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.     

Proanthocyanidins accelerate the germination of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) seeds

Proanthocyanidins (PAs) are the end products of the flavonoid biosynthetic pathway in many seeds, but their biological function is rarely unknown during seed germination. In the present study, we observed that PAs pretreatment accelerated cucumber seeds germination with maximum efficiency at 0.15% by measuring germination percentage and radical length. Using inhibitors of abscisic acid (ABA), gibberellins (GA) and alternative oxidase (AOX) and H₂O₂ scavenger pretreatment and gene expression analysis, we found that the accelerated effect of 0.15% PAs on seed germination was due to the decreased ABA biogenesis and enhanced GA production. ROS are induced by PAs pretreatment. Then, the enhanced ROS contributed to GA and ethylene accumulation and ABA decrease in seeds. Moreover, the improvement of GA was involved in the further induction of antioxidant enzymes activities. Therefore, our findings uncover a novel role of PAs in seed germination and clarify the relationships between ROS, ABA, GA and ethylene during seed germination.     

The interactions of plant growth regulators and H<Subscript>2</Subscript>O<Subscript>2</Subscript> during germination improvement of sweet corn seed through spermidine application

Low seed vigor was the main constraint on the production of sweet corn in China. Spermidine (Spd) was proved to enhance sweet corn seed germination. However, little was known about the metabolisms and interactions of plant growth regulators (PGRs) and H₂O₂ in the enhancement of Spd upon sweet corn seed germination. Spd, GA, C₂H₄ and H₂O₂ soaking treatments significantly enhanced seed vigor; while their respective biosynthesis inhibitors and ABA significantly declined seed vigor. Besides, as compared with control, seed vigor showed no significant difference in Spd+ProG (prohexadione-calcium, the inhibitor of GA), however it decreased significantly in Spd+ABA. The seed vigor treated by Spd+AVG (aviglycine hydrochloride, the inhibitor of C₂H₄) and Spd+NAC (n-acetyl-l-cysteine, a scavenger of H₂O₂) were significantly lower than those soaked in Spd solution, but still significantly higher than the control. Spd+NAC with significantly lower H₂O₂ content still up-regulated GA and C₂H₄ contents and down-regulated ABA content during seed germination. The results suggested that it was Spd rather than H₂O₂ (produced through Spd) made a direct effect on PGRs metabolism regulation in seed germination enhancement by Spd. The metabolism of GA and ABA played crucial rolesas compared with C₂H₄ and H₂O₂. Besides, complicated PGRs interactions and crosstalk between H₂O₂ and PGRs existed during sweet corn seed germination after Spd soaking, and ABA might be a key hormone in this process.     

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     

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.     

Control of rice pre‐harvest sprouting by glutaredoxin‐mediated abscisic acid signaling

Pre‐harvest sprouting (PHS) is one of the major problems in cereal production worldwide, which causes significant losses of both yield and quality; however, the molecular mechanism underlying PHS remains largely unknown. Here, we identified a dominant PHS mutant phs9‐D. The corresponding gene PHS9 encodes a higher plant unique CC‐type glutaredoxin and is specifically expressed in the embryo at the late embryogenesis stage, implying that PHS9 plays some roles in the late stage of seed development. Yeast two‐hybrid screening showed that PHS9 could interact with OsGAP, which is an interaction partner of the abscicic acid (ABA) receptor OsRCAR1. PHS9‐ or OsGAP overexpression plants showed reduced ABA sensitivity in seed germination, whereas PHS9 or OsGAP knock‐out mutant plants showed increased ABA sensitivity in seed germination, suggesting that PHS9 and OsGAP acted as negative regulators in ABA signaling during seed germination. Interestingly, the germination of PHS9 and OsGAP overexpression or knock‐out plant seeds was weakly promoted by H₂O₂, implying that PHS9 and OsGAP could affect reactive oxygen species (ROS) signaling during seed germination. These results indicate that PHS9 plays an important role in the regulation of rice PHS through the integration of ROS signaling and ABA signaling.     

Hydrogen peroxide production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The presence of 2 mM bicarbonate in the incubation medium induced stomatal closure in abaxial epidermis of Arabidopsis. Exposure to 2 mM bicarbonate elevated the levels of H₂O₂ in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H₂DCF-DA). Bicarbonate-induced stomatal closure as well as H₂O₂ production were restricted by exogenous catalase or diphenylene iodonium (DPI, an inhibitor of NAD(P)H oxidase). The reduced sensitivity of stomata to bicarbonate and H₂O₂ production in homozygous atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase is involved during bicarbonate induced ROS production in guard cells. The production of H₂O₂ was quicker and greater with ABA than that with bicarbonate. Such pattern of H₂O₂ production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H₂O₂ is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.     

