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.     

Seed Dormancy in Red Rice : III. Response to Nitrite, Nitrate, and Ammonium Ions

Sodium nitrite at 10 millimolar breaks dormancy of dehulled red rice (Oryza sativa). While germination is light independent, low pH conditions (pH 3) are required for maximum response. Water and buffer controls at pH 3 remain dormant. The response to nitrite occurs at 25 and 30°C but is reduced at 20°C, although nondormant seeds germinate readily at this temperature. The contact time for response to nitrite is less than 2 h at the start of imbibition. Seeds imbibed first in water show reduced germination when subsequently transferred to nitrite. Dehulled seeds show little or no response to nitrate and ammonium ions.

Intact seeds remain dormant in the presence of nitrite or nitrate unless partially dry-afterripened. The pH dependence of nitrite sensitivity is reduced in intact, afterripening seeds. In highly dormant seeds, vacuum infiltration experiments suggest that the hull restricts uptake of nitrite.

     

Dormancy in Cereal Seeds: I. THE EFFECTS OF OXYGEN AND RESPIRATORY INHIBITORS

A wide spectrum of respiratory inhibitors has been found tostimulate the breaking of dormancy in barley. These includecarbon monoxide, cyanide, azide, hydrogen sulphide, sodium sulphide,hydroxylamine, diethyldithiocarbamate (DIECA), fluoride, iodoacetate,malonate, monofluoroacetate, and 2,4-dinitrophenol (DNP). Inrice, only the first six of these have been shown to be effective.Apart from CO, all the above inhibitors were tested on winteroats, but in this material only cyanide, azide, and hydroxylaminewere found to increase the germination of dormant seeds. Allthe terminal-oxidase inhibitors except CO were tested on perennialryegrass, but in this case only cyanide was found to break dormancy. As compared with air, an atmosphere of 96 per cent oxygen appliedto barley during the first 24 h after the seeds have been setto germinate stimulates the breaking of dormancy. When appliedat later stages, this high oxygen tension inhibits the germinationof dormant seeds although it has no effect on nondormant seeds.Paradoxically, the stimulatory effects of respiratory inhibitorsapplied during the initial stages of germination are relatedto their ability to inhibit oxygen uptake. Thus cyanide, azide,malonate, and monofluoroacetate, while stimulating the breakingof dormancy in barley, also inhibit oxygen uptake. In rice,cyanide and azide had similar effects, but fluoride, which hadno effect on dormancy, also had no effect on the oxygen uptakeof dormant seeds. These results are compatible with the hypothesis that some oxidationreaction is necessary for germination. This oxidation is notpart of the normal respiratory pathway, and does not proceedsatisfactorily in dormant seeds. It may be stimulated, however,by increasing the oxygen tension or by reducing normal respiratorycompetition with respiratory inhibitors.     

The Role of Water Uptake in the Transition of Recalcitrant Seeds from Dormancy to Germination

In recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) maintaining a high water content during winter, dormancy is determined by the presence and influence of the seed coat, while the axial organs of the embryos excised from these seeds are not dormant. Such axial organs were capable for active water uptake and rapid fresh weight increase, so that their fresh weights exceeded those in intact seeds at the time of radicle protrusion. Fructose plays an essential role in the water uptake as a major osmotically active compound. ABA interferes with the water uptake by the axial organs and thus delays the commencement of their growth. The manifestation of seed response to ABA during the entire dormancy period indicates the presence of active ABA receptors and the pathways of its signal transduction. The content of endogenous ABA in the embryo axes doubled in the middle of dormancy period, which coincided with a partial suppression of water uptake by the axes. During seed dormancy release and imbibition before radicle protrusion, the level of endogenous ABA in axes declined gradually. Application of exogenous ABA can imitate dormancy by limiting water absorption by axial organs. Fusicoccin A (FC A) treatment neutralized completely this ABA effect. Endogenous FC-like ligands were detected in the seed axial organs during dormancy release and germination. Apparently, endogenous FC stimulates water uptake via the activation of plasmalemmal H⁺-ATPase, acidification of cell walls, their loosening, and turgor pressure reduction. FC can evidently counteract the ABA-induced suppression of water uptake by controlling the activity of H⁺-ATPase. It is likely that, in dormant intact recalcitrant seeds, axial organs, maintaining a high water content, are competent to elevate their water content and to start their preparation for germination under the influence of FC when coat-imposed dormancy becomes weaker.     

