Transgenic Cassava: The Products and Future Directions

Transgenic Cassava: The Products and
Future Directions
Richard Sayre, Dept. Plant Cellular and Molecular Biology, Ohio State Univ. Columbus, OH,
• Disease and Insect
Resistance
• Starch Production and
Modification
• Enhancing Root Storage
Proteins
• Engineering Cyanogenesis
• Engineering Protein
Expression in Roots

Disease and Insect Resistant
Cassava
• Transgenic cassava expressing an ACMV gene (in
sense) provides cross-protection to a variety of related
cassava mosaic viruses. Evidence that siRNAs are the
mechanism of resistance. Entering field trials in Kenya.
Nigel Taylor and Claude Fauquet
• Transgenic cassava expressing RNAi constructs
targeted to silencing ACMV effective in greenhouse
trials. Zhang and Gruissem
• Transgenic cassava expressing BT (control of cassava
hornworm) goes into field trials in April at CIAT. Joe
Tohme and colleagues.

Engineering Starches in Cassava
• A transgenic waxy starch (high
amylopectin) cassava was reported at
CBN V (2001) by Krit Raemakers and
Richard Visser is field trial in US Virgin Is.
• Higher yielding transgenic cassava has
been achieved by specifically expressing a
bacterial AGPase in roots.

Cassava has tremendous untapped
potential for increased starch
production
• Very high photosynthetic rate, approaching
C4 plants.
• Highest rate of sucrose synthesis measured
to date.
• In the greenhouse excess sucrose is lost
through gutation; represents a lost source of
carbohydrate.
• Large starch storage organs.

Hypothesis
Root-specific expression of a
modified (K296E/G336D)
bacterial glgC gene encoding a
more active ADP-glucose
pyrophosphorylase (AGPase
catalyzes the rate-limiting step in
starch synthesis) will result in,
1) increased carbohydrate sink
strength in roots,
2) reduced feedback inhibition
on photosynthetic carbon
fixation
3) increased starch production.
Transgenic

Plasmid WT 3D-1 3D-2 3D-3 3D-4 3D-5 Marker
The glgC gene is expressed in roots of
transgenic cassava. RT-PCR analysis
demonstrates expression of glgC gene.
350 bp

RT-PCR analysis of glgC and CYP79 (D1)
expression demonstrates that the patatin-glgC
construct is not expressed in leaves.
350 bp
650 bp
P WT 3D-1 3D-2 3D-3 M
glgC
CYP79
(D1)

PPi + ADP-Glucose Glucose-1-P +ATP
6-phosphogluconolactone Glucose-6-P
ug NADPH /mg protein/hr
0.6
0.5
0.4
0.3
0.2
0.1
0.0
NADPH NADP +
Control 3D-1 3D-2 3D-3
AGPase activity is elevated two-fold in transformed
plants expressing the glgC gene.

Plants expressing the glgC gene have more than
twice the root biomass (dry weight) of wild-type
plants.
Cultivar→
(relative AGPase
activity)
Leaves (g)
(% WT)
Stems (g)
(% WT)
Stem-Root
Junction (g)
(% WT)
Roots (g)
(% WT)
Wild Type
(100)
55.1+/-8.3 a
(100)
89.7+/-5.0 c
(100)
16.7+/-1.5 b
(100)
18.9+/-4.3 c
(100)
3D-1
(200)
68.6+/-4.0 a
(123)
148+/-8.8 a
(165)
25.4+/-2.5 a
(156)
48.2+/-9.3 a
(266)
3D-2
(100)
60.4+/-0.84 a
(109)
114+/-25.3 bc
(128)
25.2+/-2.7 a
(156)
24.9+/-7.8 bc
(133)
3D-3
(160)
62.5+/-9.4 a
(112)
134+/-14 ab
(150)
23.1+/-3.6 ab
(143)
28.5+/-8.7 b
(155)
6 month greenhouse study. Values with different letters are significantly different from each other.

Summary
• Transgenic cassava having two-fold higher
AGPase activity have been successfully
engineered.
• Transgenic plants having higher AGPase
levels produce more than twice the root
biomass of control plants.
• Total biomass of transgenic plants also
increased possibly reflecting a release of
feed-back inhibition on photosynthesis.

