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 By 20 days the four bands had been replaced by seven bands of medium mobility.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.     

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.     

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.

     

Tannins as gibberellin antagonists in the synthesis of alpha-amylase and Acid phosphatase by barley seeds

The tannins chebulinic acid or tara tannin were added to an incubation system in which GA₃ induces enzyme synthesis in endosperm half seeds of barley (Hordeum vulgare L.). The activity of amylase and acid phosphatase in the incubation medium was reduced compared to the activity in the medium after incubation with GA₃ alone. When embryo half seeds of barley were incubated with chebulinic acid or tara tannin in the absence of added GA₃, the enzyme activity of the incubation medium was also reduced. The activity of preformed enzymes obtained from endosperm half seeds previously induced with GA₃ was not reduced by the addition of tannin. Comparisons were made of the amount of enzyme activity from breis of aleurone layers incubated with GA₃ in the presence and absence of tannins. The amounts of activity were relatively small and approximately equal in both cases, indicating that secretion from the aleurone was not blocked by the tannins. The reduction of enzyme activity caused by tannins in both endosperm and embryo half seeds could be completely reversed by the addition of GA₃.     

Programmed cell death in cereal aleurone

Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis.     

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.     

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.     

The Distribution of Acidic and Neutral Peptidases in Barley and Wheat Kernels

The activity of barley and wheat peptidases which hydrolyze alpha-N-benzoyl-dl-arginine-p-nitronnilide (BAPA) and alpha-N-benzoyl-l-arginine ethyl ester (BAEE) has been measured in proximal and distal portions of ungerminated grain and in these tissues during 6 and 7 day incubations. The proximal portion of ungerminated barleys contained the major part of both the acidic (BAPA-ase and acidic BAEE-ase) and neutral (neutral BAEE-ase) peptidases. In ungcrminated wheat these acidic and neutral peptidases were nearly evenly distributed between the proximal and dislal portions. Commercial wheat embryo was very high in acidic peptidase but contained no neutral peptidase. During the germination of both wheat and barleys, acidic and neutral peptidase activity in the seedlings increased with time. No such consistent increase was observed for aleurone and starchy endosperm tissue for any of these grains. Aleurone and starchy endosperm tissue incubated in the absence of the proximal portion of the kernel showed reduced peptidase activities.     

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.     

Amylases from aleurone layers and starchy endosperm of barley seeds

Amylases from incubated aleurone layers or from starchy endosperm of barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated using acrylamide gel electrophoresis and analytical gel filtration with Sephadex G-200. Electrophoresis of amylase from aleurone layers yields seven visually distinct isozymes with an estimated molecular weight of 43,000. Because each isozyme hydrolyzes β-limit dextrin azure and incorporates calcium-45, they are α-amylases. On Sephadex G-200, amylase from the aleurone layers is separated into seven fractions ranging in estimated molecular weights from 45,000 to 3,000. Little or no activity is observed when six fractions are subjected to electrophoresis. Electrophoresis of only the fraction with the estimated molecular weight of 45,000 gave the seven isozymes. The amylases are heat labile and cannot be stabilized by the presence of substrate or by the protease inhibitor, phenylmethylsulfonylfluoride. Electrophoresis of amylase from the starchy endosperm yields nine β-amylases. Four of these β-amylases are isozymes with an estimated molecular weight of 43,000. The other five forms of β-amylase represent molecular aggregates of the four basic β-amylase monomers. A dimer, a tetramer, and an octamer of β-amylase can be identified with estimated molecular weights of about 86,000, 180,000 and 400,000, respectively. These estimated molecular weights were confirmed on Sephadex G-200. There are five additional fractions of β-amylase with estimated molecular weights ranging from 30,000 to 4,000. These fractions are not observed electrophoretically.     

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     

Kinetics of the Acidification Capacity of Aleurone Layer and Its Effect upon Solubilization of Reserve Substances from Starchy Endosperm of Wheat

The capacity of the isolated barley aleurone layer for endosperm acidification has been demonstrated (J Mikola, M Virtanen 1980 Plant Physiol 66: S-142). The kinetics of this acidification by isolated wheat aleurone layer and its effect on starchy endosperm solubilization are reported.     

Identification of thioredoxin target disulfides in proteins released from barley aleurone layers

Thioredoxins are ubiquitous disulfide reductases involved in a wide range of cellular processes including DNA synthesis, oxidative stress response and apoptosis. In cereal seeds thioredoxins are proposed to facilitate the germination process by reducing disulfide bonds in storage proteins and other targets in the starchy endosperm. Here we have applied a thiol-specific labeling approach to identify specific disulfide targets of barley thioredoxin in proteins released from barley aleurone layers incubated in buffer containing gibberellic acid.     

