Nitric oxide reduces seed dormancy in Arabidopsis

Dormancy is a property of many mature seeds, and experimentation over the past century has identified numerous chemical treatments that will reduce seed dormancy. Nitrogen-containing compounds including nitrate, nitrite, and cyanide break seed dormancy in a range of species. Experiments are described here that were carried out to further our understanding of the mechanism whereby these and other compounds, such as the nitric oxide (NO) donor sodium nitroprusside (SNP), bring about a reduction in seed dormancy of Arabidopsis thaliana. A simple method was devised for applying the products of SNP photolysis through the gas phase. Using this approach it was shown that SNP, as well as potassium ferricyanide (Fe(III)CN) and potassium ferrocyanide (Fe(II)CN), reduced dormancy of Arabidopsis seeds by generating cyanide (CN). The effects of potassium cyanide (KCN) on dormant seeds were tested and it was confirmed that cyanide vapours were sufficient to break Arabidopsis seed dormancy. Nitrate and nitrite also reduced Arabidopsis seed dormancy and resulted in substantial rates of germination. The effects of CN, nitrite, and nitrate on dormancy were prevented by the NO scavenger c-PTIO. It was confirmed that NO plays a role in reducing seed dormancy by using purified NO gas, and a model to explain how nitrogen-containing compounds may break dormancy in Arabidopsis is presented.     

Nitric oxide gas stimulates germination of dormant Arabidopsis seeds: use of a flow-through apparatus for delivery of nitric oxide

Nitric oxide (NO) is a gaseous free radical that reacts with O₂ in air and aqueous solution. NO donors have been widely used to circumvent the difficulties inherent in working with a reactive gas, but NO donors do not deliver NO at a constant rate for prolonged periods of time. Furthermore, some of the most commonly used NO donors produce additional, bioactive decomposition products. We designed and built an apparatus that allowed for the precise mixing of gaseous NO with air and the delivery of gas through sample vials at fixed rates. This experimental setup has the added advantage that continuous flow of gas over the sample reduces the buildup of volatile breakdown products. To show that this experimental setup was suitable for studies on the dormancy and germination of Arabidopsis thaliana seeds, we introduced vapors from water or sodium nitroprusside (SNP) into the gas stream. Seeds remained dormant when treated with water vapor, but gases generated by SNP increased germination to 90%. When pure NO was mixed with air and passed over dormant seeds, ∼ ∼30% of the seeds germinated. Because nitrite accumulates in aqueous solutions exposed to NO gas, we measured the accumulation of nitrite under our experimental conditions and found that it did not exceed 100 µM. Nitrite or nitrate at concentrations of up to 500 µM did not increase germination of C24 ecotype Arabidopsis seeds to more than 10%. These data support the hypothesis that NO participates in the loss of Arabidopsis seed dormancy, and they show that for some dormant seeds, exposure to exogenous NO is sufficient to trigger germination.     

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     

Nitrate, a signal relieving seed dormancy in Arabidopsis

Nitrate is an important nitrogen source for plants, but also a signal molecule that controls various aspects of plant development. In the present study the role of nitrate on seed dormancy in Arabidopsis was investigated. The effects of either mutations affecting the Arabidopsis nitrate reductase genes or of different nitrate regimes of mother plants on the dormancy of the seeds produced were analysed. Altogether, data show that conditions favouring nitrate accumulation in mother plants and in seeds lead to a lower dormancy of seeds with little other morphological or biochemical differences. Analysis of germination during seed development indicated that nitrate does not prevent the onset of dormancy but rather its maintenance. The effect of an exogenous supply of nitrate on seed germination was tested: nitrate in contrast to glutamine or potassium chloride clearly stimulated the germination of dormant seeds. Data show, moreover, that the Arabidopsis dual affinity nitrate transporter NRT1.1 (CHL1) may be involved in conveying the nitrate signal into seeds. Thus, nitrate provided exogenously or by mother plants to the produced seeds, acts as a signal molecule favouring germination in Arabidopsis. This signalling may involve interaction with the abscisic acid or gibberellin pathway.     

