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Effects of nitrogen dioxide on gas exchange in phaseolus vulgaris leaves Srivastava, Hari Shanker 1974

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EFFECTS OF NITROGEN DIOXIDE ON GAS EXCHANGE IN PHASEOLUS VULGARIS LEAVES by HARI SHANKER SRIVASTAVA B.Sc, Gorakhpur University, Gorakhpur, India 1962 M.Sc, Gorakhpur University, Gorakhpur, India 1964 M.Sc, McMaster University, Hamilton, Ontario 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department , of Plant Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1974 In presenting th is thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is for f inanc ia l gain shal l not be allowed without my wri t ten permission. Department of Ptawk SC-itj^Q The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada i i ABSTRACT The present investigation was undertaken to survey the general features of physiological responses of plants to NO,, and to understand the mechanism of inhibition of gas exchange by NIX,. To achieve these objectives, the effects of NO,, on photosynthesis, respiration and transpiration and the rate of NO,, uptake by primary bean (Phaseolus vulgaris. L. cv. 'Pure Gold wax'), leaves were examined in various environmental conditions using an open gas flow system. Apparent photosynthesis, respiration and the evolution of CO,, into CO,,-free a i r , were a l l inhibited by NO,, concentrations between 1.0 and 7.0 ppm. The degree of inhibition was increased by increasing NO,, concentration and exposure time. A 2 and 5 h exposure to 3.0 ppm NO,, inhibited the gas exchange of bean leaves at a l l plant ages and in a l l the environmental conditions examined. Photosynthesis was most inhibited in leaves of intermediate ages, at optimum temperatures, at high light intensities, at relative humidities between 45 and 80% and in leaves of plants grown without any external source of nitrogen. The inhibition was rather less affected by changing C02 or 0 2 concentration. Maximum inhibition of respiration was observed in the youngest leaves, at high temperatures and in the leaves of nitrogen deficient plants. In most cases, the maximum inhibition of C02 exchange coincided with the maximum control rate in the absence of NO,,. The inhibition of transpiration by NO,, was generally small and in a few cases either there was no effect of NO,, on transpiration or i t was slightly increased by NO,,. This indicated that the primary effects of NO,, were within the leaf mesophyll and not on the stomata. i i i The uptake of N0 2 was also modified by plant age and environmental conditions. The rate of NO^  uptake increased with increasing concentrations of N02 and decreased with increasing exposure time. It was highest in the leaves of intermediate ages, in the light, at higher temperatures, at low and O2 concentrations, and in nitrogen deficient plants. In most cases, the maximum rate of N0 2 uptake was correlated with the maximum inhibition of gas exchange, but in several cases, i t was not. Although stomatal resistance influenced the rate of N0 2 uptake to some extent, mesophyll resistance to NO2 was mainly responsible for the regulation of its absorption. In addition to Phaseolus vulgaris L.,10 other angiosperm species were examined. All species absorbed substantial amounts of N02 from an atmosphere of 3.0 ppm NOg, and a l l experienced a concomitant inhibition of photosynthesis. The rates of N02 uptake and the degree of inhibition varied according to species. The average rate of N0 2 uptake after a 2 h exposure to 3.0 ppm N0 2 -2 -1 was 0.391 mg N02 dm h and the average inhibition of photosynthesis with the same dose of N02 was 14.3%. An estimation of N02 uptake on a worldwide basis indicated that a concentration of 0.1 ppm N02 in the world's atmosphere could provide as much as 11% of the total nitrogen requirement of the terres-t i a l plants. Furthermore, the experiments reveal that the effect of N02 on plant metabolism is not restricted to a particular pathway or process; rather i t is generalized. It appears that N02 may inhibit gas exchange by disrupting the structure of cell organelles and/or by interfering with the activities of enzymes. i v TABLE OF CONTENTS Page ABSTRACT • i i TABLE OF CONTENTS . . iv LIST OF TABLES . . vi LIST OF FIGURES . . . v i i ACKNOWLEDGEMENTS ix PART I. General Introduction 1 PART II. The Effects of Plant Age, NO2 Concentration and Exposure Time on the Inhibition of Leaf Gas Exchange by NO2 INTRODUCTION . . 6 MATERIALS AND METHODS 7 RESULTS Seedling age and response to NO2 14 NO2 concentration and the time course of NO2 effects . 21 Visible symptoms of NO2 injury 24 ' DISCUSSION 28 PART II I . The Effects of Environmental Factors on the Inhibition of Gas Exchange by NO2 INTRODUCTION 34 MATERIALS AND METHODS 35 RESULTS Irradiation 35 Temperature 36 Carbon dioxide andooxygen-concentrations, 41 Humidity . . . . . . . . . . . . . . . . 4 4 DISCUSSION 52 V PART IV. The Influence of Nitrogen supply during Growth on the Inhibition of Gas Exchange and Visible Damage to leaves by N02 INTRODUCTION 5 9 MATERIALS AND METHODS . . . 60 RESULTS Soluble and insoluble nitrogen within leaves ...... 61 Leaf gas exchange 61 Time kinetics of NO2 uptake 66 Source of nitrogen supply during growth 71 Visible symptoms of NO2 injury . 71 DISCUSSION . . . 71 PART V. General Discussion 80 PART VI. Conclusions . . 92 LITERATURE CITED 93 v i LIST OF TABLES Table Tit l e Page 11 — I . Effects of NO2 concentration on rate of transpiration and the uptake of NO2 by the darkened leaves 27 11 — 11. Effects of plant age, NO2 concentration and the duration of NO2 exposure on leaf diffusion resistances in light and darkness . . . . . . . . . . 30 1 1 1 - 1. Effects of high CO2 concentration ( ^ - 2 0 0 0 ppm) on the rates of apparent photosynthesis, transpiration and NO2 uptake in the presence of 3.0 ppm NO2 45 1 1 1 - 1 1. Effects of N02 on transpiration and N 0 2 uptake in C02~free air 50 I I I — I I I . Effects of 3.0 ppm N 0 2 on the rates of apparent photosynthesis, transpiration and NO2 uptake in 0% and 20 .8% 0 2 51 IIT-IV. Effects of atmospheric humidity on the rates of apparent photosynthesis, transpiration and N.O2 uptake in the presence of 3.0 ppm NO2 . . . . . . . . 53 III-V. Effects of NO2 on leaf diffusion resistances 54 IV-I. Effect of source of nitrogen on gas exchange in bean leaves in the presence of 3.0 ppm NO2 . . . . 72 V-I. Effects of a 2 h exposure to 0 or 3.0 ppm NO2 on the apparent rate of photosynthesis and N02 uptake in different species . 84 vi i LIST OF FIGURES Figure Title Page 11—1. Leaf chamber design . . . 8 11-2. Schematic diagram of the gas circuit 12 11-3. Time course of primary bean leaf growth 15 11-4. Effects of NO2 on the rates of apparent photosynthesis, transpiration and NO2 uptake by illuminated primary leaves of different ages 17 1 1 - 5. Effects of NO2 on t i i t rates of respiration, transpiration, and NO2 uptake by darkened primary leaves of different ages 19 11-6. Effects of NO2 concentration and duration of exposure on the relative rates of apparent photosynthesis and dark respiration by primary leaves of 12 d plants 22 I I - 7 . Effects of NO2 concentration and duration of exposure on the rates of transpiration and NO2 uptake by illuminated primary leaves of 12 d plants 25 I I I - l . Effects of light intensity on apparent photosynthesis, transpiration and NO2 uptake following 0, 2 or 5 h treatments with 3.0 ppm NO2 37 III-2. Effects of temperature on apparent photosynthesis, transpiration and NO2 uptake following 0, 2 or 5 h treatments with 3.0 ppm NO2 39 III-3. Effects of temperature on dark respiration, trans-piration and NO2 uptake following a 2 h treatment with 0 or 3.0 ppm NO2 42 1 > III-4. Effects of CO2 concentration on apparent photosynthesis,transpiration and NO2 uptake following 0 , 2 or 5 h treatments with 3 . 0 ppm NO2 46 111-5. Effects of 0 , 3 . 0 or 7 . 0 ppm N 0 2 on CO2 evolution into C0 2-free a i r (20.8% O2) 48 IV-1. Effects of nitrate supply during plant growth on 80% ethanol soluble and insoluble nitrogen of the primary leaf 62 v i i i Figure T i t l e Page IV-2. Effects of nitrate supply on apparent rate of photosynthesis, transpiration and NC^  uptake rate following 0, 2 or 5 h treatments with 3.0 ppm NU2 . . . 64 IV-3. Effects of nitrate supply on rates of dark respiration, transpiration and NO2 uptake in darkened leaves after 2 h exposure to 0 or 3.0 ppm N0 2 67 IV-4. Time kinetics of N.O2 uptake by the leaves of plants grown at different nitrate levels, 69 IV-5. Morphological appearance of the primary leaves after 7 days of exposure to 3.0 ppm NO2 for 5 h in the lig h t , from the plants grown at different nitrate levels 73 IV-6. Effects of nitrate supply on the specific rates of photosynthesis and respiration, and the percent inhibition of apparent photosynthesis or dark respiration by a 5 h (for photosynthesis) or 2 h (for respiration) exposure to 3.0 ppm NO2 78 V-l. Effects of NO2 concentration on rates of apparent photosynthesis and NO2 uptake following 0 or 5 h exposure 88 ix ACKNOWLEDGEMENTS I am grateful to my research supervisors; Dr. P.A. J o l l i f f e for his ready interest, timely suggestions, encouragement and guidance, and Dr. V.C. Runeckles for his encouragement and guidance during the course of the present work. Thanks are also due to Dr. G.W. Eaton for statistical analyses and to Mr. I. Derics and Mr. A. Hoda for their assistance in building the leaf chamber and the gas exchange system. Financial support to the author was in the form of a Commonwealth Scholarship. I also wish to acknowledge the forebearanee of my wife and daughter which made this study possible. 1 PART I General Introduction 2 The accumulation of pollutants in the earth's atmosphere in association with the development of modern industrial society has long been of concern to biological and medical scientists. The phytotoxicity of air pollutants was recognized more than a century ago (6). The earliest work on the biological effects of air pollutants, however, was confined to pollutants of a complex or undefined nature, such as smokes and fumes from industrial wastes, fuel combustion and volcanism. In 1890 in Germany, SO,, was the f i r s t individual phytotoxic air pollutant to be identified (72). Phytotoxicity of other air pollutants such as fluoride, HCl gas, chlorine, iodine vapour, HI gas, oxides of nitrogen, ammonia, mercury vapour, smog, ethylene, carbon monoxide, HCN gas, H,,S and sulfur vapour were well recognized by 1951 (65). Damage to plants by smog in North America was f i r s t reported by Middleton et_ al_. (45) during 1944. Romaine lettuce and other leafy vegetables as well as ornamenlalsisuch as petunias, were found to be par-ticularly susceptible to damage by smog. Although there are eight different oxides of nitrogen, only NO and NO,,, commonly referred to as NOx, are the significant pollutants produced by man's activities. These pollutants may cause either acute or chronic injury to the plants. Acute injury is associated with the visible symptoms caused by high pollutant concentrations. Chronic injury, on the other hand, may or may not be associated with visible symptoms, and is caused by long term exposures to low levels of pollutants. Interest in NO,, was promoted by the report in 1952 by Haagen-Smit (17) that smog was the result of a reaction between nitrogen oxides