Ozone effects on plants in natural ecosystems.

Tropospheric ozone (O3 ) is an important stressor in natural ecosystems, with well-documented impacts on soils, biota and ecological processes. The effects of O3 on individual plants and processes scale up through the ecosystem through effects on carbon, nutrient and hydrologic dynamics. Ozone effects on individual species and their associated microflora and fauna cascade through the ecosystem to the landscape level. Systematic injury surveys demonstrate that foliar injury occurs on sensitive species throughout the globe. However, deleterious impacts on plant carbon, water and nutrient balance can also occur without visible injury. Because sensitivity to O3 may follow coarse physiognomic plant classes (in general, herbaceous crops are more sensitive than deciduous woody plants, grasses and conifers), the task still remains to use stomatal O3 uptake to assess class and species' sensitivity. Investigations of the radial growth of mature trees, in combination with data from many controlled studies with seedlings, suggest that ambient O3 reduces growth of mature trees in some locations. Models based on tree physiology and forest stand dynamics suggest that modest effects of O3 on growth may accumulate over time, other stresses (prolonged drought, excess nitrogen deposition) may exacerbate the direct effects of O3 on tree growth, and competitive interactions among species may be altered. Ozone exposure over decades may be altering the species composition of forests currently, and as fossil fuel combustion products generate more O3 than deteriorates in the atmosphere, into the future as well.

Demonstration of a diel trend in sensitivity of Gossypium to ozone: a step toward relating O3 injury to exposure or flux.

Plant injury by ozone (O3) occurs in three stages, O3 entrance through stomata, overcoming defences, and attack on bioreceptors. Concentration, deposition, and uptake of O3 are accessible by observation and modelling, while injury can be assessed visually or through remote sensing. However, the relationship between O3 metrics and injury is confounded by variation in sensitivity to O3. Sensitivity weighting parameters have previously been assigned to different plant functional types and growth stages, or by differentially weighting O3 concentrations, but diel and seasonal variability have not been addressed. Here a plant sensitivity parameter (S) is introduced, relating injury to O3 dose (uptake) using three independent injury endpoints in the crop species, Pima cotton (Gossypium barbadense). The diel variability of S was determined by assessment at 2h intervals. Pulses of O3 (15 min) were used to assess passive (constitutive) defence mechanisms and dose was used rather than concentration to avoid genetic or environmental effects on stomatal regulation. A clear diel trend in S was apparent, with maximal sensitivity in mid-afternoon, not closely related to gas exchange, whole leaf ascorbate, or total antioxidant capacity. This physiologically based sensitivity parameter provides a novel weighting factor to improve modelled relationships between either flux or exposure to O3, and O3 impacts. This represents a substantial improvement over concentration- or phenology-based weighting factors currently in use. Future research will be required to characterize the variability and metabolic drivers of diel changes in S, and the performance of this parameter in prediction of O3 injury.

Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field.

Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests. Although consumption of NaPI dramatically reduced the growth and development of a major insect pest, Helicoverpa punctigera, we discovered that surviving larvae had high levels of chymotrypsin activity resistant to inhibition by NaPI. We found a potato type I inhibitor, Solanum tuberosum potato type I inhibitor (StPin1A), was a strong inhibitor of the NaPI-resistant chymotrypsin activity. The combined inhibitory effect of NaPI and StPin1A on H. armigera larval growth in the laboratory was reflected in the increased yield of cotton bolls in field trials of transgenic plants expressing both inhibitors. Better crop protection thus is achieved using combinations of inhibitors in which one class of proteinase inhibitor is used to match the genetic capacity of an insect to adapt to a second class of proteinase inhibitor.

Comparison of calculated and measured foliar O3 flux in crop and forest species.

We designed a new gas exchange system that concurrently measures foliar H2O, O3, and CO2 flux (HOC flux system) while delivering known O3 concentrations. Stomatal responses of three species were tested: snapbean, and seedlings of California black oak (deciduous broadleaf) and blue oak (evergreen broadleaf). Acute O3 exposure (120-250 ppb over an hour) was applied under moderate light and low vapor pressure deficits during near steady state conditions. The rate of stomatal closure was measured when the whole plant was placed in the dark. An adjacent leaf on each plant was also concurrently measured in an O3-free cuvette. Under some conditions, direct measurements and calculated foliar O3 flux were within the same order of magnitude; however, endogenously low gs or O3 exposure-induced depression of gs resulted in an overestimation of calculated O3 fluxes compared with measured O3 fluxes. Sluggish stomata in response to light extinction with concurrent O3 exposure, and incomplete stomatal closure likewise underestimated measured O3 flux.

Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests.