Nitrite,sodium nitroprusside,potassium ferricyanide and hydrogen peroxide release dormancy of <Emphasis Type="Italic">Amaranthus retroflexus</Emphasis> seeds in a nitric oxide-dependent manner

Nitric oxide (NO) and reactive oxygen species (ROS) are important regulators involving various processes of plant growth and development. Amaranthus retroflexus L. seeds possess a relative dormancy property that means freshly collected seeds can only germinate over a limited, high temperature range. Here, we show that the relative dormancy of A. retroflexus seeds could be significantly released following treatments with exogenous NO/cyanide (CN) donors such as nitrite, gases evolved from acidified nitrite, sodium nitroprusside (SNP), potassium ferricyanide (Fe(III)CN) and gases evolved from SNP or Fe(III)CN solutions, as well as exogenously supplied ROS, hydrogen peroxide (H₂O₂). However, the effectiveness varied among these chemicals. Gases evolved from acidified nitrite displayed maximum effect while H₂O₂ had minimum effect. We also show that the effects of these compounds could be significantly inhibited by NO specific scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), indicating that NO signaling pathway might play a central role in the dormancy release and germination of A. retroflexus seeds, while both ROS and CN might act through NO-dependent signaling cascades.     

Nitric oxide accelerates seed germination in warm-season grasses

The nitric oxide (NO) donor sodium nitroprusside (SNP) significantly promoted germination of switchgrass (Panicum virgatum L. cv Kanlow) in the light and in the dark at 25°C, across a broad range of concentrations. SNP also promoted seed germination in two other warm-season grasses. A chemical scavenger of NO inhibited germination and blocked SNP stimulation of seed germination. The phenolic (+)-catechin acted synergistically with SNP and nitrite in promoting seed germination. Acidified nitrite, an alternate NO donor also significantly stimulated seed germination. Interestingly, sodium cyanide, potassium ferricyanide and potassium ferrocyanide at 200 μM strongly enhanced seed germination as well, whereas potassium chloride was without effect. Ferrocyanide and cyanide stimulation of seed germination was blocked by an NO scavenger. Incubation of seeds with a fluorescent NO-specific probe provided evidence for NO production in germinating switchgrass seeds. Abscisic acid (ABA) at 10 μM depressed germination, inhibited root elongation and essentially abolished coleoptile emergence. SNP partially overcame ABA effects on radicle emergence but did not overcome the effects of ABA on coleoptile elongation. Light microscopy indicated extension of the radicle and coleoptiles in seeds maintained on water or on SNP after 2 days. In contrast, there was minimal growth of the radicle and coleoptile in ABA-treated seeds even after 3–4 days. These data indicate that seed germination of warm-season grasses is significantly influenced by NO signaling pathways and document that NO could be an endogenous trigger for release from dormancy in these species.     

Some effects of nitrate-treated soil upon the sensitivity of buried redroot pigweed (Amaranthus retroflexus L.) seeds to ethylene, temperature, light and carbon dioxide

Abstract Fresh dormant redroot pigweed (Amaranthus retroflexus L.) seeds were buried 5 cm deep in the field at Stoneville, MS in November 1981. Potassium nitrate (200 kg ha ¹) or nothing was applied to the soil in the fall of 1981 and the late winter of 1982. Seeds were recovered at intervals under darkness during the following 2 years and tested for responses to ethylene, temperature, light and carbon dioxide. During the first overwintering, nitrate enhanced loss of primary dormancy and increases seed sensitivity to temperature, light and ethylene. The loss of dormancy reached a maximum at 25 to 30 weeks (early summer) after burial. Examination of the recovered seeds indicated that about 80% of the non-treated seeds and 98% of the nitrate-treated seeds germinated in situ during the period of maximum loss of dormancy. Thus, after one overwintering period, about 20% of the original buried seed population remained dormant in nontreted soil and 2% remained dormant in the nitratetreated soil. After the second overwintering, the percentages of dormant seeds remaining in nontreated or treated soil were both only 1–2%. Nitrate reduced dormancy and enhanced germination in early summer following the first overwintering. Regardless of treatment, the remaining 1 2% of seeds in soil after the second year were of low sensitivity to the germination stimuli (ethylene, temperature, light) and constituted the long-lived portion of the original seed population.     

The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.     

Breaking the apple embryo dormancy by nitric oxide involves the stimulation of ethylene production

Mature seeds of apple (Mallus domestica Borb. cv. Antonówka) are dormant and do not germinate unless their dormancy is removed by several weeks of moist-cold treatment. We investigated the effect of short-term (3 h) nitric oxide (NO) pretreatment on breaking of apple embryonic dormancy expressed as inhibition of germination and morphological abnormalities of young seedlings. Imbibition of embryos isolated from dormant apple seeds with sodium nitroprusside (SNP) or S-nitroso,N-acetyl penicillamine (SNAP) as NO donors resulted in enhanced germination. Moreover, NO treatment removed morphological abnormalities of seedlings developing from dormant embryo. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-1-oxyl-3 oxide (cPTIO) removed the above effects. NO-mediated breaking of embryonic dormancy correlated well with enhanced ethylene production. Inhibitor of ethylene synthesis (AOA) reversed the stimulatory effect of NO donors on embryo germination. Additionally SNP reduced embryo sensitivity to exogenously applied ABA ensuing dormancy breakage. We can conclude that NO acts as a regulatory factor included in the control of apple embryonic dormancy breakage by stimulation of ethylene biosynthesis.     

