JXB Advance Access originally published online on December 23, 2005
Journal of Experimental Botany 2006 57(3):517-526; doi:10.1093/jxb/erj060
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FOCUS PAPER |
Nitric oxide reduces seed dormancy in Arabidopsis
Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720-3102, USA
* To whom correspondence should be addressed. E-mail: pcbethke{at}nature.berkeley.edu
Received 7 September 2005; Accepted 17 November 2005
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Abstract |
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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.
Key words: Arabidopsis, cyanide, nitric oxide donor, seed dormancy, sodium nitroprusside (SNP)
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Introduction |
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The publication of this focus section devoted to nitric oxide (NO) in plant biology is a testament to the rapidly increasing number of processes in which NO has been found to play a role. NO is involved in regulating a diverse array of developmental processes in plants, and there is increasing evidence that NO is a key participant in defence against microbial attack. Attention was focused on the role of NO in seed dormancy. This investigation to discover whether NO affected dormancy was prompted by observations showing that the NO donors SNP and SNAP delayed programmed cell death in barley aleurone layers (Beligni et al., 2002
), and that barley grain had the capacity to produce copious amounts of NO from nitrite in the apoplast of the aleurone layer (Bethke et al., 2004a). The ability of the NO scavenger c-PTIO to block the effects of NO donors and its ability to accelerate aleurone cell death, led to the proposal that NO was likely to be an important endogenous regulator in aleurone tissue (Beligni et al., 2002).
Dormancy prevents seed germination under conditions that would otherwise allow germination. Many endogenous compounds reduce seed dormancy, but for any of these compounds the mechanism of action is not known. Among the compounds known to break seed dormancy are a range of apparently disparate nitrogen-containing compounds including nitrate, nitrite, cyanide, hydroxylamine, azide, and sodium nitroprusside (SNP). The ability of SNP to reduce seed dormancy of Arabidopsis (Batak et al., 2002; Bethke et al., 2004b), barley (Bethke et al., 2004b), lettuce (Beligni and Lamattina, 2000), and Paulonia tomentosa (Giba et al., 1998) led to the conclusion that NO played a role in dormancy breaking or germination in these seeds. Hendricks and Taylorson (1974) proposed that NO was a likely product of azide, hydroxylamine, and nitrite application to seeds long before NO became a fashionable research topic.
Experiments using dormant seeds of the C24 ecotype of Arabidopsis thaliana are reported here. Under these experimental conditions the data show that the NO donor SNP breaks dormancy via the production of cyanide and not NO. It is shown that cyanide is as effective as SNP in breaking dormancy of Arabidopsis, and that the NO scavenger c-PTIO prevents both cyanide- and SNP-stimulated germination. c-PTIO also strengthens seed dormancy in the absence of added NO, suggesting that endogenous NO participates in dormancy breaking in Arabidopsis. Taken together with the data showing that pure NO gas can reduce Arabidopsis seed dormancy, it can be concluded that NO plays a role in vivo in reducing seed dormancy. Some of the data in this paper have been published previously and are reviewed here (Bethke et al., 2004b, 2005; Libourel et al., 2005).
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Materials and methods |
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Plant material
Seeds of the C24 and Col-0 ecotypes of Arabidopsis thaliana (L.) Heynh. were used in these experiments. Seeds of the C24 ecotype were obtained from plants grown in a growth room. C24 seeds were sown on moist soil in 10 cm pots covered with plastic wrap. Seeds were stratified by placing pots in a cold room at 4 °C for 4 d before transferring to a 17 °C growth room with an 18 h light (300 µE m–2 s–1) 8 h dark photoperiod. Seeds were harvested 4–5 weeks after the first white petal appeared on the plants, dried at room temperature, and stored in sealed tubes at –80 °C. Seeds from several harvests were used for the experiments reported in this paper. Quantitative differences in germination percentages and rates were noted, but all harvests showed the same qualitative responses. Col-0 ecotype seeds were obtained from plants grown under fluorescent lights with an 18 h light (75 µE m–2 s–1) 8 h dark photoperiod at 21–23 °C. After harvesting, seeds were fully after-ripened at room temperature for over 6 months.
