OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records ‘-.-..v,£,-. GENETICS AND THE MECHANISM OF OZONE TOLERANCE IN SELECTED CULTIVARS OF PHASEOLUS VULGARIS L. BY Asaf Zvi Guri A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1981 ABSTRACT GENETICS AND THE MECHANISM OF OZONE TOLERANCE IN SELECTED CULTIVARS OF PHASEOLUS VULGARIS L. BY Asaf Zvi Guri The purposes of this thesis were: a) to establish the genetic background of differences in response to ozone in two tolerant and two sensitive cultivars of Phaseolus vulgaris L. and b) to suggest a physiological mechanism for the observed differences which would be consistent with the results from the genetic analysis. Four varieties, two ozone-tolerant, Nep-2 and FH, and two ozone-sensitive, PHR and 0669, were selected from among 12 cultivars initially screened, to be used in this study. The genetic study suggested that at least two major interacting dominant alleles at different loci control the expression of tolerance to ozone in these 4 varieties. In addition to the two major genes, there appeared to be an undetermined number of genes of minor effect involved in the overall genetic control system. Measurements of stomatal conductivity (prior to and after 4 hours of ozone fumigation), and stomatal density, revealed that unlike some other studies in the past, the Asaf Zvi Guri two tolerant varieties exhibited a significantly higher capacity for gaseous conductivity (both abaxial and adaxial) than the two sensitive varieties. Similar results were obtained in regard to stomatal density. These findings imply that under the conditions of this study ozone uptake of the tolerant varieties might have been even higher than that of the sensitive varieties. Production of ethane, due mainly to ozonation of lin- olenic acid, was similar in all four varieties, which suggests that the content of the most prevalent unsaturated fatty acid in bean plants (linolenic acid) is identical in the four varieties. Ethylene production, however, was higher in the ozone-injured leaves of the sensitive varieties. Since ethylene production increases after almost any kind of leaf injury, this difference in ethylene production between tolerant and sensitive varieties after ozone exposure cannot imply any particular mechanism. Production of ethylene due to ozone injury, however, can be used in the future for standardization of visual injury determinations. The measurements of the two antioxidant substances; glutathione (GSH) and ascorbic acid (AA), which might be involved in repair of ozone injury, showed that leaves of the tolerant variety Nep-2 had a higher GSH concentration (on the basis of fresh weight) than the other three varieties immediately following ozone fumigation. No such difference was detected in AA concentration. Further research has revealed that the specific activity of the enzyme GSSG reductase, which catalyzed the conversion of oxidized Asaf Zvi Guri glutathione (GSSG) to its reduced form (GSH), was signifi- cantly higher in the two tolerant varieties, both before and after ozone fumigation. The inconsistency of high enzymatic activity and low GSH concentrations after fumigation in the tolerant variety FH is not understood and can be interpreted in different ways. The fact that GSSG reductase in plants (similar to GSSG reductase in higher animals and yeast) is composed of two polypeptide subunits, could be related to the two major genes deduced from the genetic study. In memory of my grandparents, Nehama and Yehoshua Heshel Gurwitz for their caring and support. Their encouragement made this work possible. ii ACKNOWLEDGEMENTS I sincerely acknowledge the guidance of Dr. M.W. Adams and Dr. A.W. Saettler during my entire Ph.D. program at Michigan State University. The continual availability of Dr. J.B. Mudd to the biochemical part of this thesis is gratefully appreciated. The support and suggestions of Mr. L.W. LeCureux for the efficient use of the gas chromatoqraphy is appreciated. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . LIST OF APPENDICES . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . Genetic Study . . . . . . . . . . . . . . . . . Stomatal Densities and Conductivities . . . . . Ascorbic Acid (AA) and Glutathione (GSH) Assay Glutathione Reductase Activity . . . . . . . . Application of Exogenous AA and GSH . . . . . . Formation of Ethane and Ethylene . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . . Genetic Analysis . . . . . . . . . . . . . . . Stomatal Density . . . . . . . . . . . . . . . Stomatal Conductivity . . . . . . . . . . . . . Ascorbic Acid and Glutathione Assay . . . . . . GSSG Reductase, Specific Activity, and Protein content 0 O O O O O O O O O O O O O O C O O O 0 Application of Exogenous AA and GSH . . . . . . Formation of Ethane and Ethylene . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . Genetic Analysis . . . . . . . . . . . . . . . Stomatal Conductivity . . . . . . . . . . . . . iv Page vi viii 13 13 14 15 16 18 19 20 20 34 34 38 38 46 46 49 49 57 TABLE OF CONTENTS (cont'd.) Physiological Study . Ethane and Ethylene Production CONCLUSION . . . . APPENDICES . . . . LIST OF REFERENCES Page 59 65 68 72 78 Table A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 Bl LIST OF TABLES Pattern of segregation for ozone injury score in the cross (PHR x Nep-Z), as observed in results obtained from the ozone chamber . . . Pattern of segregation for ozone injury score in the cross (FH x PHR), as observed in results obtained from the ozone chamber . . . Pattern of segregation for ozone injury score in the cross (EH x 0669), as observed in results obtained from the ozone chamber . . . Pattern of segregation for ozone injury score in the cross (Nap-2 x 0669), as observed in results obtained from the ozone chamber . . . Pattern of segregation for ozone injury score in the cross (FH x Nep-Z), as observed in results obtained from the ozone chamber . . . Pattern of segregation for ozone injury score in the cross (PHR x 0669), as observed in results obtained from the ozone chamber . . . The field results in 1979-1980 . . . . . . . Test field of F3 families of the cross PHR x PH 0 I O O O O O O I O O O O O C O O O O 0 Test field of F3 families of the cross Nep-2 X PHR O O O O O O O O O O O O O O O O O O O 0 Test field of F3 families of the cross FH x 0669 O O O O O O O O O O O O O O O O C O O 0 Test field of F3 families of the cross 0669 XPHR. o o o o o o o o o O o O o o o o o o o Segregation of the F3 plants in the field . . Means of abaxial stomatal conductivity (cm/sec) of the four bean varieties at different durations of ozone exposure . . . . . . . . . vi Page 21 22 23 24 25 26 28 29 3O 31 32 33 35 vii LIST OF TABLES (cont'd.) Table B2 B3 B4 B5 C1 C2 C3 C4 C5 C6 C7 C8 D1 D2 Means of adaxial stomatal conductivity (cm/sec) of the four bean varieties at different durations of ozone exposure . . . . . . . . . . . Analysis of variance for abaxial stomatal conductivity for the four bean varieties . . . . Analysis of variance for adaxial stomatal conductivity for the four bean varieties . . . . Stomatal density in primary leaves (No./mm2) for the four bean varieties . . . . . . . . . . . Ascorbic acid content (mg/100 gr fw.) in primary leaves of the four bean varieties . . . . Analysis of variance for ascorbic acid content differences among four bean varieties prior to and after ozone fumigation . . . . . . . Glutathione (GSH) content (mg/100 gr fw.) in primary leaves of the four selected varieties of dry beans . . . . . . . . . . . . . . . . . . Analysis of variance for glutathione differences among four bean varieties prior to and after ozone fumigation . . . . . . . . . . . . . . . . Glutathione reductase specific activity (an NADPH/min/mg protein) in primary leaves of the four bean varieties before and after ozone exposure . . . . . . . . . . . . . . . . . Analysis of variance for the activity of leaf glutathione reductase in four bean varieties prior to and after fumigation with ozone . . . . Soluble proteins (mg/ml) content in the leaves of four bean varieties prior to and after fumigation with ozone . . . . . . . . . . . Application of exogenous GSH and ascorbic acid to the primary leaves of the two sensitive varieties O O O O O I O O O O O O 0 O O O O O O 0 Analysis of variance for ethane production from ozone injured leaves of four bean varieties O O O O I O O O O O O O O O O O O O O 0 Analysis of variance for ethylene production from ozone injured leaves of four bean varieties O O O O O O O O O O O O O O O O O O O O Page 35 36 36 37 39 40 41 42 43 44 45 47 48 48 Appendix I II III IV VI LIST OF APPENDICES Page Concentration of GSH and ascorbic acid in the primary leaves (mg/100g fw.) post fumigation with ozone . . . . . . . . . . . . 72 Concentration of GSH and ascorbic acid in the primary leaves (mg/100g fw.) pre fumigation with ozone . . . . . . . . . . . . 73 Ethylene production of ozone injured leaf discs (m2) 0 O I O O O O O O O O O O O O O O 74 Ethane production of ozone injured leaf discs (m2) 0 O O O O O O O O O I O O O O O O 75 Ethylene production of non ozone treated leaf discs (mm2) . . . . . . . . . . . . . . . 76 Ethane production of non ozone treated leaf discs (mm2) . . . . . . . . . . . . . . . 76 viii INTRODUCTION For two decades air pollution in urban and many agri- cultural areas has remained one of the most serious man-made problems affecting crop production. Air pollutants may reduce growth and yield in many crops, depending on the pollutants, their concentration, time of exposure, sensitivity of the plant, and environmental factors. Pollutants may also mark and discolor foliage, reducing visual appeal and thereby the marketability of both ornamental and edible products. In the U.S. alone, it is estimated that annual crop losses due to pollution amount to well over one billion dollars. Damage to natural and horticultural vegetation cannot be estimated, but certainly is extensive (Ting and Heath, 1975). Two serious types of air pollution exist in the world today. The first, commonly known as London smog, is composed of reducing components resulting largely from the combustion of fossil fuels of high sulfur content. The second type is comprised of oxidizing components in the air, primarily ozone (03), fluorine (F), nitrogen oxide (NOX), and peroxyacetyl nitrate (PAN). Most photochemical smOgs arise from the incomplete combustion of fuels by the internal combustion engine. High temperatures and insufficient combustion yield hydrocarbon fragments, which act as catalysts in oxidation reactions. Also formed are oxides of nitrogen, largely N02. The N02 is photochemically cleaved to NO and nascent oxygen. The resultant oxygen radical quickly reacts with molecular oxygen to form ozone. The ozone molecules are very reactive with a standard redox potential of approximately +2.1 V (Throp, 1954). The ozone active molecule is thought to be an ionic form (one resonance form is 6-3=0), in acidic media, in which ozone is more soluble in water than oxygen. In alkaline solutions, ozone rapidly decomposes, releasing