Purification and partial characteristic of a major gliadin-degrading cysteine endopeptidase from germinating triticale seeds

The endopeptidase of the highest electrophoretic mobility was the main endopeptidase hydrolyzing gliadin in the endosperm of germinated triticale (X Triticosecale Wittm.) grains after three days of imbibition. Activity of this endopeptidase, named EP8 starts to be detectable after two days of imbibition. The appearance of its activity in the endosperm on a second day of imbibition may suggest that EP8 is synthesized in aleurone during germination and/or secreted into the starchy endosperm as an inactive polypeptide during grains development and then activated. EP8 was isolated from the endosperm of germinating triticale seeds and purified 257-fold using ammonium sulphate, ion exchange chromatography on DEAE Sepharose CL-6B and gel filtration on Sephadex G-100. The enzyme was totally inhibited by E-64—class-specific cysteine proteinases inhibitor and activated by thiol compounds. Molecular weight estimated by SDS-PAGE was 39.5 kDa. The optimum pH for the hydrolysis of gliadin was 4.2 and for hemoglobin 5.2. High activity of EP8 against wheat gliadin in vitro suggests that this cysteine endopeptidase plays a major role in the mobilization of storage proteins in the endosperm of germinating triticale grains.     

Expression and Localization of Phosphoenolpyruvate Carboxylase in Developing and Germinating Wheat Grains

Phosphoenolpyruvate carboxylase (PEPC) activity and corresponding mRNA levels were investigated in developing and germinating wheat (Triticum aestivum) grains. During grain development PEPC activity increased to reach a maximum 15 d postanthesis. Western-blot experiments detected two main PEPC polypeptides with apparent molecular masses of 108 and 103 kD. The most abundant 103-kD PEPC subunit remained almost constant throughout the process of grain development and in the scutellum and aleurone layer of germinating grains. The less-abundant 108-kD polypeptide progressively disappeared during the second half of grain development and was newly synthesized in the scutellum and aleurone layer of germinating grains. PEPC mRNA was detected throughout the process of grain development; however, in germinating grains PEPC mRNA accumulated transiently in the scutellum and aleurone layer, showing a sharp maximum 24 h after imbibition. Immunolocalization studies revealed the presence of the enzyme in tissues with a high metabolic activity, as well as in the vascular tissue of the crease area of developing grains. A clear increase in PEPC was observed in the scutellar epithelium of grains 24 h after imbibition. The data suggest that the transiently formed PEPC mRNA in the scutellar epithelium encodes the 108-kD PEPC subunit.     

Peroxidase activity develops in the micropylar endosperm of tomato seeds prior to radicle protrusion

Peroxidase activity developed specifically in the micropylar region of the endosperm of imbibed tomato seeds prior to radicle emergence. The activity was first detected approximately 24 h after the start of imibibition (6 h before radicle emergence) and increased markedly thereafter. In the lateral portion of the endosperm, peroxidase activity was undetectable for the first 2 d after the start of imbibition. Although the activity in the lateral endosperm became detectable 3 d after imbibition, the extent of the development of the activity was slight. The localization of peroxidase activity in the micropylar endosperm 2 d after the start of imbibition was confirmed by tissue printing analyses. When the endosperm tissues were wounded, there was an enhancement of the enzyme activity at the wounded region. H2O2 was formed at the expense of NADH only in the presence of Mn2+ and dinitrophenol by the extract from the micropylar endosperm in which peroxidase activity was present. The presence of H2O2 in the micropylar portion of the endosperm was shown histochemically. The possible functions of the peroxidases that develop in the endosperm of tomato seeds are discussed.     

