Localization of carboxypeptidase I in germinating barley grain

Activity measurements and Northern blot hybridizations were used to study the temporal and spatial expression of carboxypeptidase I in germinating grains of barley (Hordeum vulgare L. cv Himalaya). In the resting grain no carboxypeptidase I activity was found in the aleurone layer, scutellum, or starchy endosperm. During germination high levels of enzyme activity appeared in the scutellum and in the starchy endosperm but only low activity was found in the aleurone layer. No mRNA for carboxypeptidase I was observed in the resting grain. By day 1 of germination the mRNA appeared in the scutellum where its level remained high for several days. In contrast, little mRNA was observed in the aleurone layer. These results indicate that the scutellum plays an important role in the production of carboxypeptidase I in germinating barley grain.     

Metabolism of Ammonium Ion and Glutamate in Relation to Nitrogen Supply and Utilization during Grain Development in Barley

Changes in the activity of a number of enzymes concerned with amino acid synthesis and metabolism were recorded for the endosperm, testa pericarp, and embryo of developing barley (Hordeum distichum L.) grains. Both glutamate-pyruvate transaminase and glutamate-oxaloacetate transaminase activities were present in all tissues and at all ages examined. Glutamate dehydrogenase activity was largely confined to endosperm while glutamine synthetase activity was mainly in the testa pericarp.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Observations on the Germ Aleurone of Barley. Morphology and Histochemistry

The germ aleurone over the embryonic axis of barley was examinedin strips of tissue peeled off harvest-ripe grains. The germaleurone is only one cell thick but resembles 'normal' aleuronein being composed of living cells with dense, lipid-rich cytoplasmand thick walls containing phenolic material. In contrast tothe cells of the 'normal' aleurone, germ aleurone cells containvery few phytin or protein deposits. When the germ aleuroneis ruptured during germination, the walls at the torn edge becomethickened with shiny golden-brown material, and 'sealed' tothe testa. Two days after germination, lignin can be detectedin the walls of a single row of germ aleurone cells adjoiningthe scutellum. The role of the germ aleurone in defence againstmicroorganisms is discussed. It is suggested that the metabolicactivities in the germ aleurone in imbibed grains compete withthe embryo for oxygen, and thus may be one of the factors whichdetermine whether a grain germinates or remains dormant.Copyright1994, 1999 Academic Press Barley, Hordeum vulgare L., germ aleurone, histochemistry, defence mechanism, lignin, dormancy, microorganisms, pre-mature germination     

Rates of Cell Division in Developing Barley Endosperms

Counts of nuclei in enzyme digested endosperms of barley cultivarBomi show that the final number of cells, 170000, is reachedbetween 18 and 21d after anthesis. Based on the number of cellprofiles in transverse mid-grain sections, starchy endospermcells divide up to day 14. Thereafter, cell proliferation isrestricted to the aleurone layer. Hordeum vulgare, starchy endosperm, aleurone, mitotic activity, light microscopy     

Amylases in developing barley seeds

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.     

Endosperm acidification and related metabolic changes in the developing barley grain

The starchy endosperm (SE) of the developing grain (caryopsis) of barley (Hordeum vulgare L.) cv Himalaya, as well as that of other barley cultivars examined, acidifies during maturation. The major decrease in pH begins with the attainment of maximum grain dry weight, onset of dehydration, and completion of chlorophyll loss. Acidification is correlated with the accumulation of malate and lesser amounts of citrate and lactate, produced and probably secreted by the pericarp/testa/aleurone (PTA). It is accompanied by large concurrent rises in phosphoeno/pyruvate carboxylase and alcohol dehydrogenase (ADH) activity in the PTA. The activity of seven other enzymes of oxaloacetate and pyruvate metabolism was found to fall or rise only slightly during acidification. Sequential changes in relative amount of ADH isozymes were found in both PTA and SE. The PTA maintained a high respiration rate and adenylate energy charge (AEC) throughout acidification, whereas the SE showed a low respiration rate and rising AEC. The data are consistent with the occurrence of hypoxia in the SE. It is suggested that the above enzyme changes are required for the development of a malate/ethanol fermentation (i.e. a mixed metabolism) in the aleurone layer during maturation.     

