Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.     

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.     

Redox-sensitive proteome and antioxidant strategies in wheat seed dormancy control

Oxidative signalling by ROS has been demonstrated to play a role in seed dormancy alleviation, but the detailed molecular mechanisms underlying this process remain largely unknown. Here, we show dynamic differences in redox-sensitive proteome upon wheat seed dormancy release. Using thiol-specific fluorescent labelling, solubility-based protein fractionation, 2-D IEF PAGE, and MS analysis in conjunction with wheat EST sequence libraries, proteins with reversible oxidoreductive changes were characterized. Altogether, 193 reactive Cys were found in 79 unique proteins responding differentially in dormant, non-dormant, abscisic, or gibberellic acid-treated seed protein extracts from RL4137, a wheat cultivar with extreme dormancy. The identified proteins included groups that are redox-, stress-, and pathogen-responsive, involved in protein synthesis and storage, are enzymes of carbohydrate metabolism, proteases, and those involved in transport and signal transduction. Two types of redox response could be detected: (i) a dramatic increase in protein thiol redox state in seeds during imbibition and hormonal treatment; (ii) higher antioxidant capacity related to sensing of a threshold redox potential and balancing the existing redox pools, in dry dormant versus non-dormant seeds. These results highlight occurrence of the antioxidant defence mechanisms required for the protection of seed during a dormancy stage.     

Hypoxia interferes with ABA metabolism and increases ABA sensitivity in embryos of dormant barley grains

Two mechanisms have been suggested as being responsible for dormancy in barley grain: (i) ABA in the embryo, and (ii) limitation of oxygen supply to the embryo by oxygen fixation as a result of the oxidation of phenolic compounds in the glumellae. The aim of the present work was to investigate whether hypoxia imposed by the glumellae interferes with ABA metabolism in the embryo, thus resulting in dormancy. In dormant and non-dormant grains incubated at 20 degrees C and in non-dormant grains incubated at 30 degrees C (i.e. when dormancy is not expressed), ABA content in the embryo decreased dramatically during the first 5 h of incubation before germination was detected. By contrast, germination of dormant grains was less than 2% within 48 h at 30 degrees C and embryo ABA content increased during the first hours of incubation and then remained 2-4 times higher than in embryos from grains in which dormancy was not expressed. Removal of the glumellae allowed germination of dormant grains at 30 degrees C and the embryos did not display the initial increase in ABA content. Incubation of de-hulled grains under 5% oxygen to mimic the effect of glumellae, restored the initial increase ABA in content and completely inhibited germination. Incubation of embryos isolated from dormant grains, in the presence of a wide range of ABA concentrations and under various oxygen tensions, revealed that hypoxia increased embryo sensitivity to ABA by 2-fold. This effect was more pronounced at 30 degrees C than at 20 degrees C. Furthermore, when embryos from dormant grains were incubated at 30 degrees C in the presence of 10 microM ABA, their endogenous ABA content remained constant after 48 h of incubation under air, while it increased dramatically in embryos incubated under hypoxia, indicating that the apparent increase in embryo ABA responsiveness induced by hypoxia was, in part, mediated by an inability of the embryo to inactivate ABA. Taken together these results suggest that hypoxia, either imposed artificially or by the glumellae, increases embryo sensitivity to ABA and interferes with ABA metabolism.     

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.     

Dormancy in Cereal Seeds: II. THE NATURE OF THE GASEOUS EXCHANGE IN IMBIBED BARLEY AND RICE SEEDS

