Properties of glutelin from mature and developing rice grain

Aminograms and SDS-polyacrylamide electrophoresis of milled rice glutelin of 12 Oryza sativa samples showed similar composition and ratio of 1 : 1 : 1 for subunits with MW 38 000:25 000: 16 000, indicating little possibility of finding variants of rice glutelins. Fractionation of S-cyanoethyl glutelin of 3 rices on polyacrylamide-agarose gels gave MW subunits differing in amino acid analysis of which the subunits with MW > 38 000 had the highest lysine content. Of the solubility fractions of endosperm glutelin, the fraction extracted by 0.5 M NaCl-0.6 % β-mercapto-ethanol-0.5% SDS was closest to glutelin in properties. In the developing grain of two varieties, appearance of protein bodies and rapid synthesis of glutelin from 7 days after flowering onward coincided with a drop in lysine content and appearance of MW 38 000 and 25 000 of crude glutelin. The MW 38 000 subunit is thus unique to endo-sperm glutelin.     

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.

     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

A structural study of germination in celery (Apium graveolens L.) seed with emphasis on endosperm breakdown

Germination of celery seed occurred after 6 d of imbibition in light. During this time the embryo enlarged at the expense of the adjacent endosperm cells and at the time of germination was 2–3 times as long as in the dry seed. Breakdown of the endosperm cells near the root cap preceeded radicle emergence. None of these changes occurred in darkness.Endosperm digestion began adjacent to the embryo and spread radially. In degrading cells, the aleurone grains often became larger and fewer in number. The cell walls were modified and appeared to undergo partial degradation. Ultimately the cells seemed to lose their contents. In cells adjacent to the root cap, similar changes occurred except there was a transient appearance of starch grains. Radial progression of endosperm breakdown also occurred in isolated endosperm treated with gibberellin A₄₊₇.The results indicate that (1) the stimulus for breakdown of celery endosperm emanates from the embryo in response to light; (2) the stimulus may be a gibberellin because changes in endosperm cells and the sequence of endosperm digestion during germination resemble the responses of isolated endosperm to gibberellin; and (3) the radial progression of endosperm breakdown during germination may be the result of a sequential response of cells to a uniformly applied stimulus rather than the result of gradual embryo expansion.     

Properties of prolamin in mature and developing rice grain

A procedure was developed for preparing lipid- and phenol-free prolamin directly from IR480-5-9 milled rice (Oryza sativa L.).The preparation consisted mainly of one protein band on analytical and SDS-polyacrylamide disc gel electrophoresis with subunit MW of 17000 and a minor fraction with subunit MW 23000. The prolamin eluted as a single peak on SDS-Sephadex G-75 gel filtration and on DEAE-cellulose column chromatography. Prolamin was poor in lysine, histidine, cystine, and methionine but rich in glutamic acid, tyrosine and proline. In dehulled developing grain of two different rices, changes in the aminogram of the prolamin fraction coincided with the start of endosperm protein body synthesis and the appearance from 7 days after flowering of a second prolamin subunit with MW 23 000.     

Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature

Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25°C, but not at 15°C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15°C and 25°C.

The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25°C than at 15°C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15°C and 25°C after the puncture force was reduced to between 0.3 and 0.4 newtons.

Application of gibberellic acid₄₊₇ (100 microliters per liter) resulted in earlier germination at 15°C and 25°C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O₂ concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid₄₊₇, but only at 25°C. At 15°C, high O₂ concentrations slowed germination and endosperm strength decline.

     

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.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

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.     

Accumulation of free ricinoleic Acid in germinating castor bean endosperm

Lipids from the endosperm of germinating castor bean (Ricinus communis var. Hale) were separated by thin layer chromatography and quantitated by gas chromatography. During the later stages of lipid breakdown (4-6 days germination at 30 C), several lipid classes were found in addition to the storage triglycerides, which are triricinoleins for the most part. One was identified as free ricinoleic acid, the proportion of which increased as germination progressed. After 6 days germination, ricinoleic acid comprised more than 30% of the total lipid. The appearance of this fatty acid implies that lipase activity (lipolysis) is not strictly coordinated with beta oxidation in this tissue.     

