Membrane turnover in imbibed dormant embryos of the wild oat (Avena fatua L.)

Germinating non-dormant (ND) embryos of wild oat incorporate [³H]glycerol into phospholipid, and a 250% increase in total extractable phospholipid occurs within 72 h. During germination, leveles of phosphatidyl inositol showed the greatest change, increasing approximately 5-fold.Imbibed dormant (D) embryos of the wild oat also incorporate [³H]gycerol into phospholipids, but there is no net synthesis. A continuous turnover of membrane phospholipids could be demonstrated in pulse chase experiments, and although the proportions of most phospholipids does not change, there was a decrease of 50% in phosphatidyl serine.The half-life of [³H]glycerol in the extracted phospholipids of D and ND embryos varies between 35 and 57 h, and in membrane fractions separated on sucrose density gradients the half-lives vary between 26 and 56 h.D embryos induced to germinate with GA and ND embryos in which germination is repressed by ABA show similar phospholipid changes to ND and D embryos respectively, with the exception that the proportion of phosphatidyl serine remained unchanged in the ND-ABA embryos.It is concluded that the continual turnover of membranes of imbibed dormant embryos is consistent with the maintenance of cellular integrity determining the longevity of the seed under natural conditions.Abbreviations D dormant - ND nondormant - ABA abscisic acid - GA gibberellic acid (GA₃)     

Changes in chromatin structure associated with germination of maize and their relation with desiccation tolerance

Morphological and physicochemical measurements of chromatin condensation were made on germinating maize (Zea mays L.) radicles to determine whether the loss of genetic activities that occurs during the loss of desiccation tolerance is linked to irreversible changes in chromatin condensation. Chromatin samples were compared at different stages of germination (0, 24 and 72 h after imbibition), before (control) and after 24 h of desiccation. Morphological changes in chromatin structure and condensation were characterized by a qualitative and quantitative electron microscope study of chromatin which was allowed to spread in 0.2 mol m^?3 EDTA and then laid on coated microscope grids. The experiments showed similar levels of chromatin condensation in quiescent embryos and 24-h-old radicles (desiccation-tolerant material). After 72 h of imbibition, when radicle emergence and desiccation intolerance had ceased, the chromatin underwent a major decondensation towards various lower order folded structures. Regardless of the desiccation tolerance stage, an in vivo drying treatment of 24- and 72-h-old radicles before chromatin extraction did not induce significant changes in the extent of condensation compared to their respective controls. Similar conclusions were drawn from measurements of several spectroscopy properties (absorbance ratios: A₂₆₀/A₂₄₀, A₂₆₀/A4₀₀; thermal denaturation, and linear electric dichroism) of chromatin fragments that were obtained after nuclease digestion and then dissolved in 0-2 mol m^?3 EDTA. In quiescent and 24-h-old material, chromatin fragments were poorly soluble but highly stable during thermal denaturation. Chromatin fragments were 3-5-fold more soluble and less thermally stable in 72-h-old material than in 24-h-old material. In vivo desiccation had no significant effects on these properties compared to the respective controls. Collectively these data suggest that desiccation did not induce irreversible changes in the condensation properties of chromatin. The likelihood that the decondensation process occurring during germination is linked to the loss of desiccation tolerance is discussed.     

Rhythmic increase in DNA content and incorporation of [3H]thymidine in the developing wheat embryo during seed germination

Summary The DNA content and incorporation of [³H]thymidine into DNA have been studied during the development of Triticum aestivum (L.) embryo at various stages of seed germination up to 102 h (18°C). The DNA content of the embryos increased in a rhythmic way, when superimposed on an increasing basal content of DNA from 12 h onwards of germination. A temporary depression in DNA content was observed before the peaks of cell division (which has earlier been published by the author). The peaks of [³H]thymidine incorporation coincided with the peaks of cell division.     