Studies on Endosperm Development and Deposition of Storage Reserves in Coix lacryma-jobi

The transition from free nuclear to cellular endosperm of Coix lacryma-jobi was eompleted 2 days after pollination. By 3 days after pollination the central cell was filled with endosperm cells. At first all cells of endosperm underwent division, later cell division was limited mainly in the peripheral region. 10 days after pollination the epidermal layer ceased its periclinal division and became the aleurone layer. Cell division persisted in the subepidermal 'cambium-like layers until the caryopsis nearly matured. Ceils of the inner region of endosperm became enlarged. Several layers of transfer cells were formed at the basal part of the endosperm. Starch grains appeared in endosperm cells on the 9th day after pollination. 10 days after pollination, lipid bodies occurred in the aleurone layer and the underlying layers. 13 and 15 days after pollination, the small vacuoles of aleurone cells contained protein and 20 days after pollenation they became aleurone grains. By 15 days after pollination pro tein bodies were formed in starch endosperm. Storage reserve deposition continued until the grain ripened. A correlation between endosperm and emoryo development was also observed.     

薏苡胚乳发育及营养物质积累的研究

薏苡 ( Coix lacryma- jobi)授粉后 2 d,游离核胚乳已转变为细胞胚乳。授粉后 3d,中央细胞被胚乳细胞充满。起初 ,全部胚乳细胞均进行分裂 ,一定时期后 ,细胞分裂主要发生在胚乳周边区。授粉后 1 0 d,表皮停止平周分裂变为糊粉层 ,内方的数层形成层状细胞行平周分裂直到颖果接近成熟。胚乳内部生长则依赖于细胞体积扩大。胚乳基部 (颖果基部的胚乳 )形成了数层传递细胞。授粉后 9d,淀粉积累。授粉后 1 0 d,糊粉层及其内方数层细胞产生了脂体 ,后者的脂体以后又消失。授粉后 1 3、1 5 d,糊粉层细胞的液泡积累蛋白质。授粉后 2 0 d,液泡变为糊粉粒。授粉后 1 5 d淀粉胚乳细胞产生蛋白质体 ,营养物质积累持续到颖果成熟。还观察了胚和胚乳发育的对应关系。     

Hormonal Regulation of Organic and Phosphoric Acid Release by Barley Aleurone Layers and Scutella

The release of acid from the aleurone layer and scutellum of barley (Hordeum vulgare L. cv Himalaya) was investigated. Aleurone layers isolated from mature barley grains acidify the external medium by releasing organic and phosphoric acids. Gibberellic acid and abscisic acid stimulate acid release 2-fold over control tissue incubated in 10 mM CACl2. Gibberellic acid causes medium acidification by stimulating the release of phosphoric and citric acids, whereas abscisic acid stimulates the release of malic acid. The accumulation of these acids in the incubation medium buffers the medium against changes in pH, particularly between pH 4 and 5. The amounts of amino acids that accumulate in the medium are low (2-12 nmol/layer) compared to other organic and phosphoric acids (100-500 nmol/layer). The scutellum does not play a major role in medium acidification but participates in the uptake of organic acids. The organic acid composition of the starchy endosperm changes after 3 d of imbibition; malic, succinic, and lactic acids decrease, whereas citric and phosphoric acids remain unchanged or increase. These results indicate that during postgerminative growth, the acidity of the starchy endosperm is maintained by acid production by the aleurone layer.     

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.

     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

大麦胚和胚乳发育的相关性及贮藏营养物质的积累

大麦(Hordeum vulgare L.)开花后1d,见合子及退化助细胞,游离核胚乳尚未形成;开花后2～3d,胚为5及10个细胞,胚乳为游离核期;开花后4及5、6d,胚为梨形及长梨形,胚乳达细胞化期;开花后8d,胚为胚芽鞘期,糊粉层原始细胞产生;开花后10d,胚具1叶,糊粉层1～2层;开花后13d胚为2叶胚,亚糊粉层发生;开花后17d,3叶胚形成,糊粉层多为3层并停止分裂,菱柱形及不规则胚乳细胞分化;开花后21～29d,胚为4叶胚,胚乳进一步分化;开花后33d,胚为5叶成熟胚,胚乳亦成熟。淀粉、蛋白质在胚中积累始于开花后13d。在盾片中由基向顶发生,在胚芽鞘及叶原基中,首先在顶端出现。成熟盾片顶端的淀粉消失。开花后6d,胚乳开始积累淀粉;开花后10d,糊粉层及胚乳细胞积累蛋白质。开花17d后胚乳的蛋白质体多聚集,29d后蛋白质体显著减少。开花后17d,在盾片及糊粉层细胞中检测到油脂。果长或果长与稃片长之比和盾片长可作为不同发育期胚和胚乳的形态指标。     