Abscisic acid-induced apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H₂O₂ production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl₃ staining, respectively, and the relationship between ABA-induced H₂O₂ production and ABA-induced subcellular activities of antioxidant enzymes was studied. H₂O₂ generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2–4 h. In mesophyll and bundle sheath cells, ABA-induced H₂O₂ accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O ₂ ⁻ scavenger Tiron and the H₂O₂ scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H₂O₂ is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O ₂ ⁻ and then H₂O₂ in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H₂O₂ produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

The involvement of a P38-like MAP kinase in ABA-induced and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated stomatal closure in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

SB203580 is a specific inhibitor of p38 mitogen-activated protein (MAP) kinase and has been widely used to investigate the physiological roles of p38 in animal and yeast cells. Here by using an epidermal strip bioassay, laser-scanning confocal microscopy and whole-cell patch clamp analysis, we assess the effects of pyridinyl imidazoles-like SB203580 on the H₂O₂ signaling in guard cells of Vicia faba L. The results indicated that SB203580 blocks H₂O₂- or ABA-induced stomatal closure, ABA-induced H₂O₂ generation, and decrease in K⁺ fluxing across plasma membrane of Vicia guard cells by application of ABA and H₂O₂, whereas its analog SB202474 had no effect on these events. Thus, these results suggest that activation of p38-like MAP kinase modulates guard cell ROS signaling in response to stress.     

GABA Enhances Thermotolerance of Seeds Germination by Attenuating the ROS Damage in Arabidopsis

Seeds germination is strictly controlled by environment factor such as high temperature (HT) through altering the balance between gibberellin acid (GA) and abscisic acid (ABA). Gama-aminobutyric acid (GABA) is a small molecule with four-carbon amino acid, which plays a crucial role during plant physiological process associated with pollination, wounding or abiotic stress, but its role in seeds germination under HT remains elusive. In this study we found that HT induced the overaccumulation of ROS, mainly H₂O₂ and O₂^- , to suppress seeds germination, meanwhile, HT also activated the enzyme activity of GAD for the rapid accumulation of GABA, hinting the regulatory function of GABA in controlling seeds germination against HT stress. Applying GABA directly attenuated HT-induced ROS accumulation, upregulated GA biosynthesis and downregulated ABA biosynthesis, ultimately enhanced seeds germination. Consistently, genetic analysis using the gad1/2 mutant defective in GABA biosynthesis, or pop2-5 mutant with high endogenous GABA content supported the potential function of GABA in improving seeds germination tolerance to HT through scavenging ROS overaccumulation. Based on these data, we propose that GABA acts as a novel signal to enhance thermotolerance of seeds germination through alleviating the ROS damage to seeds viability.     

Proteomic profiling and redox status alteration of recalcitrant tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>) seed in response to desiccation

Tea seed is believed to be recalcitrant based on its sensitivity to chilling or drying stress. Reactive oxygen species (ROS) and alterations in cytosolic redox status have been implicated in intolerance to desiccation by recalcitrant seed, but there is little information available regarding how ROS are regulated in seeds susceptible to drying stress. We investigated changes in protein expression and activity in tea embryo in response to desiccation using physiological and proteomic methods. Results showed that desiccation treatment dramatically induced the accumulation of H₂O₂ in tea embryos, accompanied by increased activities of antioxidant enzymes like ascorbate peroxidase (APX) and superoxide dismutase (SOD). Proteomic analyses also demonstrated that 23 proteins associated with defense response, metabolism and redox status were up-regulated following desiccation. Increase in antioxidants, ascorbic acid (AsA) and catalase (CAT) (H₂O₂ scavengers) partially assuaged desiccation damage to tea seed, resulting in improved germination rates. Higher accumulation of H₂O₂ aggravated desiccation damage to seeds leading to lower germination activity. We propose that desiccation causes an over-accumulation of ROS that are not efficiently scavenged by increased levels of antioxidant enzymes. High levels of ROS alter the redox status and are detrimental to seed viability. Reducing ROS to appropriate concentrations is an efficient way to reduce desiccation damage and improve germination rates of recalcitrant seeds.     

Investigating seed dormancy in cotton (Gossypium hirsutum L.): understanding the physiological changes in embryo during after‐ripening and germination

• The dormancy of seeds of upland cotton can be broken during dry after‐ripening, but the mechanism of its dormancy release remains unclear.
• Freshly harvested cotton seeds were subjected to after‐ripening for 180 days. Cotton seeds from different days of after‐ripening (DAR) were sampled for dynamic physiological determination and germination tests. The intact seeds and isolated embryos were germinated to assess effects of the seed coat on embryo germination. Content of H₂O₂ and phytohormones and activities of antioxidant enzymes and glucose‐6‐phosphate dehydrogenase were measured during after‐ripening and germination.
• Germination of intact seeds increased from 7% upon harvest to 96% at 30 DAR, while embryo germination improved from an initial rate of 82% to 100% after 14 DAR. Based on T₅₀ (time when 50% of seeds germinate) and germination index, the intact seed and isolated embryo needed 30 and 21 DAR, respectively, to acquire relatively stable germination. The content of H₂O₂ increased during after‐ripening and continued to increase within the first few hours of imbibition, along with a decrease in abscisic acid (ABA) content. A noticeable increase was observed in gibberellic acid content during germination when ABA content decreased to a lower level. Coat removal treatment accelerated embryo absorption of water, which further improved the accumulation of H₂O₂ and changed peroxidase content during germination.
• For cotton seed, the alleviation of coat‐imposed dormancy required 30 days of after‐ripening, accompanied by rapid dormancy release (within 21 DAR) in naked embryos. H₂O₂ acted as a core link between the response to environmental changes and induction of other physiological changes for breaking seed dormancy.