Evidence that a Proliferation of the Endoplasmic Reticulum Precedes the Formation of Glyoxysomes and Mitochondria in Germinating Castor Bean Endosperm

Excised castor bean endosperm halves incubated with CDP-[Me-¹⁴C]cholineactively incorporated this compound into membrane phosphatidylcholine.The capacity of the tissue to synthesize phosphatidyl-[¹⁴C]cholineincreased during the first 3 d of germination and subsequentlydeclined. At the onset of germination phosphatidyl-[^l4C]cholinewas exclusively recovered in the ER membrane fraction. The rateof incorporation into the ER membranes increased strikinglyduring the first 24 h of germination while that into mitochondriaand glyoxysomes remained low. At later developmental stagesan increasing proportion of the newly synthesized phosphatidyl-[¹⁴C]cholinewas present in mitochondria and glyoxysomes; the rate of incorporationinto the membranes of these organelles increased while thatinto the ER membrane began to level off. The kinetics of CDP-[¹⁴C]cholineincorporation into membrane phosphatidylcholine of the majororganelle fractions of 3-d-old endosperm tissue showed thatthe ER was immediately labelled, whereas a lag period precededthe labelling of mitochondria and glyoxysomes. Assuming that the incorporation of CDP-[¹⁴C]choline into phosphatidylcholineserves as a reliable indicator of membrane synthesis, the resultsobtained suggest that a proliferation of ER membranes precedesthe formation of glyoxysomes and mitochondria in germinatingcastor bean endosperm. A comparison of developmental changesin (a) total ER and glyoxysomal phospholipid content and (b)ER and mitochondrial NADH cytochrome c reductase activity providedadditional evidence supporting this conclusion.     

Ageing and Osmotic Priming in Wheat Seeds: Effects Upon Certain Components of Seed Quality

Deteriorated wheat seed lots were osmotically primed and surface-dried,dried-back, then aged for two more weeks Osmotic priming didnot affect the final germination in any of the aged seed lots,but mean germination time was decreased The response of unagedseed lot to treatments was studied in the following the timecourses of germination, as well as L-[4, 5-²H]leucine and [6-³H]thymidineincorporation (as a measure of protein and DNA synthesis, respectively)into dissected embryos during the early hours of germinationIt was found that the variation in mean germination time andthe rate of synthetic reactions were related The greatest improvementin the components of seed lot quality was achieved after primingand surface drying The beneficial effects of priming were apparenteven after dehydration followed by two further weeks of ageingCumulative correlations between mean germination time and variousbiochemical tests in variously treated aged seed lots were highlysignificant The physiological processes involved in deteriorationand recovery and the potential application of biochemical testsin detecting vigour changes are discussed Triticum durum, seed, ageing, osmotic priming, mean germination time, protein DNA synthesis     

Post-replication DNA repair in barley embryos treated with N-methyl-N-nitrosourea

In sterile cultures of free barley embryos, N-methyl-N-nitrosourea (MNU) caused a decrease in the size of both template [¹⁴C]-labeled DNA and of daughter [³H]DNA strands as determined in alkaline sucrose gradients, and inhibited the rate of [³H]thymidine incorporation. In addition, duplexes containing [³H]-daughter DNA analyzed in BND cellulose contained more single-stranded regions in MNU-treated embryos than in the corresponding control. Incubation of MNU-treated embryos in nutrient medium for up to 18 h after the [³H]-labeling permitted the recovery of small-sized daughter DNA to full-sized strands and led to the enhancement of double-strandedness of DNA duplexes containing [³H]-labeled strands. If [³H]-labeling had been carried out 8–10 h after the MNU treatment, the size of daughter DNA, the proportion of double-strandedness and the rate of thymidine uptake into DNA partially increased in comparison with rates observed when labeling had been done just after or 3 h after the MNU treatment, but these variables did not reach the values of the corresponding controls.     

DNA and histone synthesis are uncoupled during germination of maize embryos

The rate of synthesis of DNA and histones was studied in germinating maize embryos as a function of the length of the germination period. To that end excised embryos from seeds germinated for different periods of time were pulse labelled either with [¹⁴C]protein hydrolysate or with [³H]TdR. Specific activities were determined for the total cellular proteins and the total histone fraction obtained by acid-extraction of the cellular homogenate and BioRex70 ion exchange chromatography. The results show that the early germination period is characterized by a lack of coupling between the histone synthesis and that of the nuclear DNA. The early histone synthesis peak might be necessitated by the reprogramming of the embryo genome that takes place during germination.     