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.

     

Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants

Seed germination, greening of etiolated plants and inhibition of hypocotyl elongation are stimulated by light, which is sensed by various types of photoreceptor. Nitric oxide (NO) has proven to be a bioactive molecule, especially in mammalian cells and, most recently, in plants. Like some phytochrome-dependent processes, many NO-mediated ones are accomplished through increases in cGMP levels. Given these similarities, we proposed that NO could take part in light-mediated events in plants. Here we show that NO promotes seed germination and de-etiolation, and inhibits hypocotyl and internode elongation, processes mediated by light. Two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine induced germination of lettuce (Lactuca sativa L. cv. Grand Rapids) seeds in conditions in which this process is dependent on light (e.g. 26 °C). This was a dose-dependent response and was arrested by addition of an NO scavenger, carboxy-PTIO. In addition, nitrite and nitrate, two NO-decomposition products were ineffective in stimulating germination. Wheat seedlings sprayed with SNP and grown in darkness contained 30–40% more chlorophyll than control seedlings. Nitric-oxide-mediated partial greening was increased by light pulses, wounding and biotic stress. Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) and lettuce seedlings grown in the dark had 20%-shorter hypocotyls in NO treatments than in control ones. On the other hand, internode lengths of potato plants growing under low light intensity and sprayed with 100 μM SNP were also 20% shorter than control ones. These results implicate NO as a stimulator molecule in plant photomorphogenesis, either dependent on or independent of plant photoreceptors. Received: 27 April 1999 / Accepted: 16 June 1999     

Ethylene in seed dormancy and germination

The role of ethylene in the release of primary and secondary dormancy and the germination of non-dormant seeds under normal and stressed conditions is considered. In many species, exogenous ethylene, or ethephon – an ethylene-releasing compound - stimulates seed germination that may be inhibited because of embryo or coat dormancy, adverse environmental conditions or inhibitors (e.g. abscisic acid, jasmonate). Ethylene can either act alone, or synergistically or additively with other factors. The immediate precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC), may also improve seed germination, but usually less effectively. Dormant or non-dormant inhibited seeds have a lower ethylene production ability, and ACC and ACC oxidase activity than non-dormant, uninhibited seeds. Aminoethoxyvinyl-glycine (AVG) partially or markedly inhibits ethylene biosynthesis in dormant or non-dormant seeds, but does not affect seed germination. Ethylene binding is required in seeds of many species for dormancy release or germination under optimal or adverse conditions. There are examples where induction of seed germination by some stimulators requires ethylene action. However, the mechanism of ethylene action is almost unknown.
The evidence presented here shows that ethylene performs a relatively vital role in dormancy release and seed germination of most plant species studied.     

Seed after-ripening and dormancy determine adult life history independently of germination timing

? Seed dormancy can affect life history through its effects on germination time. Here, we investigate its influence on life history beyond the timing of germination. ? We used the response of Arabidopsis thaliana to chilling at the germination and flowering stages to test the following: how seed dormancy affects germination responses to the environment; whether variation in dormancy affects adult phenology independently of germination time; and whether environmental cues experienced by dormant seeds have an effect on adult life history. ? Dormancy conditioned the germination response to low temperatures, such that prolonged periods of chilling induced dormancy in nondormant seeds, but stimulated germination in dormant seeds. The alleviation of dormancy through after-ripening was associated with earlier flowering, independent of germination date. Experimental dormancy manipulations showed that prolonged chilling at the seed stage always induced earlier flowering, regardless of seed dormancy. Surprisingly, this effect of seed chilling on flowering time was observed even when low temperatures did not induce germination. ? In summary, seed dormancy influences flowering time and hence life history independent of its effects on germination timing. We conclude that the seed stage has a pronounced effect on life history, the influence of which goes well beyond the timing of germination.     

