Nonstructural carbohydrates in dormant and afterripened wild oat caryopses

Nonstructural carbohydrates were determined in both embryo and endosperm of dormant (nongerminating) and afterripened (germinating) intact caryopses of wild oat ( Avena fatua L.). No changes in endosperm starch or soluble sugar were observed at the onset of germination (18 h). No changes in glucose, fructose, sucrose or starch within dormant or afterripened embryos correlated with onset of visual germination. In afterripened embryos, depletion of raffinose (18 h), stachyose (18 h) and galactose (24 h) was correlated with germination. In contrast, raffinose-family oligosaccharide levels in dormant embryos remained constant for 7 days following imbibition. Germination of isolated dormant embryos on 88 m M galactose-containing media was accompanied by decreased endogenous levels of raffinose and stachyose. Isolated embryos from dormant caryopses incorporated ¹⁴C from ¹⁴C-fructose into both raffinose and stachyose during 24 h of imbibition. In contrast, no ¹⁴C incorporation into stachyose was observed in embryos from afterripened caryopses. No ¹⁴C incorporation into raffinose was observed at 18 and 24 h. When in vitro activities of α galactosidase were measured, no temporal differences between dormant or afterripened caryopses were detected in either embryo or endosperm tissue. Although the mechanism associated with differences in utilization of raffinose and stachyose is yet unidentified, alterations in raffinose-family oligosaccharide metabolism in the embryo appear to be a unique prerequisite for afterripening-induced germination.     

ATP Synthesis during Water Imbibition in Caryopses of Genetically Dormant and Non-dormant Lines of Wild Oat (Avena fatua L.)

Levels of ATP in dry caryopses of wild oats (Avena fatua L.)were much lower than in imbibed seeds of the seven geneticallypure lines surveyed. The ATP content of the lines with highgenetic dormancy was consistently lower than the ATP contentof genetically non-dormant lines, but no significant correlationwith depth of dormancy was found apart from this. Massive increasesin ATP content occurred within 30 min of water uptake by caryopsesof both dormant and non-dormant lines. The synthetic pathwaystudied utilized inorganic phosphate with great avidity to formATP. The ability to form ATP upon imbibition was present inboth embryo and de-embryonated caryopsis. The ATP levels attainedin imbibing caryopses appeared sufficient to support considerablesynthetic activity, and this reduced the possibility that adeficiency in ATP was responsible for the maintenance of dormancyin such imbibed seeds. The low levels of inorganic phosphatein the embryos of genetically dormant lines of wild oat couldrepresent a limiting factor, if the active formation of ATPupon water imbibition resulted in a scarcity of phosphate forother reactions essential to germination. Key words: Avena fatua, ATP synthesis, Inorganic phosphorus, Seed dormancy, Germination, Water uptake     

Dormancy-associated embryonic mRNAs and proteins in imbibing Avena fatua caryopses

The mechanisms controlling seed dormancy maintenance and release are not understood. To characterize the molecular events accompanying dormancy release, two-dimensional gel electrophoresis was used to monitor changes in soluble proteins and in vitro translation products of embryonic mRNA populations during imbibition of dormant and nondormant (after-ripened) Avena fatua L. caryopses. No differences were observed between in vitro translation products of mRNA extracted from dry dormant and nondormant embryos. However, the expression patterns of several imbibition- and germination-associated mRNAs were temporally modulated during the first 24 h of imbibition. Two dormancy-associated mRNAs, represented by polypeptides D₁ and D₂, were differentially overexpressed in dormant embryos after 3 h of imbibition. mRNA levels for D₁ and D₂ were about 8- and 3-fold higher, respectively, in dormant embryos than in nondormant embryos after 3 h of imbibition. Overexpression of D₁ continued through 12 h of imbibition, while expression of both mRNAs fell to low and equivalent amounts in dormant and nondormant embryos after 24 h. Similar dormancy-associated changes in two soluble proteins were observed during imbibition. The results demonstrate that steady-state levels of specific mRNAs and proteins change during early imbibition of dormant and nondormant A. fatua embryos and indicate that these changes may be associated with differential gene expression responsible for the maintenance of dormancy.     

