C]Sucrose Uptake and Labeling of Starch in Developing Grains of Normal and segl Barley

Previous work showed that the segl mutant of barley (Hordeum vulgare cv Betzes) did not differ from normal Betzes in plant growth, photosynthesis, or fertility, but it produced only shrunken seeds regardless of pollen source. To determine whether defects in sucrose uptake or starch synthesis resulted in the shrunken condition, developing grains of Betzes and segl were cultured in [¹⁴C]sucrose solutions after slicing transversely to expose the endosperm cavity and free space. In both young grains (before genotypes differed in dry weight) and older grains (17 days after anthesis, when segl grains were smaller than Betzes), sucrose uptake and starch synthesis were similar in both genotypes on a dry weight basis. To determine if sucrose was hydrolyzed during uptake, spikes of Betzes and segl were allowed to take up [fructose-U-¹⁴C]sucrose 14 days after anthesis and the radioactivity of endosperm sugars was examined during 3 hours of incubation. Whereas less total radioactivity entered the endosperm and the endosperm cavity (free space) of segl, in both genotypes over 96% of the label of endosperm sugars was in sucrose, and there was no apparent initial or progressive randomization of label among hexose moieties of sucrose as compared to the free space sampled after 1 hour of incubation. We conclude that segl endosperms are capable of normal sucrose uptake and starch synthesis and that hydrolysis of sucrose is not required for uptake in either genotype. Evidence suggests abnormal development of grain tissue of maternal origin during growth of segl grains.     

Dormancy in Dioscorea: Rapid Germination of Detached Embryos from Dormant Seeds of D. tokoro

The intact dormant seeds of Dioscorea tokoro germinate slowlyif at all between 11-23°C; for full and rapid germinationthey require prior chilling treatment [Okagami and Kawai (1982)Bot. Mag. Tokyo 95: 155]. The germination abilities of zygoticembryos detached from dormant seeds of this species were studiedunder various nutritional and temperature regimes. For germinationof embryos, the minimum nutritional components in Murashigeand Skoog's (1962) medium that were required were sucrose andNO^–₃ or SO^2–₄. As the source of carbohydrate forgermination of detached embryos, sucrose, mannose and maltosewere effective; glucose and fructose were less effective; andrhamnose was entirely unable to support germination. Embryos detached from dormant seeds, incubated with the sucroseplus KNO₃, germinated more rapidly with increasing temperatureup to 35°C. However, application of sucrose and KNO₃ didnot induce germination of intact seeds above 26°C. Therefore,it is very possible that the endosperm exerts an inhibitoryfunction on germination at such high temperatures. When seeds were incubated after a cut was made over a smallpart of the edge of the endosperm in which the radicle of theembryo is encased, germination occurred rapidly but the increasein germination percentage was slight. This result suggests thatthe endosperm suppots part of the germination inhibition bymeans of a mechanical barrier or its impermeability to wateror gases. Physiological features of the endosperm alone or interactionsbetween the embryo and endosperm may contribute significantlyto the characteristics of dormancy of intact seeds of this species. (Received May 30, 1988; Accepted January 11, 1989)     

Transport and Metabolism of a Sucrose Analog (1'-Fluorosucrose) into Zea mays L. Endosperm without Invertase Hydrolysis

1′-Fluorosucrose (FS), a sucrose analog resistant to hydrolysis by invertase, was transported from husk leaves into maize (Zea mays L., Pioneer Hybrid 3320) kernels with the same magnitude and kinetics as sucrose. ¹⁴C-Label from [¹⁴C]FS and [¹⁴C]sucrose in separate experiments was distributed similarly between the pedicel, endosperm, and embryo with time. FS passed through maternal tissue and was absorbed intact into the endosperm where it was metabolized and used in synthesis of sucrose and methanol-chloroform-water insolubles. Accumulation of [¹⁴C] sucrose from supplied [¹⁴C]glucosyl-FS indicated that the glucose moiety from the breakdown of sucrose (here FS), which normally occurs in the process of starch synthesis in maize endosperm, was available to the pool of substrates for resynthesis of sucrose. Uptake of FS into maize endosperm without hydrolysis suggests that despite the presence of invertase in maternal tissues and the hydrolysis of a large percentage of sucrose unloaded from the phloem, hexoses are not specifically needed for uptake into maize endosperm.     