and hydrocarbons in bright sunlight. In 1954 some effects of N02 on f i e l d plants were reported from Italy (29). Nitrogen dioxide was observed to cause necrotic stem lesions, defoliation, dieback and death of peach and black locust trees. Following that report, Bennedict and Breen (4) found that many weeds were damaged by 3 20 ppm NO,,. They observed two types of symptoms of N02 injury: (i) collapsed, white or brown irregularly shaped small necrotic lesions appearing between the large secondary veins near the leaf margin, and ( i i ) waxy shiny and green coating of leaves of certain species. More recently,Ma.cLean et a l . (39) exposed 6 citrus varieties and 6 ornamental species to high N02 concen-trations (10 to 250 ppm) for short time intervals (10 min to 8 h). These acute doses of N02 were found to cause rapid tissue degeneration, leaf necrosis and defoliation. The effects on plants of lower N02 concentrations have also been investi-gated. Middleton et al_. (44) found visible damage in pinto beans after a 4 to 8 h exposure to 3.0 ppm N02. Taylor and Eaton (61) studied the effects of exposing pinto bean or tobacco to approximately 0.5 ppm N02 for 10 to 22 days. They observed that N02 caused gradual changes in plant appearance. Growth was inhibited, foliage was more green, the leaf margin was curled downward, but there was no visible necrosis. In citrus trees, long term exposure to air containing? less than 0.5 ppm N02 has been found to reduce growth (68). In another investigation, continuous exposure of naval orange trees to 0.5 to 1.0 ppm NQ2 for 35 days caused severe defoliation and leaf chlorosis (67). At 0.5 or lower levels of N02, leaf drop was increased and f r u i t yield was reduced. Nitrogen dioxide may be introduced into the earth's atmosphere from natural or from man-made sources. These sources include microbial action, lightning in the upper atmosphere, forest f i r e s , and the burning of coal, petroleum and gas. It is l i k e l y that the N02 generated by human activities accounts for only about 5% of the N02 which enters the world's atmosphere (54). The atmospheric concentrations of N02 in urban areas are normally below 0.5 ppm, but concentrations as high as 3.93 ppm occasionally have been observed (1). It seems li k e l y that long term exposures to such N0? concentrations w i l l be 4 detrimental to the growth of some, i f not most, plant species. The chronic effects of low concentrations of NG^  may not be very apparent in terms of visual symptoms of injury; instead they may be evident as less obvious physiological changes resulting in decreased plant growth. Thus, in order to evaluate the effects of low NG^  concentrations more f u l l y , i t is necessary to study the physiological responses of plants to HO^. Such an approach was taken by H i l l and Bennett (23), who studied the effects of NO and N0 2 on photosynthesis in alfalfa and oats. At concentrations above 0.6 ppm both pollutants inhibited rates of photosynthesis during a 2 h exposure. The effects of N0 2 on other physiological processes, however, have not been investigated. The research described in this thesis is a systematic examination of the effects of N0 2 on the processes of photosynthesis, photores'piration, dark respiration and transpiration under a variety of environmental conditions. It was expected that the results of this research would provide some insight into the general mechanisms of N0 2 effects and that information bearing on the ecological significance of NO2 would also be obtained. The research is des-cribed in the following three parts. Part II of the thesis deals with the effects of NO2 concentration and exposure time on the gas exchange of primary bean leaves. That chapter also considers the effects of plant age on response to N02. Part III describes the physiological effects of N0 2 under different conditions of irradiance, temperature, humidity, CO2 concentration and O2 concentration. Part IV considers the effects of NO2 on gas exchange of leaves on plants grown at different nitrogen levels, A general discussion (Part V) which includes a survey of the susceptibility of different plant species to N0 ? and a l i s t of conclusions (Part VI), complete the thesis. 5 PART II The Effects of Plant Age, NG^  Concentration and Exposure Time on the Inhibition of Leaf Gas Exchange by NCL 6 INTRODUCTION Nitrogen oxides (NO ) are major components of photochemical smog. Large A amounts of NO are produced during high temperature combustion of coal, o i l , A natural gas or gasoline in power plants and internal combustion engines. Robinson and Robbins (54) calculated that the annual global production of nitrogen oxides from pollutant sources is 53 x 10 tons. In heavily indus-trialized areas and areas with high motor vehicle densities, such as Los Angeles, average daily concentrations of NO vary from 0.02 to 0.9 ppm and some-A times approach 4 ppm (64), with the highest levels occurring during the night (1). Among the different oxides of nitrogen, nitrogen dioxide is the most significant, since i t is regenerated by autocatalysis in sunlight and i t is highly toxic to living organisms. Some effects of NO^  on plants have been documented. Middletoh et_ al_. (44) reported damage to pinto beans by a 4 to 8 h exposure to 3.0 ppm NO,,. Taylor and Eaton (61) observed that exposure of pinto bean and tomato seedlings to 0.5 ppm NO,, for 10 to 22 days suppressed plant growth, increased total chloro-phyll concentration and caused distorted leaf development. Nitrogen dioxide has also been related to a reduction in growth of citrus trees (68). Studies with oats and alfalfa (23) and with citrus (68) have indicated that an inhibition of photosynthesis may be responsible for the suppression of growth by NO,,. The effects of N02 on other physiological processes in plants, however, have not been closely examined. In the present investigation, the effects of different exposure times with different N02 concentrations on the rates of photosynthesis, dark respir-ation, transpiration and N02 uptake by primary bean leaves have been examined. Since the sensitivity of plants to airborne oxidants (63) and smog (14) is related to plant age, the effects of age on leaf gas exchange and response to N09 have also been studied. 7 MATERIALS AND METHODS Four or five seeds of bush bean (Phaseolus vulgaris L. cv. 'Pure Gold wax') were sown in 16 cm pots containing moist vermiculite. Plants were grown 4 in Percival PGC-78 controlled environment chambers at an irradiance of 4 x 10 -2 -1 erg cm s (400 to 700 nm) during a 16 h photoperiod, and at a constant tem-perature of 25| 1°. Six days after sowing, the plants were thinned to 2 plants per pot, and they were watered daily with half-strength nutrient solution (No. 1 in reference 26) which was modified to include 10 mM KNOg as the sole source of nitrogen. At appropriate ages, primary leaves on intact plants were enclosed within a 2.0 l i t r e transparent plastic chamber (Fig. 11—1) and exposed to experimental conditions for 45 min before N02 treatments or gas exchange measurements were made. For photosynthesis or respiration, the gas exchange measurements commenced 4 or 8 h respectively after the onset of the daily photoperiod. Leaf temperature in the chamber was detected by a 24 gauge copper-constantan thermocouple placed against the lower (abaxial) side of the leaf. Leaf temperature was controlled at 25± 1.0° by the circulation of water from a Haake model FT thermoregulator through a jacket surrounding the chamber. For photosynthesis, radiant energy was supplied by three 300 W 'Cool Beam' (General Electric) incandescent lamps and the level of irradiance was controlled by adjusting the distance of the lamps from the chamber. A 14 cm water f i l t e r was interposed between the lamps and the leaf chamber to absorb excess infrared energy. During the photosyn-thesis experiments, the enclosed leaves were exposed to an irradiance of 2 x 10 erg cm" s" (400 to 700 nm). For measurements of dark respiration, the chamber was darkened by covering i t with an opaque cloth. The air within the chamber was ventilated by a stainless steel tangential blower (Lau T2-6) driven by an external motor. The atmospheric resistance to water vapour diffusion 8 Figure I I - l Leaf Chamber Design. A. Median longitudinal (lengthwise) section. B. Median transverse (widthwise) section. WJ = water jacket; RF = rotary fan; TC = thermocouple; L = leaf; MF = manifolds; NT = nylon thread. 10 11 within the chamber was 1.5 s cm" as estimated from water loss measurements on wet blotting paper (30). Fig. 11-2 shows a schematic diagram of the gas circuit used to measure the net exchange of CO,,, water vapour and NO,, by the enclosed leaves. Outdoor air was withdrawn from 15 m above ground level and was dried and passed through the sample (for photosynthesis) or reference (for respiration) cell of a Beckman IR 215A infrared gas analyzer. The air stream was then humidified by bubbling i t through d i s t i l l e d water at 17.5 ± 0.5°. If required, NO,, from a compressed cylinder containing 0.095% N02 in N 2 was metered through a Matheson R-2-15 AAA flowmeter (Fl) into the gas stream, and the resulting mixture was warmed to 25 ± 0.5° before i t entered the chamber containing 335 ± 5 ppm C02, 20.8% 0 2, 45 ± 3% relative humidity and N02 as required. The experimental con-ditions were held constant while gas exchange measurements were being carried out on any particular leaf. A differential psychrometer (58) was included in the circuit to detect the humidity difference between the gas entering and leaving the leaf chamber. The psychrometer was bypassed during measurements of leaf NO,, uptake to avoid N02 absorption by the psychrometer wet bulb wicks. The leaf chamber could also be bypassed to verify the concentration of N02 in the gas stream before i t was directed to the chamber. Near the end of the gas c i r c u i t , a valve was used to direct the gas stream to either an N02 meter (Mast model 724-11) or to the remaining cell of the IR 215A. The gas flow rate through the leaf chamber and through each side of the differential psychrometer was 1.5 1 min"^ as indicated by Gilmont R-452 (F 2 and Fg) and Matheson R-6-15 (F^) flowmeters. The output signals from the Mast meter and from the IR 215A were recorded on a Brush model 816 multipoint recorder. Full scale recorder deflection was Figure II-2 Schematic Diagram of the Gas Circuit. P-j and P^  = a i r pumps; D^ , D^  = MgClO^ drier; IRGA = infrared gas analyzer (Channel 1); IRGA2 = channel 2; H-|, H2 = humidifiers; M = mixing vessel; C = plastic c o i l ; LC = leaf chamber; PS = psychrometer; F-|, F 2 > Fg, F^  = flow meters; V = four way valve; MM = mast meter; A = signal amplifier; REC = multipoint recorder; ICE = ice dessicator; CH = charcoal f i l t e r ; T-j, T2 = temperature controlled baths. 