Genetic engineering approaches possess the advantage that gene manipulation
can include coding regions as well as regulatory elements such as promoters.
Hence, to study the regulation of lysine and threonine metabolism specifically in
developing seeds, the E. coli
feedback-insensitive AK and DHPS enzymes were
expressed in transgenic plants under the control of a seed-specific promoter
derived from a gene encoding a seed storage protein. The choice of a storage
protein gene promoter was based on the assumption that lysine biosynthesis is
spatially and temporally coordinated with storage protein production during
seed development. Whether storage protein gene promoters are the best choice
to manipulate amino acid metabolism specifically in developing seeds is still unknown and awaits detailed studies of seed development. The first studies
included the seed-specific expression of the bacterial feedback-insensitive AK
and DHPS in transgenic tobacco plants. Expression of the bacterial AK resulted
in significant elevation in free threonine in mature seeds (Karchi et al.
, 1993), but no
increase in free lysine was evident in mature seeds of transgenic plants expressing
the bacterialDHPS (Karchi et al.
, 1994). Developing seeds of these transgenic plants
also possessed over tenfold higher activity of lysine-ketoglutarate reductase
(LKR), the first enzyme in the pathway of lysine catabolism (Galili et al.
suggesting that the low lysine level in mature seeds of the transgenic tobacco
plants resulted from enhanced lysine catabolism (Karchi et al.
To study the significance of lysine catabolism in regulating free lysine accumulation
in seeds under conditions of regulated and deregulated lysine synthesis,
Galili and associates have isolated an Arabidopsis
T-DNA knockout mutant lacking
lysine catabolism (Zhu et al.
, 2001). This knockout mutant was crossed with
plants expressing a bacterial feedback-insensitive DHPS
in a seed-specific manner (Zhu and Galil, 2003). Although both parental plants
contained slightly elevated levels of free lysine compared to wild type in mature
seeds, combining both traits into the same plant synergistically boosted free seed
lysine levels by ~80-fold, rendering lysine as the most prominent free amino acid
(Zhu and Galil, 2003). Moreover, total seed lysine in these plants was nearly
doubled compared to wild-type plants (X. Zhu and G. Galili, unpublished
results). Notably, the dramatic increase in free lysine in seeds expressing the
bacterial DHPS but lacking lysine catabolism was associated with a significant
difference in the levels of several other amino acids. The most pronounced
differences were significant reductions in the levels of glutamate and aspartate
and a dramatic increase in the level of methionine (Zhu and Galil, 2003), exposing
novel regulatory networks associated with AAAM and the aspartate family
A feedback-insensitive DHPS derived from Corynebacterium glutamicum
expressed in a seed-specific manner in two additional transgenic dicotyledonous
crop plants, soybean and rapeseed (Falco et al.
, 1995; Mazur et al.
, 1999). Seeds of
these transgenic plants accumulated up to 100-fold (rapeseed) and several
hundred-fold (soybean) higher free lysine than wild-type plants, values that are
significantly higher than those obtained in transgenic tobacco plants expressing
the E. coli
DHPS (Karchi et al.
, 1994). Whether this is due to the different plant
species or to the different bacterial DHPS enzymes is still not clear, but seeds of the
lysine-overproducing soybean and rapeseed plants also contained significantly
higher levels of lysine catabolic products than wild-type nontransformed plants
(Falco et al.
, 1995; Mazur et al.
In contrast to dicotyledonous plants in which storage protein synthesis typically
takes place in the developing embryo, the synthesis of storage proteins in
cereal seeds occurs mostly in the endosperm (Shotwell and Larkins, 1989). Also,
based on in situ
analysis, the lysine catabolism pathway was suggested to function
mostly in the outer layers of the cereal endosperm (Kemper et al.
, 1999). It is thus
expected that expression of a bacterial DHPS, under control of an endospermspecific
storage protein gene promoter, will result in enhanced lysine production and perhaps also accumulation of catabolic products of lysine. This expectation
was found to be incorrect because lysine overproduction in transgenic maize
seeds was observed only when the bacterial DHPS was expressed under an
embryo-specific, but not an endosperm-specific promoter (Mazur et al.
Whether the lack of increase in lysine levels upon expressing the bacterial DHPS
in the endosperm tissue is due to factors associated with either lysine synthesis or
catabolism or both provides an interesting topic for future research.
What then are the functions of lysine catabolism during seed development and
why is this pathway stimulated by lysine? The fact that seeds of transgenic
soybean, rapeseed, and Arabidopsis
can accumulate very high levels of free lysine
without a major negative effect on seed germination (only extreme lysine accumulation
retards germination) (Falco et al.
, 1995; Mazur et al.
, 1999) suggests that
lysine catabolism is not required to reduce lysine toxicity. Also, these studies
show that the flux of lysine synthesis in developing seeds can become very
extensive when the sensitivity of DHPS activity to lysine is eliminated. It is thus
possible that during the onset of seed storage protein synthesis, lysine catabolism
and likely other amino acids catabolic pathways are stimulated to convert excessfree
lysine and other amino acids into sugars and lipids, and also back into
glutamate in the case of the lysine catabolism pathway.
The significant research advances in the regulation of lysine metabolism in
plants has made this pathway a major biotechnological target for improving the
nutritional quality of crop plants. Indeed, a high-lysine corn variety (MaveraTM,
Monsanto Inc., St. Louis, Missouri), obtained via embryo-specific expression of a
bacterial feedback-insensitive DHPS, has recently been approved for commercial
growth for livestock feeding. It is highly likely that additional varieties with
higher seed lysine content in which lysine catabolism is reduced and lysine-rich
proteins are expressed specifically in seeds will appear in the near future.