Interaction of karrikinolide and ethylene in controlling germination of dormant <Emphasis Type="Italic">Avena fatua</Emphasis> L. caryopses

Caryopses of Avena fatua L. are dormant after harvest and germinate poorly at 20 °C. Dormancy was released by after-ripening the dry caryopses in the dark at 25 °C for 3 months. Karrikinolide (butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, KAR₁), in contrast to exogenous ethylene and the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid (ACC), completely overcame dormancy. The effect of KAR₁ was not affected by aminoethoxyvinylglycine (AVG), α-aminoisobutyric acid (AIB) and CoCl₂, inhibitors of ACC synthase and oxidase, respectively. 2,5-Norbornadiene (NBD), a reversible inhibitor of ethylene binding to its receptor, counteracted the stimulatory effect of KAR₁. Ethylene, ethephon and ACC counteracted and AVG reinforced inhibition caused by norbornadiene. Inhibition due to norbornadiene, applied during the first 3 days of imbibition in the presence of KAR₁, disappeared after transfer to air or ethylene. The obtained results confirm that KAR₁ breaks dormancy and indicate that ethylene alone plays no role in releasing dormancy of Avena fatua caryopses. KAR₁ probably did not relieve dormancy via the stimulation of ethylene biosynthesis. Some level of endogenous ethylene is probably required for ethylene action, which might be required for releasing dormancy by KAR₁ or for subsequent germination of caryopses after removing dormancy.     

Characterization of cDNA clones for differentially expressed genes in embryos of dormant and nondormant Avena fatua L. caryopses

The molecular regulation of seed dormancy was investigated using differential display to visualize and isolate cDNAs representing differentially expressed genes during early imbibition of dormant and nondormant Avena fatua L. embryos. Of about 3000 cDNA bands examined, 5 cDNAs hybridized with mRNAs exhibiting dormancy-associated expression patterns during the first 48 h of imbibition, while many more nondormancy-associated cDNAs were observed. Dormancy-associated clone AFD1 hybridized with a 1.5 kb mRNA barely detectable in dry dormant and nondormant embryos that became more abundant in dormant embryos after 24 h of imbibition. Clone AFD2 hybridized with two mRNAs, a 1.3 kb message constitutively expressed in dormant and nondormant embryos and a 0.9 kb message present at higher levels in dormant embryos after 3 h of imbibition. Nondormancy-associated clones AFN1, AFN2 and AFN3 hybridized with 1.5 kb, 1.7 kb and 1.1 kb mRNAs, respectively, that were more abundant in nondormant embryos during imbibition. Expression patterns of some mRNAs in dormant embryos induced to germinate by GA₃ treatment were different than water controls, but were not identical to those observed in nondormant embryos. DNA sequence analysis revealed 76% sequence identity between clone AFN3 and a Citrus sinensis glutathione peroxidase-like cDNA, while significant sequence similarities with known genes were not found for other clones. Southern hybridization analyses showed that all clones represent low (1 to 4) copy number genes.     

Necessity of gibberellin for stimulatory effect of KAR1 on germination of dormant Avena fatua L. caryopses

Avena fatua L. florets (caryopses enclosed by lemma and palea) were partially dormant at 10–20 °C and did not germinate at temperatures outside this range. After-ripening florets at 25 °C for 12 weeks completely removed dormancy. Caryopses (florets without lemma and palea) were able to germinate totally at 20 °C. Karrikinolide (KAR₁) and gibberellic acid (GA₃) applied at 10–25 °C partially or markedly induced germination of dormant florets and caryopses, respectively. Both florets and caryopses were more sensitive to KAR₁ than to GA₃. To obtain similar effects, 1,000 to 10,000 times lower concentrations of KAR₁ than GA₃ were required. After-ripening with time gradually increased sensitivity of caryopses to these regulators. Likewise, after-ripened, non-dormant caryopses were sensitive to KAR₁ and GA₃. Inhibitors of gibberellin biosynthesis, ancymidol, paclobutrazol and flurprimidol inhibited the effect of KAR₁. This inhibition was reversed by GA₃. Caryopses pre-incubated in water with ancymidol or paclobutrazol in the presence or absence of KAR₁ germinated completely but with different rates after transfer to GA₃. KAR₁ probably requires gibberellin biosynthesis to stimulate germination of dormant Avena fatua L. caryopses. Both KAR₁ and GA₃ increased α-amylase, β-amylase and dehydrogenases activities during imbibition before visible germination occurred.     

