Uptake of Glycylglycine by the Scutellum of Germinating Barley Grain

Abstract

The scuteUa separated from germinating barley grains (Hordewn vagare L. cv. Himalaya) took up the dipeptide 14Clglycylglycine (Gly-Gly) rapidly from incubation media. The pH optimum of the process was about 4.5, and the rate of uptake conformed to Michaelis-Menten kinetics with an apparent Km of 2.3 mm and V. of 41 imole gram-l hour-'. The uptake was strongly inhibited by dinitrophenol and cyanide and by lack of 02.After incubation of the scuteUa with Gly-Gly, no intact Gly-Gly was detectable in the scuteila but the level of free glycine increased. The poorly hydrolyzed "model" dipeptide glycylsarcosine, which is actively taken up and accumulated by the scutella, was a competitive inhibitor of the uptake of Gly-Gly and completely inhibited the uptake at infinitely high inhibitor concentration. This suggests that Gly-Gly is taken up by the same mechanism as glycylsarcosine as an intact dipeptide (without hydrolysis in the membrane) and is hydrolyzed to free glycine by the abundant peptidases of the scutefla.The uptake of Gly-Gly was not affected by glycine or leucine, but was strongly inhibited by all of the 10 dipeptides tested for inhibition. The three dipeptides tested for uptake, Ala-Gly, Pro-Gly, and Gly-Pro, were all taken up by the scuteDa. Thus, the uptake mechanism for the dipeptides seems to be rather nonspecific with respect to the side chains of the amino acids. The high rates of the uptake suggest that this process has an essential role in the mobilization of reserve proteins in the germinating grain.Recent experiments (2, 6, 15) have shown that the N-methylated peptides, Gly-Sar3 and Gly-Sar-Sar, which are very resistant to hydrolysis by peptidases, were actively taken up and accumulated against a concentration gradient by the scutella of germinating barley grains. Gly-Gly, the easily hydrolyzed, unsubstituted analog of Gly-Sar, inhibited the uptake of both Gly-Sar and Gly-SarSar, presumably by competition for the peptide transport system (15). Since the uptake of Gly-Sar was not inhibited by free glycine, the peptide transport system appeared to be separate from the system(s) for the transport of free amino acids.We have now studied more closely the uptake of Gly-Gly and some other peptides by the scutellum. This paper reports an investigation of some factors affecting the uptake of Gly-Gly by the scutellum of germinating barley and its interaction with amino acids and with other peptides. data, suggest that the hydrolysis products of the reserve proteins of the starchy endosperm are taken up by the scutellum as a mixture of amino acids and small peptides and the latter are hydrolyzed to free amino acids in the scutellum before "long distance" transport to the growing tissues of the seedling. MATERIALS AND METHODSPlant Material. Grains of a huskiess variety of barley, Hordeum vulgare L. cv. Himalaya, were obtained from Agronomy Club, Washington State University, Pullman, Washington. They were surface-sterilized with sodium hypochlorite (10 g/l) for 20 mi, rinsed several times in sterile ...

An Arabidopsis peptide transporter is a member of a new class of membrane transport proteins.

“…An exception to the observed broad substrate specificity is the inability of highly basic peptides to be transported. Peptides containing three or more lysines did not support the growth of PB1X-9B transformed with either AtPTR2 or PTR2; this is also consistent with previous work on yeast (Lichliter et al, 1976;Marder et al, 1977) and plants (Higgins andPayne, 1978b, 1978c;Sopanen et al, 1978).…”

“…Consistent with this observation, transport of radiolabeled dileucine was inhibited by a number of peptides containing hydrophobic residues (Figure 3 and Results). This also supports earlier work on germinating barley seeds, which showed a strong bias toward transport of peptides composed in part or entirely of hydrophobic residues (Higgins andPayne, 1978a, 1978b;Sopanen et al, 1978). Furthermore, the hydrophobic peptides Ala-Met, Leu-Met, and Met-Met reversed the growth inhibition of Ala-Eth and Leu-Eth in roots of Arabidopsis seedlings (see Results).…”

“…However, a few reports have shown peptide transport to be involved in bacterial sporulation (Mathiopoulos et al, 1991;Perego et al, 1991), chemotaxis in bacteria (Manson et al, 1986), and recycling of bacterial cell wall peptides (Goodell and Higgins, 1987). In plants, peptide transport has been demonstrated in isolated scutella from germinating grains of barley, wheat, rice, and maize and postulated to be involved in supplying amino acids, stored in the form of peptides, from the endosperm to the germinating embryo (Higgins and Payne, 1978a;Sopanen et al, 1978;Salmenkallio and Sopanen, 1989). Peptide transport in germinating barley seeds has been demonstrated with nonhydrolyzable, nonphysiological peptide substrates, which were accumulated intact and against a concentration gradient Higgins andPayne, 1977a, 1977b;Sopanen et al, 1977).…”

Developmental Expression of Genetically Defined Peptidases in Maize

“…The scutellum is the single cotyledon of the maize seed and it completely separates the embryonic axis from the triploid endosperm which has both stored carbohydrate and protein reserves (10). A recent report has shown that glycylglycine was rapidly taken up by the scutella of germinating barley and then hydrolyzed to free amino acids (23). The specificities of both endo-and aminopeptidases using purified preparations of the maize enzymes is in progress.…”

Cloning and functional characterisation of a peptide transporter expressed in the scutellum of barley grain during the early stages of germination

“…The K m for Ala-[ 14 C]Phe transport was similar to that determined for GlyGly (2.3 mM) and GlySar (9 mM) transport in the scutellum of barley (H. vulgare L. cv. Himalaya) grain at 3 d germination (Sopanen et al, 1978). Song et al (1996) reported that S. cerevisiae expressing the cloned A. thaliana peptide carriers AtPTR2-A and AtPTR2-B transported [ 3 H]dileucine with a K m of 46.9 µM and 14.4 µM, respectively.…”