Dormancy removal in apple embryos by nitric oxide or cyanide involves modifications in ethylene biosynthetic pathway

The connection between classical phytohormone-ethylene and two signaling molecules, nitric oxide (NO) and hydrogen cyanide (HCN), was investigated in dormancy removal and germination “sensu stricto” of apple (Malus domestica Borkh.) embryos. Deep dormancy of apple embryos was removed by short-term (3–6 h) pre-treatment with NO or HCN. NO- or HCN-mediated stimulation of germination was associated with enhanced emission of ethylene by the embryos, coupled with transient increase in ROS concentration in embryos. Ethylene vapors stimulated germination of dormant apple embryos and eliminated morphological anomalies characteristic for young seedlings developed from dormant embryos. Inhibitors of ethylene receptors completely impeded beneficial effect of NO and HCN on embryo germination. NO- and HCN-induced ethylene emission by apple embryo was only slightly reduced by inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity during first 4 days of germination. Short-term pre-treatment of the embryos with NO and HCN modified activity of both key enzymes of ethylene biosynthetic pathway: ACC synthase and ACC oxidase. Activity of ACC synthase declined during first 4 days of germination, while activity of ACC oxidase increased markedly at that time. Additional experiments point to non-enzymatic conversion of ACC to ethylene in the presence of ROS (H₂O₂). The results indicate that NO and HCN may alleviate dormancy of apple embryos “via” transient accumulation of ROS, leading to enhanced ethylene emission which is required to terminate germination “sensu stricto”. Therefore, ethylene seems to be a trigger factor in control of apple embryo dormancy removal and germination.     

The beneficial effect of small toxic molecules on dormancy alleviation and germination of apple embryos is due to NO formation

Deep dormancy of apple (Malus domestica Borkh.) seeds is terminated by a 3-month-long cold stratification. It is expressed by rapid germination of seeds and undisturbed growth of seedlings. However, stimulation of germination of isolated apple embryos is also observed after applying inhibitors of cytochrome c oxidase: nitric oxide (NO) or hydrogen cyanide (HCN) during the first 3–6 h of imbibition of dormant embryos. The aim of this work was to compare the effect of yet another toxic gaseous molecule carbon monoxide (CO) with the effects of HCN and NO on germination of apple embryos and growth and development of young seedlings. We demonstrated that stimulation of germination after short-term pre-treatment with HCN, NO or CO was accompanied by enhanced NO emission from the embryo axes during their elongation. Moreover, similarly high NO production from non-dormant embryos, after cold stratification, was detected. Therefore, we propose that NO may act as signaling molecule in apple embryo dormancy break.     

Nitric oxide and hydrogen cyanide as regulating factors of enzymatic antioxidant system in germinating apple embryos

Short-term (3 or 6 h) pre-treatment of apple (Malus domestica Borkh.) embryos with nitric oxide (NO) or hydrogen cyanide (HCN) induces transient accumulation of reactive oxygen species (ROS) leading to dormancy removal and germination. We demonstrated that enhanced NO emission by apple embryos during early phase of germination “sensu stricto” is required for seed transition from dormant into non-dormant state, and may be described by the model of “nitrosative door”, analogous to “oxidative window”. Cellular ROS concentration, resulting from NO or HCN embryo pre-treatment, seems to be under severe control of antioxidant system. Activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX) and total peroxidases (Prxs) was determined during NO and HCN-mediated germination “sensu stricto” of embryos. CAT and SOD activity increased transiently 24 h after embryos pre-treatment, while GR and Prx activity was stimulated mainly after 96 h. The most evident alterations were detected in GPX activity, being more than threefold stimulated by NO or HCN. Based on this results, we conclude that these reactive molecules act simultaneously crossing their signaling pathways and we propose that ROS, reactive nitrogen species, HCN at accurate level are essential during seed germination as signaling factors.     

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.     