14 equivalent to 10 ppm NO,, or to a CO,, concentration difference of 100 ppm between the' cells of the IR 215A. Six copper-constantan thermocouples were positioned in the gas circuit for temperature measurements, and they were connected by a selector switch to a reference thermocouple at 0°C. A Weston model 315 digital panel meter indicated the thermocouple temperatures with an accuracy of ± 0.05°. After a series of gas exchange measurements was completed, the primary leaf was excised, removed from the leaf chamber, and it s area (one surface) was determined with an a i r flow planimeter (31). Leaf dry weight was determined after enclosing the leaf in an oven at 65° for 24 h. The rates of photosyn-thesis, respiration, transpiration and NO,, uptake were calculated from the change in composition of the gas stream as i t passed through the leaf chamber and from the gas flow rate through the chamber (13). Leaf diffusion resis-tances were calculated from the concentration gradients of water vapour, CO,, or NO,, and from the rates of influx or efflux of those gases per unit leaf area (one surface only). Diffusion coefficients used for water vapour, CO,, and N02 were 0.239, 0.139 and 0.135 respectively (30). In these investigations, the varia b i l i t y in gas exchange rates among plants exposed to similar treatments was quite small. As noted in the caption of Fig. II-6, the average values of the apparent rate of photosynthesis and rate of respiration for 5 replicate determinations were 15.26 ± 0.61 mg C02 -2 -1 -2 -1 dm h and 2.31 ± 0.21 mg dm h respectively. RESULTS Seedling Age and Response to NOp Measurements of leaf area a'nd leaf dry weight indicated that rapid expan-sion and dry weight increase continued for the primary leaves until 12 days 15 Figure 11-3 Time Course of Primary Bean Leaf Growth. Values for leaf area and dry weight are means from 6 leaves taken from different plants. 16 17 Figure II-4 Effects of N02 on the Rates of Apparent Photosynthesis, Transpiration and N02 Uptake by Illuminated Primary Leaves of Different Ages. Leaves were exposed to 0 (broken lines) or 3.0 ppm N02 (solid lines). Measurements were taken after 5 h of exposure. 00 19 Figure II-5 Effects of NC^  on Rates of Respiration, Transpiration and NO Uptake by IDarkened.rd Primary Leaves of Different Ages. Leaves were exposed to 0 (broken lines) or 3.0 ppm N02 (solid lines). Measurements were taken after 2 h of exposure. 21 after sowing (Fig. 11-3). Fig. 11-4 summarizes the effects of single 5 h exposures to 0 and 3.0 ppm N02 on gas exchange by illuminated primary leaves of different ages. For leaves which were not treated with N02, apparent photosynthesis and transpiration rates increased with plant age up to 11 days after planting, and thereafter the rates declined. Nitrogen dioxide inhibited apparent photosynthesis at a l l ages, but the degree of inhibition was greatest at 12 days. For example, N02 treatment depressed apparent photosynthesis by 15, 30 and 13 percent at plant ages of 8, 12 and 16 days respectively. Transpiration rate, however, was inhibited by 4 to 10 percent by NO,,, and the degree of inhibition was not strongly affected by plant age. The rate of NO,, uptake was greatest for leaves on 10 day-old plants, at 326 yg N02 dm"2 h"1-; and i t declined to 280 yg N02 -2 -1 dm h at 16 days. With darkened leaves (Fig. 11-5) the rates of transpiration and N02 uptake were much less than in the light. The effects of NO,, on transpiration in the dark were minor, and the rate of N02 uptake was again highest at the inter-mediate plant ages. The rate of respiration in untreated leaves was relatively constant with age, except for leaves from 8 day^old plants which exhibited a high rate. Nitrogen dioxide caused substantial inhibitions of respiration rate (36 to 49 percent] at a l l the ages surveyed. NOQ Concentration and the Time Course of N02 Effects Additional experiments were done with several N02 concentrations to deter-mine the time course of N02 effects on gas exchange by leaves of 12 day-old plants. After a 5 h fumigation period, apparent photosynthesis was significantly inhibited by N02 concentrations greater than 0.5 ppm (Fig. II-6). The difference between the rate of photosynthesis at 0 and 0.5 ppm NO,, was, however, not Figure 11-6 Effects of N02 Concentrationjand Duration of Exposure on the Relative Rates of Apparent Photosynthesis and Dark Respiration by Primary Leaves of 12 d Plants. For apparent photosynthesis: 100 = 15.26 ± 0.61 mg C02 -2 -1 dm h ; for dark respiration: 100 = 2.31 ± 0.27 mg -2 -1 C02 dm h (average values from determinations on 5 replicate plants). The effect of N02 above 0.5 ppm was significant at 5% level (one way anova test). 23 co CO LU X 100 CO o O u_ ^ O II CQ L U Q Q. 50| uu or Q_ < O7.0 0 Ch) z 100| o Sc Q_ oo 75| -AO.OH 50l Ol— O 0.5 1.0 1.5 2.0 DURATION OF N O g FUMIGATION Ch) 24 significant at 5% level. The response of apparent photosynthesis to NO,, concentrations above 0.5 ppm tended to be non-linear, with less response to additional NO,, at the higher NO,, concentrations tested. Above 1.0 ppm N02, the time course of the response to N02 was also non-linear, with more than half of the final inhibition of apparent photosynthesis occurring during the f i r s t hour of N02 fumigation. The general pattern of the time course of N02 effects on dark respiration (Fig. II-6) was similar to the response of apparent photosynthesis. Respiration, however, was more severely inhibited by N02 than was apparent photosynthesis. The rate of transpiration of illuminated leaves was unaffected by N02 concentrations less than 3.0 ppm. At higher N02 concentrations (Fig. 11-7), there was a slight inhibition of transpiration which increased linearly with fumigation time and which reached 17 percent after 5 h in 7.0 ppm NO,,. In the dark, 2 h exposures to NO,, concentrations from 1.0 to 7.0 ppm had minor effects on the rate of transpiration in leaves of 12 day-old plants (Table 11—I). The rate of uptake of NO,, by illuminated leaves was related to both NO,, concentration and fumigation time (Fig. 11-7). At any particular concentration there wa,s a. steady decline in N02 uptake a,scfumigation continued. At any time, however, the increase in rate of NO,, uptake with increasing NO,, concentration was linear over the concentration range surveyed. Uptake of N02 by the darkened leaves was lower than in the illuminated leaves and increased with increasing concentration of N02 (Table I I - I ) . Visible Symptoms of NOp Injury Visible symptoms of N02 injury were not immediately apparent following the N02 treatments used. When plants were returned to normal growing conditions after fumigation with 7.0 ppm N0?, however, visible symptoms of N0? damage Figure 11-7 Effects of Concentration and Duration of Exposure on the Rates of Transpiration and N09 Uptake by Illuminated Primary Leaves of 12 d Plants. 26 DURATION OF N0 2 FUMIGATION (h) Table II-I Effects of NOp concentration on rate of transpiration and the uptake of N0? by the darkened leaves.'* Concn. of NO^  Rate of transpiration Rate of NO^  uptake ppm g H20 dm"2 h _ 1 mg N02 dm"2 h - 1 0 0.70 • 1 0.76 0.054 3 0.71 0.173 7 0.60 0.301 *Measurements were made after 2 h of exposure 28 appeared within 7 to 10 days. These symptoms included a few brown and white necrotic spots on the margin of the leaf which are typical of NO,, damage (61). Leaves exposed to lower NO,, concentrations did not develop noticeable symptoms of injury. DISCUSSION Respiration, apparent photosynthesis and transpiration can a l l be inhibited by several hours of exposure to low concentrations of NO,,. In com-parison with i t s rate in the absence of NO,,, respiration was the process most strongly inhibited by atmospheric N02 (Fig. II-6), and such an effect has not previously been reported. Apparent photosynthesis, in turn, was more sensitive to N02 than was transpiration, and the responses of these processes to NO,, were related to plant age, NO,, concentration and exposure time. The effect of NO,, on photosynthesis in bean was less than that found by other workers for alfalf a and oats (23). For example, 2 h treatments with 7.0 ppm N02 inhibited apparent photosynthesis in bean by 38 percent, while oats and alfalfa (23) were inhibited by approximately 69 percent. At this time, there is insufficient evidence to indicate whether these interspecific differences in response to N02 are largely controlled by genotype, or whether they arise mainly from differences in plant age, from different conditions during growth or from different techniques of measurement. The moderate inhibition by NO,, of transpiration by illuminated leaves (Figs. II-4, 11-6) suggests that NO,, can cause partial stomatal closure. It is therefore of interest to determine whether the effects of N02 on other processes of gas exchange can be attributed to changes in stomatal diffusion resistance. For this reason, the stomatal ( r g ) or mesophyll (r ) leaf diffusion 29 resistances to water vapour, carbon dioxide or N02 were calculated for several of the experimental conditions used. Table 11—11 shows that the effects of N02 on r g were in a l l cases small. It follows that stomatal closure, which may contribute to the inhibition of photosynthesis by ozone (24), is not a major component of leaf response to NO,,. This is also indicated by the obser-vations that 1.0 ppm NO,, inhibited apparent photosynthesis but not transpiration. Furthermore, transpiration differed; from apparent photosynthesis and respiration with respect to i t s pattern of response to NO,, treatments at different ages and fumigation times. It is reasonable, therefore, to conclude that the responses of apparent photosynthesis and respiration to N02 largely reflect changes in rm ^ o r c a r' 3 0 n dioxide, even though this could not be calculated for darkened leaves. The inhibition of transpiration during N02 fumigation in the light may then be viewed as the consequence of elevated carbon dioxide concentration within the leaf caused by photosynthetic inhibition. The minor effects of N02 on transpiration in the dark (Table II-I) are consistent with this inter-pretation. The linear relationship between the rate of N02 uptake and N02 concentration (Fig. 11-7) is in accord with previous studies (22), although the modifying influence of fumigation time (Fig. 11-7) has not previously been reported. Since the responses of respiration and apparent photosynthesis to N02 concen-tration were non-linear, i t is evident that NO,, uptake was not linearly related to i t s effects. Instead, there was a tendency for the effectiveness of N02 as an inhibitor to become saturated at high uptake rates or long exposure times. The rate of NO,, uptake diminished as exposure time increased (Fig. 11-7). This could have been due to the gradual accumulation of N02 within the leaf, thereby decreasing the concentration gradient driving the N02 influx. In addition, or as an alternative, the decreased NO,, uptake may have resulted Table 11-11 Effects of plant age, N02 concentration and N02 fumigation time on leaf diffusion resistances in light and darkness. Experimental Conditions Leaf Diffusion Resistances ( s cm~^)* Illumin- Plant ation Age (days) N02 Concen-tration (ppm) Fumiga-tion Time (h ) rs SH20 rs s c o 2 \ o 2 r s SN0 2 \ o 2 Light 8 0 5 0.95 1.63 13.36 _ _ 3 5 1.08 1.86 16.23 1.91 1.91 12 0 5 0.56 0.96 10.47 - -3 2 0.73 1.25 13.67 1.29 2.12 3 5 0.79 1.36 17.04 1.40 2.70 7 2 0.83 1.43 18.39 1.47 2.28 7 5 1.00 1.72 27.49 1.77 5.09 16 0 5 1.15 1.36 14.23 - -3 5 1.44 1.70 15.84 2.55 2.93 Dark 8 0 2 5.56 9.56 ** • - -3 2 6.01 10.33 ** 10.64 0.04 12 0 2 5.15 8.85 ** - -3 2 4.71 8.10 ** 8.34 0.93 7 2 6.26 10.76 ** 11.08 1.98 16 0 2 7.46 12.82 ** - -3 2 7.67 13.18 ** 13.57 2.71 Atmospheric resistance for water vapour was 1.5 s cm" ** Since the C0 2 concentration within respiring cells was unknown, r for C02 could not be estimated for darkened leaves. 