Cytokinin Biosynthesis in Germinating Maize Embryos

Three-day-old germinating embryos of Zea mays were incubatedwith [8-¹⁴C]adenine for 8 and 24 h. Use of high performanceliquid and thin layer chromatography provided evidence for theincorporation of adenine by isolated germinating embryos intotrans-zeatin. These results are at variance with similar labellingstudies and possible reasons for this discrepancy are discussed. Key words: Adenine, germination, maize kernels, zeatin     

Repetitive Somatic Embryogenesis of Ocotea catharinensis Mez. (Lauraceae): Effect of Somatic Embryo Developmental Stage and Dehydration

Repetitive embryogenesis of Ocotea catharinensis from globular/early cotyledonary somatic embryos was successfully supported by WPM supplemented with 22.7 g l⁻¹ sorbitol, 20 g l⁻¹ sucrose, 400 mg l⁻¹ glutamine and 2 g l⁻¹ Phytagel. The best medium to induce repetitive embryogenesis in cotyledonary somatic embryos was half strength WPM supplemented with 20 g l⁻¹ sucrose, 400 mg l⁻¹ glutamine, 1.5 g l⁻¹ activated charcoal and 2 g l⁻¹ Phytagel. The mature somatic embryos gradually air dehydrated showed repetitive embryogenesis after subculture on half strength B5 medium supplemented with 20 g l⁻ sucrose, 20 g l⁻¹ Phytagel, 1.5 g l⁻¹ activated charcoal, 115.6 μM gibberellic acid and 214.8 μM naphthaleneacetic acid. The early cotyledonary, cotyledonary and mature somatic embryos tolerated respectively 95, 86 and 54% fresh weight losses without losing their repetitive embryogenesis potential. Cotyledonary and mature somatic embryos gradually air dehydrated in sealed Petri dishes showed 40–41% repetitive embryogenesis respectively after 20 days and 12 weeks desiccation storage. Repetitive embryogenesis in cotyledonary somatic embryos was significantly stimulated by chemical dehydration with 0.5 M sorbitol and 56% repetitive embryogenesis was achieved even after exposure to 2 M sorbitol for 24 h. The cotyledonary somatic embryos when alginate-encapsulated showed 47% repetitive embryogenesis even after chemical dehydration in 1.5 M sorbitol for 4 days followed by 1 h air dehydration, but failed to survive to the same dehydration conditions without encapsulation. The optimized repetitive embryogenesis and desiccation protocols offer the possibility to use in vitro techniques for continuous reliable somatic embryo production and short term germplasm storage.     

Lecithin synthesis during microbody biogenesis in watermelon cotyledons

During the normal development of watermelon seedlings, leaf peroxisomes succeed glyoxysomes as the major microbody component in the cotyledons. The possibility has thus been raised that the two organelles are ontogenetically related; that leaf peroxisomes are derived from glyoxysomes. The behavior of lecithin, an important constituent of the membranes of both kinds of organelle was examined in this study. Using labeled choline as a precursor of lecithin, its incorporation into various membrane fractions was followed during the period when glyoxysomal activity was declining and that of leaf peroxisomes increasing after exposure to light. The results showed that glyoxysomal membrane was selectively destroyed during this period. Furthermore, from double-labeling experiments using [¹⁴C]- and [³H]choline it was shown that newly synthesized lecithin was incorporated into the membranes of the developing leaf peroxisomes. These results support the thesis that leaf peroxisomes are not derived from glyoxysomes and instead represent two distinct microbody populations.     

