Later Stages Of Germination Research Articles

Polysome formation and stability in pea stem and root tissue

Polysome stability and the formation of various polysomal populations in pea stem and root tissue were examined. Both total ribosomal fraction and four polysome populations were isolated: FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). The content of above mentioned populations decreased in roots and stems during germination. In both roots and stems a gradual decrease of FP participation in the total polysomal population was also observed during germination. On the other hand, an obvious increase in participation of CMBP population in the total polysomes pool was observed in later stages of germination. Increase of CMBP participation in pea root and stem tissues in later stages of germination is probably due to intensive enzymatic protein synthesis taking place in them. These proteins may participate in elongating growth of cells. The results of investigation on polysomes stability showed that total polysomes isolated from pea roots appeared to be more resistant to digestion by exogenous ribonuclease (EC 3.1.27.5) than polysomes isolated from stems. As protein-mRNA interactions are widely known and ribosomes are also very adhesive structures, numerous non-ribosomal proteins are present in the polysome preparations. We suppose that changes in proteins bound to polysomes indicated by us previously, significantly influence both the stability and also translatability of polysomes isolated from different plant organs.

Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid.

Germination of radish (Raphanus sativus cv Eterna) seeds can be inhibited by far-red light (high-irradiance reaction of phytochrome) or abscisic acid (ABA). Gibberellic acid (GA3) restores full germination under far-red light. This experimental system was used to investigate the release of reactive oxygen intermediates (ROI) by seed coats and embryos during germination, utilizing the apoplastic oxidation of 2',7'-dichlorofluorescin to fluorescent 2',7'-dichlorofluorescein as an in vivo assay. Germination in darkness is accompanied by a steep rise in ROI release originating from the seed coat (living aleurone layer) as well as the embryo. At the same time as the inhibition of germination, far-red light and ABA inhibit ROI release in both seed parts and GA3 reverses this inhibition when initiating germination under far-red light. During the later stage of germination the seed coat also releases peroxidase with a time course affected by far-red light, ABA, and GA3. The participation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in ROI metabolism was demonstrated with specific in vivo assays. ROI production by germinating seeds represents an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens.

Complete spore-cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB.

The role of the sleB gene of Bacillus subtilis, which encodes a putative spore-cortex-lytic enzyme, and the downstream ypeB gene were investigated. Both SleB and YpeB were required for normal germination to occur. The corresponding mutants formed phase-bright, heat-resistant spores with no apparent defects in dormancy. However, mutant spore suspensions lost optical density slower than the wild-type and spores were phase-grey even 12 h after the triggering of germination. Since the loss of heat resistance and release of dipicolinic acid was similar to the wild-type, these mutants were blocked in the later stages of germination. The mutants were nevertheless capable of outgrowth on rich agar to form colonies, indicating that other spore components can compensate for their function sufficiently to allow outgrowth. The expression and regulation of the operon was examined using a lacZ transcriptional fusion. Expression of the operon began 2 h after the onset of sporulation and was under the control of RNA polymerase containing the forespore-specific sigma factor, sigmaG. The application of reverse phase HPLC revealed that the mutants do not have any structural defect in the dormant spore cortex and therefore these genes are not required for normal spore-cortex synthesis. The analysis of peptidoglycan dynamics during germination showed, however, that the cortex was only partially hydrolysed in both mutants. This analysis also revealed that the likely hydrolytic bond specificity of SleB is likely to be that of a lytic transglycosylase.

Effect of Cadmium on Activities of Some Enzymes of Glycolysis and Pentose Phosphate Pathway in Pea

Activities of alcohol dehydrogenase, hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were significantly inhibited by cadmium in germinating pea (Pisum sativum L. cv. Bonneville) seeds. The effect was concentration dependent in the range of 0.25 to 1.0 mM CdCl2. The magnitude of detrimental effect on these enzymes was reduced during later stage of germination (9 d) largely because of fall in the activities of these enzymes in the control seeds germinated in water. In vitro, activities of hexokinase, glucose-6-phosphate dehydrogenase, and alcohol dehydrogenase were inhibited at 0.5 mM Cd2+ in the reaction mixture by 62, 67, and 36 %, respectively, however, 6-phosphogluconate dehydrogenase was insensitive to Cd2+.

