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     

Secretion of basic and acidic chitinases from salt-adapted and -unadapted winged bean cells

Northern hybridizations were used to study the site of synthesis of three carboxypeptidases (Cpases I-III) which occur in the starchy endosperm of germinating barley grain ( Hordeum vulgare L.). Further evidence was obtained by studying secretion of these enzymes from scutella or aleurone layers separated from germinating grains. Messenger RNA for Cpase II was detected only in developing grain, and the bulk of the mRNA was localized in the starchy endosperm. This suggests that Cpase II is synthesized at the site of its accumulation, the starchy endosperm. In contrast, Cpase I is expressed during germination and the predominant site of synthesis is the scutellum, from which it is secreted into the starchy endosperm. Cpase III is also synthesized during germination, but the bulk of it is synthesized in and secreted from the aleurone layer. Thus, the three carboxypeptidases, all of which seem to play a role in hydrolysis of the reserve proteins in the starchy endosperm during germination, have different sites of synthesis.     

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.     

Structure and expression during the germination of rice seeds of the gene for a carboxypeptidase

The carboxypeptidase gene from rice and corresponding cDNA clones were isolated. The SalI 11.2 kb fragment of DNA cloned from a size-fractionated genome library contained eight introns and an open reading frame that encoded 500 amino acids (M _r 55445). The structure deduced for the carboxypeptidase from rice was very similar to those of type III serine carboxypeptidases from barley and wheat. The extent of homology of the amino acid sequence to that of these carboxypeptidases from barley and wheat was 92.3% and 87.2%, respectively. The accumulation of mRNA for the rice carboxypeptidase was conspicuous in germinating endosperms that contained aleurone layers, but levels were lower in leaves and roots. The abundance of the mRNA in endosperms was enhanced by gibberellic acid (GA) and accumulation of the mRNA was inhibited by abscisic acid (ABA). The rice gene for carboxypeptidase contained some pyrimidine boxes (_T ^CCTTTT_T ^C), in the 5 flanking region, which are a characteristic of a GA-responsive gene.     

Acid Carboxypeptidases in Grains and Leaves of Wheat, Triticum aestivum L

Extracts of resting and germinating (3 days at 20°C) wheat (Triticum aestivum L. cv Ruso) grains rapidly hydrolyzed various benzyloxycarbonyldipeptides (Z-dipeptides) at pH 4 to 6. Similar activities were present in extracts of mature flag leaves. Fractionation by chromatography on CM-cellulose and on Sephadex G-200 showed that the activities in germinating grains were due to five acid carboxypeptidases with different and complementary substrate specificities. The wheat enzymes appeared to correspond to the five acid carboxypeptidases present in germinating barley (L Mikola 1983 Biochim Biophys Acta 747: 241-252). The enzymes were designated wheat carboxypeptidases I to V and their best or most characteristic substrates and approximate molecular weights were: I, Z-Phe-Ala, 120,000; II, Z-Ala-Arg, 120,000; III, Z-Ala-Phe, 40,000; IV, Z-Pro-Ala, 165,000; and V, Z-Pro-Ala, 150,000. Resting grains contained carboxypeptidase II as a series of three isoenzymes and low activities of carboxypeptidases IV and V. During germination the activity of carboxypeptidase II decreased, those of carboxypeptidases IV and V increased, and high activities of carboxypeptidases I and III appeared. The flag leaves contained high activity of carboxypeptidase I and lower activities of carboxypeptidases II, IV, and V, whereas carboxypeptidase III was absent.     

Carboxypeptidases of germinating triticale grains

Presence of five carboxypeptidases was found in endosperm of germinating triticale grains, while two of them in scutellum. Changes of their activities during four days of germination suggest that carboxypeptidase II plays an important role at initial stage of germination, while carboxypeptidases I and III - at subsequent stages of the process. High activity of carboxypeptidase II both in scutellum and endosperm of dry grains accompanied by its decrease during germination, and on the other hand, the appearance of carboxypeptidases I and III activities at the 2^nd and 3^rd day of the process seems to confirm such functions of these enzymes. Experiments with GA₃ indicated that carboxypeptidase I was synthesized in scutellum, and carboxypeptidase III — in aleurone layer. Carboxypeptidases I and II cleave N-CBZ-Phe-Ala, and carboxypeptidase III — N-CBZ-Ala-Met and N-CBZ-Ala-Phe as substrates with the highest rate.     

