Differential sensitivity to ozone in two poplar clones.

The effect of acute O₃ exposure (5 h at 150 ppb) on photosynthetic electron transport and the thylakoid polypeptide pattern has been studied in 2-month-old cuttings of two poplar clones ( Populus deltoides × maximowiczii clone Eridano and P .× euramericana clone I-214), known for their differential response to O₃ in terms of visible symptoms. The more sensitive Eridano developed severe blackish bifacial necrosis concentrated in the interveinal areas of the basal and intermediate leaves, while in the less sensitive I-214 clone, visible symptoms were slight or entirely absent. A generalized, significant decrease in leaf pigment content was shown by both of the stressed clones, although the decline in β -carotene content was much more marked in Eridano than in I-214. Activation of the xanthophyll cycle was not detected in either clone. In both clones, immunoblot analysis of the thylakoid proteins showed a decrease in the levels of all the PSII polypeptides tested following O₃ exposure, whereas the PsaA/B protein in the PSI core was more severely affected in Eridano than in I-214. No changes in the chloroplast structure could be seen on electron microscopic study in either clone. The only visible difference was a great accumulation of starch grains in Eridano. All of these results suggest that O₃ exposure induces alterations in the photosynthetic apparatus in both poplar clones, Eridano being affected to a greater extent than I-214.     

Early responses to cadmium of two poplar clones that differ in stress tolerance

Soil cadmium (Cd) contamination is becoming a matter of great global concern. The identification of plants differentially sensitive to Cd excess is of interest for the selection of genotype adaptive to grow and develop in polluted areas and capable of ameliorating or reducing the negative environmental effects of this toxic metal. The two poplar clones I-214 (Populus × canadensis) and Eridano (Populus deltoides × maximowiczii) are, respectively, tolerant and sensitive to ozone (O₃) exposure. Because stress tolerance is mediated by an array of overlapping defence mechanisms, we tested the hypothesis that these two clones differently sensitive to O₃ stress factor also exhibit different tolerance to Cd. With this purpose, an outdoor pot experiment was designed to study the responses of I-214 and Eridano to the distribution of different Cd solutions enriched with CdCl₂ (0, 50 and 150 μM) for 35 days. Changes in leaf area, biomass allocation and Cd uptake, photosynthesis, chlorophyll fluorescence, leaf concentration of nutrients and pigments, hydrogen peroxide (H₂O₂) and nitric oxide (NO) production and thiol compounds were investigated. The two poplar clones showed similar sensitivity to excess Cd in terms of biomass production, photosynthesis activity and Cd accumulation, though physiological and biochemical traits revealed different defence strategies. In particular, Eridano maintained in any Cd treatment the number of its constitutively wider blade leaves, while the number of I-214 leaves (with lower size) was reduced. H₂O₂ increased 4.5- and 13-fold in I-214 leaves after the lowest (L) and highest (H) Cd treatments, respectively, revealing the induction of oxidative burst. NO, constitutively higher in I-214 than Eridano, progressively increased in both clones with the enhancement of Cd concentration in the substrate. I-214 showed a more elevated antioxidative capacity (GSH/GSSG) and higher photochemical efficiency of PSII (F_v/F_m) and de-epoxidation degree of xantophylls-cycle (DEPS). The glutathione pool was not affected by Cd treatment in both clones, while non-protein thiols and phytochelatins were reduced at L Cd treatment in I-214. Overall, these two clones presented high adaptability to Cd stress and are both suitable to develop and growth in environments contaminated with this metal, thus being promising for their potential use in phytoremediation programmes.     

Photosynthesis of two poplar clones contrasting in O3 sensitivity

 Two clones of poplar known for their phenomenological difference in response to ozone were fumigated with 150 nl l^–1 of ozone for 5 h. In both clones the treatment significantly reduced the light-saturated rate of CO₂ uptake of recently mature leaves and this was accompanied by a decrease in stomatal conductance. Intercellular CO₂ concentration of the resistant clone increased following the fumigation. After 20 h of recovery, photosynthesis recovered completely only in the resistant clone. Electrolyte leakage of leaf disks increased in both clones to indicate damage to membranes; after the recovery time this parameter only reached values of the control in the resistant clone. The photochemical efficiency of PSII slightly decreased in the resistant clone. In the other clone, the treatment caused a decline of all chlorophyll fluorescence parameters and only some of them returned to normal values after the recovery time. The physiological response appears to be different in the two clones. In the resistant one, the most probable mechanism involved in the photosynthetic reduction was a regulatory reduction in CO₂ fixation. Also data obtained by the solute leakage indicate that in the resistant clone repair mechanisms play a role. The reduction of photosynthesis observed in the sensitive clone is related both to strong stomatal closure and to an impairment in fluorescence parameters. These alterations can indicate a general disruption at the membrane level as confirmed by the solute leakage data. Received: 30 June 1997 / Accepted: 3 September 1997     

