Thiourea mediates alleviation of UV-B stress-induced damage in the Indian mustard (Brassica juncea L.)

Abstract

UV-B radiation (280–320 nm) as an environmental stress has damaging effect on plants and its overexposure can potentially interfere with growth and development. The effect of thiourea (TU) on UV-B stress tolerance of 10-day-old Brassica juncea seedlings subjected to supplementary UV-B for 5 days was investigated. An increase in the UV-B absorbing compounds anthocyanin, flavonoids and phenolic compounds was observed in UV-B + TU treated seedlings as compared to that of control. The enhanced synthesis of UV-B screening compounds resulted in lesser damage to chlorophyll and also gain in fresh weight and dry weight in UV-B + TU as compared to UV-B alone treatment. The enzymatic activities of guaiacol peroxidase (GPX) and superoxide dismutase (SOD) also increased in UV-B + TU. The expression profiling of phenylalanine ammonia lyase (pal) and chalcone synthase (chs) indicated an upregulation under UV-B + TU treatment, compared to that of control, suggesting that TU treatment ensured an early and efficient induction of flavonoid biosynthetic pathway. The results indicate that TU helps in ameliorating the damaging effects of UV-B stress by efficiently maintaining the antioxidant status and attenuating the penetration of the UV-B.     

Response of barley seedlings to water deficit and enhanced UV-B irradiation acting alone and in combination

Responses of barley seedlings to water deficit (WD) induced by polyethylene glycol (PEG 6000) and ultraviolet (UV-B; 280–320 nm) radiation and their interaction (UV-B + WD) were examined. A decrease in dry matter yield and water content of leaves and roots was observed following application of WD and UV-B + WD, while no changes were found after treating barley plants with UV-B. Proline content was increased in leaves under WD conditions and UV-B + WD. In contrast, UV-B treatment had no effect on the accumulation of proline in leaves of barley plants. Changes in root proline content showed a varied response: WD induced an increase in the level of this amino acid, while UV-B as well as UV-B + WD suppressed root proline content. The lipid peroxidation product malondialdehyde (MDA) was increased in leaves under WD and UV-B + WD stresses. Root MDA content increased in WD-stressed plants, but it decreased in the case of combined application of both stresses. The applied stress factors operated in a variable manner on phenylpropanoid metabolism. Phenylalanine ammonia-lyase (PAL) activity in leaves and roots was stimulated after exposure to WD and application of UV-B + WD stresses, while UV-B stress did not affect its activity. On the other hand, UV-B treatment enhanced the activity of 4:coumarate-CoA ligase (4CL) in leaves and this enhancement was positively correlated with the accumulation of anthocyanins and flavonols. However, the combined application of WD and UV-B reduced the positive effect of UV-B on the accumulation of these compounds and the activity of 4CL. Surprisingly, anthocyanins and flavonols were not detected in roots of examined barley seedlings despite increased 4CL activity. The results suggest that UV-B-induced activation of 4CL as well as accumulation of anthocyanin and flavonols in leaves is beneficial for the response to this stress factor. On the other hand, WD-induced reduction of the effect of UV-B on 4CL activity and the contents of anthocyanin and flavonol might be a cause of membrane damage in UV-B- and WD-stressed plants. In addition, conversely to what could be expected, the UV-B effect was perceived by the water-stressed roots, which exhibited reduced lipid peroxidation (MDA) and proline accumulation in WD-stressed plants exposed to UV-B.     