水稻籽粒发育过程中各器官镉积累量的变化及其与基因型和土壤镉水平的关系

以镉（Cd）积累潜力不同的2个籼稻品种为材料,研究了籽粒发育过程中各器官中Cd含量的动态变化及其与土壤中Cd含量的关系。结果表明,在含Cd的生长环境中,水稻根系中的Cd含量在整个生育期中保持稳定增长的趋势,而茎叶、穗轴和稻壳等器官在营养生长阶段积累了大量的Cd,然后在籽粒充实过程中向外输出,其中旗叶和稻壳中的Cd输出率在50%以上,明显高于其他营养器官。根系和叶片中的Cd含量与土壤中的Cd含量呈高度线性相关,茎秆和籽粒中的Cd含量与土壤中的Cd含量呈抛物线相关,说明根系、茎秆、叶片等营养器官对Cd有储存和“过滤”作用。低积累品种‘X24’穗轴中的Cd含量明显低于高积累品种‘T705’,说明营养器官中的Cd向籽粒中转运的速率是决定籽粒中Cd积累量的关键因素。但是,只有当土壤中的Cd含量为0.3~1.2 mg＆#183;kg-1时,低积累品种精米中的Cd含量才会显著低于高积累品种;当土壤中的Cd含量高于2.4 mg＆#183;kg-1时,2个品种精米中的Cd含量没有显著差异。     

Dormancy in Rice Seed: III. THE INFLUENCE OF TEMPERATURE, MOISTURE, AND GASEOUS ENVIRONMENT

The influence of storage conditions—temperature, moisturecontent, oxygen, nitrogen, and carbon dioxide—on dormancyin rice seed has been investigated. The effects of temperatureand oxygen when the seeds have been set to germinate have alsobeen studied. Storage in oxygen accelerates the breaking of dormancy; at lowertemperatures, the effect of oxygen is more noticeable. Carbondioxide and nitrogen have little or no effect except in so faras they exclude oxygen. It is shown that there is a negativelinear relationship between storage temperature and log. meandormancy period of intact seeds over the range 27°C. to47°C. In the variety tested, the Q₁₀ for the rate of breakingdormancy is 3.38. Variation of moisture content over the range12.0–14.5 per cent, has little effect at 27°C. andno detectable effect at higher temperatures. When dormant seeds are soaked in water, they attain a moisturecontent close to 30 per cent. Pre-soaking seeds to achieve moisturecontents in this region can stimulate the breaking of dormancywhen compared with dry storage. At laboratory temperature (meanabout 27°C.) the stimulation caused by pre-soaking intactseeds is usually small and sometimes non-existent, but at alow temperature (3°C.) the stimulation is increased; butdry storage at 3°C. markedly delays the breaking of dormancy.When seeds are dehusked, a large proportion lose their dormancy.Much of the residual dormancy of dehusked seeds can be brokenby soaking at laboratory temperature. But in the sample of dehuskedseeds used, low temperature did not increase the effect of thepre-soaking, but slightly decreased the stimulation caused bythe soaking treatment in this case. The effect of temperature on wet seed has also been investigatedwhen the seed has been set to germinate. Maximum germinationof a partially dormant population of seed is achieved at anoptimum temperature which is near or somewhat below 27°C.In the sample tested, no germination occurred at 42°C.,although in populations which have completely broken dormancyfull germination will occur at this temperature. Non-dormantpopulations of rice seed can germinate at very low oxygen tensionsor probably even in the absence of oxygen, but germination ofa partially dormant sample is reduced under these circumstances.When oxygen tension is very low there is less germination at27°C. than at 37°C. In some varieties, at least, there is evidence that the seedsgo through a stage when they will germinate in daylight butnot in the dark. A simple practical method for rapidly breakingthe dormancy of intact seeds is described. The significanceof these results is discussed in relation to the published workon seeds of other species.     