A meta-analysis of the effects of frugivory (endozoochory) on seed germination: role of seed size and kind of dormancy

Heretofore, no study has determined how germination of ingested seeds is affected by the kind (class) of dormancy nor by seed dormancy x seed size interaction. Thus, we aimed to determine the effects of seed size, kind of dormancy and their interaction on germination of defecated seeds using a meta-analysis. We collected data for 366 plant species in 97 plant families from 76 publications. In general, gut passage significantly increased germination percentage of defecated seeds by 5% compared with that of control seeds. Germination percentages of non-dormant, physiologically dormant, and morphologically/morphophysiologically dormant seeds (all water-permeable) significantly decreased after gut passage by 40, 18, and 14%, respectively, compared with control seeds (non-gut-passed). Changes in germination percentage of seeds with physical dormancy (water-impermeable) were positive, and gut passage increased germination by 69% compared with control seeds. Germination of small seeds decreased 8% after gut passage, whereas germination of both medium and large seeds increased by 18%. However, changes in germination percentage differed between categories of seed size in each class of dormancy. In physically dormant seeds, germination of all seed sizes improved after gut passage, and the magnitude of increase was higher for large than for medium and small seeds. Thus, gut passage increased germination of medium-size water-permeable seeds (physiologically dormant and morphologically/morphophysiologically dormant) more than it did for large and small seeds. However, gut-passage decreased or did not change the germination percentage of non-dormant seeds. Seed size and kind of dormancy should be included in studies on the effect of gut passage on germination.     

Proteomic analysis of seed dormancy in Arabidopsis

The mechanisms controlling seed dormancy in Arabidopsis (Arabidopsis thaliana) have been characterized by proteomics using the dormant (D) accession Cvi originating from the Cape Verde Islands. Comparative studies carried out with freshly harvested dormant and after-ripened non-dormant (ND) seeds revealed a specific differential accumulation of 32 proteins. The data suggested that proteins associated with metabolic functions potentially involved in germination can accumulate during after-ripening in the dry state leading to dormancy release. Exogenous application of abscisic acid (ABA) to ND seeds strongly impeded their germination, which physiologically mimicked the behavior of D imbibed seeds. This application resulted in an alteration of the accumulation pattern of 71 proteins. There was a strong down-accumulation of a major part (90%) of these proteins, which were involved mainly in energetic and protein metabolisms. This feature suggested that exogenous ABA triggers proteolytic mechanisms in imbibed seeds. An analysis of de novo protein synthesis by two-dimensional gel electrophoresis in the presence of [(35)S]-methionine disclosed that exogenous ABA does not impede protein biosynthesis during imbibition. Furthermore, imbibed D seeds proved competent for de novo protein synthesis, demonstrating that impediment of protein translation was not the cause of the observed block of seed germination. However, the two-dimensional protein profiles were markedly different from those obtained with the ND seeds imbibed in ABA. Altogether, the data showed that the mechanisms blocking germination of the ND seeds by ABA application are different from those preventing germination of the D seeds imbibed in basal medium.     

Hormonal and environmental regulation of seed germination in flixweed (<Emphasis Type="Italic">Descurainia sophia</Emphasis>)

Flixweed is one of the most abundant weeds in North America and China, and causes a reduction in crop yields. Dormancy of flixweed seeds is deep at maturity and is maintained in soil for several months. To identify regulators of seed dormancy and germination of flixweed, the effect of environmental and hormonal signals were examined using dormant and non-dormant seeds. The level of dormancy was decreased during after-ripening and stratification, but long imbibition (over 5 days) at 4 °C in the dark resulted in the introduction of secondary dormancy. The strict requirement of duration of cold treatment for the break of dormancy may play a role in the seasonal regulation of germination. The germination of non-dormant flixweed seeds was critically regulated by red (R) and far-red (FR) light in a photoreversible manner. Sodium nitroprusside, a donor of nitric oxide (NO), promoted germination of half-dormant seeds, suggesting that NO reduced the level of seed dormancy. As has been shown in other related species, light elevated sensitivity to GA₄ in dark-imbibied flixweed seeds, but cold treatment did not affect GA₄-sensitivity unlike in Arabidopsis. Taken together, our results indicate that seed germination in flixweed and its close relative Arabidopsis is controlled by similar as well as distinct mechanisms in response to various endogenous and environmental signals.     

SEED DORMANCY AND GERMINATION IN MELAMPYRUM LINEARE

Immature seeds of Melampyrum lineare Desr. have very high germination percentages and dormancy is induced in a variable fraction of the seed crop during ripening. Correlated with this is the endogenous gibberellin-like activity which is found in considerable amounts in immature seeds, less in batches of ripe seeds, and is not detectable in batches containing only dormant seeds. For germination dormant seeds require activation followed by cold storage. In the laboratory activation is produced by allowing moist, dormant seeds to respire freely for several weeks at 20 C, or by treatment with exogenous GA₃. Dormancy appears to be most directly related to inability of the embryo to hydrolyze the thickened, mannan-containing endosperm cell walls. Embryos excised from dormant seed can be grown on agar enriched with whole macerated dormant seeds or with the ethanol-extractable materials from these (mostly sucrose and a glycoside). However, dormant seed material does not support growth when extracted to remove benzene- and ethanol-soluble materials.