Ozone inhibits guard cell K+ channels implicated in stomatal opening.

Ozone (O3) deleteriously affects organisms ranging from humans to crop plants, yet little is understood regarding the underlying mechanisms. In plants, O3 decreases CO2 assimilation, but whether this could result from direct O3 action on guard cells remained unknown. Potassium flux causes osmotically driven changes in guard cell volume that regulate apertures of associated microscopic pores through which CO2 is supplied to the photosynthetic mesophyll tissue. We show in Vicia faba that O3 inhibits (i) guard cell K+ channels that mediate K+ uptake that drives stomatal opening; (ii) stomatal opening in isolated epidermes; and (iii) stomatal opening in leaves, such that CO2 assimilation is reduced without direct effects of O3 on photosynthetic capacity. Direct O3 effects on guard cells may have ecological and agronomic implications for plant productivity and for response to other environmental stressors including drought.

Senescence-associated gene expression during ozone-induced leaf senescence in Arabidopsis.

The expression patterns of senescence-related genes were determined during ozone (O(3)) exposure in Arabidopsis. Rosettes were treated with 0.15 microL L(-1) O(3) for 6 h d(-1) for 14 d. O(3)-treated leaves began to yellow after 10 d of exposure, whereas yellowing was not apparent in control leaves until d 14. Transcript levels for eight of 12 senescence related genes characterized showed induction by O(3). SAG13 (senescence-associated gene), SAG21, ERD1 (early responsive to dehydration), and BCB (blue copper-binding protein) were induced within 2 to 4 d of O(3) treatment; SAG18, SAG20, and ACS6 (ACC synthase) were induced within 4 to 6 d; and CCH (copper chaperone) was induced within 6 to 8 d. In contrast, levels of photosynthetic gene transcripts, rbcS (small subunit of Rubisco) and cab (chlorophyll a/b-binding protein), declined after 6 d. Other markers of natural senescence, SAG12, SAG19, MT1 (metallothionein), and Atgsr2 (glutamine synthetase), did not show enhanced transcript accumulation. When SAG12 promoter-GUS (beta-glucuronidase) and SAG13 promoter-GUS transgenic plants were treated with O(3), GUS activity was induced in SAG13-GUS plants after 2 d but was not detected in SAG12-GUS plants. SAG13 promoter-driven GUS activity was located throughout O(3)-treated leaves, whereas control leaves generally showed activity along the margins. The acceleration of leaf senescence induced by O(3) is a regulated event involving many genes associated with natural senescence.

Overproduction of Ascorbate Peroxidase in the Tobacco Chloroplast Does Not Provide Protection against Ozone.

Transgenic tobacco (Nicotiana tabacum cv Bel W3) plants were used to test the hypothesis that protection from O3 injury could be conferred by overproduction of ascorbate peroxidase (APX) in the chloroplast. The 10-fold increase in soluble APX activity in the chloroplast was expected to alleviate an implied increase in oxidative potential and prevent damage caused by O3. Three different O3 exposure experiments (one acute and two chronic) with two replicates each were conducted. APX activity in nontransgenic plants increased in response to chronic O3 exposure. However, most responses to O3 were similar between transgenic and nontransgenic plants. These included reductions in net photosynthesis and stomatal conductance, increases in ethylene emission and visible injury, and a decline in the level of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase mRNA transcripts observed in response to the air pollutant in the acute and/or chronic experiments. No O3-induced effect on ribulose-1,5-biphosphate carboxylase/oxygenase quantity was observed in the chronic experiments. O3 did not induce acceleration of senescence, as expected from studies with most other species; rather, the tobacco plants rapidly developed necrotic lesions. Thus, overproduction of APX in the chloroplast did not protect this cultivar of tobacco from O3.

Sequential expression of two 1-aminocyclopropane-1-carboxylate synthase genes in response to biotic and abiotic stresses in potato (Solanum tuberosum L.) leaves.

Plants produce ethylene in response to many biotic and abiotic stresses. In response to ozone the foliage of potato plants sequentially expressed two ACC synthase genes (ST-ACS4, ST-ACS5). The same expression pattern of the two genes also occurred in response to Cu2+ and infection with Alternaria solani. ST-ACS5 expression increases very rapidly reaching a maximum earlier than ST-ACS4 transcripts, after which ST-ACS5 expression declines. ST-ACS4 expression increases at a slower rate and reaches its maximum after ST-ACS5. The sequential nature of expression argues that the two genes have different signal transduction and gene regulatory mechanisms.

Mechanisms of fungicide resistance in phytopathogenic fungi.

The disciplines traditionally used to investigate the mode of action of fungicides have been biochemistry and physiology. Over the past decade, classical and molecular genetics have been brought to bear on this problem with increasing success. Recently, genetic studies of fungicide resistance have led to advances in our understanding of the site of action of chemicals active against plant pathogens and, in some cases, to an appreciation of additional mechanisms of resistance to fungicide action.