Dormancy alleviation by NO or HCN leading to decline of protein carbonylation levels in apple (Malus domestica Borkh.) embryos

Deep dormancy of apple (Malus domestica Borkh.) embryos can be overcome by short-term pre-treatment with nitric oxide (NO) or hydrogen cyanide (HCN). Dormancy alleviation of embryos modulated by NO or HCN and the first step of germination depend on temporary increased production of reactive oxygen species (ROS). Direct oxidative attack on some amino acid residues or secondary reactions via reactive carbohydrates and lipids can lead to the formation of protein carbonyl derivatives. Protein carbonylation is a widely accepted covalent and irreversible modification resulting in inhibition or alteration of enzyme/protein activities. It also increases the susceptibility of proteins to proteolytic degradation. The aim of this work was to investigate protein carbonylation in germinating apple embryos, the dormancy of which was removed by pre-treatment with NO or HCN donors. It was performed using a quantitative spectrophotometric method, while patterns of carbonylated protein in embryo axes were analyzed by immunochemical techniques. The highest concentration of protein carbonyl groups was observed in dormant embryos. It declined in germinating embryos pre-treated with NO or HCN, suggesting elevated degradation of modified proteins during seedling formation. A decrease in the concentration of carbonylated proteins was accompanied by modification in proteolytic activity in germinating apple embryos. A strict correlation between the level of protein carbonyl groups and cotyledon growth and greening was detected. Moreover, direct in vitro carbonylation of BSA treated with NO or HCN donors was analyzed, showing action of both signaling molecules as protein oxidation agents.     

Dormancy removal of apple seeds by cold stratification is associated with fluctuation in H2O2, NO production and protein carbonylation level

Reactive oxygen (ROS) and nitrogen (RNS) species play a signaling role in seed dormancy alleviation and germination. Their action may be described by the oxidative/nitrosative “window/door”. ROS accumulation in embryos could lead to oxidative modification of protein through carbonylation. Mature apple (Malus domestica Borkh.) seeds are dormant and do not germinate. Their dormancy may be overcome by 70–90 days long cold stratification. The aim of this work was to analyze the relationship between germinability of embryos isolated from cold (5 °C) or warm (25 °C) stratified apple seeds and ROS or nitric oxide (NO) production and accumulation of protein carbonyl groups. A biphasic pattern of variation in H₂O₂ concentration in the embryos during cold stratification was detected. H₂O₂ content increased markedly after 7 days of seeds imbibition at 5 °C. After an additional two months of cold stratification, the H₂O₂ concentration in embryos reached the maximum. NO production by the embryos was low during entire period of stratification, but increased significantly in germination sensu stricto (i.e. phase II of the germination process). The highest content of protein carbonyl groups was detected after 6 weeks of cold stratification treatment. Fluctuation of H₂O₂ and protein carbonylation seems to play a pivotal role in seed dormancy alleviation by cold stratification, while NO appears to be necessary for seed germination.     

Polyamines and Nitric Oxide Link in Regulation of Dormancy Removal and Germination of Apple (Malus domestica Borkh.) Embryos

Polyamines (PAs) belong to plant growth regulators and in complex with classical phytohormones take part in regulation of seed dormancy and germination. Although the impact of reactive oxygen (ROS) and nitrogen (RNS) species on seed germination is well described, the cross talk of PAs with ROS/RNS has never been analyzed. Due to the close connection of PAs and ethylene biosynthetic pathways to arginine (Arg)-dependent NO biosynthesis we investigated production of nitric oxide (NO), peroxynitrite (ONOO^?) and the level of O ₂ ^?? or H₂O₂ in apple embryos, germination of which was PA regulated. PAs: putrescine (Put) and spermidine (Spd) in contrast to spermine (Spm) stimulated germination of apple embryos. Among amino acids, stimulation of germination was observed in Arg and ornithine (Orn) only. Dormancy removal of embryos by PAs was associated with increased accumulation of H₂O₂ and O ₂ ^?? in embryonic axes. At the same stage of completion of sensu stricto germination the stimulatory effect of PAs (Put and Spd) and amino acids, mainly Arg and Orn, was accompanied by enhanced NO and ONOO^? production in embryonic axis. The beneficial effect of PAs (Put and Spd) and their precursors on germination of apple embryos was removed by NO scavenging, suggesting a crucial role of NO in termination of embryo germination and radicle growth. Moreover, activity of polyamine oxidase in embryo axes was greatly enhanced by embryo fumigation with NO. Our data demonstrate the interplay of RNS/ROS with PAs and point to NO action as an integrator of endogenous signals activating germination.     

