Seed Storage Proteins & Approaches For Improvement Of Their Nutritional

Quality By Genetic Engineering

• Seed storage proteins of grain crops meet major dietary protein requirement of over half of the world
population. However, Cereal proteins are generally deficient in lysine and tryptophan whereas legumes
are deficient in sulphur containing amino acids – methionine and cysteine which limits their nutritional
value.
• By convention, the ‘quality ‘of seed protein refers to how far its amino acid composition matches the
balanced amino acid composition recommended for human diet by the WHO.
• Recombinant DNA technology, has led to an enhanced interest in the means for qualitative and
quantitative improvement of seed grains by genetic engineering.

Seed proteins were classified many years ago by Osborne based on their solubility as follows:

Albumins: Water extractable (1.6S–2S);

Globulins: Extractable in dilute salt solutions (7S–13S);
Prolamins: Extractable in aqueous alcohol;
Glutelins: Most difficult to solubilize; Extractable by weakly acidic or alkaline or dilute SDS solution.

Albumins and globulins comprise the storage proteins of dicots (e.g. pulses), whereas prolamins and glutelins
are major proteins in monocots (e.g. cereals). The seed storage proteins can be distinguished from other
proteins by some of their characteristics, e.g.
1. These accumulate in high amounts in seed during mid-maturation stage of seed development and are
used up during germination.
2. These are synthesized only in the seed (in cotyledon or in endosperm) and not in other tissues.
3. They lack any other functional activity besides storage.
4. They are deposited mostly in special storage organelles called protein bodies.

Attempts To Improve The Nutritional Quality Of Seed Proteins

Two broad approaches for improving the nutritional quality of seed proteins by rDNA techniques:

(1) Gene alteration / Modification of endogenous genes - in which a seed protein gene already present and
expressed in the plant seed is isolated and its coding sequence is altered by addition, substitution or deletion
of nucleotides by site-directed mutagenesis, followed by reintroduction of the modified gene under its own
promoter into the plant itself

(2) Gene addition/ Introduction of a appropriate transgene - in which a new capability or quality is
conferred to the seed by introduction of new heterologous genes encoding higher levels of desired amino
acids, under the seed’s own promoter or more efficient heterologous promoter.

The first transgenic approach to improve Met content of seed proteins was by Saalbach et al. in 1988.
The following illustrates some attempts to improve major crops.

Improvement Of Legumes

• An ideal candidate for enhancement of methionine content of legume seeds by this strategy is the
seed-specific 2S albumin gene of Brazil nut (Bertholletia excelsa). Approx 18% of the codons of this
gene code for methionine and 8% for cysteine. This 2S albumin gene under cauliflower mosaic virus
(CaMV) 35S promoter was transferred to tobacco and the grain legume Vicia narbonensis. The 2S
albumin was expressed in all parts of the plant both in tobacco and the legume and was found to be
localized in the vacuoles of the leaf mesophyll cells of transgenic tobacco.
• In another attempt the 2S albumin gene, under a broad bean (V. faba) legumin B4 promoter for seed
tissue-specific expression was transferred to V. narbonensis. This showed that up to 3% of soluble seed
protein was 2S albumin. Some transformants had 3-fold increase in methionine content of the salt-soluble
protein fraction in seed.
• In another attempt, a maize gamma-zein gene encoding a sulphur amino acid-rich protein was used
to transform alfalfa and trefoil (Lotus corniculatus) under CaMV 35S promoter and RUBISCO small
subunit promoter. Expression level was rather low to the extent of 0.05% of alcohol soluble protein.
• In yet another attempt, gamma-zein gene was modified by the introduction of (pro-lys)n coding
sequences at different positions. High levels of protein expression were observed when (pro-lys)n
sequence was contiguous or in substitution of pro-X region of the gamma-zein. Muntzet al. summarized
the results of genetic engineering for high methionine grain legumes. The foreign protein amounted to 5–
10% of the seed proteins in the best transgenic lines.
• Transgenic soybean reached 100% and narbon bean and lupin reached 80% of the WHO standard for
nutritionally balanced proteins. These results document that the Met problem of grain legumes can be
solved by genetic engineering.
Improvement Of Cereals