31 from increased internal resistance (r ) to N0o assimilation. To evaluate these m c possibilities, time course experiments were carried out in which 7.0 ppm N02 treatments were interrupted by 30 min N02-free periods after 2 h of fumigation. Since the rate of N02 uptake was rapid, i t was presumed that the N02~free period would greatly diminish internal N02 accumulations i f such accumulations existed. When the N02 treatment was resumed, however, i t was found that the N02 uptake rate was the same as before the interruption. It was concluded from this that the changes in the rate of N02 uptake during constant fumigation were not due to internal accumulations. Instead, they were caused by changes in r m for N02 which were not quickly reversible for N02 removal. The existence of r m for N02 also indicates that the absorption of N02 by leaves involves steps in addition to the gaseous diffusion of N02 to the mesophyll cell walls. Table 11 — 11 includes estimates of r for N0o which were derived with the m 2 assumption that the N02 concentration at the end of the N02 diffusion pathway was effectively zero. Mesophyll resistance for N0o was less than r for r 2 m carbon dioxide in a l l cases. This is in accord with the high rates of N02 uptake, despite low external N02 concentration, which were consistently observed in these investigations. The r m for N02 was increased by increasing N02 concentration, increasing fumigation time, and possibly by illumination. Plants can reduce n i t r i t e to ammonia using reduced ferredoxin or reduced NADP (52). It has been suggested that N02 may inhibit photosynthetic carbon dioxide fixation by competing with carbon dioxide for reductant generated by the photosynthetic light reactions (23). The maximum rate of N02 uptake observed in this study, however, was less than 2 percent of the prevailing apparent rate of photosynthesis. Thus, a s t r i c t competition between n i t r i t e reduction and carbon dioxide reduction is insufficient to account for the large inhibitions of apparent photosynthesis which \I=. have observed with N0?. 32 Similarly, N02 is a strong oxidant, and i t s effects could be related to this property. Another strong oxidant, peroxyacetyl nitrate (PAN) can cause the direct oxidation of NADPH in vitro (47). Once again, however, the rate of N02 uptake is too slow compared with the apparent rate of photosynthesis to relate the effects of N02 to coupled oxidation of NADPH by N0 2 > There are numerous other ways, however, in which N02 could inhibit photo-synthesis. Cyclic photophosphorylation, for example, could be a susceptible si t e , since PAN has been shown to inhibit cyclic photophosphorylation in chloroplasts isolated from bean leaves (34). More dndarectly, ammonia accumul-ation following n i t r i t e reduction could inhibit photosynthetic ATP production (2). The swelling of chloroplast thylakoids in response to 3.0 ppm N02 (73) could well be associated with such effects. The mechanisms whereby N02 might influence respiration are even less certain. It is clear fltrom the present investigation that N02 exerts the greater part of i t s effects not on the diffusion pathway but on the processes of C02 exchange within the leaf i t s e l f . Perhaps i t is significant that N02 is most inhibitory to respiration and apparent photosynthesis at the ages when these processes are most active (Figs. II-3, II-4, II-5). Additional research, however, is required to determine the mechanisms of the effects of N02 on leaf gas exchange as well as the influence of other factors which regulate the magnitude of those effects. PART III The Effects of Environmental Conditions on the Inhibition of Leaf Gas Exchange by Nitrogen Dioxide 34 INTRODUCTION Several investigations have indicated that environmental conditons can modify the susceptibility of plants to oxidant air pollutants. For example, light intensity (11, 19, 20, 21), temperature (19, 20, 21), atmospheric humidity (19, 21, 51), C02 levels (19, 21) and soil nutrient levels (49, 50) can influence the visible effects of pollutants on plants. These investigations have provided insight into the environmental regulation of plant response to air pollutants. In many cases, however, previous research on this topic has involved the use of complex or undefined mixtures of pollutants, and qualitative visible symptoms of pollutant damage have been used to assess plant response. In a previous study (Part I I ) , i t was observed that concentrations above 0.5 ppm of the individual air pollutant NO,,, substantially inhibited both apparent photosynthesis and dark respiration in primary leaves of bean. While the effects of N02 on gas exchange were evident within the f i r s t hour of treatment, visible symptoms of leaf injury only occurred several days after fumigation with the highest level of N02 studied - 7.0 ppm. The magnitude of the effects of N02 on gas exchange was affected by leaf age, and i t was increased by increasing N02 concentration and exposure time. The present investigation was undertaken to evaluate the effects of light intensity, temperature, relative humidity and atmospheric C02 and 0 2 levels on plant response to NO,,. This work was intended to supply information of potential ecological value and to provide a perspective for the evaluation of possible mechanisms of N09 effects on plants. 35 MATERIALS AND METHODS Single attached primary leaves on 12-day old bush bean (Phaseolus  vulgaris L. cv. 'Pure Gold wax') plants were used for gas exchange measurements as previously described (Part I I ) , except for one modification in procedure. The concentrations of C02 and 0 2 in the gas stream entering the leaf chamber were established using an LKB 12000 Varioperpex pump to inject a gradual flow of pure C02 into a stream of C02 free a i r (20.8% 0 2) or C02 free Ng. A Beckman IR 15A infrared gas analyzer was used to minitor the st a b i l i t y of the C02 concentration in the resulting gas stream before i t was humidified in the system previously described (Part I I ) . Throughout a l l of these investigations, leaves were allowed to adjust to the environmental conditions of irradiance, temperature,.'.humidity, C02 concen-tration and 0 2 concentration for 45 min before gas exchange measurements and N02 treatments were begun. Except where otherwise indicates, these conditions were 2 x 10 5 erg cm"2 s" 1, 25 ± 1°C, 45 ± 3% R.H., 335 ppm C02 and 20.8% 0 2 respectively. These conditions as well as the N02 concentration were held constant during measurements with any particular leaf. Rates of apparent photosynthesis, dark respiration, transpiration and N02 uptake were determined as described before (Part I I ) . The rates of gas exchange were known from previous work to b<e very uniform for leaves placed in similar conditions. In a l l cases, single primary leaves from 2 different plants were used to obtain replicate measurements for each condition surveyed. RESULTS Irradiance. An i n i t i a l experiment was carried out to examine the influence of N02 on processes of gas exchange under different conditions of irradiance. In the absence of N0?, the apparent rate of photosynthesis increased with 36 increasing irradiance until saturation was achieved at about 3.5 x 10 erg cm s (between 400 and 700 nm) (Fig. 111-1). Exposure to 3.0 ppm N02 for 2 and 5 h inhibited apparent photosynthesis at a l l levels of irradiance, and the degree of inhibition was greater for the longer exposure time. While the absolute magnitude of photosynthetic inhibition by N02 was greatest at high irradiance, the percent inhibition was greatest at low irradiance. For example, 4 -2 -1 at 21 x 10 erg cm s of irradiance, a 5 h exposure to 3.0 ppm N02 inhibited -2 -1 photosynthesis by 5.09 mg of C02 dm h or 33% of the control value while the inhibition at 0.5 x 104 erg cm"2 s"1 was 2.0 mg C02 dm"2 h"1 or 54% of the control value. Also, the data suggest that N02 treatment caused the "light" 3 compensation point for apparent photosynthesis to increase from about 0.8 x 10 3 -2 -1 to about 2.2 x 10 erg cm" s" during the 5 h exposure period (Fig. I I I - l ) . The rate of transpiration was less sensitive to changes in irradiance 4 -2 -1 than was apparent photosynthesis and above 3.5 x 10 erg cm s the increase in transpiration rate with increasing irradiance was quite gradual (Fig. I I I - l ) . Exposure to N02 caused slight decreases in transpiration rate at a l l levels of irradiance. Therefore, the effect of N02 on r g H20 was also l i t t l e (Table III-V). The uptake of N02 was l i t t l e affected by irradiance, although there appeared to be a small decline in the rate of N02 uptake at the lowest levels of irradiance used (Fig. I I I - l ) . The differences in the rate of N02 uptake, however, were not significant at 5% level. Temperature. The effects of leaf temperature and N02 treatment on apparent photosynthesis, and on transpiration and N02 uptake during photosynthesis, are summarized in Fig. III-2. In the absence of N02, apparent photosynthesis was relatively rapid over the range 20° to 35° and the optimum temperature was approximately 30°. At a l l the temperatures used, N0? inhibited apparent 37 Figure I I I - l Effectsof Light Intensity on Apparent Photosynthesis, Transpiration and N0? Uptake Following 0, 2 or 5 h Treatments with 3.0 ppm NO,,. 38 Figure 111-2 Effects of Temperature on Apparent Photosynthesis, Transpiration and N02 Uptake Following 0, 2, or 5 h Treatments with 3.0 ppm NO 5 -2 -1 (Irradiance 2.0 x 10 erg cm" s" ) N0 2 UPTAKE RATE TRANSPIRATION RATE APPARENT RATE OF PHOTOSYNTHESIS Ung N0 2 dm" 2 hr 1) (g H20 dm~2 h-M (mg C 0 2 dm" 2 hr1) 41 photosynthesis, and the intensity of inhibition increased with increasing time of exposure. The inhibition was most severe near the optimum temperature. For example, the 5 h exposure to 3.0 ppm N02 inhibited photosynthesis by 36% at 30° and by 19% and 22% at 15° and 35°,respectively. At 35°, apparent photosynthesis in the absence of N02 was not stable but declined by about 7% during a 5 h test. The control rates plotted in the graph are at 0 h of fumig-ation. Thus, at 35° i t is li k e l y that the true inhibition of apparent photo-syntheis by N02 was close to 15% rather than 22%. Transpiration and N02 uptake during photosynthesis did not exhibit an optimum within the temperature range surveyed, but instead they gradually increased with increasing temperature (Fig. III-2). Transpiration was slightly depressed by the presence of N02 at a l l temperatures and this inhibition increased from about 7% at 15° to about 15% at 35°. Dark respiration also increased gradually with increasing temperature (Fig. I l l - 3 ) . The percent inhibition of dark respiration by a 2 h exposure to 3.0 ppm N02 ^steadily increased from 34% at 15° to 51% at 35°. During dark respiration, the rates of transpiration and N02 uptake were much less than previously observed during photosynthesis. Transpiration in the dark tended to increase with increasing temperature, and N02 appeared to slightly enhance transpiration rate at low temperatures and inhibit at highefctemperatures. The effect, however» was insignificant. The rate of N02 uptake in the dark was fa i r l y uniform at a l l temperatures except for a relatively low rate at 15°. Carbon Dioxide arid Oxygen Concentrations. In order to further define the effects of N02 on apparent photosynthesis, a series of N02 treatments was carried out at different C02 concentrations in a i r . Exposure to 3.0 ppm N02 for 2 or 5 h consistently decreased the apparent rate of photosynthesis at Figure 111-3 Effects of Temperature on Dark Respiration, Transpiration and NC^  Uptake following a 2 h Treatment with 0 (Broken lines) or 3.0 ppm 2 1 N0? (solid lines). (Irradiance = 0 erg cm" s~ ). N0 2 UPTAKE RATE (mg NOo dm-2 h" 1) TRANSPIRATION RATE Cg H 20 dm-ah-1) RATE OF DARK RESPIRATION CmgC0 2dm- 2 h"i) 44 each CO,, concentration (Fig. I l l - 4 ) . The actual magnitude of inhibition was almost the same at each CO^ concentration. The percent inhibition of apparent photosynthesis by NO^ , however, declined with increasing COg concentration. In the 5 h NOg treatment, for example, apparent photosyntheis was inhibited by 55% at 100 ppm COg and by only 16% at 600 ppm COg. At very high (approx. 