Etw

Exonl

363 bp

Intron 1

Exon 2

120 bp

InIran 2 I

Exon 3

86 bp

78 bp 927 bp cDNA of dhdps-2

Exon 1

Exon 2

Exon 3

1091 bp

Figure 2 Structure of the dhdps-1 and dhdps-2 genes and cDNAs. prom, promoter; bp, base pair.

chromosome 2 for dhdps-2 and chromosome 3 for dhdps-1. That dhdps-2 encodes a functional protein was shown by the growth on a nonsupple-mented medium of an E. coli DHDPS-deficient strain transformed with the dhdps-2 apoprotein coding sequence. Activity tests performed in the presence of increasing concentrations of lysine proved that the DHDPS-2 enzyme is also strongly feedback inhibited, with a 50% loss of activity at 30 /xM lysine.

Via promoter-GUS fusion, expression of the dhdps-2 gene was observed during the whole developmental cycle and appears to be quite similar to that observed for the dhdps-1 gene, although the dhdps-2 gene is in general a little more expressed. DHDPS-2 activity was strongly detected in the vasculature of stems and leaves, in carpels, and in developing seeds (33).

D. Improving Lysine and Threonine Accumulation via Transfer of Bacterial Genes

The availability of bacterial and plant genes encoding feedback-insensitive enzymes allowed redirecting the expression of these genes in plants and in particular in storage organs. Initial experiments made use of constructs monitored by the strong cauliflower mosaic virus (CAMV) promoter harboring a bacterial dapA gene that encodes a DHDPS partially or fully feedback insensitive and mutated alleles of the E. coli lysC gene encoding a feedback-insensitive AK (see Ref. 16 for review). Transgenic lines have been obtained in tobacco (34-36), potato (37), barley (38), Arabidopsis (39), and oilseed rape and soybean (40).

As previously mentioned for Nicotiana sylvestris mutants, expression in tobacco of the bacterial AK led to accumulation of free threonine not only in vegetative tissues and in tubers in the case of potato but also in reproductive organs and seeds. In the case of the bacterial DHDPS, the lysine overproduction, although present in vegetative tissues, was not expressed in mature seeds. Constitutive overexpression of the bacterial DHDPS gene was also accompanied by phenotypical alteration of the transgenic plants, as observed with the selected N. sylvestris mutants, at least when a high lysine level was reached (36). Targeting the expression of these bacterial genes to sink organs such as seeds was then proposed as a way to alleviate such deleterious effects. Expression of the chimeric AK and DHDPS gene under the control of the /3-phaseolin promoter obtained from the bean gene coding for this seed protein was thus evaluated. Transgenic tobacco seeds showed an important increase in free threonine but no significant change in the amount of lysine in mature seeds, although the activity of bacterial feedback-insensitive DHDPS was clearly higher than the DHDPS activity measured in nontransformed plants.

This lack of lysine accumulation in tobacco seeds was then ascribed to enhanced lysine catabolism as shown by higher activities of the major lysine catabolism enzyme lysine-ketoglutarate reductase (LKR) (41). We have to underline that the lysine content present in mature seeds of transgenic plants appears to be the result both of increased accumulation due to high activity of a feedback-insensitive DHDPS and of the rate of lysine catabolism. According to the balance between enhanced biosynthesis and induced catabolic degradation, the accumulation of lysine in seeds may vary among plant species (Table 1). This is best shown by the results obtained by Falco et al. (40) and Mazur et al. (42) when similar bacterial ak and dhdps genes were transformed into oilseed rape and soybean. Alone or combined with AK, expression of the bacterial DHDPS resulted in a dramatic increase of free lysine (10-100 times the level of nontransformed plants) with a significant effect on the total lysine content, which increased more than twice. In these last cases, accumulation of catabolic products such as saccharopine and a-aminoadipic acid was eventually observed but only at a minor level. However, in maize, when the bacterial AK and DHDPS were expressed with an endosperm or an embryo-specific promoter, lysine accumulation was detected only in the embryo but was sufficient to raise the overall lysine concentration in seed by 50 to 100% (42). Bacterial enzymes can thus be expressed with success in vegetative and sink organs such as seeds leading to deep modifications in lysine metabolism.

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