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.     

Effect of fusicoccin and gibberellic acid on primary dormant Avena fatua caryopses

Fusicoccin induced germination in dormant and partially afterripened dormant caryopses of Avena fatua L. The rate of caryopsis germination was slower and final percentage germination lower in the highly dormant inbred line M73 at a given concentration of fusicoccin than in the dormant caryopses of line AN265. Gibberellic acid was more effective than fusicoccin in breaking dormancy in both lines. Promotion of germination of dormant caryopses by fusicoccin was inhibited by a 6-day pretreatment with (2-chloroethyl)trimethylammonium chloride.
The basal rate of proton efflux from embryos isolated from dormant and fully afterripened line AN265 caryopses was similar. Addition of fusicoccin increased the rate of proton efflux from the isolated embryos of dormant and afterripened caryopses by nearly 400%. Gibberellic acid had no effect on the rate of proton extrusion. The uptake of ⁸⁶Rb⁺ in dormant and afterripened A. fatua embryos was similar after a 2 h uptake period. The addition of fusicoccin to the medium doubled the uptake of ⁸⁶Rb⁴ by dormant and afterripened embryos. Gibberelleic acid had no effect on the uptake of ⁸⁶Rb⁺ by isolated embryos from either dormant or afterripened caryopses. The experimental results indicate that gibberellic acid is more versatile in its action than fusicoccin, and gibberellic acid may facilitate dormant A. fatua caryopsis germination by stimulating mechanisms other than the direct H⁺ efflux and K⁺ uptake at the membrane level.     

Induction of vivipary in Avena fatua

An investigation was conducted under controlled conditions to determine whether treatments designed to maximize the availability of water during seed development could induce viviparous germination in wild oats ( Avena fatua L.). Panicles of three genetic lines, which differed in their degree of dormancy, were kept in darkness at ca 100% RH and 20±1°C and were either supplied with water through the cut end of the rachis or left attached to the plant which was exposed to light. In the non-dormant line, germination of both primary and secondary caryopses on excised panicles increased with their stage of development when treated, i.e., 0, 5 and 10 days after anthesis. Germination of primary caryopses varied between 70 and 80% and was similar on both isolated and attached panicles treated at 10 and 5 days after anthesis, respectively. The percentage germination was considerably lower in all treatments of the two dormant lines and was inversely related to the genetically determined difference in their degree of dormancy. In these dormant lines germination was significantly lower on the intact plant than on the detached panicles. Water potential measurements suggested that this difference may be due partly to the transpiration-induced negative ψ_xyin the stem which may contribute to the inhibition of embryo growth and thus to the prevention of viparous germination.     

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.     

The physiological basis of seed dormancy in Avena fatua L. I. Action of the respiratory inhibitors sodium azide and salicylhydroxamic acid

The dormancy breaking effect of sodium azide was studied in seeds of several genetically pure lines of Avena fatua L. isolated from field populations. Sodium azide (0.8 and 1 m M ) induced germination in several dormant lines (characterized by long term dormancy) after two weeks of treatment. By about five weeks, germination was nearly complete in azide treated seeds as compared to little or no germination in controls. (2-chloroethyl) trimethylammonium chloride (an inhibitor of gibberellin biosynthesis) completely inhibited the azide effect suggesting that stimulation of germination by azide requires gibberellin biosynthesis. Azide was very effective in breaking dormancy in lines AN-51, AN-86, AN-127 and AN-265, but failed to induce germination in Montana 73. In this line there was a synergism between azide and gibberellic acid in promotion of germination. Thus, at least two metabolic blocks are involved in the stimulation of germination in this line. Salicylhydroxamic acid (an inhibitor of alternative respiration) at 3 m M completely inhibited the germination induced by 1 m M azide. At this concentration, salicylhydroxamic acid did not inhibit germination in 1) genetically nondormant seeds (line SH-430), 2) afterripened seeds of a dormant line (AN-51), and 3) gibberellic acid-treated dormant seeds. These findings suggest that salicylhydroxamic acid-sensitive process(es), presumably alternative respiration, is necessary for the stimulation of germination in the presence of azide, but not in the germination of genetically nondormant, gibberellic acid-treated dormant, or afterripened seeds.     

