Transcript Altering Plants to Increase Nutritional Value

Altering Plants to Increase
Nutritional Value
Ann E. Blechl
USDA Agricultural Research Service
Albany, CA
Ways to Alter Plant Composition

Change how and/or where they are grown
– Agronomics

Change genes
– Traditional Breeding
» Introduce new variability by crosses or induced
mutations
– Genetic Engineering
» Introduce genes artificially (genetic transformation)
Advantages of Genetic Engineering
Compared to Traditional Breeding

Breeding
– Genes from limited # of
sources
» sexually compatible
relatives
– Crosses change half the
gene composition
(genome)
» Backcrosses to Adapted
Varieties Needed

Genetic Engineering
– Genes from any source
» Natural genes modified
for specific purposes
» Chemically synthesized
– Add one or a few known
genes at a time
Disadvantages of Genetic Engineering
 Unintended side effects of tissue culture
or gene insertion
– Also an issue for induced mutations in traditional
breeding
 Currently limited to varieties that
regenerate from tissue culture
 Public Acceptance
 Costly to clear regulatory and intellectual
property hurdles
Some Targets for Increased
Nutritional Value

Increased essential amino acids to make seeds
complete protein sources
– Increased lysine in cereal grains
– Increased methionine in beans

Low-Phytate Grains
– Increased bio-available iron and zinc up to 50%
– Decreased phosphate waste


Changes in fatty acid composition of oil seeds to
less saturated types
Changes in soybean anti-oxidant composition
 Vitamin E, shift tocopherol profiles to mainly -form
Changing Carotenoid Contents




Lycopene is an antioxidant
- and -carotenes
are precursors of
vitamin A
Tomato lycopene
levels have been
raised 2-3 fold
-carotene synthesis
has been engineered
in tomatoes and rice
From Rosati et al., 2000
HighCarotene
Tomatoes
Fig. 2. Phenotypic analysis of high -carotene transgenic and control
Red Setter tomato plants. Transgenic (right) and Red Setter (left).
All parts of the transgenic fruits (columella, pericarp and placenta) are
intensely orange coloured.
From D’Ambrosio et al., 2004
Engineering Vitamin A
biosynthesis in rice seeds
 Cereal plants have carotenoids in their green tissues,
but very little in their seeds
 In developing countries, about 250 million people
don’t get enough Vitamin A in their diets
 This deficiency results in retarded growth and
increased incidence of
– Blindness
– Infant and childhood mortality
 The Rockefeller Foundation funded a Swiss and a
German group in a collaborative project to increase
the -carotene (pro-vitamin A) content of rice grains
“Golden Rice”
 Peter Beyer and Ingo
Potrykus groups added 2
genes in pathway to
provitamin A
– Daffodil phytoene synthase
– Bacteria phytoene desaturase
– Added seed-specific
promoters
 0.8-1.2 g per gram
 At typical rice consumptions
levels in Asia, golden rice
would supply about 1/3
RDA of -carotene
From Hoa et al., 2003
“High-Selenium Beef,
Wheat and Broccoli: a
Marketable Asset?”
 USDA IFAFS
 One
grant
goal: Engineer wheat to
accumulate increased levels of
selenium in flour
Metabolism
of Selenate
and Selenite
in Most
Plant Cells
Glutathione
 Generally, plants
accumulate Se in
proportion to its
concentration in soil
 10 - 100 g per
gram dry weight
Adapted from LeDuc et al., 2004
Astragulus
bisulcatus
(locoweed)
can accumulate
as much as 2 mg
selenium
per gram
From Pickering et al, 2003
Metabolism of
Selenate and
Selenite in
Plant Hyperaccumulators
Adapted from LeDuc et al., 2004
Glutathione
Sequence of the
Astragalus gene
encoding
selenocysteine
methyltransferase
(SMT)
From Neuhier et al, 1999
Experimental Plan
 Modify Astragalus SMT gene for expression
in wheat seeds
 Transform wheat with modified SMT gene
 Verify transgene inheritance
 Measure amounts of SMT RNA and enzyme
activity
 Measure accumulation of Se in seeds from
transgenic wheat plants grown in selenate
and selenite
– How much Se?
– In what chemical form?
The SMT Coding Region Was Inserted
Between the Promoter and Transcription
Terminator Regions of Wheat Glutenin Genes
2945 bp
Wheat Glutenin
Promoter *
1013 bp
Astragalus
SMT Coding
Region
*Endosperm-Specific Expression
2017 bp
Wheat Glutenin
Transcript
Terminator
Biolistics (the “Gene Gun”) was used to
introduce two DNAs into wheat embryos
1. Glutenin:SMT gene
+
2. Herbicide (Bialaphos)
resistance gene
Tissue Culture Steps for
Wheat Transformation
Shoots and Roots are
Regenerated Under
Herbicide Selection
Inheritance of Glutenin:SMT
Transgene
M+- 1 23 4 567 8
1 23 45 6 78M
656 bp
Transgene Messenger RNA Levels
M
SMT
5
20 40
Actin
5
20 40
low expresser
SMT
5
20 40
Actin
5
20 40
high expresser
M
Results I
30 independent transgenic wheats
containing the Glutenin:SMT gene
 Expression ranged from 4x to 1/8x the
levels of actin
 Homozygous seeds from 2 medium- and
2 high-expressers were sent to Michael
Grusak

– USDA-ARS Children's Nutrition Research
Center, Houston, TX
Results II

Mike Grusak grew the wheats
hydroponically with selenate added from
spike emergence to harvest
– 10, 20, 30 and 40 M

Mike observed no differences between
the the four transgenic and control
plants
– Plant and seed development
– Seed set
Results from LeDuc et al., 2003


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
Same Astragulus SMT gene
Engineered to be expressed in fast-growing
mustard plants for phytoremediation
Transformed Arabidopsis and Brassica juncea
Transgenics
– Accumulated SMT enzyme
– Tolerated higher concentrations of selenate and
selenite than their non-transformed parents
– Accumulated more Se (2-4x)
– Accumulated more MethylSelenoCysteine (1.5-10x)
– Produced up to 2.5x more volatile Se
Limiting in mustards
Proposed Fates for
Selenate and
Selenite in Mustard
Plants Expressing
Astragalus SMT
Glutathione
Enzyme?
Adapted from LeDuc et al., 2004
What’s next for us?

Michael Grusak will regrow the
transgenic wheats with selenite
supplementation

John Finley will measure SMT activity,
Se amounts and forms in wheat flour

Feed rats?
Acknowledgements
 Chika Udoh
 Jeanie Lin
Acknowledgement of Support
 USDA IFAFS grant
“High-Selenium Beef, Wheat and Broccoli:
a Marketable Asset?”
 Agricultural Research Service
Dough
ViscoElasticity
The biotechnology approach: use
genetic transformation to add
HMW-glutenin genes
 Dough strength depends on flour proteins.
 Especially important are the larger type of
glutenin proteins, HMW-Glutenins.
 We have added glutenin genes to change
the proportion of these proteins in wheat
flour.
 Flours from these wheats have differing
mixing and baking properties.
Increases in native HMW-glutenin
subunits increases dough strength
T
Transgenic (T)
Control (C)
0
10
20
minutes
30
C
1.9x
Dx5
1.3x
Dy10
Mixing and Baking Results from
Field-Grown Transgenic Wheats
Protein Content
Dx5
Dy10
11.4%
1.5
2.2
11.7%
2.7
1.7