Changes in carbohydrate composition and α-amylase expression during germination and seedling growth of starch-deficient endosperm mutants of maize

Kernels of 28 different maize ( Zea mays L.) genotypes consisting of several inbreds isogenic for the starch-deficient endosperm mutations sugary 1 ( su1), sugary1 sugary enhancer 1 ( su1se1), and shrunken2 ( sh2) were used to study the effect of these mutations on carbohydrate composition and α-amylase expression during germination and seedling growth in comparison to normal starchy kernels ( Su). Sugar (fructose, glucose, sucrose, and maltose), WSP (water-soluble polysaccharide), and insoluble starch contents were determined for endosperm and scutellar tissues for each of the genotypes at four stages of seedling development. While initial levels of endosperm sucrose were higher in the endosperm mutants, they rapidly declined by radicle emergence and remained low thereafter. Mobilization of carbohydrate reserves was most reduced in the sh2 genotypes. Collectively, these factors represent a limited carbon supply for the developing sh2 seedling which may impact seedling vigor. A more significant negative correlation was observed between percentage germination and endosperm sugar content than between percentage germination and scutellar sugar content. High initial levels of endosperm sucrose in the mutants correlate with a severe reduction of α-amylase (EC 3.2.1.1) transcript abundance. The same correlation does not hold for glucose and maltose, as at later stages of germination, levels were significantly higher in the Su genotype while the α-amylase mRNA levels remained high. Furthermore, the ratio of α-amylase enzyme activity to transcript abundance was considerably lower in the sh2 aleurone compared to the Su aleurone at radicle emergence. Genotype specific differences in α-amylase gene expression strongly correlate with endosperm sugar content and composition during germination.

Starch, sugar, amylase and invertase activity in the germinating seeds of modern wheat varieties

Amylase and invertase activity was studied in germinating seeds of eight modem wheatvarieties(Akbar, Aghrani, Ananda, Balaka, Barkat, Kanchan, Pavon and Sonalika). Actiyities of the enzymes were low at the initial stages and thereafter significantly increased and further decreased at later stages of germination. The peak amylase and invertase activity was observed at 60 and 72 h of incubation for germination respectively. Protein content was signifi- cantly increased with the period of germination. Starch depletion was slow initially and thereafter fast. The degradation rate of starch was about 60% and accumulation of sugar was 4 times higher in seeds at 96 h of germination over the ungerminated seeds.

Amyl expression during wheat seed germination

The expression pattern of α- Amyl, a gene coding for high-pI α-amylase, has been studied during wheat ( Triticum aestivum) seed germination using both Northern-blot and in situ hybridisation. At early stages of germination (24 h after imbibition) this gene was exclusively expressed in the scutellar epithelium. The expression in this tissue was transient and independent of GA 3. At later stages of germination, after 48 h of imbibition, expression in the epithelium decreased while α- Amyl mRNA accumulated in the aleurone layer. In this tissue, where α- Amyl expression is regulated by GA 3, a gradient could be detected with the embryo-proximal aleurone cells showing a higher level of expression than distal cells. These results show temporal and spatial expression of α- Amyl during germination of the wheat grain.

Biosynthesis, accumulation and secretion of isoflavonoids during germination and development of white lupin (Lupinus albusL.)

