Seed Dormancy in Red Rice (Oryza sativa) (IX. Embryo Fructose-2,6-Bisphosphate during Dormancy Breaking and Subsequent Germination)

Fructose-2,6-bisphosphate (Fru-2,6-bisP) was evaluated as a potential marker for the dormancy-breaking phase or the germination phase before pericarp splitting in red rice (Oryza sativa). During 4 h of imbibition at 30[deg]C, Fru-2,6-bisP of dehulled dormant and nondormant seeds increased to 0.26 and 0.38 pmol embryo-1, respectively. In nondormant seeds, embryo Fru-2,6-bisP content remained stable until the onset of pericarp splitting (12 h) and increased rapidly thereafter. In dormant seeds, Fru-2,6-bisP declined to 0.09 pmol embryo-1 at 24 h. Embryo Fru-2,6-bisP was correlated with O2 uptake of dormant and nondormant seeds. A 24-h exposure of dehulled, water-imbibed, dormant seeds to treatments yielding >90% germination (sodium nitrite [4 mM], propionic acid [22 mM], methyl propionate [32 mM], propanol [75 mM], and propionaldehyde [40 mM]) led to changes in embryo Fru-2,6-bisP that were unrelated to the final germination percentages. Furthermore, a 2-h pulse of propionaldehyde increased Fru-2,6-bisP 4-fold but did not break dormancy. Whereas nitrite and propionaldehyde increased Fru-2,6-bisP to 0.33 pmol embryo-1 after 2 h of contact, propionic acid and methyl propionate did not increase Fru-2,6-bisP above the untreated control. In all cases, further increases in Fru-2,6-bisP occurred after pericarp splitting. However, the plateau Fru-2,6-bisP attained during chemical contact was inversely correlated with elapsed time to 30% germination (r = -0.978). Therefore, although Fru-2,6-bisP is not a universal marker for dormancy release, its rapid increase during nitrite and propionaldehyde treatments suggests that events associated with dormancy breaking can occur within 2 h of chemical treatment.     

RNA synthesis and the germination of light-sensitive lettuce seeds

Summary The germination of lettuce seeds is inhibited by the nucleotide base analogue 6-methylpurine. RNA synthesis has been measured during imbibition and germination as ³²P-phosphate incorporation into RNA species as fractionated by polyacrylamide gel electrophoresis. Seeds were surface sterilized and imbibed in the presence of various antibiotics. RNA preparations from lettuce seeds were coelectrophoresed with ³H-RNA prepared from bacteria to check for bacterial contamination of the seeds. There is a much higher rate of RNA synthesis in illuminated, germinating seeds as compared to dark, non-germinating seeds. This difference does not develop until after 12 hours of imbibition at 27°, which is the time of onset of germination and radicle growth.This investigation was supported by a contract from the United States Department of Agriculture (No. 616-15-3). Journal paper of the Purdue Agriculture Experiment Station.     

Protein Synthesis in Dormant and Non-Dormant Cocklebur Seed Segments

Using the axial and cotyledonary segments of lower cocklebur (Xanthium pensylvanicum Wallr.) seeds, protein synthesis as shown by incorporation of radioactive leucine was examined in relation to their dormant status. During the first 9 h of water imbibition, the protein synthesis was higher in the dormant axes than in the non-dormant, after- ripened ones. When imbibed for more than 12 h non-dormant axes had a higher activity than dormant ones. This was also the case with the cotyledonary segments. Cyctoheximide, an inhibitor of protein synthesis, blocked protein synthesis in the axial tissue regardless of its dormant status, and thereby inhibited germination of the non-dormant seeds. In the dormant seeds, however, cycloheximide at 3 mM slightly stimulated germination without stimulating the C₂H₄ production. Based on these results, it is suggested that in cocklebur seeds there may be some proteinaceous system which is involved in the maintenance of dormancy.     

Changes in RNA and Protein Metabolism Associated with Alterations in the Germination Efficiency of Sunflower Seeds

Precise knowledge of seed quality after harvest and during storageis of particular importance for seed producers. We analyseddifferent sunflower seed lots (Helianthus annuusL.) characterizedby extremes of germination ability. We used RNA analysis tostudy possible changes in gene expression in seeds unable togerminate. Total RNA content was very small in dry seeds showinga low germination ability. Capacity for total RNA synthesisat the onset of imbibition was also reduced in these seeds.In addition, correlations were found between these parametersand germination ability at 19 °C. We demonstrated a highcorrelation between the amount of total RNA in the dry seed,the capacity of RNA synthesis at the onset of imbibition andthe seed moisture content at the time of the harvest. The abilityof dry seed mRNAs to be translatedin vitrowas also reduced andseven polypeptides, from stored mRNAs, were characteristic ofthe cotyledons from high germinability seeds. Germination canthus be affected at several levels including membrane, enzymaticand nucleic acid deteriorations. Gene expression; germination ability; Helianthus annuusL.; marker; protein; RNA; seed; sunflower     