Cadmium interferes with auxin physiology and lignification in poplar

Cadmium (Cd) is a phytotoxic heavy metal that causes rapid growth reduction. To investigate if Cd interferes with the metabolism of auxin, a major growth hormone in plants, poplars (Populus × canescens) expressing a heterologous GH3::GUS reporter gene were exposed to 50 μM Cd in hydroponic solutions. Growth, photosynthetic performance, lignification, peroxidase activity, auxin concentration, and GUS staining were determined in order to record the activities of GH3 enzymes in the stem apex, the elongation zone, wood in the zone of radial growth, and in roots. Cd-induced growth reductions were tissue-specific decreasing in the order: roots>wood>shoot elongation and leaf initiation, whereas Cd concentrations increased in the order: leaves相似文献   

Photosynthesis of two poplar clones under long‐term exposure to ozone

The photosynthetic response was studied in two clones ( Populus deltoides × maximowiczii Eridano and Populus × euramericana I‐214), known for their differential response to ozone (O₃) in terms of visible symptoms, when exposed to O₃ (60 nl l⁻¹ 5 h day⁻¹, 7 and 15 days). The photosynthetic ability was tested using gas exchange and chlorophyll fluorescence analysis. O₃ caused a decrease in the CO₂ assimilation rate at light saturation level in mature leaves of both clones. Alterations of Chl fluorescence parameters, in particular the F_v/F_m ratio and non‐photochemical quenching were also observed. The effects were similar for both clones and it could not be concluded that differential effects on electron transport capacity were responsible for the observed reduction in photosynthesis. The reduction of photosynthetic rate in Eridano was due mainly to a reduced mesophyll activity, as evidenced by the increase in intercellular CO₂ concentration and the minimal changes in stomatal conductance. In contrast, in I‐214, stomatal effects were primarily responsible, although effects on the mesophyll cannot be excluded. Data obtained indicate that the effects observed at the mesophyll level may be attributed to indirect effects caused by membrane disorders.     

盐胁迫下白杨无性系苗木体内离子分配及比较

以白杨杂种无性系及其亲本当年生苗木为材料 ,分析了盐胁迫下无性系的离子含量 ,及 Na 、K 、Ca 在植物体内运输和分配特点 ,并对无性系间差异进行了比较。研究表明 ,随盐浓度提高 ,植物体内Na 含量迅速提高 ,K 、Ca 含量降低 ;盐分胁迫下 ,根部 Na 含量较高 ,叶片 Na 中含量最低 ,K 、Ca 含量则相反 ,特别是 Ca ,其分布顺序为叶 >茎 >根。杂种无性系 B430及其亲本新疆杨对 K 和 Ca 运输的选择性比毛白杨高 ,而对 Na 运输的选择性则比毛白杨低 ,从而导致根部存留的 Na 较多 ,叶片分配的 Na 数量较少 ,从而减轻 Na 对叶片的伤害。综合分析表明 B430和新疆杨耐盐能力最强 ,毛新杨其次 ,毛白杨最差。     

Nitrogen as an important detoxification factor to cadmium stress in poplar plants

To verify the important role of nitrogen in detoxifying plants from heavy metals in Populus, the influence of nitrogen and cadmium on growth, chlorophyll (Chl) synthesis, and the expression of the Glutamine synthetase gene (GS₂) were studied in poplar plants. Experiments were carried out in potted plants treated with (NH₄)₂CO₃, Cd(NO₃)₂, CdCl₂ and CdCl₂ plus (NH₄)₂CO₃. After treatment, plant height, biomass, chlorophyll content, the precursors content and GS₂ were investigated. Results showed that the plants treated with cadmium showed toxicity symptoms, decrease in growth and Chl content. Cd inhibited Chl synthesis seriously by blocking the site located on the steps between UrogenIII and Coprogen III. However, the plants treated with cadmium and nitrogen grew well without any toxicity symptoms. Nitrogen supplement can alleviate Cd inhibition on chlorophyll synthesis by unblocking the pathway. The results indicated that nitrogen can effectively alleviate cadmium toxicity to poplar plants.     

Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity

Cell capacity for cytosolic NADPH regeneration by NADP‐dehydrogenases was investigated in the leaves of two hybrid poplar (Populus deltoides × Populus nigra) genotypes in response to ozone (O₃) treatment (120 ppb for 17 days). Two genotypes with differential O₃ sensitivity were selected, based on visual symptoms and fallen leaves: Robusta (sensitive) and Carpaccio (tolerant). The estimated O₃ flux (POD₀), that entered the leaves, was similar for the two genotypes throughout the treatment. In response to that foliar O₃ flux, CO₂ assimilation was inhibited to the same extent for the two genotypes, which could be explained by a decrease in Rubisco (EC 4.1.1.39) activity. Conversely, an increase in PEPC (EC 4.1.1.31) activity was observed, together with the activation of certain cytosolic NADP‐dehydrogenases above their constitutive level, i.e. NADP‐G6PDH (EC 1.1.1.49), NADP‐ME (malic enzyme) (EC 1.1.1.40) and NADP‐ICDH (NADP‐isocitrate dehydrogenase) (EC1.1.1.42). However, the activity of non‐phosphorylating NADP‐GAPDH (EC 1.2.1.9) remained unchanged. From the 11th fumigation day, NADP‐G6PDH and NADP‐ME profiles made it possible to differentiate between the two genotypes, with a higher activity in Carpaccio than in Robusta. At the same time, Carpaccio was able to maintain high levels of NADPH in the cells, while NADPH levels decreased in Robusta O₃‐treated leaves. All these results support the hypothesis that the capacity for cells to regenerate the reducing power, especially the cytosolic NADPH pool, contributes to improve tolerance to high ozone exposure.     

Fertilization of poplar clones in the nursery

Summary Results of experiments with four poplar clones and various chemical fertilizers in a nursery in southern Greece are presented. At the end of the first growth period the heights of the four clones, without fertilizers, decreased in the order of I-214>I-262>cv. campeator > black poplar 1/64 with significant differences only between black poplar 1/64 and the rest of the clones.Of the fertilizer nutrients N, P, K and Mg only N improved heights of all clones significantly and especially of the clone I-214. One hundred and 200 kg of P fertilizer per ha had minimal or negative effect on height increase of all clones.Ammonium sulfate, ammonium nitrate and potassium nitrate all at 400 kg N per ha were found equally effective in improving height growth of the clone I-214 but ammonium nitrate is the N fertilizer of choice by its higher N content and relatively lower price.Ammonium nitrate at 200 kg N per ha, in two or three equal dosages, during the first growth period, June–July, gave the maximum height increase for two consecutive years of the clone I-214. Six hundred kgs, of N per ha reduced height increase of the same clone and increased losses of N, as NO₃ ^–, in drainage water.     

Differences in structure of adventitious roots in Salix clones with contrasting characteristics of cadmium accumulation and sensitivity

Glutamine synthetase (GS) genes, GS1a and GS1b, in pine ( Pinus sylvestris L.) are differentially regulated in tissue specificity and during seedling development. To gain insight into the regulatory mechanisms controlling their expression, we have analysed the 5'-flanking sequences of the gene GS1a using a transient expression system in pine protoplasts. Structural analysis of this region revealed the presence of putative regulatory elements including two AT-rich elements and a poly CT consensus sequence. A series of 5'- and 3'-deletions of the untranslated region covering the three putative elements, −800 to −626, −626 to −427 and +118 to +177 were analysed to demonstrate the functional implications of these elements in gene regulation. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from pine cotyledons interact with both AT-rich regions (−800 to −427). Interestingly, no protein binding was detected when the untranslated region (+118 to +177) was included, even if deletion of that region suppressed promoter activity in the transient expression experiments conducted. However, simultaneous deletion of both types of cis elements, A/T and CT, resulted in a recovery of promoter activity of 50%. These results suggest a key regulatory role of the CT box by the interaction with A/T stretches in the distal part of the promoter and possibly with the proximal region (−427 to −1).     