Water uptake and splitting of wild oat caryopsis

Imbibition and germination experiments were conducted on the caryopses of wild oats (Avena fatua L.). The embryo envelopes, pericarp and aleurone layer, which completely cover the embryo-endosperm, do not form barriers against water uptake. The initial uptake of water is passive and the water moves across the pericarp with ease as it contains cracks; it is, however, transported across the aleurone layer through its cell walls into the endosperm and embryo of the caryopsis. The starchy endosperm enlarges due to water uptake causing the pericarp to rupture, thus exposing the aleuronelayer-covered seed. The aleurone layer is structurally heterogenous consistings of radially compressed irregular cells and cuboidal or radiallys tretched cells; the latter contains thicker walls. The former type is present along the abaxial side of the embryo and in the crease on the adaxial side of the caryopsis; the latter type covers the endosperm. The physical distention of the endosperm due to water uptake causes the rupture of the pericarp and the aleurone layer, and facilitates the emergence of the radicle and coleorhiza of the embryo during caryopsis germination.     

Expression of the 30 kD cysteine endoprotease B in germinating barley seeds

The expression of a 30 kD cysteine endoprotease (EP-B) was studied by in situ hybridization and immunomicroscopy to clarify its role in germinating barley grains. At the beginning of germination, EP-B mRNA was expressed in the scutellar epithelium and aleurone cells next to the embryo. Later, mRNA levels were highest in the aleurone layer proceeding to the distal end of the grain. During the first day of germination, EP-B protein was strongly localized to the germ aleurone and scutellar epithelium from where the secretion into the starchy endosperm began. Secretion was also observed to proceed along the aleurone layer to the distal end. These results show that EP-B is differentially localized during germination, and both scutellum and aleurone layer are able to synthesize and secrete EP-B protein.     

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     

Changes in cell wall polysaccharides of germinating barley grains

Decorticated barley grains were germinated at 25° for 6 days, until the endosperm reserves were nearly exhausted. The neutral monosaccharide components of the hydrolysates of the cell walls and gums from the embryo, aleurone layer and starchy endosperm and the endospermic starch were determined at daily intervals. The amount of embryo cell wall polysaccharide increased 40 times and glucose became the major component, followed in abundance by xylose and arabinose. The cell wall and gum polysaccharides of the aleurone layer (plus testa) and the starchy endosperm declined during germination and their compositions altered. The endospermic starch also decreased. In the early stages of germination the apparent composition of the cell walls of the aleurone layer and starchy endosperm depended upon how they had been prepared. After 6 days the cell walls and gums had provided a significant carbohydrate supply to the living tissues, equivalent to 18.5% of the endospermic polysaccharide degraded during growth, starch having provided the remaining 81.5%.     

Isolation of Intact Protein Storage Vacuoles from Barley Aleurone (Identification of Aspartic and Cysteine Proteases)

Within the cereal aleurone reserve proteins are stored in specialized organelles, the protein storage vacuoles (PSV). We developed an aqueous method for the isolation of intact PSV. Barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts were gently lysed by passing them through a syringe needle. PSV were separated from cytoplasmic components by microfiltration and low-speed centrifugation. Isolated PSV appeared by light microscopy to be identical with those within barley aleurone protoplasts. Luminal contents were retained throughout the isolation procedure. We used isolated PSV to identify and characterize PSV-associated proteolytic activities. Isolated PSV contained cysteine proteases and aspartic proteases (APs). Gibberellic acid treatment of protoplasts increased cysteine protease activity. Protein blots probed with anti-H. vulgare aspartic proteinase (HvAP) indicated that one PSV-AP was HvAP. Immunocytochemical localization by electron microscopy confirmed the presence of HvAP within the lumen of PSV. We conclude that isolated barley aleurone PSV will be useful in further characterizing this organelle.     

The aspartic proteinase of barley is a vacuolar enzyme that processes probarley lectin in vitro.