Effects of ethylenediurea (EDU) on ozone-induced acceleration of foliar senescence in potato (Solanum tuberosum L.).

Experiments were conducted to examine the effects of the anti-ozonant ethylenediurea (EDU) and chronic ozone (O3) exposure on leaf physiology and senescence in an O3-sensitive potato cultivar (Solanum tuberosum L. cv. Norland). A dose-response experiment showed that an EDU concentration of 15 mg l(-1) soil (given as a soil drench) provided complete protection from accelerated foliar senescence induced by exposure to 0.1 microl l(-1) O3 for 5 h day(-1) for 11 days. EDU doses of 45 and 75 mg active ingredient l(-1) soil also gave protection but were associated with symptoms of toxicity and delayed senescence. In further experiments, plants were given 0 or 15 mg EDU l(-1) soil and exposed to clean air or 0.1 microl l(-1) O3 for 5 h day(-1) for 14 days. Chronic O3 exposure in the absence of EDU resulted in accelerated foliar senescence, characterized by early declines in net photosynthesis and Rubisco quantity in O3-treated plants relative to controls. EDU in the presence of O3 gave complete protection against symptoms of accelerated senescence. Senescence was not delayed in plants that received EDU in the absence of O3, and no symptoms of EDU toxicity were evident. The results suggest that EDU-induced tolerance to O3 was not based on 'anti-senescent' properties of this anti-ozonant.

Ozone-Induced Ethylene Emission Accelerates the Loss of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Nuclear-Encoded mRNAs in Senescing Potato Leaves.

The relationships among O3-induced accelerated senescence, induction of ethylene, and changes in specific mRNA and protein levels were investigated in potato (Solanum tuberosum L. cv Norland) plants. When plants were exposed to 0.08 [mu]L L-1 O3 for 5 h d-1, steady-state levels of rbcS mRNA declined at least 5-fold in expanding leaves after 3 d of O3 exposure and ethylene levels increased 6- to 10-fold. The expression of OIP-1, a 1-aminocyclopropane-1-carboxylate synthase cDNA from potato, correlated with increased production of ethylene and decreased levels of rbcS mRNA in foliage of plants treated with O3. In plants exposed to 0.30 [mu]L L-1 O3 for 4 h, rbcS transcript levels were reduced 4-fold, whereas nuclear run-on experiments revealed that rbcS transcription declined an average of 50%. The loss of rbcS mRNA may be due, in part, to posttranscriptional regulation. The levels of transcripts for other chloroplast proteins, glyceraldehyde-3-phosphate dehydrogenase, and a photosystem II chlorophyll a/b-binding protein decreased in O3-treated plants, in parallel with the decrease in rbcS mRNA. The steady-state mRNA level of a cytosolic glyceral-dehyde-3-phosphate dehydrogenase increased in O3-treated plants. The induction of ethylene and changes in transcript levels preceded visible leaf damage and decreases in ribulose-1,5-bisphosphate carboxylase/oxygenase protein levels.

A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis.

A mutant of Ustilago maydis (VR43) with single-gene resistance to the dicarboximide fungicide vinclozolin was previously isolated and characterized. A genomic library was constructed, and an 8.7-kb resistance-conferring fragment was isolated by sib selection. Sequencing this fragment, we identified an 1,218-bp open reading frame, which, if disrupted by deletion, no longer confers resistance. Analyses of the data in GenBank demonstrated a high degree of homology between the product of the 1,218-bp open reading frame, referred to as the adr-1 gene, and Ser (Thr) protein kinases.

Molecular cloning of an ozone-induced 1-aminocyclopropane-1-carboxylate synthase cDNA and its relationship with a loss of rbcS in potato (Solanum tuberosum L.) plants.

Acute or chronic exposure of potato plants to ozone (O3) induces ethylene production. We isolated a 1586 bp cDNA (pOIP-1) encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase from a cDNA library constructed with mRNA extracted from O3-treated leaves. The clone has a 1365 bp open reading frame and a 221 bp trailing sequence. The active site found in all ACC synthases and 11 of the 12 amino acid residues conserved in aminotransferases are found in pOIP-1. Northern analysis showed that the mRNA encoding ACC synthase was detectable 1 h after the onset of O3 exposure, and the message increased over time as did ethylene production. Concurrent with the increased ACC synthase mRNA was a decrease in the message for the Rubisco small subunit (rbcS) with no change in the large subunit (rbcL). When the plants were treated with aminooxyacetic acid (AOA), both ethylene production and level of ACC synthase transcript were inhibited. The decline in rbcS was also inhibited by AOA suggesting a correlation between ethylene production and loss of rbcS. Based on nuclear run-on studies it appears that the increase in ACC synthase mRNA may result from O3-induced transcriptional activity.