Dormancy of<Emphasis Type="Italic"> Arabidopsis</Emphasis> seeds and barley grains can be broken by nitric oxide

Seeds of Arabidopsis thaliana (L.) Heynh. and grains of barley (Hordeum vulgare L.) were used to characterize the affects of nitric oxide (NO) on seed dormancy. Seeds of the C24 and Col-1 ecotypes of Arabidopsis are almost completely dormant when freshly harvested, but dormancy was broken by stratification for 3 days at 4°C or by imbibition of seeds with the NO donor sodium nitroprusside (SNP). This effect of SNP on dormancy of Arabidopsis seeds was concentration dependent. SNP concentrations as low as 25 M reduced dormancy and stimulated germination, but SNP at 250 M or more impaired seedling development, including root growth, and inhibited germination. Dormancy was also reduced when Arabidopsis seeds were exposed to gasses that are generated by solutions of SNP. Nitrate and nitrite, two other oxides of nitrogen, reduced the dormancy of Arabidopsis seeds, but much higher concentrations of these were required compared to SNP. Furthermore, the kinetics of germination were slower for seeds imbibed with either nitrate or nitrite than for seeds imbibed with SNP. Although seeds imbibed with SNP had reduced dormancy, seeds imbibed with SNP and abscisic acid (ABA) remained strongly dormant. This may indicate that the effects of ABA action on germination are downstream of NO action. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (cPTIO) strengthened dormancy of unstratified and briefly stratified Arabidopsis seeds. Dormancy of three cultivars of barley was also reduced by SNP. Furthermore, dormancy in barley grain was strengthened by imbibition of grain with cPTIO. The data presented here support the conclusion that NO is a potent dormancy breaking agent for seeds and grains. Experiments with the NO scavenger suggest that NO is an endogenous regulator of seed dormancy.Abbreviations ABA Abscisic acid - cPTIO 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide - GA Gibberellin - SNP Sodium nitroprusside - NO_x Gaseous oxides of nitrogen     

Isoperoxidases in jerusalem artichoke in relation to tuberization and dormancy

Peroxidase activity and isoenzyme pattern were investigated in buds and tubers of Jerusalem artichokes in relation to induction and breaking of dormancy. Peroxidase activity per unit soluble protein is the highest in the dormant stage. Conditions leading to growth,i.e. release of dormancy by the cold, stimulation of axial growth by gibberellic acid or stimulation of radial growth (tuberization) by kinetin, cause rapid loss of total peroxidase activity together with a decrease of intensity of the most cathodic isoperoxidases. Induction of dormancy by AMO-1618 increases peroxidase activity mainly through the same cathodic isoenzymes. The role of the cathodic isoperoxidases is discussed in relation to auxin catabolism and the genesis of oxygenation products inhibitory to plant growth.     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.     

Peroxidase and α-Amylase Activities in Relation to Germination of Dormant and Nondormant Wheat

Germination capacity, and α-amylase production in relation to the peroxidase and isoperoxidase activities in the grains of three varieties of wheat have been analysed and compared. A high percentage of germination and α-amylase producation at 25°C are associated with low peroxidase activity of the isolated embryo. This correlation is lacking when the intact grain is considered. A 2-day treatment at 4°C which further increases the percentage germination and enhances α-amylase synthesis, lowers the activity of peroxidase in the embryos. A general decrease in activity of all the isoenzymes is observed. Based on the above data and on differences in the activity of the most cathodic isoperoxidasic bands, a hypothesis is put forward which suggests that a sufficiently low peroxidase activity and a minimum auxin level of the embryo are responsible for the onset of germination.     

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.     

A Total Synthesis of (±)-Pachyrrhizone

We examined whether developing cotyledons of soybean seed had photosynthetic activities. The cotyledons evolved oxygen under illumination and the activity was inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The rate of oxygen evolution decreased during the development of seeds; about 30μ miol O₂ · mg chlorophyll^-1 · hr^-1 at the early developing stage and about 10μ miol O₂-mg chlorophyll^-1 · hr^-1 at the late developing stage. The rate of oxygen uptake remained at an almost constant level of 40^mol 0₂-mg chlorophyll^-1 · hr^-1 throughout the development. Photosynthetic ¹⁴CO₂-fixation by the cotyledon was observed. Ribulose bisphosphate carboxylase was immunochemically detected in the developing cotyledons. These results show that functional photosynthetic apparatus is present in the developing cotyledons of soybean seeds.     

