The induction of freezing tolerance in jack pine seedlings: The role of root plasma membrane H+- ATPase and redox activities

The acquired freezing tolerance of jack pine seedlings (Pinus banksiana Lamb.) conditioned at low nonfreezing temperatures and short photoperiods was determined by comparison of seedling survival to that of nonconditioned (control) seedlings following exposure to ?5 and ?10°C. Compared to that of controls, survival of conditioned seedlings was markedly increased following exposure to freezing temperatures. A 1-week conditioning treatment significantly increased the survival of the seedlings after exposure to ?5°C, but was less effective on seedlings exposed to ?10°C. Conditioning periods of 2 and 4 weeks resulted in higher survival of seedlings exposed to both ?5 and ?10°C. The changes of two root-plasma-membrane-associated enzyme activities, H⁺-ATPase and NADH-dependent ferricyanide reductase, were studied in enriched plasma membrane fractions during conditioning and after freezing. Post-freezing activities of both enzymes were enhanced by conditioning at low temperatures and short photoperiods. These changes may be related to the increased frost hardiness also induced by conditioning.     

Significance of phenols in cadmium and nickel uptake

The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIP's effect on phenols.     

Effect of acetaldehyde, arsenite, ethanol, and heat shock on protein synthesis and chilling sensitivity of cucumber radicles

Exposure to a chilling temperature of 2.5°C for 96 h inhibited the subsequent growth of cucumber seedling radicles at 25°C by 92%. Exposing seedling with 5 ± 1 mm long radicles to acetaldehyde vapour (275 µl l⁻¹) or to an aqueous ethanol solution (0.6  M ) for 2 h, or to 45°C for 10 min before chilling, increased chilling tolerance so that the chilling treatment reduced growth by only 47, 39 or 36%, respectively. All of these effective treatments induced the synthesis of a number of proteins, and suppressed de novo protein synthesis (i.e. the incorporation of [³⁵S]-methionine) by about 70%. In contrast, treatment for 2 h with an aqueous arsenite solution (100 µ M ) had no effect on chilling sensitivity or the incorporation of [³⁵S]-methionine, yet it induced the synthesis of a complement of proteins that were similar to that induced by the effective heat-shock treatment. A unique protein or set of proteins may be responsible for heat-shock-induced chilling tolerance, but none was detected. The ability of various abiotic stresses to suppress protein synthesis may be more important in increasing tolerance to chilling injury than their ability to induce the synthesis of specific proteins.     

Automated sperm morphology analysis in fishes: the effect of mercury on goldfish sperm

This study is the first to examine the morphology of fish sperm using automated sperm morphology analysis (ASMA). The technique was applied to investigate the effect of an environmental pollutant, mercury, on the sperm morphology of goldfish Carassius auratus , and the effects on sperm morphology were compared with those on sperm motility. Goldfish sperm flagellar length was significantly shortened after instant exposure to 100 mg l⁻¹ (368 µM) mercuric chloride, while curvilinear velocity (VCL) and the percentage of motile sperm were significantly decreased at mercuric chloride concentrations of 1 and 10 mg l⁻¹ (3·68 and 36·8 µM), respectively. After 24 h exposure to 0·001 mg l⁻¹ (0·0037 µM) mercuric chloride, flagellar length was significantly reduced in 38% of the spermatozoa. Following exposure to 0·1 mg l⁻¹ (0·37 µM) mercuric chloride for 24 h, however, the majority of spermatozoa (98%), had significantly shortened flagella and increased sperm head length, width and area. Sperm motility was also significantly decreased at 0·1 mg l⁻¹ (0·37 µM) mercuric chloride, probably due to the significantly reduced flagellar length at this concentration. This study shows that the morphological examination of fish sperm by ASMA provides, not only, an excellent tool for monitoring reproductive disruption caused by environmental pollution, but also has applications to other areas of fish reproductive biology, such as cryopreservation and aquaculture.     