• Most of the work on maize and barley improvement was based on the characterization of ‘high
lysine’ and ‘Highproly’ lines identified by screening of several thousand collections from wild
germplasm. This Highproly mutant has reduced proportion of prolamin (which is deficient in lysine) with
an accompanying increase in other more lysine-rich proteins. However, this mutation is highly linked to
an agronomically harmful trait, namely, reduced starch content, thereby decreasing grain yield drastically.
Attempts to disrupt this linkage by classical breeding did not seem promising.
• An alternate approach is to introduce a gene for proteins having nutritionally enriched a.a
composition under strong endosperm-specific promoter like that for high mol wt. glutelin. For example,
beta-phaseolin (6% lysine) gene of common bean Phaseolus vulgaris under rice glutelin promoter was
expressed to the level of 4% of total endosperm protein in transgenic rice seeds.
• Mutant or less abundant seed storage protein zein from maize has been frequently used for quality
improvement. To increase methionine level, a new methionine-rich zein, normally expressed at low levels
was expressed at a high level using the 27 kDa zein promoter. This protein called the high sulphur zein
(HS 7) was 21 kDa and contained 37% Met.
• Soybean ferritin gene under rice glu-B-1 promoter has been introduced into rice to increase the iron
content.

Improvement Of Crucifers And Other Seeds

• The small mol. wt. albumin (2S protein or napin) of rapeseeds/mustard is one of the most important
targets for the improvement of its nutritional quality and also for‘Molecular Pharming’. There have been
several promising attempts for the genetic engineering of 2S albumins.
• Brazil nut 2S protein gene and a methionine-enriched modified napin were introduced into B. napus
and were expressed to marginally improve the methionine content of total seed protein. Altenbach et al.
reported the highest level of expression of Brazil nut protein in transgenic B. napus ranging from 1.7% to
4% of total seed protein. This 4%methionine-rich protein contributed about 33% more methionine in the
total seed protein.
• Studies on B. juncea 2S promoter dissection indicated that a suitably modified (to exclude negative
regulatory regions and by duplication of enhancer sequence) homologous promoter is more efficient in
the expression of a reporter gene. Also, the 3′ -terminator sequences used have always been from a
heterologous source. A homologous 3′ -sequence might be better for expression of foreign /modified
gene.
 • In another attempt, an indirect strategy was used in which the antisense gene for cruciferin was
introduced into B. napus, which resulted in the suppression of cruciferin and increase in the relative
proportion of napin, amore balanced protein. The antisense construct contained a part of cruA gene in
reversed orientation, driven by 5′ - flanking sequence of napin gene, such that the antisense cruciferin
mRNA was expressed in a seed-specific manner. The transgenic T1 generation seeds contained 10%, 8%
and 32% higher Lys, Met and Cys, respectively, compared to their respective levels in control seeds.

SOME PROMISING TRANSGENIC APPROACHES FOR IMPROVING NUTRITIONAL

QUALITY OF SEED PROTEINS
Gene/source Promoter/modification Transformed plant Improvement
1 2S protein (Brazil nut) CaMV 35S promoter V. narborensis Met
2 2S protein (Brazil nut) Phaseolin promoter Rapeseed Met
3 S-rich γ-zein (maize) CaMV 35S promoter Alfalfa, trefoil S-content
4 2S protein (sunflower) CaMV 35S promoter, ER-retention Clover S-content ,rumen-protected
signal
5 Beta-phaseolin (common Rice glu promoter Rice Lys
bean)
6 Ferritin (soybean) Rice glu-B1 promoter Rice Fe-content
7 Ama 1 (amaranth) Ama-1 promoter, Rice, chickpea Amino acids
Rice glu promoter
8 HS-7 zein (maize) 27 kDa zein promoter Rice Met
9 Cruciferin (rapeseed) Napin promoter, Rapeseed Met, Lys, Cys
CruA antisense gene
1 2S protein (Brazil nut) V. faba legB4 promoter V. narborensis Met
0

