Post on 29-May-2020
transcript
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