Involvement of nitrate reductase-dependent nitric oxide production in magnetopriming-induced salt tolerance in soybean

In the present study, experiments were performed to investigate the role of nitric oxide (NO) in magnetopriming-induced seed germination and early growth characteristics of soybean (Glycine max) seedlings under salt stress. The NO donor (sodium nitroprusside, SNP), NO scavenger (2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, CPTIO), inhibitors of nitrate reductase (sodium tungstate, ST) or NO synthase (N-nitro-L-Arg-methyl ester, LNAME) and NADPH oxidase inhibitor (diphenylene iodonium, DPI) have been used to measure the role of NO in the alleviation of salinity stress by static magnetic field (SMF of 200 mT, 1 h). Salt stress (50 mM NaCl) significantly reduced germination and early growth of seedlings emerged from non-primed seeds. Pre-treatment of seeds with SMF positively stimulated the germination and consequently promoted the seedling growth. ST, LNAME, CPTIO and DPI significantly decreased the growth of seedling, activities of α-amylase, protease and nitrate reductase (NR), hydrogen peroxide (H₂O₂), superoxide (O₂^•−) and NO content in roots of seedlings emerged from non-primed and SMF-primed seeds. However, the extent of reduction was higher with ST in seedlings of SMF-primed seeds under both conditions, whereas SNP promoted all the studied parameters. Moreover, the generation of NO was also confirmed microscopically using a membrane permanent fluorochrome (4-5-diaminofluorescein diacetate [DAF-2 DA]). Further, analysis showed that SMF enhanced the NR activity and triggered the NO production and NR was maximally decreased by ST as compared to LNAME, CPTIO and DPI. Thus, in addition to ROS, NO might be one of the important signaling molecules in magnetopriming-induced salt tolerance in soybean and NR may be responsible for SMF-triggered NO generation in roots of soybean.     

Sugar metabolism as related to the cyanide-mediated elimination of dormancy in apple embryos

Embryos isolated from dormant seeds of apple (Malus domestica Borb., cv. Antonówka) were treated by gaseous HCN (1 mM) for 6 h and then cultured in the light for 9 d in parallel to control non-treated embryos. Soluble sugars were quantified, and oligosaccharide hydrolysing enzyme activities were determined in axes and in cotyledons of such embryos during culture. HCN pre-treatment stimulated germination and hydrolysis of oligosaccharides in embryonic axes. Hydrolysis of sucrose in the lower cotyledon (in contact with wet medium and therefore growing and greening faster) was affected by HCN to a small extent. On the other hand, growth, greening and sucrose hydrolysis in the upper cotyledon were stimulated by HCN pre-treatment to the levels observed in lower cotyledon. It is postulated that endogenous HCN in apple seeds controls the removal of embryonic dormancy in parallel to the earlier described control by light and gibberellin. Gibberellin was demonstrated to act on the hydrolysis and further transformations of storage lipids, including gluconeogenesis, but not β-oxidation of fatty acids. It appears that hydrolysis of oligosaccharides and their catabolism is the target for cyanide.     

Triangular interplay between ROS,ABA and GA in dormancy alleviation of <Emphasis Type="Italic">Bunium persicum</Emphasis> seeds by cold stratification

Seeds of Bunium persicum (Boiss.) B. Fedtsch. have complex physiological dormancy that can be released by 15 weeks stratification. The present study revealed that cold stratification enhanced content of H₂O₂, O^{2 -·} and application of GA₃ and ROS donors (Fenton reagent, H₂O₂, methylviologen and menadione) did not affect or only slightly promoted the germination of non-stratified, fully dormant seeds. Dormancy was markedly decreased by ROS-generating reagents, GA₃ and fluridone (an inhibitor of ABA biosynthesis) and was enhanced by ROS-decreasing compounds (DMTU, Tiron, SB and DPI), diniconazole (Dinc, an inhibitor of ABA catabolism) and paclobutrazol (PAC, an inhibitor of GA biosynthesis) when dormancy was partially removed by cold stratification. The response to these compounds reduced with increasing time of stratification. ABA inhibited germination by repressing of NADPH oxidase activity and ROS accumulation and conversely, GA triggered germination by promoting an increase of NADPH oxidase activity and ROS levels. Data in this study, for the first time suggest releasing deep complex physiological dormancy by cold stratification is associated with interplay between ROS and ABA/GA.     