A combination of 14 C labelling experiments of white lupin seedlings (Lupinus a/bus L.) and high pressure liquid chromatography of tissue extracts indicated active biosynthesis of isoflavonoids during the first 2-4 d of seed germination. These were synthesized mostly as glucosides and to a lesser extent as aglycones, with trace amounts of prenylated derivatives. There was a general decrease in total isoflavonoids during later stages of germination (c. until d 13), which may be ascribed to their turnover and/or exudation into the rhizosphere. In addition, exudation of isoflavonoids by lupin seeds germinated under sterile conditions continues for 12 d with a preferential release of monoprenylated compounds. The relationship between the specific developmental events during seed germination and the accumulation of certain groups of isoflavonoids is discussed in relation to their possible role in the growth processes of lupin

Effects of Ageing on Amylase Activity and Scutellar Cell Structure during Imbibition in Wheat Seed

In wheat seed the scutellum plays an important role in the hydrolysis of stored substrate during germination. This layer is activated first, whilst the aleurone becomes activated later. A good correlation exists between the initiation of visible germination and the appearance of enzyme activity in the scutellum. Enzyme activity in the aleurone becomes apparent only when the germinating seedling reaches the rapid growth phase. Electron microscopic observations show that during the later stages of germination the scutellar cells develop finger like projections. These may serve to absorb endospermic reserves hydrolysed by aleurone amylase. The scutellum of aged non-germinating seeds showed no amylase activity and no finger like projections were produced even after prolonged imbibition.

Expression of glutamine-synthetase genes in cotyledons of germinating Phaseolus vulgaris L.

In the legume Phaseolus vulgaris L., glutamine synthetase (GS; EC.6.3.1.2.) is encoded by four actively transcribed genes, gln-α, gln-β, gln-γ and gln-δ. We have studied the expression of these genes in cotyledons during seed germination and have studied the effect of light and nitrate on this process. An RNase-protection method, used to detect the abundances of GS mRNAs, revealed that the four GS genes are differentially expressed in the germinating cotyledons. The gln-α. mRNA was present in dry seeds and was the most abundant GS mRNA during early stages of germination. The gln-β and gln-γ mRNAs were first detectable 2 d after sowing and their abundances differed in light- and dark-grown cotyledons at later stages of germination. The gln-δ mRNA (which encodes the plastid-located GS) was detectable only in light-grown cotyledons, at a low abundance. A nitrate supply of 2 mM had only a minor effect on the expression of the GS genes. Western immunodetection and ion-exchange high-performance liquid chromatography demonstrated that the α polypeptide and isoenzyme were present in extracts of dry seeds and represented the major GS products at 2 d and 4 d. Both the β and γ polypeptides appeared at the 2-d stage. The role of differential GS gene expression in controlling cotyledonary GS activity is discussed.

The effect of the etched (et) mutation on the amylolytic enzyme activities in germinating kernels and seedlings of Zea mays

The etched (et) mutation in maize causes distinct depressions and structural gaps in the endosperm and also gives rise to virescent seedlings, α- and β-Amylase activities were observed to be higher in et (+) et (+) kernels and seedlings as compared to that of the et et mutant. The total amylase and β-amylase trends during germination also differed between normal and mutant kernels and seedlings (it increases in the wildtype and decreases in et et). On the contrary, the overall α-amylase trend was found to be similar in both genotypes (slight decrease during germination). The native gel electrophoresis of crude enzyme extracts did not reveal any qualitative differences in α and β amylases during germination. The germinating et et kernels initially showed lower levels of starch compared with the wild type kernels, whereas no such difference was found at later stages of germination. It is concluded that et gene associated endosperm lesions lead to an impairment of starch degradation in germinating kernels resulting in virescent seedlings.

Sterol and phospholipid changes during alfalfa seed germination

Spinasterol, dihydrospinasterol, 24-methylcholest-7-enol, sitosterol, stigmasterol, and campesterol are in decreasing order the major sterols of Medicago sativa seeds. The first three are Δ 7-sterols and account for 94.4% of the total, while the latter three are Δ 5-sterols and represent 5.6%. During germination the total sterol level increased and the Δ 7-to Δ 5-sterol ratio remained constant. The steryl esters were the major sterol form and they increased during the first two days, while the free sterols increased at later stages of germination. During germination, spinasterol increased while dihydrospinasterol decreased. The major seed phospholipid was phosphatidylcholine, and its level increased during germination. Phosphatidylethanolamine and phosphatidylinositol occurred in about equal quantitties; the former increased slightly while the latter decreased with germination. No correlation could be established during germination between various sterols and phospholipids and chlorophyll accumulation.