Development of aleurone and sub-aleurone layers in maize

Electron-microscope studies indicate that the aleurone tissue of maize (Zea mays L.) starts developing approximately 10–15 days after pollination in stocks that take ca. 40 days for the aleurone to mature completely. Development commences when specialized endosperm cells adjacent to the maternal nucellar layer start to differentiate. Differentiation is characterized by the formation of aleurone protein bodies and spherosomes. The protein bodies of the aleurone layer have a vacuolar origin whereas the protein bodies of the immediate underlying endosperm cells appear to develop from protrusions of the rough endoplasmic reticulum. Thus, two morphologically and developmentally distinct types of protein bodies are present in these adjacent tissues. The spherosomes of the aleurone layer form early in the development of this tissue and increase in number as the tissue matures. During the final stages of maturation, these spherosomes become closely apposed to the aleurone grains and the plasma membrane. No further changes are apparent in the structure of the aleurone cells after 40 days from pollination when the caryopsis begins to desiccate.     

Development of Endopeptidase Activities in Maize (Zea mays L.) Endosperms

An activity stain was used after native polyacrylamide gel electrophoresis, and at least 17 different endopeptidase activities were detected in maize (Zea mays L.) endosperm extracts prepared during the first 6 d after imbibition. The enzymes detected were classified into four groups based on their time of appearance and on their mobility in polyacrylamide gels. The first group, which included two enzymes present in dry endosperms, disappeared soon after imbibition. The second group, comprising five activity bands, appeared during the first 2 to 3 d after imbibition and then disappeared. The third set of enzymes increased continuously throughout the experimental period. The fourth group appeared after d 3 and remained at a constant level after that time. The endopeptidase activities were characterized by the effect of specific inhibitors on their activities. The two enzymes of the first group are metalloendopeptidases based on their sensitivity to ethylenediaminetetracetate (EDTA). Enzymes of the second, third, and fourth groups are sulfhydryl-endopeptidases as judged by their sensitivity to antipain, chymostatin, leupeptin, and E-64 and by their requirement for 2-mercaptoethanol. Pepstatin, phenylmethylsulfonyl fluoride, or EDTA had no effect on these enzymes. Many of the second, third, and fourth group enzymes cleaved [alpha]-zein-rich proteins as well as such easily obtained proteins as gelatin (used in our standard assay) and hemoglobin. The second group had a high affinity for [gamma]-zein, whereas none of the bands in the fourth group of enzymes cleaved this type of zein. The two metalloenzymes of the first group cleaved neither [alpha]- nor [gamma]-zeins.     

Changes in Endogenous Cytokinin Levels in Kernels of Zea mays L. during Imbibition and Germination

In order to assess the potential role of endogenous cytokininsin the germination of Zea mays L. caryopses, cytokinin activitywas determined in mature kernels and in kernels 1, 2, and 3d after imbibition. Cytokinin activity was also recorded inthe endosperm and embryo tissue of mature kernels and of kernels3 d after imbibition to establish the localization and changesin the levels of endogenous cytokinins during imbibition andearly germination. Using chromatographic, chemical, and enzymic techniques, compoundswith chromatographic properties resembling those of zeatin,zeatin riboside, zeatin glucoside, zeatin riboside glucoside,and their respective dihydro derivatives were detected in extractsfrom mature maize kernels. From analyses of the endosperm andembryo tissue at the two stages, it appears that the cytokininglucosides present in the endosperm are transported to the embryonicaxis for utilization in growth and development of the seedling.This concept is supported by the fact that the levels of ß-glucosidaseactivity detected were highest in the embryos, particularlyin the radicles, 3 d after imbibition.     