Temperature control of germination and its possible role in the survival of a non-dormant population of Avena fatua

Germination of freshly harvested seeds of a non-dormant (ND) line (Stonehouse 319) of wild oats ( Avena fatua L.) was inhibited by incubation of the seeds at relatively high temperatures of 25 and 30°C. The germination inhibition in these seeds appeared to be a case of thermo-inhibition which was the direct effect of hightemperature treatment (HIT), since it did not persist after transferring the seeds to an optimum germination temperature of 20°C. Even a prolonged HTT of 30°C for over 5 weeks did not prevent germination of about 80% of the seeds transferred to 20°C. However, in a significant proportion of the seeds, thermo-dormancy was induced by 10 days of HTT at 30°C if the seeds were then incubated at sub-optimal temperatures of 5 to 15°C. This thermo-dormancy would appear to be 'restrictive' in form, since its expression was restricted to very specific conditions. Relatively low inclubation temperaturs of 5 and 10°C markedly slowed germination whether HTT was applied or not. The results suggest that thermo-inhibition and thermo-dormancy, induced during seasonal temperature fluctuations, may provide a survival mechanism for seeds of such ND lines as Stonehouse 319.     

The physiological basis of seed dormancy in Avena fatua. IX. Characterization of two dormancy states

The dormancy-breaking effect of several known germination promoters was studied in 9 genetically pure lines of Avena fatua L. during a period of controlled after-ripening. Changes in the germination response show at least two dormancy states in the caryopses of these lines. The first state is overcome by a short period of after-ripening and is insensitive to nitrate and azide, while the second state is more persistent and is sensitive to nitrate and azide. Both states are sensitive to gibberellic acid (OA,) and ethanol. In the most dormant lines a third ethanol-insensitive dormancy state is present. The duration of both major dormancy states was related to several environmental factors influencing plant growth and seed storage. Duration was increased in caryopses produced from plants matured under low temperatures (15°C) and decreased in caryopses produced from plants matured under high temperatures (25°C). Duration was increased in caryopses after-ripened under low temperatures (4°C) and decreased in caryopses after-ripened under high temperatures (45°C). Dehulling the seeds prior to after-ripening reduced the duration of both major dormancy states. The multiple state dormancy system and its environmentally induced plasticity are discussed with reference to previous explanations of the dormancy mechanism in wild oats.     

The physiological basis of seed dormancy in Avena fatua. VII. Action of organic acids and pH

Citric, succinic, fumaric, malic, pyruvic and lactic acids induced germination in two genetically pure dormant lines of Avena fatua L. The sensitivity to these acids was low immediately after harvest and increased markedly after a period of dry after-ripening. Because the acids could only overcome dormancy in partly after-ripened caryopses, the mode of their action in these caryopses differed from that of another germination promotor, ethanol, and was similar to that of the germination promoter, sodium nitrate. The mode of action of the organic acids on the partly after-ripened caryopses through lowering pH was indicated by the observation that other non-metabolic weak acids could also break dormancy while neutral pH value salt solutions of some of the tested acids were inactive. The dose-response curves of citric acid for the stimulation of germination and for oxygen uptake were similar, indicating that this organic acid may stimulate germination by promoting oxygen uptake. A time sequence study showed that citric acid stimulated oxygen uptake before the first visible signs of germination. Stimulation of germination and oxygen uptake over a range of pH values showed that those values of pH which stimulated germination also stimulated oxygen uptake, indicating that the ability to stimulate oxygen uptake was not confined to organic acids. The stimulation of both germination and oxygen uptake by citric acid was not inhibited by salicylhydroxamic acid, an inhibitor of alternative respiration, therefore stimulation of both germination and oxygen uptake by citric acid does not require the operation of the alternative pathway of respiration. The function of weak acids as promoters of oxygen uptake is discussed with reference to the breakage of dormancy in partly after-ripened caryopses and the involvement of various respiratory pathways is indicated.     

