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Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.

The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration, including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat, levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight, suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern, being low at 50 mg and progressively increased until 150 mg, coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars, ∼2-fold higher levels of asparaginase B1 protein and activity in seed coat, were associated with high protein concentration, reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene, ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.

Several approaches were taken to examine the role of N-assimilate supply in the control of soybean (Glycine max) seed composition. In the first study, developing seeds were grown in vitro with D-[U-14C]sucrose (Suc) and different concentrations of Gln. Light stimulated carbon flux into oil and protein, and was required to sustain Suc uptake and anabolic processes under conditions of elevated nitrogen supply. High Gln supply resulted in higher transcript levels of beta-conglycinin and oleosin. In the second study, analyses of soluble amino acid pools in two genetically related lines, NC103 and NC106 (low- and high-seed protein, respectively) showed that, in the light, NC106 accumulated higher levels of Asn and several other amino acids in developing cotyledons compared with NC103, whereas at the seed coat and apoplast levels both lines were similar. In the dark, NC103 accumulated Gln, Arg, and its precursors, suggesting a reduced availability of organic acids required for amino acid interconversions, while NC106 maintained higher levels of the pyruvate-derived amino acids Val, Leu, and Ile. Comparing NC103 and NC106, differences in seed composition were reflected in steady-state transcript levels of storage proteins and the lipogenic enzyme multi-subunit acetyl CoA carboxylase. In the third study, a positive correlation (P < or = 0.05) between free Asn in developing cotyledons and seed protein content at maturity was confirmed in a comparison of five unrelated field-grown cultivars. The findings support the hypothesis that high seed-protein content in soybean is determined by the capacity of the embryo to take up nitrogen sources and to synthesize storage proteins. Asn levels are probably tightly regulated in the embryo of high-protein lines, and may act as a metabolic signal of seed nitrogen status.

The effect of colonization of tissue-cultured strawberry (Fragaria×ananassa Duch. cv. Kent) plantlets in vitro by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices on plantlet response to poly(ethylene glycol) (PEG)-8000-induced water stress was investigated. The plantlets were inoculated axenically and co-cultured with the AMF for 4 wk, then transferred to 15% PEG-8000 solutions for 4, 8 and 12 h. Relative water content, water potential, osmotic potential, leaf conductance for water vapour diffusion and photosynthetic efficiency as estimated by chlorophyll a fluorescence were all affected by the PEG treatment and its duration but not by the presence of the intraradical phase of the AMF. However, distinct differences in PEG-induced changes in amino acid content were observed between nonmycorrhizal and mycorrhizal plantlets. In the latter, the treatment with PEG caused a substantial decrease in asparagine levels in leaves that was accompanied by a marked increase in asparagine concentration in roots. The opposite was observed in nonmycorrhizal plantlets. Furthermore, concentrations of aspartic acid, serine, threonine, amino-N-butyric acid, alanine and starch increased in roots of mycorrhizal and decreased in nonmycorrhizal plantlets. Our results suggest the presence of a mobile pool of asparagine that can be translocated from leaves to roots or vice versa in response to PEG-induced water stress, depending on the mycorrhizal status of the plantlets. These opposite patterns suggest different strategies of mycorrhizal and nonmycorrhizal plantlets to water stress, which seem to involve different adjustments in nitrogen and carbon metabolism.