EFFECTS O3 SOIL FIJOODTNG 0N ETHANOL CONTENT OF TOMATO PLANTS RELATED TO CERTAIN ENVLR-ONMEEEETAL ‘CGNDITtONS Thesis for flat: fiagms of 931. D. MfCHiGAN STATE flNi‘JERSETY Evemfl F. Bait-on 1.966 ¥fi¥fic¥§ This is to certifg that the thesis entitled Effects of soil flooding on ethanol content of tomato plants related to certain environmental conditions presented by Everett F. Bolton has been accepted towards fulfillment of the requirements for PhoDo degree in 8011 SCience Major professor Date October 17, 1966 0-169 ABSTRACT EFFECTS OF SOIL FLOODING ON ETHANOL CONTENT OF TOMATO PLANTS RELATED TO CERTAIN ENVIRONMENTAL CONDITIONS by Everett F. Bolton Effects of certain environmental conditions associ- ated with soil flooding were related to the metabolic status of tomato plants. Ethanol, an indicator of celluar oxygen deficiency, was used as a parameter of these ef— fects. Concentration of ethanol responded markedly to environmental factors under anaerobic conditions result- ing from flooding. Simulated environments were provided with a growth chamber, a transpiration chamber and mist chambers. Ethanol concentration was determined in xylem exudate, transpired condensate and in plant tissue samples by gas chromatographic procedures. A method was developed for distilling samples from tissue. Air temperature markedly influenced ethanol con- centration in xylem exudate of flooded tomato plants. High air temperature resulted in the largest ethanol concentration compared with lower temperatures. At high temperature, concentration approached a maximum in xylem exudate at the 2A-hour flooding period. At medium air Everett F. Bolton temperature an intermediate ethanol concentration re- sulted and reached a maximum at the 12—hour period of flooding. Low air temperature produced the lowest ethanol concentration. Light intensity influenced plant ethanol concen- tration under certain conditions of flooding. At medium air temperature high light increased ethanol over low light for the 12- and 24-hour periods. Under high air temperature high light intensity increased ethanol at only the 2A-hour flood period. Under flooded conditions soil temperature showed little effect on ethanol concentration at low air temper- ature and low light. At medium air temperature and high light intensity a decrease in soil temperature delayed the rate of ethanol accumulation. Transpiration losses of ethanol were small but were proportional to exudate concentration for each flooding period. A significant concentration of ethanol occurred in root excretions after 12 hours of flooding. Concentration of ethanol in anaerobic plants was high- est in the bottom stem and top roots and decreased in the foliage and in the bottom roots. Carbon dioxide at 20.1 percent, combined with oxygen at 20.1 percent, did not increase ethanol concentration over that of aerobic plants. Everett F. Bolton Ethanol concentration was higher in plants flooded in 1.9 liter soil volumes than for larger volumes. A reduced rate of ethanol exudation for larger soil volumes only occurred, however, during the early flooding period. Reduced ethanol exudation was attributed to entrapped air. Field flooding of tomatoes resulted in ethanol con- centrations that agreed closely with growth chamber data for equivalent environmental conditions. The investigation indicated the usefulness of ethanol as a measure of environmental effects associated with soil flooding of tomato plants. Since tomato plant growth and yield previously have been related to soil oxygen supply during short flooding periods ethanol measurement pro— vides a potential means of correlating flood damage inten— sity with environmental conditions. EFFECTS OF SOIL FLOODING ON ETHANOL. CONTENT OF TOMATO PLANTS RELATED TO CERTAIN ENVIRONMENTAL CONDITIONS By \c) . 0’ «'JJ ( Everett F. Bolton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Soil Science 1966 ACKNOWLEDGMENTS The author wishes to sincerely thank Dr. A. E. Erickson for his encouragement and for his enthusiastic discussions which made the program an extremely valuable and enjoyable research experience. The author thanks Dr. R. L. Cook, Dr. A. R. Wolcott, Dr. R. S. Bandurski, Dr. B. G. Ellis and Dr. N. E. Tolbert for serving as members of the guidance committee. The author also wishes to express appreciation to the above committee members for their excellent courses. Appreciation is also expressed to the Department of Soil Science, Michigan State University for their generous financial support and to the Canada Department of Agri- culture for granting educational leave and assistance during the course of study. ii TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES INTRODUCTION LITERATURE REVIEW MATERIALS AND METHODS Description of Growth Chamber. Description of Plant Growth Media and Growth of Plants for Air and Soil Temperature Studies . . . Growth Medium . Care of Plants. Description of Plant Growth Media and Growth of Plants for Transporation Studies. Growth Medium Careof Plants. Description of Soil Temperature Control Water Baths Greenhouse Water Baths . . Growth Chamber Water Baths. Description of the Transpiration Chamber Description of Mist Chambers Measurement of Ethanol . . Entrapment of Transpiration Samples. . Distillation of Ethanol from Plant Tissue. Statistical Analysis. . . . . iii Page ii vi vii ix 12 12 14 IA 15 l5 l5 l6 l6 l6 17 18 19 2O 2O 22 EXPERIMENTAL RESULTS Air Temperature and Light Intensity Effects on Ethanol Accumulation in Flooded Tomato Plants Description of Experiment. Results. Soil Temperature Effect on Ethanol Accumu- lation in Flooded Tomato Plants. Description of Experiment in Greenhouse Results of Soil Temperature Experiment-- Greenhouse. Description of Soil Temperature Experi- ment in Growth Chamber. . . Results of 8011 Temperature Experiment in Growth Chamber . . . Transpiration Losses of Ethanol from Tobacco and Tomato Plants. Description of Experiment. Results. Effect of Flooding Tomato Plants in Differ- ent Soil Volumes. . . . . . . Description of Experiment. Experimental Results Distribution of Ethanol in Anaerobic Tomato Plants . . . . . . . . . Description of Experiment. Results and Discussion. Effect of Carbon Dioxide on Ethanol Con- centration in Tomato Plants Under Aerobic Conditions Description of Experiment. Experimental Results Quantities of Ethanol Accumulated and Eliminated from Tomato Plants iv Page 2A 2A 2A 26 U8 “9 A9 A9 Page Flooding of Tomato Plants in 3.8 Liter Con- tainers in the Field . . . . . . . . 52 Description of Experiment . . . . . . 52 Results . . . . . . . . . . . . 53 DISCUSSION OF RESULTS . . . . . . . . . . 56 APPENDICES . . . . . . . . . . . . . . 6A BIBLIOGRAPHY . . . . . . . . . . . . . 76 Table LIST OF TABLES -1 Oxygen Diffusion Rates gm x 10-8 cm—2 min , in Soils Flooded at Three Cyclic Air Temperatures and Two Light Intensities . . . . . . . . . Effect of Flooding with Nitrogen Gas on Ethanol Distribution in Tomato Plants . . . . . . . . Effect of Flooding with Nitrogen Gas on Root Excretion of Ethanol . . . . . . . . . . . . . Effect of Carbon Dioxide on Ethanol Distribution in Tomato Plants Under Aerobic Conditions . . . Effect of Carbon Dioxide on Root Excretion of Ethanol Under Aerobic Conditions . . . Ethanol Accumulation and Elimination from Anaeros bic Tomato Plants in ugm Per Plant . . . . . . . vi Page 26 A7 A7 50 ,51 LIST OF FIGURES Page Ethanol Concentration in Exlem Exudate of Tomato Plants Flooded Under Three Ranges of Air Temperature Cycled to Indicate Maxima and at Two Light Intensities . . . . . . . Ethanol Concentration in Xylem Exudate of Tomato Plants Flooded at Three Soil Tempera— tures in the Greenhouse at an Air Temperature of 18.3 — 21.1° C and Low Light Intensity. . Rate of Ethanol Exudation From Tomatoes Flooded at Three Soil Temperatures in the Greenhouse at an Air Temperature of 18.2 — 21.1° C and Low Light Intensity. . . . . . . . . . . . . . Ethanol Concentration in Xylem Exudate of Tomato Plants Flooded at Two Soil Temperatures Under Medium Air Temperature and High Light Intensity. . . . . . . . . . . . Ethanol Concentration in Transpired Samples From Tobacco Plants Flooded at a Constant Tempera- ture in the Laboratory and at a Cyclic Temperature in the Growth Chamber. . . . . . . Rate of Ethanol Loss by Transpiratioanrom Tobacco Plants Flooded at a Constant Tempera- ture in the Laboratory and at a Cyclic Temper- ature in the Growth Chamber. . . . . . . . . . Ethanol Concentration in Transpired Samples From Tomato Plants Flooded at a Constant _ Temperature in the Laboratory and at a Cyclic Temperature in the Growth Chamber. . . . . . . Rate of Ethanol Loss by Transpiration From Tomato Plants Flooded at Constant Temperature in the Laboratory and at a Cyclic Temperature in the Growth Chamber. . . . . . . . . . . . . Ethanol Concentration in Xylem Exudate of Tomato Plants Flooded in Three Different Soil Volumes at Medium Air Temperature and High Light Intensity. . . . . . . . . . . . . . . . vii 28 31 32 35 38 39 A0 Al A3 Figure Page 10. Rate of Ethanol Exudation From Tomato Plants Flooded in Three Different Soil Volumes at Medium Air Temperature and High Light Intensity . . . . . . . . . . . . . . . . . . AA 11. Ethanol Concentration in Xylem Exudate of Tomato Plants Flooded in the Field in Comparison with Data Obtained in Growth Chamber Experiments . . 