festuc

a

Helian

thus

Stellar

ia

Mysoti

s

Trifoliu

m

Medica

goLo

tus

Tim

e at

max

imum

rate

t 1 (d

)

0

5

10

15

20

25

30

non-planted controlLegumesDicotsMonocots

* * * * ** * * * *

Shaw and Burns (2005) Environ. Microbiol. 7, 191

Specific objectives:1. To investigate the impact of plant and soil properties on the enhanced biodegradation2. To compare the number and diversity of 2,4-D degradativemicroorganisms in Trifolium, Lolium and non-planted systems

Production of the active component is dependent on the presence of microorganisms

Shaw and Burns (2005) Environ. Microbiol. 7, 191.

t 1 (d

)

020

30

40

50a

b

max

imum

rate

(d-1

)

0

2

4

6

a

b

non-

amen

ded

a

a

gnot

obio

tic-

grow

n ro

ots

non-

ster

ile-

grow

n ro

ots

non-

amen

ded

gnot

obio

tic-

grow

n ro

ots

non-

ster

ile-

grow

n ro

ots


6

The production of the rhizodeposit component that stimulates 2,4-D mineralization:

Has specificity to legumesIs dependent on presence of microorganismsIs dependent on plant age


The production of the rhizodeposit component that stimulates 2,4-D mineralization:

Has specificity to legumesIs dependent on presence of microorganisms

Implies plant-microbe signallingIs dependent on plant age


7

Rhizodeposit composition

O

CH2OH

H

HO

H

OH

H OH

HO

O

OH

O

NH2 C

CH3

H

COOH

OH

OH

N N OH O

HO

O

NH

OHO

OCH2OH

H

HO

O

OH

H OH

OCH2OH

H O

OH

H OH

OCH2OH

H H

OH

H OH

mucilage

sloughed border cells

flavonoids

MODULES – REMEDIATION and SOIL MICROBIOLOGY & BIOTECHNOLOGY

Role of flavonoids: nod factor inducers

E.g. 7,4’-dihydroxyflavone

from Broughton et al. (2003) Plant and Soil 252, 129

OHO

OH

O

8

The mechanism?The stimulatory rhizodeposit (isoflavonoid?) component is acting as:

A significant additional growth substrate for 2,4-D degradersAn inducer of 2,4-D catabolism

Is the stimulatory rhizodeposit enhancing 2,4-D mineralization by acting as a growth substrate?

If this was the case, would expect:Increased numbers of 2,4-D degraders in the rhizosphere of Trifolium compared to Lolium or non-planted pristine soilRhizosphere-dependent shift in diversity of 2,4-D degrading microorganisms

9

104

105

106

107

Plant age (d)0 25 60 116

Log

(Med

ian

MP

N g

-1)

1

10

100Non-plantedLoliumTrifolium

1.7 LoD

After 2,4-D(+ve control)


No effect of plant species or plant age on numbers of 2,4-D degraders

Shaw and Burns (2004) Appl. Env. Microbiol. 70, 4766

3 6 8 2 5 9 4 7 1 10 15 20 18 14 19 21 11 13 12 16 17 22

0.67

1

Mat

chin

g si

mila

rity

mea

sure

nn n ll lt tt t NN NN LLL LT TTT

N,n = non-plantedL,l = LoliumT,t = Trifoliumn,l,t = before 2,4-DN,L,T = after 2,4-D

No effect of plant species on 2,4-D degrader diversity: Hierarchical cluster analysis of SSCP profiles: tfdAα from soil



10

The mechanism?The stimulatory rhizodeposit component is acting as:

A significant additional growth substrate for 2,4-D degradersAn inducer of 2,4-D catabolism ?

Suggested mechanism for the role of flavonoids as inducers in enhanced 2,4-D mineralization (1)

O

O

HO

OH

OH

OH

O

O

HO

OH

OH

O

A C

B

HO

OH

OH

OH

A C

OHHO

OH

B

OH

A

A

OH

C

OH

B

O

O

B

OH

Quercetin

Naringenin

Phloroglucinol 3,4-Dihydroxy cinnamic acid

11

OH

OH

O

O

OH

OH

OH

OH

CHO

OH

OH

COOH

Cl

Cl OH

Cl

COO-COO-

COO-

3,4-dihydoxycinnamic acid

3,4-dihydoxystyrene

Cl Cl

O

O

O

B-carboxymucanate

2,4-dichloromuconate

Cl

HO

OH

protocatechuicaldehyde

COO-COO-Cl

Cl

2,4-dichlorophenol

2,4-D

protocatechuicacid

3,5-dichlorocatechol

TCA cycle TCA cycle

Suggested mechanism for the role of flavonoids as inducers in enhanced 2,4-D mineralization (2)

TfdA can accept hydroxy cinnamicacids as substrates1

1 Dunning Hotopp and Hausinger, 2001 J. Mol. Catal. B 15: 155

2,4-D pathway in W. eutropha JMP134 pJP41 kb

A S R DII CII EII FII BII K T C D E F B

OH COOHCOOH

OH

Cl

Cl Cl

ClOCH2COOH

Cl

Cl

OH

Cl

Cl

tfdA tfdC(II)tfdB(II)

tfdR gene product activates expression and the effectormolecule for TfdR is 2,4-dichloro-cis,cis-muconate.

