Hypoglycin A Content in Blood and Urine Discriminates Horses with Atypical Myopathy from Clinically Normal Horses Grazing on the Same Pasture

Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7–319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8–8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8–926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17–0.65 mmol/L (controls < 0.01), and 0.34–2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis.     

Sorbitol-6-Phosphate Dehydrogenase Expression in Transgenic Tobacco : High Amounts of Sorbitol Lead to Necrotic Lesions

We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 μmol g⁻¹ fresh weight were found. Plants that accumulated up to 2 to 3 μmol g⁻¹ fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 μmol g⁻¹ fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 μmol g⁻¹ fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.     

Effect of Short-Chain Fatty Acids Extracted from Beef Cattle Manure on Germination and Seedling Development

Composted and fresh beef cattle manure samples were extracted with distilled water, acetone, methanol, 2 N sodium hydroxide, 2 N hydrochloric acid, and ether. Bioassay techniques, using the extracts, showed that composted manure extracts had limited effect on seed germination and seeding development of wheat and sorghum. All the extracts of fresh manure, other than distilled water, retarded germination. Acetic, butyric, propionic, valeric, and isovaleric acids were found in ether extracts of fresh manure at average concentrations of 348, 876, 578, 34, and 19 μg/g, respectively, on a dry-weight basis. However, only trace amounts of these acids were present in composted manure. Propionic acid up to the 200-μg/ml level stimulated seedling growth. Acetic and butyric below the 200-μg/ml level had no detrimental or beneficial effects on seedling development. When acetic, butyric, and propionic acids were mixed in equal parts, germination and seedling growth were reduced at all levels (50 to 500 μg/ml).     

Translocation of iron from soybean cotyledons

Soybean seeds, Glycine max L. Merrill, were produced by plants treated from anthesis to seed maturity with ⁵⁹Fe supplied as ferric ethylenediaminedi (o-hydroxyphenylacetate). Seed coats accounted for 7.4% of dry seed weight and had Fe concentrations 5 times greater than the embryos. After germinating 2 days, cotyledons contained 69.6% and radicles 5.0% of original seed Fe. Fractions of seed Fe unavailable to seedlings were: 19.8% in seed coats, 1.7% in germination paper, 0.1% in the water under germinating seeds, and 3.8% unaccounted for. Every 3 days seedlings received nutrient solution without Fe or with 10 μm ferric ethylenediaminedi (o-hydroxyphenylacetate) and developed as deficient Fe or normal Fe plants. The deficient Fe cotyledons on day 18 retained 13% of the labeled Fe originally present. Cotyledons of normal Fe plants retained 50 to 70% of their original Fe. Moreover, cotyledons of the normal Fe plants accumulated externally supplied Fe and finally contained twice the quantity of Fe originally present. Stem exudate collected above cotyledons of deficient Fe plants contained 5.3 μm⁵⁹Fe. Electrophoresis of exudate showed that most of the ⁵⁹Fe migrated anodically as a single band and was in the position of ferric citrate.     

Salinity Promotes Accumulation of 3-Dimethylsulfoniopropionate and Its Precursor S-Methylmethionine in Chloroplasts

Wollastonia biflora (L.) DC. plants accumulate the osmoprotectant 3-dimethylsulfoniopropionate (DMSP), particularly when salinized. DMSP is known to be synthesized in the chloroplast from S-methylmethionine (SMM) imported from the cytosol, but the sizes of the chloroplastic and extrachloroplastic pools of these compounds are unknown. We therefore determined DMSP and SMM in mesophyll protoplasts and chloroplasts. Salinization with 30% (v/v) artificial seawater increased protoplast DMSP levels from 4.6 to 6.0 μmol mg⁻¹ chlorophyll (Chl), and chloroplast levels from 0.9 to 1.9 μmol mg⁻¹ Chl. The latter are minimum values because intact chloroplasts leaked DMSP during isolation. Correcting for this leakage, it was estimated that in vivo about one-half of the DMSP is chloroplastic and that stromal DMSP concentrations in control and salinized plants are about 60 and 130 mm, respectively. Such concentrations would contribute significantly to chloroplast osmoregulation and could protect photosynthetic processes from stress injury. SMM levels were measured using a novel mass-spectrometric method. About 40% of the SMM was located in the chloroplast in unsalinized W. biflora plants, as was about 80% in salinized plants; the chloroplastic pool in both cases was approximately 0.1 μmol mg⁻¹ Chl. In contrast, ≥85% of the SMM was extrachloroplastic in pea (Pisum sativum L.) and spinach (Spinacia oleracea L.), which lack DMSP. DMSP synthesis may be associated with enhanced accumulation of SMM in the chloroplast.     