NUTRITIONAL ENHANCEMENT OF PLANTS (CARBOHYDRATES)

Genetic Modification Of Plant Carbohydrate Metabolism

Considerations When Modifying Carbohydrate Metabolism :-

1. Source – Sink Interactions

Adequate translocation of sucrose from source to sink tissues ( meristems , immature leaves, seeds, roots
etc) is essential to ensure optimal plant growth and crop yield. For eg potato plants transformed with an
antisense construct of the sucrose transporter cDNA under the control of the CaMV 35S promoter showed
reduction in the expression of the sucrose transporter (SUT1) RNA & hence reduction in the export of
carbohydrates from the source leaves as compared to wild-type plants thus reduced growth, root
development & tuber yield.
2. Flexibility Of Carbohydrate Metabolism

A characterisitic feature of plant metabolism, is that alternative routes to the same product often exist
within the same tissue that makes it harder to manipulate carbohydrate metabolism because attempts to
change flux through one pathway may be compensated by changes in another.

3. Sugar- Induced Signaling In Plants

Carbohydrates such as glucose, fructose,& sucrose also function as signaling molecules and many genes
have been characterized of which transcription is regulated by sugars. The expression of the genes
encoding the storage proteins Patatin , VSPA , VSPB, & chalcone synthase is induced in presence of
glucose, fructose , mannose & sucrose . The transcription of photosynthetic genes, such as those encoding
the chlorophyll a/b binding protein, ribulose biphosphate carboxylase small subunit etc is repressed in
presence of glucose, fructose or other substrates of hexokinase.

4. Expression Of Introduced Genes

Most frequently used promoter for the expression of genes in transgenic plant tissues is the 35S promoter
from Cauliflower Mosaic Virus which is constitutively expressed in all tissues throughout development.
In some cases the expression of a certain protein in particular cell types might have detrimental effects
e.g. is when tobacco or potato plants constitutively express the E.coli enzyme pyrophosphorylase in the
cytosol. Pyrophosphorylase reduces the cytosolic pyrophosphate conc , thereby stimulating sucrose
synthesis at the expense of starch biosynthesis.
To evade the “translocation” problems the ectopic expression of genes should be controlled either by
using a tissue specific promoter (eg granule-bound starch synthase promoter) , a developmentally
regulated promoter or a promoter which can be induced ( pathogen related promoter PR-1a).

5. Manipulations Of Starch Metabolism

Starch & its derivatives have various uses- in making paper products, baking and other food uses, textile
manufacture, paints, rubbers etc. Many of the starch derivatives are obtained by post- harvest
modification of the isolated starch which is expensive & environmentally polluting, so to evade these
problems, there is great interest in using transgenic plants for the production of modified starch.
6. Starch Biosynthesis

Starch biosynthesis occur in the plastids of leaves & in amyloplasts of storage organs. Upon entry of
sucrose in the sink cells , sucrose is cleaved by sucrose synthase & converted to Glu-1-P which can be
used for the synthesis of ADP-glucose, the major substrate for starch biosynthesis. This reaction is
catalysed by ADP –glucose- pyrophosphorylase & is thought to be rate limiting step in starch
biosynthesis.

7. Manipulation Of Starch Content

The amount of starch deposited in the potato tuber is affected by the rate of photosynthesis, the
translocation of photoassimilates & the sink strength (capacity to attract photoassimilates). In potato
tubers the sink strength is dependent on the mechanism by which sucrose is cleaved & converted into
starch & the enzyme sucrose synthase plays a pivotal role in degradation of sucrose. Experiments show
that it is possible to manipulate crop yield & amount of key metabolites by affecting regulatory steps in
biosynthetic pathways.