A novel endosperm transfer cell-containing region-specific gene and its promoter in rice

The endosperm of cereal grains is an important resource for both food and feed. It contains three major types of tissue: starchy endosperm, the aleurone layer, and transfer cells. To improve grain quality and quantity using molecular methods, control of transgene expression directed by distinct temporal and spatial promoter activity is necessary. To identify aleurone layer-specific and/or transfer cell-specific promoters in rice, microarray analyses were performed, comparing the aleurone layer containing transfer cells and the other reproductive and vegetative tissues. After confirmation by RT-PCR analysis, we identified two putative aleurone layer and/or transfer cell-specific genes, AL1 and AL2. The promoter regions of these genes and β-glucuronidase (GUS) fusion constructs were stably transformed into rice. The GUS expression patterns indicated that the AL1 promoter was active exclusively in the dorsal aleurone layer adjacent to the main vascular bundle. In rice, transfer cells are differentiated in this region. Therefore, the promoter of the AL1 gene exhibits transfer cell-containing region-specific activity. The AL1 gene encodes a putative anthranilate N-hydroxycinnamoyl/benzoyltransferase. The promoter of this gene will be useful for enhancing uptake of nutrients from the mother cells and protecting filial seeds from pathogen attack.     

Differences in the activity and distribution of peroxidases from three different portions of germinating Brassica oleracea seeds

Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.     

Amino acids and quaternary nitrogen compounds in the germinating wheat grain

The tissues of the quiescent wheat grain contained free amino acids and quaternary nitrogen compounds. During germination the amino acid levels increased several fold. In the aleurone tissue and starchy endosperm glutamine was the predominant amino acid. Asparagine was predominant in the seedling tissues. Choline and glycine betaine were the principal quaternary nitrogen compounds present. The aleurone tissue and the embryo/seedling contained large quantities of glycine betaine. The increase in free amino acid levels in the aleurone tissue during the first 2 days of germination occurred independently of the embryo. After the second day, the further increase in levels was dependent upon the presence of the embryo and of gibberellic acid (GA). Estimation of the individual amino acids and quaternary nitrogen compounds released from incubating aleurone layers into aqueous media revealed a selective release of some compounds and retention of others. The process was regulated by GA. Possible mechanisms for the release of amino acid and its control by GA are discussed.     

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.     

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     

The thick aleurone1 mutant defines a negative regulation of maize aleurone cell fate that functions downstream of defective kernel1

The maize (Zea mays) aleurone layer occupies the single outermost layer of the endosperm. The defective kernel1 (dek1) gene is a central regulator required for aleurone cell fate specification. dek1 mutants have pleiotropic phenotypes including lack of aleurone cells, aborted embryos, carotenoid deficiency, and a soft, floury endosperm deficient in zeins. Here we describe the thick aleurone1 (thk1) mutant that defines a novel negative function in the regulation of aleurone differentiation. Mutants possess multiple layers of aleurone cells as well as aborted embryos. Clonal sectors of thk1 mutant tissue in otherwise normal endosperm showed localized expression of the phenotype with sharp boundaries, indicating a localized cellular function for the gene. Sectors in leaves showed expanded epidermal cell morphology but the mutant epidermis generally remained in a single cell layer. Double mutant analysis indicated that the thk1 mutant is epistatic to dek1 for several aspects of the pleiotropic dek1 phenotype. dek1 mutant endosperm that was mosaic for thk1 mutant sectors showed localized patches of multilayered aleurone. Localized sectors were surrounded by halos of carotenoid pigments and double mutant kernels had restored zein profiles. In sum, loss of thk1 function restored the ability of dek1 mutant endosperm to accumulate carotenoids and zeins and to differentiate aleurone. Therefore the thk1 mutation defines a negative regulator that functions downstream of dek1 in the signaling system that controls aleurone specification and other aspects of endosperm development. The thk1 mutation was found to be caused by a deletion of approximately 2 megabases.