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

Abstract

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...

Uptake of Glutamine by the Scutellum of Germinating Barley Grain

“…The uptake of glutamine into barley scutellum resembles in several respects the uptake of leucine into this organ (26). Both processes appear to be carrier-mediated active transport, have a pH optimum near pH 5, and are inhibited by 100 mM NaCl.…”

“…We reported earlier that the Hofstee plot for the uptake of leucine is not linear (26). We have now repeated the experiment in the conditions of the present study and with a correction for the nonmediated uptake.…”

“…These are taken up into the scutellum, most probably into the epithelial cells (11,26, and references cited therein), from where the amino acids taken up or liberated from the peptides are transferred to the growing seedling.…”

“…Previously, some properties of the uptake of amino acids into the scutellum were studied using leucine as substrate (18,26). The uptake of leucine is separate from the uptake of peptides and is due to active transport.…”

“…The standard procedure was modified from the leucine uptake assay used earlier (26). Samples of four separated scutella were weighed and incubated in 3 ml of 1 mM L-["4C] glutamine (about 30 nCi/flask) in 5 mm sodium 2,2'-dimethylglutarate buffer (pH 5) in a shaking water bath at 30°C, the time of incubation being now 10 min instead of 1 h. The scutella were then rinsed with water (about 1 min), transferred into scintillation vials containing 0.5 ml of6% sulfosalicylic acid, and warmed for 20 min at 50C.…”