Isolation and identification of proteins from the peptide-transport carrier in the scutellum of germinating barley (Hordeum vulgare L.) embryos

Peptide-transport proteins, intrinsic to the epithelial plasmalemmae of the scutella of germinating barley (Hordeum vulgare L.) embryos, have been selectively labelled with p-chloro-[(203)Hg]mercuribenzenesulphonate using both a substrate-screening technique and a procedure developed to label exclusively vicinal dithiol groups, which were shown previously (Walker-Smith and Payne, 1983, FEBS Lett. 160, 25-30) to be essential components of the peptide-transport system. After radioactive labelling, proteins from the scutellar membranes have been solubilised with lithium diiodosalicylate plus sodium dodecyl sulphate and separated by using polyacrylamide gel electrophoresis. Fluorography and silver staining of these gels has for the first time allowed identification of two presumptive components of the peptide-transport system. These components only become detectable in an extract of the scutellar epithelia after 15 h imbibition, concomitant with a dramatic increase in peptide-transport activity, and they remain present at least 3 d after the onset of germination. [(35)] Methionine was shown to be incorporated into these proteins between 15-20 h after imbibition, but its incorporation during a similar 5 h period into scutella isolated after 3 d was undetectable, implying a slow turnover of these proteins during the later stages of germination.

RNA synthesis during germination of UV-irradiated Dictyostelium discoideum spores.

UV irradiation to the spores of Dictyostelium discoideum NC4 resulted in a more prolonged delay of amoeba-emergence from swollen spores with increasing UV fluence. During the germination, an inhibition of total RNA synthesis and a shift of stage of maximum RNA synthesis to the later period were observed. The maximum poly(A)+ RNA synthetic activity was found on an early stage of amoeba-emergence prior about 1 h to the beginning of rRNA synthesis in unirradiated spore germination; but, in UV-irradiated spore germination, the stage of maximum poly(A)+ RNA synthesis shifted to the later stage of germination with increasing UV fluence. A decreased synthesis of poly(A)+ RNA and a severe inhibition of rRNA synthesis were observed on UV-irradiated and germinated spores, but no significant inhibition of 4-5 S RNA synthesis was detected. Actinomycin D suppressed almost completely the rRNA synthesis of emerged amoebae but the drug apparently did not affect the emergence of amoebae at any stage of germination. It was postulated that the delay of amoeba-emergence in UV-irradiated spore must be mainly due to the shift of the stage of maximum synthesis of poly(A)+ RNA to the later stage of germination.

Polyadenylated RNA metabolism and loss of vigour and viability in germinating wheat embryos

The quiescent wheat (Triticum aestivum L. cv. Hobbit) embryo contains polyadenylated RNA species (polyA+ RNA, presumptive mRNA species) which are rapidly degraded upon water imbibition by the embryo even when the embryo has lost viability. The level of polyA+ RNA species in the quiescent embryo does not reflect the vigour or viability rating of the seed lot. Embryos which have lost viability appear to have lost the capacity for de novo transcription of polyA+ RNA species during the hours following water imbibition by the embryo. Embryos from seed lots of differing vigour ratings can be distinguished by their very different patterns of polyA+ RNA metabolism during germination at a sub‐optimal temperature. In embryos of reduced vigour there is a delayed onset of degradation of polyA+ RNA species during germination at 10°C and a reduced rate of polyA+ RNA synthesis through the first 6 h of germination compared with these processes in high vigour embryos. However, at later germination stages embryos of reduced vigour synthesise polyA+ RNA species at rates similar to those found in high vigour embryos yet newly synthesised polyA+ RNA species accumulate in embryos of reduced vigour only to a level which is 50% of that found in high vigour embryos of comparable viability. Results suggest that additional lesions affecting the stability and/or turnover of the messenger ribonucleo‐protein complex containing polyA+ RNA are present in embryos of reduced vigour at later stages of germination at a sub optimal temperature.