Releasing primary dormancy in <Emphasis Type="Italic">Avena fatua</Emphasis> L. caryopses by smoke-derived butenolide

The effect of smoke and smoke-derived butenolide in releasing dormancy of caryopses (referred to as seeds) of the economically important weed Avena fatua L. was studied. Seeds of A. fatua are dormant after harvest. Both smoke-water and butenolide, applied continuously, removed dormancy in darkness at 15, 20 and 25°C and slightly at 30°C. Butenolide was very active at a concentration of 10⁻⁸ M. Butenolide at 10⁻⁸ M was also able to remove dormancy at 20°C when applied for 12 or 24 h at 4°C or for 3 to 24 h at 20°C. Sensitivity to butenolide decreased with longer preincubation times in water. This compound was less effective in releasing dormancy in the light than in darkness. Dormancy release by butenolide involves induction of cell-cycle activity just before coleorhiza protrusion. Stimulatory effects of smoke-water and butenolide were also observed in respect of seedling growth and vigor.     

Water uptake and distribution in non-dormant and dormant wild oat (Avena fatua L.) caryopses

The influence of seed coat modification and light quality onwater uptake and distribution in caryopses of dormant and non-dormantlines of wild oat (Avena fatua L.) was determined using NMRmicroimaging. Non-dormant seeds absorbed water more rapidlythan dormant seeds during imbibition on distilled water. Thiseffect was detected first in the embryo-scutellar region (8h) and later in the proximal endosperm (12 h). Cutting the testaand pericarp close to the embryo or scarification with KOH promotedrapid embryo/scutellum hydration and germination. Cutting atthe middle part of the caryopsis did not enhance embryo hydrationnor did it greatly improve germination. The sensitivity of waterdistribution to the phytochrome germination effect was examined.Significant differences in imbibitional water uptake by embryos-scutellumtissue were detected by 18 h following red-light (germinationpromoter) compared with far-red (germination inhibitor) treatment.The results indicated that both the rate and the sequence ofembryo/scutellum hydration were important in initiating germinationin dormant seeds. A refinement of the model that describes waterimbibition in wild oat seeds during the early stages of germinationis discussed. Key words: Water uptake, water distribution, Avena fatua, seed coat modification, light quality, dormant and non-dormant seeds     

Participation of the tightly-bound (putative cytoskeleton-bound) polysomes in translation during germination of dormant and non-dormant cereal caryopses

Research was done on dormant and non-dormant barley cv. Ars caryopses and triticale cv. Grado caryopses treated and non-treated with abscisic acid (ABA). During germination higher participation of populations of so-called tightly-bound polysomes (TBP) in embryos of dormant barley caryopses was observed, as well as their high metabolic activity. In embryos of triticale caryopses of which dormancy was imposed in an artificial way by ABA (100 microM), the strongest incorporation of 14C-amino acids into nascent polypeptide chains in vivo was found in population of TBP, as well as the highest participation among three of the studied fractions (free polysomes, membrane-bound polysomes and tightly-bound polysomes). These results may indicate the significant role of TBP (putative cytoskeleton-bound polysomes--CBP) in maintaining dormancy during imbibition of cereal caryopses.     

Studies in Wild Oat Seed Dormancy: I. THE ROLE OF ETHYLENE IN DORMANCY BREAKAGE AND GERMINATION OF WILD OAT SEEDS (AVENA FATUA L.)

Seed of Avena fatua were shown to exhibit a characteristic loss of dormancy during dry storage at 25 C, whereas similar seed stored at 5 C maintained dormancy. 2-Chloroethylphosphonic acid was shown to increase germination of partly dormant seed imbibed under certain temperature regimes; a similar effect could not be established for fully dormant or fully nondormant seed. Using gas-liquid chromatography, natural ethylene levels were followed during imbibition of fully dormant and nondormant seed. A large peak in production was observed in the period prior to radicle emergence in the case of the nondormant seed. Measurements of ethylene production taken at 15 C, following periods of after-ripening in moist soil at either 5 or 25 C, indicated that endogenous production was unlikely to be a main cause of dormancy breakage in this species. The possibility that endogenous ethylene could play a role in natural dormancy breakage in aged seeds is discussed. The practical possibilities of 2-chloroethylphosphonic acid as a dormancy breaking agent in a field situation are outlined.     