molecular oxygen (Alder and Hill, 1950). The use of the term ozone injury is common and it includes several symptoms, like "water-logging", "bronzing", "flecking", and "necrosis". These symptoms refer to a generalized dullness of the leaf surface, although each may represent slightly different processes occurring within the leaf. Under most conditions, exposure to ozone is likely to be expressed as a combination of the above symptoms. Visible injury is associated with a decrease in total leaf photo- synthetic activity, and hence reduced leaf productivity (Todd, 1958), and ultimately cell death. In addition, the following cryptic symptoms occur within the leaf after exposure to ozone: l) a reduction of the plant's photosynthetic activity, 2) a build-up of the pollutant's by-products within the leaf, 3) overall unhealthy appearance without necrotic lesions, 4) reduced growth or yield over a considerable length of time, and 5) increased susceptibility to disease, parasite or insect invasion (McCune et al., 1967). It is quite obvious that visible injury is the result of a series of events beginning at the primary site of damage within the leaf and leading to the final collapse of whole cellular regions. It is generally accepted that ozone, like other pollutant gases, enters the plant through the stomates. Stomatal closure results in little or no injury. Ozone then reacts with water in the substomatal cavity and can be modified to other active species like hydrogen peroxide and hydroxyl ions, both of which can be detrimental to leaf cells (Heath, 1980). Next, ozone may react with the charged groups, such as cellulose, amino acids, galacturonic acid residues, lignic acid and bound Cat in the cell wall or with the few enzymes present (Somers, 1973). Reactions of ozone at the plasmalemma are likely to be with the: (a) unsaturated fatty acid residues, (b) aromatic residues and primary amines, and (c) exposed sulfhydryl groups (Ting and Heath, 1975). Reaction at the membrane may alter binding sites of various plant pathogens and alter membrane permeability. Changes in permeability would, in turn, affect the movement of ions through membranes, affecting the net osmotic potential across the membrane. Chloroplasts and mitochondria may be particularly sensitive to osmotic changes (Heath and Frederick, 1979). Consequently, regulatory mechanisms of normal cellular metabolism begin to fail and eventually this leads to tissue collapse and death. The objectives of this thesis are: 1) To continue attempts to explain the heritable basis of ozone tolerance in selected cultivars of Phaseolus vulgaris. 2) To try to identify a physiological mechanism which might be compatible with data obtained in the genetic study: a) To determine whether ozone-tolerant plants absorb less ozone into their leaves (due to lower stomatal conductivities) than ozone- sensitive varieties. b) To determine whether leaf tissues of tolerant varieties have higher concentrations than sensitive varieties of antioxidant compounds, such as ascorbic acid and glutathione. c) To determine whether ethane and ethylene production in the leaves may be related to ozone injury. LITERATURE REVIEW The role of stomata in ozone injury has been recognized since the beginning of air pollution study with plants, due to the fact that ozone must enter the leaf before it can elicit the physiological responses of cells and bring about the characteristic necrotic spotting. Treatments which cause the stomata to close or partially close, such as withholding irrigation, reduce damage from weather fleck (Dean, 1972). Application of the phytohormone abscisic acid (ABA) which closes stomata also reduces ozone injury in the treated plants (Flecher et al., 1972). The stomata of resistant onion varieties were reported to close upon exposure to low concentrations of ozone (Engel and Gabelman, 1966). Ozone at low concentrations caused a loss of differential perme- ability in the guard cells, resulting in stomatal closure. The guard cells recovered soon after the ozone level was lowered. In addition, Dean (1972) in tobacco, and Butler and Tibbitts (1979) in beans, showed that ozone-resistant cultivars have lower stomatal densities than ozone—sensitive cultivars. Evans and Ting (1973) and Perchorowicz and Ting (1974), reported that ozone had a considerable effect on cell perme- ability. Following ozone fumigation of bean leaves, membrane permeability to titrated water decreased, but increased for internal solutes and labeled rubidium and glucose. The authors believed that cellular membranes might be a primary target for ozone. Tomlinson and Rich (1968) believe that sulfhydryl compounds are critically involved in ozone injury, based on evidence that an ozone-resistant tobacco variety had fewer sulfhydryl groups than an ozone-susceptible variety. They found a slight drop in total sulfhydryl content after ozone exposure of beans and spinach plants. Furthermore, tobacco varieties treated with sulfhydryl-binding agents, such as iodoacetate and iodoacetamide, developed symptoms similar to those produced by ozone. Since sulfhydryl groups are essential for fatty acid synthesis, it was suggested that ozone affected membrane permeability by inhibiting fatty acid synthesis (Tomlinson and Rich, 1969). Many workers believe that the critical sites for ozone injury are the unsaturated fatty acid residues of the membrane lipids and that damage occurs by a process similar to lipid peroxidation. Lipid peroxides are formed by a cyclic reaction involving: a) extraction of a hydrogen atom from a methylene carbon between the double bonds, b) attack of the free radical by molecular oxygen, and c) a further extraction of hydrogen from another fatty acid, in a cyclic reaction (Lundberg, 1962). The first reaction of ozone with unsaturated fatty acids is believed to involve the production of an ozonide (ozone addition across the double bond) with one possible breakdown product being melanodialdehyde upon multiple ozonide forma- tion (Ting and Heath, 1975). The loss of fatty acid material from ozonated tissue would be evidence of ozone attack of fatty acid residues. Actually, such losses are reported to be very low. Swanson et a1. (1973) showed that while relative concentrations of C16:2 (fatty acid with 16 carbon atoms and 2 double bonds) and C16:3 declined slightly (5-10%), the concentration of C16:0, C16:1, C18:0, and C18:l increased in leaves of ozone- treated plants as compared with control. Tomlinson and Rich (1969) reported a decline in all fatty acids in ozonated tobacco leaves, with the largest decline in Cl6:0 and C18:3. There appears to be some relationship between levels of some nitrogenous compounds and ozone injury. MacDowell (1965) reported that tobacco leaves were most sensitive to ozone just after full leaf expansion and that sensitivity was associated with a decline in total protein. Ting and Mukerji (1971) suggested that free amino acids play a role in ozone sensitivity, since their concentration declines at about the same time as maximum ozone sensitivity. In addition, many amino acids increased after ozone exposure while protein content decreased. Therefore, ozone may affect protein metabolism either by enhancing protein hydrolysis, resulting in an increase of free amino acids, or by interfering with protein synthesis without affecting amino acid accumulation (Ting and Heath, 1975). The decline in protein synthesis could occur if the endoplasmic reticulum was disrupted, or if the internal ionic medium (K+ or Mg++) was altered (Pestka, 1971). Dugger et a1. (1962) first noted that concentrations of reducing and soluble sugars in beans were lowest in leaves of greatest ozone sensitivity. External application of a simple hexose solution reduced ozone sensitivity of leaves, although it was not clear how much entered the leaf. In addition, the ozone sensitive developmental stage in cotton leaves was correlated with a depletion of soluble sugars (Ting and Mukerji, 1971). Both Tomlinson and Rich (1968) and Pell and Brennan (1973) have observed a small decline in ATP levels in bean plants exposed to ozone. This decline was observed within one hour of exposure and was interpreted as an initial response. In addition, Mudd et a1. (1974) have shown that the nicotine-amide ring of NADH is cleaved when ozone is bubbled through an aqueous system containing this compound. Since the ratio of NADH : NAD+ regulates cellular metabolism, it is likely that metabolism would be affected by the reduced nucleotide loss. Cracker and Starbuck (1972) found, in beans, that ozone fumigation decreased RNA content in primary leaves due to a corresponding increase in the level of RNAase. On the other hand, Tingey et a1. (1975) could find no increase in the activity of RNAase in soybean plants after ozone fumigation. Ozone has a deleterious affect on photosynthesis. Nobel (1974) reported that ozone fumigation can reduce the chloro- phyll content of pea chloroplasts. Nobel and Wang (1973) found that ozone fumigation could inhibit photophosphoryla- tion in pea chloroplasts. This was apparently related to an increase in chloroplast membrane permeability. In contrast, Coulson and Heath (1974) found that ozone fumigation inhibited the electron transport of photosystem I and photosystem II without uncoupling photophosphorylation. The authors postu- lated that ozone, unlike detergents, disrupted the normal pathway of energy flow from light-excited chlorophyll into the electron transfer compounds by "loosening" but not completely disrupting the membrane. The importance of the age at which tissue is exposed to ozone has become evident. Early studies showed that older, more mature leaves were injured more readily than young leaves (Dugger et al., 1962). Bobrov (1955) observed in oats that neither young nor mature leaves were ozone susceptible, and that only leaves which had just completed expansion were injured. Engel and Gabelman (1966) observed that an ozone sensitive inbred onion line maintained open stomata after ozone exposure, whereas stomata of the resistant inbred line closed. They hypothesized that the guard cells of the resistant plants became leaky after ozone exposure, resulting in subsequent closure; later, stomata functioned normally. Genetic crosses in this case suggested that resistance was controlled by a dominant genetic system, but it was not ascertained that resistance was due to a single gene difference. Butler et 10 a1. (1979) in beans, Huang et a1. (1975), and Povilaitis (1967) in tobacco, reported that ozone resistance is recessive and cytoplasmic factors are not involved. Other studies by Hanson et a1. (1976) in petunia, Cameron (1975) in sweet corn, and Saettler (1975) in beans showed that resistance to ozone is partial or completely dominant involving one or a few genes. Hucl and Beversdorf (1979) suggested that genetic control of ozone tolerance in beans is rather complex and could involve many genes. The factors of quality, quantity, and duration of light appear to be very important in governing