2000 ppm) CO^  concentrations, NO,, s t i l l inhibited apparent photosynthesis, but the rates of gas exchange were much reduced by partial stomatal closure (Table 111-I). The rate of transpiration was only slightly inhibited by N0^ at the COg concentrations tested (Fig. I l l - 4 ) . High CO^ concentrations also decreased the rate of NOg uptake (Fig. III-4), and i t appears from the results that NO^  uptake was more strongly affected by COg concentration than was transpiration. In COg-free a i r (Fig. III-5), there was net evolution of CO^  from the experimental leaves and this efflux was decreased by treatments with NOg. This effect of NO^  was increased by increasing NOg concentration and treatment time. Transpiration rate in COg-free air was similar to its rate in normal a i r , but the rates of NO^  uptake were considerably greater than those previously obtained in normal air (Table I I I — I I ) . The absence of Og enhanced the apparent rate of photosynthesis but did not alleviate the inhibition caused by NO^  (Table I I I — I I I ) . The percent inhibition of apparent photosynthesis by NOg was unaffected by changing the atmospheric Og level from 20.8% to zero percent. The effects of Og on transpiration were minor, but N02 uptake was appreciably greater in Og-free air than in 20.8% Og. Humidity. Measurements were also carried out to determine whether atmospheric humidity influenced the uptake or effects of NO^ . Preliminary tests confirmed that changes in humidity did not introduce errors in the measurement of leaf 45 Table '-. Effects of high CO2 concentration (~-2000 ppm) on apparent rate III-I of photosynthesis, transpiration and NO2 uptake in the presence of 3.0 ppm N 0 ? . Measurements Time after exposure to N02 % Inhibition Oh 2 h 5 h in 5 h Apparent rate of photosynthesis (mg CO2 dm-2 h -1) 4.50 4.25 3.81 15% Rate of transpiration (g H20 dm-2 h -1) 0.73 0.76 0.70 4% Uptake rate of N0 2 (mg NO2 dm-2 h -1) - 0.199 0.174 Figure 111-4 Effects of GOg Concentration on Apparent Photosynthesis, Transpiration and N02 Uptake Following 0, 2, or 5 h Treatments with 3.0 ppm N09. N 0 2 U P T A K E R A T E T R A N S P I R A T I O N R A T E A P P A R E N T R A T E OF P H O T O S Y N T H E S I S C m g N 0 2 d m - 2 h - 1 ) ( g H 2 0 d m " 2 h r 1 ) ( m g C O P d m " 2 h r 1 ) P o o h* » t- H* t-Figure II1-5 Effects of 0, 3.0 or 7.0 ppm NO,, on CO,, Evolution into C02-free Air (20.8% 0 2) ppm NQJ 0 0.5 1,0 1.5 2.0 DURATION OF N02 FUMIGATION Ch) Table ; Effects of NO? on Transpiration and NO? Uptake in C0?-free Air * III-II N02 Rate of Rate of Concentration Transpiration NO2 Uptake (ppm) (g H20 dm-2 h-1) (mg N02 dm"2 h - 1) 0 2.33 3 2.26 0.571 7 2.16 0.836 * Measurements were made after 2 h exposure to NO2 in the light. 51 Table 1 1 1 - 1 11 Effects of 3.0 ppm N0? on the rates of apparent photo-synthesis, transpiration and N02 uptake in 0% and 20.8% 0,,. Concen-tration (%) Characteristic Duration of exposure to N02 (h) % Inhibition in 5 h 20.8 Apparent rate of photosynthesis 15.26 12.21 10.22 33 CO (mg C02 dm"2 h"1) 19.90 16.40 13.58 32 20.8 0 Rate of transpiration (g H20 dm"2 dm"2 h - 1) 2.26 2.48 2.09 2.42 2.07 2.40 8 3 20.8 Rate of N02 Uptake 0 (mg N02 dm"2 h"1) 0.334 0.300 0.421 0.365 52 NOg uptake. As shown in Table 111-IV, the rate of transpiration decreased moderately with increasing relative humidity (significant at 5% level), and was l i t t l e affected by NO,,. Variations in relative humidity from 20% to 80% did not strongly affect apparent photosynthesis. Inhibition of photosynthesis by NO,, in 20% relative humidity was slightly lower than in 45% or 80% relative humidity. The rate of NO,, uptake was highest at 80% relative humidity, and after 5 h of exposure the rates in different relative humidity were significantly different. DISCUSSION The preceding results include information on plant response to NO,, under many different circumstances, and some of they may assist in the interpretation of the effects of N02 in ecological systems. Nitrogen dioxide was absorbed by and exerted its effects on primary bean leaves over a wide range of environ-mental conditions. Whenever a N02 treatment was applied, apparent photosynthesis or dark respiration was inhibited to some degree. The environmental conditions under which N02 caused the greatest decline in the magnitude of apparent photo-synthesis or respiration tended to coincide with the conditions under which those processes of gas exchange were most rapid in the absence of N02. In this respect, i t may be pertinent to recall the earlier findings that N02 was most inhibitory at the ages during leaf development when apparent photosynthesis or darkl'respiration were maximal (Part I I ) . Compared with processes of C02 exchange, transpiration was less susceptible to N02 and this is in agreement with earlier results (Part I I ) . Nevertheless, transpiration was somewhat depressed in a l l conditions used except at low temperature in the dark. As shown in Table III-V, the presence of N0« caused Table 111-IV Effects of atmospheric humidity on the apparent rate of photosynthesis, rate of transpiration and rate of uptake of N09 in the presence of 3.0 ppm N0?. R e l a t i v e Characteristic Duration of Exposure to N0g (h) Humidity % 0 2 5 -N02 -N02 +N02 -N02 +N02 20 Apparent rate of 16.57 16.20 14.73 15.60 13.12 45 fmf2gyndm?i1h-i) 1 5 ' 2 6 1 5 ' 0 0 1 2- 2 1 1 5 ' 2 0 1 0- 2 2 80 2 15.80 15.60 13.28 15.18 11.90 20 Rate of transpira- 2.40 2.46 2.43 2.31 . 2.26 4 5 ( g ^ O dnf 2 h"1) 2 ' 2 6 2' 2 1 2 ' 1 9 2 ' 2 3 2 ' 0 9 80 L 1 .95 1.98 1.98 2.00 1.89 20 45 80 N0o Uptake Rate (mg N0o dm"2 h"1) 0.342 0.346 0.505 0.290 0.308 0.355 Table III-V. Effects of NO2 on the leaf resistances. Resistances, s cm-.' * Radiant flux density Stomatal Resistance Mesophyll Resistance Mesophyll Resistance x 104 ergs s " ' cm-2 ( r s H2O) ( r m CO2) ( r m N02) -NO2 +N02 -NO2 +NO2 +N02 0.52 2.77 3.11 50.15 115.67 -1.18 1.05 2.38 2.55 25.84 48.22 -0.35 1.57 1.54 1.65 17.82 32.38 0.31 2.10 1.73 1.87 12.25 19.63 1.01 3.15 1.09 1.19 10.29 16.61 1.36 5.25 0.96 1.03 9.54 16.74 1.65 21.00 0.56 0.74 10.50 17.22 2.85 * Atmospheric diffusion resistance was 1.5 sec cm' 55 only a small increase in stomatal diffusion resistance under a variety of light regimes. The major effect of N02 was to increase mesophyll resistance to CG"2 exchange. The results appear to be consistent with an earlier sugges-tion (Part II) that NO^-induced variations in transpiration and stomatal dif-fusion resistance may be the outcome of changes in internal leaf C02 concen-tration following alterations in the activity of C02 exchange processes within the leaf. The uptake of N02 was enhanced by high temperature, low C02 concentration and high humidity. The rate was higher in light than in darkness, but the intensity of the irradiance did not influence the N02 uptake rate. The variations were partly the result of changes in stomatal diffusion resistance, but they also originated from changes in mesophyll resistance to N02 uptake. For example, decreasing irradiance was associated with increasing stomatal diffusion resistance but decreasing mesophyll resistance for N02 (Table III-V). Because of the wide range of conditions under which N02 uptake occurred, however, the results of this study do not provide a specific indication of the possible site(s) of N02 absorption within the leaf. Nitrogen dioxide differs from some other air pollutants with respect to the regulation of i t s effects by environmental conditions. Peroxyacetyl nitrate, for example, has been found to damage pinto bean only i f i t is applied during illumination (11), while the present experiments found N02 to be inhib-itory both in light and in darkness. Also, variations in humidity exert a relatively large influence of plant susceptibility to "oxidants" (10, 51), but only minor effects on sensitivity to N02 were observed in the present study. On the other hand, there appear to be some similarities in the influence of temperature on the responses of plants to various a i r pollutants. Tobacco plants have been found to be more sensitive to 0^  at 25° than at 5° (43). In 56 bean, endive and cotton, intermediate temperatures were associated with greatest damage from oxidant (propylene - N02 mixture) treatments (18). In the present experiments, the inhibition of apparent photosynthesis by N02 was greatest near the optimum temperature. For dark respiration, however, the inhibition by N02 was most severe at the highest temperature used. Such con-trasting results imply that the mechanism of action or assimilation of N02 differs from that of some other air pollutants. L i t t l e work has been done concerning the sensitivity of plants to air pollutants in relation to C02 and 0 2 concentrations. In one study, Heck and Dunning (20) found that the susceptibility of tobacco and pinto bean to 0^  decreased when the C02 concentration of normal a i r was increased by 500 ppm. It is possible that this decrease may have been the outcome of decreased 0^  uptake due to partial stomatal closure at the higher C02 concentration. In the present research, stomatal closure caused low rates of photosynthesis and transpiration at high (% 2000 ppm) C02 concentration, but the present inhibition of photosynthesis by N02 was approximately the same as at 600 ppm C02. In addition to defining many of the relationships between environment, N02 uptake and N02 effects, the present work provides some general information relevant to the mechanism of N02 effects. As noted earlier, the primary effects of N02 appear to occur within the leaf mesophyll and are not on the stomata. Nitrogen dfoxide affected dark respiration and i t imposed the same degree of inhibition on apparent photosynthesis in both 20.8% and 0% 0 2. Since photorespiration is not sustained in darkness or in the absence of 0 2 (28), the effects of N02 are not dependent on the occurrence of photorespiration. The evolution of C02 into C0 2~free a i r , however, was decreased by N02. This leads to the conclusion that photorespiration, true photosynthesis and dark respiration can a l l be inhibited by N0?. In addition, apparent photosynthesis 57 was affected by NOg under conditions where either radiant energy or COg was limited. This is evidence that NOg can inhibit processes associated with the photosynthetic conversion of radiant energy as well as processes in the path-way of C02 fixation. In other investigations, addition of ni t r i t e to spinach chloroplasts inhibits CO^  uptake possibly by inhibiting NADPH production (15). At the present time, i t is uncertain whether N02 exerts i t s effects directly or whether i t must f i r s t be converted to another form,Tfor^example, ammonia (2). That NOg is inhibitory to several major cellular processes under a wide variety of environmental conditions suggests that NOg may cause relatively general transformations in the physiological characteristics of leaf c e l l s . Such changes may not be associated with the metabolism of NOg i t s e l f , but could be caused by NOg induced alterations in the properties of cellular membranes (73) or modifications in the activities of a number of enzyme systems. 