The aspartate family amino acids, lysine, methionine, and threonine, and the
aromatic amino acid tryptophan are the most important essential amino acids
required in human foods and livestock feeds. They are the most limiting essential
amino acids in the major crop plants that serve as human foods and animal feeds,
particularly cereals and legumes that are supplied as grain and/or as forage
(Galili et al.
, 2002). Cereals are deficient mainly in lysine and tryptophan, while
legumes are mainly deficient in methionine (Syed Rasheeduddin and Mcdonald,
1974). Thus, many of the commonly used diet formulations based on these crops
contain limiting amounts of these essential amino acids.
Livestock that are consumed as human foods are nonruminant animals, such
as poultry or pigs, and ruminants, such as cattle or sheep, which differ in feed
requirements for optimal incorporation of essential amino acids. The nonruminants
or monogastric animals, like humans, cannot synthesize essential amino
acids and thus depend entirely on the external supply of essential amino acids.
Ruminant animals also cannot synthesize these essential amino acids; however,
the microbial flora inhabiting their rumen can metabolize nonessential into essential
amino acids and incorporate them into microbial proteins that later become
nutritionally available. Nevertheless, these microbial proteins, although of better
nutritional quality than plant proteins, do not provide sufficient essential amino
acids for optimal growth and milk production (Leng, 1990). Moreover, although
the rumen microflora can produce essential amino acids, it can also oppositely
metabolize essential amino acids into nonessential ones. Hence, in contrast to
nonruminant animals that can utilize either free or protein-incorporated essential
amino acids, ruminant feeds should contain the essential amino acids in proteins
that are highly stable in the rumen to minimize their degradation by the rumen
Improving Lysine Levels in Crops: A Comprehensive Approach
Although free lysine content could be significantly improved in legume and cereal
grain crops by expression of a bacterial feedback–insensitive DHPS (Avraham and
Amir, 2005), such transgenic plants may not be optimal foods and feeds. These
plants accumulate relatively high levels of intermediate products of lysine catabolism,
such as α-amino adipic acid, which may act as neurotransmitters in animals
and can be toxic at high levels (Bonaventure et al.
, 1985; Karlsen et al.
Reichenbach and Wohlrab, 1985; Welinder et al.
, 1982). In addition, these plants
overaccumulate free lysine rather than lysine-rich proteins and are therefore not
suitable for feeding of ruminant animals (National Research Council, 2001). To
address this issue, Jung and Falco (2000) used a composite approach to generate
lysine-overproducing transgenic maize grains. This included combined expression
of two transgenes. One encoded a bacterial feedback-insensitive DHPS under
an embryo-specific promoter since lysine overproduction is achieved only in
maize embryos (see above). The second encoded a lysine-rich protein (either
hordothionine HT12 or the barley high-lysine protein BHL8, containing 28% and
24% lysine, respectively) under an endosperm-specific promoter since the endosperm
consists a major part of the maize grain. Two types of maize plants were
transformed with these genes, wild-type maize and a maize mutant lacking lysine
catabolism due to a knockout of the maize LKR/SDH gene. The HT12 and BHL8
proteins accumulated between 3% and 6% of total grain proteins, and when
introduced together with the bacterial DHPS resulted in a marked elevation of total lysine to over 0.7% of seed dry weight (Jung and Falco, 2000), for example, as
compared to around 0.2% in wild-type maize. Combination of these genes into a
homozygous LKR/SDH knockout background increased grain lysine level further
and alleviated the problem of high-level accumulation of lysine catabolic products
(Jung and Falco, 2000).
The additive effect of free lysine overproduction in the maize embryo and its
incorporation into lysine-rich proteins in the endosperm on total grain lysine
content suggests that free lysine is effectively transported between the two tissues.
Should the dramatic elevation of lysine levels, obtained by this composite
approach, not interfere with yield and other grain quality factors, the commercial
application of such high-lysine transgenic maize plants for feeding human and
nonruminant livestock looks very promising. Maize is also a suitable crop for
ruminant feeding because maize seed proteins are on average highly resistant to
rumen proteolysis (National Research Council, 2001). Moreover, the endogenous
maize seed proteins may protect transgenic high-lysine proteins from rumen
Improving Methionine Levels in Plant Seeds: A Source–Sink Interaction
Most attempts to improve the methionine contents of seeds have focused on
overexpression of methionine-rich seed storage proteins, such us Brazil nut
2S albumin, sunflower 2S albumin (SSA), and maize methionine-rich zeins (for
review see Avraham and Amir, 2005). The SSA was also found highly resistance
to rumen proteolysis (Mcnabb et al.
, 1994), suggesting that transgenic plants
overexpressing it may be beneficial not only for nonruminants but also for ruminant
feeding. Indeed, feeding experiments with transgenic lupin grains, which
expressed the SSA gene, enhanced both rat growth (Molvig et al.