Age-related sensitivity to lung oxidative stress during ozone exposure

As immature and aged rats could be more sensitive to ozone (O(3))-linked lung oxidative stress we have attempted to shed more light on age-related susceptibility to O(3) with focusing our interest on lung mitochondrial respiration, reactive oxygen species (ROS) production and lung pro/antioxidant status. For this purpose, we exposed to fresh air or O(3) (500 ppb 12 h per day, for 7 days) 3 week- (immature), 6 month- (adult) and 20 month-old rats (aged). We determined, in lung, H(2)O(2) release by mitochondria, activities of major antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)], heat shock protein (HSP(72)) content and 8-oxodG and dG-HNE nDNA contents, as DNA oxidative damage markers. In adult rats we did not observe alteration of pro/antioxidant status. In contrast to adults, immature rats exposed to O(3) higher nDNA 8-oxodG content and HSP(72) and without antioxidant enzymes modification. Aged rats displayed mild uncoupled lung mitochondria, increased SOD and GPx activities, and higher 8-oxodG content after O(3) exposure. Thus, in contrast to adults, immature and aged rats displayed lung oxidative stress after O(3) exposure. Higher sensitivity of immature to O(3) was partly related to ventilatory parameters and to the absence of antioxidant enzyme response. In aged rats, the increase in cytosolic SOD and GPx activities during O(3) exposure was not sufficient to prevent the impairment in mitochondrial function and accumulation in lung 8- oxodG. Finally, we showed that mitochondria seem not to be a major source of ROS under O(3) exposure.     

Copper- and arsenate-induced oxidative stress in Holcus lanatus L. clones with differential sensitivity

The biochemical responses of Holcus lanatus L. to copper and arsenate exposure were investigated in arsenate‐tolerant and ‐non‐tolerant plants from uncontaminated and arsenic/copper‐contaminated sites. Increases in lipid peroxidation, superoxide dismutase (SOD) activity and phytochelatin (PC) production were correlated with increasing copper and arsenate exposure. In addition, significant differences in biochemical responses were observed between arsenate‐tolerant and ‐non‐tolerant plants. Copper and arsenate exposure led to the production of reactive oxygen species, resulting in significant lipid peroxidation in non‐tolerant plants. However, SOD activity was suppressed upon metal exposure, possibly due to interference with metallo‐enzymes. It was concluded that in non‐tolerant plants, rapid arsenate influx resulted in PC production, glutathione depletion and lipid peroxidation. This process would also occur in tolerant plants, but by decreasing the rate of influx, they were able to maintain their constitutive functions, detoxify the metals though PC production and quench reactive oxygen species by SOD activity.     

杨树无性系光合特征的研究

对新培育的金科系列的4个杨树无性系（3^#、6^#、8^#、9^#）的光合特征及净光合速率、水分利用效率与主要影响因子的关系进行了研究。结果表明：不同月份4个无性系的净光合速率的日进程一般在10:00左右达到峰值,然后逐渐降低;蒸腾速率日进程不尽一致;气孔导度在7:00～9:00出现峰值后,缓慢下降。在6～8月,4个无性系的净光合速率的总平均值(μmol CO₂·m^-2·s^-1)排序为：无性系9^#（8.53）>6^#>（7.21）3^#（6.47）>8^#（4.98）;蒸腾速率的总平均值(mmol H₂O·m^-2·s^-1)排序为：无性系9^#（3.74）>3^#（2.76）>6^#（1.76）>8^#（1.47）;水分利用效率的总平均值(mmol CO₂·mol^-1 H₂O)排序为：无性系8^#（4.77）>6^#（4.35）>3^#（2.99）>9^#（2.40）。4个无性系的净光合速率与水分利用效率的排序并不一致。9^#属于高光合、高蒸腾、低水分利用效率类型,8^#属低光合、低蒸腾、高水分利用效率类型。在6月份6^#无性系的净光合速率和水分利用效率与温度、湿度显著相关,3^#、8^#、9^#无性系与光合有效辐射和气孔导度密切相关。     

Factors affecting Agrobacterium tumefaciens-mediated transformation in several black poplar clones