58 PART IV The Influence of Nitrogen Supply during Growth on the Inhibition of Gas Exchange and Visible Damage to Leaves by N02 59 INTRODUCTION A number of investigations have indicated that nutrient status (8, 9, 49, 50) including nitrogen supply, can influence plant response to air pol-lutants. In 1950, Brennan ejt aV. (7) reported that plants grown at either deficient or excessive levels of nitrogen were less susceptible to injury by atmospheric fluoride than plants grown at adequate nitrogen levels. Similarly, foliage injury by SOg was later found to be reduced i f plants were grown at deficient or excessive nitrogen levels (36). Also, damage to mangels by ozonated hexene (8), and the suppression of radish growth by 0g (50) were generally less at deficient or low levels of nitrogen than when nitrogen was abundant. On the other hand, the susceptibility of tobacco to damage by 0g was greater at low or excessive (41) levels of nitrogen than at adequate levels. In addition, visible damage to pinto bean by 0g was found to decrease with increasing nutrient supply (42). Such investigations have provided much useful information concerning the regulation of plant susceptibility to ai r pollutants. The physiological origins of such plant responses, however, are not well understood. The effects on plants of the ai r pollutant NOg, in particular have not been examined in connection with plant nutrition. Because of possible relationships between N02 uptake and nitrogen metabolism, and as a part of a larger study of the influence of N02 on plants (Parts II and I I I ) , i t was f e l t appropriate to investigate the physiological effects of NOg on plants of different nitrogen status. The following research, therefore, determines N02 assimilation and the effects of NOg on gas exchange and leaf appearance in bean plants grown at different levels of nitrogen. 60 MATERIALS AND METHODS Bean (Phaseolus vulgaris L. cv. 'Pure Gold wax1) plants were grown from seeds sown in plastic pots containing moist vermiculite as described earlier (Part II). During growth, the plants were watered daily with half strength nutrient solution (No. 1 in 26) which was modified to include 0 to 50 mM KNOg or 5 mM urea as sole source of nitrogen. Nitrogen fixing nodules were not visible on the roots of plants used for these investigations. Single, attached primary leaves on 12-day old plants were, used for measure-ments of the rates of apparent photosynthesis, dark respiration, transpiration and NOg uptake as described previously (Part I I ) . The environmental conditions during these measurements were 25 ± 1.0° leaf temperature, 335 ± 5 ppm COg, 20.8% 0 2, 45 + 3% relative humidity, and darkness (for dark respiration) or 5 -2 -1 2.1 x 10 erg cm" s" radiant flux density (for apparent photosynthesis). Since the variability in gas exchange characteristics among replicate plants measured under similar conditions was small (Part I I ) , i t was possible to use small sample sizes. The gas exchange results presented here are the average of two replicate experiments under each condition. For determining nitrogen content of the leaves, the leaves were dried in a hot air oven at 65° for 24 h. About 200 mg of dry tissue were extracted with excess of 80% aqueous ethanol and the extract was centrifuged for 10 min at 12,000 x g. The supernatant and residue were separated and evaporated to dryness by bubbling air through the Kjeldahl flask at 30-35°. Each fraction was then digested with cone. HgSO^ , in a modified microkjeldahl procedure (35), ammonia produced being determined by Nesslerization with Folin and Wu Nessler's reagent. Nitrogen determinations were made on leaves from 3 replicate plants for each treatment of nitrogen concentration during growth. Values for the protein were obtained from multiplying insoluble nitrogen by a factor of 6.25, 61 RESULTS Soluble and Insoluble Nitrogen Within Leaves. The measurements of the distribution of nitrogen between 80% ethanol soluble and insoluble fractions (Fig. IV-1) give a general indication of the nitrogen status of the leaves used for the gas exchange experiments. Nitrate supply during growth influenced the concentration and composition of nitrogen within the 12-day primary leaves. The concentration of nitrogen in the insoluble fraction, which was considered to be protein-N, increased sharply with increasing nitrate level up to 5 mM nitrate, and then was constant for high nitrate levels. Soluble nitrogen increased in concentration with increasing nitrate supply throughout the range examined, but the increase was most pronounced up to 10 mM nitrate. Leaf Gas Exchange. In the absence of NO,,, apparent photosynthesis and trans-piration were stable during the 5 h experimental period, and their rates were almost the same in leaves of plants grown at 5, 10, 25 or 50 mM nitrate (Fig. IV-2). At 0 and 2 mM nitrate, the rates of apparent photosynthesis and transpiration were greatly reduced. Exposure to 3.0 ppm N02 inhibited apparent photosynthesis in leaves of plants grown at a l l nitrate levels, and the inhib-ition increased with increasing exposure time. The degree of inhibition by NO,, was greatest at zero nitrate. During photosynthesis, transpiration rate was also inhibited by N02 in al l cases, but the effects of N02 on transpiration were relatively small. At 50 mM nitrate, i"sH,>0 in the presence of N02 increased to 0.93 s cm"1 from 0.58 s cm"1 in the absence of NO,,. The observed variations in transpiration rate strongly suggest that nitrogen deficiency and N02 treatments can cause partial stomatal closure. It is also possible that nitrate supply affects the develop-ment and distribution of stomata. Despite this, the rate of NO,, uptake during photosynthesis was highest in leaves of plants grown at zero nitrate level. Figure IV-1 Effects of Nitrate Supply During Plant Growth on 80% Ethanol Soluble and Insoluble Nitrogen of the Primary Leaf. Circles (0 - 0) insoluble nitrogen; triangles (A - A) soluble nitrogen. 63 NITRATE CONCENTRATION (mM) Figure IV-2 Effects of Nitrate Supply on Apparent Rate of Photosynthesis, Transpiration and NOg Uptake Rate Following 0, 2 or 5 h Treatments with 3.0 ppm NOg. The effect of nitrate supply on rate of photosynthesis and NOg uptake was significant at 5% level (regression analysis). 65 66 At 5% level the uptake of NO^  was significantly different in different nitrate treatments. The effects of a 2 h treatment with 3.0 ppm NOg on gas exchange by dar-kened leaves are shown in Fig. IV-3. In the absence of NOg, the rate of dark respiration was highest in leaves of plants grown at zero nitrate, and i t was not affected by nitrate levels above 5 mM. Exposure to NOg inhibited dark respiration in a l l cases, and the inhibition was most severe for 0 mM nitrate during growth. The rate of transpiration during respiration (Fig. IV-3) was much less than during photosynthesis (Fig. IV-2) but i t s response to nitrate supply during growth was similar. The effects of NO,, on transpiration in the dark were small. Except at 50 mM nitrate, transpiration tended to be slightly inhibited in the presence of NOg. In contrast to the results with illuminated leaves, the rate of NOg uptake in darkness (Fig. IV-3) was lowest at 0 mM nitrate and reached a peak at 2 to 5 mM nitrate. Between 10 and 50 mM nitrate, the rate of N0^ uptake was f a i r l y constant. The rates, however, did not differ significantly at 5% level. Time Kinetics of NO,, Uptake. Uptake of N02 by the illuminated leaves as i n f l u -enced by exposure time and nitrate supply is presented in Fig. IV-4. In the leaves of plants grown at zero nitrate the uptake rate increased to a maximum in about 1 h and then i t declined gradually with further exposure time. The time course of NOg uptake by the leaves of 2 mM nitrate plants was similar but the rates of N02 uptake were lower. For other nitrate treatments, there was a gradual deline in the rate of NO,, uptake after the start of exposure, and the rate also declined with increasing nitrate supply during growth. Figure IV-3 Effectsof Nitrate Supply on Rates of Respiration, Transpiration and N02 Uptake in Darkened Leaves after 2 h Exposure to 0 or 3.0 ppm N02. Broken lines (----) 0 ppm; solid lines ( — 3 . 0 ppm. NOo UPTAKE RATE TRANSPIRATION RATE RATE OF RESPIRATION (mg N0 2 dm-2 h-i) C g H 20 dm-2 h-i") (mg C02dm~2 h"1) CO Figure IV-4 Time Kinetics of NO,, Uptake by the Leaves of Plants Grown;:. Different Nitrate Levels. RATE OF N0 2 UPTAKE (mg N0 2 dm-2 h-i ) 71 Source of Nitrogen Supply During Growth. An experiment was also carried out to determine i f the nature of the nitrogen source during growth affects plant response to N0 2 # Leaves of plants grown on 5 mM urea, in place of 10 mM nitrate, exhibited higher rates of apparent photosynthesis and lower rates of transpiration (Table IV-I). Exposure to 3.0 ppm NO^  inhibited apparent photosynthesis in leaves of plants grown on both sources of nitrogen. The response of apparent photosynthesis to NO^ , however, was not greatly modified by nitrogen source during growth. A more pronouneedLeffect of nitrogen source during growth was on the rate of N02 uptake, which was much higher in leaves of plants grown on urea compared with those grown on nitrate. Visible Symptoms of NOp Injury. The visible appearance of bean leaves exposed to 0 or 3.0 ppm N02 or 5 h in the light was observed for 7 days follow-ing treatment. The N02 treatments were applied to primary leaves on plants grown at nitrate levels from 0 to 50 mM and following exposure the plants were kept under the same conditions as before treatment. For the 0 mM nitrate plants, the N02 treatment was followed by the appearance of yellow and brown spots near the leaf margins starting about 2 days after exposure. Subsequently, such spots developed over the rest of the surface of the leaves. By 7 days following treatment, the leaves were patched and mottled in appearance with some marginal necrosis (Fig. IV-5). The severity of visual injury caused by N02 treatments was decreased by increasing nitrate supply during growth and leaves from plants grown at 10 and 50 mM nitrate did not exhibit visible symptoms of injury at 7 days after N02 treatment (Fig. IV-5). DISCUSSION It is evident from the preceding results that limitations to nitrogen supply during plant growth promote increased susceptibility to the harmful 72 Table IV-1. Effect of source of nitrogen on gas exchange in bean leaves in the presence of 3.0 ppm N0?. Characteri sric Apparent rate of photosynthesis (mg CO^  dm" h-1) Rate of transpiration (g H20 dm-2 h-1) Rate of N02 uptake (mg N09 dm-2 h-1) % Inhibition Nitrogen Source Time after exposure to N02 (h) at 5 h Nitrate Urea Nitrate Urea Nitrate Urea 0 15.00 19.62 2.14 1.89 11.40 16.42 2.09 1.86 0.342 0.657 10.68 13.00 2.01 1.75 0.316 0.446 28.8 33.7 6.5 7.4 The seedlings were supplied with T/2 strength modified Hoagland's solution which included either 10.MTIM KNOg or 5 mM urea as sole source of nitrogen. Figure IV-5 Morphological Appearance of the Primary Leaves after 7 Days of Exposure to 3.0 ppm NOg for 5 h in the Light from the Plants Grown at Different Nitrate Levels. The plants were grown at 0, 2, 5, 10 or 50 mM nitrate. 