Monoterpene levels in needles of Douglas fir exposed to elevated CO2 and temperature

Monoterpene levels in current year needles of Douglas fir ( Pseudotsuga menziesii (Mirb.) Franco) seedlings were measured at the end of 4 years of exposure to ambient or elevated CO₂ (+179 µmol mol⁻¹), and ambient or elevated temperature (+0.3.5^C). Eleven monoterpenes were identified and quantified using gas chromatography/flame ionization detector/mass spectroscopy, with eight of these compounds regularly occurring in all trees examined. Elevated CO₂ exposure significantly reduced the levels for four of the eight main compounds in needles. Total monoterpene production was reduced by 52% ( P  < 0.05). Elevated temperature also reduced monoterpene levels ( P  < 0.07). The combination of elevated temperature and elevated CO₂ resulted in a 64% reduction in total monoterpenes compared with needles on ambient temperature trees. Two-way anova showed no significant temperature-CO₂ interaction. It is hypothesized that seasonal reductions in needle monoterpene pools under elevated CO₂ and temperature conditions may be due to a combination of competing carbon sinks, including increased carbon flux through the roots.     

Restoration of secondary metabolism in birch seedlings relieved from PAL-inhibitor

Phenylalanine ammonia lyase (PAL) plays a key role in phenylpropanoid metabolism, catalyzing the deamination of phenylalanine (Phe) to form trans-cinnamic acid. Inhibitors of PAL have been used to study the physiological role of the different compounds derived from trans-cinnamic acid, and to test theories about a trade-off between growth and defence in plants. In a previous study with birch (Betula pubescens Ehrh.) seedlings, the PAL inhibitor 2-aminoindane-2-phosphonic acid monohydrate (AIP) caused an accumulation of Phe and a strong decrease in the quantity of simple phenolics, soluble condensed tannins and growth, whereas flavonol glycosides were generally not affected. The present study demonstrates restoration of secondary metabolism in the previously AIP treated birch seedlings. Our results indicate that Phe accumulated during PAL inhibition could be partly used to increase the content of the phenolic acids, flavan-3-ols and to some extent the soluble condensed tannins. Seedling growth also increased when the supply of PAL inhibitor ceased. We thereby show that the inhibition of PAL by AIP in vivo is reversible, at least for moderate AIP concentrations and the rate of restoration is dependent on the inhibitor concentration.     

Phenolics during early development of <Emphasis Type="Italic">Betula pubescens</Emphasis> seedlings: inhibition of phenylalanine ammonia lyase

We studied phenolic metabolism and plant growth in birch seedlings at the beginning of their development by inhibiting phenylalanine ammonia lyase (PAL), which is the first committed step in phenylpropanoid metabolism. Betula pubescens (Ehrh.) seeds were germinated in inhibitor-free media and the seedlings were transferred to hydroponic culture at the cotyledon stage. They were 6 days old at the start of the experiment, which lasted for 3 weeks. PAL activity was inhibited by three different concentrations of 2-aminoindane-2-phosphonic acid monohydrate (AIP) in the growing media. At the end of 3 weeks, phenolics in all plant parts (roots, stem, cotyledons, first, second and third true leaves) were determined. AIP inhibited strongly the accumulation of phenolic acids, salidroside, rhododendrins, ellagitannins and their precursors, flavan-3-ols, and soluble condensed tannins. The accumulation of lignin and flavonol glycoside derivatives was moderately inhibited. The accumulation of flavonol glycosides, such as quercetin glycosides and kaempferol glycosides, was not generally inhibited, even in leaves that emerged during the experiment, while the accumulation of insoluble condensed tannins was inhibited only slightly and not in all plant parts. This suggests that flavonol glycosides, which may have a UV-B protective role, and insoluble condensed tannins, which may have structural functions, are prioritized in seedling development. Inhibition of PAL with AIP decreased seedling growth and possible reasons for this are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Responses of Pinus clausa, Pinus serotina and Pinus taeda seedlings to anaerobic solution culture. I. Changes in growth and root morphology