Effect of Solar UV-B Radiation on a Phyllosphere Bacterial Community

The effect of solar UV-B radiation on the population dynamics and composition of the culturable bacterial community from peanut (Arachis hypogeae L.) was examined in field studies using plants grown under UV-B−transmitting (UV-B+) or UV-B−excluding (UV-B−) plastic filters. Our data demonstrate that solar UV-B selection alters phyllosphere bacterial community composition and that UV tolerance is a prevalent phenotype late in the season. The total bacterial population size was not affected by either UV-B treatment. However, isolates from the UV-B+ plots (n = 368) were significantly more UV tolerant than those from the UV-B− (n = 363) plots. UV sensitivity was determined as the minimal inhibitory dose of UV that resulted in an inhibition of growth compared to the growth of a nonirradiated control. The difference in minimal inhibitory doses among bacterial isolates from UV-B+ and UV-B− treatments was mainly partitioned among nonpigmented isolates, with pigmented isolates as a group being characterized as UV tolerant. A large increase in UV tolerance was observed within isolate groups collected late (89 and 96 days after planting) in the season. Identification of 200 late-season isolates indicated that the predominant UV-tolerant members of this group were Bacillus coagulans, Clavibacter michiganensis, and Curtobacterium flaccumfaciens. We selected C. michiganensis as a model UV-tolerant epiphyte to study if cell survival on UV-irradiated peanut leaves was increased relative to UV survival in vitro. The results showed an enhancement in the survival of C. michiganensis G7.1, especially following high UV-C doses (300 and 375 J m⁻²), that was evident between 24 and 96 h after inoculation. A dramatic increase in the in planta/in vitro survival ratio was observed over the entire 96-h experiment period for C. michiganensis T5.1.     

Inhibition of hypocotyl elongation by ultraviolet-B radiation in de-etiolating tomato seedlings. II. Time-course, comparison with flavonoid responses and adaptive significance

UV-B radiation inhibits hypocotyl elongation in etiolated tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) seedlings acting through a photoreceptor system with peak apparent effectiveness around 300 nm. In order lo further characterize the response and gain insight into its potential ecological significance, the time-course of inhibition was measured and compared with the time-course of flavonoid accumulation in the same seedlings. When a background of strong (> 620 μmol m^?2 s^?1) white light (WL) was supplemented with low irradiance UV-B (～ 3 μmol m^?2 s^?1). substantial (～ 50%) inhibition of elongation occurred within 3 h of the light treatment. The magnitude of UV-B-induced elongation inhibition was similar in wild type (WT) and au-mutant seedlings, in spite of the large differences between genotypes in rate and temporal pattern of elongation. In comparison to the effect of UV-B on elongation, induction of flavonoid accumulation in WT and au seedlings undergoing de-etiolation was a much slower response. Several UV-absorbing compounds appeared to be specifically induced by light, and some of them accumulated faster under the WL + UV-B treatment than under WL alone. However, there was little or no delectable effect of WL on flavonoid levels until up to 3 h of treatment, and the specific UV-B effect was measurable only after 6 h of continuous treatment. Indeed. UV-B-screening properties of crude alcoholic extracts were not different between WL and WL + UV-B treatments until after 9 or 24 h. When the light treatments were applied to seedlings that were just breaking through the soil surface. UV-B was found to consistently retard seedling emergence. These results suggest that the rapid inhibition of elongation in de-etiolating seedlings is an evolved response lo UV-B, which may serve to minimize seedling exposure to sunlight until protective pigmentation responses (triggered by WL and UV-B) have taken place in the seedlings epidermis.     

Purification and biochemical characterization of proteins which bind to the H-box cis-elementimplicated in transcriptional activation of plant defense genes

The H-box (CCTACC(N)₇CT(N)₄A), which occurs three times within the −154 to −42 region of the bean chalcone synthase chs15 promoter, is important for developmental regulation of chs15 , and induction of chs15 and coordinately regulated defense genes by elicitors and other stress stimuli. Two protein factors, KAP-1 and KAP-2, which recognize conserved features in the H-box motif, were purified from bean cell suspension cultures by a combination of ion exchange chromatography and DNA affinity chromatography. KAP-1 is a 97 kDa polypeptide, whereas KAP-2 comprises two polypeptides of 76 and 56 kDa. KAP-1 and KAP-2 also differ in the sensitivity of their DNA-bound forms to trypsin. Dephosphorylation of KAP-1 or KAP-2 affects the mobility of the protein/H-box binding complex in gel shift assays but does not inhibit DNA binding. Elicitation of bean cell suspensions with glutathione does not affect the total cellular activities of KAP-1 or KAP-2, but causes a rapid increase in the specific activities of both factors in the nuclear fraction, consistent with a role for these factors in the signal pathway for elicitor induction of chs15 and related defense genes.     

