Download Reducing Cyanide*Dependent ROS Production in Transgenic

Reducing Cyanide–Dependent ROS
Production in Transgenic Cassava Roots:
Impact on Post-Harvest Physiological
Deterioration
Tawanda Zidenga1, Dimuth Siritunga2 and
Richard Sayre1
1.Department of Plant Cellular and Molecular Biology, The Ohio State University
2. Department of Biology, University of Puerto Rico
Post-harvest physiological deterioration is a
major problem in cassava farming
Deterioration occurs in 72 hrs
Post-harvest physiological deterioration is a
major problem in cassava farming
PPD affects quality of crop for
consumption and marketing
There are land use implications
to harvesting cassava per piece
meal
There are several methods for
controlling PPD
1. Harvest when needed
2. Wax the roots
3. Process as soon as harvested
4. Use long shelf life cassava lines
Summary
Linamarin as a transportable form
of reduced nitrogen in cassava
The oxidative burst in wounded
cassava roots is induced by cyanide
release
Reducing cyanide-induced ROS
production via expression of
Alternate oxidase
Reducing cyanide-induced ROS may
be a strategy to control postharvest physiological deterioration
in cassava
PPD is a biochemically active process
PPD is associated with an oxidative
burst in cassava roots
There is also production of
phenolic compounds like scopoletin
And, increase ethylene
biosynthesis
Cyanide release in higher plants
Ethylene Biosynthesis
methionine
S-adenosyl methione
Degradation of cyanogenic glucosides
Linamarin
Acetone cyanohydrin
ACC
Acetone + HCN
Ethylene + HCN + CO2 + H2O
All plants
Cyanogenic plants
Linamarin is made in the leaves and transported to
the roots
Linamarin
Unprocessed cassava releases about 53 mg HCN
per 100g fresh weight.
Minimal lethal dose of HCN 0.5-3.5mg per kg body
weight.
The fate of cyanide in the root may have
important implications on single nitrogen
metabolism in cassava
DETOXIFICATION
Rhodanese
Thiocyanate
ASSIMILATION
β-Cyanoalanine synthase
Amino acids, Ammonia
Hypothesis: Assimilatory pathway, which incorporates cyanide
into nitrogen metabolism, is preferred in cassava roots
Rhodanese is barely detectable in cassava roots
Leaf Activities
Root Activities
Root β-cyanoalanine synthase activity is 3X
the shoot activity
Cyanogenic glucosides are assimilated via βCyanoalanine synthase in cassava roots
ASSIMILATION
β-Cyanoalanine synthase
Amino acids, Ammonia
Over-expression of Nitrilase 4 and β-cyanoalanine
synthase to enhance assimilation of cyanide in
cassava roots
ROOT
Linamarin
Hypothesis:
Root-specific
expression of βCAS and NIT4 will
increase amino acid
pool sizes in
cassava roots and
reduce cyanideinduced ROS
production
Linamarase
Acetone cyanohydrin
HNL
Cyanide + Cysteine
β-cyanoalanine synthase
Cyanoalanine + H2O
Nitrilase/hydrase
Asparagine
Aspartate + NH3
Current and future work on cyanide assimilation
Screening NIT4 and
CAS plants
Analysis of amino acid
pool sizes in the
transgenics compared to
wild type plants
Analysis of levels of
linamarin in transgenics
compared to wild type
plants
Using Low Linamarin transgenics to understand cyanide
metabolism in cassava roots
100
99
98
97
96
3.40
3.36
3.32
3.28
% Linamarin
0.08
2.0
0.98
0.73
0.06
1.5
0.04
1.0
0.29
0.5
0.33
0.02
0.26