Spatial and temporal regulation of the metabolism of reactive oxygen and nitrogen species during the early development of pepper (Capsicum annuum) seedlings

Background and Aims The development of seedlings involves many morphological, physiological and biochemical processes, which are controlled by many factors. Some reactive oxygen and nitrogen species (ROS and RNS, respectively) are implicated as signal molecules in physiological and phytopathological processes. Pepper (Capsicum annuum) is a very important crop and the goal of this work was to provide a framework of the behaviour of the key elements in the metabolism of ROS and RNS in the main organs of pepper during its development.Methods The main seedling organs (roots, hypocotyls and green cotyledons) of pepper seedlings were analysed 7, 10 and 14 d after germination. Activity and gene expression of the main enzymatic antioxidants (catalase, ascorbate–glutathione cycle enzymes), NADP-generating dehydrogenases and S-nitrosoglutathione reductase were determined. Cellular distribution of nitric oxide (^·NO), superoxide radical (O₂^·–) and peroxynitrite (ONOO^–) was investigated using confocal laser scanning microscopy.Key Results The metabolism of ROS and RNS during pepper seedling development was highly regulated and showed significant plasticity, which was co-ordinated among the main seedling organs, resulting in correct development. Catalase showed higher activity in the aerial parts of the seedling (hypocotyls and green cotyledons) whereas roots of 7-d-old seedlings contained higher activity of the enzymatic components of the ascorbate glutathione cycle, NADP-isocitrate dehydrogenase and NADP-malic enzyme.Conclusions There is differential regulation of the metabolism of ROS, nitric oxide and NADP dehydrogenases in the different plant organs during seedling development in pepper in the absence of stress. The metabolism of ROS and RNS seems to contribute significantly to plant development since their components are involved directly or indirectly in many metabolic pathways. Thus, specific molecules such as H₂O₂ and NO have implications for signalling, and their temporal and spatial regulation contributes to the success of seedling establishment.     

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.     

Dormancy and Impotency of Cocklebur Seeds: VII. Inability of Dormant Cotyledons to Form Chlorophyll

Dormant seeds of cocklebur (Xanthium pennsylvanicum Wallr.) were characterized by the lack of ability to form chlorophyll. Such an inability of cotyledons of the dormant seeds was improved by the application of various factors and reagents which were capable of breaking the dormancy and of increasing cotyledon enlargement. Of these, ethylene, benzyladenine, and high temperature treatments were particularly effective, and, in turn, oxygen enrichment, gibberellic acid, thiourea, carbon doxide, and potassium nitrate were also promotive to the greening of the dormant cotyledons. The effects of benzyladenine, oxygen enrichment, and high temperature were reduced in the presence of absorbents for endogenously evolve carbon dioxide and ethylene. δ-Aminolevulinic acid could not restore their greening ability.     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

A protocol for direct somatic embryogenesis of Protea cynaroides L. using zygotic embryos and cotyledon tissues

We describe a protocol for somatic embryogenesis of Protea cynaroides, with potential for high frequency production of this important horticultural species. Somatic embryos formed directly on both P. cynaroides mature zygotic embryos and excised cotyledons cultured on MS medium without growth regulators. The addition of growth regulators such as naphthalene acetic acid (NAA) (5; 13 and 27 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (5; 11 and 23 μM), in combination with thidiazuron (TDZ) (1 μM), benzylaminopurine (BAP) (1 μM) or kinetin (1 μM) suppressed the formation of somatic embryos. After eight weeks in culture, formation of somatic embryos was observed. Zygotic explants formed the most embryos when cultured in a 12-h photoperiod in comparison to explants cultured in the dark. Up to 83% of these embryos germinated after transferal to the germination medium containing 0.3 μM GA₃. Significantly fewer embryos germinated in MS medium with no growth regulators, or supplemented with higher concentrations of GA₃, while low germination percentages were also observed in MS media containing casein hydrolysate and coconut water. The germination of normal somatic embryos (two separate cotyledons and a single radicle) was observed only in media containing either no growth regulators, 0.3 μM GA₃ or 1 μM GA₃. All embryos that germinated in high concentrations of GA₃ were malformed.     

Gas Content of Berries of the Australian Mistletoe Amyema preissii and the Effect of Maturity, Viscin, Temperature and Carbon Dioxide on Germination

Experiments conducted at light intensities equivalent to thosereaching the embryo through the intact pericarp showed that: (a) excised embryos from green berries would not germinate,but those from transitional and pink (mature) berries begangermination immediately; (b) the viscid layer neither stimulated nor inhibited germination; (c) there was 100% germination at 10–25°C but maximumelongation at 25–30 °C, with a minimum at 4 °Cand death at 40°C; (d) there was 100% germination at 1–5% CO₂ in air, butmaximum elongation at 1%. with>90% germination in air minusCO₂ and negligible at 20% CO₂, 16% O₂, 64% N₂|| or higher concentrationsof CO₂; (e) the average gas concentration in mature berries, as measuredby gas spectrometry, was 127% CO₂, 12% O₂, 61% N₂||. Minor fluctuationsin CO₂/O₂ content indicated limited photosynthesis as well asrespiration in both green and mature berries. The high CO₂ concentrationin the berry is sufficient in itself to explain the maintenanceof dormancy until the seed is excised by birds; (f) contrary to previous assumptions, most elongation of theembryo was due to growth of the cotyledons rather than of thehypocotyl or radicle.     