Perception of solar UV radiation by plants: photoreceptors and mechanisms

About 95% of the ultraviolet (UV) photons reaching the Earth’s surface are UV-A (315–400 nm) photons. Plant responses to UV-A radiation have been less frequently studied than those to UV-B (280–315 nm) radiation. Most previous studies on UV-A radiation have used an unrealistic balance between UV-A, UV-B, and photosynthetically active radiation (PAR). Consequently, results from these studies are difficult to interpret from an ecological perspective, leaving an important gap in our understanding of the perception of solar UV radiation by plants. Previously, it was assumed UV-A/blue photoreceptors, cryptochromes and phototropins mediated photomorphogenic responses to UV-A radiation and “UV-B photoreceptor” UV RESISTANCE LOCUS 8 (UVR8) to UV-B radiation. However, our understanding of how UV-A radiation is perceived by plants has recently improved. Experiments using a realistic balance between UV-B, UV-A, and PAR have demonstrated that UVR8 can play a major role in the perception of both UV-B and short-wavelength UV-A (UV-A_sw, 315 to ∼350 nm) radiation. These experiments also showed that UVR8 and cryptochromes jointly regulate gene expression through interactions that alter the relative sensitivity to UV-B, UV-A, and blue wavelengths. Negative feedback loops on the action of these photoreceptors can arise from gene expression, signaling crosstalk, and absorption of UV photons by phenolic metabolites. These interactions explain why exposure to blue light modulates photomorphogenic responses to UV-B and UV-A_sw radiation. Future studies will need to distinguish between short and long wavelengths of UV-A radiation and to consider UVR8’s role as a UV-B/UV-A_sw photoreceptor in sunlight.

In sunlight, UVR8 mediates the perception of both UV-B and short-wavelength UV-A radiation with its sensitivity moderated by blue light perceived through cryptochromes.     

Action of ultraviolet radiation (UV-B) upon cuticular waxes in some crop plants

The surface structure and composition of surface lipids were examined in leaves of barley, bean, and cucumber seedlings grown in a growth chamber under white light and low levels of ultraviolet (UV-B; 280–320 nm) radiation. The cuticular wax of cucumber cotyledons and bean leaves appeared as a thin homogeneous layer, whereas on barley leaves crystal-like structures could be observed under these irradiation conditions. Principally, the amount of cuticular wax found in barley leaves was five times greater than in bean or cucumber leaves. The prediominant wax components were primary alcohols in barley, primary alcohols and monoesters in bean, and alkanes in cucumber cotyledons. Irradiation with enhanced UV-B levels caused an increase of total wax by about 25% in all plant species investigated. Aldehydes, detected as a minor constituent of cucumber and barley wax, increased twofold. Distribution patterns of the homologs within some wax classes were different at low and enhanced UV-B levels. In general, the distribution of the homologs was shifted to shorter acyl chain lengths in wax of leaves exposed to enhanced UV-B levels. This was most apparent in cucumber wax, less in bean or barley wax. The UV-B-caused effects upon cucumber wax were mainly due to a response by the adaxial surface of the leaf.Abbreviation UV-B Ultraviolet radiation (280–320 nm)     

Heat Shock Increases the Tolerance of Plants to UV-B Radiation: 1. Growth,Development, and Water Supply to Tissues

The effects of heat shock (HS); UV-B irradiation; and the consecutive action of these factors on the growth, development, and water supply of seven-day-old melon (Melo sativusSager., cv. Torpeda) seedlings were investigated. Depending on the HS severity, we observed growth stimulation (after treatment at 45°C for 1 h), growth retardation (after treatment at 45°C for 2 h or at 48°C for 1 h), or complete growth inhibition and cell death (after treatment at 45°C for 3 h or at 55°C for 1 h). UV-B irradiation (18.3 kJ/(m²h)), depending on its duration, stimulated (5–10 min), retarded (60 min), or resulted in complete growth inhibition and plant death (90 min). HS treatment (at 45°C for 1 h) prior to UV-B irradiation (for 1 h) favorably affected both the growth and water balance of seedlings. Apparently, the HS pretreatment increases the tolerance of seedlings to high doses of UV-B radiation.     