5A viii Table l. 10. ll. 12. LIST OF APPENDICES Page Ethanol Concentration in Xylem Exudate of Tomato Plants Flooded Under Three Ranges of Air Temper- ature and at Two Light Intensities . . . . . . . 65 Ethanol Concentration in Xylem Exudate of Tomato Plants Flooded at Three Soil Temperatures in Greenhouse . . . . . . . . . . . . . . . . . . . 67 Rate of Ethanol Exudation by Tomato Plants at Three Soil Temperatures in the Greenhouse . . . . 68 Ethanol Concentration in Xylem Exudate of Tomato Plants Flooded at Two Soil Temperatures Under .Medium Air Temperature and High Light Intensity . 69 TranSpiration Loss of Ethanol From Flooded Tobacco Plants 0 o o c e o o o o e o o o a e o o o o o 0 70 Effect of Flooding on Rate of Ethanol Transpiration From Tobacco Plants . . . . . . . . . . . . . . 7O Transpiration Loss of Ethanol From Flooded Tomato Plants 0 o o o o o o o o o o o o o c o o o o o o 71 Effect of Flooding on Rate of Ethanol Transpiration From Tomato Plants .. . . . . . . . . . . . . . 71 Ethanol Concentration in XylemExudate of Tomato Plants Flooded in Three Soil Volumes at Medium Temperature and High Light Intensity . . . . . . 72 Rate of Ethanol Exudation From Tomato Plants Flooded in Three Soil Volumes at Medium Tempera- ture and High Light Intensity . . . . . . . . . . 73 Oxygen Diffusion Rates in Three Soil Volumes'- Flooded at Medium Temperature and High Light Intensity . . . . . . . . . . . . . . . . . . . . 74 Ethanol Concentration in Xylem Exudate of Tomato Plants Flooded in the Field at a Medium-High Air Temperature and High Light Intensity . is. . . . ‘75 ix INTRODUCTION Soil flooding recurrently inflicts severe damage on many cultivated crops. Short periods of flooding often reduce growth and yield while extended flooding periods may result in death of plants and complete loss of crop (7). Soil physical characteristics predispose some soils parti- cularly to flooding conditions and, in addition, plant species have been observed to differ in susceptibility to flood damage. It has frequently been observed, also, that concurrent climatic conditions affect the severity of flood damage. Specialization and intensity of cropping in cer- tain soil regions focus increased attention on the flood- ing factor in agriculture. This problem demands a search for greater knowledge of the plant reaction to flooding conditions. The soil physical system has been defined considerably with respect to flooding and water saturated soils are now considered to be synonymous with oxygen deficiency (ll, 33). Early work showed that oxygen was essential for plant growth in nutrient solutions and extension of studies to field conditions supported this conclusion. In the field soil oxygen supply, within the available soil moisture range, is normally dependent upon the large pore spaces in the soil, which represent a capacity factor. In addition 1 to the capacity factor it is now known that a continuous supply of oxygen to the plant, representing a rate factor, is essential for normal growth. 'The platinum micro- electrode technique of Lemon and Erickson (24) has con- tributed greatly in defining the rate of oxygen supply for different soil pore space and moisture conditions. As soil moisture increases within the available moisture range the air capacity is decreased and the increasing thickness of moisture films reduces the rate of oxygen supply. In a completely saturated soil, constituting flooded conditions, the air pore space is eliminated and the oxygen diffusion rate is greatly reduced. Under these conditions oxygen supply is dependent upon solubility of oxygen in water and oxygen solubility in water under prevailing soil tempera~ tures is low. Rapid advances in biochemistry and related fields have extensively clarified the role of oxygen in the plant cell and the nature of oxygen deficiency. Oxygen is known' to be a final electron acceptor in the electron transport chain within the cell. In this capacity oxygen enables degradation of energy rich products of photosynthesis to carbon dioxide and water with the concomitant release and transient storage of energy for carrying out cell functions. A deficiency of oxygen alters metabolic routes and, as a consequence, reduces energy production and re— sults in the formation of reaction products that differ from those of aerobic cells (8). Ethanol is one of the resultant cellular products of metabolism produced under oxygen deficient conditions (A2). In the aerobic cell, hexose products of photosyn— thesis are degraded via the glycolytic sequence to pyruvic acid and thence through the tricarboxylic acid cycle to CO2 and water. In anaerobic cells the tricarboxylic acid cycle is restricted since it is dependent upon oxygen and the electron tranSport chain to reoxidize the coenzymes essen— tial in its enzyme systems. The glycolytic sequence can continue to function under anaerobic conditions since the coenzyme, nicotinamide adenine dinucleotide, which is re- duced early in the sequence, is subsequently reoxidized when acetaldehyde, the decarboxylated product of pyruvic acid, is reduced to ethanol. Most plant cells have enzyme systems (l6)exhibiting the ethanol, or fermentation, reac— tion. Since ethanol production in the cell has been thus shown to be oxygen dependent, measurement of ethanol in the plant has been investigated as an index of anaerobic conditions. Studies by Kenefick (20), using an enzyme as— say procedure, and investigations by Grineva (13), using an alcohol oxidant, showed an increase in alcohol under anaerobic conditions in sugar beets, corn and sunflowers. The alcohol was presumed to be predominantly ethanol. Fulton (12), using gas chromatographic analysis, showed that ethanol concentration increased markedly in root xylem exu- date of tomatoes under flooded conditions. In addition the ethanol concentration of tomato exudate was highly cor— related with oxygen diffusion rate, at least during the early periods of flooding. It is considered that the ethanol assay can be used to provide valuable and extensive information on the re- action of plants to the anaerobic environment of flooded soils. Soils research has advanced considerably on the basis of chemical analysis of plants in relation to the chemical and physical characteristics of soils. Chemical analyses of nitrogen in plants, for example, have been re- lated to the many environmental factors of soils. These factors have included fertility level of this nutrient within the soil and soil physical factors influencing the amount of nitrogen released for plant use (3). In an analogous manner the measurement of ethanol in plants should provide a means of determining oxygen requirement and supply to the plant under given environmental conditions. In principle, the ethanol assay should provide a more sensi- tive measure with respect to oxygen supply than plant analyses provide for nutrient supply since the ethanol is part of a short term reaction while chemical analyses measure the integrated product of long term effects. Soil physicists, Baver and Russell, (5, 33), among others, have repeatedly pointed to the need for such measurements to indicate plant response to soil conditions. The objective of this investigation was to measure effects of certain soil environmental conditions on ethanol \I'l accumulation in plants under conditions of flooding. Tomato plants were used primarily throughout this study since this species is known to be damaged extensively by flood- ing. Tobacco plants, another sensitive species, were used in certain phases of the investigation. The primary objectives include: 1. Determination of the effect of air temperatures and light intensities on the accumulation of ethanol in xylem exudate of tomato plants flooded for short periods of time. Determination of the soil temperature effect on the accumulation of ethanol in xylem exudate of roots under certain air temperatures. Measurement of transpired ethanol from tomato and tobacco plants to determine the effectiveness of transpiration as a means of eliminating ethanol under flooded conditions. I Evaluation of the magnitude of root excretion as a means of eliminating ethanol from flooded plants. Determination of ethanol distribution in an- aerobic plants. Comparison of ethanol accumulation in tomato plants flooded in the field with results from plants flooded in the growth chamber to test the applicability of simulated conditions. LITERATURE REVIEW Flooding reduces plant growth principally due to reduced oxygen supply. This effect was originally demon- strated through aeration of plant culture solutions (33). Studies extended to soil systems also indicated low oxygen supply to be a causative factor in reduced growth under these conditions (7, 9). Oxygen supply in soil has been attributed to the amount of large soil pores (AA) which contain air at mois- ture contents within the available soil moisture