Asparagine Synthesis in Pea Leaves, and the Occurrence of an Asparagine Synthetase Inhibitor

Abstract

Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, '4Claspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label).Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of I'4Claspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.Asparagine is a major transport form of N in many plants, including legumes such as lupin (1) and pea (20). Asparagine synthetase (EC 6.4.5.4), catalyzing the glutamine-dependent amidation of aspartate, is active in germinating seedlings (9, 15, 17), but there is some question about the synthesis of asparagine in older, nongerminating tissue. '5N-Labeling patterns in pea leaves (2, 3) and spinach (21) for example in Asparagus (4). However, the enzymic properties and physiological concentrations of substrates have lead a number of workers to doubt that this can be a major route for asparagine synthesis (17-19). The principal argument in favor of the cyanide pathway is the inability to detect asparagine synthetase in Asparagus tissue (4).Although the level of asparagine decreases in mature pea leaves, presumably due to selective export (20), labeling data suggests that pea leaf tissue is capable of asparagine synthesis (2). This has been confirmed here by analysis of detached shoots; aspartate can be shown to be an asparagine precursor if a transaminase inhibitor is used to prevent the rapid equilibration of aspartate carbon with the organic acid pool. The presence of a potent asparagine synthetase inhibitor, detected in pea leaves and Asparagus shoots, may account for the inability to detect the enzyme in these and other tissues. MATERIALS AND METHODSPlants of Pisum sativum ...

The Role of Transamination in the Synthesis of Homoserine in Peas

“…Again, the labeling of homoserine bears little relationship to the labeling of aspartate. It is interesting to note that succinate provided carbon for the synthesis of asparagine which is increased in darkness (3). AOA prevented homoserine synthesis from succinate (Table V).…”

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

“…The classic example of Asn accumulation in response to stress is documented in asparagus spears in which concentrations increase five-to 1 O-fold within 24 hr of harvest (King et al, 1990). Pea leaves darkened for 72 hr have an -15-:old increase in free Asn compared with leaves exposed to the normal photoperiod (Joy et al, 1983). Similarly, sugar-starved maize root tips (Brouquisse et al, 1992) and sycamore suspension cells (Genix et al, 1990) have markedly higher amounts of free Asn compared with control tissues.…”

“…Previous studies have shown that AS enzyme activity in pea leaves (Joy et al, 1983) and mRNA in leaves of pea (Tsai and Coruzzi, 1990), Nicofiana spp (Tsai and Coruzzi, 1991), and Arabidopsis (Lam et al, 1994) are repressed by light. In contrast, AS expression in asparagus (Davies and King, 1993) and maize roots (Chevalier et al, 1996) appears to be unaffected by light.…”

“…Two forms of AS are found in Escherichia coli One, dependent on ammonia, is encoded by the asnA gene (Nakamura et al, 1981); the other, dependent on Gln, is encoded by the asnB gene (Scofield et al, 1990). Plant AS enzyme activity is known to be quite unstable and has only been partially purified from pea (Joy et al, 1983), lupin (Lea and Fowden, 1975;Rognes, 1975), maize (Oaks and Ross, 1984), and soybean (Huber and Streeter, 1985). However, recently, alfalfa root nodule AS was purified to near homogeneity, and antibodies were raised against the protein (Twary et al, 1994; S.N.…”

Overexpression of the ASN1 Gene Enhances Nitrogen Status in Seeds of Arabidopsis

“…Precise study of ASN1 enzyme activities from plant extracts is hampered by instability of the AS enzyme in vitro (Sieciechowicz et al, 1988); copurification of a heat-stable, dialyzable inhibitor (Kern and Chrispeels, 1978;Joy et al, 1983); and the presence of contaminating asparaginase activity in plant extracts (Huber and Streeter, 1985) and the existence of multiple isoenzymes . Besides, our ultimate goal is to study the changes in amino acid metabolism and nitrogen status; therefore, we directly determined the steady-state levels of free Asn (product of AS enzymes) in leaves and siliques of 35S-ASN1 plants and compared them with control wild type (Col-0) or wild type transformed with an empty vector (359-2C-8; Fig.…”