Response of antioxidant defense system to laser radiation apical meristem of Isatis indigotica seedlings exposed to UV-B

To determine the response of antioxidant defense system to laser radiation apical meristem of Isatis indigotica seedlings, Isatis indigotica seedlings were subjected to UV-B radiation (10.08 kJ m⁻²) for 8 h day⁻¹ for 8 days (PAR, 220 µmol m⁻² s⁻¹) and then exposed to He-Ne laser radiation (633 nm; 5.23 mW mm⁻²; beam diameter: 1.5 mm) for 5 min each day without ambient light radiation. Changes in free radical elimination systems were measured, the results indicate that: (1) UV-B radiation enhanced the concentration of Malondialdahyde (MDA) and decreased the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in seedlings compared with the control. The concentration of MDA was decreased and the activities of SOD, CAT and POD were increased when seedlings were subjected to elevated UV-B damage followed by laser; (2) the concentration of UV absorbing compounds and proline were increased progressively with UV-B irradiation, laser irradiation and He-Ne laser irradiation plus UV-B irradiation compared with the control. These results suggest that laser radiation has an active function in repairing UV-B-induced lesions in seedlings.Key words: Isatis indigotica, laser, UV-B lesion     

Beneficial effects of enhanced UV-B radiation under field conditions: improvement of needle water relations and survival capacity of Pinus pinea L. seedlings during the dry Mediterranean summer

The possible mechanism(s) by which supplemental UV-B radiation alleviates the adverse effects of summer drought in Mediterranean pines (Petropoulou et al. 1995) were investigated with seedlings of Pinus pinea. Plants received ambient or ambient plus supplemental UV-B radiation (biologically equivalent to a 15% ozone depletion over Patras, 38.3° N, 29.1° E) and natural precipitation or additional irrigation. Treatments started on 1 February, 1994 and lasted up to the end of the dry period (29 September). In well-watered plants, UV-B radiation had no influence on photosystem II photochemical efficiency and biomass accumulation. Water stressed plants suffered from needle loss and reduced photosystem II photochemical efficiency during the summer. These symptoms, however, were less pronounced in plants receiving supplemental UV-B radiation, resulting in higher total biomass at plant harvest. Laboratory tests showed that enhanced UV-B radiation did not improve the tolerance of photosystem II against drought, high light, high temperature and oxidative stress. Enhanced UV-B radiation, however, improved the water economy of water stressed plants, as judged by measurements of needle relative water content. In addition, it caused an almost two-fold increase of cuticle thickness. No such UV-B radiation effects were observed in well-watered pines. The results indicate that the combination of water stress and UV-B radiation may trigger specific responses, enabling the plants to avoid excessive water loss and, thereby, maintain a more efficient photosynthetic apparatus during the summer. The extent of this apparently positive UV-B radiation effect would depend on the amount of summer precipitation. Abbreviations: DW – dry weight, F_v/F_m – ratio of variable to maximum fluorescence, A ₃₀₀ – absorbance at 300 nm, PAR – photosynthetically active radiation, PS II – photosystem II, RWC – relative water content, TCA – trichloroacetic acid, UV-B_BE – biologically effective ultraviolet-B radiation     

Solar UV-B radiation affects leaf quality and insect herbivory in the southern beech tree<Emphasis Type="Italic"> Nothofagus antarctica</Emphasis>