The physiological basis of seed dormancy in Avena fatua VI. Respiration and the stimulation of germination by ethanol

Ethanol induced germination in several partly after-ripened dormant lines of Avena fatua L. The dose-response curves for the stimulation of germination and for oxygen uptake were similar, indicating that ethanol may stimulate germination by promoting oxygen uptake. A time-sequence study showed that ethanol stimulated oxygen uptake by as much as 70% prior to the first visible signs of germination. A similar methanol treatment failed to induce germination or significantly elevate oxygen uptake, indicating that the promotive effects of ethanol are not common to all alcohols. The stimulation of both germination and oxygen uptake by ethanol was not inhibited significantly by salicylhydroxamic acid, an inhibitor of alternative respiration. Thus, stimulation of germination and oxygen uptake by ethanol does not require the operation of the alternative pathway of respiration. Similarly, the stimulation of germination and oxygen uptake by ethanol were not inhibited by sodium azide, an inhibitor of cytochrome-mediated respiration. However, both germination and oxygen uptake were prevented when salicylhydroxamic acid and sodium azide were administered together. Thus, stimulation of these events by ethanol requires only the operation of one or other of these pathways of respiration; a specific requirement for the operation of the alternative pathway of respiration does not exist. The function of ethanol as a promoter of respiration is discussed with reference to dormancy and involvement of the Krebs cycle.     

Effects of salicylhydroxamate on respiration, seed germination and seedling growth in Avena fatua

The effects of inhibitors of alternative respiration [salicylhydroxamate (SHAM) and propyl gallate (PG)] on germination, seedling growth and O₂ uptake in Avena fatua L. (wild oats) were studied. SHAM did not inhibit germination or O₂ uptake prior to germination. SHAM-sensitive (alternative) respiration, therefore, cannot be a pre-requisite for germination. Following germination, both chemicals inhibited seedling growth with the root being more susceptible than the shoot. SHAM concentrations that inhibited root growth by 90 to 95%, inhibited O₂ uptake of 1 cm root apices by less than 15%. While sodium azide (a cytochrome-oxidase inhibitor; 1 m M ) alone inhibited O₂ uptake by only 40 to 50%, in the simultaneous presence of SHAM (or PG), O₂ uptake was inhibited by 90 to 99%. Thus: 1) respiration of wild oat seedling root apices is predominantly cytochrome-mediated and incomplete inhibition of O₂ uptake in the presence of azide alone is due to diversion of electrons to the alternative pathway and 2) even though these roots have little alternative respiration, they maintain the capacity to support a much greater flux of electrons via this path way. SHAM and PG at concentrations (0.05 to 0.4 m M ) which inhibited O₂ uptake significantly in the presence (but not in the absence) of azide had little effect on root growth suggesting that an effect(s) other than that on respiration is involved in the inhibition of root growth at higher concentrations. The effect of SHAM on wild oat root growth is not selective as it also inhibits growth of a number of crop species.     

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)     

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

A comparative study of protein synthesis has been carried out with embryos excised from dormant (D) and non-dormant (ND) caryopses of the wild oat. Although D embryos imbibed in water or ND embryos imbibed in abscisic acid do not germinate, they incorporate [¹⁴C]leucine into TCA-insoluble material for the first 48 h as readily as embryos that do germinate (ND embryos imbibed in water, or D embryos imbibed in gibberellic acid). Pulsechase experiments with [¹⁴]leucine show that in both D and ND embryos the proteins associated with the membranes undergo turnover. The rates of decay of incorporated radioactivity are similar in both dormant and germinating embryos up to 98 h following embryo excision. Fractionation of the membrane proteins in SDS-polyacrylamide gels indicates that the different polypeptides have different rates of turnover. It is concluded that membrane proteins in imbibed D embryos are in a state of constant turnover, and that this is a part of the replacement processes necessary to maintain the integrity of hydrated cells. The continuation of such synthetic events could account for long term survival of dormant Avena fatua in the imbibed state.Abbreviations CCRSE cytochrome relative stain equivalents - D dormant - ND nondormant - ABA abscisic acid - GA gibberellic acid GA₃     

Dormancy and enzyme levels in seeds of wild oats

Nine enzymes were compared in dry and steeped mature dormant and non-dormant seeds of wild oats. In dry seeds only glutamate-pyruvate transaminase and phosphoglycerate kinase were greater in non-dormant seeds. In steeped non-dormant seeds glucose-6-phosphate dehydrogenase activity doubled while the enzyme declined sharply in dormant seeds. Increases in isocitrate dehydrogenase, glutamate-oxaloacetate transaminase and acid phosphatase in non-dormant seeds, during steeping, are consistent with the hypothesis that the pentose phosphate and glycolysis-tricarboxylic acid pathways are involved in the control of dormancy of wild oat seed.     