Cotyledonary mRNA Species and Germinability of Immature Vigna unguiculata Seeds

We have defined four stages in the development of cowpea seeds:I(9–11 days after flowering), II (13–15), HI (17–19)and IV (22–24). Poly A⁺ RNA fractions were prepared fromcotyledons of developing (stages I–IV) and germinating(0, 12, 24 and 48 h after imbibition) seeds. Poly A⁺ RNAs fromstages I–III exhibited high translation activities witha maximum at stage II, and the activity was markedly reducedat stage IV. In cotyledons of germinating seeds, the translationactivity was low until 12 h after the onset of imbibition butrose thereafter. Analysis of in vitro translation products withSDS-polyacrylamide gel electrophoresis and fluorography showedthat the abundant mRNA population underwent a distinct changebetween stages II and III of seed development. Since the mRNApopulation at stage III was very similar to that of stage IV(mature seeds), it appears that, as far as mRNA species areconcerned, the prerequisites for germination are fully availablein the developing seeds by stage III. This assumption was supportedby the fact that immature seeds at stage III exhibited highgermination rates and normal axial growth and produced -amylaseat levels similar to those produced by mature seeds. Severalpolypeptides which have been regarded as translation productsof stored mRNA (poly A⁺ RNA from dry seeds) were detected atearlier stages of germination. (Received September 29, 1988; Accepted January 25, 1989)     

Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

Background and Aims

α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone.

Methods

α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties.

Key Results

The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population.

Conclusions

The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.     

Long-lived Messenger RNA and its Relationship to Protein Synthesis During Germination of Pea (Pisum sativum L.) Seeds

Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Results from in vitro and in vivo protein synthesis experimentsand from studies of polysome formation suggest that much ofthe long-lived mRNA present in the embryo axis does not directprotein synthesis. The increase in the rate of protein synthesisduring germination is thus dependent on recruitment of newlysynthesized mRNA molecules. Pea, Pisum sativum L., germination, mRNA, protein synthesis     

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

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

Untersuchungen über die Regulation der DNS-Synthese während des Aktivitätswechsels vonAgrostemma githago-Samen

The relationships between DNA synthesis and germination capacity ofAgrostemma seeds have been studied. Protein synthesis and RNA synthesis are activated at the very beginning of imbibition, whereas DNA synthesis starts in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30° C), or aged seeds with a low germination capacity are characterized by a remarkably reduced protein synthesis. DNA synthesis is also reduced. The inhibition of protein-synthesis ofAgrostemma embryos fed with cycloheximid or actinomycin D causes a depression of DNA synthesis. These results indicate that the initiation of DNA synthesis of imbibingAgrostemma seeds depends on the synthesis of special proteins. Abscisic acid inhibits growth as well as DNA synthesis of isolatedAgrostemma embryos. Mitomycin inhibits germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also show a reduced incorporation of³H-thymidine in DNA. We suggest that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, is involved in the mechanism of afterripening of theAgrostemma seeds.     

Release of heat pretreatment-induced dormancy in lettuce seeds by ethylene or cytokinin in relation to the production of ethylene and the synthesis of 1-aminocyclopropane-1-carboxylic acid during germination

The germination of lettuce (Lactuca sativa L.) seeds was greatly reduced when the seeds were heated at 97°C for 30 h prior to imbibition. This dormancy was effectively released when ethylene (1–100 ppm) or benzyladenine (BA) (0.005–0.05 mM) was applied during the imbibition period. Ethylene was not required during the early part of imbibition, but was essential during the period immediately prior to radicle protrusion. Treatment with 1-aminocyclopropane-1-carboxylic acid (ACC) (0.1–10 mM) stimulated germination, but was not as effective as ethylene or cytokinin treatment. During the germination of nondormant lettuce seeds, ethylene production increased rapidly and reached a peak at 24 h, which coincided with the emergence of the radicle, and then declined; the level of ACC increased as ethylene production rate increased, but remained at a high level after radicle protrusion. In heat-pretreated dormant lettuce seeds, the increases in percent germination, ethylene production, and ACC levels were all delayed and lower than those of nondormant seeds, and these increases were accelerated by treatment with ethylene or cytokinin.     

Induction of dormancy during seed development by endogenous abscisic acid: studies on abscisic acid deficient genotypes of Arabidopsis thaliana (L.) Heynh.