We examined the effects of solar ultraviolet-B (UV-B) radiation on plant-insect interactions in Tierra del Fuego (55°S), Argentina, an area strongly affected by ozone depletion because of its proximity to Antarctica. Solar UV-B under Nothofagus antarctica branches was manipulated using a polyester plastic film to attenuate UV-B (uvb–) and an Aclar film to provide near-ambient UV-B (uvb+). The plastic films were placed on both north-facing (i.e., high solar radiation in the Southern Hemisphere) and south-facing branches. Insects consumed 40% less leaf area from north- than from south-facing branches, and at least 30% less area from uvb+ branches than from uvb– branches. The reduced herbivory on leaves from uvb+ branches occurred for both branch orientations. Leaf mass per area increased and relative water content decreased on north- versus south-facing branches, while no differences were apparent between the UV-B treatments. Solar UV-B did lead to lower gallic acid concentration and higher flavonoid aglycone concentration in uvb+ leaves relative to uvb– leaves. Both the flavonoid aglycone and quercetin-3-arabinopyranoside were higher on north-facing branches. In laboratory preference experiments, larvae of the dominant insect in the natural community, Geometridae Brown (Lepidoptera), consumed less area from field-grown uvb+ leaves than from uvb– leaves in 1996–97, but not in 1997–98. Correlation analyses suggested that the reduction in insect herbivory in the field under solar UV-B may be mediated in part by the UV-B effects on gallic acid and flavonoid aglycone.     

Photomorphogenic regulation of increases in UV-absorbing pigments in cucumber (Cucumis sativus) and Arabidopsis thaliana seedlings induced by different UV-B and UV-C wavebands

Brief (1–100 min) irradiations with three different ultraviolet-B (UV-B) and ultraviolet-C (UV-C) wave bands induced increases the UV-absorbing pigments extracted from cucumber ( Cucumis sativus L.) and Arabidopsis . Spectra of methanol/1% HCl extracts from cucumber hypocotyl segments spanning 250–400 nm showed a single defined peak at 317 nm. When seedlings were irradiated with 5 kJ m⁻² UV-B radiation containing proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm; full-spectrum UV-B, FS-UVB), tissue extracts taken 24 h after irradiation showed an overall increase in absorption (91% increase at 317 nm) with a second defined peak at 263 nm. Irradiation with 1.1 kJ m⁻² UV-C (254 nm) caused similar changes. In contrast, seedlings irradiated with 5 kJ m⁻² UV-B including only wavelengths longer than 290 nm (8% of UV-B between 290 and 300 nm; long-wavelength UV-B, LW-UVB) resulted only in a general increase in absorption (80% at 317 nm). The increases in absorption were detectable as early as 3 h after irradiation with FS-UVB and UV-C, while the response to LW-UVB was first detectable at 6 h after irradiation. In extracts from whole Arabidopsis seedlings, 5 kJ m⁻² LW-UVB caused only a 20% increase in total absorption. Irradiation with 5 kJ m⁻² FS-UVB caused the appearance of a new peak at 270 nm and a concomitant increase in absorption of 72%. The induction of this new peak was observed in seedlings carrying the fah 1 mutation which disrupts the pathway for sinapate synthesis. The results are in agreement with previously published data on stem elongation indicating the existence of two response pathways within the UV-B, one operating at longer wavelengths (>300 nm) and another specifically activated by short wavelength UV-B (<300 nm and also by UV-C).     

The sensitivity of transgenic spruce (Picea glauca (Moench) Voss) cotyledonary somatic embryos and somatic seedlings to kanamycin selection

Transformed white spruce cultures containing immature Stage I somatic embryos, were developed after particle bombardment of somatic embryos with pBI 426, carrying an expression cassette with a gus::nptII fusion gene. These Stage I cultures did not show tolerance to kanamycin concentrations above 3 to 5 mg l–1, although assays for GUS and NPTII showed that functional enzymes were present in the transgenic tissue. Embryonic liquid suspension cultures were initiated from this transformed tissue. After treatment on agar-solidified maturation medium with 48 m (±)-ABA high numbers of Stage III (cotyledonary) somatic embryos were produced. When subjected to an embryogenesis re-induction process with 2,4-D and BA, these Stage III embryos produced a new generation of Stage I embryogenic tissues which could tolerate 5--10 mg l–1 kanamycin. Stage III somatic embryos could alternatively be placed onto germination medium for the development of somatic seedlings. When germinated in the presence of 20 mg l–1 kanamycin, 77% of inoculants were resistant. The stability of integration of the gus::nptII fusion gene in the genome of white spruce Stage III somatic embryos and somatic seedlings was confirmed through Southern blot analysis     

