OxidativeStress Related Genes in Cassava Postharvest Physiological Deterioration

1
Oxidative-Stress Related Genes in Cassava
Post-harvest Physiological Deterioration

• 1Kim Reilly, 1Yuanhuai Han, 1Holger Buschmann, 2
  Maria Rodriguez, 1Rocio Gomez Vasquez, 2 Diego
  Cortes,2Joe Tohme, 1John Beeching
• 1University of Bath,Claverton Down, UK. 2
  CIAT, Cali, Colombia

2
Postharvest Physiological Deterioration (PPD)

• Endogenous root disorder
• Similarities to plant wound
• and defence responses
• Some genetic components,
• subject to environmental
• conditions

3
Oxidative Stress and Post-harvest Physiological
Deterioration

• Molecular oxygen is required
• Low temperature storage, high temperature
  treatment
• and cyclohexamide can inhibit or reduce PPD
• Total peroxidase activity increases during PPD
• Decreases in the non-enzymatic antioxidants
  b-carotene and ascorbate reported during PPD
• High root carotene content is correlated with
  reduced
• susceptibility to PPD

4
Reactive Oxygen Species in Plants
O2 O2i H2O2 iOH H2O

• Superoxide Dismutase
• 2O2- 2H H2O2 O2
• Catalase
• 2H2O2 2H2O O2
• Peroxidase
• H2O2 2AH 2A 2H2O

SOD CAT PX
Reducing equivalents
5
Production of Superoxide (O2-) in Wounded Cassava
Roots
Time after wounding (hours/minutes)
control 0.15 1.15 2.15 3.15
4.15 5.15 6.15 7.15 In situ
detection of O2- production by vacuum
infiltration with 0.05 NBT in 10mM K2HPO4
buffer (pH 6)
6
In situ Detection of Hydrogen Peroxide (H2 O2)
in Wounded Cassava Roots
Time after wounding (hours) control 3 6
9 12 15 18 21
24 27 30 33 36 In situ
detection of H2O2 production by vacuum
infiltration with 2mg/ml DAB and incubation at RT
for 3 hours. Control reactions were infiltrated
with DAB (2mg/ml) and 10mM ascorbate.
7
In situ Localisation of Hydrogen Peroxide (H2
O2) in Wounded Cassava Roots
40 x magnification 200 x magnification
cortical parenchyma ? xylem vessel ? ?
storage parenchyma
In situ localisation of H2O2 production in roots
examined by light microscopy. Time after injury
3 hours.