SEED DORMANCY AND GERMINATION IN MELAMPYRUM LINEARE

Immature seeds of Melampyrum lineare Desr. have very high germination percentages and dormancy is induced in a variable fraction of the seed crop during ripening. Correlated with this is the endogenous gibberellin-like activity which is found in considerable amounts in immature seeds, less in batches of ripe seeds, and is not detectable in batches containing only dormant seeds. For germination dormant seeds require activation followed by cold storage. In the laboratory activation is produced by allowing moist, dormant seeds to respire freely for several weeks at 20 C, or by treatment with exogenous GA₃. Dormancy appears to be most directly related to inability of the embryo to hydrolyze the thickened, mannan-containing endosperm cell walls. Embryos excised from dormant seed can be grown on agar enriched with whole macerated dormant seeds or with the ethanol-extractable materials from these (mostly sucrose and a glycoside). However, dormant seed material does not support growth when extracted to remove benzene- and ethanol-soluble materials.     

Sugar uptake and starch biosynthesis by slices of developing maize endosperm

¹⁴C-Sugar uptake and incorporation into starch by slices of developing maize (Zea mays L.) endosperm were examined and compared with sugar uptake by maize endosperm-derived suspension cultures. Rates of sucrose, fructose, and d- and l-glucose uptake by slices were similar, whereas uptake rates for these sugars differed greatly in suspension cultures. Concentration dependence of sucrose, fructose, and d-glucose uptake was biphasic (consisting of linear plus saturable components) with suspension cultures but linear with slices. These and other differences suggest that endosperm slices are freely permeable to sugars. After diffusion into the slices, sugars were metabolized and incorporated into starch. Starch synthesis, but not sugar accumulation, was greatly reduced by 2.5 millimolar p-chloromercuribenzenesulfonic acid and 0.1 millimolar carbonyl cyanide m-chlorophenylhydrazone. Starch synthesis was dependent on kernel age and incubation temperature, but not on external pH (5 through 8). Competing sugars generally did not affect the distribution of ¹⁴C among the soluble sugars extracted from endosperm slices incubated in ¹⁴C-sugars. Competing hexoses reduced the incorporation of ¹⁴C into starch, but competing sucrose did not, suggesting that sucrose is not a necessary intermediate in starch biosynthesis. The bidirectional permeability of endosperm slices to sugars makes the characterization of sugar transport into endosperm slices impossible, however the model system is useful for experiments dealing with starch biosynthesis which occurs in the metabolically active tissue.     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

Proteinbiosynthesen in dormanten und nachgereiften embryonen und samen von Agrostemma githago

Seed germination of Agrostemma githago is prevented by inhibitors of protein and RNA synthesis. Thus protein as well as RNA synthesis are essential prerequisites for germination. Early protein synthesis of Agrostemnia embryos can be completely inhibited by cycloheximide and cordycepin. During the aging of seeds there is a considerable decrease in germination capacity and protein synthesis. In dormant and afterripened embryos of Agrostemma githago¹⁴C-leucine and ¹⁴C-uracil are incorporated in protein and RNA respectively with nearly the same intensity, whereas RNA and protein synthesis of dormant seeds and embryos starts earlier than in those subjected to afterripening. ³H-uracil-labelled RNA from dormant and afterripened embryos are able to hybridize on oligo-dT-cellulose to the same extent. There is a similarity in the protein pattern of dormant and afterripened embryos revealed by electrophoresis in polyacrylamide gels of double-labelled proteins. According to these results dormancy of Agrostemma githago is not caused by a general but by a specific metabolic block.     

Kernel Abortion in Maize : II. Distribution of C among Kernel Carbohydrates

This study was designed to compare the uptake and distribution of ¹⁴C among fructose, glucose, sucrose, and starch in the cob, pedicel, and endosperm tissues of maize (Zea mays L.) kernels induced to abort by high temperature with those that develop normally. Kernels cultured in vitro at 30 and 35°C were transferred to [¹⁴C]sucrose media 10 days after pollination. Kernels cultured at 35°C aborted prior to the onset of linear dry matter accumulation. Significant uptake into the cob, pedicel, and endosperm of radioactivity associated with the soluble and starch fractions of the tissues was detected after 24 hours in culture on labeled media. After 8 days in culture on [¹⁴C]sucrose media, 48 and 40% of the radioactivity associated with the cob carbohydrates was found in the reducing sugars at 30 and 35°C, respectively. This indicates that some of the sucrose taken up by the cob tissue was cleaved to fructose and glucose in the cob. Of the total carbohydrates, a higher percentage of label was associated with sucrose and a lower percentage with fructose and glucose in pedicel tissue of kernels cultured at 35°C compared to kernels cultured at 30°C. These results indicate that sucrose was not cleaved to fructose and glucose as rapidly during the unloading process in the pedicel of kernels induced to abort by high temperature. Kernels cultured at 35°C had a much lower proportion of label associated with endosperm starch (29%) than did kernels cultured at 30°C (89%). Kernels cultured at 35°C had a correspondingly higher proportion of ¹⁴C in endosperm fructose, glucose, and sucrose. These results indicate that starch synthesis in the endosperm is strongly inhibited in kernels induced to abort by high temperature even though there is an adequate supply of sugar.     