range. Baver (5) and others (1, 6) have shown that the amount of large pores, referred to as air pore space, is important for plant growth and that reduction of this pore space be- yond certain limits markedly reduces growth and yield de- pending on plant species. The oxygen supply to the large pores and eSpecially to plant roots has been shown to depend upon a diffusion process. A platinum microelectrode technique, developed by Erickson and Lemon (2A, 25, 26, 27), enabled measurement of oxygen movement by diffusion in the liquid phase of soil. Since the platinum microelectrode must be in contact with soil moisture films to be operative it is considered to simulate the conditions under which plant roots contact oxygen. The amount of space available for storage of air con- stitutes a capacity factor while the rate of diffusion rep- resents the rate of oxygen supply (22, 26, 27). It has been shown (19) that the effect of each of these factors on oxygen supply is dependent upon moisture content within and above the available soil moisture range. As moisture con- tent increases the moisture films around the soil particles increase in thickness with a resultant decrease of oxygen diffusion across the films. Concurrently, the amount of soil pore space available for air decreases with increasing moisture content. Where the soil is completely saturated with water, as occurs during flooding, the supply of oxygen decreases rap— idly (38) and the rate of oxygen diffusion has been shown (19) to diminish very quickly. Under these conditions the oxygen supply depends upon the amount of oxygen dissolved in water and the amount of oxygen dissolved is very small under field conditions. The effect of increasing soil temperature, within plant growth limits, increases oxygen diffusion rate slightly and decreases solubility (17) but the alterations are negligible with respect to plant oxygen demands. Erickson and co-workers (11) have shown that short periods of flooding accompanied by decreased oxygen dif— fusion rate, reduced plant growth and yield. Oxygen de— ficient periods of twenty-four hours decreased growth of tomato plants in the field, eSpecially when the oxygen deficient period occurred during the early growth periods. A greater reduction in growth occurred when the oxygen de— ficient period was applied under high light intensities than under low light, an effect that the authors attributed to higher rates of photosynthesis and transpiration under these conditions. The nature of reduced oxygen supply within the plant has been considerably clarified by rapid advances in bio- chemistry. Limitation of oxygen supply within the cell has been shown (8, A2) to alter the metabolic pathways available for degradation. Energy within the cell is derived from enzymatic ox- idation of reduced products of photosynthesis. It is used to effect synthesis and carry out cell functions. The energy release occurs as electrons are transferred from degraded photosynthetic products through a highly organ- ized series of compounds arranged in order of decreasing electronegative potential. Ferredoxin is probably the in- itial electron acceptor and is followed by oxidized pyri— dine nucleotides and other components of a system collec- tively known as the electron transport chain. Oxygen is normally the final electron acceptor in this electron transfer system. This transfer process mediates the for- mation of a high energy compound, adenosine triphosphate. Where an adequate supply of oxygen exists, glucose is degraded to pyruvic acid by the glycolytic reaction and H sequence and is subsequently oxidized to CO 0 by 2 2 way of the tricarboxylic acid cycle. Under anaerobic conditions the tricarboxylic acid cycle is inhibited since inadequate oxygen supply limits operation of the electron transport chain and thus restricts reoxidation of pyridine nucleotides. The glycolytic sequence can continue anaero— bically since the NAD, which is reduced early in the se- quence, in turn reduces acetaldehyde, the decarboxylated product of pyruvic acid, with the formation of ethanol. As a consequence, a low oxygen supply to the plant cells results in reduced energy production and in both a quali- tative and quantitative change in reaction products (3A). Ethanol accumulation in plants has been determined as an index of anaerobic conditions. Kenefick (20), using an enzyme redox reaction, showed that alcohols pres- ent in sugar beets increased under anaerobic conditions. TranSpired samples also indicated that alcohol, considered to be ethanol, was tranSpired from the leaves. Grineva (13) using potassium dichromate as an oxidant showed the presence of alcohol in samples from corn and sunflower plants subjected to anaerobic conditions. Fulton (12) found a very high correlation between ethanol content of root exudate from tomato plants and ox- ygen supplying power of the medium during the early stages of flooding. In this investigation ethanol was identified and quantitatively measured by gas chromatographic tech- niques. The results indicated that for short term flood- ing periods, at least, measurement of ethanol provided a satisfactory means of measuring the extent to which lO anaerobic soil conditions could influence the fermenta- tion reaction. Ethanol occurred at a higher concentration in plants flooded in light than in the dark. Morphologi- cal age of tomato plants also affected ethanol accumula- tion to some extent and plants in flower contained more ethanol than younger plants. Alcohols, including ethanol, have been shown to have a stimulating effect on plant growth at very small concentrations (1A). On the other hand ethanol has pro- ven toxic to plants (12, 1A) when supplied from external sources in concentrations equivalent to those occuring in anaerobic plants. It is well known that plants, edaphically adapted and grown under favorable moisture conditions within the available soil moisture range, are markedly influenced by environmental factors (29). Rate of metabolite production by means of photosynthesis is dependent on light intensity and temperature while rate of degradation through respiraé tion depends upon air and soil temperature. The metabolic behavior of upland plants has received considerably less attention at soil moisture conditions in excess of the available range. Work by Erickson cited above has indicated that deleterious effects of soil an— aerobic conditions on plants are associated with light in- tensity. In addition, Fulton showed a difference for ethanol content of plants flooded in the light compared with those flooded in the dark. The above considerations 11 would suggest the feasibility of using the ethanol assay to characterize certain environmental effects with respect to plants subjected to flooded conditions for short periods of time. MATERIALS AND METHODS Description of Growth Chamber Where other factors permitted, a large Sherer-Gillett walk—in type growth chamber was used for control of environ- mental conditions in these experiments. The inside dimen- sions of the chamber were 3.35 meters square by 2.80 meters high. The unit was equipped to enable simulation of field conditions with respect to temperature, light intensity and humidity. Temperature was controlled by a cam-shaped template which programmed temperature in-a daily cycle. With this equipment temperature cycles could be chosen within a tem— perature range from near freezing to slightly more than A0° C. Air was continuously recirculated through the chamber from bottom to top. The unit was designed to provide a range Of quality and intensity of light (30) through the use of separately controlled banks of fluorescent and incandescent bulbs. As a result it was possible to select combinations of fluores- cent and incandescent light intensities. A further control of light intensity was achieved by means of the plant plat- form, within the chamber, which could be raised or lowered. The fluorescent banks consisted of A0, 20, and 12 bulb units while the incandescent lights consisted of A0, 25, and 16 12 l3 bulb units, where each unit was controlled by a separate time clock. With this equipment it was possible to obtain a programmed cycle of light intensities for periods of the day that approached light conditions during the main summer growing season. Light intensity was measured within the growth chamber for combinations of light sources referred to as "low" and "high" light intensities. The low light intensity consisted of a 16 bulb bank of incandescent lights combined with a 12 bulb bank of fluorescent lights. The high intensity light source contained 16, 25, and AC bulb banks of incandes- cent lights combined with 12, 20, and A0 bulb banks of fluorescent lights. The light energy emitted for each intensity was measured with a Beckman and Whitley thermal radiometer at- tached to a Sargent recorder. *Measurement was made at 3 distances from the light sources by placing the black sur- face sensing element of the radiometer at 25, 56, and 127 cm from the incandescent light bulbs. The distances cor— responded, respectively, to the top and mid points of the tomato