Seedlings of pond pine ( Pinus serotina Michx.), sand pine [ P. clausa (Engelm.) Sarg.] and two edaphic seed sources of loblolly pine ( P. taeda L., dry- and wet-site seed sources) were grown in non-circulating, continuously flowing solution culture under aerobic (250 μ M O₂) and anaerobic (≤ 23 μ M O₂) conditions. Survival was 100% for all seedlings at 11 weeks. Although shoot height, biomass and leaf emergence of loblolly and pond pine seedlings were not significantly affected by 15 or 30 days of anaerobic growth conditions, root biomass was significantly reduced. Sand pine suffered the largest reduction in biomass, showing extensive root dieback and shoot chlorosis with retarded leaf development. These anaerobically induced symptoms of flooding injury were less severe in dry-site loblolly pine, and absent in wet-site loblolly and pond pine seedlings. Adventitious or new, secondary roots, and stem or taproot lenticels were particularly abundant under the 30-day anaerobic treatment in wet-site loblolly and pond pine seedlings, present to a lesser degree in dry-site loblolly pine, and nearly absent in sand pine seedlings. These results indicate that much of the immediate damage from flooding is due to the anoxic condition of the root rather than to the build-up of phytotoxins or soil nutrient imbalances. On the basis of overall seedling vigor, root plasticity and growth, we suggest the following flooding-tolerance/intolerance species (seed source) gradient: pond pine ≥ wet-site loblolly pine > dry-site loblolly pine > sand pine.     

Requirement for far-red light to maintain secondary needle extension growth in northern but not southern populations of Pinus sylvestris (Scots pine)

Extension growth of secondary needles is under photoperiodic control in Pinus sylvestris . To test for the effects of far-red light on maintaining this extension growth, seedlings of six populations originating from latitudes between 57° and 67°N were raised for 11 weeks in continuous incandescent (metal halogen) light at 300 µmol m⁻² s⁻¹ and 20°C and then transferred at the same temperature to a daily regime of 8 h incandescent light (230 µmol m⁻² s⁻¹) followed by a 16 h day extension with cool white fluorescent light (40 µmol m⁻² s⁻¹, R/FR ratio 7.5) or with incandescent lamps (20 µmol m⁻² s⁻¹, R/FR ratio 2.0). For the seedlings from the three populations north of 64°, needle extension growth over 42 days in the FR-poor day extension treatment was lower by up to 40% than in the FR-rich day extension treatment, whereas for the seedlings from the three southern populations the needle extension growth was similar in both day extension treatments. The requirement for FR in day extensions is characteristic of 'light-dominant' photoperiodic control mechanisms. It appears that P. sylvestris changes from dark-dominant night timekeeping to light-dominant day timekeeping with increasing latitude, as with the photoperiodic control of budset in Picea abies .     

Photobiological properties of the inhibition of etiolated Arabidopsis seedling growth by ultraviolet-B irradiation

Alteration of 'normal' levels of ultraviolet-B light (UV-B, 280–320 nm) can affect plant chemical composition as well as growth; however, little is known about how plants perceive UV-B light. We have carried out fluence response curves, and demonstrated that the growth inhibition of etiolated Arabidopsis thaliana seedlings by low fluence UV light is specific to UV-B and not UV-A (320–390 nm). The response shows reciprocity between duration and intensity, at least over a limited range, and thus depends only on photon fluence and not on photon flux. The action spectrum for this response indicates a peak of maximum effectiveness at 290 nm, and response spectra at different fluences indicate that the most effective wavelength at 30 000  µ mol m^–2 is 290 nm, whereas 300 nm light was the most effective at 100 000  µ mol m^–2. This response occurs in mutant seedlings deficient in cryptochrome, phytochrome or phototropin, suggesting that none of the known photoreceptors is the major UV-B photoreceptor. Some null mutants in DNA repair enzymes show hypersensitivity to UV-B, suggesting that even at low fluence rates, direct damage to DNA may be one component of the response to UV-B.     