When barley seeds imbibe water, the O₂ uptake of non-dormantseeds is considerably less than that of dormant seeds for atleast the first 6 h, irrespective of the rate at which the seedshad previously lost dormancy. During the initial 6 h of imbibition, the CO₂ output of dormantbarley seeds is usually only slightly greater than and sometimesno different from that of nondormant seeds. The CO₂ output ofdormant seeds is reduced by about 66 percent by millimolar KCN,whereas that of non-dormant seeds is decreased by about 12–13per cent only. The CO₂ output of dormant barley in nitrogenis considerably less than the CO₂ output of non-dormant seedsunder the same conditions. Dormant rice seeds also show a higher initial O₂ uptake thannon-dormant seeds, though this is not generally as marked asin barley. Similarly, the initial CO₂ output of dormant seedsis distinctly greater than that of non-dormant seeds, but inmillimolar KCN it is depressed to a greater extent than in non-dormantseeds. In nitrogen, the CO₂ outputs of dormant and non-dormantseeds were found to be the same. Consequently, unlike barley,dormant rice seeds appear to be as capable of carrying out alcoholicfermentation under anaerobic conditions as nondormant seeds. In barley, increasing the O₂ tension from 21 per cent to 100per cent increased the oxygen uptake of dormant seeds more thanthat of non-dormant seeds (an increase of 53 per cent as against20–23 Per cent). In dormant seeds there was a concomitantincrease in CO₂ output (about 50 per cent), but the CO₂ outputof non-dormant seeds was hardly affected. High concentrations of CO₂ are inhibitory to the germinationof both dormant and non-dormant barley seeds. At a concentrationof 10 per cent, however, CO₂ is inhibitory only to dormant seeds,although at 2.5–5 per cent it is sometimes stimulatoryto the germination of dormant seeds. A 24–h treatmentwith appropriate concentrations of ethanol, lactic acid, oracetaldehyde is also stimulatory to the germination of dormantbarley seeds. Histochemical investigations in barley indicated the presenceof peroxidase, cytochrome oxidase, and -glycero-phosphate dehydrogenasein the embryo, aleurone layer, and in a layer associated withthe testa. A number of other redox enzymes were detected inthe embryo and aleurone layer only. No differences in distributionor intensity of activity were detected between dormant and nondormantseeds.     

Abscisic acid-regulated responses of dormant and non-dormant embryos of Helianthus annuus: Role of ABA-inducible proteins

Embryos of Helianthus annuus L. became dormant 3 weeks after anthesis and their dormancy was lifted during storage in dry conditions. The objectives of this study were to investigate changes in the pattern of soluble proteins associated with the release of embryo dormancy. Sunflower dehydrins and group 3 late embryogenesis-abundant (LEA) proteins were studied in developing embryos. Three dehydrins (17, 21 and 26 kDa) and two group 3 LEA polypeptides (17 and 23 kDa) appeared during dormancy induction. Their levels remained steady until maturity. After imbibition, these polypeptides disappeared within 24 h except for the 23-kDa protein whose levels remained stable for a further 4 d, whatever the culture condition. Analysis of radiolabelled proteins by two-dimensional gel electrophoresis revealed that among dormancy-associated proteins other than dehydrin and group 3 LEA, several low molecular mass (18, 19, 20 and 21 kDa) proteins were expressed in dormant embryos but not detected in non-dormant embryos. After a treatment with fluridone, which inhibits ABA synthesis, or with GA₃, which allows germination to occur, the 19-kDa protein could not be detected. In contrast, application of ABA to non-dormant embryos arrested germination and enhanced the synthesis of the 18- and 21-kDa proteins, but not that of the 19- and 20-kDa polypeptides. These results demonstrate that steady-state levels of specific proteins change during early imbibition of dormant and non-dormant sunflower embryos and indicate that these changes may be associated with differential gene expression responsible for the maintenance of dormancy.     

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     

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.     