Germination
For most experiments, Arabidopsis seeds were germinated in open, 3.5 cm plastic Petri dishes, referred to as receiver dishes, containing 3 ml of 0.6% aqueous agarose and the appropriate additions. Two receiver dishes were enclosed in a sealed glass, 10 cm Petri dish together with one open, 3.5 cm Petri dish, referred to as a donor dish, containing the solution to be tested. The solution in the donor dish was replaced with water after 3 d. For experiments with c-PTIO, nitrate, and nitrite, those compounds were incorporated into the agarose on which seeds were sown. Seeds (approximately 20–30 per dish) were allowed to germinate in the light (120 µE m–2 s–1) at 24 °C. Germination was scored daily by counting seeds with the aid of a dissecting microscope. For experiments using purified nitric oxide, Arabidopsis seeds (approximately 20 per vial) were imbibed on 3 ml of 1% agarose in dH2O in 5 ml serum vials (Wheaton, Millville NJ) that were then capped with an airtight stopper. Seeds were allowed to germinate on the bench-top at 24 °C under continuous illumination (45 µE m–2 s–1). Seeds were judged to have germinated when the radicle pierced the seed coat.
Chemicals
A 50 mM stock of c-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide, Molecular Probes, Eugene OR) was made in dH2O and stored at –20 °C. A 500 µM stock solution of (+)-ABA (Sigma, St Louis, MO) was prepared by dissolving it in dH2O containing sufficient KOH to keep the pH near 7 and was stored at –20 °C. Sodium nitroprusside (SNP), potassium cyanide, potassium ferricyanide, and potassium ferrocyanide were freshly made for each experiment.
Gas flow control
The flow rate and composition of gas mixtures was regulated using Tylan (FC-280 AV) mass flow controllers (MFCs). A computer-controlled setpoint controller was custom-made to allow for simultaneous control of 8 MFCs using an RS232-controlled analogue-to-digital converter (WTADC-M) and two digital-to-analogue converters (WTDAC-M) cards (Weeder Technologies, Walton Beach, FL). A regulated power supply (Sola 3030–15T, Mouser Electronics, Mansfield, TX) was used to power the MFCs and the data communication cards. Set NO concentrations were obtained by mixing NO with N2 using appropriate flow rate ratios. Breathing air and NO-nitrogen mixtures were then combined in similar fashion. The gas mixture was bubbled through a sparger containing 0.5 M KOH (500 ml Pyrex brand, Fisher Scientific, Pittsburg, PA) to humidify the gas and scrub the gas mixture of nitrous acid. Nitric oxide was applied to seeds in 5 ml serum vials (Wheaton, Millville, NJ), which had a total airspace volume of 9 ml. Vials were capped using 20 mm straight plug butyl stoppers and disposable tear-off aluminium seals (Wheaton, Millville, NJ).
Nitrite detection
Nitrite was determined colorimetrically using the Griess reagent (Hageman and Reid, 1980). Absorbance measurements at 543 nm were performed on a Shimadzu UV-160 spectrophotometer (Shimadzu Corp., Kyoto, Japan) and concentrations were calculated relative to a 100 µM NaNO2 standard.
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Results |
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The nitric oxide scavenger c-PTIO strengthens Arabidopsis seed dormancy
The NO scavenger c-PTIO is routinely used to remove NO from biological tissues and from solution, and it was shown that c-PTIO effectively scavenges the NO produced by isolated barley aleurone layers incubated with nitrite (Bethke et al., 2004a
). To investigate a possible involvement of NO in dormancy release, Arabidopsis seeds of the C24 ecotype were imbibed on filter paper moistened with 50 µM or 100 µM c-PTIO. Imbibition with the NO scavenger strengthened seed dormancy, as shown in Fig. 1A. As the concentration of c-PTIO increased from 0 µM to 100 µM the final germination percentage decreased from 20% to 5%. These effects of c-PTIO did not result from a non-specific inhibition of germination. When fully after-ripened, non-dormant Arabidopsis seeds were imbibed with 100 µM or 200 µM c-PTIO, neither the rate of germination nor the final extent of germination was reduced relative to controls imbibed with water alone (Fig. 1B). Similar data have been obtained for barley grain (Bethke et al., 2004b).