Modulation of Calmodulin mRNA and Protein Levels in Barley Aleurone

Changes in calmodulin (CaM) mRNA and protein were investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) incubated in the presence and absence of calcium, gibberellic acid (GA3), and abscisic acid (ABA). CaM mRNA levels increased rapidly and transiently following incubation of aleurone layers in H2O, CaCl2, or GA3. The increase in CaM mRNA was prevented by ABA. This increase in CaM mRNA was brought about by physical stimulation during removal of the starchy endosperm from the aleurone layer. CaM protein levels did not increase in response to physical stimulation. Only incubation in GA3 plus CaCl2 brought about a rapid increase in CaM protein levels in the aleurone cell. ABA reduced the level of CaM protein below that found at the beginning of the incubation period. The rise in CaM protein preceded increases in the synthesis and secretion of [alpha]-amylase. Immunocytochemistry with monoclonal antibodies to carrot and mung bean CaM was used to localize CaM in aleurone protoplasts. Monoclonal antibodies to tubulin and polyclonal antibodies to tonoplast intrinsic protein and malate synthase were used as controls. CaM was localized to the nucleus, the vacuolar membrane, and the cytosol, but was not associated with microtubules.     

The NADPH-dependent thioredoxin reductase/thioredoxin system in germinating barley seeds: gene expression, protein profiles, and interactions between isoforms of thioredoxin h and thioredoxin reductase

The NADPH-dependent thioredoxin reductase (NTR)/thioredoxin (Trx) system catalyzes disulfide bond reduction in the cytoplasm and mitochondrion. Trx h is suggested to play an important role in seed development, germination, and seedling growth. Plants have multiple isoforms of Trx h and NTR; however, little is known about the roles of the individual isoforms. Trx h isoforms from barley (Hordeum vulgare) seeds (HvTrxh1 and HvTrxh2) were characterized previously. In this study, two NTR isoforms (HvNTR1 and HvNTR2) were identified, enabling comparison of gene expression, protein appearance, and interaction between individual NTR and Trx h isoforms in barley embryo and aleurone layers. Although mRNA encoding both Trx h isoforms is present in embryo and aleurone layers, the corresponding proteins differed in spatiotemporal appearance. HvNTR2, but not HvNTR1, gene expression seems to be regulated by gibberellic acid. Recombinant HvNTR1 and HvNTR2 exhibited virtually the same affinity toward HvTrxh1 and HvTrxh2, whereas HvNTR2 has slightly higher catalytic activity than HvNTR1 with both Trx h isoforms, and HvNTR1 has slightly higher catalytic activity toward HvTrxh1 than HvTrxh2. Notably, both NTRs reduced Trx h at the acidic conditions residing in the starchy endosperm during germination. Interspecies reactions between the barley proteins and Escherichia coli Trx or Arabidopsis thaliana NTR, respectively, occurred with 20- to 90-fold weaker affinity. This first investigation of regulation and interactions between members of the NTR/Trx system in barley seed tissues suggests that different isoforms are differentially regulated but may have overlapping roles, with HvNTR2 and HvTrxh1 being the predominant isoforms in the aleurone layer.     

Location of a Protease and its Inhibitor in the Barley Kernel

Tissues of barley caryopsis and seedling were examined for the protease, BAPAase, and an inhibitor. The enzyme was present in extracts of alevn-one but was absent from aleurone incubation media and extracts of: embryo with scutellum; seedling with scutellum and rootlets, and endosperm that was free of aleurone tissue. The enzyme was present in non-incubated aleurone and did not increase significantly during incubation under conditions where alpha-amylase increased in the medium and tissue. Addition of gibberellie acid produced no detectable increase in BAPAase. Extracts of endosperm had weak BAPAase-inhibitory activity; embryo or seedling extracts produced strong inhibition. The inhibitor present in these extracts was dialyzable.     

Effects of the barley mutants Risø 1508 and 527 high lysine genes on the cellular development of the endosperm

The dry grain weight of the Risø barley ( Hordeum vulgare L. var. disticum ) high lysine mutants 1508 and 527 at maturity was 32 and 37% lower, respectively, than the grains of the cultivar Bomi. Dry grain weight of the double mutant 527/1508 was reduced by 57%. Total number of endosperm nuclei from cv. Bomi, mutants 1508, 527 and 527/1508 at 24 days after anthesis was 173 000,156 000,121 000 and 111 000, respectively. Transverse mid-grain sections from mutants 1508 and 527 contained fewer aleurone cells and approximately the same number of starchy endosperm cells as cv. Bomi. The cellular organization of the endosperm of the double mutant deviated substantially from the normal. Cell volume in the central starchy endosperm of cv. Bomi, mutants 1508 and 527 at 33 days after anthesis averaged 390 000, 270 000 and 180 000 um5, respectively. Cell volume in the double mutant was smaller than in 527, but could not be accurately estimated due to the irregular shape of the cells. The mean section area of individual large starch granules in the central endosperm of mutants 1508, 527 and 527/1508 at 33 days after anthesis was 30, 48 and 72% smaller, respectively, than those of cv. Bomi. The average aleurone cell volume in cv. Bomi at 33 days after anthesis was 6 200 μm³.     

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%.