plants under treatment and to the surface of the table upon which the containers were placed. At the low light intensity, energies measured at the top and mid point of the plants were .512 and .A80 gm-cal cm-2 min-l, respectively. Corresponding energy values at the high light intensity were l.Al7 and 1.286 gm-cal 1A cm_2 min—l. The energy of direct sunlight, measured in July, was 1.A72 gm—cal cm"2 min—l, Humidity could also be controlled widhin the chamber by means of moisture nozzles which were activated by a wet bulb control. It was possible to obtain relative humidities from approximately 50 percent to those near saturation al— though only higher relative humidities were maintained for these experiments. Description of Plant Growth Media and Growth of Plants for Air and Soil Temperature Studies Growth Medium A coarse sand material was used as the plant growth medium in all experiments where air temperatures, light intensities and soil temperatures were varied. Total pore space of this material, calculated.from bulk density measure— ments (5), approximated A0 percent of the volume and about 36 percent of the total pore Space represented air pore space. This medium was used to insure aerobic conditions for plants prior to flooding. The sand was placed in 15 cm diameter pipe containers, of galvanized metal, which were inserted within 20 cm di- ameter pipes constructed of similar material. Both pipes were 61 cm in height. The outside container was con— structed with a bottom while the inside pipe containing the sand was open at both ends. This arrangement permitted rapid entry and exit of water to and from the sand. Water 15 was added to the sand as required and was pumped daily from the outside container. This technique was used to ensure adequate aeration for the plants during the entire growth period. Care of Plants Fireball variety tomatoes, Lycopersicum esculentum Mill” were used in all experiments involving tomatoes. The plants were grown from seeds planted in greenhouse flats containing a potting soil mixture and were transplanted when the first true leaves appeared. The plants were grown in the greenhouse until flower buds were initiated and were then placed in the growth chamber for two days under medium temperature and high light conditions. The plants were watered twice daily. Major and minor nutrients were supplied to the soil at planting and subsequently at weekly intervals. Description of Plant Growth Media and Growth of Plants for Trangpiration Studies Growth Medium A greenhouse potting soil composed of 2 parts loam soil: 1 part sand: 1 part sphagnum peat moss was prepared for growing tomato and tobacco plants to be used in the transpiration studies. When this soil mixture was placed in 13 cm clay pots it reached a bulk density of approxi- mately 1.20 gm cc_l, equivalent to 55.0 percent total pore space. .16 Care of Plants Tomato and tobacco seedlings were tranSplanted to the 13 cm clay pots at the first true leaf stage. Subsequently these plants were grown in the greenhouse and received-appli- -cations of water and nutrients as required for normal growth. Description of Soil Temperature Control_WaterBaths Greenhouse Water Baths Insulated water baths were used to control soil tem- perature in the greenhouse. These tanks were 132-cm long, 86-cm wide and 69-cm deep, inside dimensions- They were sufficiently large to;accommodate_six tomato_plants in galvanized pipe containers. Temperatures of 10.0, 18.2 and 26.7°C were maintained in the three tanks, respectively, by means of thermostatically controlled heating and cooling equipment. The water baths provided a satisfactory degree of temperature control within the plant root medium and temperature varied only within one degree of the bath tem— perature. Growth Chamber Water Baths Cylindrical galvanized containers were adapted for use with the soil containers used in the growth chamber, since the growth chamber could not accommodate the large temperature control baths. The small baths were 61 cm in height, the same as the soil containers, and were 30 Cm in diameter so that one soil container could be l7 placed inside each bath. Water pipe attachments, 13 mm I. D., were attached at the top and bottom of each bath and the baths were connected in series with garden hose so that a complete replicate could be run simultaneously. Description of the Transpiration Chamber A chamber was constructed of 6—mm plexiglass for ob- taining a transpiration sample from tomato and tobacco plants. The chamber was 33-cm square and 38—cm in height with a 13-cm diameter opening on the bottom to enable place— ment over the plant. Solid plastic tubing, 6-mm in diameter, was installed in the side, near the bottom, for entry and, near the top on the opposite side, for removal of air which was fed through the chamber from a compressed air line to sweep out the transpired sample. I A separate container of l5—cm cubic dimensions was constructed of plexiglass, also, and was used to contain the l3—cm clay pot with the plant. This chamber had an in— let and outlet tube for water. The pot containing the plant and one inch of the stem was sealed off with a plastic lid and secured with tape. The chamber enclosing the top of the plant was sealed onto the bottom container with stop— cock grease and held in place by the weight of the light cooling bath. Light was supplied to the plant by five 300-watt bulbs that were immersed in water which removed some far infrared light and was recirculated to reduce radiant heat. 18 Fluorescent lights were placed to the side to extend the range of light quality. The light source supplied 1.21 n— gm—cal cm.2 min.l at the mid-point of the chamber. Description of Mist Chambers Mist chambers, designed and constructed by Erickson, were used for growth and treatment of plants in experiments where root secreted ethanol and ethanol concentration within the plant were measured. These chambers were constructed of 6-mm plexiglass and were 122-cm in length, 32-cm wide and 6l~cm deep at the mid-section. The bottom sloped from each end in order that the condensed mist would return rapidly to the liquid chamber for recirculation. A commercial room humidifier, equipped with fan, was installed in a cen- tral compartment in the base of the chamber. This pro- vided a continuous mist of Hoagland's solution to the plant roots that was adequate for growth. Openings were spaced at intervals in the top of the chamber for the young plants. Tomato plants were trans— planted to the mist chamber at the first leaf stage by wrapping each plant in cotton and attaching it to the open- ing with masking tape. An opening was left in the tOp for addition of nutrient solution and for aeration of the plants during growth. Other openings were available for admitting nitrogen gas that was used to effect anaerobic conditions in the chamber and for extraction of gas and vapor samples used for ethanol 19 measurement. A pump also was attached to one opening to permit oxygen measurement with a Beckman oxygen analyzer, in order to ensure that anaerobic conditions were main— tained during treatment. Openings were also placed in the bottom of the chamber, directly beneath the plants, so that plastic cups with tubes attached could be used to col- lect mist condensate samples from the roots. Measurement_9£_Eth§ngl_ The ethanol assay procedure, develOped by Fulton (12), was used to measure ethanol in samples obtained from xylem exudation, transpiration and root excretion. The sampling techniques are described below. Ethanol was measured with the same Beckman GC2A gas chromatograph and hydrogen flame attachment described by Fulton. The method involved injection of a 20 ul sample into the chromatograph and elution of the sample through a 1.83 meter stainless steel column to the flame. Column tempera- ture was maintained at 100° C and helium was used as the carrier gas with a flow rate of 80 cc min—l. Chromosorb W‘ was used as solid support material in the column. Diethy~ lene glycol succinate* comprised the liquid partition phase and phosphoric acid was used as activator in earlier ex- periments. Cartowax A00 was used as the partition phase in later experiments. Elution time and peak characteristics *Columns with these materials were prepared by Beck- man Instruments Inc., Fullerton, Calif., U.S.A. 20 were similar for both materials but more sensitive quanti- tative detection was provided by the carbowax. Elution peaks were integrated with a Sargent model SR recorder. Standard curves were prepared from a series of ethanol standards ranging in concentration from 5 to 300 parts per million on a weight basis. Determination of ethanol in condensed tissue samples necessitated a different column for separation of ethanol and methanol. A 1.83—meter x 6-mm column was prepared for this purpose using a commercial material designated as Pora— pak type Q.