Overexpression of heat shock protein 70 inhibits epithelial-mesenchymal transition and cell migration induced by transforming growth factor-β in A549 cells

Heat shock protein 70 (HSP70) is a key member of the HSP family that contributes to a pre-cancerous environment; however, its role in lung cancer remains poorly understood. The present study used geranylgeranylacetone (GGA) to induce HSP70 expression, and transforming growth factor-β (TGF-β) was used to construct an epithelial-mesenchymal transition (EMT) model by stimulating A549 cells in vitro. Western Blot was performed to detect protein levels of NADPH oxidase 4 (NOX4) and the EMT-associated proteins E-cadherin and vimentin both before and after HSP70 expression. Cell morphological changes were observed, and the effect of HSP70 on cell migration ability was detected via the wound healing. The results demonstrated that GGA at 50 and 200 μmol/L could significantly induce HSP70 expression in A549 cells (P < 0.05). Furthermore, HSP70 induced by 200 μmol/L GGA significantly inhibited the changes of E-cadherin, vimentin, and cell morphology induced by TGF-β (P < 0.05), while HSP70 induced by 50 μmol/L GGA did not. The results of the wound healing assay indicated that 200 μmol/L GGA significantly inhibited A549 cell migration induced by TGF-β. Taken together, the results of the present study demonstrated that overexpression of HSP70 inhibited the TGF-β induced EMT process and changed the cell morphology and migratory ability induced by TGF-β in A549 cells.     

Stereoselective Regulation of P-gp Activity by Clausenamide Enantiomers in Caco-2, KB/KBv and Brain Microvessel Endothelial Cells

The (−)- and (+)-clausenamide (CLA) enantiomers have different pharmacokinetic effects in animals, but their association with putative stereoselective regulation of P-glycoprotein (P-gp) remains unclear. Using three cells expressing P-gp—Caco-2, KBv and rat brain microvessel endothelial cells(RBMEC), this study investigated the association of CLA enantiomers with P-gp. The results showed that the rhodamine 123 (Rh123) accumulation, an indicator of P-gp activity, in Caco-2, KBv and RBMECs was increased by (−)CLA (1 or 5 μmol/L) at 8.2%–28.5%, but reduced by (+)CLA at 11.7%–25.9%, showing stereoselectivity in their regulation of P-gp activity. Following co-treatment of these cells with each CLA enantiomer and verapamil as a P-gp inhibitor, the (+)-isomer clearly antagonized the inhibitory effects of verapamil on P-gp efflux, whereas the (−)-isomer had slightly synergistic or additive effects. When higher concentrations (5 or 10 μmol/L) of CLA enantiomers were added, the stimulatory effects of the (+)-isomer were converted into inhibitory ones, leading to an enhanced intracellular uptake of Rh123 by 24.5%–58.2%; but (−)-isomer kept its inhibition to P-gp activity, causing 30.0%–63.0% increase in the Rh123 uptake. The biphasic effects of (+)CLA were confirmed by CLA uptake in the Caco-2 cells. (+)CLA at 1 μmol/L had significantly lower intracellular uptake than (−)CLA with a ratio[(−)/(+)] of 2.593, which was decreased to 2.167 and 1.893 after CLA concentrations increased to 2.5 and 5 μmol/L. Besides, in the non-induced KB cells, (+)CLA(5 μmol/L) upregulated P-gp expression at 54.5% relative to vehicle control, and decreased Rh123 accumulation by 28.2%, while (−)CLA(5 μmol/L) downregulated P-gp expression at 15.9% and increased Rh123 accumulation by 18.0%. These results suggested that (−)CLA could be a P-gp inhibitor and (+)CLA could be a modulator with concentration-dependent biphasic effects on P-gp activity, which may result in drug—drug interactions when combined with other P-gp substrate drugs.     

Induction of somatic embryogenesis in gum arabic tree [Acacia senegal (L.) Willd.]

Factors affecting somatic embryogenesis from immature cotyledon of gum arabic tree [Acacia senegal (L.) Willd.] were investigated. Induction of somatic embryogenesis was influenced by plant growth regulator concentrations and addition of amino acids in medium. Best induction of somatic embryogenesis was obtained on MS medium supplemented with 0.45 μM 2, 4-D, 2.32 μM Kin and 15 mM L-glutamine. L-glutamine plays a significant role in the maturation of somatic embryos and most of embryos attained maturity only on L-glutamine (15 mM) containing medium. Maximum percent (75.0 ± 2.5) germination of somatic embryos was recorded on medium containing 0.22 μM BAP.     

