The effect of ammonium ions on uptake of glutamine and other amino compounds by cultured cells of rapeseed

The scutella separated from nongerminated grains of barley (Hordeum vulgare L. cv. Pirkka) took up labeled glycylsarcosine (Gly-Sar), glycylglycine, and leucyUeucine at a rate of about 2 nanomoles per scutellum per hour. About 55% of the uptake of Gly-Sar (2 millimolar) was due to nonmediated uptake, but the active component was similar to that operating in the scutela from germinating grains (pH optimum about 4.5, Km about 10 millimolar).When the grains were allowed to germinate aseptically on agar, the rate of uptake of Gly-Sar began to increase after a lag of about 5 hours. The increase continued until a maximum value of 70 nanomoles per scutellum per hour (35-fold increase) was attained after 3 days of germination. After this the activity began to decrease slowly. The uptake of glycylglycine and leucyileucine changed in a similar way. When embryo-containing halves of seeds or separated embryos were allowed to germinate for 12 hours on agar containing various inhibitors of RNA and protein synthesis, the increase in the peptide uptake activity was completely or partially inhibited.The increase was not affected by removal of the "embryo proper" or endosperm, or by additions of gibberellic or abscisic acid.Root tips and aleurone layers took up Gly-Sar very slowly; the rate of the mediated component of uptake was less than 1% of the uptake by scutella.The preferential localization of the peptide transport system in the scutelium and its rapid development at the beginning of germination strengthen the earlier conclusion that peptide transport plays an essential role in the mobilization of reserve proteins during germination.

Section: Resultsmentioning

confidence: 99%

Development of Peptide Transport Activity in Barley Scutellum during Germination

Sopanen

1979

“…1-4, [11][12][13][15][16][17][18]. Leucine was selected as the first amino acid to be tested; it comprises about 7% of all amino acid residues in the main reserve protein, hordein (10), and is generally metabolized slower than most amino acids.…”

Section: Introductionmentioning

confidence: 99%

Characteristics and Development of Leucine Transport Activity in the Scutellum of Germinating Barley Grain

Sopanen

Uuskallio²,

Nyman³

et al. 1980

Scutelia separated from grains of Himalaya barley after germination for 3 days rapidly took up L-leucine from aerated incubation media; with 1 millimlar leucine the rate varied between 4 and 14 micromoles per gram per hour and the pH optimum was at 3.5 to 5, both depending on buffer composition and prewashing time. The rate of the uptake increased with increasing concentration of leucine in a complex manner, which could be interpreted as multiphasic kinetics with apparent Km values of 3.4 and 15.5 millilar below and above 3 mflimlar leucine, respectively. The uptake took place against a concentration difference (highest estimated ratio 270: 1) and was strongly inhibited by dinitrophenol. Uptake was apparently due to active transport requiring metabolic energy.The development of the uptake activity during germination was studied using Pirkka barley. A low actinty was present in the scutelia of ungerminated grains. It began to increase after 6 hours imbibition, and the increase was biphasic, the major changes occurring during days 0 to 3 and 4 to 6. The total increase was about 20-fold.The regulation of the development was studied by allowing separated embryos to germinate on agar gel. The increase of uptake activity was strongly inhibited by inhibitors of RNA or protein synthesis. Increase did not require the presence of the embryo proper, and was not affected by gibberellic or abscisic acid. Removal of the endosperm greatly accelerated the increase of uptake activity, and the presence of 5 or 20 mfllimolar glutamine counteracted the removal of the endosperm. The results suggest that the availability of glutamine or amino acids in general in the endosperm may regulate the development or the activity of the transport system.In germinating barley grain the main site of reserve protein degradation is the nonliving storage tissue, the starchy endosperm, where the insoluble reserve proteins are hydrolyzed apparently to a mixture of amino acids and oligopeptides (14). As this "digestive compartment" of the seed is separated from the growing seedling by the scutellum, the hydrolysis products must be taken up by the scutellum before "long distance transport" to the seedling. An active transport system for oligopeptides has recently been shown to function in barley scutellum (6-9, 19-21); its properties include high transport capacity, wide specificity, and a pH optimum of 4 to 5, which is near to the pH of the endosperm.To add to the picture of reserve protein mobilization in barley we wanted to see how free amino acids are taken up by the scutella at different stages ofgermination. An uptake system for one amino ' This work was supported by grants from the National Council for Sciences.2 Resarch Assistant of the National Council for Sciences.acid, glutamine, has been described in slices of maize scutella (22), and several other plant tissues have been shown to take up amino acids from incubation media (e.g. 1-4, [11][12][13][15][16][17][18]. Leucine was selected as the first amino acid to be tested; it comprise...

“…However, when rapeseed or soybean cells are cultured in suspension in the presence of NH4NO3, they take up amino acids, e.g. glutamine and leucine, very slowly (9,10). When the cells are transferred to a medium without nitrogen nutrients, their capacity for uptake of many amino acids increases several-fold in 24 h. The authors suggested that one possible explanation for the inhibition caused by NH4NO3 is that NH4' increases the intracellular concentration of glutamine, which would then inhibit amino acid uptake through transinhibition (9).…”

Section: Discussionmentioning

confidence: 99%

“…glutamine and leucine, very slowly (9,10). When the cells are transferred to a medium without nitrogen nutrients, their capacity for uptake of many amino acids increases several-fold in 24 h. The authors suggested that one possible explanation for the inhibition caused by NH4NO3 is that NH4' increases the intracellular concentration of glutamine, which would then inhibit amino acid uptake through transinhibition (9). It is quite possible, however, that amino acid uptake in these cells is regulated by a repression-like mechanism similar to that in the barley scutellum.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Development of Leucine Uptake Activity by Glutamine in the Scutellum of Germinating Barley Grain

Nyman

Sopanen²,

Mikola³

1983

Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the gains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system. Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and-possibly to a lesser extent-by some other amino acids taken up from the endosperm.The hydrolysis of reserve proteins in the starchy endosperm of a germinating barley grain results in a mixture of free amino acids and short oligopeptides (7,13,14,20). The amino acids and peptides are taken up into the scutellum by separate transport systems (6,11,(20)(21)(22). We have previously studied some properties of amino acid uptake into the scutellum using leucine as the substrate (24). Leucine is rapidly taken up by an active transport system(s), the rate of uptake being highest near pH 5, the pH of the starchy endosperm (14).The uptake of leucine is slow in scutella separated from ungerminated grains, but increases about 10-fold during the first 3 d ofgermination of whole grains (24). This increase is followed by a lag phase of about 1 d, after which there is another increase.Removal of the embryonic axis has no effect on the initial ' Supported by the Research Council for Natural Sciences of which T. S. is a Research Fellow. The work was also supported by a grant from the Emil Aaltonen foundation to S. N.2 Abbreviations: CHI, cycloheximide; Sar, sarcosine; COR, cordycepin; AIB, a-amino-iso-butyric acid.increase in the rate of uptake (24). On the other hand, removal of the endosperm either after imbibition for 4 h or after germination for 3 d enhances the rate ofthe increase during subsequent incubation. In both cases, the increase is completely prevented by CHI2 and markedly slowed down by glutamine. It was therefore su...