plant sensitivity to ozone. First, light affects stomatal opening which regulates the amount of ozone uptake by leaves. Numerous studies show that high light intensity treatment tends to protect plants against ozone injury; e.g., bean plants on 8 hour photoperiods are less sensitive to ozone at 30,000 ft.c. than at 20,000 ft.c. (Heck and Dunning, 1967). Similar results were reported by Ting and Heath (1975) and by Ting and Dugger (1971), in tobacco. To account for this, Dugger et a1. (1962) claimed that high light intensity results in high soluble sugar levels which then protect against ozone injury. There is some circumstan- tial evidence that plant sensitivity to ozone is influenced by certain wave lengths (Heck, 1968). Temperature controls stomatal aperture, which then indirectly influences sensitivity to ozone (Ting and Heath, 1975). Plants with adequate nitrogen levels are generally more sensitive to ozone and other oxidants than are those with deficient or excess nitrogen (MacDowell, 1965) and (Leone et 11 al., 1966). Brewer (1960) reported that increased phospho- rous decreased spinach and mango leaf weight and reduced oxidant injury. Potassium had no effect on leaf weight but did increase injury when available phosphorous was low. When nitrogen was high, potassium reduced injury, suggesting a significant interaction among the macro-elements. Mass at al. (1973) and Hoffman et a1. (1973) reported that salinity reduced both growth and ozone injury to pinto beans. The authors concluded that greater ozone tolerance was related to lower uptake of ozone. There are a few reports concerning the effects of oxi- dants on plants infected with pathogens. Resh and Runeckles (1973) found that bean leaves infected and noninfected with Uromyces phaseoli did not respond differentially to low ozone levels. On the other hand, wheat leaves infected with Puccinia helcaniki were less injured by ozone than healthy leaves (Heagle and Key, 1973). The protection afforded by pathogens seems to be specific, viz. meSOphyll cells directly below stomata with visible appressoria and cells adjacent to inoculated areas were protected. For these reasons, the authors suggested that protection of cells was due to a diffusible material from the fungus. Brennan and Leone (1969), observed reduced ozone injury in tobacco leaves infected with mosaic virus and suggested that virus infection may alter susceptibility to ozone by hastening maturity. As mentioned previously, ABA application leads to stomatal closure, which then leads to reduced ozone uptake and decreased leaf injury. Certain other chemicals which 12 reduce stomatal aperture including phosphon D, 8-hydroxy- quinolin sulfate, and phenylmercuric acetate tend to protect against injury from ozone as well as other pollutants (Seidman et al., 1965). Unfortunately, plant productivity is reduced by these chemicals due to lack of C02 absorption. Attempts to offset the adverse effect of ozone in beans has been successful by using antioxidants such as ascorbic acid, and nickel-N-dibutyl dithiocarbamate (NBC) or prevention of SH bond oxidation by treatment with glutathione (Dass and Weaver, 1968). In tobacco, herbicides like iSOpropalin and pebulate reduced ozone sensitivity (Sung and Moore, 1979), and in turfgrasses the systemic fungicides benomyl were effective in reducing ozone injury (Papple and Ormrod, 1977). Tomlinson and Rich (1973) found that free sterol content of bean leaves decreased after ozone fumigation. However, treatment with cytokinins such as kinetin, N-6 benzyladenine, and benzimidazole resulted in less ozone injury, accompanied by higher levels of free sterol in the leaf. Howell (1974), suggested that increased phenol synthesis by ozone inhibits ATP synthesis, oxidative phosphorylation and SH-dependent enzyme activity. MATERIALS AND METHODS Genetic Study Twelve bean varieties were exposed to ozone 12-14 days after planting. The ozone concentration was maintained at 0.28-0.32 ppm for 10 hours. Of the 12 varieties, the follow- ing four varieties, French Horticulture (F.H.), a cranberry type; Pink Half Runner (P.H.R.), an old dry bean variety; Nep-2 and MSU #0669, both navy bean varieties, expressed the highest stability for the studied trait, and were selected for further studies. To eliminate genetic variability within cultivars, seeds from only one mother plant per variety were utilized. Twelve crosses (including reciprocals) were made among the ozone-sensitive P.H.R. and 0669 and ozone-tolerant F.H. and Nep-Z. The majority of the F1 seeds were planted to obtain the F2 generations. The parents, F1 and F2 genera- tions were fumigated with ozone inside a plexiglas exposure chamber 12-14 days after planting (primary leaf stage) at 0.28-0.34 ppm for 10 hours. The exposure chamber was large enough to contain 36 plants. Ozone was regenerated by passing ambient air over a u.v. tube and was carried to the chamber by a flow of ambient air. The moisture content inside the chamber was adequate to maintain normal growth and photosynthesis. Plants were watered prior to fumigation and were planted in 13 14 plastic pots to decrease ozone absorption by the pots. Each group of plants included F and F2 plants from the 1 same cross and their two parents. Plants were arranged in a completely randomized design, since no detectable gradient in ozone injury had been found in a preliminary test. Amount of leaf injury on each plant was visually evaluated on a scale of 0 to 4, 2 days post-fumigation, where 0 represents no damage, 1 - minor damage, 2 - moderate damage, 3 - severe damage, and 4 — very severe damage. In addition, F1 and F2 generations and the four varieties were planted in the field in two consecutive summers (1979 and 1980). A space of 15 cm was maintained between plants and 30 cm between rows, in order to prevent interaction between neigh- boring plants. Plants after anthesis were visually evaluated for presence or absence of ozone injury. The F2 segregants from the crosses PH x PHR, FH x 0669, PHR x 0669, and Nep-Z x PHR (30 plants per cross) were sepa- rately exposed to ozone in the chamber at 12-14 days, and injury recorded 2 days later. They were then allowed to yield F3 seeds. Ten seeds from each plant were planted in the field in 1980, and the amount of injury was recorded after anthesis, as mentioned previously. In all field experiments, the groups of plants (group = variable number of plants from the same variety, cross or generation) were arranged in completely randomized designs. Stomatal Densities and Conductivities Stomatal densities were determined by the leaf impres- 15 sion method. A cellulose acetate film and acetone were used to obtain an accurate impression of the primary leaf surface. Primary impressions were made on 8 plants each of the four varieties at 13 days after seeding. One adaxial and two abaxial leaf surface impressions were taken on both primary leaves. The impressions were made at about the same position between major veins on all leaves. Stomata number per mm2 was determined from the cellulose acetate replicates using a Ziess light microscope (x41). Thirteen-day old plants of each variety (seven plants per variety) were placed inside the chamber 2 hours prior to the exposure to ozone. Plants were watered and exposed to ozone (0.28-0.34 ppm) for 8 hours. Stomatal resistance was measured with an autoporometer (Li-65 automatic diffusive resistance meter) immediately before the fumigation and at 4 and 6 hours after the beginning of fumigation. Four measure- ments were taken per plant; two in each primary leaf, one on the abaxial and one on the adaxial surface. The experiment was replicated three times and the stomatal resistance figures obtained were converted to their reciprocals, viz. stomatal conductivities. Ascorbic Acid (AA) and Glutathione (GSH) Assay Ascorbic acid assay was conducted by using the 2,6- dichloro-phenollindophenol (DCIP) photometric method described by Hanson et a1. (1971). One 9. fresh weight of primary leaves of uniform size were quickly detached and ground with sorvall omni mixture in 10 ml of an ice cold 0.0005 M di-sodium EDTA 16 solution containing 3% TCA (trichloro-acetic acid) for 1-2 minutes. The homogenate was quickly filtered through Whatman #4 paper and brought up to 20 ml with EDTA-TCA extracting solution. One ml distilled water, 2 ml DCIP reagent and 20 m1 filtered leaf extract were added to each test tube and their optical densities at 600 nm were determined from a standard curve which was prepared previously, using various determined concentrations of AA. In the GSH assay, 2 g. of primary leaves were chopped and homogenized with 10 ml EDTA—TCA in an ice bath, the homogenate was quickly filtered through Whatman #4 paper and brought to 15 ml with EDTA-TCA. Each homogenate (for each treatment) was titrated with 1.5 ml 0.1N NaOH to pH range of 6.0-8.0. Each test tube con— tained 0.5 ml distilled water, 2.0 ml leaf homogenate, 0.5 m1 0.2M potassium phosphate buffer pH 7.0, and 0.1 ml the reagent Dithiobis-Z-nitrobenzoic acid (DTNB). The optical densities for each set of test tubes was determined at 412 nm from a standard curve of known concentrations of GSH. A test tube (one per each set) containing all the above ingredients and concentrations but without the reagent DTNB was used as blank. This was done because of the high absorption in 412 nm by the leaf homogenate prior to the reagent application. Similar assays were made for each one of the 4 varieties simultaneously at pre— and post-10 hours of ozone fumigation. Glutathione Reductase Activity Prior to and immediately after ozone fumigation, 2 g. fresh weight leaves were chOpped, ground and homOgenized 17 with 25 ml 0.1M of Tris HCl buffer, pH 7.5, and 2 g. Poly Vinyl Pyrrolidone (PVP) in an ice bath. The homogenate was centrifuged at 12,500 RPM (20,000 g.) for 10 minutes and the supernatant collected and recentrifuged at 17,500 RPM (37,000 g.) for 10 minutes. Supernatants were collected and used as a crude leaf enzyme extract. To determine enzymatic activi- ties, 0.7 ml distilled water, 0.2 m1 0.1M Tris HCl buffer pH 7.5 and 0.15 ml of the supernatant were added to one cuvette used as blank, while the same ingredients plus 0.01 ml 0.1M EDTA and 0.03 ml 0.005M NADPH were added to a second cuvette. The reading of the second cuvette at 340 nm (peak for NADPH absorbance) was registered on a chart recorder for 5 minutes. Then 0.005 ml of 0.1M oxidized glutathione (GSSG) was added as an electron acceptor as in the following equation: GSSG + NADPH + H+ --------£> 2 GSH + NADP+. This reaction is catalyzed by glutathione reductase and its velocity can be determined from the slope of the line (absorbance units/min) which represents the drop in absorbance at 340 nm due to conversion of NADPH to NADP+. The slope of the line before GSSG addition was subtracted from the slope after GSSG addition. The reaction rate was then calculated as follows. The molar extinction of NADPH is 6.22 x 103 (mol/liter at 340 nm). Therefore, 6.22 x 103 absorbance units per minute are decreased due to the oxidation of 1 mol of NADPH by GSSG. In this case, 0.00622 absorbance units per minute were used as the extinction coefficient. This corresponds to 1 an of NADPH oxidized to NADP+ by GSSG. In addition, the amount of soluble proteins in each sample was determined 18 using 1 mg/ml Bovin serum albumin (BSA) as standard and Coomassie Blue (Bradford assay) as a reagent. Soluble protein content was determined spectrophotometrically at 595 nm from daily prepared standard curve of BSA. All the elements were inserted in the following formula: A x B C x D x E = GSH reductase specific activity rate where A* = the slope of the line represents the decrease in 340 nm absorbance B = the volume of all the ingredients in the 2 cuvettes = 1.095 ml C = the volume of the plant extract in the 2 cuvettes = 0.15 ml D* = the concentration of soluble proteins in the plant extract E = NADPH molar extinction coefficient = 0.00622 The GSH reductase catalyzation rate units are: anNADPH/min/ mg protein. 