Localization and activity of various peptidases in germinating barley

Summary Germinating barley grains contain at least eight different peptidases: three carboxypeptidase (pH optima 4.8, 5.2, and 5.7), three aminopeptidases which act on aminoacyl--naphthylamides (pH opitima in the hydrolysis of di- and tripeptides at pH 5.8–6.5), and two peptidases which hydrolyse Ala-Gly and Leu-Tyr optimally at pH 7.8 and 8.6 respectively. We have determined the activities of these enzymes in the different tissues of non-germinated grains and followed the changes in the activities during a 5-day germination at 16°C.The aleurone layers contain high activities of all three groups of peptidases; there are no changes in the activities of the five aminopeptidases on germination, while the carboxypeptidases exhibit a small increase of activity. The starchy endosperms contain high carboxypeptidase activities, which increase during germination, but are totally devoid of the five aminopeptidases.All the peptidases exhibit high activities in the scutella; the carboxypeptidases and the enzymes acting on Ala-Gly and Leu-Tyr increase in activity during germination, while the naphthylamidase activities remain constant.The three peptidase groups occur in the seedling as well, but compared to the other tissues the carboxypeptidase activities are very small and the naphthylamidase activities are very high. The last-named enzymes seem to be characteristic for growing tissues.The starchy endosperm contains about two thirds of the total reserve proteins of the grain. Its internal pH during germination is 5.0–5.2, a value at which all the carboxypeptidases are highly active. As these enzymes are present in high concentrations in this tissue, it is probable that they have a central role in the mobilization of the reserve proteins during germination. The high peptidase activities of the scutellum, on the other hand, suggest that some of the hydrolysis products are absorbed as peptides and these are further hydrolysed to amino acids in this tissue.Abbreviations used DTT dithiothreitol - GA₃ gibberellic acid - -NA -naphthylamide - TNBS 2,4,6-trinitrobenzene sulphonic acid - Z- N-carbobenzoxy     

Mobilization of proline in the starchy endosperm of germinating barley grain

In germinating grains of barley, Hordeum vulgare L. cv. Himalaya, free proline accumulated in the starchy endosperm during the period of rapid mobilization of reserve proteins. When starchy endosperms were separated from germinating grains and homogenized in a dilute buffer of pH 5 (the pH of the starchy endosperm), the liberation of proline continued in these suspensions. The process was completely inhibited by diisopropylfluorophosphate, indicating that it was totally dependent on serine carboxy-peptidases. The carboxypeptidases present in the starchy endosperms of germinating grains were fractionated by chromatography on DEAE-cellulose. Four peaks were obtained, all with different activity spectra on the seven carbobenzoxydipeptides (Z-dipeptides) tested. Two of the peaks corresponded to previously known barley carboxypeptidases; these as well as a third peak hydrolyzed substrates of the types Z-X-Y and Z-X-Pro (X and Y denote any amino acid residue except proline). The fourth peak corresponded to a proline carboxypeptidase specific for substrates of the Z-Pro-X type. Apparently, in the hydrolysis of longer proline-containing peptides there must be sequential cooperation between the two carboxypeptidase types. The carboxypeptidases in extracts of starchy endosperms also liberated proline from the peptides Ala-Ala-Ala-Pro and Ala-Ala-Pro while Ala-Pro and Pro-Ala were not attacked. The dipeptides, however, were rapidly hydrolyzed around pH 7 by extracts prepared from the scutella of germinating grains. It is concluded that one part of the proline residues of the reserve proteins is liberated in situ in the starchy endosperm through the combined action of acid proteinases and carboxypeptidases, while another part is taken up in the form of small peptides by the scutellum, where proline is liberated by amino- and/or dipeptidases in some neutral compartment.Abbreviations DFP diisopropylfluorophosphate - DTT dithiothreitol - TNBS 2,4,6-trinitrobenzenesulphonic acid - Z N-carbobenzoxy - TLC thin layer chromatography A preliminary account of these results was given at the Meeting of the Federation of European Plant Physiological Societies in Edinburgh in July 1978. Abstract No. 181     