The ornamental tobacco (Nicotiana alata) produces one 6-kDa chymotrypsin inhibitor and four 6-kDa trypsin inhibitors from a single 40.3-kDa precursor protein. Three different approaches have been used to assess the potential of these proteinase inhibitors (PIs) in insect control. The first was an in-vitro approach in which all five inhibitors, the single chymotrypsin inhibitor or three of the four trypsin inhibitors were tested for their ability to inhibit gut protease activity in insects from four orders. The second approach was to incorporate the N. alata PIs in the artificial diet of the native budworm (Helicoverpa punctigera) and the black field cricket (Teleogryllus commodus). H. punctigera larvae and T. commodus nymphs had a significant (P<0.01) reduction in growth after ingestion of the PI and were more lethargic than insects on the control diet. Several of the H. punctigera larvae also failed to complete moulting at the third or fourth instar. The third approach was to express the N. alata PIs in transgenic tobacco under the control of the 35S CaMV promoter. When H. punctigera larvae were fed tobacco leaves expressing the N. alata PIs at 0.2% soluble protein, significant (P<0.01) differences in mortality and/or growth rate were observed.

Structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of Nicotiana alata.

The three-dimensional structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of the ornamental tobacco Nicotiana alata have been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The proteins, T1-T4, are proteolytically cleaved from a 40.3-kDa precursor protein, NA-proPI, together with a chymotrypsin inhibitor, C1, the structure of which was reported recently [Nielsen, K.J., Heath, R.L., Anderson, M.A., & Craik, D.J. (1994) J. Mol. Biol. 242, 231-243]. Each of the proteinase inhibitors comprises 53 amino acids, including 8 cysteine residues which are linked to form 4 disulfide bridges. The proteins have a high degree of sequence identity and differ mainly in residues around the putative reactive sites. The structure of T1 was determined using a set of 533 interproton distance restraints derived from NOESY spectra, combined with 33 dihedral restraints derived from 3JNH-H alpha coupling constants and 16 hydrogen bonds. The structures of the remaining inhibitors (T2-T4) were deduced to be almost identical to T1, on the basis of their similar chemical shifts and 2D spectra. The current study demonstrates that the structures of the trypsin inhibitors (T1-T4) are similar to that previously found for the chymotrypsin inhibitor, C1. Despite differences in sequence, there is conservation in backbone geometry between the reactive site loops of the two classes of inhibitors. From this, it is clear that the nature of the side chain on the primary binding residue, rather than the backbone fold, is the main determinant of the enzyme specificities of these proteinase inhibitors.

Prosodic characteristics of speech pre- and post-right hemisphere stroke.

Case-control studies have shown right hemisphere specialization in the production of intonation in speech. We examined spontaneous prosody in audiotapes of interviews with a 77-year-old right-handed woman recorded 6 months before and 6 weeks after she suffered a stroke affecting the right frontotemporo-parietal regions and the right basal ganglia. Post-stroke, the patient had a normal Mini-Mental Status Examination Score of 29, hemispatial neglect, and impairments in the comprehension of facial expression and prosody. Self-rated mood was within normal limits. We compared beginning, peak, and ending fundamental frequencies (fo) in breath groups, the timing of these fo changes, rate of speech, pause duration, and breath-group duration. We found that post-stroke, the patient had a more restricted fo contour, no changes in the timing of peak fo, an increased rate of speech, less variability in pause duration, and no changes in breath-group duration.

Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata.

A gene encoding a 40.3-kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, Nicotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N-terminal sequencing, electrospray mass spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease-sensitive linker region is selectively cleaved by the endoproteinases Asp-N, Glu-C and Lys-C to release fully active approximately 6-kDa PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.

The three-dimensional solution structure by 1H NMR of a 6-kDa proteinase inhibitor isolated from the stigma of Nicotiana alata.

The three-dimensional structure and disulfide connectivities of a 6-kDa protein isolated from the stigma of the ornamental tobacco Nicotiana alata has been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The protein, termed C1, is a chymotrypsin inhibitor and is one of five homologous proteinase inhibitors that are proteolytically cleaved from a 40.3-kDa precursor protein. The other four proteinase inhibitors (T1 to T4) contain reactive sites for trypsin. The three-dimensional structure of C1 is generally well defined and contains a triple stranded beta-sheet as the dominant secondary structural feature. Several turns and a short region of 3(10) helix are also present. The putative chymotrypsin reactive site is present on an exposed loop which is less defined than the rest of the protein. The overall shape of C1 is disc-like and the N and C termini are exposed, supporting the proposal that this protein results from post-translational processing of the 40.3-kDa precursor protein.

Possible mechanisms for the inhibition of photosynthesis by ozone.