12

OH

OH

O

O

OH

OH

OH

OH

CHO

OH

OH

COOH

Cl

Cl OH

Cl

COO-COO-

COO-

3,4-dihydoxycinnamic acid

3,4-dihydoxystyrene

Cl Cl

O

O

O

B-carboxymucanate


Cl

HO

OH

protocatechuicaldehyde

COO-COO-Cl

Cl

2,4-dichlorophenol

2,4-D

protocatechuicacid


TCA cycle TCA cycle

Suggested mechanism for the role of flavonoids in enhanced 2,4-D mineralization (2)

Involvement of flavonoids as inducers requires further testing ?

Further research to investigate the role of isoflavonoids and inducers

Establish the role of isoflavonoidsSpiking experiments with commercially available isoflavonoidsUse of legume mutants (e.g. Medicago truncatula) deficient in isoflavonoid biosynthesis

Establish whether rhizodeposits (isoflavonoids) act as inducers of the 2,4-D pathway

Use of transcriptional fusions to quantify gene expression in model bacterial strains

13

Arrangement of tfd genes in plasmid pEST4011 of Achromobacter xylosoxidans subsp. dentrificansEST4002

From Vedler et al. (2004) J. Bacteriol. 186, 7161.

tfdA tfdK tfdB tfdE tfdC tfdR iaaH tfdD

Role of plant-microbe interactions in the evolution of xenobiotic catabolic pathways

Arrangement of tfd genes in plasmid pEST4011 of Achromobacter xylosoxidans subsp. dentrificansEST4002

From Vedler et al. (2004) J. Bacteriol. 186, 7161.

tfdA tfdK tfdB tfdE tfdC tfdR iaaH tfdD

Indole acetamide hydrolase(Bradyrhizobium japonicum USDA110)

Role of plant-microbe interactions in the evolution of xenobiotic catabolic pathways

14

Thank you

HERBICBIOREM

15

tfd A, B, and C primer sets

tfdC(Calvaca et al. (1999) FEMS Micro. Ecol. 29, 45. )

Bacillus B11JMP 228 (pEMT8)RASCJMP134-ve

OH COOHCOOH

OH

Cl

Cl Cl

Cl

tfdB(This study)

OH

Cl

Cl Cl

Cl

OH

OHJMP228 (pEMT8)Bacillus B11RASCJMP134-ve

tfdA(Vallaeys et al. (1998) FEMsMicrob. Ecol. 20, 163.)

OCH2COOH

Cl

Cl

OH

Cl

Cl

RASCJMP134-ve

1000 bp 100 bp

Used these primer sets to PCR amplify from DNA extracted directly from soil or from 2,4-D degrading enrichment cultures

NO PRODUCTS

tfdA-like genes in 2,4-D-degrading bacteria belonging to the BANA cluster in α-ProteobacteriaItoh et al. (2002) AEM 68, 3449.

Bacillus B11

RASC

RD5-C2

HW13

-ve

100 bp ladder

Japanese upland soil α-Proteobacterial isolate RD5-C2 tfdAα gene 56-60% homologous to canonical tfdA genesDesigned primers from alignments of tfdAα genes in RD5-C2, HW13, BTH and HWK12

16

N L T N L T N L T N L T N L T N L T N L T N L T

A

B

Before 2,4-D After 2,4-D

PCR amplification (A) and subsequent SSCP analysis (B) of tfdAα genes in Sourhope soil before and after 2,4-D application

2,4-dichlorophenoxyacetic acid canonical pathway in β-Proteobacteria Wautersia eutropha JMP134 pJP4

tfdA encodes 2,4-D/α-ketoglutarate dioxygenase

tfdA

O

ClCl

2,4-D

OH

ClCl

2,4-dichlorophenol

OH

ClCl


OH

COOHCOOH

ClCl


COOH

TCA cycle

17

The mycorrhizosphererh

izos

pher

e

Hypho-sphere

dead/dyinghyphae

root

hyphosphere

Addition of chopped Trifolium roots enhances 2,4-D mineralization

non-a

mende

d

roots

100 m

g

autoc

laved

roots

100 m

g

roots

200 m

g

max

imum

rate

(d-1

)

0123456

non-a

mende

d

roots

100 m

g

autoc

laved

roots

100 m

g

roots

200 m

g

t 1 (d

)

02025303540 a

b bb

b

a

bb

18


0 12 25 60 116

Max

imum

rate

(d-1

)

4

5

6

7

8

9

10

11

12

Plant age at rhizosphere harvest (d)

0 12 25 60 116

Tim

e at

max

imum

rate

(t1)

12

14

16

18

20

22

24



The rhizosphere effect depends on plant species and plant age

Flavonoids of leguminous plants: a tentative identity for the stimulatory rhizodeposit?

From: Aoki et al. (2000) J. Plant Res. 113, 475

19

Role of flavonoids (2): phytoalexinsE.g. medicarpin produced by Medicago spp.

Data from Blount et al. (1992) Plant Pathol. 41, 333.

OHO

O

H

H

OCH3

Medicarpin (mM)

0.10 0.25 0.50

% d

iffe

renc

e in

rad

ial g

rowt

h-100

-80

-60

-40

-20

0

Antifungal activity of medicarpin on Nectria haematococca

Initial mineralisation kinetics are linear

Time (days)

0 2 4 6 8 10 12 14 16

% m

iner

alis

atio

n

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Non-plantedLinear regression

accelerated biodegradation of 2,4-d in legume rhizospheres ... · pdf file1 accelerated...

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