* Entrapment of Transpiration Samples TranSpiration samples from tobacco and tomato plants were entrapped in a U-tube and Erlenmeyer flask assembly which was submersed in a salt—ice bath at —15° c. The condensing apparatus was connected by tygon tubing to a solid plastic inlet tube in the wall near the top of the plexiglass tranSpiration chamber described above. Come pressed air from an air line was fed through the chamber continuously at a flow rate of A-5 liters min—1 for the 2A- hour period. The condensing trap was connected tithe cham- ber only for two-hour periods when samples were obtained. Distillation of Ethanol from Plant Tissue A distillation method was developed to extract tis- sue-free samples from plant material. Such a method was *This material was obtained from Waters Associates, Inc., Framingham, Mass., U.S.A. 21 chosen in preference to the use of expressed tissue samples since methods to express tissue result in a release of large molecular weight components in addition to cellular structures. Contamination by these high molecular weight and particulate materials was found to alter column charac- teristics. The present method was adapted to extract plant sap, containing ethanol, from stems, leaves and roots of tomato plants. Fresh tissue samples ranging in weight from 2 to 5 gm were placed within a glass tube annealed to the inside of a 125-ml Erlenmeyer flask. The flask was sealed with a foil-covered rubber stopper and the sample was distilled for two hours under a bank of 300-watt flood lamps. Initial attempts to measure ethanol in the distillate. using the carbowax and diethylene glycol succinate parti- tioning materials proved unsuccessful. In addition to the ethanol peak, which eluted at 1.8 minutes, another com- pound eluted at 1.7 minutes. It was not possible to re- solve the ethanol peak under these conditions. Preliminary work with suspected compounds showed that methanol was the interfering agent. In view of the tempera- ture (80° C) used to distill the samples from plant tissue it was concluded that methanol was formed non-enzymatiCally during the distillation process. It was necessary to use a partitioning material that could separate the two components. The commercial material, Porapak Q, described above was used for ethanol analysis. 22 With this material methanol formed a peak at 2.2 minutes while ethanol formed a peak at 5.6 minutes with column temperature at 160° C and a carrier gas flow rate of 100 cc min-l. To insure that the measured ethanol was a metabolic product rather than a product of autolysis recovery of known standardsvwnsmeasured. Dried tomato stem tissue was placed in aliquots of 15, 25 and 50 ppm ethanol stan- dards. The saturated stem tissue was distilled by the same procedure used for fresh tissue. A greater amount of methanol was released from the dried tissue than from fresh tissue and this resulted in some interference from the methanol peak even with this column. However, duplicate ethanol standard samples were quite reproducible and the peaks aggreed closely with stan- dards. It was readily concluded that the ethanol meaSured by this procedure was the metabolic product. Statistical Analysis Analysis of variance was used to determine statis- tical differences for treatments in all experiments ex- cept the transpiration and mist-chamber experiments. Where analysis of variance was conducted F values were calculated and are included in the appendices along with the data for each experiment. A randomized block design was used for all experiments to which statistical analyses were applied. Treatments were 23 replicated A to 6 times, the number of replications depend— ing primarily on preliminary results. Considerable variation due to error occurred in these experiments and this was attributed to biological variability. Although this variability was present the treatments signifi- cantly influenced ethanol content as indicated below. Transpiration and mist-chamber data were not treated statistically in this investigation. Only two replicates were used in the transpiration study and consequently the average values were used. In the mist-chamber experiment also, the design to give the desired information was not prediSposed to statistical analysis and average values were used. EXPERIMENTAL RESULTS Air Temperature and Light Intensifiy Effects on Ethanol Accumulation in Flooded Tomato Plants Description of Experiment The effects of air temperature and light intensity on ethanol accumulation in xylem exudate of flooded tomato plants was investigated by treatment of plants in the growth chamber described above. A set of flooding treatments, each replicated in quintuplicate, was established within each of six simulated environments, with each set of plants being subjected to only one environment during flooding. Three air temperatures, each combined with a low and high light intensity comprised the six environments. The growth cham— ber was maintained at approximately 75 percent relative hu— midity within each temperature and light intensity. The three air temperatures were cyclic and were se— lected to represent a range of temperatures under which plants may be subjected to field flooding conditions in Michigan (A). A temperature gradient was present in the growth chamber but thermometer readings indicated that temperature was maintained at the desired level within the immediate plant zone. The high air temperature was pro- grammed from a minimum of 16.7 to 35.6° C for the 2A-hour 2A R 3 \I I flooding period and during the l2-hour day period ranged from 31.7 to 35.6° C. Minimum and maximum temperatures for the medium temperature range were 9.1 and 30.00 C respec- tively, for the 2A-hour period. Air temperature for the medium cycle ranged from 22.2 to 300°C for the l2-hour day period at full light intensity. The low temperature cycle increased from 16.0 to 20.60 C for the l2-hour day period and ranged from 10.0 to 20.60 C for the full day. The plants used in the experiment were tranSplanted as seedlings into the sand containers and were grown in the greenhouse until they approached treatment stage where blossom clusters appeared. Transplantings were carried out in sequence in order that the plants for each experiment would be at a similar morphological age at the time of treatment. As plants reached the early bloom stage the con- tainers with plants were placed in the growth chamber for a two-day period at the medium temperature cycle and high light intensity in order that the condition of the plants would be constant at the time when that group of plants was subjected to a Specific temperature and light regime during treatment. Flooding treatments within each combination of air temperature and light intensity consisted of an unflooded control and flooding with water to the sand surface for A— and 12—hour periods. Additional 2A—hour flood periods were subsequently applied to plants at the high temperature cycle combined with low and high light intensities and at the 26 medium temperature cycle combined with the high light intensity. Xylem exudate samples were obtained by decapitating plants at the end of the flooding period and collecting samples in tygon tubing attached to the decapitated stem. The flood water was pumped from the outside container at the end of the designated flood period. Results Table 1 shows that the oxygen diffusion rates of the flooded soils were sufficiently low for all environments to harm tomato plant growth (11, 2A). The slightly higher rate for the shorter flooding period is attributed to en- trapped air. TABLE l.-—0xygen diffusion rates gm x 10'"8 cm"2 min"1 soils flooded at three cyclic air temperatures and two light intensities. in Flooding Period Hours High Light Low Light High Temperature A 17.1 15.2 12 10.7 8.8 Medium Temperature 14 18.3 111.3 12 11.6 10.7 Low Temperature A 13.3 1A.3 12 11.8 7-7 27 Results in Fig. 1 indicate that air temperature had a pronounced effect on ethanol accumulation in xylem exudate of flooded tomato plants, eSpecially beyond the A-hour flooding period. At the high temperature cycle, where a maximum of 35.6° C was reached, ethanol concentration con— tinued to increase at 12 hours and subsequent measurements at 2A hours showed a considerably greater ethanol concen- tration. The shape of the curve, however, indicated that, at the 2A-hour flood period, ethanol concentration was ap- proaching a maximum at the 35.6 ° C. At the 30.0° C range, ethanol concentration reached a maximum at the 12-hour flooding period. This is in agreement with results obtained by Fulton. At the low temperature, where a maximum of 20.6° C was reached, ethanol concentration was considerably lower at the l2-hour flooding period than for the medium tempera- ture at high light intensity. A Light intensity had a less consistent effect on ethanol concentration in xylem exudate than air temperature, in these experiments. In the high temperature range ethanol concentrations were nearly identical for both levels of light intensity up to and including the l2—hour flooding period. At the 2A-hour flood period, however, ethanol con— centration decreased from the l2-hour period where light intensity was low while concentration continued to rise under high light. The level of light intensity had its greatest effect on ethanol concentration within the medium temperature range. In the medium temperature experiments, 28 A.mopmofiaoou m>am mo ommho>m on» ma ozam> cowmv cocoon madman cumsou no osmosxc anzx an soaumnuccocoo Hocmnpmll.a .me masomileofipom weapooam .mmauamcoucfi unwfia 03» no use msaxms pcumofioca on oedema caspmnodsmp has no mcwcmn ocean moods an on as NA m + T m 1+ A I d: a O semen soH o oc.om nm (in new: swan u soon ml a saw: 33 0 poem 4.) . 