Role of Endogenous Growth Regulators in Seed Dormancy of Avena fatua: I. Short Chain Fatty Acids

The hypothesis that endogenous short chain fatty acids (C 6-C 10) are important in maintaining seeds of wild oat (Avena fatua L.) in the dormant state by acting as natural germination inhibitors (Berrie, Buller, Don, Parker, 1979 Plant Physiol 63: 758-764) was investigated. When germination of nondormant seeds was inhibited by treatment with short chain fatty acids, the seeds did not revert to a similar biochemical and physiological state as exhibited by dormant seeds. First, nonanoic acid-induced inhibition of seed germination was not reversed by hormone treatments which normally break dormancy in wild oat seeds. Second, nondormant seeds treated with short chain fatty acids maintained similar relative proportions of the pentose phosphate pathway and the Embden-Meyerhoff-Parnas pathway for respiratory glucose metabolism as that found in the nondormant controls. Seeds imbibed in the presence of nonanoic acid lost more amino acids and proteins into the imbibition solution than did the untreated controls, suggesting membrane damage had occurred. Inasmuch as increasing concentrations of nonanoic acid also progressively reduced the growth of the coleoptile and roots of intact seedlings until all growth ceased and no germination occurred, the inhibition of seed germination could be due to a nonspecific inhibition of growth of the embryo, perhaps because of disruption of membrane structure and function. Finally, no correlation between endogenous levels of short chain fatty acids in seeds or isolated embryonic axes and seed dormancy could be demonstrated.     

Secondary dormancy in Avena fatua: Induction and characteristics in genetically pure dormant lines

The induction of secondary dormancy in caryopses of genetically pure dormant lines of Avena fatua L. is described. Seeds harvested from mature plants were after-ripened under controlled conditions (26°C, 25% relative humidity) until fully non-dormant. Secondary dormancy was then induced into these caryopses by incubation on moist filter papers in an aspirated nitrogen atmosphere at 20°C over periods from 3 h to 14 days. These caryopses failed to germinate when returned to an aerobic environment. The dose-response curves for gibberellic acid, sodium azide, sodium nitrite, sodium nitrate and ethanol show that all of these treatments can overcome the induced secondary dormancy. Drying increased the sensitivity of secondary dormant caryopses to these treatments. These treatments overcame secondary dormancy at all times, indicating the presence of only one of the two known blocks to germination that exist during primary dormancy. Similarities between primary and secondary dormancy in A. fatua are discussed.     

Correlative Aspects of Imposition of Dormancy in Caryopses of Aegilops kotschyi

Caryopses of Aegilops kotschyi Boiss. from different positions within the same spikelet differ greatly in their degree of dormancy. Imposition of this dormancy pattern is a correlative phenomenon within the spikelet, inasmuch as the uppermost developing caryopsis is least dormant and its development is associated with the dormancy status imposed on the lower (one or two) caryopses. Differences in pigmentation of the caryopses follow a corresponding pattern.     

Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy

Freshly harvested seeds of Arabidopsis thaliana, Columbia (Col) accession were dormant when imbibed at 25°C in the dark. Their dormancy was alleviated by continuous light during imbibition or by 5 weeks of storage at 20°C (after-ripening). We investigated the possible role of reactive oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25°C, non-dormant seeds produced more ROS than dormant seeds, and their catalase activity was lower. In situ ROS localization revealed that germination was associated with an accumulation of superoxide and hydrogen peroxide in the radicle. ROS production was temporally and spatially regulated: ROS were first localized within the cytoplasm upon imbibition of non-dormant seeds, then in the nucleus and finally in the cell wall, which suggests that ROS play different roles during germination. Imbibition of dormant and non-dormant seeds in the presence of ROS scavengers or donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly harvested seeds of the mutants defective in catalase (cat2-1) and vitamin E (vte1-1) did not display dormancy; however, seeds of the NADPH oxidase mutants (rbohD) were deeply dormant. Expression of a set of genes related to dormancy upon imbibition in the cat2-1 and vet1-1 seeds revealed that their non-dormant phenotype was probably not related to ABA or gibberellin metabolism, but suggested that ROS could trigger germination through gibberellin signaling activation.     