1 *The values are variable and depends on the cultivar and the timing (before or after fumigation with ozone) when extractions were prepared. Application of Ex0genous AA and GSH Primary leaves of 60 plants, 30 each of PHR and 0669, were sprayed 6 and 12 hours before ozone exposure (0.28-0.34 ppm) with distilled water (control), 0.005M AA or 0.005M GSH. Ozone damage was determined visually (by classifying to 0-4 groups as mentioned above) one day after exposure. l9 Formation of Ethane and Ethylene Fourteen-day old plants of each variety (24 per variety) were fumigated with ozone as described previously and when injury symptoms appeared on the leaves, a disc of approxi- mately 1.5 cm diameter was removed from the center of each primary leaf. Discs were placed inside 25 ml flasks with 0.5 ml distilled water and sealed with rubber caps. All flasks including empty control flasks were incubated 24 hours above a source of high intensity light (1-1.5 mv/cmz). Subsequently, amounts of ethane and ethylene in each flask were measured with gas chromatography techniques whereby samples of flask gases were inserted by syringe into a 0.318 cm by 100 cm stainless steel column packed with Porpak R and held at 80°C in a Varian model 2400 gas chromatograph. Gas flows were: N2, 35 ml min—l; air, 300 1; H2, 30 m1 min-1. Ethylene and ethane could be ml min- detected down to 5 ml liter-l, ethylene appearing as the first peak. Identities of ethylene and ethane were based on order of appearance and retention time. RESULTS Genetic Analysis Results from the ozone exposure chamber indicate that the varieties FH (tolerant) and 0669 (sensitive) in this experiment showed more stability in the expression of the trait than the other two varieties, Nep-Z (tolerant) and PHR (sensitive) (Tables Al-A6). The data of the F1 plants implies that cytoplasmic factors are not involved in the genetic control of tolerance or sensitivity to ozone "attack", since no differences have been found between Fl progenies from reciprocal crosses (Tables Al-A6). The fact that Fl progenies from crosses between tolerant and sensitive varieties were, in the majority of the cases, ozone tolerant (Tables A1-A4) implies that most of the alleles which condi- tion the expression of tolerance to ozone are partially or completely dominant. The fact that most of the F1 progenies from the cross between the two sensitive varieties (Table A6) were unexpectedly ozone tolerant, indicates that alleles of different genes interact with each other in a complementary way. It is possible that the trait is controlled by several genes since F2 segregants have shown a wide assortment of reaction classes in all the crosses, excluding the cross between the two tolerant varieties (Table A5) in which a 20 21 Table A1. Pattern of segregation for ozone injury score in the cross (PHR x Nep-2), as observed in results obtained from the ozone chamber. Number Level of Ozone Injury Varieties and Crosses Plgfits _2__ _l_ _2_ _3_ _4_ Nep-Z 56 27 19 10 PHR 69 3 6 25 28 a (PHR x Nep—2) Fl 17 ll 4 2 b (PHR x PHR) Fl 15 12 l 2 (PHR x Nep-2) F2 233 133 50 21 27 Observed 183 50 Expected 174.75 58.25 2 _ X ldf— 2 2 [183 - 174.75 (-0.5)] + [50 - 58.25 (-0.5)] = 1.96 N. . 174.75 58.25 S a = 0.05 22 Table A2. Pattern of segregation for ozone injury score (FH x PHR), as observed in results in the cross obtained from the ozone chamber. Number Level of Ozone Injury Parents and Crosses Plgfits 0 _l_ _2_ _3_ _4_ PHR (sensitive) 53 1 4 19 12 17 FH (tolerant) 38 24 9 5 (PHR x FH) Fl 16 9 6 1 (EH x PHR) Fl 15 9 4 1 (PHR x FH) F2 201 136 21 15 20 9 Observed 157 44 Expected 150.75 50.25 x2 ldf = [157 - 150.75 (-o.5)]2 + [44 - 50-25 ('0-5)12 = 0,377 N.s. 150.75 a = 0.05 50.25 Table A3. the cross (FH x 0669), 23 obtained from the ozone chamber. Pattern of segregation for ozone injury score in as observed in results Number Level of Ozone Injury of Parents and Crosses Plants 0 l 2 3 4 0669 (sensitive) 40 2 7 12 19 FH (tolerant) 43 34 9 (0669 x FH) Fl 18 8 8 2 (PH x 0669) Fl 16 10 5 l (0669 x FH) F2 224 105 48 30 28 13 Observed 153 71 Expected 168 56 2 = X 1df _ 2 _ _ 2 [153 - 168 ( 0.5)] + [71 56 ( 0.5)] = 5.004 168 (I: 0.05 56 24 Table A4. Pattern of segregation for ozone injury score in the cross (Nep-2 x 0669), as observed in results obtained from the ozone chamber. Number Level of Ozone Injury Parents and Crosses Plgfits _0_ _1_ A_2_ _3_ _4_ Nep-Z (tolerant) 48 19 16 5 0669 (sensitive) 43 1 2 13 18 9 (Nep-2 x 0669) F1 21 12 9 2 (0669 x Nep-2) Fl 17 9 5 3 (Nep-2 x 0669) F2 181 100 31 27 16 7 Observed 131 50 Expected 135.75 45.25 2 _ X ldf" 135.75 45.25 = 0.532 N.S. a = 0.05 25 Table A5. Pattern of segregation for ozone injury score in the cross (FH x Nep-Z), as observed in results obtained from the ozone chamber. Number Level of Ozone Injury Parents and Crosses Plgits _Q_ _l_ _2_ _3_ _4_ Nep-Z (tolerant) 28 13 9 4 2 PH (tolerant) 35 19 13 2 1 (Nep-2 x FH) Fl 16 ll 5 (PH x Nap-2) F1 16 13 3 (Nep-2 x FH) F2 61 33 16 9 3 26 Table A6. Pattern of segregation for ozone injury score in the cross (PHR x 0669), obtained from the ozone chamber. as observed in results Number Level of Ozone Injury of Parents and Crosses Plants 0 l 2 3 4 PHR (sensitive) 55 2 6 15 20 12 0669 (sensitive) 54 13 15 25 (PHR x 0669) F1 22 15 3 4 (0669 x PHR) F1 9 4 5 (PHR x 0669) F2 197 41 75 53 13 15 Observed 116 81 Expected 110.8 86.187 2 = x 1df _ _ 2 _ _ 2 [116 110.8 ( 0.5)] + [81 86.187 ( 0.5)] = 0.452 N.S. 110.8 a = 0.05 86.187 27 narrow range of genetic variation probably exists. The F3 families analysis (Tables A8-A11) indicated that in the majority of the cases (approximately 85%), visual classification in the F2 was essentially correct. In addition, it was judged acceptable for genetic analysis that the line between tolerance and sensitivity lies between classes 1 and 2 (Table A12), inasmuch as the ratio of tolerants to sensitives, comparing F3 plants with F2 parents, changes sharply at that point. Therefore, in order to facilitate genetic evaluation, plants with a degree of injury of 0 to 1 were considered as ozone tolerant while the remainder (classes 2 to 4) were considered ozone sensitive. As a result of this rearrangement, segregation in F from 3 crosses between tolerant and sensitive 2 varieties (Tables A1, A2, and A4) suggests that only a single dominant allelic difference is involved. In one cross (Table A3), the X2 value for monogenic inheritance was significantly greater than zero. The ratio 9:7 (tolerant:sensitive) in the F2 generation of the cross between the two sensitive varieties (Table A6), suggests that two co-dominant complementary alleles in two different loci are involved in the regulation of tolerance to ozone. Results from the field nursery (Table A7) indicate that the majority of the alleles that express tolerance to ozone injury in bean plants are partially to completely dominant. Since results from the chamber implied nonexistance of maternal effects, F1 and F2 seeds from reciprocal crosses were composited and analyzed together. The test ratio 9:7 (tolerant:sensitive) in the F2 generation of the cross 28 Table A7. The field results in 1979-1980. Varieties and Crosses PHR Nep-2 0669 PH (0669 x Nep-2) Fl (FH x PHR) F1 (0669 x PHR) F1 (PHR x Nep-Z) F1 (FH x 0669) F1 (FH x Nep-2) F1 (0669 x Nep-2) F2 (0669 x FH) F2 (Nep-Z x FH) F2 (PHR x 0669) F2 (Nep-2 x PHR) F2 (PHR x FH) F2 Total Number Plants 146 126 118 122 16 18 17 22 15 19 199 206 175 279 292 226 Number Ozone Tolerant Plants 10 108 16 118 14 15 13 16 14 19 159 172 164 153 185 153 Number Ozone Sensitive Plants 136 18 102 Home-cow“: x2 Value 2.68 N.S. S. 2.06 N.S. S. S. 29 Table A8. Test field of F3 families of the cross PHR x FH. F2 Plants F3 Plants Number Number Serial Ozone Ozone Tolerant Number Level of Injury_ Tolerant Sensitive Total 1 0 5 5 0.5 2 1 7 3 0.7 3 2 4 6 0.4 4 0 7 3 0.3 5 0 8 2 0.8 6 2 5 5 0.5 7 l 6 4 0.6 8 l 7 3 0.7 9 3 l 9 0.1 10 0 10 0 1.0 11 3 died -- - --- 12 0 8 2 0.8 13 4 died -- - --- 14 1 6 4 0.6 15 2 3 7 0.3 16 0 7 3 0.7 17 1 9 1 0.9 18 0 7 3 0.7 19 l 8 2 0.8 20 4 died -- - --- 21 0 died -- - --— 22 3 5 5 0.5 23 0 7 3 .3 24 4 died -- - --- 25 2 4 6 0.4 26 2 3 7 0.3 27 3 2 8 0.2 28 1 6 4 0.6 29 2 1 9 0.1 30 0 9 l 0.9 30 Table A9. Test field of F3 families of the cross Nep-2 x PHR. F2 Plants F3 Plants Number Number Serial Ozone Ozone Tolerant Number Level of Injury Tolerant Sensitive Total 1 0 6 4 0.6 2 l 8 2 0.8 3 2 5 5 0.5 4 0 10 0 1.0 5 l 7 3 0.7 6 3 3 7 0.3 7 1 7 3 0.7 8 l 7 3 0.7 9 4 died -- - --- 10 0 9 1 0.9 11 1 6 4 0.6 12 4 3 7 0.3 13 l 5 5 0.5 14 l 7 3 0.7 15 4 died -- - --- 16 1 8 2 0.8 17 0 9 l 0.9 18 1 7 3 0.7 19 3 4 6 0.4 20 2 2 8 0.2 21 0 8 2 0.8 22 1 8 2 0.8 23 0 8 2 0.8 24 3 3 7 0.3 25 0 9 l 0.7 26 1 7 3 0.7 27 0 8 2 0.8 28 2 5 5 0.5 29 1 6 4 0.6 30 3 2 8 0.2 31 Test field of F3 families of the cross FH x 0669. Table A10. F3 Plants Number Number F2 Plants Serial Ozone Tolerant Sensitive Ozone Tolerant Level of Injury Total Number 52782471309969 00000000010000 738653984098889 000000000100000 372457126012221.58328639701141 738653984098889 — 52782471309969 1 _ 1 died 130212012000000013102202201110 123456789 32 Table A11. Test field of F3 families of the cross 0669 x PHR. F2 Plants F3 Plants Number Number Serial Ozone Ozone Tolerant Number Level of Injury Tolerant Sensitive Total 0.8 died - \DGJQGU'IDWNH mflHmflflNWQWqumNmflflml—‘Nolm 0000000000.....0000000 U‘QHWQQNWWWHQCGNQNQO‘HNW OOOOOOOOOOOOOOOOOOOOOO died H \ooooom H f—‘NOO-b I UIWKDNwwmi-‘UJQKDWl—‘bmbWWubKDCDI-‘I N l—' m OOUJOI-‘UJNowHHHwOHWNHHHNWP-‘Hl-‘wa-‘wH 33 Table A12. Segregation of the F3 plants in the field. Level of Injury of the Parental Plants (F2) O 1 Number F3 Ozone Tolerant Plants 288 270 62 38 5 Number F3 Ozone Sensitive Tolerant: Plants Sensitive 62 4.65:1 112 2.4:1 118 1:1.9 112 1:2.9 15 1:3 34 2 value not between the two sensitive varieties produced a X significantly different from zero. Only in one cross (out of four) between tolerant and sensitive varieties (Nep-2 x 0669) the Mendelian monogenic ratio of 3:1 (tolerant:sensitive) was statistically (XZN.S. from 