7 4 75 effects of N02. In contrast to these results, nitrogen deficiency has been found to decrease the sensitivity of plants to some other air pollutants, for example, fluoride (7), S0 2 (36), and Og (50). Thomas (66), however, has reported an increased sensitivity of plants to London type smog when the plants were grown on poor s o i l . As in earlier studies (Parts II and I I I ) , the effect of N02 on transpir-ation was not great and in this study the size of effect was not strongly modified by nitrogen supply during growth. When N02 is dissolved in water, NO^  and N02 are formed (32). Plants are capable of reducing nitrate and n i t r i t e to ammonia (3) and ammonia in turn can be incorporated into amino acids and eventually into proteins. This general biochemical route, therefore, seems a plausible pathway for the assimilation of at least some of the N02 absorbed by the leaves. According to this line of reasoning, N02 uptake might be expected to be influenced by factors affecting N02 transfer into leaves from atmosphere, by the nitrogen require-ment of a plant, and by the efficiency of the pathway responsible for nitrate and n i t r i t e metabolism. Some of the results of the present research relate to these considerations. Changes in stomatal diffusion resistance can influence N02 uptake as was pre-viously observed (Parts II and II I ) . Rate of N02 uptake in the urea grown plants was higher than those grown on nitrate. Plants grown at nitrate nitrogen may be "saturated" with nitrate-nitrite system, while urea grown plants may not and thus permit higher uptake of N02. Also, the rate of N02 uptake was generally high for plants grown at low levels of nitrogen. One exception to this was the slow N02 uptake by darkened leaves of 0 mM nitrate plants, and the low rate of transpiration by those leaves suggest that this exception may have resulted from high stomatal diffusion resistance (Fig. IV-3). 76 The possible link between high NOg uptake and nitrogen shortage during growth is strengthened by the results in Fig. IV-2. One can further speculate that the variations in the time course for HO^ uptake may be associated with changes in the activities or concentrations of enzymes participating in nitrate and ni t r i t e reduction. Nitrite reductase activity is relatively low in nitrogen deficient cells and i t is induced by the presence of ni t r i t e (33). Such changes might relate to the i n i t i a l rise in NOg uptake rate in the nitrogen deficient (leaves (Fig. IV-4). The increase in NOg by illumination might also be incorporated into this framework. Light promotes the induction of nitrate reductase in leaves (71), and Miflin (46) has found that the rate of reduc-tion of ni t r i t e in darkness is low. Furthermore, limitations to the supplies of reduced NADP and ferredoxin in the dark may also limit the assimilation of NO^ . All of this evidence, however, is circumstantial and i t neither proves the existence of such a pathway for NOg uptake nor excludes the possibility of other uptake routes. Nevertheless, i t does encourage examination of this aspect as an early part of future studies on NOg absorption by plants. Whatever the mechanism of NOg uptake may be, i t is interesting to point out that large responses of gas exchange to NOg were not always correlated with high rates~of NOg uptake. For example, at 0 mM nitrate, dark respiration was most severely inhibited by NOg but the rate of NOg uptake was relatively low. This matter wi l l be considered in more detail later (Part V). During the analysis of these results, i t was noticed that nitrate supply during growth limited leaf protein (i.e. 80% ethanol insoluble nitrogen) content a,t levels below 10 mM nitrate. At the same time, the rates of apparent photosynthesis or dark respiration were not restricted unless nitrate level was less than 5 mM. When expressed on a per unit protein basis, therefore, the rates of apparent photosynthesis and dark respiration must increase in 77 leaves of plants grown at less than 10 mM nitrate. The nitrogen deficient plants were also the most susceptible to inhibitions by NO^ . This encouraged comparisons of photosynthetic or respiratory activity per unit protein with the inhibition caused by N02, as shown in Fig. IV-6. There is a clear correspondence between the inhibitory effects of N02 on C02 exchange processes and the activity of those processes per unit protein in the absence of NO,,. It is easy to suggest from this result that the general mode of action of NO,, is related to the concentration and activity of leaf proteins. The correspon-dence, however.',':does not prove such a mode of action because other leaf parameters may be affected by nitrate supply during growth in the same manner as leaf protein content. Nevertheless, this result does point the way for future work, and the possible mode of action i t suggests is reasonable in the light of the very general effects caused by N0o. 78 Figure IV-6 Effects of Nitrate Supply on the Specific Rates of Photosynthesis and Respiration and Percent Inhibition by a 5 h (for photosynthesis) or 2 h (for respiration) Exposure to 3.0 ppm NO^ . Circles ( 0 - 0 ) control rates; Squares ( ° ) percent inhibition. At 5% level, the effect of nitrate supply on the rate of photosynthesis was significant, while that on the rate of respiration was not. 79 I NITRATE CONCENTRATION (mM) PART V General Discussion 81 This research has shown that N02 at concentrations from 1.0 to 7.0 ppm inhibits apparent photosynthesis, dark respiration and the evolution of CO,, into COg-free a i r by primary bean leaves. The inhibitions were observed at a l l ages of plants and in a l l environmental conditions used. The inhibitions were increased with increasing concentrations of N02 and increasing exposure time. In most instances, N02 treatments also inhibited the rate of transpiration. The effects of N02 on transpiration, however, were not as severe as on other processes of gas exchange. The results indicated that changes in stomatal diffusion resistances contributed to the effects of N02 on gas exchange, but the primary effects of NO,, were exerted within the leaf mesophyll. Particular aspects of this research have been discussed at several earlier points in this thesis. The following discussion w i l l consider the overall implications of this research with respect to the mechanisms of N02 absorption and i t s effects, and w i l l attempt to evaluate the importance of atmospheric levels of N02 to plants at a global level. The rate of N02 uptake was promoted by l i g h t , high temperature, low C02 and 0 2 levels and high humidity and i t was greatest in leaves of intermediate ages and in leaves of plants grown at low nitrogen levels. Several results indicated that internal (mesophyll) resistances as well as stomatal diffusion resistances helped to regulate N02 uptake rate differently from transpiration rate (Fig. I I I - l ) . Elevation of the C02 concentration to approx. 2000 ppm affected N02 uptake less than transpiration. In leaves of urea grown plants, the uptake of N02 was higher than for leaves of nitrate grown plants, while transpiration was higher in the latter (Table IV-1). It is also possible that some N02 absorption may occur on the cuticle. While this cannot be ruled out, i t was considered to be of secondary importance, since N02 uptake was so often influenced by stomatal diffusion resistance and by the physiological 82 activity of the leaf mesophyll. In many cases, high rates of NOg uptake coincided with high inhibitions of COg exchange. An exception to this was the low rate of NOg uptake associated with severe inhibition of dark respiration in leaves of 0 mM nitrate plants (Fig. IV-3). Also, the maximum inhibition of respiration was observed in the youngest leaves, while the rate of NOg uptake was higher in the leaves of inter-mediate ages (Fig. I1-4). These observations may mean that the effectiveness of NOg as an inhibitor may depend on steps which are isolated from the main route of NOg assimilation. On the other hand, the exceptions noted above may have developed because the particular conditions where they occurred in some manner predisposed the leaves to greater susceptibility to inhibition by NOg. Several workers (15, 16, 40) have studied the assimilation of ni t r i t e and i t s related inhibition of CO,, uptake in isolated chloroplasts. Inhibition of photosynthesis by nitrous acid has been observed in Chlorella pyrenoidosa also (25). Reduced ferredoxin and NADPH are required for photosynthetic C02 fixation and also participate in n i t r i t e reduction (3). Thus the metabolism of N02 by n i t r i t e reduction might limit the rate of photosynthesis by res-tricti n g the supply of reduced cofactors for CO,, fixation (23). This concept was supported circumstantially by observations of Yung and Mudd (74) who found that inhibition of acetate metabolism by low concentrations of n i t r i t e can be reversed by illumination. Furthermore, the inhibition of the NADPH-requiring conversion of acetate to lipids was greater than the inhibition of synthesis of non-volatile soluble compounds which does not require NADPH. In the present study, however, the percent inhibition of apparent photosynthesis by N02 did 4 4 not vary appreciably over a range of irradiances from 4 x 1 0 to 20 x 10 erg cm s (Fig. I I I - l ) . Also, after 5 h of exposure to 3.0 ppm NO,,, about 15 mg of C0o was excluded from C0„ assimilation for every mg N09 absorbed. Thus, NO,, 83 can inhibit photosynthesis under conditions when reduced cofactors ought to be in abundant supply, and a s t r i c t competition between N02 and C02 for reduc-tant does not seem l i k e l y . Besides this, the addition of NADP to a chloroplast system .Twhich isareducing n i t r i t e has been shown to suppress n i t r i t e reduction in favour of the reduction of NADP (55). Accumulation of ammonia produced by the reduction of N02 may also inhibit metabolism related to C02 exchange. Chloroplasts are relatively impermeable to ammonia (59) and ammonia accumulated inside the chloroplast can inhibit photo-synthesis by blocking ATP formation (2). Ammonia is also known to induce structural changes in tomato leaf chloroplasts (53) which may interfere with the normal photosynthetic process. Grant and Canvin (15), however, have demon-strated that low concentrations of NH^+ had no effect either on 0 2 evolution or COr, fixation by isolated spinach chloroplasts, while higher concentrations of NH^+ always resulted in a slight stimulation of both 0 2 evolution and C02 fixation. Perhaps, the high rates of photosynthesis in the leaves of urea grown plants (Table IV-I), which may contain high levels of NH^+ is a reflection of a similar situation. Since N02 inhibited apparent photosynthesis under conditions of low C02 concentrations, i t appears that N02 can inhibit photosynthetic carbon metab-olism as well as the photosynthetic light reactions. The effect of N02 does not seem to be specific to the