Tropospheric ozone produced by industrial civilization is widespread. Although the levels are not clearly life threatening, they do have the potential to inhibit normal plant productivity, thought to be by an inhibition of photosynthesis. While the mechanism for this inhibition is not yet clear, there are several hypotheses for its cause. It is unlikely that ozone can penetrate the cell membrane unreacted; therefore, reactions at the plasma membrane either causing general ionic and metabolic disturbance within the cell or causing the production of unidentified toxic products must ultimately produce the alterations within the chloroplasts. While model systems, such as individual biochemicals, isolated chloroplasts, and algae, can give some understanding of possible reactions, they cannot provide the full story. One continuing controversy revolves about the role of stomata in the inhibition process-they play an important role, but the full interaction between stomatal closure and inhibition of photosynthesis has not yet emerged. In order to reach a political compromise on air quality standards, we need to have a good understanding of the fundamental mechanisms by which ozone causes any decline in plant productivity.

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.

Fluorescence Quenching in the Varied Photosynthetic Modes of Portulacaria afra (L.) Jacq.

The kinetics of chlorophyll fluorescence were measured in Portulacaria afra (L.) Jacq. when the plants were functioning in either Crassulacean acid metabolism (CAM) or C(3)/CAM cycling (called cycling) modes, as determined by fluctuation in titratable acidity and gas exchange properties. Cycling plants showed primarily daytime CO(2) uptake typical of C(3) plants, but with a slight diurnal acid fluctuation, whereas CAM plants showed nocturnal CO(2) uptake, daytime stomatal closure, and a large diurnal acid fluctuation. Results from fluorescence measurements indicated no significant differences in photochemical quenching between cycling and CAM plants; however, sizable differences were detected in nonphoto-chemical quenching (q(n)), with the largest differences being observed during the middle of the day. Cycling plants had lower q(n) than CAM plants, indicating altered photosynthetic regulation processes. This q(n) difference was believed to be related to reduced internal CO(2) concentration in the CAM plants because of daytime stomatal closure and reduced deacidification rates in the late afternoon when most of the malic acid has been utilized. Experimentally, higher external CO(2) given to plants in the CAM mode resulted in a decline in q(n) in comparison to that measured in plants in the cycling mode. No changes were observed in photochemical quenching when CO(2) was added.

Ca transport in membrane vesicles from pinto bean leaves and its alteration after ozone exposure.

The influence of ozone on Ca(2+) transport in plant membranes from pinto bean (Phaseolus vulgaris L. var Pinto) leaves was investigated in vitro by means of a filtration method using purified vesicles. Two transport mechanisms located at the plasma membrane are involved in a response to ozone: (a) passive Ca(2+) influx into the cell and (b) active Ca(2+) efflux driven by an ATP-dependent system, which has two components: a primary Ca(2+) transport directly linked to ATP which is partially activated by calmodulin and a H(+)/Ca(2+) antiport coupled to activity of a H(+)-ATPase. The passive Ca(2+) permeability is increased by ozone. A triangular pulse of ozone stimulates a higher influx of Ca(2+) than does a square wave, even though the total dose was the same (0.6 microliter per liter x hour). Leaves exposed to a square wave did not exhibit visible injury and were still able to recover from oxidant stress by activation of calmodulin-dependent Ca(2+) extrusion mechanisms. On the other hand, leaves exposed to a triangular wave of ozone, exhibit visible injury and lost the ability of extruding Ca(2+) out of the cell.

Alteration of Extracellular Enzymes in Pinto Bean Leaves upon Exposure to Air Pollutants, Ozone and Sulfur Dioxide.

Diamine oxidase and peroxidase, associated with the wall in pinto bean (Phaseolus vulgaris L. var Pinto) leaves, can be washed out by vacuum infiltration and assayed without grinding the leaf. The diamine oxidase activity is inhibited in vivo by exposure of the plants to ozone (dose of 0.6 microliters per liter x hour), whereas the peroxidase activity associated with the wall space is stimulated. This dose does not cause obvious necrosis or chlorosis of the leaf. These alterations are greater when the dose of ozone exposure is given as a triangular pulse (a slow rise to a peak of 0.24 microliters per liter followed by a slow fall) compared to that given as a constant square wave pulse of 0.15 microliters per liter for the same 4 hour period. Exposure of the plants to sulfur dioxide (at a concentration of 0.4 microliters per liter for 4 hours) does not result in any change in the diamine oxidase or peroxidase activities, yet the total sulfhydryl content of the leaf is increased, demonstrating the entry of sulfur dioxide. These two pollutants, with different chemical reactivities, affect the activities of the extracellular enzymes in different manners. In the case of ozone exposure, the inhibition of extracellular diamine oxidase could profoundly alter the movements of polyamines from cell to cell.