Biochemical factors affecting protein accumulation in the rice grain

Rice grains (Oryza sativa L.) from three varieties and three pairs of lines with different protein content were collected at 4-day intervals from 4 to 32 days after flowering. The samples were analyzed for protein, free amino nitrogen, ribonucleic acid, protease activity, and ribonuclease activity. In addition, the capacity of the intact grain to incorporate amino acids was determined for the three pairs of lines. The maximal level of free amino nitrogen and the capacity of the developing grain to incorporate amino acids were consistently found to be higher in the samples with the high protein content in the mature grain. The ribonucleic acid content of the grain tended to be higher in the high protein samples.     

Effect of sucrose level on rice grain deposition in detached panicle system

In detached IR36 rice panicles incubated in liquid medium for 10 days in 1.0–2.0% sucrose and 0.6% glutamine, final panicle weight increased but percentage and weight of grain protein decreased with 1.0–1.75% sucrose. Soluble sugars increased in stem and hull but not in developing grain. With five other rice panicles and IR36 panicles differing in grain size incubated in liquid culture containing 0.075% glutamine for 7 days, percentage grain protein was again lower in 1.5% sucrose than in 1.0% sucrose, with correspondingly heavier grain weight in four cases. Free-sugar levels of developing grains were lower in detached panicles than in the field grain samples in both experiments. Thus, sucrose level has a depressing effect on protein accumulation in the developing rice grain. Lysine content of grain protein decreased with increase in protein content.     

Different properties of the inward rectifying potassium conductance of aleurone protoplasts from dormant and non-dormant barley grains

Isogenic dormant and non-dormant barley grains provide a useful system to study the molecular mechanisms of grain dormancy and the role of plant hormones in this process. As ion fluxes are associated with dormancy-related plant hormone responses, we compared the properties of the inward rectifying potassium conductance in aleurone protoplasts isolated from dormant and non-dormant Triumph grains and in germinating Himalaya grains. Maximal conductance, voltage dependency of steady-state activation, activation and deactivation kinetics were studied in the whole-cell patch-clamp configuration. Activation and deactivation time courses were single exponential. No differences in the above described properties were found between the protoplasts isolated from non-dormant Triumph and Himalaya grains. However, the maximal conductance (corrected for cell size) in protoplasts from dormant Triumph grains was much smaller (65%), and activation time constants were much larger as compared to protoplasts from non-dormant grains. No differences were found in the deactivation kinetics in the three different types of protoplasts. The half-maximal activation potential was slightly more negative in protoplasts from dormant grains than from non-dormant grains.     

A QTL located on chromosome 4A associated with dormancy in white- and red-grained wheats of diverse origin

Improved resistance to preharvest sprouting in modern bread wheat (Triticum aestivum. L.) can be achieved via the introgression of grain dormancy and would reduce both the incidence and severity of damage due to unfavourable weather at harvest. The dormancy phenotype is strongly influenced by environmental factors making selection difficult and time consuming and this trait an obvious candidate for marker assisted selection. A highly significant Quantitative Trait Locus (QTL) associated with grain dormancy and located on chromosome 4A was identified in three bread wheat genotypes, two white- and one red-grained, of diverse origin. Flanking SSR markers on either side of the putative dormancy gene were identified and validated in an additional population involving one of the dormant genotypes. Genotypes containing the 4A QTL varied in dormancy phenotype from dormant to intermediate dormant. Based on a comparison between dormant red- and white-grained genotypes, together with a white-grained mutant derived from the red-grained genotype, it is concluded that the 4A QTL is a critical component of dormancy; associated with at least an intermediate dormancy on its own and a dormant phenotype when combined with the R gene in the red-grained genotype and as yet unidentified gene(s) in the white-grained genotypes. These additional genes appeared to be different in AUS1408 and SW95-50213.     

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.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.