0) acceptable. Stomatal Density The data in Table BS suggests that 0669, a sensitive 2 on the variety, has significantly fewer stomata per mm abaxial (lower) leaf surface than do the other 3 varieties. FH, a tolerant variety, has significantly higher stomata per 2 mm on the adaxial (upper) leaf surface in comparison to the other 3 varieties. Stomatal Conductivity With reference to the data in Tables Bl and B2, the abaxial stomatal conductivity is about 8 to 10-fold greater than the adaxial conductivity, which implies that most ozone uptake occurs through the lower surfaces of the leaf canopy. In general, both abaxial and adaxial conductivity decreased significantly in all 4 varieties as ozone exposure time increased (Tables Bl-B4). The two tolerant varieties exhib- ited higher stomatal conductivity (both abaxial and adaxial) prior to ozone fumigation and in the two periods of fumigation in contrast to the two sensitive varieties (Tables Bl and B2). The two main factors; variety and timing (period of fumigation) were significant in both abaxial and adaxial analyses of variance (Tables B3 and B4). The interaction between the 35 Table B1. Means of abaxial stomatal conductivity (cm/sec) of the four bean varieties at different durations of ozone exposure. Length of Ozone Fumigation (Hours) FH PHR Nep-Z 0669 Mean 0 0.420 0.417 0.437 0.347 0.405 4 0.290 0.192 0.225 0.190 0.224 6 0.086 0.070 0.082 0.047 0.071 Mean 0.266 0.227 0.248 0.195 Table BZ. Means of adaxial stomatal conductivity (cm/sec) of the four bean varieties at different durations of ozone exposure. Length of Ozone Fumigation (Hours) 0 4 6 Mean FH 0.050 0.057 0.020 0.042 PHR 0.020 0.014 0.008 0.014 13223 0.044 0.023 0.013 0.026 _Me_an_ 0.038 0.028 0.012 Table B3. Analysis of variance for abaxial stomatal conductivity for the four bean varieties. 36 Source d.f. SS MS Total 503 16.2 Block 2 0.286 0.146 Variety 3 0.861 0.287 Timing 2 9.1 4.55 Variety x Timing 6 0.463 0.077 Error 22 0.335 0.015 Sample 216 3.691 Det. 252 1.464 Table B4. Analysis of variance for adaxial stomatal conductivity for the four bean varieties. Source d.f. SS MS Total 503 0.294 Block 2 0.0072 0.0036 Variety 3 0.063 0.021 Timing 2 0.058 0.029 Variety x Timing 6 0.019 0.003 Error 22 0.0348 0.016 Sample 216 0.09 Det. 252 0.022 37 .ucmam mom mucmEmusmme “sow mo mcmwze ow now we mam m.mm m.mma we Sam M mma mam boa mmm mam mma mma mmm mma mam me me mom baa moa mwm me mma he mam we Sea mo mmm ww mam me ewm mm mma mm NmH em mom me mmm mm mwa mm oom mm ham mm eHm we mha mm mHm a? 33 gamma Emma mmo muomz mooo mm .mmepmflum> comb “50m on» new AmEE\.ozv mm>mma mumEHum ca anemcmc HmumEoume .mm dance 38 variety and timing was significant only in abaxial conduc- tivity (Table B3). Ascorbic Acid and Glutathione Assay When the 4 varieties were considered together, there was a significant decline in the amounts of ascorbic acid (AA) detected after ozone fumigation. On the other hand, no differences in AA content were detected among the varie- ties before and after exposure to ozone (Tables Cl and C2). Glutathione (GSH) contents among the four varieties prior to fumigation were not significantly different. However, after ozone treatment, there was a significant decline in the amount of GSH in the varieties PHR, 0669 (both sensitive) and FH (ozone tolerant), while Nep-2 (ozone tolerant) showed a significant increase. GSSG Reductase, Specific Activity, and Protein Content GSH reductase activity was significantly greater in primary leaves of the two tolerant varieties than the activ- ity in the two sensitives (Tables C5 and C6). Also, it appears that ozone within the leaves significantly altered GSH reductase activity. Enzymatic activity recovered slightly in both Nep-2 (tolerant) and 0669 (sensitive), and decreased in PB (tolerant) and PHR (sensitive). Ozone treatment decreased the amount of soluble protein in the primary leaves of all varieties (Table C7). 39 Table C1. Ascorbic acid content (mg/100 gr fw.) in primary leaves of the four bean varieties. Variety and Treatment Nep-2 0669 PHR FH Average Pre-fumigation with ozone 560.4 554.8 548.6 542.5 551.6 Post-fumigation with ozone 515.2 520.7 508.5 528.2 518.1 Average 537.8 537.7 528.6 535.3 Table 40 C2. Analysis of variance for ascorbic acid content differences among four bean varieties prior to and after ozone fumigation. Source QLEL 88 MS Treatment 7 31,439 Q1 1 26,773 26,773 Q2 1 1,246 1,246 Q3 1 1,608 1,608 Q4 1 684 684 Q5 1 630 630 Q6 1 223 223 Q7 1 273 273 Error 16 65,759 4,110 Sample 72 12,912 180 Q1 = contrast between pre and post fumigation with ozone. Q2 = within pre contrast between Nep-2 and the other three varieties. Q3 = within post contrast between FH and the other three varieties. Q4 = within pre contrast between 0669 and the average of PHR and FH. Q5 = within post contrast between 0669 and the average of PHR and Nep-2. Q6 = within pre contrast betwwen PHR and FH. Q7 = within post contrast between PHR and Nep-2. Table C3. Glutathione (GSH) primary leaves of of dry beans. 41 content (mg/100 gr fw.) in the four selected varieties Variety and Treatment Nep-2 Pre-fumigation with ozone 4.67 Post-fumigation with ozone 7.24 Average 5.95 0669 PHR FH Average 4.69 4.82 4.66 4.71 2.71 2.13 2.28 3.59 42 Table C4. Analysis of variance for glutathione differences among four bean varieties prior to and after ozone fumigation. Source QL£L SS MS Treatment 7 466.55 01 1 145.64 145.64 Q2 1 0.194 0.194 Q3 1 301.02 301.02 Q4 1 0.003 0.003 05 1 4.56 4.56 Q6 1 0.002 0.002 Q7 1 0.135 0.135 Error 16 336.33 22.9 Sample 72 130.11 1.8 Q1 = contrast between pre and post fumigation with ozone. Q2 = within pre contrast between PHR and the average of the other three varieties. Q3 = within post contrast between Nep-2 and the average of the other three varieties. Q4 = within pre contrast between FH and the average of Nep-2 and 0669. QB = within post contrast between 0669 and the average of PHR and FH. Q6 = within pre contrast between Nep-Z and 0669. Q7 = within post contrast between PHR and FH. 43 Table C5. Glutathione reductase specific activity (an NADPH/min/mg protein) in primary leaves of the four bean varieties before and after ozone exposure. GSH Reductase Activity Before GSH Reductase Activity After Number Variety Fumigation Fumigation l Nep-Z 7.29 , 8.31 2 Nep-Z 5.55 9.66 Average 6.59 9.04 1 FH 8.05 6.82 2 FH 6.59 5.85 3 FH 7.31 6.02 Average 7.32 6.23 1 PHR 4.43 3.49 2 PHR 4.32 3.22 3 PHR 4.12 3.20 Average 4.29 3.30 l 0669 3.18 4.35 2 0669 2.06 3.40 3 0669 2.91 3.65 Average 2.71 3.80 44 Table C6. Analysis of variance for the activity of leaf glutathione reductase in four bean varieties prior to fumigation with ozone. Source QLEL SS MS Total 11 43.68 Varieties 3 40.24 13.41 Error 8 3.44 0.43 According to Duncan's New Multiple Range Test = 0.05 Nep-Z FH PHR 0669 Analysis of variance for the activity of leaf glutathione reductase in four bean varieties after fumigation with ozone. Source d4f;_ SS MS Total 11 64.16 Varieties 3 62.17 20.72 Error 8 1.99 0.25 According to Duncan's New Multiple Range Test = 0.05 Nep-2 FH PHR 0669 45 Table C7. Soluble proteins (mg/ml) content in the leaves of four bean varieties prior to and after fumigation with ozone. Proteins in the Plant Leaf Proteins in the Plant Leaf Number Variety Pre Fumigation Post Fumigation 1 FH 1.149 1.054 2 EH 1.145 1.069 3 EH 1.212 1.046 1 Nep-2 1.278 1.228 3 Nep-2 1.291 1.241 1 PHR 1.149 0.906 2 PHR 1.158 0.921 3 PHR 1.193 0.972 1 0669 1.149 0.697 2 0669 1.063 0.683 3 0669 1.114 0.745 46 Application of Exogenous AA and GSH Application of either AA or GSH to leaves significantly increased tolerance to ozone in the two sensitive varieties (Table C8). Generally, GSH appeared to increase tolerance to ozone more efficiently. Treatment with AA and GSH 6 hours prior to fumigation reduced ozone injury to a greater degree than did treatment 12 hours before fumigation. Formation of Ethane and Ethylene_ In general, leaf discs taken from ozone fumigated plants produced higher amounts of ethane and ethylene as compared with discs from untreated plants (see Appendices III-VI). Sensitive PHR and 0669 varieties produced much more ethylene after exposure to ozone than tolerant FH and Nap—2 varieties (Table D2). There were no significant differences between the two sensitive or the two tolerant varieties. Although ethane is a natural product of plant metabolism, it is usually produced in smaller quantities as compared to ethylene. Unlike ethylene, there were no significant differences in ethane production among the four varieties after fumigation with ozone (Table D1). 47 Table C8. Application of exogenous GSH and ascorbic acid to the primary leaves of the two sensitive varieties. Injury Level Injury Level 6 Hour Pre 12 Hour Pre Variety Treatment Fumigation Fumigation 0669 H20 4 4 0669 H20 4 4 0669 H20 4 4 0669 H20 4 4 0669 H20 4 4 0669 A.A. (0.005M) 2 3 0669 A.A. (0.005M) 1 4 0669 A.A. (0.005M) 1 3 0669 A.A. (0.005M) 2 4 0669 A.A. (0.005M) 2 3 0669 GSH (0.005M) 0 2 0669 GSH (0.005M) 1 l 0669 GSH (0.005M) 1 1 0669 GSH (0.005M) l 2 0669 GSH (0.005M) 0 l PHR H20 4 4 PHR H20 4 4 PHR H20 4 4 PHR H20 4 3 PHR H20 3 3 PHR A.A. (0.005M) O 0 PHR A.A. (0.005M) 2 2 PHR A.A. (0.005M) 1 3 PHR A.A. (0.005M) 0 2 PHR A.A. (0.005M) l 3 PHR GSH (0.005M) 0 0 PHR GSH (0.005M) 0 2 PHR GSH (0.005M) 0 2 PHR GSH (0.005M) 1 0 PHR GSH (0.005M) 0 2 48 Table D1. Analysis of variance for ethane production from ozone injured leaves of four bean varieties. Source QLEL SS MS Total 99 166,222 Variety 3 3,619 1,206 N.S. Error 96 162,603 1,694 Table D2. Analysis of variance for ethylene production from ozone injured leaves of four bean varieties. Source QLEL SS MS Total 99 215,490,000 Variety 3 184,440,000 61,466,667 Error 96 31,050,000 323,428 According to Duncan's New Multiple Range Test = 0.05 FH Nep-2 PHR 0669 a a b b DISCUSSION Genetic Analysis Previous studies which have dealt with genetic regulation of tolerance to ozone injury in plants are very contradictory. This lack of agreement among previous workers can probably be explained as due to their research being conducted in many different crops in which overall genetic backgrounds were different, and in which experimental factors such as ozone concentration, length of exposure, and plant age were variables. Because ozone apparently enters leaves only through stomata, variables such as temperature, humidity, soil moisture, and others which directly affect stomatal aperture, therefore, may affect amount of ozone uptake into the leaves. This situation, therefore, adds to the difficulty of reaching unanimous inter- pretations of the genetic basis involved. In fact, there is no fundamental reason why ozone tolerance in a large array of species has to be based upon a single genetic interpretation. There is probably more than one way in which plants can be protected from ozone injury. Rapid stomatal closure in response to ozone exposure could decrease ozone uptake by the leaves (Engel and Gabelman, 1966). A compact arrangement of cells within the leaf could reduce ozone flecking by reducing the volume