Synthesis of human rotavirus polypeptides in cell culture.

The replication strategy of a human serotype 1 rotavirus, adapted to rapid growth in CV-1 cells, was investigated. A single cycle growth curve revealed eclipse and latent periods of 3 and 4 hours, respectively. Although the extent of reduction of host cell protein synthesis was directly related to the multiplicity of infection of the virus, incorporation of actinomycin D and excess NaCl into the medium resulted in significant reduction in host cell background and enabled observation of viral polypeptides as early as 2 hours post infection. Five polypeptides were found to be structural components of the virion, and a further eight appeared to be nonstructural proteins or intermediates. Five polypeptides were glycosylated during virus replication, but only one of these, VP7, was a definite structural glycoprotein. Pulse-chase experiments revealed that four low molecular weight polypeptides underwent post-translational modifications.

Use of serotype-specific monoclonal antibodies to study the epidemiology of rotavirus infection.

The development of an enzyme immunoassay (EIA) capable of serotyping human rotavirus (HRV) in faecal extracts has enabled us to retrospectively study the epidemiology of rotavirus infection in Melbourne. Of 552 stored specimens obtained from individuals with rotavirus-associated gastroenteritis between 1975 and 1986, the serotype could be determined in 62%. Infection was most prevalent in two groups, neonates and children aged between 12 and 24 months. In these groups infection was due to different serotypes, type 1 in older children and an untypable virus in infants. Serotype 1 strains were detected in greater numbers than the other serotypes and circulated in each year of the study. Serotype 2 rotaviruses were associated with a large epidemic in 1978, but have been detected only rarely since.

Isolation of feline rotaviruses and their relationship to human and simian isolates by electropherotype and serotype.

Rotaviruses were detected by electron microscopy in the faecal specimens of six clinically well cats and virus was subsequently isolated from four of them. Analysis of the RNA of the isolates showed the existence of three electrophoretic types characteristic of the 'long' RNA electrophoretic pattern exhibited by rotaviruses. All feline isolates were neutralized only by antiserum to SA11 rotavirus, indicating that these isolates were serotype 3 rotaviruses. Antiserum prepared against a feline strain neutralized all the feline isolates as well as SA11 but showed no neutralizing activity against human isolates of serotype 1, 2 or 4.

Photosynthetic water oxidation. A new chemical model.

A sequential four-step chemical model for the water oxidation process in photosystem II is presented, based on the observation that a peroxide-linked biquinone complex can be chemically formed as a result of hydroxide ion addition to quinone. In our model, the hydroxide ion intermediate is generated in photosystem II as a result of proton abstraction from water. In the model, the first two flashes of light raise the oxidation state of the bimanganese center, while the third and fourth flashes of light sequentially generate the peroxide-linked biquinone which is then directly oxidized by the bimanganese center to produce oxygen and regenerate quinone.

Effects of polyethylene-glycol-induced osmotic stress on transpiration and photosynthesis in pinto bean leaf discs.

A new leaf disc chamber allows measurements of chlorophyll fluorescence and CO(2) and H(2)O vapor exchanges during infusion of solution into the cut edge of the disc. Polyethylene glycol (molecular weight, 6000) was used to apply a mild external osmotic stress to the leaf disc within this chamber. This stress rapidly caused a temporary increase in transpiration. This increase was reversed (5-6 minutes later) and after 20 to 25 min, the stomates nearly completely closed. Internal CO(2) (calculated) and leaf temperature followed the transpiration measurements. However, chlorophyll fluorescence (small rise) followed internal CO(2) (small rise). This complete sequence of events resembles those caused by exposure of leaves to certain air pollutants which have been seen to cause such a transient increase followed by a decrease in stomatal closure.

Inhibition of the k-stimulated ATPase of the plasmalemma of pinto bean leaves by ozone.

Three varieties of Phaseolus vulgaris which differ in their sensitivity to ozone were examined for changes in some physiological and structural plasma membrane characteristics. Plasma membrane vesicles were prepared from control and ozone-treated (0.2 to 0.5 microliters per liter ozone for 5 hours) leaf tissue, and the (K(+) + Mg(2+))-ATPase activity determined and compared. No major changes were observed in the resistant varieties. The sensitive variety showed a severe inhibition of ATPase activity which was largely due to a decrease in the K(+)-stimulated component. This inhibition was completely reversed by the addition of sulfhydryl compounds.Ozone-induced plasma membrane permeability changes may be effected by damage to membrane proteins, perhaps by oxidation of amino acid sulfhydryl groups to disulfide and sulfenic moieties.