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Sodium nitroprusside vapours reduce Arabidopsis seed dormancy
Sodium nitroprusside (SNP), a commonly used NO donor, effectively reduced dormancy of Arabidopsis seeds imbibed in the light (Bethke et al., 2004b
, 2005), but not in the dark (data not shown). SNP undergoes photolysis when illuminated and releases NO (Feelisch, 1998). The mechanism of NO release from SNP has been examined by others and it is clear that compounds other than NO are generated from SNP that might have biological activity. Only a few of these compounds are expected to be volatile. Therefore, the experimental system shown in Fig. 2A was used to determine if vapours from a solution of SNP could reduce the dormancy of Arabidopsis seeds. When seeds were imbibed on water agarose and exposed to vapours from SNP, dormancy was strongly reduced and approximately 90% of the seeds germinated (Fig. 2B). This effect of SNP on seed dormancy was counteracted when seeds were imbibed on agarose containing the NO scavenger c-PTIO at 100 µM. Control seeds exposed to water vapour remained dormant and only 5% germinated.
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The effect of SNP on dormancy loss depended on the time of exposure to SNP vapours and the amount of SNP, as shown in Fig. 3. Exposure of seeds to vapours generated from SNP for 3 d resulted in a greater germination percentage than exposure for 1 d. Likewise, exposure to vapours from two dishes containing 3 ml of 100 µM SNP increased germination relative to exposure to one dish containing 3 ml of 100 µM SNP (Fig. 3).
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SNP decreases the sensitivity of seeds to ABA
Abscisic acid (ABA) has been shown to be essential for the establishment of dormancy, and ABA or ABA synthesis is required for the maintenance of dormancy (Koornneef et al., 2002
). It was hypothesized that seed dormancy in Arabidopsis might be maintained by continued ABA synthesis and that seed dormancy might decrease if seeds were imbibed in the presence of an ABA synthesis inhibitor. To test this hypothesis, dormant Arabidopsis seeds were imbibed with the ABA synthesis inhibitor norfluorazon at concentrations from 0 to 30 µM and the extent of germination was measured 7 d later. As shown in Fig. 4, imbibition with norfluorazon did not result in a loss of dormancy, and germination in the presence of norfluorazon was no greater than that observed for control seeds imbibed in water. Seeds treated with 1 µM norfluorazon or more that did germinate had white cotyledons and hypocotyls, indicating that norfluorazon was acting as expected. To show that norfluorazon was not acting as a non-specific inhibitor of germination, dormant Arabidopsis seeds were imbibed with norfluorazon and then exposed to vapours from SNP for 1 d. In the absence of norfluorazon, approximately 45% of seeds lost dormancy and germinated following treatment with SNP vapours. Imbibition with norfluorazon did not inhibit SNP-stimulated germination. Instead, imbibition with norfluorazon at 10 µM to 30 µM resulted in higher rates of germination than those observed for controls (Fig. 4).
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The hypothesis that SNP vapours decrease the sensitivity of Arabidopsis seeds to ABA was tested using two seed lots that differed in their degree of dormancy. When a population of seeds having low dormancy was imbibed with ABA, germination after 7 d decreased from 60% with no ABA to 10% with 10 µM ABA as shown in Fig. 5A. When the same seeds were imbibed with ABA and exposed to vapours from SNP, germination percentages after 7 d were approximately 80%, regardless of ABA concentration. Similar results were obtained with more dormant seeds. Control seeds imbibed with water gave 5% germination, and this percentage decreased to 0% as the concentration of ABA increased to 10 µM (Fig. 5B). SNP vapours, however, were still able to elicit substantial rates of germination. The germination percentage 7 d after imbibition was 65% when seeds were imbibed on water and exposed to SNP vapours. Imbibition with ABA decreased the final germination percentage, but even at 10 µM ABA, approximately 30% of the seeds germinated (Fig. 5B). These data support the hypothesis that SNP vapours decrease the sensitivity of Arabidopsis seeds to exogenous ABA. Whether this is apparent depends on the concentration of ABA and on the concentration of SNP. In a previous report, SNP at 25 µM stimulated germination of weakly dormant seeds but was not sufficient to overcome the affect of 1 µM or 10 µM ABA (Bethke et al., 2004b
).