Production of Salicylic Acid Precursors Is a Major Function of Phenylalanine Ammonia-Lyase in the Resistance of Arabidopsis to Peronospora parasitica

Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (PAL1)-[beta]-glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The PAL1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-staining procedure was developed to distinguish between GUS activity and fungal structures. The PAL1 promoter was activated in cells undergoing lignification in the incompatible interaction in response to the pathogen. Pretreatment of the seedlings with 2-aminoindan-2-phosphonic acid (AIP), a highly specific PAL inhibitor, made the plants completely susceptible. Lignification was suppressed after AIP treatment, and surprisingly, pathogen-induced PAL1 promoter activity could not be detected. Treatment of the seedlings with 2-hydroxyphenylaminosulphinyl acetic acid (1,1-dimethyl ester) (OH-PAS), a cinnamyl alcohol dehydrogenase inhibitor specific for the lignification pathway, also caused a shift toward susceptibility, but the effect was not as pronounced as it was with AIP. Significantly, although OH-PAS suppressed pathogen-induced lignification, it did not suppress pathogen-induced PAL1 promoter activation. Salicylic acid (SA), supplied to AIP-treated plants, restored resistance and both pathogen-induced lignification and activation of the PAL1 promoter. Endogenous SA levels increased significantly in the incompatible but not in the compatible combination, and this increase was suppressed by AIP but not by OH-PAS. These results provide evidence of the central role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility.     

Photomorphogenic responses to UV radiation IV: a comparative study of UVB effects on growth and pigment accumulation in etiolated and de-etiolated hypocotyls of wild-type and aurea mutant of tomato Lycopersicon esculentum Mill)

ABSTRACT

The question of how de-etiolation of tomato seedling under continuous monochromatic yellow light exerts an influence on UV radiation-induced responses has been studied. Hypocotyl extension and the accumulation of anthocyanins and UV-absorbing compounds was compared in the aurea mutant of tomato and its isogenic wild type. The data of the present paper provide evidence that, during de-etiolation of tomato seedlings, yellow light exerts its effects over seedlings responsiveness to subsequent UV irradiation through several mechanisms: 1) a significant enhancement of shorter UVB wavelength efficiency in both the genotypes; 2) the abolition of UVA -blue light-induced accumulation of UV-absorbing compounds that does not involve pnyA; 3) the disappearance of UVA-blue light-induced hypocotyl growth inhibition that does not involve phyA; 4) higher anthocyanin accumulation rate in response to UV radiation mediated by phyA. Yellow light-induced growth inhibition and accumulation of UV-absorbing compound both mediated by phyA and present only in wild type tomato, appear to be completely separate from the action of UV radiation on the same responses.     

Changes in leaf expansion and epidermal screening effectiveness in Liquidambar styraciflua and Pinus taeda in response to UV-B radiation

Absorption or screening of ultraviolet-B (UV-B) radiation by the epidermis may be an important protective method by which plants avoid damage upon exposure to potentially harmful UV-B radiation. In the present study we examined the relationships among epidermal screening effectiveness, concentration of UV-absorbing compounds, epidermal anatomy and growth responses in seedlings of loblolly pine (Pinus taeda L.) and sweetgum (Liquidambar styraciflua L.). Seedlings of each species were grown in a greenhouse at the University of Maryland under either no UV-B radiation or daily supplemental UV-B radiation levels of 4, 8 or 11 kJ m^?2 of biologically effective UV-B (UV-B_BE) radiation. Loblolly pine seedlings were subsequently grown in the field under either ambient or supplemental levels of UV-B radiation. At the conclusion of the growing season, measurements of epidermal UV-B screening effectiveness were made with a fiber-optic microprobe. In loblolly pine, less than 0.5% of incident UV-B radiation was transmitted through the epidermis of fascicle needles and about 1% was transmitted in primary needles. In contrast, epidermal transmittance in sweetgum ranged from about 20% in leaves not preconditioned to UV-B exposure, to about 10% in leaves grown under UV-B radiation. The concentration of UV-absorbing compounds was unaffected by UV-B exposure, but generally increased with leaf age. Increases in epidermal thickness were observed in response to UV-B treatment in loblolly pine, and this accounted for over half of the variability in UV-B screening effectiveness. In spite of the low levels of UV-B penetration into the mesophyll, delays in leaf development (both species) and final needle size (loblolly pine) were observed. Seedling biomass was reduced by supplemental UV-B radiation in loblolly pine. We hypothesize that the UV-induced growth reductions were manifested by changes in either epidermal anatomy or epidermal secondary chemistry that might negatively impact cell elongation.     