4 new: swan o ooom d c. unwed soH o oc.mm my ilu o ocwaa case 0 oc.mm er. ,lo T1 [OW om .IOOH Toma .r 9: Iowa Iowa loom .tomm Roam omm Ill Tomm .[O_Jm mdd-uotqeaqueouoa Ioueqqg 29 the ethanol concentrations in plants flooded for 12— and 2A—hdur periods under low light were less than for high light. Under the low light and medium air temperature regime ethanol ccncentrations approximated values for low air temperature. Light is reported (29) to be limiting in photosyn- thesis for tomato plants when light is less than 1/3 to l/A full light intensity. It is probable that the low light intensity used here only approached the limiting value. Nevertheless, the low light level used in this ex- periment, is less than values generally obtained in cloudy conditions during the growing season for this latitude(AA). Soil Temperature Effect on Ethanol Accumulation in Flooded Tomato Plants Description of Egperiment in Greenhouse The review of Richards g£_§l, (31) has indicated the importance of soil temperature on plant growth and physio- logical processes. Plant roots, like plant tops, have been shown to have optimum temperature ranges for growth (10) and for accumulation or depletion of constituents (31). In addition,increased soil temperature has been calculated to impose greater oxygen demands on respiring roots (28). Consequently, measurement of ethanol should indicate the intensity of anaerobiosis at different soil temperatures. The present experiment was established to measure the effect of ethanol accumulation in xylem exudate of tomato 30 plants flooded at three soil temperatures. Water bath temperatures employed were 10.0, 18.2 and 26.70 C and soil temperatures within the pipe containers were maintained within one degree of these values. Air temperatures in the greenhouse during treatment varied from 18.2 to 21.10 C, and a low light intensity was established with fluorescent light. The plants used in the study were grown in the greenv house and placed in the growth chamber for a conditioning period before being placed in the water baths. Results of Soil Temperature Experiment- Greenhouse Fig. 2 shows a slight increase in average ethanol con- centration at the l2—hour flood period with increasing soil temperature. All values, however, were low and there was high variability within treatments. 0n the basis of these results it is concluded that soil temperature was a factor influencing ethanol accumulation in roots but that the effect was not great compared with the effect of high air temperatures. Soil temperature influenced rate of root exudate in accordance with results reported elsewhere (18, 35). Rates of ethanol exudate were expressed as ugm hr"1 and are pre— sented in Fig. 3. Results expressed in this form appear more meaningful with regard to ethanol accumulation under these conditions. They do not appear to alter the earlier conclusion, however that soil temperature effect was small under low air temperature and low light intensity. Ethanol Concentration ppm 60 55 50 10 31 26.70 C 18.2° C $10.00 c Flooding Period — Hours Fig. 2.--Ethanol concentration in xylem exudates of tomato plants flooded at three soil temperatures in the greenhouse at an air temperature of18-3-21.l° C and low light intensity. (Each value is the average of six replicates.) 32 A.mmpmoflaomh xflm mo mtho>m ocp ma ozam> sommv .zpfimcopCH pcwfia 30H ocm o oa.amlm.wa mo oLSPM Imooeop nfim am pm omsogcoopw 05» CH mopspwpoQEop afiom mopcp pm oooooag moOmeOp Eopm coapmosxo Hocmcpm mo commul.m .wam masom I UOfipom msfiuooam a a l. _ o r m J «V [OH 0. o n Ima «QT o om.mH J ION [mm . N o 0N 0 Tom rmm l O J (I_JU m3“) UOIqepnxs toueqqs JO aqea 33 Description of_Soil Temperature Experiment iniGrowth Chamber Results of the greenhouse soil temperature experiment suggested that effect of soil temperature may be more im- portant at a higher air temperature and greater light inten- sity. In the air and light intensity experiments, reported above, soil temperature in the containers followed air temperature closely in a similar cycle at the 7— and lS-cm depths. An experiment was established at the medium tempera— ture in the growth chamber to determine the effect of soil temperature controlled at 18.2° C while air temperature followed its regular cycle. The cylindrical water baths, described above, were used for this experiment and water was circulated through the pipes to maintain soil temperature within one degree of 18.2° C. The baths were insulated with commercial insulating material during treatment. Results of Soil Temperature Experiment in Growth Chamber Ethanol concentration Fig. 4 was numerically lower up to 12 hours flooding where soil temperature was maintained at 18.2° C than where soil temperature followed the medium temperature cycle (30.0° C maximum). Rates of exudation were similar for the two soil temperatures and it is con- cluded that soil temperature had some influence under these air environmental conditions. In the field, soil tempera— tures tend to follow air temperatures closely in a similar 34 but delayed diurnal cycle within surface depths (31). Consequently, it is considered that the growth chamber data obtained from the 9.1—30.0°C soil temperature range would apply more readily to field situations than would data for the constant soil temperature of 18.2 centigrade. Transpiration Losses of Ethanol From Tobacco and Tomato Plants Description of Experiment The small molecular size and relatively high volatility of ethanol would suggest that this compound could be readily removed by the transpiration stream. Investigations by Kenefick (20) showed that transpiration served as a means of eliminating alcohol from sugar beet leaves under anaerobic conditions. Fulton (12) showed that intact tomato plants had less ethanol than decapitated plants flooded for identical periods of time. This effect was attributed in part to elimination capacity of the foliage. The following experiment was conducted to determine the effectiveness of transpiration for removal of ethanol from tomato and tobacco plants. Tobacco plants were in— cluded in this experiment since these plants have large leaves and have also been shown to accumulate large ethanol concentrations when flooded.* *Erickson, A. E., J. M. Fulton and G. H. Brandt. New Techniques for Relating soil Aeration and Plant Response. Trans. 8th International Cong. Soil Sci. Bucharest (in press). 35 l BOI .k ~o C l2G« 311d g lOG« 3 90a EU :3 an H :3 Soil Temp. Cycle i, {99.1 - 30.0° C O 2?, Soil Temp. :3 18.2° C LL] 0' I I I I I I ’4 8 12 16 2o 24 Flooding Period - Hours Fig. U.—-Ethanol concentration in xylem exudate of tomato plants flooded at two soil temperatures under medium air temperature and high light intensity. (Each value is the average of five replicates.) 36 Tomato and tobacco plants were grown in lB-cm clay pots in a greenhouse potting soil and were treated when the plants yet could be accommodated in the plastic chamber described above. At this time the tomato plants were in the early bloom stage with one or two blossom clusters. The tobacco plants were in the foliar stage. Two replicates of each plant species were subjected to flooding in the laboratory while another set was flooded in the growth chamber. Light intensities were 1.20—1.30 gm-cal cm"2 min.1 at the mid-plant zone in both experiments. Air temperature in the lab experiment was constant at 26.7° C within the chamber while chamber temperature within the Sherer—Gillett unit reached 35.6° C in a cyclic manner. Transpiration samples were trapped by the method described previously. Results Ethanol concentration in tranSpiration condensates of both tobacco and tomato plants, Fig. 5 and Fig. 7 increased with duration of flooding period. Maximum tranSpiration concentration was reached at the 12-hour flooding period for tomato plants in both the laboratory and growth chamber experiments. The maximum concentration for tobacco plants was also reached at 12 hours in the constant temperature experiment but continued to increase at 24 hours in the growth chamber. Concentrations for tomato plants followed the pattern established in exudate accumulation, although concentrations were much 37 lower in transpired condensates. Tomato plants had a slightly higher ethanol concentration than tobacco plants at both the constant and cyclic temperatures. Since concentrations were small, rates of ethanol loss in the tranSpired samples were calculated and presented in Fig. 6 and Fig. 8 for tobacco and tomatoes. Results expressed in this manner disclosed a cyclic pattern for ethanol loss in transpired samples under the cyclic temperature and light environment. Loss rate was high at the u-hour flood period when transpiration was high and was low at 12 hours when tranSpiration was negligible. Transpiration loss increased again at the 24-hour period. Effect of Flooding Tomato Plants in Different Soil Volumes Description of Experiment Flooding of tomato plants in the greenhouse and growth chamber involved the use of relatively small soil volumes. It is known that saturation of soil is associated with entrapment of air in the soil (5). The amount of air entrapped would depend on volume. In addition the work of Williamson (M3) has indicated that oxygen diffusion rates are dependent to some extent on soil volume. Such an effect could presumably result from decreased oxygen concentration due to increased plant requirement per unit soil volume. 