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Cyanide is the active compound in SNP with regard to dormancy loss
To determine if NO was the compound in SNP vapours that reduced Arabidopsis seed dormancy, control experiments were carried out with potassium ferrocyanide (Fe(II)CN) and potassium ferricyanide (Fe(III)CN), two compounds that have structures similar to SNP but which lack the ability to produce NO. As seen in Fig. 6A, Fe(II)CN and Fe(III)CN were as effective as SNP in reducing Arabidopsis seed dormancy. For both Fe(II)CN and Fe(III)CN vapours, the rate of germination and the final germination percentage were comparable with those observed for seeds treated with SNP vapours. To determine if NO was being produced, the presence of nitrite was assayed in the donor dishes containing SNP, Fe(II)CN, and Fe(III)CN, and in dummy receiver dishes containing water. Nitrite is a breakdown product of NO in an aerobic environment (Ignarro et al., 2002
). Given the reactive nature of NO, assays for nitrite report the approximate amount of NO that accumulated in a given location whenever the half-life of NO is much less than the sampling time. As expected, nitrite accumulated in donor dishes containing SNP and this is seen in Fig. 6B. Receiver dishes near a donor dish of SNP, however, accumulated very little nitrite. This suggests that very little of the NO generated by SNP was transferred through the gas phase to the receiver dishes. Neither donor dishes containing Fe(II)CN or Fe(III)CN, nor receiver dishes near them accumulated nitrite, indicating that NO was not produced by these compounds to any appreciable extent (Fig. 6B).
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SNP, Fe(II)CN, and Fe(III)CN can all produce cyanide (CN) by way of photolysis (Meeussen et al., 1992
; Feelisch, 1998), and the amount of CN– in donor and receiver dishes was also determined. As seen in Fig. 6C, CN– accumulated in donor dishes containing SNP, Fe(II)CN, and Fe(III)CN at concentrations between 30 µM and 60 µM. Significantly, receiver dishes also accumulated CN– and the rate of accumulation was comparable to that observed in donor dishes. These data suggest that CN– produced in the donor dishes moves readily through the gas phase to receiver dishes, most likely as HCN. Because SNP, Fe(II)CN, and Fe(III)CN all produced volatile CN– and all reduced Arabidopsis seed dormancy through the gas phase, the hypothesis was tested that CN– was the active compound in SNP vapours by exposing Arabidopsis seeds to vapours from donor dishes containing KCN. The data from these experiments are presented in Fig. 7 and show clearly that volatile CN– effectively breaks dormancy of Arabidopsis seeds. Germination 7 d after imbibition increased from 0% to 90% as the concentration of KCN in the donor dish increased from 0 to approximately 400 µM.
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The loss of seed dormancy promoted by cyanide, nitrite, and nitrate depends on NO
The data in Fig. 7 show that CN– is highly effective in reducing Arabidopsis seed dormancy. Two other compounds that reduce dormancy in a range of monocot and dicot species are nitrite and nitrate (Hilhorst and Karssen, 1992
; Debeaujon et al., 2000).The NO scavenger c-PTIO was used to find out if NO was integral to the process of dormancy loss initiated by CN–, nitrite, and nitrate. When dormant Arabidopsis seeds were imbibed with c-PTIO and treated with vapours from KCN, the effect of KCN vapours on dormancy was reduced as the concentration of c-PTIO increased from 100 µM to 500 µM, as shown in Fig. 8A. Likewise, c-PTIO strongly maintained dormancy of seeds imbibed with either nitrite or nitrate (Fig. 8B). In the absence of c-PTIO, approximately 50% of previously dormant Arabidopsis seeds germinated when imbibed with 0.5 mM to 1.5 mM nitrite. As shown in Fig. 8B, however, dormancy was retained when seeds were also imbibed with 100 µM c-PTIO. As the nitrate concentration increased from 0.5 mM to 5.0 mM, the final germination percentage increased from 25% to 65%, but at all nitrate concentrations c-PTIO maintained dormancy, and germination percentages after 7 d were 5% or less (Fig. 8B).