丛枝菌根真菌对西瓜嫁接苗生长和根系防御性酶活性的影响

在温室盆栽条件下,研究丛枝菌根(AM)真菌地表球囊霉(Glomus versiforme)对连作土壤中西瓜自根苗和嫁接苗生长、根系膜透性、丙二醛(MDA)含量和防御性酶活性的影响.结果表明： 接种AM真菌能显著增加西瓜自根苗和嫁接苗的生物量,提高根系活力,降低根系膜透性和MDA含量.接种AM真菌的自根苗地上部鲜质量、地上部干质量和根系活力分别增加了57.6%、60.0%和142.1%,而接种AM真菌的嫁接苗分别增加了26.7%、28.0%和11.0%;自根苗（C）、嫁接苗（G）、接种AM真菌自根苗（C+M）和接种AM真菌嫁接苗（G+M）的根系细胞膜透性为C>G>C+M>G+M,根系MDA含量为C>G>G+M>C+M.接种AM真菌能提高西瓜自根苗和嫁接苗根系的苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、过氧化物酶(POD)、几丁质酶和β 1,3 葡聚糖酶活性,而且接种AM真菌的西瓜自根苗和嫁接苗根系POD、PAL和β-1,3-葡聚糖酶活性的峰值比不接种的提前2周出现.接种AM真菌能激活西瓜自根苗和嫁接苗与抗逆性有关的防御性酶反应,使根系对逆境产生快速反应,从而提高其抗连作障碍的能力.     

UV radiation-responsive proteins in rice leaves: a proteomic analysis

Depletion of stratospheric ozone has led to increased UV radiation reaching the surface of the Earth. This may damage plants. Using physiological, proteomic and quantitative real-time PCR (qPCR) methods, we systematically studied the response of 16-day-old rice seedlings to UV [0.67 W m(-2) biologically effective UVB (UVB(BE)) and 0.28 W m(-2) UVA] exposure for 6, 12 and 24 h. UV exposure resulted in the appearance of light brown patches on leaves, a decrease in the net photosynthetic rate (Pn), lipid peroxidation, accumulation of UV-absorbing compounds (including flavonoids and other phenolic pigments) and differential expression of 22 proteins. Both physiological and molecular responses became stronger with increasing UV exposure time, indicating the effects of UV accumulation on plants. UV-induced responses included (i) phytohormone-regulative responses (up-regulation of proteins related to phytohormone synthesis such as IAA and ethylene); (ii) injurious responses (photosynthesis suppression, lipid peroxidation and visible injury); and (iii) protective responses (accumulation of UV-absorbing compounds and differential expression of proteins involved in detoxification/antioxidation, defense, protein processing, RNA processing, carbohydrate metabolism and secondary metabolism). The identification of UV-responsive proteins provided a better understanding of the molecular mechanism of plant responses to UV stress. Proteomic and qPCR analysis identified one up-regulated and two induced proteins with important functions: tryptophan synthase α chain (production of radical oxygen species), glyoxalase I (detoxification/antioxidation) and a Bet v I family protein (defense). These results will contribute to future research into their roles in UV stress responses in plants.     

