Ozone-sensitive Clone Research Articles

Ultrastructural alterations induced by tropospheric ozone: comparison between resistant and sensitive clones of Trifolium repens L. CV. Regal

The effects of ozone on ozone-sensitive (NC-S) and ozone-resistant (NC-R) clones of Trifolium repens L. cv. Regal are compared at ultrastructural level by Transmission Electron Microscopy (TEM). Each clone were exposed to ambient ozone levels in the Botanical Garden of Rome, Italy, according to the ICP Vegetation Experimental Protocol (2005). TEM analyses showed that ultrastructural injuries appeared to be widespread in NC-S leaves. On the contrary, the NC-R clones showed much less damage: the chloroplast maintained an almost intact organisation of thylakoids, but the chloroplast side facing the apoplastic space appeared with no thylakoids. Interestingly, Hsp70 levels, an important stress marker, were only slightly increased in the ozone-sensitive clone with respect to the ozone-resistant clone; in contrast, a strong decrease (−60%) in phosphoenolpyruvate carboxylase (PEPCase) protein levels was measured in the NC-R clone. These data are discussed in the frame of the proposed ozone toxicity mechanism.

Differences in leaf characteristics between ozone-sensitive and ozone-tolerant hybrid aspen (Populus tremula x Populus tremuloides) clones

The authors analyzed a suite of leaf characteristics that might help to explain the difference between ozone-sensitive and ozone-tolerant hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) clones. An open-field experiment comprising ambient ozone and 1.5x ambient ozone concentration (about 35 ppb) and two soil nitrogen regimes (60 and 140 kg N ha(-1) year(-1)) was conducted over two growing seasons on potted plants of eight hybrid aspen clones. Four of the clones had previously been determined to be ozone sensitive based on impaired growth in response to elevated ozone concentration. Photosynthetic rate, chlorophyll fluorescence, and concentrations of chlorophyll, protein and carbohydrates were analyzed three times during the second growing season, and foliar phenolic concentrations were measured at the end of the second growing season. Nitrogen amendment counteracted the effects of ozone, but had no effect on growth-related ozone sensitivity of the clones. Ozone-sensitive clones had higher photosynthetic capacity and higher concentrations of Rubisco and phenolics than ozone-tolerant clones, but the effects of ozone were similar in the sensitive and tolerant groups. Nitrogen addition had no effect on phenolic concentration, but elevated ozone concentration increased the concentrations of chlorogenic acid and (+)-catechin. This study suggests that condensed tannins and catechin, but not salicylates or flavonol glycosides, play a role in the ozone tolerance of hybrid aspen.

Profiling Secondary Metabolites of Needles of Ozone-Fumigated White Pine (Pinus strobus) Clones by Thermally Assisted Hydrolysis/Methylation GC/MS

Plant secondary metabolites have an important role in defense responses against herbivores and pathogens, and as a chemical barrier to elevated levels of harmful air pollutants. This study involves the rapid chemical profiling of phenolic and diterpene resin acids in needles of two (ozone-tolerant and ozone-sensitive) white pine (Pinus strobus) clones, fumigated with different ozone levels (control, and daily events peaking at 80 and 200 ppb) for 40 days. The phenolic and resin acids were measured using thermally assisted hydrolysis and methylation (THM) gas chromatography/mass spectrometry. Short-term fumigation affected the levels of two phenolic acids, i.e., 3-hydroxybenzoic and 3,4-dihydroxybenzoic acids, in that both showed a substantial decrease in concentration with increased ozone dose. The decrease in concentration of these THM products may be caused by inhibition of the plant's shikimate biochemical pathway caused by ozone exposure. The combined occurrence of these two ozone-sensitive indicators has a role in biomonitoring of ozone levels and its impact on forest productivity. In addition, chromatographic profile differences in the major diterpene resin acid components were observed between ozone-tolerant and ozone-sensitive clones. The resin acids anticopalic, 3-oxoanticopalic, 3beta-hydroxyanticopalic, and 3,4-cycloanticopalic acids were present in the ozone-sensitive pine; however, only anticopalic acid was present in the ozone-tolerant clone. This phenotypic variation in resin acid composition may be useful in distinguishing populations that are differentially adapted to air pollutants.

Effects of long-term open-field ozone exposure on leaf phenolics of European silver birch (Betula pendula Roth).