Assessment of the Germination Potential of Brassica oleracea Seeds Treated with Karrikin 1 and Cyanide,Which Modify the Ethylene Biosynthetic Pathway

The relationship between ethylene and cyanide (HCN) and karrikin 1 (KAR1) in dormancy release was studied in secondary dormant Brassica oleracea L. (Chinese cabbage) seeds. Freshly harvested seeds of Brassica oleracea usually have poor germination potential. Karrikin1 (KAR1) and cyanide (HCN) are able to stimulate seed germination. However, the stimulatory effects of these two chemicals depend on the activation of the ethylene biosynthesis pathway and on ethylene perception. In this study, KAR1 and HCN application increased the activity of ethylene and of two ethylene biosynthesis enzymes, ACC synthase (ACS) and ACC oxidase (ACO). KAR1 and HCN collectively promoted the accumulation of 1 aminocyclopropane-1-carboxylic acid (ACC). In the presence of NO (nitric oxide) and KAR1, ACS and ACO activities reached their maximum levels after 36 and 42 h, respectively. Ethylene inhibitors suppressed seed germination by approximately 55%, whereas the respiratory inhibitors SHAM and NaN3 inhibited seed germination by 5–10% in the presence of HCN and KAR1. KAR1 and HCN collectively reduced the abscisic acid (ABA) content in seeds, increased the gibberellic acid (GA) content and released seed dormancy. The expression of ethylene biosynthesis genes and ethylene receptor genes (BOACO1, BOACS1, BOACS3, BOACS4, BOACS5, BOACS7, BOACS9, BOACS11, BOETR1 and BOETR2) provided further evidence of the involvement of ethylene in KAR1 and HCN-induced germination. BOACO1, BOACS1, BOACS5, BOACS7, BOACS9, BOACS11, BOETR1 and BOETR2 genes were up regulated in the presence of KAR1 and HCN, while the remaining genes were down regulated. The expression of various ethylene biosynthesis and ethylene receptor genes suggested functional diversification and variations in seed sensitivity in the presence of KAR1 and HCN. Therefore, in the current study, KAR1 and HCN application effectively induced the germination of B. oleracea seeds (approximately 97% germination rate) after 6 days by modifying the ethylene biosynthetic pathway.

     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.     

Response of Amaranthus retroflexus L. seeds to gibberellic acid, ethylene and abscisic acid depending on duration of stratification and burial

Freshly harvested, dormant seeds of Amaranthus retroflexus were unable to germinate at 25 and 35 °C. To release their dormancy at the above temperatures, the seeds were stratified at a constant temperature (4 °C) under laboratory conditions or at fluctuating temperatures in soil or by outdoor burial in soil. Fully dormant, or seeds stratified or buried (2006/2007 and 2007/2008) for various periods were treated with exogenous gibberellic acid (GA₃), ethephon and abscisic acid (ABA). Likewise, the effects of these regulators, applied during stratification, on seed germination were determined. The results indicate that A. retroflexus seed dormancy can be released either by stratification or by autumn–winter burial. The effect of GA₃ and ethylene, liberated from ethephon, applied after various periods of stratification or during stratification, depends on dormancy level. GA₃ did not affect or only slightly stimulated the germination of non-stratified, fully dormant seeds at 25 and 35 °C respectively. Ethylene increased germination at both temperatures. Seed response to GA₃ and ethylene at 25 °C was increased when dormancy was partially removed by stratification at constant or fluctuating temperatures or autumn–winter burial. The response to GA₃ and ethylene increased with increasing time of stratification. The presence of GA₃ and ethephon during stratification may stimulate germination at 35 °C. Thus, both GA₃ and ethylene can partially substitute the requirement for stratification or autumn–winter burial. Both hormones may also stimulate germination of secondary dormant seeds, exhumed in September. The response to ABA decreased in parallel with an increasing time of stratification and burial up to May 2007 or March 2008. Endogenous GA_n, ethylene and ABA may be involved in the control of dormancy state and germination of A. retroflexus. It is possible that releasing dormancy by stratification or partial burial is associated with changes in ABA/GA and ethylene balance and/or sensitivity to these hormones.