Growth and development of embryo parts during the germination of caryopses of the wild oat (Avena fatua L.)

Wild oat (Avena fatua L.) caryopses were germinated on moist filter paper and under water in the presence and absence of hydrogen peroxide (H₂O₂). The sequential growth and development of embryo parts were studied. Germination, as indicated by radicle emergence, was least and slowest in caryopses submerged in deoxygenated water. The coleorhiza in such caryopses elongated much earlier than the root, in contrast to the other treatments where the coleorhiza and the root emerged at about the same time. In caryopses incubated on moist filter paper all embryo parts showed considerable growth. In H₂O₂ treated caryopses only the epicotyl showed substantial growth over the experimental period. In all treatments the first mitotic peaks were noticed at the same period. The occurrence of these early nuclear divisions may be due to release of 4 C nuclei from inhibition by the uptake of water during caryopsis imbibition. The mitosis continued in the radicle of the embryo in those caryopses germinating on moist filter paper, indicating occurrence of DNA synthesis. In the other two treatments, however, few divisions were detected. Here the early growth of the root, causing caryopsis germination, was due to cell elongation, especially in the proximal part of the root.     

Avena fatua L. seed and seedling nutrient dynamics as influenced by mycorrhizal infection of the maternal generation

Abstract. The objective of this study was to determine how mycorrhizal infection of one generation of plants influences the nutrient dynamics of seeds and seedlings comprising the subsequent generation. We showed that, for Avena fatua L., seeds produced by mycorrhizal (M) plants consistently contained significantly more phosphorus (particularly the phytate P and residual P fractions) than seeds produced by non-mycorrhizal (NM) plants. We also followed the development of spikelets produced by M and NM plants. The rates of increase in spikelet dry weight and nitrogen content were largely unaffected by mycorrhizal infection. However, the rate of P accumulation into spikelets was significantly increased by mycorrhizal infection. Greater endosperm P reserves in seeds produced by M plants were associated with greater rates of P accumulation in resultant seedlings. Moreover, offspring plants (all NM) produced by M mother plants had significantly higher root and rhizosphere phosphatase, ATPase and phytase activities than offspring plants produced by NM mother plants. This persistent maternal effect has never before been described. Our results suggest that mycorrhizal infection of one generation of plants may have substantial positive effects on the offspring generation, and thus, may influence plant population dynamics.     

Identification of differentially expressed cDNA sequences in ovaries of sexual and apomictic plants of Brachiaria brizantha

The isolation of genes associated with apomixis would improve understanding of the molecular mechanism of this mode of reproduction in plants as well as open the possibility of transfer of apomixis to sexual plants, enabling cloning of crops through seeds. Brachiaria brizantha is a highly apomictic grass species with 274 tetraploid apomicts accessions and only one diploid sexual. In this study we have compared gene expression in ovaries at megasporogenesis and megagametogenesis of sexual and apomictic accessions of B. brizantha by differential display (DD-PCR), with 60 primer combinations. Specificity of 65 cloned fragments, checked by reverse northern blot analysis, showed that 11 clones were differentially expressed, 6 in apomictic ovaries, 2 in sexual and 3 in apomictic and sexual, but at different stages. Of the 6 sequences isolated that were preferentially expressed in the apomictic accession: one sequence was from ovaries at megasporogenesis stage; three were from megagametogenesis stage; two were from both stages. Of the two sequences isolated from the sexual accessions, one showed expression in ovaries at megagametogenesis, while the other sequence was shown to be specific to both stages. Three sequences were from megasporogenesis stage in apomicts but were also detected at megagametogenesis in sexual plants. Sequence analysis showed that 5 of the 11 clones had no apparent homologues in the protein database. Some of the clones identified as apomictic-specific shared homology with known genes enabling their functional annotation. The relationships of these functions to the generation of the apomictic trait are discussed.     

Recovering and reamplifying of the differentially expressed cDNA bands isolated from mRNA differential display

Methods for retrieving and reamplifying the differentially expressed cDNA bands have been modified. Direct reamplification of differentially expressed bands after cutting from a polyacrylamide gel (PAG) followed by a simple rinse and crush step has proved to be more convenient and effective than the traditional glycogen-precipitation method. Combination of 30 cycles of differential display (DD) polymerase chain reaction (PCR) and 20 cycles of standard PCR reaction also yielded higher reamplification rates.