Regulation of Alcohol Dehydrogenases in Maize Scutellum during Germination

The pattern of change in the activity of alcohol dehydrogenase in maize (Zea mays L.) scutellum during seed germination is not altered by 10 μg/ml cycloheximide or 50 μg/ml actinomycin D. The enzyme does not become density labeled when maize seeds are germinated in the presence of D₂O and ¹⁵NH₄Cl, indicating that no new alcohol dehydrogenase molecules are synthesized after the onset of germination. However, the activity of an endogenous inhibitor for alcohol dehydrogenase is increased after germination. The increase of this inhibitor is concomitant with the decline of alcohol dehydrogenase activity, indicating that the activity of alcohol dehydrogenase during seed germination is controlled by the level of the inhibitor.     

The Effect of 5OH-1,4-Naphthoquinone on Norway Spruce Seeds during Germination

The effect of 5-OH-1,4-naphthoquinone (5OH-NQ), a known inhibitor of germination and growth and an inducer of oxidative stress, on seeds from Norway spruce (Picea abies) during germination was studied. 5OH-NQ was activated by homogenate from seeds to reactive species that reduce oxygen to superoxide radicals in vitro. Increasing concentrations of 5OH-NQ increased lipid peroxidation during this activation. Small effects of 5OH-NQ on germination of seeds were observed at concentrations up to 200 μm. However, higher concentrations, e.g. 500 and 1000 μm, exerted more pronounced effects on seeds. These results suggest that the effect of 5OH-NQ was a delay rather than an inhibition of germination. However, the effect of 5OH-NQ on postgerminative growth was more potent than that on germination, and higher concentrations inhibited growth >97%. These results suggest that the seeds have a very effective defense system against quinone and reactive oxygen species, since the small effects of 5OH-NQ on germination and postgermination at concentrations up to 200 μm can be explained by the formation of a metabolite of 5OH-NQ that is not as reactive with oxygen as the original quinone. The 5OH-NQ metabolite collected during germination experiments showed differences in its absorption spectrum in comparison with 5OH-NQ, which suggest changes in structure. This metabolite was reduced by quinone reductase, but reduction of oxygen to superoxide radicals was not detected during its activation with homogenate from seeds. This metabolite may arise via a conjugation reaction, since the addition of 500 μm uridine 5′-diphosphoglucuronic acid or 3′-phosphoadenosine-5′-phosphosulfate to the incubation mixture during activation of this metabolite by homogenate from seeds in vitro inhibited reduction of oxygen to superoxide radicals by 50 and 64%, respectively. The constitutive levels of superoxide dismutase and catalase were sufficient to prevent oxygen toxicity during activation of 5OH-NQ, since these enzymes were not induced when the seeds were treated with 200 μm 5OH-NQ.     

Changes in the Activity of Some Hydrolases, Peroxidase, and Catalase in the Rice Seed during Germination

A study was made of the changes in activity of enzymes involved in the breakdown of stored phytin, lipid, and hemicellulose in the aleurone layer of rice seed (Oryza sativa L., variety IR8) during the 1st week of germination in the light. Enzyme assays were made on crude extracts from degermed seed, and activities were expressed on a per seed basis. Phytase activity increased within the 1st day of germination. The increase in activity of most other enzymes—phosphomonoesterase, phosphodiesterase, esterase, lipase, peroxidase, catalase, β-glucosidase, and α- and β-galactosidase—closely followed the increase in protein content. Their peak activities occurred by the 5th to the 7th day. Some enzymes, such as β-1, 3-glucanase and α-amylase, continued to increase in activity after the 7th day. Phytase, β-1, 3-glucanase, and α-amylase followed a similar sequence of production in embryoless seed halves incubated in 0.12 μM gibberellin A₃, but the production of lipase was delayed.     

Nitrate Reduction in a Sulfate-Reducing Bacterium, Desulfovibrio desulfuricans, Isolated from Rice Paddy Soil: Sulfide Inhibition, Kinetics, and Regulation