Expression of an Artificial Storage Protein 1
(ASP1) in cassava leaves and roots.
Amino Acid
Composition
Arg 3.2%
Glu 16.8%
Gly 6.3%
Ile 9.5%
Leu 8.4%
Lys 17.9%
Met 12.6%
Phe 4.2%
Pro 3.2%
Thr 9.5%
Trp 3.2%
Val 5.3%
Zhang Peng and Willi Gruissem, ETH, Zurich, Switzerland
ASP1 is enriched in essential amino acids that
are limiting in cassava and was designed to
mimic stable storage albumin proteins.
ASP1 traits:
• Four, 20 amino acid, amphipathic helical
repeats connected together by three β-turns.
• Tertiary structure is stabilized by Glu-Lys salt
bridges.
• Predicted size = 7.5 kD; pI = 9.35.
Zhang et al. (2003) Transgen. Res. 12: 243-250

Western blot analysis of ASP1
expression (35S) in leaves and roots.
Leaves
HIGH
Roots
LOW
Zhang et al. (2003) Transgen. Res. 12: 243-250

Changes in leaf protein amino acid content of
plants expressing ASP1.
• Amino acids having elevated levels in leaves of ASP1
transgenic plants included:
– Arg (5-30%)
– Ile (2-5%)
– Pro (5-10%)
– Ser (10%)
– Thr (2-8%).
• Amino acids having reduced levels in leaves of ASP1 trangenic
plants included:
– Tyr (5%)
– Met (2-8%)
– Ala (6-12%)
– Glu (5%)
– Asn (20-22%)
• Expected results: Higher Arg, Glu, Ile, Leu, Met, Lys, Phe, Thr,
Pro, Trp, Val, Gly
• Results from roots presented on poster by Zhang et al..

Engineering cyanogenesis in
transgenic cassava
Dimuth Siritunga and Richard Sayre, Dept. of Plant Cellular and
Molecular Biology, Ohio State Univ., Columbus, OH 43210 USA
• Inhibition of cyanogenic glycoside
synthesis may be lethal to cassava.
• Acceleration of cyanogen turnover.
Siritunga D and Sayre RT (2003) Planta 217: 367-373.
Siritunga et al. (2004) Plant Biotechnology Journal 2: 37-43

Target enzymes for regulating cyanogen synthesis
and turnover in transgenic cassava
Valine
N-hydroxyvaline
2-Methyl proponol oxime
Acetone cyanohydrin + UDPG
Linamarin
Glucose + Acetone cyanohydrin
Acetone + HCN
CYP79D1
CYP79D2
CYP71E
UDPG-glucosyl
transferase
Linamarase
Hydroxynitrile lyase
(or spontaneously)

Agrobacterium-mediated Agrobacterium mediated transformation
Antisense inhibition of
CYP79D1/D2 expression
using leaf-specific Cab1
promoter
Antisense inhibtion of
CYP79D1/D2 expression
using tuber-specific
Patatin promoter
Siritunga and Sayre (2003) Planta 217: 367-373

LEAF linamarin levels were reduced between 60%
and 94% in Cab1-CYP79D1/D2 Cab1
transformants
% Linamarin
100
80
60
40
20
0
11.3
29.4
23.5
40.5
6.2
WT Cab1-1 Cab1-2 Cab1-3 Cab1-4 Cab1-5
80
70
60
50
40
30
20
10
0
Linamarin (umoles/gdw)

Surprise, root linamarin levels in Cab1-CYP79D1/D2
transformants were < 1% of wild-type levels!
% Linamarin
100
99
98
97
96
2.5
2.0
1.5
1.0
0.5
0.0
0.98
0.29
0.26
0.33
0.73
WT Cab1-1 Cab1-2 Cab1-3 Cab1-4 Cab1-5
3.40
3.36
3.32
3.28
0.08
0.06
0.04
0.02
0.00
Linamarin (umoles/gdw)

How can ROOT linamarin levels be reduced
in Cab1-CYPD1/D2 antisense plants?
Two options……….
1. Cab1 promoter, although leaf-specific, might be
‘on’ on’ in cassava.
Or
2. Root linamarin is synthesized in leaves and
transported to the roots.

• No suppression of CYP79 expression in roots driven by
cab1 promoter (not shown).
• No reduction in leaf or root linamarin levels in patatin-
CYP79D1/D2 antisense plants. Linamarin is transported!
% Linamarin
100
80
60
40
20
0
Pat1 Pat2 Pat3 WT
Leaves
Roots

Summary
• Inhibition (> 60%) of linamarin synthesis in leaves
results in no linamarin accumulation in roots.
• Inhibition of CYP79D1/D2 expression in roots has
no effect on root linamarin levels.
• Root linamarin is made in leaves and transported to
roots.
• The function of root CYP79D1/D2 activity is
unclear.

