Analysis of Primary and Secondary Plant

Metabolites in Root Exudates of

Arabidopsis thaliana

Katja Witzel

Leibniz Institute of Vegetable and Ornamental Crops

(IGZ), Großbeeren, Germany

Primary Plant Metabolites

Carbohydrates

Amino Acids

Organic Acids

Fatty Acids

Secondary Plant Metabolites

Flavonols

Lignins

Coumarins

Glucosinolates

Proteins and Peptides

Mediation of positive

and negative

interactions in the

rhizosphere

Content and Function of Root Exudation

Up to 30 % of fixed carbon released as root exudates in seedlings

Primary Plant Metabolites

Carbohydrates

Amino Acids

Organic Acids

Fatty Acids

Secondary Plant Metabolites

Flavonols

Lignins

Coumarins

Glucosinolates

Proteins and Peptides

Mediation of positive

and negative

interactions in the

rhizosphere

Content and Function of Root Exudation

Up to 30 % of fixed carbon released as root exudates in seedlings

Is exudation genetically determined

Absolute characterisation of quality and quantity

What compounds act in defense against pathogens ?

Primary Plant Metabolites

Carbohydrates

Amino Acids

Organic Acids

Fatty Acids

Secondary Plant Metabolites

Flavonols

Lignins

Coumarins

Glucosinolates

Proteins and Peptides

Mediation of positive

and negative

interactions in the

rhizosphere

Content and Function of Root Exudation

Up to 30 % of fixed carbon released as root exudates in seedlings

Is exudation genetically determined

Absolute characterisation of quality and quantity

What compounds act in defense against pathogens ?

Günter Neumann‘s Talk on Tuesday

Collection of Root Exudates from Arabidopsis thaliana

Plant growth in sand culture,

watering with nutrient solution

5-6 week-old plants are

digged up and sand is

removed carefully

Roots are incubated in

destilled water for 1 h

Roots are replaced in

bidestilled water for 4 h

Sterile filtration of

exudates (0.2 µm) Metabolomics via

GC-MS,

LC-MS,

HPLC

88 compounds (16 currently not identified):

Amino acids, sugars, fatty acids, phenylpropanoids

GC-MS Analysis of Arabidopsis thaliana Root Exudates

88 compounds (16 currently not identified):

Amino acids, sugars, fatty acids, phenylpropanoids

Glc

Frc

Suc

GC-MS Analysis of Root Exudates

Validation of measurements using enzymatic assays:

Glc 0,976 µg/mL Frc 0,283 µg/mL Suc 0,099 µg/mL

Has the Collection Procedure Influence on Metabolite Patterns?

- comparative analysis of metabolite profiles from control roots or exuded

roots

- does bidestilled water lead to a non-specific release of metabolites?

- non-targeted LC-MS analysis: alteration of 98 entities (≈ 3.5 %) out of

2,800 detected ones

Red: control roots Blue: exuded roots

http://homepage.ruhr-uni-bochum.de/Markus.Piotrowski/Research_Glucosinolate.html

Cruciferous species with high content in glucosinolates: multiple functions in plant-pest interactions: -Feeding and ovipositioning stimulants -Toxins -Feeding deterrents to generalist herbivores -Flavouring compounds, cancer prevention

Detection of Important Defence Compounds

Metabolite Extraction and Analysis

Glucosinolates Enzymatically formed glucosinolate degradation products

- 500 ml of exudates loaded onto DEAE-Sephadex A-25 ion-exchanger column - washing step - incubation with aryl sulfatase for desulfatation - elution, HPLC-based separation

- 500 ml of exudates extracted with 50 ml methylene chloride in a separatory funnel - separated methylene chloride layer dried using anhydrous NaSO4 - extract dried under nitrogen to 300 µl and subjected to GC-MS analysis

Glucosinolates and their Degradation Products are Present in Root Exudates

Intact GLS

0

0,5

1

1,5

2

2,5

3

nm

ol/

g d

ry w

eig

ht

GLS breakdown products

I3M 3-indolylmethyl GLS 8MSOO 8-(methylsulfinyl)octyl GLS 4MOI3M 4-methoxyindole-3-ylmethyl GLS 1MOI3M 1-methoxyindole-3ylmethyl GLS 7MTH 7-(methylthio)heptyl GLS 8MTO 8-(methylthio)octyl GLS 7MSOH 7-(methylsulfinyl)heptyl GLS 4MTB 4-(methylthio)butyl GLS

4MTB-CN 5-(methylthio)butylnitrile 4MTB-ITC 4-(methylthio)butyl ITC 8MTO-CN 9-(methylthio)nonylnitrile 8MTO-ITC 8-(methylthio)octyl ITC 8MSOO-ITC 8-(methylsulfinyl)octyl ITC IAN 3-indoleacetonitrile 4MO-IAN 4-methoxy-3-indoleacetonitrile

0

2

4

6

8

10

12

14

nm

ol/

g fr

esh

we

igh

t

Root tissue: 1,1 µmol/g fresh weight Root tissue: 22 µmol/g dry weight

- Cooperation B.A. Halkier, University of Copenhagen

- GLS transporter GTR1/2: retention and storage of GLS in roots

- GTR1/2 knock-out [µmol g-1 dry weight]:

Root Glucosinolates Contents Can Be Modified

0

1

2

3

4

5

6

7

[nm

ol/

g]