of intercellular space available for flow of ozone 49 50 molecules. Close spacing of cells could imply an increase in the number of chloroplasts per unit area which may increase the number of still functional chloroplasts in the presence of ozone (Uharing, 1978). Many believe that ozone alters the permeability of membranes in the cell due to oxidizing properties (Ting et al., 1974; Beckerson and Hofstra, 1980; and Heath, 1980). Sulfhydryl groups, unsaturated fatty acids and aromatic residues of amino acids are primary targets of ozone and its by-products in the cell membranes (Heath, 1980). The ability to chemically reduce rapidly the oxidized components in the membrane in the presence of ozone, and thus to repair membrane injury, endows plant with ozone tolerance (Sutton and Ting, 1977). Tingey et a1. (1975), have found that ozone affects numerous metabolic enzymes in leaf tissues of sensitive soybean cultivars. Therefore, tolerance may be associated with adaptive enzymes which function normally in the presence of ozone. Different reactions to ozone activity may be found in different developmental stages of the leaf. Dugger et a1. (1962), already showed that older, more mature leaves were injured more easily than younger leaves. Uharing (1978) suggested that stomata in young leaves are not fully devel- oped and functional. Therefore, ozone uptake is higher in mature leaves. In addition, the greater intercellular volume in mature leaves is more favorable for flow of ozone. In contrast, Dugger et a1. (1962) and Ting and Mukerji (1971), found that stomatal conductivity for gas exchange in pinto 51 beans and in cotton leaves remained constant throughout leaf maturation, and the period of maximum sensitivity was corre- lated with low soluble sugar and amino acid content. Evans and Ting (1973) concluded that leaf sensitivity throughout the developmental period was a function of internal conditions rather than to variation in stomatal resistance. The results obtained in this research and others suggest that the trait "tolerance-to-ozone-flecking" in higher plants is controlled by several genes, and that it is highly affected by the environment to which the plants are subjected. Due to its physiological complexity it was impossible in this study to assess the genetic basis of ozone tolerance rigorously. As a result of the FZ-F3 data (Tables A8-A12), I grouped the 5 injury classes (0 to 4) obtained in the chamber study into two major classes, tolerant (0 to l) and sensitive (2 to 4). According to this rearrangement, it is suggested that basically at least two major interacting dominant alleles (in two different loci) arbitrarily denoted as A and B, account for ozone tolerance in the varieties of this study. Assuming that the four varieties used in this study were 100% homozygous, the two sensitive varieties, PHR and 0669, are assigned genetic symbols AAbb and aaBB, respectively. On the other hand, the two tolerant varieties, FH and Nep-2, must both be AABB. Such genotypes would account for ozone tolerant F1 plants in crosses between the two sensitive varieties, and for the 9:7 (tolerant:sensitive) ratio among the F2 segre- gants. The proposed system would also account for the appearance of tolerant F1 plants in the crosses between 52 tolerant and sensitive varieties, and for a 3:1 (tolerant: sensitive) ratio, in three of the four crosses. Handling the situation in this way allowed some clarifi- cation of the ambiguity represented by this trait. However, in the F3 generation there was not complete genetic fixation viz. the F3 progenies for almost every F2 parental plant still appeared to continue to segregate. In addition, there was some difference between the two tolerant varieties in the level of response to ozone (FH was somewhat more tolerant than Nep-2 and more stable). Similar differences were found between the two sensitive varieties. Such phenomena may be explained by introducing in addition to the two major genes, several genes with minor effect and non-genetic factors into the picture. These two circumstances may account also for the variability within each of the two main classes, namely, tolerant and sensitive. The available data do not permit us to determine how many minor genes are involved in the expres- sion of the trait, whether the minor alleles (which increase the expression of ozone tolerance) are partially or fully dominant, or strictly additive, whether non-allelic inter- action exists between some of the minor genes, and whether or to what extent they might be affected by external factors of the environment. Non-genetic factors definitely affect the phenotypic expression because of the observed variability within each parent and among F1 plants from the same cross. Such non- genetic factors could be due to: environmental variables such as temperature, soil moisture, humidity, etc., which 53 alter plant response to ozone, and developmental non— uniformity within fumigated plants, unequal distribution of ozone inside the chamber, and human error in estimating scores on plants subjected to ozone damage. The four cultivars apparently possess slightly different minor alleles, therefore exhibit slightly different responses to ozone, and are affected and interact differently by and with the various environmentals variables. This can explain the slight difference in response to ozone within the two tolerant and the two sensitive varieties, and the difference in phenotypic stability. The segregation in the F2 generation in the cross between the two tolerant varieties (Table A5) may be due partially to the segregation of these minor alleles for which the parents differ. Continuous segregation within F3 families strengthens the assumption that the minor alleles are still segregating, although the effect of environmental variables in addition cannot be eliminated. The presence of both minor genes and environmental variables may explain why at least 5 response classes were necessary to visually classify ozone-treated plants. My attempts to find basic genetic interpretation by grouping the 5 classes into the two main categories were not fruitful in the cross FH x 0669 (Table A3). It is possible that this particular cross is one out of 20 cases in which probability theory states that the 3:1 ratio will be acceptable. At any rate, this suggestion should be rechecked. The results in this study agree with those of previous studies (Hanson et al., 1976; Cameron, 1975; and Butler 54 et al., 1979) that cytOplasmic genes or extra-chromosomal factors are probably not involved as a determinant of ozone tolerance. Environmental conditions in the field during this experiment were not under close experimental control. According to Taylor (1974), the complex interaction between climatic factors, ozone concentrations, exposure duration, soil conditions, and physiological characteristics of the plant determine the reaction of plant tissues. Therefore, the phenotype as observed in the field will be an imperfect indicator of the genotype. Ambient air pollution levels were not measured during the experiment. While ozone is the primary pollutant involved in the bio-injury of many beans, the effect of other pollutants such as 502, N02, and peroxyacetyl nitrate (PAN) cannot be ignored. Under field conditions it was impractical to classify the whole plant (due to large variability of ozone injury within each plant) into more than two categories; tolerant and sensitive. Nevertheless, the X2 for testing the good- ness of fit of the 9:7 ratio within the F2 generation of the cross between the two sensitive varieties (PHR x 0669) was statistically not significantly different from zero and similarly the 3:1 ratio in just one of four crosses between tolerant and sensitive varieties (Nep—Z x 0669). It is plausible that under the uncontrolled conditions in the field, and because of the imprecise evaluation of the whole plant reaction it was impossible to identify successfully 55 the genetic segregation of such a trait as ozone tolerance. However, the fact that the two major alleles (A and B) which express ozone tolerance, are dominant, and the presence of complementary interaction between them were demonstrated even under uncontrolled conditions. It is customary to perform progeny tests in breeding for traits with low heritability. Since ozone tolerance is affected to a great extent by environmental factors, its heritability is probably low. Information on the F2 plants gained via their F3 families increases the value of the heritability in this case which leads to an increase in the precision of genetic determination. Previous analysis of the inheritance of tolerance to ozone has been conducted primarily in a quantitative fashion. Hanson et a1. (1976) crossed seven petunia inbreds (ozone tolerant and ozone sensitive) in all possible combinations to yield a complete 7 x 7 diallel set. In addition, they transformed the visual injury levels (from 1 to 5) into number of hours prior to appearance of each particular level of injury. Hence, class 1 = no visible damage was transformed into 4.1 hours, whereas, the highest level of leaf damage corresponded to 6.8 hours. Working in this way, they constructed an analysis of variance to test for presence of additivity, dominance, and maternal effect. They concluded that the alleles which contribute to ozone tolerance seem to act primarily in an additive manner. Furthermore, they estimated component of variation including D, H, F, E, the dominance ratio, and heritability. In many papers like 56 Engel and Gabelman (1966) in onion, Papple and Ormrod (1977) in turfgrasses, Rasput and Ormrod (1976) in eggplant, Huang et a1. (1975) in tobbaco, and Butler et a1. (1979) in beans, the visual scores of ozone injury were transformed into percentages of injury area of leaf surface. By using such quantitative transformed data in typical quantitative genetic assays they determined also in what fashion (additivity, dominance, etc.) alleles for ozone tolerance act. Knudson et a1. (1977) suggested correlating the amount of ozone injury with decreasing chlorophyll concentration. In my opinion, the above three methods do not faith- fully represent overall ozone injury. Injured area on different leaf surfaces can vary greatly from each other in regard to the severity of the damage, e.g., the number of dead cells in cross section. On the other hand, chloro- phyll concentration does not tell us how much of the leaf area was damaged. Perhaps a combination of both methods, while tedious, would give a better estimation. The method which was used by Hanson et a1. (1976), is subjected to a great amount of error in its measurements, and in addition, they checked only one sensitive variety and then used the results on the other varieties, which probably increased the amount of imprecision. In addition, the term ozone- resistant which is widely used is not accurate, since even so-called ozone-resistant plants exhibit typical ozone injury symptoms under appropriate conditions. 