Synthesis of salt-soluble proteins in barley. Pulse-labeling study of grain filling in liquid-cultured detached spikes

The accumulation of salt-soluble proteins in the endosperm of developing barley (Hordeum vulgare L.) grains was examined. Detached spikes of barley were cultured at different levels of nitrogen nutrition and pulse-labeled with [¹⁴C] sucrose at specific times after anthesis. Proteins were extracted from isolated endosperms and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Fluorography revealed an early, middle and late synthesis of specific proteins during grain filling. Synthesis of proteins appearing at the later stages responded to increased nitrogen nutrition. Two major components, -amylase and protein Z in particular, had a synthesis profile almost identical to that of the endosperm storage protein, hordein.Abbreviations CIE Crossed immunoelectrophoresis - SDSPAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis,cotyledons and endosperm

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), -galactosidase and -mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo--mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - EDTA ethylenediamine tetraacetic acid disodium salt - TCA trichloroacetic acid Part 2 of a series, of which the first was published in Planta 139, 1–8 (1978)     

Proteolytic activities in the developing seeds of ×Haynaldoticum sardoum

Aminopeptidase, carboxypeptidase and proteinase activities were measured in endosperms from unripe and ripe seeds of ×Haynaldoticum sardoum. Aminopeptidase and proteinase activities were high during the early maturation stages and then decreased. In contrast, carboxypeptidase activity increased with maturation. Localization studies demonstrated that aminopeptidase and carboxypeptidase activities were present in the three tissues examined (pericarp, green layer plus aleurone, and starchy endosperm). Proteinase activity against gliadin was located in the pericarp and in the green layer plus aleurone, but was absent in the starchy endosperm. The presence of proteolytic activities in the outer kernel layers might be correlated to the hydrolysis of transitory protein reserves during the senescence of the seed coat?. Aminopeptidase and carboxypeptidase activities located in the starchy endosperm could participate in the breakdown of protein reserves during the early phases of seed germination.     

Asparaginyl endopeptidase during maturation and germination of durum wheat

Asparaginyl-endopeptidase activity was detected in endosperms of maturing and germinating wheat seeds. The highest activity was found during maturation before the maximal accumulation of storage proteins. The enzyme activity then decreased in the dry seeds and increased again during germination. The increase of activity during germination required the presence of the embryo. In fact, the activity found in detached endosperms was lower than that found in attached ones. The localization at tissue level of the enzyme reveals differences between maturation and germination: the enzyme was about equally located in the aleurone layer and starchy endosperm during maturation, but solely in the aleurone layer during germination. The asparaginyl enzymes from maturing and germinating seeds had many similar properties, such as pH optimum, pH stability, thermal stability and sensitivity to thiol reagents and to thiol compounds. The results suggest that asparaginyl endopeptidases may be involved in the modification of proproteins of storage proteins during seed maturation and in the degradation of storage proteins deposited in the aleurone layer during germination.     

Occurrence of acid and neutral carboxypeptidases in germinating cereals

High neutral metallocarboxypeptidase activity (EC 3.4.17) has earlier been detected in young seedlings of rice ( Oryza sativa L.) using benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala) as substrate at pH 7. This finding was confirmed, and it was observed that the activity could be assayed with higher specificity and sensitivity by using Z-Gly-Ala or Z-Gly-Phe as substrate at pH 6.5–7. No corresponding activity was detected in seedlings of barley ( Hordeum vulgare L. cv. Himalaya), oats ( Avena sativa L.) or maize ( Zea mays L.). The seedlings of the four cereals possessed similar activities of acid carboxypeptidases (EC 3.4.16; hydrolysis of Z-Phe-Ala and Z-Ala-Phe at pH 5.2 and of Z-Ala-Arg at pH 5.7). However, in endosperms of germinating rice and maize these activities were only about 1–5% of those in barley and oats. A corresponding, although less pronounced, difference was evident between the scutella of the two pairs of cereals. The possible relationship between neutral carboxypeptidase activity and ability to grow in anaerobic conditions is discussed.     

Localization and Activity of a Carboxypeptidase in Germinating Seeds of Scots Pine, Pinus sylvestris

Extracts prepared from the endosperm of germinating seeds of Scots pine, Pinus sylvestris L., hydrolysed two typical carboxypeptidase substrates, Z-Phe-Ala and Z-Phe-Phe, with pH optima at 4.2 and 5.0. The activities were completely destroyed by diisopropylfluorophosphate. Identical heat inactivation curves and elution patterns in gel chromatography on Sephadex G-200 suggest that the two activities are due to a single enzyme. In resting seeds very low carboxypeptidase activity was present in both the endosperm and the embryo. During germination on agar gel at 20°C in the dark the activities, expressed as enzyme units per seed, increased in the seedling and particularly in the endosperm up to the stage when the reserves of the endosperm were completely depleted. The time of rapid increase of activity in the endosperm did not coincide with the onset of storage protein mobilization. On the contrary, the major part of the increase occurred after the bulk of endosperm nitrogen had already been transferred to the seedling. The results suggest that the carboxypeptidase does not play a major role in the mobilization of storage proteins in germinating pine seeds. On the other hand, it probably functions in the proteolytic reactions associated with the senescence of the reserve-depleted endosperm.     