Oxidative DNA damage in cucumber cotyledons irradiated with ultraviolet light

DNA was isolated from the cotyledons of cucumber seedlings irradiated with ultraviolet (UV)-C (254 nm) or UV-B+UV-A (280–360 nm; maximum energy at 312 nm) at various fluence rates and durations. Following enzymatic hydrolysis of DNA, the content of 8-hydroxy-2-deoxyguanosine [(8-OHdG), 8-oxo-7,8-dihydro-2-deoxyguanosine], a well-established biomarker closely identified with carcinogenesis and aging in animal cells, was determined using a high-performance liquid chromatograph equipped with an electrochemical detector. The levels of 8-OHdG increased with UV-C and UV-B irradiation in a fluence-dependent manner. This increase was also observed in etiolated cotyledons that had been excised from dark-grown cucumber seedlings and then cultured in vitro under UV light: monochromatic UV light at 270 nm or 290 nm increased the 8-OHdG level considerably, while UV at wavelengths above 310 nm had only small effects. In situ detection of H₂O₂ and quantification of H₂O₂ in plant extracts revealed that H₂O₂ accumulated in cotyledons irradiated with UV light. These results suggest that UV irradiation induces oxidative DNA damage in plant cells.     

Growth and morphological responses to different UV wavebands in cucumber (Cucumis sativum) and other dicotyledonous seedlings

We examined the influence of short-term exposure of different UV wavebands on the fine-scale kinetics of hypocotyl growth of dim red light-grown cucumbers (Cucumis sativus L.) and other selected dicotyledonous seedlings to evaluate: (1) whether responses induced by UV-B radiation (280-320 nm) are qualitatively different from those induced by UV-A (320-400 nm) radiation, and (2) whether different wavebands within the UV-B elicit different responses. Responses to brief (30 min) irradiations with 3 different UV wavebands all included transient inhibition of elongation during irradiation followed by wavelength specific responses. Irradiations with proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm) induced inhibition of hypocotyl elongation within 20 min of onset of irradiation, while UV-B including only wavelengths longer than 290 nm (and only 8% of UV-B between 290 and 300 nm) induced inhibition of hypocotyl elongation with a lag of 1-2 h. The response to short wavelength UV-B was persistent for at least 24 h, while the response to long wavelength UV-B lasted only 2-3 h. The UV-A treatment induced reductions in elongation rates of approximately 6-9 h following exposure followed by a continued decline in rates for the following 15-18 h. Short wavelength UV-B also induced positive phototropic curvature in both cucumber and Arabidopsis seedlings, and this response was present in nph-1 mutant Arabidopsis seedlings defective in normal blue light phototropism. Reciprocity was not found for the response to short wavelength UV-B. The short wavelength and long wavelength UV-B responses differed in dose-response relationships and both short wavelength responses (phototropic curvature and elongation inhibition) increased sharply at wavelengths below 300 nm. These results indicate that different photosensory processes are involved in mediating growth and morphological responses to short wavelength UV-B (280-300 nm), long wavelength UV-B (essentially 300-320 nm) and UV-A. The existence of two separate types of hypocotyl inhibition responses to UV-B, with one that depends on the intensity of the light source, provides alternate interpretations to findings in other studies of UV-B induced photomorphogenesis and may explain inconsistencies between action spectra for inhibition of stem growth.     

