Reactive oxygen species,ABA and nitric oxide interactions on the germination of warm-season C<Subscript>4</Subscript>-grasses

Hydrogen peroxide (H₂O₂) as a source of reactive oxygen species (ROS) significantly stimulated germination of switchgrass (Panicum virgatum L.) seeds with an optimal concentration of 20 mM at both 25 and 35°C. For non-dormant switchgrass seeds exhibiting different levels of germination, treatment with H₂O₂ resulted in rapid germination (<3 days) of all germinable seeds as compared to seeds placed on water. Exposure to 20 mM H₂O₂ elicited simultaneous growth of the root and shoot system, resulting in more uniform seedling development. Seeds of big bluestem (Andropogon gerardii Vitman) and indiangrass [Sorghastrum nutans (L.) Nash] also responded positively to H₂O₂ treatment, indicating the universality of the effect of H₂O₂ on seed germination in warm-season prairie grasses. For switchgrass seeds, abscisic acid (ABA) and the NADPH-oxidase inhibitor, diphenyleneiodonium (DPI) at 20 μM retarded germination (radicle emergence), stunted root growth and partially inhibited NADPH-oxidase activity in seeds. H₂O₂ reversed the inhibitory effects of DPI and ABA on germination and coleoptile elongation, but did not overcome DPI inhibition of root elongation. Treatment with H₂O₂ appeared to enhance endogenous production of nitric oxide, and a scavenger of nitric oxide abolished the peroxide-responsive stimulation of switchgrass seed germination. The activities and levels of several proteins changed earlier in seeds imbibed on H₂O₂ as compared to seeds maintained on water or on ABA. These data demonstrate that seed germination of warm-season grasses is significantly responsive to oxidative conditions and highlights the complex interplay between seed redox status, ABA, ROS and NO in this system.     

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.     

ABA,ROS and NO are Key Players During Switchgrass Seed Germination

Seed dormancy and germination are complex physiological processes usually under hormonal control. Germination of seeds from many plants including switchgrass, are inhibited by ABA and promoted by NO or ROS. However, ABA apparently requires both ROS and NO as intermediates in its action, with ROS produced by membrane-bound NADPH-oxidases responsive to ABA. In switchgrass seeds, externally supplied hydrogen peroxide (ROS), but not NO will overcome ABA-imposed inhibition of germination. Stimulation of germination by external ROS can be partially blocked by NO-scavengers, suggesting that NO is required for seed germination in switchgrass as well as for ABA-induced inhibition of germination. Collectively, these data suggest that multiple mechanisms might be required to sense and respond to varying levels of ABA, NO and ROS in switchgrass seeds.Key Words: switchgrass, seed germination, ROS, hydrogen peroxide, ABA, nitric oxide     

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     

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     

Sodium nitroprusside, cyanide, nitrite, and nitrate break Arabidopsis seed dormancy in a nitric oxide-dependent manner

The seeds of many plant species are dormant at maturity and dormancy loss is a prerequisite for germination. Numerous environmental and chemical treatments are known to lessen or remove seed dormancy, but the biochemical changes that occur during this change of state are poorly understood. Several lines of research have implicated nitric oxide (NO) as a participant in this process. Here, we show that dormant seeds of Arabidopsis thaliana (L.) Heynh. will germinate following treatment with the NO donor sodium nitroprusside (SNP), cyanide (CN), nitrite or nitrate. In all cases, the NO scavenger c-PTIO effectively promotes the maintenance of seed dormancy. c-PTIO does not, however, inhibit germination of fully after-ripened seeds, and c-PTIO does not interact directly with nitrite, nitrate or CN. We also show that volatile CN effectively breaks dormancy of Arabidopsis seeds, and that CN is the volatile compound in SNP that promotes dormancy loss. Our data support the hypothesis that NO is a signaling molecule that plays an important role in the loss of seed dormancy.     