8. Genetic Modification Of Starch Structure & Properties

The physical properties of starch granules are mainly determined by granule size, lipid content, amylose
content, chain length, degree of branching & amylase/ amylopectin ratio which can be modified by
altering the expression of endogenous enzymes or by introduction of foreign genes.

9. Cyclodextrin Production In Potato Tubers

Cyclodextrin are produced from starch by cyclodextrin glycosyltransferases (CGT). Cyclodextrins are
cyclic oligosaccharides joined by α-D-(1-4) linkages & used for various applications. They are
commercially produced in batch fermentors, using purified starch as a substrate. Production in potato
tubers would omit the starch purification since this will directly be used by the CGT for cyclodextrin
production.

MOLECULAR FARMING

It is a term coined to describe the application of molecular biology techniques to the synthesis of
commercial products in plants.
 Major Targets For Carbohydrate Molecular Farming
Compound Origin Of Genes Application Transgenic Plant
1. Amylose-free starch Potato Food, industrial Potato
2. High-amylose starch Potato Food, industrial Potato
3. Cyclodextrin Klebsiella pneumoniae Food, pharmaceutical Potato
4. Fructans Bacillus subtilis Industrial Tobacco, potato, maize
Jerusalem artichoke Food Sugarbeet
5. Trehalose E.coli Food Tobacco

STARCH MODIFICATION BY GENETIC MODIFICATION

Genetic engineering offers possibilities for creating novel starch with new functional properties. The
genes encoding the main enzymes of starch biosynthesis have been cloned & used to produce transgenic
lines.

Granule-bound genes starch synthase I (GBSS I) – is the main enzyme of amylose biosynthesis.
Terefore, increased activity of GBSS I is expected to increase the amylose content of starch, while
suppression of GBSS I should enhance the amylopectin content. Suppression of GBSS I activity in potato
(by antisense RNA technology) yielded an amylose-free or “waxy” starch in potato.This was the first
successful genetic modification of starch reported in 1991.

Soluble Starch Synthase (SSS) – is the main enzyme of amylopectin biosynthesis; it has 2 isoforms
called SSS I & SSS II. A freeze-thaw stable potato starch has been created by simultaneous down
regulation of all the 3 starch synthase, viz, GBSS I & SSS I & SSS II, genes using anti sense RNA
technology. This manipulation yielded an amylose - free short chain amylopectin. This starch is
extremely freeze – thaw stable & shows no syneresis( separation of starch gel &water phases) even after 5
freeze-thaw cycles . The use of this starch has potential for environmental & consumer benefits as its
production requires no chemical modification.

Starch Branching Enzyme (SBE) – activity is required for amylopectin biosynthesis. An increased SBE
activity is expected to increase branching , while its suppression should reduce the amount of
amylopectins. In potato, SBE activities were suppressed to < 1% of the wild type by using antisense
constructs of the genes concerned. This resulted in a very high amylose starch; this unique starch has
novel food & industrial applications.
 Metabolic Pathway For The Biosynthesis Of Carbohydrate Products For Molecular Farming

CYCLODEXTRINS FROM STARCH

• One type of high value product that could be made from starch is the cyclodextrins.
• These compounds are typically 6, 7 or 8 membered rings comprising glucopyranose subunits attached in
α (1-4) linkage.
• These compounds are normally produced by bacterial fermentation of maize starch, particularly for
pharmaceutical applications(for solubilization of hydrophobic pharmaceuticals) .
• Having been identified as a suitable target for molecular farming, it is striking that only one
report in 1991, of an attempt to produce cyclodextrins has been published.

CYCLODEXTRIN
• A bacterial cyclodextrin glycosyltransferase gene from Klebsiella pneumoniae was fused to a plastid-
targetting sequence & placed under the control of the promoter from the patatin gene.
• Patatin is a protein that accumulates in potato tubers & the promoter directs high levels of expression in
the tubers of transgenic potatoes.
• Transformation of potatoes with this construct resulted in very little conversion (0.001-0.01%) of starch
to cyclodexrins. It was concluded that the insoluble starch granules may have been inaccessible to the
bacterial enzyme, or that the enzyme became trapped in the growing granule.