8
In vivo Quantification of Hydrogen Peroxide (H2
O2) in Wounded Cassava Roots
In vivo detection of H2O2 by the luminol
injection method of Warm Laties (1982)
9
Superoxide Dismutase (SOD) Expression in Wounded
Cassava Roots
Time after injury (days)
Time after injury (days) 0 1
2 3 4 0
1 2 3 4
MecCuZnSOD 18S rDNA
IEF PAG of cassava root SOD isoforms (left)
northern analysis of total root RNA using clone
MecCuZnSOD as probe (right)
10
MecCuZnSOD Expression in Different Tissues
in Response to Various Treatments
Root Petiole Leaf CK Pruned
CK Ethylene CK JA
MecCuZnSOD 18S rDNA
Northern analysis of total root, leaf or petiole
RNA using MecCuZnSOD as the hybridisation probe.
ROOT TREATMENTS Pruning treatment plants were
pruned 2 weeks prior to harvest. Ethylene
treatment 0.02 ethephon for 24 hours. JA
treatment 500mm MJA for 24 hours.
11
Peroxidase (PX) Expression during PPD
Time after harvest (days) 0 1 2
3 4 M pI
Time after harvest (days) 0 2
4 6
9.3 8.7 8.5 8.2 7.4 6.9 6.5 5.8 5.2 4.6 3.5
MecPX1 18S rDNA
IEF PAG of root peroxidase isoforms (left)
northern analysis of total root RNA using clone
MecPX1 as probe (above)
12
MecPX1 Transcript Expression in Different
Tissues
Root Petiole Leaf
MecPX1 18S rDNA
Northern analysis of total root, leaf or petiole
RNA using MecPX1 as the hybridisation probe.
13
MecPX1 Transcript Expression in Response to
Various Treatments
Control Pruned Control
Ethylene Control JA
MecPX1 18S rDNA
Northern analysis of total root RNA using MecPX1
as the hybridisation probe. Pruning treatment
plants were pruned 2 weeks prior to harvest.
Ethylene treatment 0.02 ethephon for 24 hours.
JA treatment 500mm MJA for 24 hours.
14
Localisation of Peroxidase Expression in
Wounded Cassava Roots
A B C
Tissue print localisation of peroxidase activity
in cassava roots. A tissue print showing
peroxidase activity in non deteriorated root. B
tissue slice stained with phluroglucinol.
Lignified and suberised tissues stain red. C
peroxidase activity in deteriorated root.
15
Localisation of Peroxidase Expression in
Wounded Cassava Roots
100X 200X
200X
Stained with guiacol Stained with
toluidine blue
16
Catalase (CAT) Expression in Wounded Cassava
Roots
Time after injury (days) 0 2 4
6 8 Leaf
MecCAT1 Total RNA
Northern analysis of total root RNA using clone
MecCAT1 as the hybridisation probe.
17
MecCAT1 Transcript Expression in Response to
Various Treatments
Control Pruned Control
Ethylene Control JA
MecCAT1 18S rDNA
Northern analysis of total root RNA using MecPX1
as the hybridisation probe. Pruning treatment
plants were pruned 2 weeks prior to harvest.
Ethylene treatment 0.02 ethephon for 24 hours.
JA treatment 500mm MJA for 24 hours.
18
Localisation of Catalase Expression in Wounded
Cassava Roots
A B C
Tissue print localisation of catalase activity in
cassava roots. A tissue print showing
catalase activity (T 0 days). B tissue slice
from which the print was prepared. Panel C
control tissue print stained with Coomassie blue.
19
Summary

• Rapid transient production of O2- and H2O2
• A possible signalling role?
• Catalase up-regulated in pruned (less
  susceptible)
• plants.
• A protective role via scavenging of of H2O2 ?
• In initial stages after harvest H2O2 and
  peroxidase
• are localised primarily to the xylem tissues.
• Implications of this localisation re. vascular
  
• streaking?

20
Model 1 Mechanistic Level
Peroxidase H2O2
Scopoletin Vascular

Streaking

Localisation data provide support for the
hypothesis of Tanaka et al. and Wheatley et al.
that PPD results from peroxidase mediated
oxidation of scopoletin.
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Model 2 Molecular Level



wounding








-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-
pre
-
harvest pruning?



oxidative burst

other signals?

O

2
exclusion
of O

-

-

-

-

-

-

-

-

-

-

-

-

-

2
-
O

H
O

2
2
2
OH
formed via

SOD

-

?

Haber
-
Weiss reaction


-

-

-

-

-

-

-

-

-

-

-

-

-

-

-
Cycloheximide

Induction of PPD related gene expression

Membrane breakdown.


Cell death.

Lipid based signal
s JA?

Defence related genes

Antioxidant genes

PCD genes?

PAL

Catalase


Aspartic protease


Peroxidase
ACC oxidase





Serine protease


Phenylpropanoid

H
O

H
O


2
2
2
2


s
cavenging?

s
cavenging?


metabolism

Cysteine protease



ethylene


Inhibitor
(cystati
ns)

Wound healing?

Modulation of


H
O
signalling?


2
2

Interaction with
Scopoletin



Promotion / modulation
scopoletin?
of
PCD responses?


















22
Acknowledgements

• DFID ( Department for International
• Development ), UK
• Colciencias, Colombia
• CIF, Colombia
• University of Bath, UK