Involvement of ABA in induction of secondary dormancy in barley (Hordeum vulgare L.) seeds

At harvest, barley seeds are dormant because their germination is difficult above 20 degrees C. Incubation of primary dormant seeds at 30 degrees C, a temperature at which they do not germinate, results in a loss of their ability to germinate at 20 degrees C. This phenomenon which corresponds to an induction of a secondary dormancy is already observed after a pre-treatment at 30 degrees C as short as 4-6 h, and is optimal after 24-48 h. It is associated with maintenance of a high level of embryo ABA content during seed incubation at 30 degrees C, and after seed transfer at 20 degrees C, while ABA content decreases rapidly in embryos of primary dormant seeds placed directly at 20 degrees C. Induction of secondary dormancy also results in an increase in embryo responsiveness to ABA at 20 degrees C. Application of ABA during seed treatment at 30 degrees C has no significant additive effect on the further germination at 20 degrees C. In contrast, incubation of primary dormant seeds at 20 degrees C for 48 and 72 h in the presence of ABA inhibits further germination on water similarly to 24-48 h incubation at 30 degrees C. However fluridone, an inhibitor of ABA synthesis, applied during incubation of the grains at 30 degrees C has only a slight effect on ABA content and secondary dormancy. Expression of genes involved in ABA metabolism (HvABA8'OH-1, HvNCED1 and HvNCED2) was studied in relation to the expression of primary and secondary dormancies. The results presented suggest a specific role for HvNCED1 and HvNCED2 in regulation of ABA synthesis in secondary seed dormancy.     

Molecular cloning and characterization of an inorganic pyrophosphatase from barley

A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During imbibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.     

Tolerance of kikuyu grass to long term salt stress is associated with induction of antioxidant defences

Primary dormancy in A. retroflexus seeds wascompletely broken by dry storage or ethylene treatment and partially removedwith GA₃. Norbornadiene counteracted the dormancy breaking action ofethylene and GA₃. The GA₃ effect was lowered bycobaltous ions. ABA increased the ethylene requirement in primary dormant seeds.Dormant seeds had a similar or different ability to produce ethylene and ACCoxidase in vivo activity than did non-dormant seeds,depending on the period of incubation. Dormant seeds contained less endogenousACC than non-dormant seeds. Thus, ethylene seems to play an essential role inthe release of primary dormancy in A. retroflexus seeds.Ethylene also participates in the release of dormancy achieved by GA₃treatment. The results indicate that both ethylene biosynthesis and action isinvolved in the control of primary dormancy in Amaranthusretroflexus seeds.     

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.     

Fructose 2,6-bisphosphate in germinating oat seeds. A biochemical study of seed dormancy

When dormant oat seeds were imbibed at the non-permissive temperature of 30 degrees C, the concentration of phosphoenolpyruvate and of glycerate 3-phosphate, which are two inhibitors of phosphofructokinase 2, increased almost linearly during 30 h. By contrast, these metabolites increased only after a lag period of about 10 h in non-dormant seeds imbibed at the same temperature. As a consequence of this, the concentration of the C3 derivatives remained always remarkably lower in non-dormant than in dormant seeds. Accordingly, the concentration of fructose 2,6-bisphosphate, which increased similarly in the two types of seeds during the first 8 h after the start of inhibition, then reached a plateau in dormant seeds but continued to increase for another 8 h in non-dormant seeds, reaching a maximal value a few hours before the beginning of radicle protrusion. When the dormant seeds were imbibed at the permissive temperature of 10 degrees C, the evolution of all metabolites was slowed down but behaved like that of non-dormant seeds imbibed at 30 degrees C. Experiments in which the dormant seeds were submitted to a jump from 10 degrees C to 30 degrees C and vice versa, always provoked reverse changes in the concentration of the C3 derivatives and of fructose 2,6-bisphosphate, the latter being increased in all conditions that allowed germination. Dormant seeds were also allowed to germinate at 30 degrees C by imbibition during 24 h in the presence of 3% ethanol. Again, this permissive treatment caused an arrest in the accumulation of C3 derivatives and an increase in fructose 2,6-bisphosphate. Another, apparently unrelated, biochemical difference between dormant and non-dormant oat seeds was their inorganic pyrophosphate content, which was approximately five-fold higher in non-dormant than in dormant seeds. This difference was observed before and persisted during imbibition as long as measurement could be made and was not affected by the temperature jumps or by ethanol. In contrast to the phosphoric esters under investigation, pyrophosphate was not preferentially located in the embryo.