Eva J. Pell

Investigations into the Role of the Plastidial Peptide Methionine Sulfoxide Reductase in Response to Oxidative Stress in Arabidopsis

Peptidyl Met residues are readily oxidized by reactive oxygen species to form Met sulfoxide. The enzyme peptide Met sulfoxide reductase (PMSR) catalyzes the reduction of Met sulfoxides back to Met. In doing so, PMSR is proposed to act as a last-chance antioxidant, repairing proteins damaged from oxidative stress. To assess the role of this enzyme in plants, we generated multiple transgenic lines with altered expression levels of the plastid form of PMSR (PMSR4). In transgenic plants, PMSR4 expression ranged from 95% to 40% (antisense) and more than 600% (overexpressing lines) of wild-type plants. Under optimal growing conditions, there is no effect of the transgene on the phenotype of the plants. When exposed to different oxidative stress conditions—methyl viologen, ozone, and high light—differences were observed in the rate of photosynthesis, the maximum quantum yield (Fv/Fm ratio), and the Met sulfoxide content of the isolated chloroplast. Plants that overexpressed PMSR4 were more resistant to oxidative damage localized in the chloroplast, and plants that underexpressed PMSR4 were more susceptible. The Met sulfoxide levels in proteins of the soluble fraction of chloroplasts were increased by methyl viologen and ozone, but not by high-light treatment. Under stress conditions, the overexpression of PMSR4 lowered the sulfoxide content and underexpression resulted in an overall increase in content.

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.

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.

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 enzymes 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.

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.

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.

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.

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.

Role of Carbon Dioxide in Modifying the Plant Response to Ozone

Publisher Summary This chapter discusses the potential responses of plants to interacting effects of Ozone (O 3 ) and elevated Carbon dioxide (CO 2 ). Both CO 2 and O 3 have direct effects on the growth and vitality of plants. Carbon dioxide is a limiting factor to photosynthesis in C3 plants. Therefore, increases in ambient CO 2 concentrations can result in increased rates of photosynthesis and stimulate growth and biomass production. O 3 is potentially damaging for plants and, therefore, may counteract or prevent positive responses to enhanced atmospheric CO 2 concentrations. With respect to carbon fluxes, the interactive effects of elevated CO 2 and O 3 on growth and vitality of plants are important because vegetation is a major sink for CO 2 . Its sink strength, particularly the maintenance of enhanced growth under elevated CO 2 concentrations, depends on a range of edaphic, climatic, and species-inherent factors. Further limitations to the sink strength of the vegetation are imposed by man-made environmental constraints, including O 3 as an important air pollutant. Primary reactions of O 3 in plants initially cause subtle biochemical changes, which may initiate a sequence of reactions leading to reductions in photosynthesis, changes in carbon allocation patterns, symptoms of visible injury on leaves, accelerated senescence, reduced growth, and loss of yield.