Previous work suggested that the aspartic proteinase from Hordeum vulgare (HvAP) would be a vacuolar protein in plant cells. Based on N-terminal sequencing we show that the in vitro-translated protein was translocated into the lumen of microsomal membranes, causing a concomitant removal of 25 amino acid residues from the protein. Vacuoles were purified from barley leaf protoplasts and were shown to contain all of the aspartic proteinase activity found in the protoplasts. This vacuolar localization of HvAP was confirmed with immunocytochemical electron microscopy using antibodies to HvAP in both barley leaf and root cells. In an attempt to discern a function for this protease, we investigated the ability of HvAP to process the C-terminal proregion of barley lectin (BL) in vitro. Prolectin (proBL), expressed in bacteria, was processed rapidly when HvAP was added. Using several means, we were able to determine that 13 amino acid residues at the C terminus of proBL were cleaved off, whereas the N terminus stayed intact during this incubation. Immunohistochemical electron microscopy showed that HvAP and BL are co-localized in the root cells of developing embryos and germinating seedlings. Thus, we propose that the vacuolar HvAP participates in processing the C terminus of BL.     

Aspartic proteinase from barley grains is related to mammalian lysosomal cathepsin D

Resting barley (Hordeum vulgare L.) grains contain acid-proteinase activity. The corresponding enzyme was purified from grain extracts by affinity chromatography on a pepstatin-Sepharose column. The pH optimum of the affinity-purified enzyme was between 3.5 and 3.9 as measured by hemoglobin hydrolysis and the enzymatic activity was completely inhibited by pepstatin a specific inhibitor of aspartic proteinases (EC 3.4.23). Further purification on a Mono S column followed by activity measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the affinity-purified enzyme preparation contained two active heterodimeric aspartic proteinases: a larger 48k Da enzyme, consisting of 32-kDa and 16-kDa subunits and a smaller one of 40 kDa, consisting of 29-kDa and 11-kDa subunits. Separation and partial amino acid sequence analysis of each subunit indicate that the 40-kDa enzyme is formed by proteolytic processing of the 48k Da form. Amino-acid sequence alignment and inhibition studies showed that the barley aspartic proteinase resembles mammalian lysosomal cathepsin D (EC 3.4.23.5).     

Enhancement by gibberellic acid and asymmetric distribution of lysophospholipase in germinating barley

Germination of whole barley seeds for 4 and 6 days followed by measurement of lysophospholipase (lysolecithin acyl hydrolase, LAH) in the embryo-containing and embryo-free halves revealed a gradient of activity between the two halves of the seed. Most of the activity appeared in the embryo-containing half. This gradient decreased slightly in the aleurone and dramatically in the starchy endosperm during the 2 day germination interval. Embryo-containing and embryo-free half seeds of surface sterilized barley were placed separately on sterile agar plates. After 4 and 6 days LAH was observed in both the aleurone and starchy endosperm of the embryo-containing halves. In the embryo-free halves, LAH appeared at low levels in the aleurone and was virtually absent in the starchy endosperm. The scutellum of germinating seeds contains LAH activity. Exposure of embryo-free half seeds to GA₃ for 24 hr showed enhancement of acidic and alkaline LAH activities in the aleurone fraction and in the GA₃-medium in which the half seeds were treated. The LAH activity of the starchy endosperm of these half seeds was little changed by GA₃ treatment. Exposure of isolated aleurones to GA₃ for 24 hr resulted in substantial enhancement of acidic and alkaline LAH activities in the bathing medium and in fractions prepared from the aleurone. The physiological significance of the influence of GA₃ on LAH activity during barley germination is discussed.     

The role of phytic Acid in the wheat grain

The concentrations of adenosine triphosphate and phytic acid in testa, embryo plus scutellum, aleurone, and endosperm fractions from grain of Triticum vulgare cv. Insignia have been determined during development under both normal conditions and those of water stress. Phytic acid was not detected in the endosperm. In the embryo plus scutellum and aleurone fractions there was a rapid build-up of phytic acid, but the adenosine triphosphate level did not change markedly at this time. These results are not consistent with physiological roles previously suggested for phytic acid other than the role of phytin as a phosphorus and cation store for the germinating seed.