The Use of Abscisic Acid to Synchronize Carrot Seed Germination Prior to Fluid Drilling

Abscisic acid (ABA) was used as a reversible block to the progressof carrot seed germination in a practical seed treatment. Pre-treatingseeds with 10^–4M ABA solution at 15 °C for 12 d gave93% germination of viable seeds on subsequent transfer to waterbefore radicle lengths became too long for fluid drilling. Thiscompared with only 31 % without pre-treatment ABA pretreatment significantly increased the synchrony of carrotseed germination and did not affect final percentage germinationor early seedling growth rates. Seedling emergence from ABA-treatedgerminating seeds was earlier and more uniform than from untreatedgerminating seeds and seedlings from both these treatments emergedbefore those from ungerminated seeds Daucus carota L., carrot, germination, seed treatment, fluid drilling, abscisic acid, radicle extension     

Water-soluble phenolic compounds in the coat control germination and peroxidase reactivation in <Emphasis Type="Italic">Triticum aestivum</Emphasis> seeds

In this work, we investigated the inhibitory effects of water-soluble phenolic compounds (WSPCs) in the coat of after-ripening wheat (Triticum aestivum L.) seeds on the processes of germination and peroxidase reactivation. Wheat bran has a WSPC content of 862.5 μg gallic acid equivalent g⁻¹ dry weight. When seeds were incubated in the water extract of bran, germination, peroxidase reactivation, and coleoptile and radicle growth were suppressed in a WSPC concentration-dependent manner. The inhibitory effects were significantly ameliorated by removing WSPCs from bran extract by treating with 1% insoluble polyvinylpolypyrrolidone. Pretreatment of seeds with 0.1% H₂O₂ reduced the WSPC content in the coat, which was confirmed using Fourier transform infrared microspectroscopy. With H₂O₂ pretreatment, seed germination, peroxidase reactivation, and post-germination seedling growth were significantly stimulated. Application of the known phenolics caffeic acid, feruic acid, or vanillin to the germination medium blocked seed germination and suppressed peroxidase reactivation. The results described here indicate that WSPCs act as endogenous inhibitors in the coat to control germination of Triticum aestivum seeds, and that inhibition of germination is at least partially caused by suppressing peroxidase reactivation.     

Gibberellin-mediated changes in carbohydrate metabolism during flower stalk elongation in tulips

We tested the effects of cold stratification, temperature, light and NaCl on seed germination and germination recovery and of NaCl on radicle growth and radicle elongation recovery of Kalidium caspicum, a small leafy succulent shrub dominant in saline deserts in northwest China. In all conditions of temperature and light/darkness, germination percentages and rates of cold-stratified seeds were significantly higher than those of nonstratified seeds. Germination of a high percentage of both nonstratified and stratified seeds was inhibited by 0.2 M NaCl, and 0.6 M NaCl completely inhibited germination. Nongerminated seeds germinated after they were transferred from NaCl solutions to distilled water. Radicle elongation significantly decreased with increase in salinity, and it was completely inhibited by ≥1.0 M NaCl; radicle elongation recovered in young seedlings pretreated by 10 days of incubation in ≤0.4 M NaCl. Results show that seed germination and early seedling growth of K. caspicum are salt tolerant, and these characteristics help explain why this species can survive and dominate salt habitats, such as those in the Junggar desert in Xinjiang, northwest China.     

Seed vigour and water relations in wheat

The laboratory germination (criterion radicle emergence) of seven seed lots of winter wheat cv. Slejpner was similar. However, they differed in vigour as demonstrated by differences in germination after controlled deterioration carried out at a range of seed moisture contents, at two temperatures and for different times. A vigour assessment for each lot was quantified by calculating a value for the seed lot constant K_i, of the viability equation. Germination in lower water potentials reduced the uptake of water, radicle and coleoptile emergence and radicle and coleoptile extension. There was no difference in the water uptake of seed lots differing in vigour. However, seed lots of lower vigour showed less radicle emergence, less coleoptile emergence and shorter radicles than higher vigour seed lots in low water potentials. Similarly, controlled deterioration resulted in reduced radicle and coleoptile emergence and growth compared to unaged seed, and also to a greater sensitivity to low water potentials. The implications for field establishment are discussed.     

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.     

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.     