Mutant lines of Arabidopsis thaliana (L.) Heynh., which are characterized by symptoms of withering and the absence of seed dormancy, showed much lower levels of endogenous abscisic acid (ABA) in developing seeds and fruits (siliquae) than the wild type. Reciprocal crosses of wild type and ABA-deficient mutants showed a dual origin of ABA in developing seeds. The genotype of the mother plant regulated a sharp rise in ABA content half-way seed development (maternal ABA). The genotype of the embryo and endosperm was responsible for a second ABA fraction (embryonic ABA), which reached much lower levels, but persisted for some time after the maximum in maternal ABA. The onset of dormancy correlated well with the presence of the embryonic ABA fraction and not with the maternal ABA. Dormancy developed in both the absence and presence of maternal ABA in the seeds. In this respect maternal ABA resembled exogenously applied ABA which did not induce dormancy in ABA-deficient seeds. However, both maternal and applied ABA stimulated the formation of a mucilage layer around the testa, which could be observed during imbibition of the mature seeds. In the mature state, ABA-deficient seeds germinated in the siliquae on the plant, but only when the atmosphere surrounding the plant was kept at high relative humidity. In younger stages germination in siliquae occurred after isolation from the plants and incubation on wet filter paper. Therefore, it seems that limited access to water is the primary trigger for the developmental arrest in these seeds.     

Relation between Water Stress and DNA Synthesis during Germination of Zea mays L.

Mitotic index, percent nuclei in DNA synthesis and the relativeDNA content per nucleus were determined from cells of the Zeamays radicle at various times after the beginning of germination.Nuclear DNA synthesis was initiated after 45 h and mitosis wasfirst observed after 74 h from sowing. Most of the dormant nucleiwere in the pre-synthetic or G₁ phase of the mitotic cycle.By 72 h most cells were in S and 77 h after the beginning ofgermination, the cells of the primary root were observed inall phases of the mitotic cycle. Dehydration of karyopses after45–74 h of imbibition progressively reduced the percentof germination to zero upon dehydration and subsequent replantingdemonstrating that drought sensitivity was related to the onsetof nuclear DNA synthesis and genome duplication.     

菜用大豆种子活力与DNA,RNA及蛋白质合成的关系

菜用大豆种子随着其活力的下降,对DNA,RNA和蛋白质前体的吸收,以及合成这些大分子的能力都明显下降,已丧失合成DNA和蛋白质能力的失活种子,仍能进行微弱的RNA合成。高活力种子在吸胀初期DNA合成速率较低,然后增加,至16h达高峰;RNA的合成速率在吸胀一开始就很高,在整个吸胀过程中均保持较高水平;蛋白质的合成速率则在开始较高,并随着吸胀过程呈增强趋势。     

Involvement of phytohormones in germination of dormant and non-dormant oat (Avena sativa L.) seeds

Oat seeds are susceptible to high temperature dormancy. Dormant grainsdo not germinate at 30 °C unless afterripened, dry, for severalweeks. Isolated embryos of dormant grains do germinate, especially ifGA₃ is added to the germination medium. ABA inhibits germinationproportionally to the concentration applied and GA₃ can overcome theABA inhibitory effect. Measurements of endogenous ABA and several GAs revealedthat the initial levels of ABA in dormant and non-dormant grains were quitesimilar. But, endogenous ABA in non-dormant seeds almost disappeared within thefirst 16 h of imbibition, while the amount in dormant grains haddecreased by less than 24%. The level of GA₁₉ in non-dormant seedswas higher, and GA₁₉ appears to be converted to GA₂₀ within the first 16h. The GA₂₀ was converted to GA₁ at leastduring the first 48 h of the germination process. Bothphytohormones thus appear to be involved in the germination process ofnon-dormant seeds. ABA first declines, while GA₁ is producedduring the first 16 h of imbibition to allow proper germination.Indormant grains the level of ABA remained high enough to prevent germinationduring at least a week and precursor GAs were not converted to GA₁.     

In vivo Studies on the Occurrence of Stored mRNA in Embryonic Axes of Vigna unguiculata Seeds

-Amanitin and cordycepin at various concentrations were testedfor their inhibitory effect on the fresh weight increase ofVigna unguiculata embryonic axes after the onset of imbibitionand on the incorporation rate of ³H-labeled leucine into proteinin axes of the 36–38 h stage. -Amanitin at 0.5–5µ/Kg/ml clearly exerted an inhibitory effect on both thefresh weight increase and the protein synthesis. This drug at1 µg/ml, however, showed no significant effect on theprotein synthesis at an early stage of imbibition (4 h), whereascycloheximide was a very potent inhibitor. By experiments inwhich ‘dry’ axes were allowed to imbibe 3H-lebeledadenosine solution for 4 and 12 h in the presence of -amanitin,it was found that poly A⁺RNA was newly synthesized to some extentin axes as early as 4 h after the onset of imbibition and thatthe drug effectively inhibited the poly A⁺RNA synthesis. Theresults may indicate the occurrence of stored mRNA in embryonicaxes of V. unguiculata seeds. (Received June 11, 1983; Accepted August 16, 1983)