39 A.mmps0fiaqmn 03» mo mmmpm>m on» ma wzam> nommv .nonsmno nuzohw on» Ca manpmanEmp oaaozo m pm cam mLOpmpoomH on» Ca mndumnmoEop pcwpmsoo a pm cocooam mmeHQ ooomn0p Eomm coapmuflamcmpp an mmoa Hocmcpm no opmmll.m .me mpzom I anpom wcHUOOHm 2m om ma NH m a _ _ _ h _ -c 0 Am H o u I m 3 o 1.m 1.: \ 0 1m \. Q\\. nonsmco zpzonw OIIIIIIAV v.0 zmopmmoomq “WIIIIII@ I p ssoq JO aqag Iouequ Jq mBn) (I- 42 The present experiment was established to measure ethanol concentration in xylem exudate of tomato plants flooded in three different soil volumes. Tomato plants were grown in a potting soil mixture in 1.9, 3.8 and 7.6 liter glazed crooks. Flooding treatments were applied when the plants were in the bloom stage and each flooding treatment was replicated in quadruplicate within each soil volume. The treatments consisting of a control and 3-, 6- and 9-hour flooding periods were carried out in the growth chamber at the medium air temperature combined with high light intensity. Experimental Results Ethanol concentration, Fig. 9,increased during the 9—hour period within each soil volume and appeared to reach a maximum concentration at 6-hours in the 7.6 liter containers, while ethanol continued to increase in the smaller containers. Plants in the 1.9 liter containers had considerably more ethanol than plants in larger soil volumes at the 6- and 9—hour periods. Ethanol concentration in 3.8 liter containers was slightly greater than in 7.6 liter volumes at 9—hours. Plants in the three soil volumes differed in exudation rate and results in Fig. 10 show this effect. On this basis, rate of ethanol exudation was slightly greater at 3- and 6—hour periods from plants in 1.9 liter containers than from plants in larger volumes. At the osam> nommv mnsom I coauom wchooam A.mmuMoHHaoh 930m no mwmnm>m on» ma .mpfimcmpcfi panH swan cam manpmnoQEmu paw Esavoe pm mmESHo> HHom acmAmMMfiU omnnp ca cocooau mpcwaa cause» no mumpsxm Emamx CH cofipwnpcmocoo Hocmnpmln.m .wfim a. m i .l. m .w m. m H. II 3 u. nee: e; o 0 H33 w.m 9 o see: a; o 6 OOH com com 00: com uotqeaquaouoo Iouaqqg wdd — All A.ncpsoaaomh Lsom mo mwmpm>m can ma msam> sommv .zuwmcoucfi ucmwa :mH; 6:3 opspwpoesop Lam Ezflcos pm moESHo> HHow ocoLoMMHo omen» :H nooooHu mucmHQ oumEOp Sosa :owpwosxo Hocmcum mo opwmII.oH .mHm mpzo: I oofihom mcfiooon o m h. . P III 0 ON foo I o co rOOH Ide \\\\\ I Ieefl Idwa qufia 0.x. I b T owH Lop: m.m ¢ 49 mefia m.a 0 I0 Ian (I_Jq an) snag uotqepnxg Ioueuig 45 9-hour period ethanol exudation rate was increasing more rapidly on the plants in larger soil volumes. The results would suggest that soil volume altered the pattern of ethanol concentration and exudation through the amounts of entrapped air in the soil. Such an effect would be feasible during short term flooding periods. Entrapped air is commonly referred to (5, 33), as a factor effecting the wetting characteristics in soils. Oxygen diffusion rate was slightly higher in the 7.6 liter pots during flooding than on smaller soil volumes although all oxygen diffusion rates for flooding periods were low. It is to be noted that plants in the 1.9 liter con— tainers were smaller than in the larger soil volumes. The plants were at the same morphological stages, however, on the basis of flowering. Distribution of Ethanol in Anaerobic Tomato Plants Description of Experiment The present experiment was established to determine the distribution of ethanol in anaerobic tomato plants and to measure ethanol secretion by the roots. The work of Grineva (13) showed that pyruvic acid, an ethanol pre- cursor, was in higher concentration in sunflower tops than in the roots when the plants were subjected to anaerobic conditions. Measurements of oxygen in plants (23), (39) have indicated an increasing concentration from bottom A6 to top during conditions of photosynthesis. In addition, it has been shown that plants excrete many organic compounds through the root (32). Grineva showed that alcohols were excreted by anaerobic plants. The plants in this experiment were grown and treated in a mist chamber described before. This culture technique was chosen to enable measurement of root excreted ethanol and to obtain clean roots for determination of ethanol content. Nutrients were supplied in the mist by Hoagland's solution and the plants were grown to the early bloom stage before anaerobic treatment was applied. One plant was selected to serve as a control while four plants were subjected to anaerobic treatment. The anaerobic condition was established in the mist chamber, which contained the roots, by supplying a stream of nitrogen gas to the chamber for a l2-hour period. After treatment, the anaerobic plants, along with the control plant, were cut into sections designated as top leaves, bottom leaves, top stem, bottom stem, top roots and bottom roots. In addition to plant tissue samples, effluent samples were obtained from the root environment and condensed mist samples were collected from under each plant root. Results and Discussion Anaerobic conditions, established in the mist chamber, increased ethanol concentration, (Table 2), throughout the plant in comparison with the aerobic A7 IABIE 2.--Effect of flooding with nitrogen gas on ethanol distribution in tomato plants. Treatment Part of ,.NO, l2-Hour Flood with Nitrogen Gas Flood Plant Control ' Plant 2 3 4 Av. Ethanol ppm Iop Leaves 3 19 24 26 17 22 Bottom Leaves 4 25 2O 29 E2 24 Top Stem 5 12 38 36 4O 32 Bottom Stem 7 23 46 78 41 47 Top Root 7 28 38 25 28 30 Bottom Root 6 l3 17 3O 15 19 TABLE 3.--Effect of flooding with nitrogen gas on root excretion of ethanol. Treatment Sample No I l2-Hour FlOOd Flood Ethanol ppm Nitrogen Effluent 8 24 Plant 1 5 9 2 u 24 3 1 14 u 2 20 Average Root 3 l7 48 control. In nitrogen treated plants, ethanol was in highest concentration in the bottom portion of the stem and decreased toward the top of the plant and decreased also in the bottom roots. Ethanol concentration was considerably lower than in the previous experiments where sand or soil constituted the root medium. Ethanol concentration, (Table 3), determined on condensed vapor samples from the root atmOSphere, was increased three-fold by the l2-hour flood period. In addition, ethanol was increased in condensed mist samples collected under the roots at the l2-hour flood period. Under the anaerobic conditions of this experiment it is indicated that ethanol is excreted to the external root environment in considerable concentrations. Effect of Carbon Dioxide on Ethanol Concentration in Tomato Plants Under Aerobic Conditions Descriptioppof Experiment Carbon dioxide has been investigated (33) as a deleterious factor affecting plants on saturated soils. Carbon dioxide concentration in soils has often been associated reciprocally with low oxygen content. An experiment was established, using the mist chamber technique, to determine whether high carbon dioxide concentration influences the fermentation reaction in tomato plants under aerobic conditions. Tomato plants were grown in the mist chamber by means of identical 49 culture techniques used in the nitrogen flooding experiment. Treatment of the plants in this experiment consisted of enclosing the roots in a gaseous mixture composed of 20.1 percent carbon dioxide, 20.1 percent oxygen and 59.8 percent nitrogen. Plant tissue, chamber effluent and mist condensate samples from below the plants were collected as in the previous experiment. Experimental Results Ethanol concentrations throughout the plants were low (Table 4) and showed no effect due to carbon dioxide concentration under the aerobic conditions provided. The concentrations of ethanol in the atmosphere surrounding the roots and in the condensed mist samples (Table 5), collected below the roots, were also low. Difficulty was experienced in obtaining accurate measurements of ethanol at concentrations below 5 parts per million. The data indicate that carbon dioxide concentration did not influence the fermentation reaction under aerobic conditions. The effect of carbon dioxide concentration under anaerobic conditions was not investigated. Quantities of Ethanol Accumulated and Eliminated from Tomato Plants Total quantities of ethanol in tomato plants and the amounts eliminated from plants by tranSpiration and root excretion were determined for a l2-hour anaerobic period. The amounts of ethanol in plants and quantities excreted by roots were calculated from mist chamber data. 50 TABLE 4.