POLYFRUCTANS
• These compounds are soluble polymers of fructose that are synthesized& stored in the vacuole.
It is another carbohydrate targeted for production in transgenic plants
• They have typical structure of glucose-fructose-(glucose)n (G-F-Fn).
• The use of fructans as a carbohydrate reserve is widespread throughout the plant kingdom.
Inulins are found in bulbs & storage roots, Levans in leaves & stems.
• The biosynthetic pathway of fructans in plants is a 2 stage process. The first step involves the
transfer of fructose from a donor sucrose molecule to an acceptor sucrose molecule to form
kestose by the enzyme sucrose-sucrose fructosyltransferase (SST). In the second step the
kestose (GFF or GF2) acts as the fructose donor to the growingfructan chain, via fructan-
fructan fructosyltransferase activity & a sucrose molecule is recycled.

• A number of transgenic plants producing polyfructans have been developed. In earlier

experiments the sacB gene of B. subtilis or B. amyloliquifaciens which encodes a levan-sucrase
catalyzing a 6-2β linkage was transformed into maize, tobacco, potato,& sugarbeet. sacB gene
was modified with a vacuolar targeting sequence from the yeast carboxypeptidase gene(cpy).
• In tobacco, expression of the sacB gene was responsible for the accumulation of significant
amounts of fructans. The transgenic tobacco plants were found to be more tolerant to drought
stress induced by growth in medium containing polyethylene glycol to reduce the water
concentration.
• Expression of the sacB gene in sugarbeet has also been shown to improve tolerance to
polyethylene glycol-mediated drought stress.
• In potato, the formation of a new sink, by diversion of sucrose away from starch accumulation
in the tuber & towards fructans in the vacuole has provided a useful model system for studying
regulation of sucrose metabolism & the partitioning of the assimilates.
• The levan sucrase gene from Erwinia amylovora was fused to different targeting sequences
(apoplast,vacuole& cytosol) & used to transform starch deficient potatoes. The apoplast &
vacuole targeted enzymes produced fructans to level of 12 & 19 % of tuber dry wt resp.
• In maize a B.amyloliquifaciens sacB gene was expressed in the endosperm under the control of
a zein promoter. The enzyme was targeted to vacuole of the endosperm cells using two
different vacuolar targeting sequences. In one construct the sweet potato sporamin signal
peptide & vacuolar targeting sequences were fused to the N-terminal end of the enzyme. In the
other , the barley lectin signal peptide was fused to the N-terminal end & the barley lectin
vacuolar targeting sequence was fused to the C-terminal end of the enzyme.
• Fructan accumulated to 7-9 mg/g of seed in several different lines from either construct. This
level could be increased by upto 8-9 fold when transgene was crossed into maize starch-mutant
lines, which accumulate much higher levels of sucrose.
• Short oligofructans (GF2, GF3 &GF4) have been produced in sugarbeet using a gene encoding
the 1-SST enzyme from Jerusalem artichoke. This enzyme catalyses the production of not only
GF2(ketose) but also GF3 &GF4. It was found that the sugar stored in the storage root of
sugarbeet was almost totally converted into these oligofructans. This transgenic sugarbeet has
therefore been renamed fructan beet (has direct applications in the nutraceuticals market).
• Larger inulin molecules have been produced in potato tubers transformed with the 1-SST & 1-
FFT genes of globe artichoke
• It has been possible to produce inulin of the neoseries in chicory by transformation with the
barley 6-SFT gene to produce branched fructans of the graminan type.
• The production of fructans in transgenic plants has been critically reviewed by Cairns(2003).
• Cairns considers a number of possible explanations for the low fructan accumulation, including
codon usage, low transgene mRNA concentration, low expression of enzyme protein., low
enzyme activity, kinetic properties of transgenic enzymes inappropriate for host environment,
product hydrolysis & fructan toxicity.