Germination of Rye Embryos Following Hydration-Dehydration Treatments: Enhancement of Protein and RNA Synthesis and Earlier Induction of DNA Replication

On germination of low viability embryos of rye, var. LovaszPatonai, the rate of protein synthesis increases during theearly hours of imbibition and high rates of DNA replicationcommence around the 9th hour. If embryos are imbibed for 3 or6 h then dehydrated back to their original weight, their rateof protein synthesis when next imbibed closely corresponds tothat of embryos germinated for a period equal to that of thehydration pre-treatment plus the duration of the second imbibition.Pre-treatment also enhances subsequent RNA synthesis and embryoshydrated for 9 h then dehydrated start major DNA synthesis atonce as water is again supplied. Many changes occurring duringthese periods of hydration pre-treatment must therefore be stableto subsequent dehydration. Damage occurs to areas that are firstactive in protein and RNA synthesis if pre-treatments extendbeyond 9 h and subsequent germination of the embryo is thenimpaired. The implications of these results are discussed inrelation to the effects of hydration pre-treatments upon enhancedgermination and the stability to dehydration of the productsof early RNA and protein synthesis.     

Incorporation of D-[14C]galactose into organelle glycoprotein in castor bean endosperm

Excised casto bean (Ricinus communis L.) endosperm tissue supplied with [¹⁴C]galactose incorporates radioactivity into particulate cell components. Fractionation of homogenates established that ¹⁴C-labeled trichloroacetic acid-insoluble material was located primarily in the microsomal and glyoxysomal fractions. The capacity of the tissue to incorporate [¹⁴C]galactose into organelle glycoprotein varied during seedling development, increasing during the first 3 days of germination and subsequently declining. The kinetics of incorporation into the major organelle fractions of 2-day old endosperm tissue showed that the endoplasmic reticulum was immediately labeled whereas a lag period preceded the labeling of glyoxysomes. Sub-fractionation of the isolated organelles established that the greatest proportion of the [¹⁴C]-galactose labeled glycoprotein was located in the membrane, although a significant incorporation into the matrix protein was also observed.The results indicate that the addition of the carbohydrate moiety to the polypeptide cores occurs in the endoplasmic reticulum during or immediately after their synthesis on membrane-bound ribosomes.Abbreviations ER endoplasmic reticulum - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Protein synthesis patterns in barley embryos during germination

Summary The incorporation pattern of [¹⁴C] amino-acid into protein during the first 8 h of germination in isolated barley embryos (Hordeum vulgare) is described. Two maxima were recognised. The first, at 4 h, was entirely accounted for by scutellum activity and the second, at 8 h, coincided with active radicle elongation. An intervening minimum was situated at 5.5 h. The first peak was insensitive to actinomycin-D but the second showed a partial inhibition by this compound. Only slight changes in enzyme activity were associated with these periods of increased synthesis. Incorporation of [17-¹⁴C] kaurenoic acid into compounds co-chromatographing with gibberellins was followed over the same period in both embryos and scutella and high activity was found after only 2–4 h. It is concluded that, on the basis of protein synthetic activity, the scutellum is the most probable source of the initial gibberellin stimulus.     

Early DNA synthesis during the germination of wheat embryos

Both [³H]thymidine and [³H]deoxyadenosine were found to be incorporated into the nuclear DNA of wheat embryos immediately after dry embryos were allowed to imbibe aqueous solutions of the radioactive precursors. The early labelled DNA sedimented in a manner suggesting that replicative intermediates were already formed within the first 90 min of germination. However, aphidicolin remained without any effect on this early DNA synthesis. Likewise, a cell-free system derived from early embryos incorporated [³H]dCTP into DNA independently of the presence of aphidicolin. On the contrary, dideoxyTTP inhibited the DNA synthesis considerably. It is concluded that a proportion of the resting wheat embryo cells is able to initiate a replicative DNA synthesis immediately upon imbibition. The synthesis seems, however, to proceed with the participation of a γ-like, rather than an α-like, DNA polymerase.     