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.     

Movement of C-compounds from Maternal Tissue into Maize Seeds Grown in Vitro

Uptake from nutrient media into the cob and translocation of various ¹⁴C-compounds from maternal tissue (cob) into developing maize seeds was examined by using caryopsis cultures. Based on relative ¹⁴C concentrations in the cob and the endosperm, it was concluded that the relative efficiencies of movement of amino acids (leucine, phenylalanine, proline), vitamins (thiamine HCl, nicotinic acid), and nucleic acid bases (adenine, thymine) from the cob to the endosperm were 11 to 250 times lower than that of sucrose. Thiamine was unique in that it was concentrated in the embryo at a level that was almost 10 times higher than in the endosperm. The absence of auxotrophic mutants requiring an organic supplement in higher plants (other than thiamine auxotrophs) may be explained by inadequate translocation of these essential metabolites into the mutant zygotes (embryos) to enable their development to mature seeds.     

The cellular pathway of photosynthate transfer in the developing wheat grain. III. A structural analysis and physiological studies of the pathway from the endosperm cavity to the starchy endosperm

The cellular pathway of sucrose transfer from the endosperm cavity to the starchy endosperm of developing grains of wheat (Triticum turgidum) has been elucidated. The modified aleurone and sub-aleurone cells exhibit a dense cytoplasm enriched in mitochondria and endoplasmic relicilium. Significantly, the sub-aleurone cells are characterized by secondary wall ingrowths. Numerous plasmodesmata interconnect all cells between the modified aleurone and starchy endosperm. The pro-tonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) slowed [¹⁴C]sucrose uptake by grain tissue slices enriched in modified aleurone and sub-aleurone cells but had no effect on uptake by the starchy endosperm. The fluorescent weak acid sulphorhodamine G (SRG) was preferentially accumulated by the modified aleurone and sub-aleurone cells, and this uptake was sensitive to CCCP. The combined plasma membrane surface areas of the modified aleurone and sub-aleurone cells appeared to be sufficient to support the in vivo rates of sucrose transfer to the starchy endosperm. Plasmolysis of intact excised grain inhibited [¹⁴C]sucrose transfer from the endosperm cavity to the starchy endosperm. The sulphydryl group modifier p-chloromercuribenzenesulphonie acid (PCMBS) decreased [¹⁴C]sucrose uptake by the modified aleurone and sub-aleurone cells but had little effect on uptake by the starchy endosperm. In contrast, when PCMBS and [¹⁴C]sucrose were supplied to the endosperm cavity of intact excised grain, PCMBS slowed accumulation by all tissues equally. Estimates of potential sucrose fluxes through the interconnecting plasmodesmata were found to be within the published range. It is concluded that the bulk of sucrose is accumulated from the endosperm cavity by the modified aleurone and sub-aleurone cells and subsequently transferred through the symplast to the starchy endosperm.     

Early Actions of Gibberellic Acid on the Embryo and on the Endosperm of Avena fatua Seeds

Gibberellic acid at 0.1 μm stimulates amylase synthesis in dormant Avena fatua seeds without inducing germination; at 0.5 mm it enhances biosynthesis of proteins and RNA in both the embryo and the endosperm and utilization of the endosperm sugars by the embryo. These events occur in early hours (0-14th hour) and prior to germination, which begins 24 hours after gibberellic acid application. These observations are in agreemeent with the concept that in cereal grains gibberellic acid has two morphological sites of actions: the embryo and the endosperm, and that germination (radicle protrusion) is not caused by gibberellic acid-induced amylase synthesis in the endosperm.     

Carbon-14 Distribution in Carbohydrates of Immature Zea mays. Kernels Following CO(2) Treatment of Intact Plants

Shortly after Zea mays L. plants were exposed to ¹⁴CO₂, most of the radioactivity in the kernel occurred in the free monosaccharides, glucose and fructose. Later the proportion of ¹⁴C in sucrose increased and that in the monosaccharides declined. These data have been interpreted as showing that the translocated sugar is hydrolyzed prior to or during its movement into the storage cells of the endosperm. This hydrolysis appears to occur in the “pedicel region” of the kernel. After entry into the endosperm tissue, sucrose was rapidly resynthesized from the monosaccharides prior to its utilization in starch synthesis.     

Lysophosphatidylcholine biosynthesis in developing barley

Acetate-2-[¹⁴C] and choline-Me-[ ¹⁴C], absorbed through the stems of isolated barley heads, were used to label lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) of the endosperm tissue. Labelling of LPC occurred in barley heads at almost all stages of development but was at a maximum when the fr. wt of the seeds had attained ca 60–70% of their maximum wt. In time-course experiments labelling of PC from each substrate reached a maximum after 50 hr and then declined. Label in LPC, however, continued to accumulate throughout 72 hr. Stimulation of labelling of LPC from choline-Me-[¹⁴C] by sucrose was observed. A bound form of LPC (starch lipid) and a free form were distinguished by differential solvent extraction.     