     

Gain of function of stomatal movements in rooting <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. plants: regulation by H<Subscript>2</Subscript>O<Subscript>2</Subscript> is independent of ABA before the protruding of roots

Reactive oxygen species (ROS), namely superoxide radical (O₂ ⁻) and hydrogen peroxide (H₂O₂) are generated when plant tissues endure a variety of environmental stresses, including light stress. The extremely short life times of ROS makes the study of their production in planta very difficult. The use of ROS-specific tracer dyes, 3-3′ diaminobenzidine and nitroblue tetrazolium, together with high-resolution imaging provides the opportunity to identify sites of photooxidative stress response by ROS accumulation. This technique was applied to grapevine during the first 7 days after transfer from in vitro to ex vitro under an irradiance 4-fold higher than in vitro. ROS accumulation was detected in the first days of analysis, which gradually decreased to levels comparable to greenhouse leaves. O₂ ⁻ was uniformly distributed while H₂O₂ accumulated preferentially in veins, wounds and stomatal guard and surrounding cells. To evaluate the role of H₂O₂ in stomatal functioning and its crosstalk with abscisic acid (ABA) we focused on the percentage of coloured structures, stomatal aperture and ABA concentration. We propose that the high H₂O₂ level triggered by increased light is responsible for the activation of a signalling pathway over stomatal cells, in a process apparently irrespective of ABA regulation prior to root protrusion. This could explain the gain of function of a low yet consistent percentage of stomatal cells, essential for plant survival during the ontogenic period in analysis.     

Identification of a hydrogen peroxide signalling pathway in the control of light-dependent germination in Arabidopsis

Germination is controlled by external factors, such as temperature, water, light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and demonstrated that ROS in addition to their role as cell wall loosening factor are essential signalling molecules in this process. Indeed, we showed that ROS are released prior to endosperm rupture, that their production is required for germination, and that class III peroxidases, as ROS level regulators, colocalized with ROS production. Among ROS, H₂O₂ modifies, during germination early steps, the expression of genes encoding for enzymes regulating ROS levels. This pointing out a regulatory feedback loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels. We followed the early visible steps of germination (testa and endosperm rupture) in Arabidopsis seeds treated by specific ROS scavengers and as the light quality perception is necessary for a regular germination, we examined the germination in presence of exogenous H₂O₂ in different light qualities. H₂O₂ either promoted germination or repressed germination depending on the light wavelengths, showing that H₂O₂ acts as a signal molecule regulating germination in a light-dependent manner. Using photoreceptors null-mutants and GA-deficient mutants, we showed that H₂O₂-dependent promotion of germination relies on phytochrome signalling, but not on cryptochrome signalling, and that ROS signalling requires GA signalling.     

Cell death and reactive oxygen species metabolism during accelerated ageing of soybean axes

In this paper, Glycine max L. seeds under accelerated ageing condition (40°C and 100% relative humidity) were used as experimental material to study the relationships between seed viability and cell death, production and scavenging of reactive oxygen species (ROS) during accelerated ageing. Water content of seeds gradually increased, while the final germination percentage, germination rate of seeds and fresh weight of seedlings produced decreased with increasing accelerated ageing time. The accelerated ageing time (T ₅₀) when final seed germination decreased to 50% was about 10.5 days. During the period of accelerated ageing, the viability of root cells was lost gradually as manifested by the increase in staining with Evans blue. The respiration rate of seeds, ^·O₂ production rate, and H₂O₂ content of axes increased, peaked at the 10 days of accelerated ageing, and then decreased. Activities of superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase of axes decreased; and malondialdehyde contents of axes markedly increased. A sceme to explain relationships between seed vigor, cell death, and production and scavenging of ROS during accelerated ageing was suggested. This text was submitted by the authors in English.     

Chromotoxic effects of exogenous hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) in barley seeds exposed to salt stress

The effect of exogenous hydrogen peroxide (H₂O₂) on mitotic activity and chromosomal aberrations in root tip meristems of barley (Hordeum vulgare L. var. Tokak 157/37) germinated under salinity was analyzed. The inhibitory effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration (0.00 control, 0.35, 0.40, 0.45 M, molal NaCl). The frequency of chromosomal aberrations of seeds germinated in medium with 0.40 M NaCl after pretreatment with H₂O₂ (30 μM, micromolal) was significantly higher than the control group. The highest concentration of NaCl (0.45 M) together with H₂O₂ caused total inhibition of germination. In this study, the intention was to determine the performance of H₂O₂ in alleviating detrimental effect of salt stress on mitotic activity and chromosomal aberrations. However, H₂O₂ did not reduce the detrimental effect of NaCl on these parameters. Also, it caused higher chromotoxic effect compared to those of control groups.