Enhancing carrot somatic embryos survival during slow dehydration, by encapsulation and control of dehydration

In order to obtain dry artificial seeds, carrot somatic embryos were encapsulated and dehydrated. Encapsulation in some hydrogels delayed the dehydration and preserved the water content of carrot somatic embryos. In particular, a matrix made of alginate with gellan gum was found to be the most efficient in maintaining a high water activity (a_w) around somatic embryos. By delaying dehydration, and also rehydration, encapsulation seemed to protect somatic embryos against desiccation and imbibition damages, giving better germination and emergence of cotyledons. Matrices made of alginate mixed with kaolin or gellan gum were particularly adapted to protect the embryos during the dehydration. Apart from the matrix composition, the control of dehydration speed enhanced the survival and regeneration of encapsulated-dehydrated somatic embryos. Using a slow dehydration protocol (95-15% RH—relative humidity into the chamber—in 11.5 days), it was possible to exert different dehydration speeds. Slowing the dehydration between 70 and 45% RH stabilized the water activity (a_w) of the encapsulation matrix, and enhanced the survival and regeneration frequencies of encapsulated-dehydrated embryos. In the absence of any maturing pretreatment, alginate-gellan gum encapsulated carrot somatic embryos, dehydrated to 15% RH, and rehydrated in moistured air (90% RH), germinated up to 72.9%. Therefore, encapsulation in alginate-gellan gum, combined with a slow dehydration, leads to enhance the somatic embryos' desiccation tolerance.     

Template active chromatin structures: Degradation by deoxyribonuclease I

Chicken embryos were pulse-labelled in vivo with [³H]uridine (10 min), the chromatin isolated and treated with DNAse I. The residual chromatin was separated from the degradation products by centrifugation. The nascent pulse-labelled RNA is completely recovered in the residual chromatin even after prolonged incubation with DNAase I, whereas the DNA is completely degraded to 80 base polynucleotide fragments and smaller fragments.Abbreviations cDNA DNA complementary to mRNA - DOC deoxycholate - EDTA ethylendiamintetraacetic acid - SDS sodium-dodecylsulfate     

Effects of sodium tungstate on the nuclear uptake of glucocorticoid-receptor complex from rat liver

Effects of sodium tungstate on the nuclear uptake of rat liver cytosolic glucocorticoidreceptor complex were examined at pH 7. The nuclear uptake of heat-activated [³H]triamcinolone acetonide-receptor complex was blocked completely in the presence of 1 mm tungstate. A preincubation of nuclear preparation with tungstate (>0.1 mm) blocked the subsequent uptake of [³H]triamcinolone acetonide-receptor complex. When the tungstate-treated nuclear preparation was washed with 0.3 M KCl, its [³H]triamcinolone acetonide-receptor complex binding capacity recovered to 50% of that of control samples with no tungstate treatment. A preincubation of chromatin with tungstate yielded similar results. The nuclear-bound [³H]triamcinolone acetonide-receptor complex, formed either by an in vivo administration of [³H]triamcinolone acetonide or by an in vitro incubation of glucocorticoid-receptor complex with isolated nuclei, was extracted by tungstate in a concentration-dependent manner. The majority of nuclear-bound [³H]triamcinolone acetonide could be extracted with 0.1 and 1 mm tungstate from in vitro- and in vivo-labeled nuclei, respectively. The tungstate-extracted steroid-receptor complexes sedimented in 4–5 S and 3.3–3.5 S region in 10 mm KCl- and 0.3 mm KCl-containing sucrose gradients, respectively. Tungstate treatment caused an irreversible loss of the nuclear binding capacity of [³H]triamcinolone acetonide-receptor complex which could not be recovered after dialysis. These studies indicate that tungstate affects both glucocorticoidreceptor complex and certain nuclear or chromatin proteins.     

Redistribution of Tritium during Germination of Grain Harvested from myo-[2-H]Inositol- and scyllo-[R-H]Inositol-Labeled Wheat

Wheat kernels from myo-[2-³H]inositol- or scyllo-[R-³H]inositol-labeled plants (Sasaki and Loewus 1980 Plant Physiol 66: 740-745) were used to study redistribution of ³H into growing regions during germination. Most of the labeled 1-α-galactinol (or the analogous scyllo-inositol galactoside) was hydrolyzed within 1 day. Water-soluble phytate was dephosphorylated within 3 days. A large reserve of bound phytate continued to release myo-inositol over several days. Translocation of free myo-inositol to growing regions provided substrate for the myo-inositol oxidation pathway and incorporation of ³H into new cell wall polysaccharides.