Increased hexokinase levels in endosperm of mutant maize

Hexokinase activity was measured in endosperms of shrunken-2 (sh₂) and starchy maize. Initial increases in hexokinase were observed for developing endosperms of both genotypes, and the enzyme declined in both as the seeds matured. A higher level of hexokinase was observed in developing sh₂ than in starchy endosperm. This difference persisted throughout maturation and occurred also in germinating seeds. Soluble hexokinase activity per endosperm continued to increase in sh₂ for about 8 days (22–30 days after pollination) after the enzyme in starchy endosperm had attained maximum activity and begun to decline. Hexokinase was predominantly soluble in both genotypes so the differences observed are not due to altered distribution of enzyme between particulate and soluble fractions.     

Amino Acid and Peptide uptake in the scutella of germinating grains of barley, wheat, rice, and maize

Scutella separated from germinating grains of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) took up the four amino acids and the three peptides tested from incubation media. The uptake of amino acids by wheat scutella was similar to that of barley scutella and was via at least four uptake systems: two nonspecific amino acid uptake systems, one system specific for proline, and another system specific for basic amino acids. The scutellum of rice apparently has two nonspecific systems and a system specific for the basic amino acids, but the proline-specific system is lacking. The scutellum of maize seems to have the same systems as the scutellum of rice, but one (or both) of the nonspecific systems differs from that of the other species studied in taking up arginine only slowly. No great differences were observed in the uptake of peptides in the four species studied. The rates of uptake of different amino acids and peptides were of the same order of magnitude in the four cereals. The fact that carboxypeptidase activities in the endosperms of wheat and barley are 20-to 100-fold higher than those in rice and maize, does thus not seem to be reflected in the uptake properties of the scutella.     

Tissue-specific localization of aspartic proteinase in developing and germinating barley grains

Resting seeds of several plant species, including barley grains, have been reported to contain aspartic proteinase (EC 3.4.23) activity. Here, the expression of the Hordeum vulgare L. aspartic proteinase (HvAP) was studied in developing and germinating grains by activity measurements as well as by immunocytochemical and in-situ hybridization techniques. Southern blotting suggests the presence of one to two HvAP-encoding genes in the barley genome, while Northern analysis reveals a single 2.1-kb mRNA in grains and vegetative tissues. Western blotting with antibodies to HvAP shows the same subunit structure in different grain parts. In developing grains, HvAP is produced in the embryo, aleurone layer, testa and pericarp, but in the starchy endosperm HvAP is present only in the crushed and depleted area adjacent to the scutellum. During seed maturation, HvAP-encoding mRNA remains in the aleurone layer and in the embryo, but the enzyme disappears from the aleurone cells. The enzyme, however, remains in the degenerating tissues of the testa and pericarp as well as in resting embryo and scutellum. During the first three days of germination, the enzyme reappears in the aleurone layer cells but is not secreted into the starchy endosperm. The HvAP is also expressed in the flowers, stem, leaves, and roots of barley. The wide localization of HvAP in diverse tissues suggests that it may have several functions appropriate to the needs of different tissues.Abbreviations DAA days after anthesis - DTT dithiothreitol - HvAP Hordeum vulgare aspartic proteinase Both authors have contributed equally to this workWe thank Mart Saarma, Pia Runeberg-Roos, Alan Schulman and Yrjö Helariutta for helpful discussions during the study, Tiina Arna and Sari Makkonen for their help in proteinase activity experiments as well as Jaana Korhonen (Department of Pathology, University of Helsinki), Salla Marttila and Ilkka Porali (Department of Biology, University of Jyväskylä, Jyväskylä, Finland) for their advice on microscopical techniques. We also thank Liisa Pyhälä and Leena Liesirova for the production of the antibodies to HvAP at the National Public Health Institute, Helsinki. This study was supported by grants from the Ministry of Agriculture and Forestry and the Academy of Finland.     

