The nutritional quality of crop plants is determined by their content of essential amino acids provided as proteins in foods for humans or in feed for monogastric animals. Plant proteins are often deficient in some of the 10 essential amino acids that are required in the human and animal diet. In genera], cereals are deficient in lysine and legumes in the sulfur amino acids, methionine and cysteine.
Several approaches have been presented and used with the goal of increasing the relative content of essential amino acids in plants, in particular the aspartate-derived amino acids, lysine and threonine.
One of the earliest approaches consists of screening genotypes and induced mutants to identify plants with higher lysine levels in the seed. This method was first applied successfully to maize and led to the high-lysine opaque-2 type (12) . Later, similar types of mutants were reported in barley and Sorghum (see review in Ref. 13). The increase in total lysine content was due to a shift in the spectrum of storage proteins to the benefit of lysine-rich albumins and globulins instead of lysine-poor prolamines. However, this type of mutation was accompanied by a drastic reduction in yield and protein content.
The introduction of gene transfer technologies opened new ways to alter the amino acid composition of the seed proteins, in cereals and legumes in particular. In this case, genes encoding storage proteins naturally rich in essential amino acids or engineered genes leading to the production of "synthetic" proteins displaying a high content of defined essential amino acids were expressed in transgenic plants under the control of suitable promoters. In order to modify the nutritional value significantly, the foreign proteins must constitute a high percentage of the total proteins, so that in addition to high levels of transcription and translation, the stability of the additional proteins is crucial. As underlined by Galili et al. (14), although numerous attempts founded on such strategies have been reported, a significant, reliable accumulation of a stable nutritionally valuable protein still has to be convincingly demonstrated. In that context, it is worthwhile to mention the work of Chakraborty et al. (15) on the expression in potato of an AmA-1 gene coding for a seed-specific albumin from Amaranthus hypochondriacus with a well-balanced amino acid composition. The authors reported that when this gene was put under the control of a granule-bound starch synthase promoter, a striking increase in most essential amino acids present in potato tubers was observed. Unexpectedly, the growth and production of tubers as well as the total protein content of this accumulation organ were significantly increased.
An alternative method consists of producing plants containing elevated levels of specific amino acids by manipulating their metabolism. This can be achieved through alteration of key enzymes from those biosynthetic pathways by mutation and transformation. We have focused on the aspartate-derived amino acid biosynthetic pathway producing the four essential amino acids lysine, threonine, isoleucine, and methionine. Considerable research, aided by genetic engineering, has been devoted to this pathway, its enzymes, and their regulation with the aim of optimizing amino acid content (16).
A. Biochemical Regulation of Lysine and Threonine Biosynthesis
Higher plants synthesize lysine, threonine, isoleucine, and methionine from the common precursor aspartate via a complex branched pathway (Fig. 1) (17). Understanding the inner regulation taking place during this process is essential with respect to knowledge of basic plant metabolism but also to possibly modifying the nutritional value of crops.
Close examination of the lysine and threonine biosynthesis revealed the existence of several enzymatic steps feedback controlled by these two end products. Essentially, two enzymes play key regulatory roles in the pathway: aspartate kinase (AK), the first enzyme of the overall pathway, which is retroinhibited by either lysine or threonine, and dihydrodipicolinate synthase (DHDPS), the first enzyme of the lysine-specific branch, which is severely inhibited by lysine. The main limiting step in lysine production appears to occur at the DHDPS level via the feedback control exerted by lysine (K, between 5 and 20 juM). Most of the biosynthetic enzymes of the pathway have been localized in the plastids on the basis of biochemical data (17) and molecular evidence from genes encoding the majority of the bio-
ß Aspartyl phosphate
* ASD Aspartate semialdehyde
Lysine Cystathionine ^ Threonine
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