0.0
0.00
WT
Siritunga and Sayre, 2003
Cab1-1
Cab1-2
Cab1-3
Cab1-4
Cab1-5
Linamarin (umoles/gdw)
2.5
Low cyanide cassava roots are significantly
smaller
CYP79D1/CYP79D2
antisense with
PATATIN promoter
CYP79D1/CYP79D2
antisense with CAB
promoter
P8
C3-12
P6
C3-5
P4
C3-1
Cyanide is an inhibitor of cytochrome oxidase in
mitochondrial respiratory chain
Cyanide sensitive
Biochemistry and Molecular Biology of Plants
Tissue disruption in cassava releases cyanide
Linamarin
Linamarase
Acetone cyanohydrin
HNL
Acetone + Cyanide
Analyzing ROS production in low and high
cyanide cassava
• Analysis of reactive oxygen
species production in wild type
and low cyanide cassava using
fluorescent probe
• Root-specific expression of
Alternative oxidase as a strategy
to control PPD in cassava
Hypothesis: Cyanide initiates post-harvest
physiological deterioration through increased
reactive oxygen species production
Over-expression of Nitrilase 4 and β-cyanoalanine
synthase to enhance assimilation of cyanide in
cassava roots
ROOT
Linamarin
Hypothesis:
Root-specific
expression of βCAS and NIT4 will
increase amino acid
pool sizes in
cassava roots and
reduce cyanideinduced ROS
production
Linamarase
Acetone cyanohydrin
HNL
Cyanide + Cysteine
β-cyanoalanine synthase
Cyanoalanine + H2O
Nitrilase/hydrase
Asparagine
Aspartate + NH3
Plants over-expressing Nitrilase 4 show reduced
production of ROS
NIT1
NIT1
NIT1
60444
60444
60444
The oxidative burst in wounded cassava roots is
cyanogen induced
CONTROL (no dye)
CAB (low cyanide)
WILD TYPE
Mean
18
16
14
12
10
Mean
8
6
4
2
0
Control
Low cyanide
Wild Type
Cyanide complements ROS production in low cyanide
plants
CAB (low cyanide)
WILD TYPE
CAB + 5mM NaCN
Mean
25
20
15
Mean
10
5
0
Low cyanide
Wild Type
Low cyanide + 5mM NaCN
The ROS-induced fluorescence is mitochondrial
WILD TYPE
WILD TYPE + DPI
Mean
.
20
18
16
14
12
10
8
6
4
2
0
Mean
Wild Type
Wild type + DPI
The NADPH oxidase
inhibitor, diphenyl
iodonium chloride
(DPI), does not inhibit
ROS production in wild
type cassava roots
Possible strategy to reduced cyanide-induced
ROS – express Alternative oxidase
Cyanide insensitive
Biochemistry and Molecular Biology of Plants
Cyanide sensitive
Why Aox
• Overproduction of
AOX in transgenic
cell lines reduces
ROS production
• Antisense cells with
reduced levels of
the AOX accumulate
five times more ROS
than control cells
Annu. Rev. Plant Biol. 2004. 55:373–99
Alternative oxidase reduces ROS production in
cassava roots
Wild type
AOX-3
AOX-1
18
16
14
12
10
8
6
4
2
0
60444
AOX1
AOX3
Current and future work on cyanide, ROS and PPD
Generating more transgenics
Evaluating confirmed
transgenics for ROS
Transferring transgenics to
Greenhouse
Evaluating AOX transgenics
for PPD
Evaluating low cyanide plants
for PPD
Summary of strategies for controlling PPD
ROS scavenging enzymes
Amino acids, NH3
ROS
Cyt ox
Assimilation
PPD
HCN
2
Cyanide
3
assimilation
Cyanide elimination
Aox
1
Sayre Lab Cassava Group
Dr. Uzoma Ihemere
Dr. Narayanan N. Narayanan
Dr. Hangsik Moon
Elisa Leyva –Guerero
Anthonia Soboyejo
Reid Rice
Sayre Lab Chlamy Group
Dr Shayani
Dr Vanessa Falcao
Anil Kumar
Zoe Gokhale
Dr. Mary-Ann Abiado
Ohio State University Microscopy Core Lab Facility
Biao Ding Microscopy Lab
BioCassava Plus