Phosphorylases I and II of Maize Endosperm

Two phosphorylases have been found in the endosperm of Zea mays. Phosphorylase I is found through all stages of endosperm development and seed germination investigated. The other enzyme, phosphorylase II appears only at the stage of rapid starch biosynthesis and is not found during germination. At 22 days after pollination, the activity of phosphorylase II is 10 times that of phosphorylase I. These 2 phosphorylases are separable by column chromatography and behave differently in several respects.     

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.     

Structure and biochemistry of endosperm breakdown in date palm (Phoenix dactylifera L.) seeds

Summary The zone of endosperm breakdown in the germinated date seed (Phoenix dactylifera L.) is a narrow area immediately adjacent to the surface of the enlarging cotyledon, or haustorium. The zone width is correlated with the amount of cell division in the adjacent region of the haustorium. The sequence of endosperm breakdown is: 1. protein bodies vacuolate, 2. storage cell walls become electron-transparent immediately adjacent to the protoplast of each endosperm cell, 3. all remaining cytoplasm and lipid bodies disappear, and 4. the remaining cell walls become electron-transparent and collapse against the haustorium surface. Two cell wall hydrolases are present—endo-mannanase (EC3.2.1.78) and -mannosidase (EC3.2.1.25). -mannosidase is detectable in the endosperm before germination. At germination, the major portion of activity is found in the softened endosperm. -mannanase is only detectable from germination and there is always hundreds of fold greater activity in the softened endosperm than elsewhere. Proteinase is detectable in trace amounts at germination in the softened endosperm but is also found in the haustorium at later stages. Isolated haustoria, incubated in extracted ivory nut (Phytelephas macrocarpa) mannan in buffer, cause no mannan breakdown. Haustoria, incubated in a solution of locust bean galactomannan, cause no decrease in galactomannan viscosity. Our observations suggest that although haustoria probably regulate mannan breakdown in the endosperm, they do not seem to secrete the hydrolytic enzymes concerned.     

Physiological effects of gibberellic Acid. X. The release of gibberellin-like substances by germinating barley embryos

Barley embryos, completely free from endosperm, were excised from germinating grain at various times and allowed to diffuse into an aqueous medium for varying lengths of time. At the end of this time, the embryos and ambient solutions were separately extracted. Gibberellin-like activity in the extracts was determined with the barley endosperm bioassay using seed from the same variety, harvest and treatment schedule as was employed for the embryo diffusion experiments. Gibberellin-like substances were released by embryos throughout the 60 hour germination period, though at no time during this period could sufficient activity be extracted from the embryos themselves to account for the observed release. Solvent partitioning and chromatography identified at least one major acidic component migrating at an Rf similar to that of GA₃.

It is concluded that the endogenous gibberellin-like substance(s) originates within the embryo during germination, and that the release of this substance(s) is temporally consistent with, and quantitatively sufficient to account for the in vivo endosperm mobilization response syndrome. A gibberellin-like substance is undoubtedly the endosperm mobilizing hormone.

     

Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds

A spectrophotometric assay was devised to characterize the asparaginyl (Asn) endopeptidase activity from the endosperm of castor oil seeds. (Ricinus communis L. var. Baker 296). The assay measures the release of p-nitroaniline from the hydrolysis of benzoyl-l-Asn-p-nitroanilide. Assay sensitivity was improved through diazotization of the reaction product with N(]-napthy])-ethylenediamine dihydrochloride: diazotized p-nitroaniline was determined spectrophotometrically at 548 nm (?₅₄₈= 1.64 × 10^?1M^?1 cm^?2). By using this assay. Asn endopeptidase activity was detected in endosperm extracts of developing, mature and germinating castor seeds. Comparison of the Asn endopeptidase activities of developing and germinating castor endosperms revealed that they: 1) have identical pH-activity profiles with optimal activity occuring at pH 5.4: 2) are heat-labile proteins displaying comparable thermal stability profiles, and 3) are activated and inhibited by dithiothreitol and thiol modifying reagents, respectively. Thus, the Asn endopeptidases of developing and germinating castor seeds are very similar, if not identical, cysteine proteases. The most significant increase in the activity of endosperm Asn endopeptidase occurs during the full coryledon to maturation stage of seed development, this period coincides with the most active phase of reserve protein accumulation by ripening castor oil seeds. Asn endopeptidase activity of fully mature (dry) castor seeds was about 2-fold lower than that of muturation stage ripening castor oil seed. Asn endopeptidase activity showed a slight reduction over the inicial 2-day period following seed imbibition, and then rapidly decreased over the next several days of germination. The results are compatible with the proposal that Asn endopeptidase functions both to process storage preproteins following their import into protein bodies of developing seeds, as well as to participate in the mobilization of storage proteins during the early phase of seed germination.     