Uptake of proline by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up 1 millimolar l-[¹⁴C]proline at an initial rate of about 6.5 micromoles gram⁻¹ fresh weight hour⁻¹ (pH 5, 30°C). The uptake had a pH optimum at 5. The bulk of the uptake (93%) was via carrier-mediated active transport. All of the 19 l-amino acids tested at 10 millimolar concentration inhibited the mediated uptake of 1 millimolar proline, the inhibitions varying from 18 to 76%. By studying how large a fraction of the mediated uptake was inhibitable by asparagine, alanine, glutamine, and leucine, the mediated uptake was shown to be due to three components. Two of these are most probably attributable to the two nonspecific uptake systems proposed earlier to act in the uptake of glutamine and leucine. The third component was not inhibited by glutamine, asparagine, or alanine, but was inhibited by unlabeled proline and leucine. The uptake by this system was apparently carrier-mediated active transport. d-Proline inhibited this system as strongly as l-proline. Nine of the 16 l-amino acids tested at 50 millimolar concentrations did not inhibit the uptake of 1 millimolar proline by this system. Valine, leucine, isoleucine, and the basic amino acids were inhibitory, but in spite of this, they did not appear to be taken up by this system. It seems therefore that in addition to two nonspecific amino acid uptake systems the scutella have an uptake system which is specific for proline. It is likely that this proline-specific system accounts for the bulk of proline uptake in a germinating grain.     

Uptake of glutamine by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up [¹⁴C]glutamine at an initial rate of about 10 micromoles·gram⁻¹·hour⁻¹ in the standard assay conditions (pH 5, 30°C, 1 millimolar glutamine). Inhibition by unlabeled glutamine and by dinitrophenol indicated that about 95% of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was 5, and after correction for a nonmediated component the uptake appeared to conform to Michaelis-Menten kinetics with an apparent K_m of about 2 millimolar and a V_max of about 25 micromoles·gram⁻¹·hour⁻¹.

The uptake of glutamine was inhibited by all of the 18 amino acids tested; the mode of inhibition was studied only with proline and was competitive. Eight of the ten amino acids tested at high concentrations appeared to be able to inhibit the mediated uptake of glutamine virtually completely. However, when the inhibitory effect of asparagine was extrapolated to an infinitely high concentration of asparagine, about 24% of the mediated uptake of glutamine remained uninhibited. These results suggest that glutamine is taken up by two (or more) rather unspecific amino acid uptake systems, the minor one having no affinity for asparagine.

Glutamine and alanine could completely inhibit the mediated uptake of 1 millimolar leucine, but about 12% of the mediated uptake appeared to be uninhibitable by asparagine. Furthermore, the ratio of the mediated uptake of glutamine to that of leucine changed from 0.9 to 1.7 between days 1 and 3 of germination. These results give further support for the presence of two unspecific amino acid uptake systems in barley scutella.

     

Uptake of glycylglycine by the scutellum of germinating barley grain

The scutella separated from germinating barley grains (Hordeum vulgare L. cv. Himalaya) took up the dipeptide [¹⁴C]glycylglycine (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 K_m of 2.3 mm and V_max of 41 μmole gram⁻¹ hour⁻¹. The uptake was strongly inhibited by dinitrophenol and cyanide and by lack of O₂.     

Endogenous gibberellins and kauranoids identified from developing and germinating barley grain

Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA₁ (trace), GA₄ (trace), GA₈ (trace), GA₁₂, GA₁₇, GA₂₀ (tentative) (trace), GA₂₅, GA₃₄, GA₄₈, 18-hydroxy-GA₄, 12-hydroxy-GA₉, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₁₇, GA₂₀, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA₁, GA₃ (possibly a contaminant), GA₁₇, GA₁₉, GA₂₀, GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1-Hydroxylated GAs previously found in wheat were not found in barley in this study.This work has been reported in a poster demonstration (Gaskin et al. 1982).     