From the second-highest dilution in a most-probable-number dilution series with lactate and sulfate as substrates and rice paddy soil as the inoculum, a strain of Desulfovibrio desulfuricans was isolated. In addition to reducing sulfate, sulfite, and thiosulfate, the strain also reduced nitrate to ammonia. The latter process was studied in detail, since the ability to reduce nitrate was strongly influenced by the presence of sulfide. Sulfide inhibited both growth on nitrate and nitrate reduction. A 70% inhibition of the nitrate reduction rate was obtained at 127 μM sulfide, and growth was inhibited by 50% at approximately 320 μM sulfide and was not detectable above 700 μM sulfide. In contrast, sulfate reduction was not affected at concentrations of up to 5 mM. After growth with sulfate, an induction period of 2 to 4 days was needed before nitrate reduction started. When nitrate and sulfate were present simultaneously, only sulfate was reduced, except when sulfate was present at very low concentrations (4 μM). At higher sulfate concentrations (500 μM), nitrate reduction was temporarily halted. The affinity for nitrate uptake was extremely high (K_m = 0.05 μM) compared with that for sulfate uptake (K_m = 5 μM). Thus, at low nitrate concentrations this bacterium is favored relative to denitrifiers (K_m = 1.8 to 13.7 μM) or other nitrate ammonifiers (e.g., Clostridium spp. [K_m = 500 μM]).     

Suboptimal Serum α-Tocopherol Concentrations Observed among Younger Adults and Those Depending Exclusively upon Food Sources,NHANES 2003-20061-3

Vitamin E is an essential nutrient for human health, with an established function as a lipid-soluble antioxidant that protects cell membranes from free radical damage. Low vitamin E status has been linked to multiple health outcomes, including total mortality. With vitamin E being identified as a ‘shortfall nutrient’ because >90% of American adults are not consuming recommended amounts of vitamin E, we aimed to determine the prevalence of both clinical vitamin E deficiency (serum α-tocopherol concentration < 12 μmol/L) and failure to meet a criterion of vitamin E adequacy, serum α-tocopherol concentration of 30 μmol/L, based on the Estimated Average Requirement (EAR) and lowest mortality rate in the Alpha-Tocopherol Beta-Carotene (ATBC) study. The most recent nationally-representative cross-sectional data (2003–2006) among non-institutionalized US citizens with available serum concentrations of α-tocopherol from the National Health and Nutrition Examination Survey (NHANES); Centers for Disease Control and Prevention were analyzed. Serum α-tocopherol distributions were compared between those reporting consumption of food without supplement use (FOOD) and food and supplement use (FOOD+DS) by sex, age, and race/ethnicity. Only 1% of the US population is clinically deficient. FOOD consumers have lower average α-tocopherol levels (24.9± 0.2 μmol/L) than FOOD+DS users (33.7 ± 0.3 μmol/L), even when adjusted for total cholesterol. Using a criterion of adequacy of 30 μmol/L, 87% of persons 20-30y and 43% of those 51+y had inadequate vitamin E status (p<0.01). A significant greater prevalence of FOOD compared to FOOD+DS users did not meet the criterion of adequacy which was based on the EAR and low ATBC mortality rate consistently across age, sex, and race/ethnic groups. The prevalence of inadequate vitamin E levels is significantly higher among non-users of dietary supplements. With declining usage of vitamin E supplements, the population should be monitored for changes in vitamin E status and related health outcomes.     

一氧化氮对水分胁迫下玉米种子萌发及幼苗生理特性的影响

以0.4mol/L的甘露醇(M)模拟水分胁迫状况,研究了外源一氧化氮(NO)供体硝普钠(SNP)对水分胁迫下玉米种子萌发、幼苗生长和生理特性的影响。结果表明:(1)水分胁迫下,玉米种子萌发和幼苗生长受到抑制,叶片丙二醛(MDA)含量、质膜相对透性、脯氨酸含量均显著增加;(2)SNP能显著提高水分胁迫下玉米种子的发芽率、发芽势、发芽指数和活力指数,增加玉米幼苗的根长、茎长、根重和整株干重,抑制水分胁迫下玉米幼苗叶片MDA含量的上升,降低叶片质膜相对透性,降低脯氨酸含量。其中以100μmol/L和200μmol/LSNP对水分胁迫的缓解效果最佳。     

A water relations analysis of seed germination rates

Seed germination culminates in the initiation of embryo growth and the resumption of water uptake after imbibition. Previous applications of cell growth models to describe seed germination have focused on the inhibition of radicle growth rates at reduced water potential (Ψ). An alternative approach is presented, based upon the timing of radicle emergence, to characterize the relationship of seed germination rates to Ψ. Using only three parameters, a `hydrotime constant' and the mean and standard deviation in minimum or base Ψ among seeds in the population, germination time courses can be predicted at any Ψ, or normalized to a common time scale equal to that of seeds germinating in water. The rate of germination of lettuce (Lactuca sativa L. cv Empire) seeds, either intact or with the endosperm envelope cut, increased linearly with embryo turgor. The endosperm presented little physical resistance to radicle growth at the time of radicle emergence, but its presence markedly delayed germination. The length of the lag period after imbibition before radicle emergence is related to the time required for weakening of the endosperm, and not to the generation of additional turgor in the embryo. The rate of endosperm weakening is sensitive to Ψ or turgor.     

Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus

Nitric oxide (NO) is a bioactive molecule, which in plants was found to function as prooxidant as well as antioxidant. In the present study, we found that NO donor sodium nitroprusside (SNP) stimulates seed germination and root growth of lupin (Lupinus luteus L. cv. Ventus). Seed germination is promoted at concentrations between 0.1 and 800 μM SNP in a dose-dependent manner. The stimulation was most pronounced after 18 and 24 h and ceased after 48 h of imbibition. The promoting effect of NO on seed germination persisted even in the presence of heavy metals (Pb, Cd) and sodium chloride. Pretreatment of lupin seedlings for 24 h with 10 μM SNP resulted in efficient reduction of the detrimental effect of the abiotic stressors on root growth and morphology. The inhibitory effect of heavy metals on root growth was accompanied by increased activity of superoxide dismutase (SOD, EC 1.15.1.1.), which in roots preincubated with SNP was significantly higher. Some changes in the activity of other antioxidant enzymes, peroxidase (POX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) were also detected. Using the superoxide anion (O₂^•–)-specific indicator dihydroethidium (DHE), we found intense DHE-derived fluorescence in heavy metal-stressed roots, whereas in those pretreated with SNP the fluorescence was very low, comparable to the level in unstressed roots. On the basis of the above data, we conclude that the protective effect of NO in stressed lupin roots may be at least partly due to the stimulation of SOD activity and/or direct scavenging of the superoxide anion.     

Analysis of arsenic induced physiological and biochemical responses in a medicinal plant,Withania somnifera

Withania somnifera has been an important herb in the Ayurvedic and indigenous medical systems for centuries in India. However, these grow as weeds mostly in the wastelands, which receive contaminated water from municipal and industrial sources. In the present investigation, plants of Withania somnifera were exposed to various concentrations of arsenate (AsV) and arsenite (AsIII) (0, 10, 25, 50, 100 μM) for 10 days and analysed for accumulation of arsenic (As) and physiological and biochemical changes. Plants showed more As accumulation upon exposure to AsIII (320 μg g⁻¹ DW in roots and 161 μg g⁻¹ DW in leaves) than to AsV (173 μg g⁻¹ DW in roots and 100 μg g⁻¹ DW in leaves) after 10 days of treatment. Consequently, AsIII exposure caused more toxicity to plants as compared to that AsV, as evaluated in terms of the level of photosynthetic pigments and oxidative stress parameters (superoxide, hydrogen peroxide and lipid peroxidation), particularly at higher concentrations and on longer durations. Plants could tolerate low concentrations (variable for AsIII and AsV) until longer durations (10 days) and high concentrations for shorter durations (1–5 days) through increase in antioxidant enzymes and by augmented synthesis of thiols. In conclusion, As tolerance potential of Withania plants on one hand advocates its prospective use for remediation under proper supervision and on the other demonstrates possible threat of As entry into humans due to medicinal uses.     

Selenium nutrition of green plants. Effect of selenite supply on growth and selenium content of alfalfa and subterranean clover

Alfalfa and subterranean clover plants were grown in highly purified nutrient solutions to which selenite selenium had been added at 0, 0.025, 0.25, 2.5 or 25.0 μg-atoms/liter. In both species, yields of tops and roots were significantly less at 25.0 μg-atoms/liter than at lower selenium concentrations (p < 0.01). The results indicated that growth was adversely affected when the concentration of selenium in mature leaf tissue reached 0.2 to 0.8 μg-atom/g dry weight.     

Factors Affecting the Germination of Akinetes of Nodularia spumigena (Cyanobacteriaceae)

Nutritional and physical factors which influence the germination of akinetes of Nodularia spumigena (Cyanobacteriaceae) were examined. Low concentrations of phosphorus (<0.9 μM) were required for germination. Nitrate had no effect, but ammonia, at concentrations of >45 μM, inhibited germination. Salinities of >20‰ were inhibitory to germination. Optimum temperatures were 22°C or greater. Germination did not take place in the dark, but only very low light intensities (0.5 microeinstein m⁻² s⁻¹) were necessary to initiate germination. Red light (620 to 665 nm) was required. More than 24 h of continuous exposure to light was necessary for any significant germination to occur. The conditions for germination corresponded with conditions in the Peel-Harvey Estuary, Western Australia, 2 to 3 weeks before large summer Nodularia blooms.     

Flavonols stimulate development, germination, and tube growth of tobacco pollen

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 μm), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.