Biochemical and molecular basis for impairment of photosynthetic potential.

Ozone induces reductions in net photosynthesis in a large number of plant species. A primary mechanism by which photosynthesis is reduced is through impact on carbon dioxide fixation. Ozone induces loss in Rubisco activity associated with loss in concentration of the protein. Evidence is presented that ozone may induce oxidative modification of Rubisco leading to subsequent proteolysis. In addition, plants exposed to ozone sustain reduction in rbcS, the mRNA for the small subunit of Rubisco. This loss in rbcS mRNA may lead to a reduced potential for synthesis of the protein. The regulation of O3-induced loss of Rubisco, and implications of the decline in this protein in relation to accelerated senescence are discussed.

O3 -induced degradation of Rubisco protein and loss of Rubisco mRNA in relation to leaf age in Solanum tuberosum L.

The effect of ozone (O3 ) on Rubisco degradation was investigated by dark incubation of potato plants (Solanum tuberosum L. cv. Norland) following exposure to charcoal-filtered air or 0.30µl l-1 O3 for 6 h. Rubisco small subunit (rbcS) mRNA levels declined dramatically in control foliage within 10.16 h of dark incubation; thus declines in Rubisco protein following a 48-h dark period were ascribed to proteolytic degradation. Foliage sampled from the 4th and 6th leaves from the apex, designated immature and mature, respectively, showed no effect of O3 on Rubisco protein content immediately following the exposure. However, the decline in Rubisco quantity during the dark incubation was significantly enhanced by prior treatment with O3 in the mature leaves. The immature leaves sustained a similar decline in Rubisco quantity in both O2 and control treatments. O3 had a significant effect on the relative quantity of rbcS mRNA in the immature leaves (sampled immediately following the O3 exposure). Levels of rbcS mRNA in mature leaves and Rubisco large subunit (rbcL) mRNA in both leaf ages were not significantly affected by O3 , There was no effect of O3 , on Rubisco quantity in immature or mature leave's of plants maintained in the greenhouse under a 16 h photoperiod far 48 h following the exposure. Thus the effect of short-term O3 exposure on processes affecting Rubisco synthesis and degradation may have been transient, and a more prolonged exposure would he necessary to effect a decline in Rubisco protein quantity in plants grown under a 16 h photoperiod. We concluded that O3 caused enhanced degradation of Rubisco in mature leaves of plants induced to senesce by dark incubation following O3 exposures. The potential for O3 -induced reduction in synthesis of Rubisco also exists.

Modification of Rubisco and Altered Proteolytic Activity in O3-Stressed Hybrid Poplar (Populus maximowizii x trichocarpa).

Exposing hybrid poplar (Populus maximowizii x trichocarpa) plants to ozone (O3) resulted in an acceleration of the visual symptoms of senescence and a decrease in the activity and quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Whole plants, crude leaf extracts, and isolated intact chloroplasts of hybrid poplar clone 245 were used to test the hypothesis that O3-induced structural modifications of Rubisco affect the activity of this key photosynthetic enzyme. Proteolytic activity, per se, could not account for losses in Rubisco; acidic and alkaline protease activities declined or were unaffected in foliage of O3-treated poplar saplings. In vitro treatment of leaf extracts with O3 decreased total Rubisco activity and binding of the enzyme's transition-state analog, 2-carboxyarabinitol bisphosphate. Additionally, O3 increased the loss of Rubisco large subunit (LSU) when extracts were incubated at 37[deg]C. Treatment of isolated intact chloroplasts with O3 accelerated both the loss of the 55-kD Rubisco LSU and the accumulation of Rubisco LSU aggregates, as visualized by immunoblotting. The time-dependent modification in Rubisco structure was the primary response of the isolated organelles to O3 treatment, with little proteolytic degradation of the LSU detected.

Response of radish to multiple stresses: I. Physiological and growth responses to changes in ozone and nitrogen.

Experiments were conducted to determine the impact of nitrogen and ozone (O3 ) stress on the growth of domestic radish Raphanus sativus L. cv. Cherry Belle. Plants were grown in field chambers with sub-, optimal and supra-optimal levels of nitrogenous fertilizer. Chamber air was either charcoal-filtered, or supplemented with one of two levels of O3 . The highest O3 treatment resulted in significant reduction in weight of hypocotyls and roots while elevated nitrogen treatments resulted in increased weight of all plant parts. Ozone did not affect the weight of plant foliage at any nitrogen level. Plants grown with lower levels of nitrogen had less leaf biomass but the tissue accounted for a greater percentage total weight than did the foliage of higher nitrogen treatments. Relative growth rate of whole plants was not affected by O3 or nitrogen treatments reflecting compensation in response to both stresses. Ozone-induced depression in biomass was observed in O3 -treated plants grown with higher nitrogen supply but not in those grown with limiting nitrogen. This observation could reflect compensation at the lower levels of nitrogen supply or inability to detect changes in biomass due to reduced weights of plants grown at the lowest nitrogen supply. The dry weight ratio of sink organs (hypocotyl plus root)/shoot was significantly correlated with the total non-structural carbohydrate (TNC) content of these organs, regardless of treatment. Initially, O3 induced a significant decrease and nitrogen an increase in percent TNC of sink organs. At later sampling times, plants adjusted to stress as effects on percent TNC were no longer evident.

