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yet been tested. Very recently, Wu and Mess-
ing (2011) reported a potential accelerated QPM
selection scheme, which is based on an RNAi
construct that is directed against 22 and 19 kda
zeins, fused with visible green fluorescent pro-
tein (GFP) marker gene. When such RNAi lines
were crossed with QPM lines, carrying
o2
ker-
nel hardness modifier genes, green and vitre-
ous progenies could be selected in the segregat-
ing generations, thereby demonstrating that high
lysine content and hard endosperm traits could
be selected in dominant fashion.
An alternative option for enhancing the Lys
content is to manipulate the critical rate limit-
ing steps in the aspartate pathway, which plays
an essential role in the regulation of the biosyn-
thesis of several essential amino acids, includ-
ing lysine in higher plants. Increases in seed
lysine content have been achieved through engi-
neering of both lysine anabolism and lysine
catabolism. The first key step of this pathway is
regulated by aspartate kinase (AK), which catal-
yses the conversion of aspartate to b-aspartyl
phosphate and is feedback-regulated by several
end products, including lysine and threonine
(Galili 1995; Wang et al. 2001). At least two
isoforms of AK exist in plants - lysine-sensitive
and threonine-sensitive enzymes (Dotson et al.
1989; Muehlbauer et al. 1994a, b; Azevedo et al.
1997). In maize, two mutant loci encoding mono-
functional AK,
ask1
and
ask2
, were identified by
genetic screening (Diedrick et al. 1990; Dotson
et al. 1990b; Muehlbauer et al. 1994a), and the
enzymes they encode are feedback-inhibited by
lysine (Dotson et al. 1989). Mutations at these
loci result in AKs that are less sensitive to lysine
feedback inhibition and result in the overpro-
duction of free lysine, threonine, methionine,
and isoleucine (Dotson et al. 1990b; Muehlbauer
et al. 1994a). While
ask1
is located on short
arm of chr. 7,
ask2
is mapped and cloned on
long arm of chr. 2 in maize (Muehlbauer et al.
1994a; Wang et al. 2007). DHDPS (dihydrodipi-
colinate synthase), a key regulatory enzyme in
Lys biosynthesis, catalyzes the formation of
dihydrodipicolinic acid by condensing pyruvate
and ASA (aspartate beta semialdehide). DHDPS
is highly sensitive to Lys feedback regulation.
Plants with a mutant DHDPS are less sensitive
to Lys feedback inhibition and overproduce the
amino acid (Ghislain et al. 1995). Because bacte-
rial DHDPS is less sensitive than plant DHDPS
to Lys, genes encoding bacterial DHDPS have
been used to genetically engineer plants that
overproduce Lys (Falco et al. 1995). Another
possible intervention point in the Asp metabolic
pathway is the Lys degradation reaction, where
LKR (Lysine ketoglutaric acid reductase) cataly-
ses the first step of Lys degradation. LKR activity
is dramatically reduced in
o2
maize and hence it
is believed that depressed LKR activity is one of
the significant factors resulting in enhanced Lys
in
o2
maize endosperm. A two-pronged approach
(Frizzi et al. 2008) of introducing a feedback
insensitive DHDPS (CordapA) and suppressing
the LKR by an intron-embedded suppression
construct through a single transgene has yielded
impressive increase in seed lysine content (4000
ppm of Lys), representing
100-fold enhance-
ment, the highest ever reported in maize kernels.
Although RNAi technology has emerged as a
powerful tool for overcoming the pleiotropic and
secondary effects of the desirable mutant genes,
social acceptance and biosafety concerns regard-
ing GM (genetically modified) food crops for
large scale adoption still exist in some countries,
and high-lysine GM maize may be no exception.
∼
HighProvitaminAMaize
Vitamin A deficiency in a majority of the chroni-
cally hungry nations is responsible for a number
of disorders, such as impaired iron mobilization,
severe growth retardation, blindness, depressed
immunological responses, increased susceptibil-
ity to infections, and increased childhood mor-
tality (FAO 2000; Sommer and Davidson 2002;
WHO 2009; Wurtzel et al. 2012). Interestingly,
maize happens to be the predominant staple
food in those areas such as sub-Saharan Africa
and Latin America, where vitamin A-deficiency
symptoms abound. While carotenoids are absent
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