Accelerating cyanogen turnover in processed
cassava maintains the benefits of cyanogens
The major cyanogen present in poorly processed cassava roots is
acetone cyanohydrin, not linamarin.
Cassava roots have virtually no HNL activity (0 - 6% of leaf
levels) possibly accounting for high acetone cyanohdrin levels
Elevated expression of HNL in roots is predicted to accelerate
the conversion of acetone cyanohydrin to cyanide and the
detoxification (cyanide volatilization) of cassava root starch.
LB
2X35S Rbcs Ter Nos Ter Kan r Nos Prom
RB
Siritunga et al. (2004) Plant Biotechnology Journal 2: 37-43

Accelerating cyanogenesis in cassava
Valine
N-hydroxyvaline
2-Methyl proponol oxime
Acetone cyanohydrin + UDPG
Linamarin
Glucose + Acetone cyanohydrin
Acetone + HCN
CYP79D1
CYP79D2
Putative
CYP71E
UDPG-glucosyl
transferase
Linamarase
Hydroxynitrile lyase
(or spontaneously)

Root hydroxynitrile lyase (HNL) activity in wildtype
and transformed plants
Specific Activity (umoles CN/mg protein/hr)
350
300
250
200
150
100
50
0
WT HNL-1 HNL-2 HNL-3

Reduction in root acetone cyanohydrin levels in
processed roots of plants expressing elevated HNL
activity.
ACN (umoles/gfw)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Wild type
HNL-1
HNL-2
HNL-3
0 20 40 60 80 100 120
Time (minutes)
100 %
59 %
49 %
35 %

Summary
• The absolute increase in HNL activity was 10-fold
greater in leaves than in roots of transgenic plants.
• A 13-fold increase in root HNL activity was
achieved, however, the absolute increase in enzyme
activity was small.
• Over-expression of HNL in roots accelerates
cyanogenesis by more than 3-fold and facilitates
cyanogen removal and food detoxification.

Why is it difficult to express proteins in
roots of cassava?
• Transcriptional control: Strong root promoters
(1-2X 35S and patatin) do not deliver
expression levels comparable to leaves.
• Post-translational: No evidence for
accelerated protein turnover from westerns.
• Translational: Limiting free amino acid pool
sizes for protein synthesis.

It is easier to over-express proteins in
leaves than roots when using strong
Enzyme Activity
(mMol CN/mg protein/hr)
Hydroxynitrile lyase
(HNL)
HNL-transgenic
2X35S promoter)
promoters (2X35S).
Root
Activity
0.025
0.325
Leaf
Activity
1.7
4.0
Leaf
minus
Root
Activity
1.68
3.68
Root/Leaf
Ratio
0.02
0.08

Free amino acid pool sizes in cassava roots
are low and may limit protein production
• The root/leaf free amino acid pool size
ratio in cassava is 1:6. Cassava roots and
leaves typically have 1% and 6-10%
protein, respectively.
• The root/leaf ratio of free amino acids in
wild cassava relatives having high (8%)
root protein content is 1:4.2
(Martin Fregene, personal communication).

Invariably, enzymes that are expressed in cassava leaves and
roots have 10-20 fold higher activity levels in leaves than in
roots with the exception of CN-assimilating enzymes.
Enzyme
Hydroxynitrile lyase (HNL)
HNL-transgenic (35S promoter)
Linamarase
β-cyanoalanine synthase
β-cyanoalanine hydrase
Asparaginase
Root/Leaf
Activity Ratio
0.02
0.08
0.05
1.39
2.84
0.70

Linamarin is a transportable form of reduced
nitrogen for amino acid and protein synthesis in
cassava roots
• Linamarin accounts for 60% of the total nitrogen in phloem
exudates of wild-type plants.
• Roots of Cab1 - CYP79D1/D2 antisense plants will not grow
if supplied nitrate as the sole source of nitrogen.
• Addition of ammonia to growth media restores root growth to
Cab1 – CYP79D1/D2 antisense plants.
• In contrast, Patatin – CYP79D1/D2 antisense plants grow as
well as WT plants in media containing nitrate as the sole
source of nitrogen.
• Conclusion: The absence of root linamarin necessitates
an alternate source of reduced nitrogen for root growth

Biochemical evidence for cyanide as a source
of reduced nitrogen for amino acid synthesis
in cassava
• Nartley observed in 1969 that germinated cassava
seedlings exposed to 14 CN incorporated 49% of the label
into the amide C of asparagine and 6% in aspartate.
• Some 14 CN was also incorporated into glutamine and
glutamate via aspartate deamination and cycling of carbon
through the TCA cycle.
• The most abundant free amino acids in cassava seedlings
are asparagine and glutamine. Cysteine could not be
detected.
• Conclusion: CN is probably assimilated into free
amino acids concomitant with the consumption of
cysteine.
Nartley F. (1969) Physiol. Plantarum 22: 1085-96.