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The increase in germination at higher nitrite levels in the presence of c-PTIO suggests that the activity of c-PTIO could be partially saturated with a product resulting from nitrite. Similarly, high concentrations of SNP relative to the concentration of c-PTIO also lessened the effect of c-PTIO, as indicated by the data in Fig. 8C. c-PTIO at 100 µM maintained Arabidopsis seed dormancy, even in the presence of vapours from 100 µM SNP and germination was approximately 10%. In the presence of vapours from 200 µM SNP, however, 100 µM c-PTIO was relatively ineffective, and 60% of the seeds germinated (Fig. 8C).
Purified NO gas reduces Arabidopsis seed dormancy
The data showing that c-PTIO strengthens dormancy in seeds imbibed in the absence of an NO donor, and maintains dormancy in seeds treated with compounds that would otherwise reduce dormancy, are strong evidence for the involvement of NO in the process of dormancy release. To further establish a role for NO in this process, purified, gaseous NO was applied to dormant Arabidopsis seeds and the effect on dormancy was determined by scoring germination. Because NO is unstable in aerobic environments, a system was designed and fabricated that allowed for the controlled application of gaseous NO at precisely regulated concentrations and flow rates. This apparatus was used to apply either pulses of NO-containing gas mixtures or a continuous stream of NO-containing gas to Arabidopsis seeds imbibed on water agarose. In both cases, purified NO reduced Arabidopsis seed dormancy. The data from the pulsed flow configuration are shown in Fig. 9A. When seeds were exposed to 5 min pulses of NO-containing gas every hour for 48 h, seed germination increased as the concentration of NO in the gas mixture increased. Control seeds exposed to pulses of air had approximately 2% germination, but seeds exposed from 25 ppm to 100 ppm NO had 7% to 40% germination, with higher final germination percentages at higher concentrations of NO. It is inevitable that nitrite will accumulate in a system such as this, but the measured amounts of nitrite, <200 µM with 100 ppm NO, were insufficient to account for the observed rates of germination. For the seed lot used in these experiments, 200 µM nitrite gave less than 10% germination (Libourel et al., 2005). A continuous flow of NO-containing gas at 45 ppm also reduced Arabidopsis seed dormancy as seen in Fig. 9B. Final germination percentages varied substantially, with a mean for 15 separate experiments of approximately 25%. These data provide direct evidence for the involvement of NO in a process that leads to a reduction in seed dormancy.
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Discussion |
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The data presented here strongly indicate that NO is involved in the process of dormancy release in Arabidopsis. In particular, it has been shown that the NO scavenger c-PTIO strengthened dormancy (Fig. 1A) but did not inhibit germination (Fig. 1B). Dormancy loss stimulated by exogenous cyanide (Fig. 7), nitrite, or nitrate was also inhibited by c-PTIO (Fig. 8). These data suggest to us that NO is a shared, required component of the dormancy breaking response that is initiated by each of these compounds. Application of purified, gaseous NO reduced Arabidopsis seed dormancy (Fig. 9), and these data give further support to the hypothesis that NO acts to inhibit dormancy.
These data are consistent with those obtained using seeds of other species. c-PTIO strengthened dormancy of barley grain (Bethke et al., 2004b) and prevented the germination of Lactuca sativa that was stimulated by the NO donor S-nitroso-N-acetylpenicillamine (Beligni and Lamattina, 2000). Conversely, NO donor compounds promoted germination of Arabidopsis (Batak et al., 2002), Paulownia tomentosa (Giba et al., 1998), and Lactuca sativa (Beligni and Lamattina, 2000). Taken together, these data raise the intriguing possibility that an involvement of NO in the loss of seed dormancy is a common feature of many angiosperms.
Although these data show that NO is a component of the dormancy loss process, they provide little evidence as to where and how NO is acting. Because seed dormancy is intimately tied to ABA production (Koornneef et al., 2002), the possibility was examined that there might be an interaction between ABA signalling and NO signalling. Inhibiting ABA synthesis during imbibition with norfluorazon was not sufficient to reduce seed dormancy (Fig. 4). These data argue against the hypothesis that cyanide or SNP vapours inhibited a metal-containing enzyme involved in the synthesis of ABA. On the other hand, SNP vapours desensitized weakly dormant and dormant seeds to exogenous ABA (Fig. 5). Whether this represents a direct or an indirect interaction between the ABA signalling pathway and NO or cyanide is unknown. Given the central role that ABA plays in seed dormancy, further experiments in this area are likely to be highly informative.