Exogenous putrescine increases the responsiveness of thermodormant <Emphasis Type="Italic">Avena fatua</Emphasis> L. caryopses to karrikinolide and gibberellic acid

A natural feature of freshly harvested Avena fatua L. caryopses is primary dormancy which, however, was relieved partially by putrescine (Put) (10^?2 M) and completely by karrikinolide (KAR₁) (3 × 10^?9 M) or gibberellic acid (GA₃) (10^?5 M). The sensitivity of A. fatua caryopses to these stimulators was adversely affected by supraoptimal temperature (SOT) (30 °C). A reduced germinability of caryopses due to high temperature even after transferring them to lower temperatures (10 or 20 °C) indicated the induction of thermodormancy. The maintenance of relatively constant levels of abscisic acid (ABA) in embryos but not surrounding tissues during SOT treatment was observed. The application of Put either during the SOT treatment or afterwards counteracted the effects of high temperature but had no significant impact on ABA content. The action of exogenous Put in alleviating the loss of responsiveness to KAR₁ and GA₃ imposed by SOT treatment in A. fatua PD caryopses is discussed in reference to the interconnection between ABA and GA metabolism and signaling pathways.     

Possible involvement of the cytoskeleton in the regulation of barley caryopsis dormancy and germination

This study was conducted on barley cv. Ars. caryopses collected at full ripeness and divided into two batches. From one batch (dormant caryopses) polysomes were isolated from embryos immediately after harvesting and after two days of germination. From the other batch (non-dormant caryopses) the same was done after eight months storage in a dry state. A low ionic strength cytoskeleton-stabilizing buffer was used for the isolation of polysomes. Four different fractions of polysomes were examined: free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP). In germs grown from non-dormant caryopses, the first two fractions (FP + MBP) made up about 78 % of the total ribosomal material, whereas in embryos of dormant, imbibed caryopses, two last fractions (CBP + CMBP) made up about 71 %. The percentage of polysomes after 48 hours of imbibition of dormant caryopses in the FP, MBP and CBP was only about 13 % (i.e., 87 % monosomes), whereas a greater proportion (19.4 %) was found in the CMBP. The highest incorporation of ³H-uridine and ¹⁴C-amino acids (after 48 hours of germination and 0.5, 3 and 6 hrs incubation with precursors) took place in trhc CMBP both in dormant and non-dormant caryopses The major amount of the two polysome fractions associated with the cytoskeleton (CBP and CMBP) and the higher activity of CMBP in protein synthesis in embryos of dormant, imbibed triticale caryopses may indicate a significant role for polysomes associated with the cytoskeleton in the control of protein synthesis in dormant and germinating caryopses.     

Evidence of active NADP(+) phosphatase in dormant seeds of Avena sativa L

Freshly-harvested seeds of Avena sativa L. do not germinate when imbibed at temperatures higher than 25 degrees C. This high temperature dormancy is due to the seed coats, and to the low activities of glycolysis and the oxidative pentose phosphate pathway (OPP) in the embryo. The analysis by exclusion chromatography of soluble NADP(+) phosphatase activities of embryos revealed two isoforms: a 37 kDa isoform present in both dormant and after-ripened caryopses, and a second isoform, with an apparent molecular weight of 160 kDa, five times more active in embryos of dormant seeds than in the after-ripened ones, after 6 h of imbibition at 30 degrees C. Moreover, the activity of this 160 kDa isoform was three times less in embryos from dormant caryopses when they were grown at 10 degrees C, a permissive temperature for radicle protrusion. These results suggest a correlation between the activity of the 160 kDa NADP(+) phosphatase and the dormancy state of the caryopsis. The two isoforms differed in the pH required for optimal activity: pH 5.7 and 6.5 for the 37 kDa and the 160 kDa phosphatases, respectively. Furthermore, the 160 kDa NADP(+) phosphatase displayed a strong specificity for NADP(+), whereas the 37 kDa isoform was able to hydrolyse numerous other phosphorylated compounds.