Induction of chilling tolerance in wheat during germination by pre-soaking seed with nitric oxide and gibberellin

Chilling depresses seed germination and seedling establishment, and is one major constraint to grain yield formation in late sown winter wheat. Seeds of winter wheat (Triticum aestivum L.) were separately pre-soaked with sodium nitroprusside (SNP, as nitric oxide donor) and Gibberellic acid (GA₃) before germination and then germinated under low temperature. SNP and GA₃ pre-treatment increased seed germination rate, germination index, weights and lengths of coleoptile and radicle, while they decreased mean germination time and weight of seeds germinating under low temperature. Exogenous NO and GA₃ increased seed respiration rate and promoted starch degradation along with increased amylase activities. In addition, efficient antioxidant systems were activated by NO, and which effectively reduced concentrations of malondialdehyde and hydrogen peroxide (H₂O₂). Seedling growth was also enhanced by exogenous NO and GA₃ as a result of improved seed germination and maintenance of better reactive oxygen species homeostasis in seedling growing under chilling temperatures. It is indicated that exogenous NO was more effective than GA₃ in alleviating chilling stress during seed germination and seedling establishment in wheat.     

Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root

Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.     

珍稀濒危植物珙桐种子休眠萌发过程中内源激素的变化

珙桐是我国特有珍稀濒危植物,休眠期长且具二次休眠现象。将处于休眠萌发过程中的珙桐种子依据胚根长度划分为4个阶段,利用高效液相色谱(HPLC)测定各阶段种子及其内果皮中ABA(脱落酸)、GA(赤霉素)、KT(细胞分裂素)、IAA(3-吲哚乙酸)4种内源激素含量,分析其比值动态变化,并与幼苗阶段进行比较。结果显示:未破壳种子的内果皮中内源激素含量以ABA最高,其次是GA、IAA、KT,随着种子破壳后四种激素含量显著降低。除ABA外,种子中GA、IAA和KT含量随着胚根的伸长逐渐升高,但仍低于幼苗阶段。此外,随着胚根伸长,种子中GA/ABA、IAA/ABA、KT/ABA比值逐渐增大,其中以GA/ABA的变化最显著。因此,珙桐种子的休眠和萌发可能主要受ABA和GA的平衡和拮抗来调控。     

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.     

Proanthocyanidins Inhibit Seed Germination by Maintaining a High Level of Abscisic Acid in Arabidopsis thaliana

Proanthocyanidins (PAs) are the main products of the flavonoid biosynthetic pathway in seeds, but their biological function during seed germination is still unclear. We observed that seed germination is delayed with the increase of exogenous PA concentration in Arabidopsis. A similar inhibitory effect occurred in peeled Brassica napus seeds, which was observed by measuring radicle elongation. Using abscisic acid (ABA), a biosynthetic and metabolic inhibitor, and gene expression analysis by real-time polymerase chain reaction, we found that the inhibitory effect of PAs on seed germination is due to their promotion of ABA via de novo biogenesis, rather than by any inhibition of its degradation. Consistent with the relationship between PA content and ABA accumulation in seeds, PA-deficient mutants maintain a lower level of ABA compared with wild-types during germination. Our data suggest that PA distribution in the seed coat can act as a doorkeeper to seed germination. PA regulation of seed germination is mediated by the ABA signaling pathway.     

Measurement of the Water Potential of Intact Plant Tissues2PISUM SATIVUM L.)

Through the use of a microthermocouple psychrometer it has becomepossible to measure the water potential over part of the surfaceof a pea seed, starting about 19 hours after sowing in distilledwater, or later in the case of seeds germinating at lower osmoticpotentials, in both instances until well after radicles haveemerged. It has been shown that the potential of air-dry seedsis well below –6,000 joules/kg but increases rapidly duringimbibition, depending upon the water potential of the germinationmedium. Pea seeds subjected to lower external water potentialsgerminate at lower internal water potentials than they exhibitin distilled water. The water potentials of the seeds decreasejust after radicle emergence till the radicle establishes contactwith its germination medium, possibly as a result of demandfor moisture during that period due to incipient cell elongation.No detectable amounts of osmotically active substances are exudedfrom the seed during germination; the pea seed coat restrictsthe entry of polyethylene glycol molecules into the seed untilemergence of the radicle.