Gaseous delivery of dormancy-breaking compounds is an attractive experimental approach, and it has been used to apply purified NO (Fig. 9) and vapours from SNP, Fe(II)CN, Fe(III)CN (Fig. 6), and KCN (Fig. 7). The data show that, under the conditions used here, the NO donor SNP produced very little volatile NO, and its effects were derived from volatile cyanide. That NO was required for dormancy loss initiated by cyanide was demonstrated in experiments showing that the effect of volatile cyanide could be prevented by c-PTIO at 200 µM or more (Fig. 8).
Although c-PTIO maintained dormancy in the presence of SNP (Figs 2, 8), CN–, nitrite, and nitrate (Fig. 8), it is worth noting that the effect of c-PTIO could be partially overcome by increased concentrations of nitrite (Fig. 8B), SNP (Fig. 8C), or CN– (Fig. 8A) relative to the concentration of c-PTIO. These data suggest that SNP, CN– and nitrite lead to increased amounts of NO, which then react with c-PTIO and diminish its effectiveness. Nitrite is a known substrate for NO production (Yamasaki, 2000). That NO may be produced in response to CN– is an intriguing possibility.
The observation that nitric oxide gas was not as effective as SNP vapours in reducing dormancy does not exclude the possibility that NO is the only requirement for the breakage of dormancy. The seed coat of Arabidopsis is highly impermeable and NO diffusion rates may limit the effect of applied NO on dormancy. The data do not provide evidence that NO penetrated the seeds sufficiently for maximal effectiveness.
The data presented here do not point to a single model for how NO functions to reduce seed dormancy. Figure 10A shows a simple four-component, logical model that is consistent with the data presented in this paper. (i) Nitric oxide gas is sufficient to reduce dormancy (Fig. 9), and experiments with c-PTIO show that NO is required for the breakage of dormancy by cyanide, nitrate, and nitrite (Fig. 8). In addition, c-PTIO alone strengthens dormancy of partially after-ripened seeds (Fig. 1). These data lead to the firm suggestion that NO is required, and may be sufficient, to break dormancy. (ii) The data showing that cyanide and nitrite treatment can saturate c-PTIO (Fig. 8) suggest that NO concentrations may increase in response to the dormancy-breaking treatments that were used here. Elevation of NO concentration could result either from an increase in its synthesis, or from reduced NO breakdown. (iii) It is unknown whether NO levels need to rise in order for seeds to respond to cyanide. Steady-state NO levels may be sufficient for seeds to be able to respond to cyanide, making the response to cyanide essentially independent of NO, provided that the steady-state NO concentration is sufficiently high. (iv) Cyanide may potentiate the sensitivity of seeds to NO and, therefore, act downstream of NO production but upstream from NO perception. This model provides a useful framework for future research and the possibility is currently being tested that CN– brings about an increase in NO concentrations in seeds.
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Many molecular models could explain a cyanide-induced increase in NO, but models in which NO and CN– compete for a common binding site are particularly attractive. An example of such a model is depicted in Fig. 10B. NO in seeds is subject to enzymatic and non-enzymatic breakdown. For example, plants such as Arabidopsis contain non-legume haemoglobin (Trevaskis et al., 1997
; Hill, 1998), and non-legume haemoglobin converts NO to nitrate (Duff et al., 1998; Dordas et al., 2003). Applied CN– might bind to haemoglobin and prevent haemoglobin-dependent detoxification of NO, thereby bringing about an increase in NO concentration within the seed (Dordas et al., 2003). In this context it is interesting to note that NO signalling was indeed found to be modulated by overexpression of haemoglobin in Arabidopsis (Perazzolli et al., 2004).