The response of phenolic compounds as a result of long-term low open-field ozone exposure was studied in ozone-sensitive and ozone-tolerant clones of European silver birch (Betula pendula Roth). The saplings were exposed to 1.5-1.6 times the ambient (elevated) ozone and ambient air (as control) over three growing seasons from May 1996 until August 1998. Quantification by modified Folin-Ciocalteau assay showed a 16.2% increase in total phenolics in elevated ozone plants as compared to that in controls and a corresponding 9.9% increase of 10 phenolic compounds quantified by HPLC. Five nonflavonoids and five flavonoids showed 8.4% and 11.4% increases, respectively. The phenolic results indicated slightly higher ozone sensitivity of clone 5 as compared to clone 2. The most ozone-responsive phenolic compounds in clone 2 and clone 5 were (+)-catechin (CT), chlorogenic acid (CGA), 5-p-coumaroylquinic acid (5CQA), 3-p-coumaroylquinic acid (3CQA), myricetin galactopyranoside (MG), quercetin-3-O-glucuronopyranoside (QGR), and quercetin-3-O-arabinofuranoside (QA). Increased phenolic content in ozone-exposed plants was related to impaired growth and accelerated leaf senescence, indicated by enhanced autumn leaf yellowing and lower chlorophyll and Mg content. The change in carbon allocation towards defensive phenolics at the expense of growth was greater in the ozone-sensitive clone as compared to tolerant clone.

Gene structure and expression of the aspen cytosolic copper/zinc-superoxide dismutase (PtSodCc1)

Genomic and cDNA clones, corresponding to an ozone-induced cytosolic copper–zinc superoxide dismutase, were isolated from quaking aspen ( Populus tremuloides Michx.). The cytosolic superoxide dismutase (SOD) appears to be part of a multi-gene family in aspen and is interrupted by five introns in the coding region. Northern blot analysis with a gene-specific probe revealed an increase in the expression of this gene in response to ozone in the leaves of an ozone-tolerant aspen clone, compared with an ozone-sensitive clone. Cytosolic SOD transcript expression levels in leaves were also found to increase significantly within 6 h of mechanical wounding, after which the level of the transcript decreases. Under normal growing conditions, immature male and female aspen floral bud tissues contained the highest levels of the cytosolic SOD gene transcript, whereas transcript levels were almost undetectable in older leaves.

Ozone Sensitivity in Hybrid Poplar Is Correlated with a Lack of Defense-Gene Activation

Ozone is a major gaseous pollutant thought to contribute to forest decline. Although the physiological and morphological responses of forest trees to ozone have been well characterized, little is known about the molecular basis for these responses. Our studies compared the response to ozone of ozone-sensitive and ozone-tolerant clones of hybrid poplar (Populus maximowizii x Populus trichocarpa) at the physiological and molecular levels. Gas-exchange analyses demonstrated clear differences between the ozone-sensitive clone 388 and the ozone-tolerant clone 245. Although ozone induced a decrease in photosynthetic rate and stomatal conductance in both clones, the magnitude of the decrease in stomatal conductance was significantly greater in the ozone-tolerant clone. RNA-blot analysis established that ozone-induced mRNA levels for phenylalanine ammonia-lyase, O-methyltransferase, a pathogenesis-related protein, and a wound-inducible gene were significantly higher in the ozone-tolerant than in the ozone-sensitive plants. Wound- and pathogen-induced levels of these mRNAs were also higher in the ozone-tolerant compared with the ozone-sensitive plants. The different physiological and molecular responses to ozone exposure exhibited by clones 245 and 388 suggest that ozone tolerance involves the activation of salicylic-acid- and jasmonic-acid-mediated signaling pathways, which may be important in triggering defense responses against oxidative stress.

Growth responses and related biochemical and ultrastructural changes of the photosynthetic apparatus in birch (Betula pendula) saplings exposed to low concentrations of ozone

Saplings of ozone-sensitive and ozone-tolerant birch (Betula pendula Roth.), clones B and C, respectively, were exposed to ozone concentrations that were 1.7-fold higher than ambient for one growing season under open-field conditions. Ambient air was used as the control treatment. In the ozone-sensitive clone B, there was an initial stimulation of leaf area growth in response to the ozone treatment, but further ozone exposure caused reductions in leaf and stem biomass growth, Rubisco and chlorophyll a contents, net photosynthesis, water use efficiency and chloroplast size. It also caused an alteration in chloroplast shape and injury to thylakoid membranes. In the ozone-tolerant clone C, ozone fumigation did not affect growth rate, and there were no consistent changes in chlorophyll content, photosynthesis or water use efficiency. There were also fewer ultrastructural abnormalities in the chloroplasts of clone C than of clone B. Based on the observed biochemical, physiological and structural changes in chloroplasts of clone B in response to low concentrations of ozone, we conclude that the increasing concentration of tropospheric ozone represents a risk to natural birch populations.

