We will now focus on an approach based, as previously mentioned, on the deregulation of biosynthetic pathways of such essential amino acids, in particular in the case of lysine and threonine.
In various species, mutants accumulating lysine or threonine in the soluble amino acid pool have been isolated by selecting for growth in the presence of the lysine amino acid analogue 5-2-(aminoethyl)-1-cysteine (AEC) or toxic combinations of lysine and threonine in the culture medium (18). Although threonine overproduction was detected in all tissues analyzed and in particular in seeds, the increase of free lysine was noticeable only in leaves and calli. Moreover, an aberrant phenotype characterized by reduction of the foliar surface, absence of shoot elongation, and sterility was observed when high levels of free lysine accumulated (25% and more of the total free amino acid pool) (19). The accumulation of both amino acids in leaves is also dependent on the developmental stage, reaching a maximum at shoot elongation.
The biochemical characterization of these mutants has shown that we are dealing with deregulation at the level of the two key enzymes, DHDPS in the lysine overproducer mutant and AK in the threonine overproduces These mutated forms are both fully insensitive to feedback inhibition by the respective amino acid (19).
C. Key Genes of the Lysine and Threonine Pathways
At least five forms of aspartokinase have been identified in plants and may be classified on the basis of their differential feedback properties. One gene coding for a threonine-sensitive form (aki-hsd,) was isolated by heterologous hybridization in Arabidopsis thaliana. The deduced amino acid sequence revealed the presence of a second region corresponding to the enzyme activity of homoserine dehydrogenase (HSD), the first committed enzyme in the branch of the pathway that leads to threonine synthesis. We are thus dealing with a gene coding for a bifunctional protein with an AK activity at the NH2-side and an HSD activity at the COOH-terminus (20). The expression of the A. thaliana akrhsd! gus gene was elevated in an array of young plant tissues containing actively growing cells (meristems, young leaves, cortical and vascular stem tissues, anthers, gynoecium, developing seeds). During the development of the embryo its expression appeared coordinated with the initiation and onset of storage protein synthesis (21). A second gene showing a high identity (82%) with the ak,-hsd, nucleotide sequence was identified in a bacterial artificial chromosome (BAC) clone [European Molecular Biology Laboratory (EMBL bank)] and localized as ak2-hdh2 on chromosome 4 of the A. thaliana genome (V. Frankard, per-sonnal communication).
As the major part of the AK activity is sensitive to feedback inhibition by lysine, attempts have been made to clone the corresponding genes. Degenerated primers corresponding to conserved motifs between lysine-sensi-tive bacterial AKs were used to clone two genes (ak-lysl and ak-lys2) encoding monofunctional AKs in A. thaliana (22,23). The presence of two nuclear genes both encoding AK-lys proteins and both targeted to the chlo-roplast, as shown by the presence of a transit peptide, but with only 70% identity at the deduced amino acid level raises questions about their respec tive roles in plant development. Therefore, A. thaliana and Nicotiana tabacum have been transformed with the 5' upstream region of either ak-lys gene fused with the uidA reporter gene encoding glucuronidase (GUS). In A. thaliana, a comparison of the GUS patterns of ak-lysl and ak-lys2 revealed strongly predominant expression of ak-lys2 over ak-lysl. Throughout the vegetative phases, ak-lys2-GUS expression reached a high level, especially in the vasculature, whereas ak-lysl-GUS plants showed a markedly reduced intensity of the histochemical staining. In the reproductive phase, both genes were well expressed in flowers, ak-lys2 was the only one expressed in the fruits, and no staining could be detected in seeds in both cases. The physical position of ak-lysl and ak-lys2 could be assigned to chromosome 5 at two different loci at a distance of 2 cM.
The Arabidopsis ak-lys gene family is thus composed of at least two different members. We identified a third ak-lys gene located on chromosome 3 of Arabidopsis using the Blast program to screen databases. The deduced ak-lys3 amino acid sequence showed higher identity with the ak-lys2 (82.8%) than with the ak-lysl (68%) peptide sequence. The ak-lys3 gene was reported to be highly expressed in leaf vein tissues (24). The isolation and characterization of A. thaliana mutants displaying an AK-HSD isozyme less sensitive to threonine inhibition or an AK-lys isozyme less sensitive to lysine feedback inhibition (25,26) will make it possible to identify at the nucleotide level mutations that lead to threonine accumulation. After incorporation into the corresponding copy DNA (cDNA), these mutated plant genes can be respectively expressed in an appropriate construct to obtain threonine accumulation in specific tissues, especially in seeds.
2. Cloning and Characterization of Wild-Type and Mutant Genes Encoding Dihydrodipicolinate Synthase
A mutant of Nicotiana sylvestris (RAEC-1) was shown to overproduce lysine because of a mutation in the DHDPS gene that causes the DHDPS enzyme to be insensitive to the normal feedback inhibition of lysine. The dhdps-rl mutation was identified as a substitution of two nucleotides changing as-paragine in isoleucine in a conserved region of the protein (27). In maize, a series of single amino acid substitutions were found to eliminate lysine inhibition of DHDPS (28). DHDPS-encoding sequences were cloned by functional complementation in a bacterial DHDPS-deficient strain. This first isolated clone made it possible to obtain and sequence a full-length Arabidopsis DHDPS cDNA (29). Constructs derived from the Arabidopsis cDNA allowed the generation through ethyl methane sulfonate in vitro mutagenesis of clones encoding fully insensitive forms of the DHDPS protein (30). Furthermore, the clones successfully isolated by functional complementation in a DHDPS-deficient E. coli mutant were all found to encode insensitive en zyme forms, which means that complementation selects for insensitive DHDPS plant enzymes. In soybean, three mutants were constructed containing specific amino acid substitutions that lead to lysine-desensitized DHDPS (31).
In a further step, the Arabidopsis promoter has been isolated and fused with the reporter gene gus to study the transcription properties of this upstream sequence. Expression of GUS was detected in meristems and vascular tissues of roots, in vascular tissues of stems and leaves, and in the meristems of young shoots. In flowers, high expression was found in the carpels, pollen grain, and young embryos but not in endosperm of mature seeds. No lysine-induced repression of the dhdps gene could be detected (32).
An Arabidopsis genomic sequence encoding a second DHDPS enzyme was identified by screening the EMBL database. The dhdps-2 coding sequence shows 84% identity with the nucleotide sequence of dhdps-1. The genomic dhdps-2 sequence contains three exons and two introns, whereas only one intron and two exons are present in dhdps-1 (Fig. 2). Comparison of the promoter regions of dhdps-1 and dhdps-2 did not reveal boxes with any significant conservation. The two Arabidopsis genes were localized on dhdps-1 gene genomic DNA
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