carboxylation reactions of photosynthesis, however, since N02 also inhibited apparent photosynthesis when C02 was plenti-ful and irradiance in short supply. In addition, photorespiratory metabolism may be susceptible to inhibition by N02, as was indicated by the measurements of C02 evolution into C0 2-free a i r (Fig. III-4). Photosynthesis in species which apparently lack photorespiration, however, is also inhibited by N02 (Table V-l). Table V-I Effects of a 2 h exposure to 0 or 3.0 ppm N02 on the apparent rate of photosynthesis and N0o uptake in different species. Apparent rate of Percent R a t e o f NOg uptake Species photosynthesis. Inhibition (mg N0? dm" h" ) (mg C0o dm-2 h"1) L •N02 +N02 Phaseolus vulgaris L. Pure Gold wax * 15.26 12.21 19.9 0.334 Phaseolus vulgaris L. Tender crop * 16.04 14.00 12.7 0.349 Medicago sativa L. 17.22 13.58 21.1 0.657 Helianthus annus L. 23.10 19.90 13.8 0.487 Amaranthus albus L. 17.69 14.91 15.6 0.528 Gomphrena mixed 21.34 18.70 12.4 0.394 Avena sativa L. 16.00 13.02 18.6 0.301 Hordeum vulgare L. 18.00 15.09 16.2 0.229 Triticum aesticum L. 21.28 18.38 13.6 0.319 Zea mays L. 26.85 24.09 10.3 0.339 Sorghum vulgare L. 24.35 22.27 8.5 0.364 Average 19.74 16.92 14.3 0.391 * only attached primary leaves were used. In others detached shoots were used. 85 The effects of NO,, on photosynthesis, respiration and evolution of CO,, into CC^-free a i r , which have different biochemical pathways, suggest that the effect of NO,, is generalized. This effect might not be associated with the metabolism of NO,, i t s e l f and may be brought about either by disrupting the cell membranes or by the inhibition of enzyme ac t i v i t i e s . Of these two, the f i r s t possibility has previously been explored to some extent. Biological membranes contain a high proportion of unsaturated fatty acids. Exposure of l i p i d monolayers to NO,, causes increases in surface tension, possibly by saturating the unsaturated fatty acids (12). Changes in chloroplasts of Vicia  faba leaves exposed to 3.0 ppm N02 have been observed by Wei 1 burn et al_. (73). Similarly, nitrogen dioxide may affect the structural integrity of mitochondria, peroxisomes and possibly other cell organelles to interfere with their normal acti v i t i e s . Nitrogen dioxide when dissolved in water forms nitrous and n i t r i c acids (32). The absorption of N02 may thus decrease the pH of the cell sap. In addition to this, the acids formed may form complexes with biological molecules. The cis-trans isomerization of oleic acid exposed to nitrous acid has been reported (38). Nitrite has been shown to react with secondary amines to form nitrosamines which have carcinogenic properties (37). As an oxidant, N02 can also oxidize important biological molecules such as ascorbic acid and pyridine nucleotides and the -SH groups of enzymes. All these reactions of N02 w i l l interfere with enzyme activities either directly or indirectly by changing their chemical environment. Changes in enzyme activities may in turn lead to inhibitions of gas exchange and other physiological processes. The gas flow system employed to study the physiological responses of plants to N02 has therefore provided some data which can be interpreted to understand the mechanism of N0? assimilation and the inhibition of photosynthesis 86 and respiration by N02 to a limited extent. For a complete understanding of the mechanism of NO^  assimilation and i t s inhibitory effects on photosynthesis and respiration, a more direct biochemical as well as histological approach will be required. Because of the generalized effects of N02 on the physiological processes of plants, and of the methodology used in the present investigation, i t is d i f f i c u l t to ascertain the specific mode of inhibitory effects of NO,,. Hence this discussion suggests that inhibitory effects of NO,, are produced by causing either changes in the properties of the cell membrane or enzyme a c t i v i t i e s . Effects of NO,, on membrane structure have been demonstrated by electron microscopy (73). A study of the induction of enzyme activities as affected by NO,, may provide some evidence concerning the pathway of N02 uptake and possibly the mechanism of NO,, effects. The work in this thesis also relates to the question of the present sig-nificance of atmospheric N02 to vegetation of the world. Results have been presented here concerning the effects of N02 concentrations, NO,, exposure time, irradiance, temperature, C02 concentration, 0,, concentration, humidity, nitrogen nutrition and age on plant response to NO,,. These results should be valuable in the interpretation of ecological studies involving NO,, and plants. In addition, i t seems from this work that nitrogen f e r t i l i z a t i o n may be a convenient way of minimizing plant injury from NO,,. Whether other nutrients have similar effects is not known. In the experiments discussed so far, beans were the only plant material used. To assess the sensitivity of other plants to N02, 9 other species of angiosperms were also exposed to 3.0 ppm NO,, for 2 h. This survey included representative monocots and dicots, as well as plants possessing either the C» dicarboxylic acid pathway or the C_ pathway of photosynthesis. As shown 87 in Table V-I, a l l species experienced an inhibition of apparent photosynthesis which varied from 8.5% to 21% of the N02-free rates. A ll species absorbed N02 but there did not seem to be a direct relationship between the rates of N02 absorption and the inhibitory effects of N02 among the different species. In bean leaves, the inhibition :of apparent photosynthesis and the rate of N02 uptake increased linearly with increasing N02 concentration (Fig. V-l). There was very l i t t l e effect of 0.5 ppm N02 on apparent photosynthesis and this inhibition did not increase i f the exposure time was extended beyond 2 h. A linear relationship between N02 uptake rate and N02 concentration has also been found at low concentrations of N02 in al f a l f a (70). The rate of N02 uptake in the dark was about 20 to 35% of the rate in the light for alfalfa (22) and about 50% for bean (Part II ) . It is d i f f i c u l t to estimate the present approximate world wide average atmospheric N02 concentration. Although the atmospheric level of N02 has reached local concentrations of 3.93 ppm, the average level is normally below 0.5 ppm (1). In industrial areas, an average concentration of N02 of 0.2 ppm to 0.3 ppm in the winter and 0.1 ppm in the summer has been determined by Shuck et_ al_. (56). According to the results of the present research, concen-trations in the range of 0.1 ppm should have negligible effects on photosyn-thesis and respiration, although concentrations above 0.5 ppm have noticeable effects. However, other physiological processes, not pexamined in this study, might be affected by 0.1 ppm N02. Long term exposure to less than 0.5 ppm N02 has resulted in reduced growth of citrus trees (68). If the inhibitory effects of N02 are small at average atmospheric N02 concentrations, perhaps the absorption of N02 is a more significant outcome of the presence of N02 in the earth's atmosphere. Because of the linear relationship between N0? uptake and N0? concentration (70 and Fig. V-l), i t 88 Figure V-l Effects of N02 Concentration on Rates of Apparent Photosynthesis and Uptake Following 2 or 5 h Exposure. Triangles ( A - A ) 2 h; circles (0-0) 5 h. 89 N0 2 C O N C E N T R A T I O N ( p p m ) 90 is possible to estimate the approximate rate of NOg uptake at low NOg con-centrations. From the present data (Table V-I), the rate of uptake from an -2 -1 atmosphere of 0.1 ppm by plants:, may be estimated at 0.013 mg N0^ dm h . If the average rate of uptake in the dark is one third of the rate in the light and i f the area of the metabolically active leaves on terrestial 15 2 plants is 9.892 x 10 dm a year (5), the total N02 uptake by t e r r e s t i a l x o plants from an atmosphere of 0.1 ppm NOg wil l be 7.51 x 10 tons of N0g or o 2.28 x 10 tons of nitrogen (photoperiod = 12h). The worldwide annual pro-g duction of biomass has been estimated to be about 100 x 10 tons and about 50% of this production may be from terrestial plants (48). About 2% of plant P dry matter is nitrogen (3) so terrestial plants may contain about 10 x 10 tons of nitrogen. It is quickly apparent from this rough calculation that N0g from an atmosphere containing 0.1 ppm N0g may satisfy about 23% (2.28 x 8 8 10 tons T 10 x 10 tons x 100) of the nitrogen requirement of terrestial plants. Even i f half of the atmospheric N0g is deposited in the ocean, approximately 11.4% of the nitrogen requirement of the terrestial plants could be obtained from atmospheric NOg. Admittedly the present rate of N0g uptake has been calculated under optimum laboratory conditions. Under the f i e l d conditions, the actual rate of uptake may be lower because of the canopy resistance and adverse environmental conditions which favour stomatal closure. Although different estimates can be chosen for the above calculation, i t is clear that atmospheric NO^  may be a significant source of nitrogen for terrestial plants. From our point of view, the efficiency of plants in absorbing atmos-pheric N0^ should not be surprising. Vegetation has.been shown to be a good sink for other air pollutants (22, 27, 69) and plants have evolved in atmos-pheres which contained pollutants from many natural sources. Even in the present technological period, the total annual production of h^S, C02, NO, N02, NHg, N20, C02, S0 2 and hydrocarbons from human ac t i v i t i e s , constitutes only a few percent of the total natural production (54). The present research does suggest, however, that i f the atmospheric N02 concentrations become higher than their present average levels, even for short periods of time, serious decreases in primary production may occur. 92 CONCLUSIONS 1. Nitrogen dioxide in a concentration range of 1.0 to 7.0 ppm inhibited photosynthesis, respiration and photorespiration; the degree of inhi-bition is affected by plant age, temperature, light intensity, relative humidity, nitrogen status of the plant, to some extent by CO,, and 0,, concentration, and by the species. 2. The primary effect of NO,, seems to be inside the leaf mesophyll rather than on the stomata, since inhibition of transpiration by NO,, in a l l conditions was small. 3. The rate of NO,, uptake increases with NO,, concentration and decreases with exposure time. The rate is also modified by plant age, light intensity, temperature, CO,, and 0 2 concentration and the nitrogen nutrition of the plant. 4. In some cases, the rate of NO,, uptake does not coincide with the rate of maximum inhibition. The rate of NO,, uptake seems to be primarily gover-ned by mesophyll resistance although stomatal resistance also exerted some control over N02 uptake. 5. The effects of N02 on various physiological processes suggest that the effect of N02 is generalized. This effect is probably due to the dis-ruption of cellular membranes or inhibition of enzyme ac t i v i t i e s . 6. A survey of different species indicates that the average atmospheric level of 0.1 ppm N02 can f u l f i l as much as 11% of the total nitrogen require-ment of the terrestial plants, without inhibiting plant productivity significantly. 93 LITERATURE CITED 1. Anonymous. 1962. 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