Growth and biomass allocation of symptomatic and asymptomatic Populustremuloides clones in response to seasonal ozone exposures

The effect of single-season ozone exposures on growth and biomass production of numerous trembling aspen (Populustremuloides Michx.) clones was studied, using open-top chambers, in the Upper Peninsula of Michigan. All clones studied had previously been ranked according to foliar sensitivity to short-term, high-dose ozone exposures. For this study, comparisons were made between plants grown in charcoal-filtered, nonfiltered, and ozone-added (consisting of 80 ppb ozone for 6 h per day, 3 days per week) chambers and in open-air plots. For the plants grown in ozone-added chambers, as compared with those grown in the charcoal-filtered air, stem biomass decreases were 0% for the 18 ozone-tolerant clones and 46.4% for the 18 ozone-sensitive clones, in 1988. In 1989, they were 5% for the tolerant clones and 74% for the sensitive clones. Thus, our results suggest that ozone can have either a negative effect on growth or no effect, depending on the clones tested. However, it is clear from this study that ozone consistently affected biomass production in sensitive clones in a negative manner. These results strengthen the case for ozone being a strong selective force, even at relatively low total doses.

Natural selection for ozone tolerance in Populustremuloides: an evaluation of nationwide trends

Relative ozone sensitivity was evaluated among populations of Populustremuloides Michx. from 15 locations, mostly in national parks, that spanned the United States. Seven to 15 clones were selected from each population, greenhouse grown, fumigated with 150 ppb ozone for 6 h, and evaluated for visible injury. Differences among populations were statistically significant, with the most tolerant populations being obtained from the West Coast, the northeast, and the industrialized portions of the Great Lakes. Populations sampled from areas that failed to achieve the National Ambient Air Quality Standard for ozone had significantly less injury than populations from areas that achieved this standard. There was a significant negative correlation between the amount of injury and maximum daily ozone averages at localities where the populations were collected. There were also significant correlations between the amount of injury and two climatic variables, annual precipitation and minimum temperature. Differences among clones within populations were highly significant, and clonal variance was negatively correlated with maximum daily ozone average for the area where the populations were collected. These findings support our hypothesis that ambient levels of ozone may be eliminating ozone-sensitive clones from natural populations of this species, but indicate that climatic variables play a role as well.

Natural selection for ozone tolerance in Populustremuloides: field verification

Field plantings of Populustremuloides Michx. cuttings in two areas with elevated levels of ambient ozone (New York and Michigan) were evaluated for differences in relative ozone sensitivity among five natural populations from areas that differed in air quality. At least fivefold differences in leaf injury were measured among population means in each planting, and the means were negatively correlated with ozone concentrations for each area. The relative sensitivity rankings among populations and among clones within populations agreed closely with rankings derived from laboratory fumigations of the same plant material. The hypothesis that ozone-sensitive clones of this species are less common in relatively polluted parts than they are in pristine parts of the eastern United States is therefore further supported by field verification of laboratory results. Data from a 10-year-old aspen planting in New York showing impaired growth of ozone-sensitive clones suggest that sensitive individuals can be eliminated from populations by the mechanism of competition.

Growth and premature leaf fall in American aspen as bioindications for ozone

Several clones of the American aspen ( Populus tremuloides Michx.) differing in ozone sensitivity were fumigated continuously for 105 days with different ozone concentrations, producing different ozone doses. The suitability of the clones as possible replacements for tobacco as bioindicators in forested areas was tested. The life span of leaves of the ozone-sensitive clones was distinctly reduced by ozone. The difference to controls was significant even in the relatively clean air at Birmensdorf. Growth was diminished, too, but the decrease in height was much less pronounced than that in weight. In sensitive clones an after-effect was detectable, since the weight of new shoots (grown from reserves) decreased with increasing ozone stress in the previous year. Since the tolerant clone grew less, it is—at first sight—of decreased economic interest from the point of view of rapid productivity. In the long run, however, it might be economically interesting because it may be able to maintain productivity (although at a lower level) due to better competitive strength.