Alternative targets for NO and CN– include cytochrome c oxidase and catalase. Like haemoglobin, cytochrome c oxidase has been shown to metabolize NO in mammals (Borutaite and Brown, 1996; Torres et al., 2000; Giuffre et al., 2005) and binding of CN– to cytochrome c oxidase strongly inhibited NO catabolism by this enzyme (Borutaite and Brown, 1996). Data showing that NO is produced in plant mitochondria (Planchet et al., 2005) and that NO inhibits mitochondrial function in plants (Casolo et al., 2005) indicate that a detoxification system for NO in mitochondria is likely to exist.
These models lead to the intriguing possibility that application of cyanide could result in elevated intracellular NO levels by interfering with enzymes that detoxify NO. If this is so, then cyanide application may be effective by increasing the concentration of NO within a seed.
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  J. M. Barrero, M. J. Talbot, R. G. White, J. V. Jacobsen, and F. Gubler Anatomical and Transcriptomic Studies of the Coleorhiza Reveal the Importance of This Tissue in Regulating Dormancy in Barley Plant Physiology, June 1, 2009; 150(2): 1006 - 1021. [Abstract] [Full Text] [PDF]
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T. Matakiadis, A. Alboresi, Y. Jikumaru, K. Tatematsu, O. Pichon, J.-P. Renou, Y. Kamiya, E. Nambara, and H.-N. Truong The Arabidopsis Abscisic Acid Catabolic Gene CYP707A2 Plays a Key Role in Nitrate Control of Seed Dormancy Plant Physiology, February 1, 2009; 149(2): 949 - 960. [Abstract] [Full Text] [PDF]
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 M. Moreau, G. I. Lee, Y. Wang, B. R. Crane, and D. F. Klessig AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase J. Biol. Chem., November 21, 2008; 283(47): 32957 - 32967. [Abstract] [Full Text] [PDF]
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 S. Jasid, M. Simontacchi, and S. Puntarulo Exposure to nitric oxide protects against oxidative damage but increases the labile iron pool in sorghum embryonic axes J. Exp. Bot., October 1, 2008; (2008) ern235v1. [Abstract] [Full Text] [PDF]
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 U. Lee, C. Wie, B. O. Fernandez, M. Feelisch, and E. Vierling Modulation of Nitrosative Stress by S-Nitrosoglutathione Reductase Is Critical for Thermotolerance and Plant Growth in Arabidopsis PLANT CELL, March 1, 2008; 20(3): 786 - 802. [Abstract] [Full Text] [PDF]
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S. Neill, R. Barros, J. Bright, R. Desikan, J. Hancock, J. Harrison, P. Morris, D. Ribeiro, and I. Wilson Nitric oxide, stomatal closure, and abiotic stress J. Exp. Bot., February 1, 2008; 59(2): 165 - 176. [Abstract] [Full Text] [PDF]
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S. Neill, J. Bright, R. Desikan, J. Hancock, J. Harrison, and I. Wilson Nitric oxide evolution and perception J. Exp. Bot., January 1, 2008; 59(1): 25 - 35. [Abstract] [Full Text] [PDF]
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F. Chopin, M. Orsel, M.-F. Dorbe, F. Chardon, H.-N. Truong, A. J. Miller, A. Krapp, and F. Daniel-Vedele The Arabidopsis ATNRT2.7 Nitrate Transporter Controls Nitrate Content in Seeds PLANT CELL, May 1, 2007; 19(5): 1590 - 1602. [Abstract] [Full Text] [PDF]
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P. C. Bethke, I. G.L. Libourel, N. Aoyama, Y.-Y. Chung, D. W. Still, and R. L. Jones The Arabidopsis Aleurone Layer Responds to Nitric Oxide, Gibberellin, and Abscisic Acid and Is Sufficient and Necessary for Seed Dormancy Plant Physiology, March 1, 2007; 143(3): 1173 - 1188. [Abstract] [Full Text] [PDF]
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F. Beisson, Y. Li, G. Bonaventure, M. Pollard, and J. B. Ohlrogge The Acyltransferase GPAT5 Is Required for the Synthesis of Suberin in Seed Coat and Root of Arabidopsis PLANT CELL, January 1, 2007; 19(1): 351 - 368. [Abstract] [Full Text] [PDF]
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