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.     

α-d-Galactosidase activity and galactomannan and galactosylsucrose oligosaccharide depletion in germinating legume seeds

Germinating seeds of lucerne, guar, carob and soybean initially depleted raffinose series oligosaccharides and then galactomannan. This depletion was accompanied by a rapid increase and then a decrease in α-galactosidase levels. Lucerne and guar contained two α-galactosidase activities, carob three and soybean four. One of these in each plant, from its location in the endosperm, time of appearance and kinetic behaviour, appeared to be primarily involved in galactomannan hydrolysis. This enzyme in lucerne had MW of 23 000 and could not be separated from β-mannanase by (NH₄)₂SO₄ fractionation, DEAE, CM or SE-cellulose chromatography or gel filtration, but only by polyacrylamide gel electrophoresis. In guar, carob and soybean, it could be separated by ion-exchange chromatography and gel filtration. In lucerne, carob and guar most of the total increase in activity was due to this enzyme. The other α-galactosidases had MWs of about 35 000 and could be separated from β-mannanase by dissection, ion exchange cellulose chromatography and gel filtration. They were located in the cotyledon-embryo and appeared to be primarily involved in galactosylsucrose oligosaccharide hydrolysis.     

Changes in Ultrastructure and Subunit Composition of Protein Body in Rice Endosperm during Germination

Changes in ultrastructure of protein bodies in subaleurone cells of rice endosperm during germination were studied by transmission electron microscopy. The subaleurone cells contained two different types of protein bodies: PB-I (spherical) and PB-II (crystalline). Both types of protein bodies were deconstructed during germination. But there was a considerable difference in digestibility between PB-I and PB-II. PB-II which did not have a dense core was easily digested from the central portion when germination began. At 6 days of germination, PB-II was almost deconstructed. On the other hand, PB-I which displayed concentric rings with a dense core was digested from the outside after 3 days of germination. At 9 days of germination, many kernels of the spherical protein bodies remained.

Changes in subunit composition of protein bodies during germination were investigated by SDS-polyacrylamide gel electrophoresis. Protein body fractions were isolated from germinating grains at various stages by enzymatic digestion and two-phase system, then subjected to SDS-polyacrylamide gel. As germination proceeded, 15 (b₁), 20 (d₁), 24 (e), 35 (f₁) and 37 (f₃) kdaltons subunits decreased. On the other hand, 16 (b₂), 21 (d₂) and 36 (f₂) k daltons subunits remained at the later stage of germination. We think that PB-I contains b₂, d₂ and f₂ subunits and is attacked only from the outside at middle and later stages of germination by de novo protease. On the contrary, PB-II contains b₁ d₁ e, f₁ and f₃ subunits is utilized at an early stage of germination. PB-II may possibly contain latent protease. The breakdown process of PB-I was by exo-type digestion, on the contrary, that of PB-II was by endo-type digestion.     

Action of Phytochrome During Prechilling of Amaranthus retroflexus L. Seeds

Dark germination of Amaranthus retroflexus L. seeds at 35° increased after several days of prechilling at 20° or lower. Irradiation with far-red light for short periods during the early hours of a prechilling period at 10° inhibited subsequent dark germination at 35°. The inhibition was completely reversible with red light. Far-red irradiation in the latter part of the prechilling period was less effective. Increased dark germination of A. retroflexus seeds following a prechilling period at 20° or less is attributed to action of preexistent P_FR, the far-red absorbing form of phytochrome, within the seeds. Inactivation of P_FR was found to proceed ca. 4 times more rapidly at 25° than at 20°. Failure of imbibition temperatures above 20° to increase dark germination of A. retroflexus seeds is attributed to the rapid thermal reversion of pre-existent P_FR. We suggest that the action of prechilling (layering) on many other seed kinds arises in a similar way.