Rhamnolipid biosurfactants decrease the toxicity of chlorinated phenols to Pseudomonas putida DOT-T1E

Aims:  To investigate the effect of a mixture of rhamnolipid R1 and R2 biosurfactants produced by a Pseudomonas aeruginosa strain on the toxicity of phenol and chlorophenols to Pseudomonas putida DOT-T1E.
Methods and Results:  Toxicity was quantified by the effective concentration 50% (EC50), that is the concentration that causes a 50% inhibition of bacterial growth. The presence of 300 mg l⁻¹ rhamnolipids, that is at about twice their critical micelle concentration (CMC), increased the EC50 of phenol, 4-chlorophenol, 2,4-dichlorophenol and 2,4,5-trichlorophenol by about 12, 19, 32 and 40%, respectively, and consequently reduced the bioavailability and the freely dissolved concentration of the toxic phenolic compounds. The reduction was related to the phenols' octanol–water partition coefficients ( K _ow).
Conclusions:  The reduction in toxicity of the phenols can be explained by a combination of toxin accumulation in biosurfactant micelles and hydrophobic interactions of the phenols with rhamnolipid-based dissolved organic carbon.
Significance and Impact of the Study:  Results provide evidence that next to the effect of the micelle formation also hydrophobic interactions with rhamnolipid-based dissolved organic carbon affects the bioavailability of the phenols. Quantifying the effect of biosurfactants on the toxicity of hydrophobic compounds such as phenols thus appears to be a useful approach to assess their bioavailable equilibrium concentration.     

Cadmium stimulates the accumulation of salicylic acid and its putative precursors in maize (Zea mays) plants

The effect of 10, 25 and 50 µ M Cd(NO₃)₂ on the salicylic acid (SA) metabolism was investigated in young maize seedlings ( Zea mays L., hybrid Norma). Cadmium (Cd) was translocated into the leaves and induced oxidative damage, as indicated by the reduced chlorophyll content, the decreased quantum efficiency of photosystem II and the enhanced malondialdehyde (MDA) content, especially after 7 days. The activity of glutathione reductase (EC 1.6.4.2) increased from the fourth day and that of guaiacol peroxidase (EC 1.11.1.7) after 7 days of Cd stress compared with the control leaves. These effects of Cd exhibited a correlation with the concentration. Under these conditions, Cd did not affect the MDA content or the antioxidant enzyme activities in the roots. After 7 days, Cd increased the levels of free and bound forms of benzoic acid (BA), O -coumaric acid ( O -hydroxy-cinnamic) ( O- HCA) and SA in the leaves, but in the roots, only the 50 µ M rate of Cd caused changes in the free O -HCA acid and bound BA content.     

Influence of ultraviolet-B radiation on the drift response of stream invertebrates

1. Recent studies have shown that ultraviolet (UV) radiation (280–400 nm) has increased by ≈ 8% in temperate regions over the past decade, but little effort has been devoted to understanding the ecological effects on temperate ecosystems. This research examined the effects of artificial ultraviolet-B (UVB; 280–320 nm) radiation on the drift response of immature stream insects in laboratory microcosms.
2. Two experiments involved natural populations of stream invertebrates, collected from the Cache la Poudre River (September 1994) and the Arkansas River (October 1995) in Colorado. UVB lamps were turned on from 10.00 to 14.00 h each day, and drifting animals were collected on days 1, 3, 5 and 7 during the exposure period. Levels of artificial UVB used in these experiments were similar to levels that stream organisms experience during clear, mid-day conditions at Fort Collins, Colorado (longitude 105°30'; latitude 40°35').
3. Drift was significantly higher in microcosms exposed to UVB than in controls and was dominated by Baetis sp. (Ephemeroptera: Baetidae), Trichoptera (caddisflies) and Simulium sp. (Diptera: Simuliidae). The increased drift of some stream invertebrates in UVB-exposed streams may be a behavioural response and/or a result of injury.
4. Stream organisms may be particularly sensitive to predicted increases in UV radiation, because streams are generally shallow with clear water. As a result of this potential sensitivity, we recommend that research be directed to understanding the ecological effects of UV radiation on these habitats.