Expression of antisense chalcone synthase RNA in transgenic hybrid walnut microcuttings. Effect on flavonoid content and rooting ability

Walnut somatic embryos (Juglans nigra × Juglans regia) were transformed with a vector containing a neomycin phosphotransferase II, a -glucuronidase and an antisense chalcone synthase (chs) gene. This antisense construct included a 400 bp cDNA fragment of a walnut chs gene under the control of the duplicated CaMV-35S promoter. Molecular, biochemical and biological characterizations were performed both on transformed embryos propagated by secondary somatic embryogenesis and on microshoots developed by in vitro culture of embryonic epicotyls from somatic embryos. Thirteen transformed lines with the vector containing the antisense chs gene, one line with only the gus and nptII genes and one untransformed line were maintained in tissue culture. Six of the antisense lines were shown to be flavonoid-deficient. They exhibited a strongly reduced expression of chs genes, very low chalcone synthase activity and no detectable amounts of quercitrin, myricitrin, flavane-3-ols and proanthocyanidins in stems. Rooting tests showed that decreased flavonoid content in stems of antisense chs transformed lines was associated with enhanced adventitious root formation. Free auxin and conjugated auxin contents were determined during the latter phase of the micropropagation, and no variations were detected between control and antisense chs transformed lines. The in vitro plants developed a large basal callus and apical necrosis upon auxinic induction and the transformed lines highly deficient in flavonoids were more sensitive to exogenous application of indolebutyric acid (IBA).     

Heat Shock Increases the Tolerance of Plants to UV-B Radiation: 2. Ethylene and Carbon Dioxide Evolution

The effects of heat shock (HS, 45°C) and UV-B radiation (280–320 nm, 18.3 kJ/(m²h)) and the consecutive action of the combination of these factors on ethylene production, gas exchange, and the growth of intact melon (Melo sativusSager.) seedlings were investigated. The changes in ethylene production and carbon dioxide exchange were described by a single-peaked curve. In the course of UV-B irradiation, the time of maximum ethylene and CO₂evolution coincided (the first 5 min) and comprised 0.36 nl/(seedling h) for ethylene and 146.2 l/(seedling h) for carbon dioxide. After HS, the maximum of ethylene production (0.37 nl/(seedling h)) was reached within 10 min, and that of carbon dioxide production (313 l/(seedling h)), within 45 min. The rate of ethylene production (0.22 nl/(seedling h)), carbon dioxide production (97.7 l/(seedling h)), and oxygen consumption (162.5 l/(seedling h)) in the control seedlings did not change in the course of experiment. Throughout the experiment, the respiratory quotient of seedlings was ca. 0.6 regardless on the nature and duration of the acting factor. Preliminary heating at 45°C for 1 h increased the tolerance of seedlings to the subsequent UV-B radiation for 1 h. The protective effect of HS manifests itself in alleviating the inhibiting action of UV-B radiation on seedling growth and development, and this effect was preceded by an increase in ethylene production and respiration. The possible mechanisms of cross-tolerance of plants to overheating and UV-B radiation are discussed.     

Plant responses to ultraviolet-B (UV-B: 280–320 nm) stress: What are the key regulators?

Depletion of the stratospheric ozone layer is leadingto an increase in ultraviolet-B (UV-B: 280–320 nm)radiation reaching the earth's surface. This hasraised interest in the possible consequence ofincreased UV-B levels on plant growth and developmentand the mechanisms underlying these responses. Although the effects of UV-B are now wellcharacterised at the physiological level, little isknown about the cellular and molecular mechanismsinvolved. Recent studies have shown that UV-B affectsa number of important physiological processes, such asphotosynthesis, through effects on gene expression. In addition, induction of a number of defensemechanisms, such as production of UV-B screeningpigments, increase in antioxidant enzymes andinduction of pathogenesis-related proteins, are alsomediated at the level of gene expression. The signaltransduction pathways by which UV-B regulates geneexpression are at present poorly understood. Thestudies carried out to date have, however, indicateda pivotal role for reactive oxygen species as keysecond messengers acting up-stream of a number ofpathways involving the plant hormones salicylic acid,jasmonic acid and ethylene. The transduction pathwaysidentified to date and the role of intermediates inregulating tolerance to UV-B damage are discussed inthis review.     

Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence –190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (–677 to –453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the -glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.