Endogenous gibberellins and kauranoids identified from developing and germinating barley grain

Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA₁ (trace), GA₄ (trace), GA₈ (trace), GA₁₂, GA₁₇, GA₂₀ (tentative) (trace), GA₂₅, GA₃₄, GA₄₈, 18-hydroxy-GA₄, 12β-hydroxy-GA₉, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₁₇, GA₂₀, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA₁, GA₃ (possibly a contaminant), GA₁₇, GA₁₉, GA₂₀, GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1β-Hydroxylated GAs previously found in wheat were not found in barley in this study.     

Characteristics and development of leucine transport activity in the scutellum of germinating barley grain

Scutella separated from grains of Himalaya barley after germination for 3 days rapidly took up l-leucine from aerated incubation media; with 1 millimolar 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 K(m) values of 3.4 and 15.5 millimolar below and above 3 millimolar 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 activity was present in the scutella 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 millimolar 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.     

Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain

α-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20°C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.

When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl₂, the α-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of α-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of α-amylase activity in the starchy endosperm in a whole grain.

Scutella separated after 1 day of germination released small amounts of α-amylase activity into 10 millimolar CaCl₂. This release was linear for at least 1 hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of α-amylase activity in the starchy endosperm in a whole grain.

Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total α-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

     

Selenium-containing peroxidases of germinating barley

Germinating barley grown on an artificial medium was exposed to⁷⁵Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and⁷⁵Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.     

Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain

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 grains 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.

     

Turnover of starch-bound lysophosphatidylcholine in germinating barley

Free and starch-bound lysophosphatidylcholine (LPC) in germinating barley was isolated and quantified during the first 8 days of germination. During the first 4 days the starch-bound LPC remained at a relatively constant level (ca 0.4,μmol/seed) and then declined during the next 2 days to ca 0.1 μmol/seed. There appeared to be no further loss of this starch-bound lipid on further germination. The decrease in the content of starch-bound LPC is not due to the action of phospholipase C and/or D on the starch lipid because there was no corresponding accumulation of starchbound lysophosphatidic acid or monoacylglycerol. The free LPC remained relatively constant at 0.02 to 0.04 μmol/seed during the entire germination period indicating that the LPC released from the starch during days 5 and 6 is further metabolized. Amylase activity was also measured in the germinating seed and increased 20-fold between days 2 and 4 which just precedes the rapid decline in starch-bound LPC. The starch content of the seed however declined to ca one third of the original value by day 5. LPC represents 65–70 % of the starch-bound lipid phosphorus in the dry seed. Through days 5 and 6 when the loss of LPC is most rapid there is no marked change in this percentage. After 8 days, however, the LPC is only ca 30%. of the starch-bound lipid phosphorus. Ofthe two major populations of starch-bound LPC, the one bearing a linoleyl group appears to decline more rapidly during days 4–6 than does that carrying a palmitoyl group. The role of starch-bound LPC in barley development and germination is discussed.     

Glutamine synthetase isozymes in germinating barley seeds

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of ammonia assimilation in higher plants. In the present study the subunit composition and localization of GS in germinating barley ( Hordeum vulgare ) seed have been clarified. Analysis of the GS polypeptide composition by immunoblotting revealed two different polypeptides. A and B, with a molecular mass of 42 and 40 kDa, respectively. In the scutellum subunit A was already present in the ungerminated seed and remained unchanged, whereas subunit B appeared on day 2 and increased about 5-fold during germination. Polypeptide B also appeared later during germination in the aleurone layer, roots and weakly in the etiolated shoots. By immunogold microscopy, GS was detected in the scutellum and the aleurone layer of barley seeds during germination. Subcellular localization of GS on ultrathin cryosections showed that a cytosolic isozyme was present in the scutellum. Our study confirms that only a cytosolic GS is expressed in barley seed, and its subunit composition changes during germination.     

The role of alpha-glucosidase in germinating barley grains

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.     

Peptide transport by germinating barley embryos

Glycylsarcosine, a dipeptide which is resistant to peptidase activity, was accumulated intact against a concentration gradient by germinating embryos of Hordeum vulgare L., var. Maris Otter, Winter. This is the first clear evidence for the presence of a dipeptide transport system involved in the movement of protein reserves across the scutellum from the endosperm to the embryo during germination.Abbreviations gly-sar glycylsarcosine - TCA trichloroacetic acid