The Mechanism of Low Temperature Dormancy in Mature Seeds of Syringa Species

The mechanism of seed dormancy at low temperatures (15-9°C) was investigated in the seeds of Syringa josikaea, S. reflexa and S. vulgaris. Low temperature dormancy in Syringa species was mainly imposed by endosperm embedding the radicle. Different degrees of embryo dormancy may occur in S. reflexa seeds. In most cases the low temperature dormancy was broken completely by removing the endosperm around the radicle. The endosperm did not seem to contain significant quantities of germination inhibitors, and the results indicate that it prevents germination mainly due to its mechanical resistance. The mechanical resistance of endosperm did not change during chilling or during induction of dormancy by high temperature incubation. The strength of the endosperm decreased rapidly in non-dormant seeds before visible germination. Similar changes were not observed in dormant seeds. Generally, the strength of the endosperm was lower in the non- (or less) dormant species S. josikaea and S. vulgaris than in the more dormant S. reflexa seeds. The growth potential of the embryos, measured as their ability to germinate in osmotic solutions (mannitol or polyethylen glycol 4000), was increased by chilling and by GA₃-treatment. The growth potential of untreated S. josikaea and S. vulgaris embryos was generally higher than that of S. reflexa embryos. Acid ethyl acetate fractions of methanol extracts from embryos of all three species contained substances with GA³-like activity in the lettuce hypocotyl test. The activity was found at Rf 0.9–1.0 on paper chromatograms run in distilled water. No significant changes in the activity were detected during chilling or prior to visible germination.     

Patterns of food utilization by the germinating lettuce seeds

The embryo excised from seed of Grand Rapids lettuce (Lactuca sativa L.) can be cultured in distilled water. Complete digestion of the endosperm and transfer of nutrients from the endosperm to the embryo occur in the germinating seed with fat as the source of food. The fat is utilized for respiration, synthesis of amino acids, and to a degree, converted to sucrose. ¹⁴C-Glucose administered to the seed is quickly converted to sucrose in the endosperm and translocated to the embryo. Radioactivity associated with the glucose remains predominantly in the carbohydrate fraction, and much of it is incorporated into what is believed to be cell wall polysaccharides. Relatively little isotope is distributed in the amino or organic acids.     

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a "seed coat bedding" assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.     

The Role of Hormones in Controlling the Mechanically Induced Dormancy of Suaeda spp.

Seeds of several taxa in the genus Suaeda with a mechanically resistant testa were stimulated to germinate with applications: of 1 × 10^?4M gibberellic acid (GAg). The barley endosperm bioassay indicated that the non-dormant taxa, S. macrocarpa, had high initial gibberellin-like activity while the dormant varieties, S. flexilis and S. vulgaris, had not. Soaking for 2 days increased the gibberellin-like activity of the dormant taxa. At 20 days hormonal activity was repressed, and few seeds germinated. The barley endosperm bioassay also revealed an inhibitor activity in the dormant varieties. The wheat coleoptile bioassay and UV irradiation of thin layer chromatograms indicated that an inhibitor occurred at the Rf of cochromatographed ABA and that it had similar inhibitory characteristics.     

Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

Effect of gibberellin on protein and non-structural carbohydrate in dormant Avena fatua caryopses

Application of gibberellic acid (GA₃) to dormant Avena fatua L. caryopses resulted in the termination of dormancy within 24 h as indicated by germination between 24 and 48 h. During the period of imbibition from 0 to 24 and 24 to 48 h changes occurred in protein and carbohydrate metabolism in GA-treated and untreated caryopses. Germination did not occur in untreated caryopses, therefore physiological changes in these caryopses were not associated with the termination of dormancy. GA-treatment increased the concentration of soluble and SDS-extractable protein in the endosperm tissue by 4 and 5%, respectively, over the 24 h untreated material; no changes were apparent when the protein profiles of GA-treated and untreated tissues were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 0, 24 and 48 h after imbibition. The concentration of hexose and sucrose in the GA-treated endosperm tissue increased 189 and 151 μmol, respectively, over the untreated material at 24 h. Gibberellic acid had no effect on starch metabolism in the endosperm tissue in the first 24 h, the period associated with the termination of dormancy. The concentration of hexose increased by 57 μmol and starch decreased by 80 μmol in the GA-treated embryo tissue within 24 h. Our results demonstrate that exogenously applied GA influences sucrose and hexose metabolism in the endosperm tissue. The specific effect of GA on starch and hexose metabolism in the dormant A. fatua caryopsis embryo tissue may be associated with the termination of dormancy.