57 Stomatal Conductivity Flecking on the upper surface (adaxial) of bean leaves is a common symptom of ozone injury on dicotyledonous plants although most of ozone uptake by the leaf occurs in the lower surface (abaxial) (Rich and Tomlinson, 1974). The authors assumed that the random pattern of stomata in the lower surface, the different geometry of the air passages through the spongy parenchyma, and the greater tolerance of spongy parenchyma cells than palisade cells to ozone make it unlikely that ozone injury would appear on the abaxial surface of the leaf. Often the first visible symptom of ozone toxicity is the death of the palisade parenchyma cells that line the cavities directly beneath the adaxial stomata (Heath, 1980). Stomatal conductivity has always been considered directly related to ozone injury, since ozone enters leaves through the stomata (Ting and Heath, 1975). According to research with different species including onion (Engel and Gabelman, 1966), tobacco (Turner et al., 1972), petunia (Thorne and Hanson, 1976), and beans (Butler and Tibbitts, 1979), ozone- tolerant varieties, due to their lower stomatal conductivity during ozone exposure, absorbed much less ozone in their leaves than sensitive varieties, and therefore, avoided leaf injury. Engel and Gabelman (1966), suggested that rapid regulation of the membranes of guard cells accounts for tolerance to ozone. In addition, differences in stomatal density which were found in tobacco by Dean (1972) and in beans by Butler and 58 Tibbitts (1979) between tolerant (lower density) and sensitive (higher density) varieties, according to the authors, may be part of the mechanism that protects plants from ozone injury. In this study, such differences in stomatal conductance and density between tolerant and sensitive varieties have not been found. On the contrary, the two tolerant varieties had larger values of both abaxial and adaxial stomatal conductance than the two sensitive varieties. Similar findings were reported in beans by Hucl and Beversdorf (1979), and in COWpea by Adepipe and Tingey (1979). Although in all the varieties stomatal conductance was significantly decreased due to ozone exposure, conductivity of the tolerant varieties was less affected by ozone after four hours of exposure, in contrast to the sensitive varieties. In View of the fact that alter- ation of the electric potential of guard cell membranes and permeability due to ozone could affect stomatal closure, it is reasonable that the guard cell membranes of the tolerant varieties were less affected by ozone than membranes in the sensitive varieties. This may be due to biochemical differ— ences, or due to a better ozone injury repair mechanism. The lower stomatal density mainly in the abaxial surface of the sensitive variety 0669 may partially account for its low stomatal conductance values. FH, on the other hand, had the highest stomatal density which can partially account for its high stomatal conductance. It is important to say that the total variability in the values of stomatal conductance was high mainly due to its dependence on many environmental variables. In conclusion, the above-suggested mechanism, 59 although appealing because of its simplicity, is not supported by the data of this study. The possibility that differences between tolerant and sensitive varieties are due to changes in the capacity to repair biochemical ozone injury was suggested already by Dugger et a1. (1962), Evans and Ting (1973), and Sutton and Ting (1977), and others. Physiological Study \ Foyer and Halliwell (1976), found that high concentra- tions of ascorbic acid (AA) in chloroplasts can react with oxidant radicals like superoxide (05'), and hydrogen peroxide (H202). One would assume that AA might react with ozone as well, and possibly prevent or alleviate the damage caused by the ozone. Such an interpretation was suggested by Freebairn and Taylor (1960), and by Dass and Weaver (1968). Hanson et a1. (1971), reported that ozone tolerant petunia varieties had higher AA concentrations than sensitive varieties on an area basis. On the fresh weight basis however, no such differences were found. Later, Thorne and Hanson (1976) con- cluded that the correlation of variety sensitivity to ozone with AA concentrations per unit area was not significantly different from zero. In the present study, AA concentrations were determined on a fresh weight basis and were much greater (lO-fold) than concentrations in petunia leaves. Although a significant reduction of AA concentrations occurred after ozone exposure the remaining concentrations were still high. This implies that in beans perhaps only a small amount of AA reacts directly with ozone or with its by-products, possibly 60 because endogenous AA in beans is not in a highly mobilized form. In addition, AA content of each of the four varieties (in comparison) was not significantly different prior to, and after ozone fumigation. These two facts indicate that AA plays a minor role in protection from ozone injury. Mountain (1963) demonstrated that ozone exposure caused an i2_yiyg_oxidation of reduced glutathione (GSH) in mouse lung. The simple tripeptide GSH is found in almost all living cells and takes part in numerous biochemical reactions, as well as helping stablize certain enzymes by preventing oxidation of thiol groups (Jocelyn, 1972). Most of the GSH in beans and other legumes is structurally different from ordinary GSH since it contains the amino acid B-alanine instead of glycine (Carnegie, 1963). However, the catalytic behavior of both is similar. It is possible that GSH, which can reduce oxidized thiol groups such as disulfide bonds or sulfonic acid (-——— SOZH) back into sulfhydryl groups (Ting and Heath, 1975), could play an important role in an ozone injury repair mechanism. Similar to previous studies with AA, application of exogenous GSH to different plant organs prior to ozone fumigation significantly reduced the level of injury. The amount of GSH in the four varieties of the present study was 100-fold smaller than AA. No differences in GSH concentrations were detected among the four varieties prior to ozone fumigation; however, after fumigation, GSH concentrations of Nep—2 (tolerant) rose significantly, while the concentrations in the remaining varieties significantly decreased. Therefore, it is possible that GSH takes part in 61 ozone injury repair. Furthermore, Foyer and Halliwell (1976) found that GSH will non-enzymatically reduce dehydroascorbate (the oxidized state of AA) back to AA. This implies that GSH may play an important role, too, in the limited contribution of endogenous AA to tolerance against ozone injury. Quite surprisingly, FH, the highly ozone tolerant variety, unlike the other tolerant variety Nep-2, exhibited reduction in GSH content after exposure to ozone. There are two possi- bilities for this outcome: a) that FH is endowed with another mechanism to tolerate chronic ozone concentrations, and b) that the extraction of GSH from FH leaves was not complete. Leaves of PH are substantially thicker than leaves of the other varieties (this was verified by using the electronic leaf area meter) which implies that perhaps FH after exposure to ozone had more GSH per unit area than the sensitive varieties or possibly more mesophyll cells and chloroplasts per unit area than the other three varieties, which would enable it to tolerate high concentrations of ozone. Dass and Weaver (1968) and Freebairn and Taylor (1960), significantly reduced ozone injury in beans by spraying leaves and roots with ex0genous AA and GSH prior to ozone fumigation. The data in the present study confirmed their results; however, it was found here that GSH sprays are more efficient than AA sprays in reducing injury which again suggests that GSH is a major component in ozone flecking repair mechanism. In addition, application of 62 both compounds 6 hours prior to ozone fumigation was more efficient than application 12 hours prior to fumigation, which suggests that the exogenous AA and GSH were subjected to an overall oxidation after application. Due to the oxidation, GSH is converted to its oxidized form, abbreviated GSSG, which can be converted back to the reduced form, GSH, by the enzyme glutathione reductase (GSSG reductase) in the following reaction: + GSSG reductasegy GSSG + NADPH + H // ZGSH + NADP In the present study, specific activity of GSSG reductase was much higher in the two tolerant varieties than in the two sensitives. Since GSH conversion to GSSG is due to the reduc- tion of S-—-S- bonds (bonds previously oxidized due to ozone) by GSH back to SH- groups, the higher GSSG reductase activity in the two tolerant varieties can be interpreted as higher ability to repair ozone damage of cell membranes. Sutton and Ting (1977), based on previous experiments by Dugger et a1. (1962) and Dugger and Palmer (1969), found that dipping bean leaves in glucose solution immediately following ozone fumiga- tion significantly reduced ozone injury. They suggested that glucose probably acts by providing the necessary energy to repair oxidized cell components. It is possible that glucose utilization via the pentose phosphate pathway which, according to Tingey et a1. (1975), is activated by ozone, yields NADPH molecules which increase the rate of GSSG reduction. The different activities of GSSG reductase between tolerant and sensitive varieties could be: 63 a) due to the presence of two isozymes that differ in rates of catalysis, which means that the isozymes which are formed by the structural genes of the tolerant varieties have higher rates of catalysis than the isozymes formed by the sensitive varieties, and/or b) due to quantitative differences in the forma- tion of GSSG reductase, viz., the regulatory genes of tolerant varieties produce more GSSG reductase molecules than the sensitive varie- ties. Schedle and Bassham (1977) found that the activity of glutathione reductase is inhibited by Zn+2,; therefore, higher Zn+2 concentration in leaves of the sensitive varie- ties in contrast to the tolerant varieties also could account for their low GSSG reductase activity. In general, the activity of GSSG reductase will vary due to alterations of the NADPH/NADP+ ratio, which increases during illumination (Lendzian and Bassham, 1975). This may explain why response to ozone flecking as reported by Heck (1968), and Taylor (1974) was affected by the length of the photoperiod prior to fumigation with ozone. The results from this study showed that exposure to ozone induced changes in GSSG reductase activity. Such changes were not consistent among susceptible and tolerant varieties; however, these changes did not alter the overall picture, viz., the tolerant varieties always exhibit (prior to and after fumigation) significantly higher levels of GSSG 64 reductase activity. The study of GSSG specific activity supports the hypothesis that the two tolerant varieties, FH and Nep-2, during ozone "invasion", due to their higher enzymatic activity, regenerate GSH faster from GSSG, and therefore, are able to more quickly reduce essential ozone- oxidized compounds in the leaf. This possibility led me to believe that probably the method which was used to extract GSH (quantitative assay) which was originally used in spinach leaves was only partially effective with FH leaves; viz., not all the GSH from FH leaves was released. Therefore, it is possible that FH leaves had a high or the highest GSH content prior to ozone fumigation. This condition is plausible considering the fact that FH is also the most ozone-tolerant variety; however, a conclusive answer to this matter may only be obtained through further studies. As reported by workers in cotton (Ting and Mukerji, 1971) and in beans (Cracker and Starbuck, 1972), it was found in this study, too, that there is a significant decrease in soluble protein levels within the leaf immediately after exposure to ozone. This suggests that ozone may affect protein metabolism by promoting protein hydrolysis. Generally, it seems that a good correlation may exist between the content of soluble protein in the leaf and a visual estimation of ozone injury. A continuous study of this aspect, which will yield scientific support for such correlation, will increase the precision in ozone injury determination. 