Diversion of linamarin from storage to amino acid synthesis could
increase amino acid pool sizes and protein synthesis in roots
Linamarin
Linamarin
Export
Linamarin
Storage
Valine
Linamarin
Storage
Acetone cyanohydrin
Linamarin can be stored in roots
or used for protein synthesis.
CN - + Cysteine
β-cyanoalanine + H 2 O
Asparagine
Aspartate + NH 3
β-cyanoalanine
synthase
β-cyanoalanine
hydrase
Asparaginase

1. RT-PCR RT PCR analysis of ROOT CYP79D1/D2 transcript
levels in Cab1 transformants indicates the Cab1
promoter is not ‘on on’ in roots.
Wild Type
Cab1-1
Cab1-2
Cab1-3
Cab1-4
Cab1-5
- control
+ control
700bp CYP79D1
550bp CYP79D2
SBE II gene

RT-PCR RT PCR demonstrates the reduction or complete loss of
CYP79D1/D2 transcripts in leaves of Cab1- Cab1
CYP79D1/D2 antisense plants.
WT
Cab1-1
Cab1-2
Cab1-3
Cab1-4
Cab1-5
- control
+ control
700bp CYP79D1
550bp CYP79D2
SBE II gene
RT-PCR on 8µg of Cassava Cab1 transformant total leaf RNA. CYP79D1/D2
gene specific primers amplify the 3’-end of the respective genes.

2. CYP79D1/D2 expression is inhibited in ROOTS of patatin- patatin
CYP79D1/D2 antisense plants.
Pat-2
Pat-1
Pat-3
Pat-2
Pat-1
Pat-3
WT - +
WT - +
CYP79D1
CYP79D2
SBE II
CYP79D1
CYP79D2
SBE II
Root RT-PCR RT PCR
shows
elimination of
root
transcripts
Leaf RT-PCR
shows little or
no effect on
leaf transcript
levels

PCR amplification of patatin/glgC gene border
in transgenic (3D) cassava
Plasmid WT 3D-1 3D-2 3D-3 3D-4 Marker
.
• the fidelity of the PCR product was confirmed by DNA sequence
analysis.
850 bp

UDP-Glucose +
Fructose-6-P
Rate Limiting Step
ADP-Glucose
sucrose phosphate synthase
AGPase
Sucrose-6-P +
UDP + H +
Sucrose
invertase
Fructose + Glucose
Glucose-1-P

Leaves and roots of transgenic plants have
greater mass per unit than wild type.
Plants Ratio of leaf
weight/leaf
number
WT 2.40 10.57
3D-1 2.72 16.58
3D-2 2.6 14.13
3D-3 2.62 11.20
Ratio of root
weight/root
number

T-DNA containing modified glgC gene with an Nterminal
chloroplast transit peptide.
RB
850bp
Patatin TP
glgC nos polyA
1.2kb 1.3kb
The red box corresponds to the unique T-DNA region
amplified by PCR for identification of transgenic
plants.
LB

Limiting free amino acid pool sizes
affect protein synthesis in plants
• Due to limiting amino acid pool sizes, expression of a sulfurrich
sunflower seed albumin gene (accounting for 7% of
total seed protein) in transgenic rice resulted in no net
change in the total amount of sulfur-containing amino acids.
(Hagan et al., (2003) Pl. J. 34: 1-11).
• Enhancing the expression of sulfur-rich proteins in peas was
dependent on the pool sizes of free Cys and Met. (Tabe et al.
(2002) Cur. Opin. Pl. Biol. 5: 212-217).
• Expression of threonine and methionine rich proteins in
transgenic tobacco having 15-fold and 3-fold higher
threonine and methionine levels, respectively, was limited
by the pool sizes of other amino acids. (Karchi et al. (1993) Pl. J. 3:
721-727).

Cyanogen catabolism and amino acid synthesis in
roots
Linamarin
Linamarin
Valine
Linamarin
Acetone cyanohydrin
Linamarin can be stored in roots
or used for protein synthesis.
CN - + Cysteine
β-cyanoalanine + H 2 O
Asparagine
Aspartate + NH 3
β-cyanoalanine
synthase (1.4x)
β-cyanoalanine
Hydrase (3X)
Asparaginase
(0.7X)

GBSS
SSS
ADP-Glucose
sucrose phosphate synthase
UDP-Glucose +
Fructose-6-P
Rate Limiting Step
In Starch Production
AGPase
Sucrose-6-P +
UDP + H +
Sucrose
invertase
Fructose + Glucose
Glucose-1-P
+ ATP

ENGINEERING CASSAVA FOR INCREASED
STARCH PRODUCTION
Uzoma E. Ihemere 1 and Richard T. Sayre 2
Dept. of Horticulture & Crop Science 1 and Dept. of Plant Cellular and Molecular Biology 2
Ohio State University, Columbus, OH 43210 USA
Ihemere et al. (2004) in preparation for Plant Physiology