, 1997) and sheep
live weight gain and wool production (White et al.
Although transgenic methionine-rich proteins can accumulate to high levels in
plant seeds, in most cases the total methionine is still less than necessary for
optimal feeding (Avraham and Amir, 2005; Demidov et al.
, 2003; Galili and
Hofgen, 2002). This is largely because production of transgenic methionine-rich
protein is associated with a compensatory decrease in the levels of endogenous
sulfur-rich proteins. This phenomenon implies the presence of limiting levels of
free methionine, whose synthesis in the seeds may be regulated by limited
availability of its precursor metabolites cysteine, O-p
N-methyltetrahydrofolate. Combined seed-specific overexpression of a bacterial
feedback-insensitiveAK (apparently to increase the level ofO-p
as well as Brazil nut 2S albumin in transgenic narbon beans resulted in an additive
increase of seed methionine, compared to the parental plants expressing each of
these transgenes alone (Demidov et al.
, 2003). This suggests that methionine accumulation in seeds depends on the pool size of O-p
addition, when the SSA was expressed in seeds of transgenic lupin and rice plants
(Hagan et al.
, 2003; Molvig et al.
, 1997; Tabe and Droux, 2002), although seed
methionine levels were increased, there was no increase in seed cysteine and the
total seed sulfur content, implying that the vegetative cysteine pool and the extent
of sulfur transport from the canopy to the seeds represent two additional ratelimiting
factors. Thus, possible additional target genes for genetic engineering of
plants with high seed methionine would be genes controlling the assimilation,
metabolism, and transport of sulfur. Indeed, constitutive overexpression of serine acetyl transferase, an important regulatory enzyme in cysteine biosynthesis
(Fig. 3.2), enhanced seed methionine content in transgenic maize (Tarczynski et al.
Limited levels of sulfur-containing metabolites in seeds retard the synthesis of
endogenous sulfur-rich proteins by negatively regulating the expression of their
genes (Tabe and Droux, 2002; Tabe et al.
, 2002). One way to overcome this negative
regulation is by replacing regulatory elements of endogenous genes encoding
sulfur-rich proteins with analogous elements derived from endogenous genes
whose expression is not responsive to sulfur availability. In a recent study, the
promoter and 50 untranslated regions of a maize gene encoding a methionine-rich
d-zein were substituted with analogous sequences derived from another gene
encoding a γ-zein gene and transformed back into transgenic maize plants (Lai
and Messing, 2002). Expression of this chimeric transgene caused an ~30%
increase in total seed methionine.
Improving the Nutritional Quality of Hay for Ruminant Feeding
Improving the nutritional quality of hay for ruminant feeding requires the expression
of proteins, which are both nutritionally balanced and resistant to rumen
proteolysis in vegetative tissues. When genes encoding vacuolar methionine-rich
seed storage proteins, which stably accumulate in seeds, were constitutively
expressed in various transgenic plants, their encoded proteins failed to accumulate
in the protease-rich vegetative vacuoles because of extensive degradation (see
Avraham and Amir, 2005 for review). This was partially overcome by preventing
the trafficking of these proteins from the endoplasmic reticulum (ER) to the
vegetative vacuole, by engineering of an ER retention signal (KDEL or HDEL)
into their C-terminus (see Avraham and Amir, 2005 for review).
Vegetative storage proteins (VSPs) may be preferred alternatives to seed
storage proteins because they are nutritionally balanced and also stably accumulate
in vacuoles of vegetative cells (Staswick, 1994). Galili and associates tested the
potential of constitutive expression of genes encoding the α- and β-subunits of
soybean VSPs to improve the nutritional quality of vegetative tissues of heterologous
plants. The soybean VSPα-subunit accumulated to high levels (up to 3% of
total leaf soluble proteins) and its levels remained stable also in mature leaves of
transgenic tobacco plants (Guenoune et al.
, 1999). However, this subunit was
totally unstable to rumen proteolysis (Guenoune et al.
, 2002b). The soybean
VSPb was however more resistance to rumen proteolysis (Guenoune et al.
2002b), but accumulated only in young leaves and its levels declined with leaf
age (Guenoune et al.
, 2003). Coexpression of both subunits in the same transgenic
plant resulted in stable accumulation of both proteins in older leaves and also
improved their stability to rumen degradation (Guenoune et al.
Accumulation of transgenic proteins in vegetative tissues may be further
improved by their targeting to more than one organelle. Directing the soybean
VSPα to plastids resulted in a similar level to that of the vacuole-targeted counterpart
(Guenoune et al.
, 2002a). Targeting of the soybean VSPα to these two organelles in a single transgenic plant resulted in its significantly high
accumulation to up to 7.5% of the total soluble proteins (Guenoune et al.