Transient expression of the uidA reporter gene was used in preliminary experiments with two oncogenic and two disarmed Agrobacterium tumefaciens strains in order to test the efficiency of T-DNA transfer to N084 x Populus nigra and N107 x P. nigra clones. The oncogenic strain A281 pKIWI105 produced the highest average number of GUS spots per leaf disc. In order to optimize the production of transgenic plantlets from different P. nigra clones (San Giorgio, Jean Pourtet, N084 x P. nigra and N107 x P. nigra, respectively), two A. tumefaciens strains (GV2260 p35S GUS, A281pKIWI105) and bacterial concentrations (7×10⁸; 1.2×0⁹ bacteria ml^-1) were used. Following co-cultivation with A281 pKIWI105, the frequency of leaf discs producing kanamycin-resistant calli was not significantly different between the clones and bacteria concentrations used. Transformed shoots were regenerated from all clones, except for Jean Pourtet. Co-cultivation of leaf discs with GV2260 p35S GUS produced very few calli which died when transferred to selective regeneration medium. In addition, the effects of acetosyringone and leaf wounding were evaluated for the San Giorgio and Jean Pourtet clones, using the same strains. Factors which significantly affected the transformation efficiency of leaf explants were the P. nigra clone, the A. tumefaciens strain, and the presence of acetosyringone. Genetic transformation of calli and regenerated plantlets was confirmed by their ability to grow and root on Woody Plant Medium containing kanamycin, by histochemical -glucuronidase assays, and Southern blot hybridization analyses.Abbreviations BA benzyladenine - GUS -glucuronidase - IBA indolebutyric acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - nptII neomycin phosphotransferase II gene - uidA -glucuronidase gene - WPM Woody Plant Medium     

水稻叶片对镉胁迫响应的蛋白质差异表达

为揭示水稻镉抗性的分子机理,以抗镉水稻品种P1312777和镉敏感水稻品种IR24为材料,在镉离子浓度为0(对照)、50和100 μmol·L-1条件下水培处理7 d,应用蛋白质组学方法分析了2种水稻叶片对镉胁迫响应的蛋白质差异表达.结果表明:镉胁迫下水稻PI312777叶片中共检测到差异表达蛋白质点31个,通过MALDI-TOF/MS分析,鉴定了其中的24个蛋白质(包括20个不同蛋白质,4个重复检出蛋白质);IR24叶片中共检测到差异表达蛋白质点19个,其中15个蛋白质得到鉴定.PI312777叶片鉴定出的20个蛋白质覆盖了IR24叶片鉴定的15个蛋白质,前者有4个与光合作用相关,11个与细胞防御代谢相关,3个与其他代谢相关,2个为功能未知蛋白.与对照相比,不同浓度镉胁迫下,抗镉水稻PI312777叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型、果糖1,6-二磷酸醛缩酶、硫氧还蛋白和DNA重组修复蛋白均上调表达;镉敏感水稻IR24叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型的表达无显著差异,果糖1,6-二磷酸醛缩酶和硫氧还蛋白则下调表达.此外,DNA重组修复蛋白仅在镉胁迫的PI312777叶片中表达.水稻PI312777比IR24具有更强的镉抗性与这些差异表达的蛋白质密切相关.     

Condensed lignins are synthesized in poplar leaves exposed to ozone

Poplar (Populus tremula x alba) trees (clone INRA 717-1-B4) were cultivated for 1 month in phytotronic chambers with two different levels of ozone (60 and 120 nL L(-1)). Foliar activities of shikimate dehydrogenase (EC 1.1.1.25), phenylalanine ammonia lyase (EC 4.3.1.5), and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) were compared with control levels. In addition, we examined lignin content and structure in control and ozone-fumigated leaves. Under ozone exposure, CAD activity and CAD RNA levels were found to be rapidly and strongly increased whatever the foliar developmental stage. In contrast, shikimate dehydrogenase and phenylalanine ammonia lyase activities were increased in old and midaged leaves but not in the youngest ones. The increased activities of these enzymes involved in the late or early steps of the metabolic pathway leading to lignins were associated with a higher Klason lignin content in extract-free leaves. In addition, stress lignins synthesized in response to ozone displayed a distinct structure, relative to constitutive lignins. They were found substantially enriched in carbon-carbon interunit bonds and in p-hydroxyphenylpropane units, which is reminiscent of lignins formed at early developmental stages, in compression wood, or in response to fungal elicitor. The highest changes in lignification and in enzyme activities were obtained with the highest ozone dose (120 nL L(-1)). These results suggest that ozone-induced lignins might contribute to the poplar tolerance to ozone because of their barrier or antioxidant effect toward reactive oxygen species.