Aspartate

Figure 2 The pathway of lysine biosynthesis in plants showing feedback regulatory steps as arrows. AK, aspartate kinase; DHDPS, dihydrodipicolinate synthase.

Consequently, there is interest in increasing the methionine content of sulfur-deficient legume seeds.

A range of sulfur-rich proteins has been characterized from plant seeds including the /3-zeins (4.4 mol % Cys, 11.4 mol % Met), y-zeins (over 7 mol % Cys), and <5-zeins (23-27 mol % Met) of maize (17). In addition, Chui and Falco (44) have described the molecular cloning of a protein that appears to be related to S-zeins but contains over 40 mol % methionine. Similarly, the Mr 10,000 prolamins of rice contain about 10 mol % Cys and 20 mol % Met (45).

The methionine-rich 8-zein gene has been used to increase the methionine content of maize seeds by up to 30% (46), but neither this nor any other sulfur-rich cereal prolamin genes have so far been used in transgenic legumes.

Instead, the main interest in improving the sulfur amino acid content of legume seeds has been focused on the 2S albumins. Youle and Huang (47) demonstrated that the 2S albumin fraction from Brazil nut (Bertholletia excelsa) contained over 17 mol % Met and 13 mol % Cys, and subsequent studies showed the presence of at least six closely related components (48,49). Methionine-rich 2S albumins have since been reported in sunflower, cotton, and amaranthus and cysteine-rich components in quinoa and pea (20).

Expression of the Brazil nut albumins has resulted in significant increases in the methionine content of seeds of oilseed rape, tobacco (by up to 30%) (50,51), Arabidopsis (by 20%) (52), narbon bean (Vicia narbonen-sis) (by up to threefold) (53), and soybean (R. Jung, personal communication). However, it is now known that the Brazil nut protein is allergenic to humans (54,55) and the commercial development of seeds expressing this protein has therefore been suspended.

Although a number of other (i.e., methionine-poor) 2S albumins are also allergenic (e.g., from mustards, castor bean, cotton), there is no evidence that this applies to the methionine-rich albumin (SFA8) of sunflower, which contains 16 Met out of 103 total residues (but see note added in proof). Molvig et al. (56) reported that the expression of this protein in seeds of lupin resulted in an increase in methionine by 94%, although there was no effect on total seed sulfur, the increase being at the expense of cysteine (reduced by 12%) and sulfate. Feeding trials with rats showed that the nutritional quality of the seeds was improved significantly, with increases in live weight gain, true protein digestibility, biological value, and net protein utilization. These results demonstrate that the methionine content of legume seeds can be increased by genetic engineering but that the extent of this may ultimately be limited by the availability of sulfur within the seed. Further increases may require the manipulation of the sulfur economy of the plant to increase the transport of sulfur into the developing grain. This will require further basic studies to identify the underlying mechanisms and their regulation.

Other approaches can also be proposed to increase the methionine content of legume seeds, some of which have been tested in seeds of model species or other crop species. Saalbach et al. (53,57,58) showed that a me-thionine-enriched 7S globulin from Vicia was stably accumulated at levels of 1.5 to 2.2% of the total protein in seeds of transgenic tobacco plants, whereas methionine-enriched 11S globulin from the same species did not accumulate and was subsequently shown to be degraded (59). Similarly, Utsumi and co-workers (60,61) have shown that wild-type and methionine-enriched 11S globulins of soybean accumulate stably in seeds of transgenic rice. De Clercq et al. (62) constructed modified 2S albumins from Arabidopsis and chimeras comprising parts of the Brazil nut and Arabidopsis albumins and expressed them in seeds of tobacco, Arabidopsis, and oilseed rape. Down-regulation of the sulfur-poor 11S globulins (cruciferins) in oilseed rape has also been shown to result in compensatory increases in more sulfur-rich proteins (29). Similarly, Kjemtrap et al. (63) demonstrated that mutant phytohemagglutinins of bean containing four additional methionine residues were stably accumulated in seeds of transgenic tobacco although the level was not reported. However, none of these approaches has yet been successfully applied to grain legumes.

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