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     

Protein Body Degradation in the Starchy Endosperm of Germinating Sorghum

Taylor, J. R. N., Novellie, L. and Liebenberg, N. v. d. W. 1985.Protein body degradation in the starchy endosperm of germinatingsorghum.—J. exp. Bot. 36: 1287–1295. Transmission electron micrographs of starchy endosperms of germinatingsorghum indicate that the protein bodies are degraded predominantlyby progressive hydrolysis of prolamin from their surface. Theappearance of holes within partially degraded protein bodiesindicates that some internal hydrolysis also takes place. Chemicalanalyses of protein bodies isolated at different stages duringgermination showed that their amino acid composition and electrophoreticpattern remained relatively unchanged during hydrolysis. Theend result of protein body degradation was that the organellescompletely disappeared leaving empty starchy endosperm cells.The protein bodies did not swell prior to or during degradation.This mode of protein body degradation differs from that in germinatingdicotyledonous seeds and in the aleurone layer and embryo ofcereal seeds but was identical to the mode of prolamin proteinbody degradation in the starchy endosperm of germinating riceseeds. Key words: Sorghum bicolor, protein body degradation, prolamin     

Agrobacterium tumefaciens-Mediated Transformation of Maize Endosperm as a Tool to Study Endosperm Cell Biology

Developing maize (Zea mays) endosperms can be excised from the maternal tissues and undergo tissue/cell-type differentiation under in vitro conditions. We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors. We show that both aleurone and starchy endosperm cells can be successfully transformed using a short cocultivation with A. tumefaciens cells. The highest transformation rates were obtained with the A. tumefaciens EHA101 strain and the pTF101.1 binary vector. The percentage of aleurone cells transformed following this method varied between 10% and 22% whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells. Cultured endosperms undergo normal cell type (aleurone and starchy endosperm) differentiation and storage protein accumulation, making them suitable for cell biology and biochemical studies. In addition, transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.The endosperm is a unique plant tissue that arises from a second fertilization event between a male gamete and the central cell. Its main function is to provide nutrients to the embryo either during seed development or during germination. In cereals, the endosperm consists of three main cell types: the starchy endosperm cells, which constitute the bulk of the endosperm and accumulate large quantities of storage proteins and starch; the epidermal aleurone cells; and the transfer cells, which are in contact with the maternal vascular tissues (Olsen, 2004). The cereal endosperm is important as a model system to study plant development, cell differentiation, programmed cell death, and synthesis, trafficking, and accumulation of storage compounds. In addition, it is a major source of carbohydrate and proteins for human and animal nutrition.In spite of its importance, cell biology studies on the cereal endosperm using modern imaging approaches such as expression of fluorescent subcellular markers are very scarce because: (1) the endosperm is deeply immersed in maternal tissues and therefore, not readily available for imaging analysis and (2) the long time required for transformation and regeneration of stable transgenic plants. Although several approaches for culturing maize (Zea mays) endosperm in vitro have been reported in the past years (Shimamoto et al., 1983), only recently a novel method developed by Odd-Arne Olsen and colleagues (Gruis et al., 2006) has proven to be successful in retaining endosperm tissue and cell type identity in in vitro conditions. Cultures derived from transgenic maize lines in which endosperm cell types are identified by the activity of specific promoters have shown that aleurone and starchy endosperm cell identity continues to be established in vitro (Gruis et al., 2006).Although Agrobacterium tumefaciens is not a natural pathogen of most monocots (Cleene, 1985; Binns and Thomashow, 1988), it has been successfully used to transform many cereals, including maize, wheat (Triticum aestivum), Sorghum, barley (Hordeum vulgare), and rice (Oryza sativa; Grimsley et al., 1989; Gould et al., 1991; Chan et al., 1993; Ishida et al., 1996, 2007; Gurel et al., 2009; Harwood et al., 2009; Hensel et al., 2009). In the case of maize, stable transgenic plants can be obtained by A. tumefaciens-mediated transformation using either super-binary or standard-binary vectors (Frame et al., 2002; Mohanty et al., 2009a, 2009b). However, transformation of isolated maize endosperms have been only possible using transient transformation approaches such as biolistic methods (Torrent et al., 1997; Gruis et al., 2006) and protoplast transfection (Gallie and Young, 1994). Unfortunately, these two methods are not always ideal for cell biology studies. On one hand, biolistic methods often result in high-copy number transgenic events and on the other, protoplasts are usually highly stressed cells not suitable for detailed protein localization studies. A. tumefaciens-mediated transformation methods circumvent these disadvantages by resulting in a low-copy number of transgenes in intact tissues.We have developed a method to transform in vitro-grown endosperms using a brief incubation time with A. tumefaciens cells carrying standard binary vectors. We present here a detailed explanation of the method and quantitative information on the transformation efficiency using different A. tumefaciens strains, culture density, and incubation time. We also provide evidence that the in vitro-differentiated aleurone and starchy endosperm cells are comparable to the corresponding cell types differentiated in planta and therefore, suitable for cell biology studies. In addition, we show that transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling imaging techniques.