Secretion of α-Amylase by the Aleurone Layer and the Scutellum of Germinating Barley Grain

alpha-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20 degrees 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 CaCl(2), the alpha-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 alpha-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 alpha-amylase activity in the starchy endosperm in a whole grain.Scutella separated after 1 day of germination released small amounts of alpha-amylase activity into 10 millimolar CaCl(2). This release was linear for at least 1 hour and did not occur at 0 degrees 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 alpha-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 alpha-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.

Changes in Activities of Purine Salvage and Ureide Synthesis during Germination of Black Gram (Phaseolus mungo) Seeds

Summary The relative rate of purine salvage and its degradation in the cotyledons and embryonic axes of black gram ( Phaseolus mungo L.) seeds during germination were determined by tracer experiments using [8- 14 C]adenine, [8- 14 C]adenosine, [8- 14 C]guanine and [8- 14 C]hypoxanthine. In the cotyledons, the salvage of adenine, adenosine and guanine was higher at 12–48 h of germination than at 0–12 and 48–96 h. In contrast, appreciable ureide formation was found after 48 h of germination. In the embryonic axes, a higher incorporation of labelled purines into nucleic acids and nucleotides was observed during the early stage of germination, whereas significant incorporation into ureide was detected in the later stage of germination. [8- 14 C]Adenine absorbed into the cotyledons during imbibition was translocated to the embryonic axes and radioactivity was found to be associated with ureides. Allopurinol, an inhibitor of xanthine dehydrogenase, depressed the incorporation of [8- 14 C]adenine into ureides as well as the total ureide content in the embryonic axes. The results suggest that purine derivatives stored in the cotyledons of black gram seeds were utilized for nucleotides and nucleic acids especially during the early stage of germination, and that they were degraded to allantoin and allantoic acid in the later stage and reutilized as a nitrogen source for seedling growth.

Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination.

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

The γ-aminobutyric acid shunt in germinating Sinapis alba seeds

The metabolism of glutamic acid via the γ-aminobutyric acid (GABA) shunt was investigated during the germination of Sinapis alba by comparing the 14CO 2 evolved from germinating seeds after feeding samples with either L-[U- 14C] glutamate, [U- 14C]GABA or L-[1- 14C]glutamate. Glutamate appeared to be metabolised mainly via GABA during imbibition but was metabolised via 2-oxoglutarate during later stages of germination. Glutamate decarboxylase may be more active than other enzymes at low levels of hydration so that initially glutamate was converted to γ-aminobutyrate and was then oxidised in the Krebs tricarboxylic acid cycle via succinate. Later, at higher levels of hydration glutamate was metabolised to 2-oxoglutarate which was then oxidised, again via the Krebs tricarboxylic acid cycle.

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES, ALECTRA VOGELII AND STRIGA GESNERIOIDES. II. DNA SYNTHESIS AND DEVELOPMENT OF THE QUIESCENT CENTER IN THE RADICLE

DNA synthetic activity in the radicle meristem of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides was followed by autoradiography of 3H‐thymidine incorporation. Incorporation of 3H‐thymidine occurred in the nuclei of cells destined to form the vascular tissues, ground meristem and epidermis. An analysis of the distribution of labeled nuclei demonstrated the presence of a quiescent center of 2‐4 cells in the radicle at the beginning of seed germination, becoming more prominent at later stages of germination. During continued growth of the radicle which resulted in a reduction in size of the meristem, cells of the original quiescent center were activated to undergo DNA synthesis.