65 Ethane and Ethylene Production John and Curtis (1977) showed that the unsaturated fatty acid, linolenic acid, is the main precursor of ethane in bean plants. Ethane production by plants was found to be associ- ated with tissue injury, which may be related to membrane destruction and peroxidation of linolenic acid (Liberman, 1979). Ting and Heath (1975) mentioned that exposure to ozone may cause peroxidation (ozonation) of double bonds in unsaturated fatty acids within membrane lipids. Since linolenic acid is very prevalent in leaf tissues, it probably serves as a target for ozone "attack". Therefore, quantitative differences in linolenic acid content in the narrow sense or unsaturated fatty acids content in the broad sense between cell membranes of tolerant versus sensitive varieties may account for the differences in ozone injury. If this assumption is correct, sensitive varieties (with higher linolenic acid content) will be expected to produce more ethane after ozone fumigation than the tolerant varieties with lower content of linolenic acid. Methionine, the sulfur-containing amino acid, is gener- ally the accepted precursor for ethylene, although both ethylene and ethane can be produced from linolenic acid hydrOperoxide (Dumelin and Tappel, 1977). Wilson et a1. (1978), reported that an analogue of rhizobitoxin, an inhibitor of ethylene from methionine, partially inhibited the emission of ethylene but not the emission of ethane from curcurbit leaf tissues maintained in bisulfite solution. This observation supports the idea that the two gases are 66 produced largely by different pathways; however, Wilson's results do not exclude the possibility that some ethylene may arise from peroxidized linolenic acid. Tingey et a1. (1976), found in a wide array of plant species that plants exposed to ozone produced more ethylene in the dark than in the light. On the other hand, Wilson et a1. (1978), reported that light enhanced both ethylene and ethane production. In full agreement with the results obtained by Wilson et a1. (1978) in curcurbits, Filner (personal communication) in cucumber, and Peisner and Young (1979) in alfalfa in which the production rates of both ethylene and ethane increased after exposure to 502, in this study production of ethane and ethylene was increased due to exposure to ozone (in comparison with non-fumigating plants). However, unlike curcurbits (Bressan et al., 1978), the two ozone sensitive bean varieties in the current study did not produce signifi- cantly higher amounts of ethane than the two tolerant varieties after ozone exposure. In spite of the fact that the leaf discs were incubated (after ozone fumigation) under high light intensity, which was claimed by Tingey et a1. (1976), to inhibit or slow ethylene production, ethylene production was significantly higher in leaf discs from sensitive varieties. In conclusion, the lack of consistency between ethane production and ozone injury suggests that in beans ozone- tolerant and sensitive varieties have similar concentrations of linolenic acid or in general similar unsaturated fatty acids content in the cell membrane. Therefore, tolerance to ozone in beans is probably not due to reduced content of 67 linolenic acid in particular or unsaturated fatty acids in general. Since ethylene production is promoted by almost any type of plant tissue injury, including ozone flecking, it is hard to prOpose a mechanism for ozone tolerance based on ethylene evolution. However, this burst of evolved ethylene following an ozone exposure suggests that ethylene is involved in the trigger mechanism of plant responses to ozone injury. Abeles (1973) found that in the presence of ethylene there is an increase in the activity of phenylalanine ammonia lyase, polyphenol oxidase, and peroxidase which are involved in the synthesis of phenolic compounds, and which are known to be activated during ozone exposure (Howell, 1974). The increase in the activity of these enzymes could be mediated through an ozone enhancement in the rate of ethylene production. In spite of the fact that one of the primary objectives of this study was to prOpose a mechanism for ozone tolerance, a distictive positive correlation between levels of visual ozone injury and ethylene production has been observed in this study. Such a correlation was studied by Tingey et a1. (1976), and as a result they suggested that ethylene measurements provide a more sensitive and accurate measure of plant response to ozone than the common visual injury determination. CONCLUSION The idea in this study has emerged that glutathione (GSH) and glutathione reductase (GSSG reductase) might be involved as a repairing mechanism of ozone injury. Halliwell and Foyer (1978), using affinity chromatography, found that spinach GSSG reductase, similar to animal and yeast GSSG reductase, is a dimer with subunits of similar size; therefore, it is possible that GSSG reductase in bean plants has a similar structure. This fact could be associated with the results from the genetic analysis in which at least two interacting major genes appear to be involved in the response of plants tolerant to ozone. Hence, by this hypothesis, the two dominant alleles A and B which increase ozone tolerance, code for the two "active" polypeptides of GSSG reductase, resulting in higher enzymatic activity in Nep-2 and FH (both tolerant and both AABB). In contrast, the lower enzymatic activity in the two sensitives, PHR and 0669 (AAbb and aaBB) in which dominant alleles of only one gene are present, is due to the formation of only one active polypeptide. The appearance of one dominant allele per each one of the two major genes (AaBb), as exists in F1 plants from the cross between the two sensitive varieties, would endow these plants with the active GSSG reductase isozyme. 68 69 The role of the minor genes in the expression of ozone tolerance is undefined, and they might be involved in determining the: number of mes0phyll cells and chloroplasts per unit of leaf area, intercellular space in the leaf, carriers involved in GSH and GSSG mobilization within leaf cells, structural entities in cell membranes, mainly in the plasmalemma, and a large array of regulatory genes which control in leaf tissues the amount of soluble proteins, SH dependent enzymes, structural proteins, saturated and unsaturated fatty acids, glycolipids, phospholipids, etc. The environmental variables which affect the expression of ozone tolerance can be classified into two major catego- ries: a) affecting directly or indirectly stomatal aperture which control ozone uptake into the leaf. b) affecting within the leaf reaction or processes which are associated with the presence of ozone or its by-products. In fact, the activity of GSSG reductase can be affected by environmental variables such as length of photoperiod (Lendzian and Bassham, 1975), and temperature (Esterbauer and Grill, 1978). The other suggested mechanisms for tolerance to ozone injury which were examined in this study, namely, differ- ences in response of guard cells to ozone, stomatal density, and variation in the amount of the unsaturated fatty acid linolenic acid, were not supported by the data from this 70 study. However, it is possible that these mechanisms may account for the variability (in regard to ozone tolerance) which exist among different species and genera. APPENDICES 72 mam m.~ Hem e.~ Hem m.~ mmm m.m «mm a.H Hem o.~ mmm m.~ on H.m awm e.~ emw e.m Hmm o.~ owe m.m mam m.~ mam a.~ oom m.m New o.~ mew e.m mam w.~ mew H.m eom m.~ oom o.~ wem o.~ Nam m.H me mom o.~ aooo oom o.~ awm m.o mmm o.m ham o.e mam o.~ «mm a.e men o.~ Hem m.e mam m.m ooe ~.e mam w.~ mes w.e Nae e.~ aae m.e mew ~.~ mom o.o New m.~ Hmm H.e mme ~.~ mam o.e Ham m.a mmm o.e mam m.H mmd ewm ~.e mudmz .nz among oooa .nz among moon sumanm> .ne among oooa .nz nmono oo3 samenm> .«.« no as mom 00 me .<.¢ mo me mmo no me .wcoNo QDflB coflummHEDM “mom A.3m mooa\mEv mm>mmH mumeflud may CH oflom oenuoowm can mmo mo coeumuucwocou "H XHDZMQAfi 73 omm m.w mmm m.w eom e.e mmm w.e mam a.w Hmm o.e eam w.w Ham ~.w mam H.m mam m.e mom ~.m Hmm m.w Ham m.e pom ~.w men a.e Ham o.w omm m.w mmm a.w New m.e omm o.m Sam o.e hem o.m mam o.w mm eam ~.m mooo oom e.e Hem H.m Hmm o.w wom m.m mam ~.w mum m.m mam m.w Hmm e.w mam w.w mom ~.w mom o.w mam o.e amm m.w Nmm m.w wem m.w oom e.e mam m.m oao w.w Hem m.m Hem o.w New o.o ham m.e mom m.m mud omm H.m mudmz .nz among ooos .ns among oooa snoanm> .83 Boone moon .03 among oooa sumnum> .¢.< no me now mo me .4.¢ no ms and mo me .wcoNo nuflz coflummHEsm mum A.3m mooa\mEv mm>mma SumEHud ca oflom venuoomm paw mmw mo coflumuucmocou “HH xHDmemd XI APPENDIX III: Ethylene discs (m 74 production of ozone injured leaf m ). Nep-2 FH PHR 0669 254 249 1515 1813 474 346 3966 1905 236 280 2865 2384 385 156 3747 2286 466 417 1817 2452 249 258 2953 3113 354 321 2073 4022 218 292 3251 4221 349 415 1929 3120 265 353 2003 2525 374 243 1816 3081 115 575 4312 4715 500 110 2677 3219 425 315 3233 4160 332 496 3870 3510 174 311 4022 2872 256 222 3021 2466 440 205 2866 4155 502 255 3032 4432 557 480 2843 3190 383 293 3611 4154 470 512 3416 2656 492 503 2430 3819 508 410 2931 2726 610 376 3115 2934 375 335 2932 3197 APPENDIX IV: Ethane production of ozone injured leaf discs (mmz). 75 Nep-2 FH PHR 0669 126 141 211 293 144 132 129 116 132 205 130 177 188 214 130 184 100 250 144 206 115 181 155 131 132 116 229 115 199 173 181 219 171 244 136 125 111 155 123 127 130 221 104 211 125 113 229 185 116 122 191 109 184 167 157 166 153 155 166 174 132 164 138 218 114 139 174 130 176 211 215 219 210 166 212 147 225 174 163 172 230 209 207 211 242 188 136 150 133 145 252 166 158 125 202 145 191 183 166 118 157 172 171 169 APPENDIX V: Ethylene production of non ozone treated leaf discs (mm ). 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