Col-0

GTR 1/2

Leaf Root

Col-0 GTR1/2 Col-0 GRT1/2

Methylthioalkyl glucosinolates

4MTB 0.87±0.32 n.d.*

7MTH 0.74±0.37 0.03±0.05*

8MTO 4.35±1.95 0.62±0.47*

Methylsulfinylalkyl glucosinolates

3MSOP 0.58±0.15 0.01±0.02*

4MSOB 4.24±1.01 n.d.*

7MSOH 0.42±0.36 0.10±0.14

8MSOO 3.92±1.09 0.76±0.09*

Indole glucosinolates

I3M 1.53±0.19 0.84±0.10*

4OHI3M 0.41±0.03 0.10±0.05*

1MOI3M 4.79±1.68 4.93±2.62

4MOI3M 1.02±0.48 0.75±0.38

In Roots: In root exudates:

Microsclerotia as

survival state in soil

http://www.apsnet.org/edcenter/intropp/lessons/fungi/ascomycetes/Pages/VerticilliumWilt.aspx

- hosts: over 400 plant species

(pepper, tomato, potato and eggplant)

- Biofumigation approaches to

sustainable control Verticillium

disease

Verticillium Wilt Threatening Vegetables

Root Glucosinolates May Confer Resistance to Verticillium Wilt

- Inoculation with Verticillium longisporum

- Root glucosinolates are critical for plant defense

- Isolated from leaves of winter wheat

Root surface

Characterisation of a Growth-Promoting Endophyte: Kosakonia radicincitans

Ruppel S et al. Plant Soil. 1992 Arabidopsis thaliana

Growth promotion in a wide range of crops

- Similar growth-promoting effects were observed

for wheat (Triticum aestivum), maize (Zea mays),

rape seed (Brassica napus), kohlrabi (Brassica

oleracea)

- Trails in greenhouse, growth chamber and field

Plant health status: • Bacterial production of antibiotics,

vitamines, hormones

• Induction of plant systemic resistance

• Suppression of pathogens by enhanced

colonisation rate

Plant nutritional status: • Biological nitrogen fixation

• Ca-, Al-, Fe-phosphate solubilization by bacterial carboxylic

acid production

• Iron chelating

Plant hormonal status: • Decrease in plants ethylene level via ACC deaminase

• Bacterial production of Auxine, Cytokinine, Gibberelline

Modes of Growth-Promotion by Endophytic Bacteria

Does Genetic Variation in Arabidopsis Determines Endophytic Responses?

Genetic resource: Arabidopsis thaliana MAGIC (Multiparent Advanced Generation Inter-Cross) population = 19 parental lines

0

20

40

60

80

100

120

140

160

Oy-0 Bur-0 Edi-0 Hi-0 Col-0 Sf-2 Tsu-0 Po-0 Ct-1 No-0 Kn-0 Wil-2 Wu-0 Rsch-4 Ws-0 Mt-0 Can-0 Ler-0

Ro

sett

e w

eig

ht

[%]

Kontrolle

Inokuliert

Plant Growth in Response to Microbial Colonisation

Screening of parental lines of the MAGIC population in response to bacterial inoculation

0

20

40

60

80

100

120

140

160

Oy-0 Bur-0 Edi-0 Hi-0 Col-0 Sf-2 Tsu-0 Po-0 Ct-1 No-0 Kn-0 Wil-2 Wu-0 Rsch-4 Ws-0 Mt-0 Can-0 Ler-0

Ro

sett

e w

eig

ht

[%]

Kontrolle

Inokuliert

Plant Growth in Response to Microbial Colonisation

Screening of parental lines of the MAGIC population in response to bacterial inoculation

Analysis of exudate quality and quantity

LC-MS analysis of collected exudates: 2449 m/z-RT-signals differentially abundant

Genotypic Alterations in Exudate Patterns

GC-MS analysis of collected exudates: 80 m/z-RT-signals differentially abundant

Genotypic Alterations in Exudate Patterns

Endophytic Colonisation Affects Host Exudation

- Inoculation of A. thaliana Oy-0 with K. radicincitans

- Collection of root exudates

- Metabolic profiling: 57 differentially abundant compounds (GC-MS, LC-MS)

42 identified metabolites

- Decreased abundance of GLS-related metabolites in root exudates:

Metabolite Elemental

Composition Substance Class B/C p-Value

Tetrahomomethionine C9H19NO2S aliphatic GLS precursor amino acid 0.4 4.9E-03

Pentahomomethionine C10H21NO2S aliphatic GLS precursor amino acid 0.3 2.0E-03

Methylthiooctanoic acid C9H18O2S

aliphatic GLS degradation product 0.4 4.1E-04

Methylthiononanoic acid C10H20O2S aliphatic GLS degradation product 0.2 3.7E-04

Methylthioheptylamine C8H19NS aliphatic GLS degradation product 0.3 5.1E-04

Methylthiooctylamine C9H21NS aliphatic GLS degradation product 0.3 3.3E-03

B/C: ratio between the median intensity in the exudate of inoculated plants and the median intensity in

the exudate of non-inoculated plants

Measurements of other Arabidopsis genotypes

Acknowledgements

Dr. Franziska Hanschen

Prof. Monika Schreiner

PD Dr. Silke Ruppel

Dr. Rita Grosch

Dr. Anja Buhtz

Mandy Heinze

Prof. Dierk Scheel

Dr. Nadine Strehmel

Prof. Barbara A. Halkier

Dr. Deyang Xu

Funding:

Mechanisms of root exudation

Badri & Vivanco, Plant, Cell and Environment, 2009

Transport of Compounds into the Rhizosphere