Decline of activity and quantity of ribulose bisphosphate carboxylase/oxygenase and net photosynthesis in ozone-treated potato foliage.

The effect of ozone (O(3)) on ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity and quantity and net photosynthesis in greenhouse-grown Solanum tuberosum L. cv ;Norland' foliage was studied in relation to oxidant-induced premature senescence. Plants, 26 days old, were exposed to 0.06 to 0.08 microliters per liter O(3) from 1000 to 1600 hours for 4 days in a controlled environment chamber. On day 5, plants were exposed to a 6-hour simulated inversion in which O(3) peaked at 0.12 microliters per liter. Net photosynthesis declined in response to O(3) but recovered to near control levels 3 days after the exposure ended. Rubisco activity and quantity in control potato foliage increased and then decreased during the 12-day interval of the study. In some experiments foliage studied was physiologically mature and Rubisco activity had peaked when O(3) exposure commenced. In those cases, O(3) accelerated the decline in Rubisco activity. When less mature foliage was treated with O(3), the leaves never achieved the maximal level of Rubisco activity observed in control foliage and also exhibited more rapid decline in initial and total activity. Percent activation of Rubisco (initial/total activity) was not affected significantly by treatment. Quantity of Rubisco decreased in concert with activity. The decrease in activities is most likely due to a decrease in available protein rather than a decrease in the percentage of Rubisco activated in vivo. The reduction in the quantity of Rubisco, an important foliar storage protein, could contribute to premature senescence associated with toxicity of this air pollutant.

Qualitative and quantitative effects of ozone and/or sulfur dioxide on field-grown potato plants.

Solanum tuberosum L. cv Norchip plants were grown in open-top chambers in the summer of 1986. Plants were treated with charcoal-filtered air, nonfiltered air, or nonfiltered air supplemented with 33, 66, or 99% of the ambient ozone (O3) concentrations from 1000 to 2000 h eastern daylight time daily. In addition, plants received charcoal-filtered air plus 0, 0.15 (393 microg m(-3)), 0.34 (891 microg m(-3)), or 0.61 (1598 microg m(-3)) ppm sulfur dioxide (SO2) from 0900 to 1200 h once every 14 d for a total of four treatments. Ozone induced a linear reduction in number and weight of Grade One (> 6.35-cm diameter) potato tubers and in total weight of tubers. Ozone also induced linear reductions in the percentage of dry matter of tubers and linear decreases in glucose and fructose content of Grade One tubers. Sulfur dioxide induced a stimulation and then decline of the number, percentage of dry matter, and sucrose content of Grade One tubers. The SO2 response best fit a quadratic curve. No O3 x SO2 interactions were detected for any of the yield or quality functions measured.

Ozone-Induced Reduction in Quantity of Ribulose-1,5-bisphosphate Carboxylase in Alfalfa Foliage.

The concentration of ribulose-1,5-bisphosphate carboxylase was measured in the foliage of ;Moapa 69,' ;Ladak,' and ;Vernal' cultivars of alfalfa (Medicago sativa L.) after plants were exposed to 0.25 or 0.30 microliters per liter ozone for 2 hours. The quantity of ribulose-1,5-bisphosphate carboxylase decreased 80%, 68%, and 36% in leaves which did not exhibit ozone symptoms and to undetectable levels, 99% and 79% in symptomatic foliage for each cultivar, respectively.

Changes in respiration, photosynthesis, adenosine 5'-triphosphate, and total adenylate content of ozonated pinto bean foliage as they relate to symptom expression.

The effect of 0.25 to 0.30 microliter per liter ozone on photosynthesis and respiration and on the ATP and total adenylate content of the primary leaves of pinto beans (Phaseolus vulgaris L.) was examined. Changes in these parameters over a 72-hour time period were correlated with the development of symptoms of ozone toxicity. Toxicity symptoms normally appeared within 24 hours. The content of ATP and total adenylates increased immediately following a 3-hour exposure to ozone. Photosynthesis was depressed initially, but returned to normal within 24 hours. Respiration was not always altered initially, but it was significantly stimulated within 24 hours. We interpret the results to mean that the changes in adenylate content and photosynthesis are early events in the initiation of ozone damage and that the change in respiration is a consequence rather than a cause of cellular injury.