--Effect of carbon dioxide on ethanol distribution in tomato plants under aerobic conditions. Treatment No l2-Hour Treatment with Gas Mixture Part of Flood Plant Control Plant 1 2 3 4 Av. Ethanol—ppm Top leaves 2 4 2 4 5 5 Bottom leaves 2 2 4 2 7 4 Top stem 1 4 2 2 5 3 Bottom stem 3 4 2 2 5 3 Top root 3 2 2 2 4 3 Bottom root 2 2 4 2 3 3 TABLE 5.--Effect of carbon dioxide on root excretion of ethanol under aerobic conditions. Treatment Sample No l2-Hour Flood Flood Ethanoleppm Cas Efflueit 2 2 Plant 1 3 O 2 O 0 3 O 5 4 O O 51 The quantities of ethanol eliminated by transpiration were calculated from the tomato transpiration results. Plants used in both experiments were similar in size and morpho- logical stage of growth. Table 6 shows that the rate at which ethanol was eliminated by plants was considerably less than the rate at which it accumulated. This result is expected since otherwise the compound would not accumulate in such read— ily measurable concentrations. The data does show that root excretion served more effectively than transpiration in eliminating some of this anaerobic product. In addi- tion the amount of ethanol in aerobic plants is sufficiently high to suggest that this compound is normally metabolized, at least to a small extent, in the plant. TABLE 6.——Ethanol accumulation and elimination from anaero- bic tomato plants in ugm per plant. w Flooding Period - Hours Amount of Ethanol None 12 ugm Total Plant 380 1829 Transpiration 2 127 Root Excretion 4 453 52 Flooding of Tomato Plants in 3.8 Liter Containers in the Field Description of Experiment Experiments in the growth chamber under simulated environments had shown Specific responses to air temperature and light intensity using ethanol measurements as an indicator of anaerobic conditions. Soil temperature was also shown to influence the anaerobic effect to a lesser extent. To further investigate the usefulness of ethanol measurements as indicators of flooding damage it was considered important to obtain measurements under field environmental conditions. Initially an experiment was established for this purpose by growing plants in a field location on a loam textured soil. In this earlier experiment the tomato seedlings were transplanted into the field at the first true leaf stage as was done in the greenhouse experiments. The plants were flooded by enclosing each plant in a 91-cm perimeter of aluminum sheeting inserted to a 10-cm depth in the soil. The entire soil area was extremely dry at the time the plants reached flooding stage and difficulty was experienced in attaining flooded conditions. The first flooding attempts consisted of applying water within each aluminum enclosure until aboutEScnn of water remained on the soil surface around the plant. Drainage characteristics of the soil were rapid and it was necessary to provide a continuous supply of water to the plant area. Oxygen supply in the soil surface decreased to levels known to limit plant growth as indicated by ox- ygen diffusion rates. Ethanol concentration, however, was very low in root exudates from the plants and it was con- cluded that soil flooding was not achieved throughout the root zone, and the oxygen supply did not become limiting. An experiment was designed to measure the field en- vironmental effect under conditions where soil flooding could be assured. To do this plants were grown in 3.8 liter glazed pots in a potting soil mixture similar to that used in the soil volume experiments. The plants were grown outside until they reached the early bloom stage. Two days before treatment the plants were transferred to the field and enclosed in the soil in order that tempera— ture in the plant root zone could equilibrate with that of the soil environment. Beam In Figure 11 data from this field experiment are com— pared with data obtained at two temperatures and at high light intensity in the growth chamber (of, Figure 1). Ethanol concentrations in the field flooding experiment followed a pattern for flooding duration similar to that obtained in the growth chamber. In addition the average value at the 24—hour flood period was between the high tem- perature and low temperature concentrations. 54 .mpcmEHmexm schemes cpzopw CH omcflmp Ipo some Spas ComaanEoo CH vamam one CH oooooam mucmaa oumE I0p mo mumpsxo Scams CH soapmpucmocoo HocmcpMII.HH .wflm mpdom I coahom mafioooam em om ma ma w z .1 . b _ b _ \2 tobacco cpzono T Iflwjlnwe: e u ooIom w\ GM saw .mm a» .9 o 0 eye .4 «7 fix was e8 0 0o runway \\% o\ r OOH com com uotqeaquaouoo Iouequ wdd — 55 Air temperature in the field reached a maximum of 33.40 C which was close to the high temperature cycle maximum of 35.60 C in the chamber. Light intensity was high in both locations except that a gradient would not exist in the field as occurred in the simulated conditions. Soil temperature in the field did not reach the maximum temperature recorded in the chamber but was 3 to 50 C lower. It is evident from the data that the simulated conditions in the growth chamber were quite similar to the field conditions and it is concluded that results ob- tained in the growth chamber are applicable for field situations. DISCUSSION OF RESULTS Air temperature had a pronounced influence on ethanol concentration in xylem exudate of flooded tomato plants. Within each air temperature environment, ethanol concen- tration increased with flooding period during the first 12 hours, in agreement with results reported elsewhere (12). At the high air temperature level, in the presence of high light intensity, ethanol concentration continued to increase at 12 hours but appeared to reach a maximum at the 24-hour period. For all other air and light environments maximum ethanol concentration was reached at 12 hours. Light intensity, within the range supplied in these experiments, had a smaller effect than air temperature on ethanol accumulation. High light intensity, under medium air temperature, increased ethanol concentration at 12— and 24-hour periods of flooding. At the high air temperature ethanol concentration was lower at the 24-hour period under low light than under high light intensity. Since the low light intensity provided in these experiments was lower than light intensity values for cloudy daytime periods in the field it is concluded that light intensity would be of less practical significance than air temperature with re— gard to the fermentation reaction. 56 57 Soil temperature, in these experiments, had a smaller effect on ethanol concentration in tomato root exudate than air temperature. Where light intensity and air tempera- ture were low, in the greenhouse experiment, ethanol con— centration was-also low, although the amount exuded was dependent on soil temperature. At medium air temperature and high light intensity in the growth chamber, where a maximum air temperature of 30.0° C was reached, soil tem- perature had a greater.effect on the concentration of eth— anol in xylem exudate than in the greenhouse experiment. This effect was especially evident during the early periods of flooding. Where soil temperature followed the air tem— perature cycle ethanol concentration reached a maximum at 12 hours. Where soil temperature was maintained constant at a lower level of 18.2° C ethanol concentration approached the same maximum but the maximum was not reached until the 24-hour period. Also, ethanol concentration at the 18.20 C soil temperature reflected a linear relationship with flood— ing period. The amount of ethanol transpired from tomato and tobacco plant foliage was small in relation to the amount of this compound exuded by the roots. Concentration of transpired ethanol, however, was proportional to exudate accumulation with respect to duration of flooding conditions. In addition, a maximum transpiration concentration appeared to be reached that coincided with the maximum of root exu- date for similar conditions (21). 58 Results of the above experiments indicated that the fermentation reaction as measured by ethanol concentration was dependent upon a continuous supply of photosynthetic products (41) rather'than upon degradation of stored com- pounds. At high air'temperature and high light intensity 'ethanol concentration continued to increase up to the 24- hour period while at low light intensity and high air tem- perature ethanol concentration decreased from the 12- to the 24-hour period. The soil temperature experiment, con- ducted in the greenhouse, indicated that ethanol concentra- tion was increased with soil temperature but the amounts of ethanol involved were small. The effect of lower soil temperature in the growth chamber was to produce a linear increase in ethanol concentration that approached the maximum obtained earlier where soil temperature followed the air temperature cycle. The cyclic nature of amounts cf ethanol transpired would also indicate the dependency of the reaction upon the photosynthetic supply rather than upon stored metabolites. Ethanol was excreted in small amounts by aerobic tomato plants and in considerably larger quantities by anaerobic plants. This compound was present in the root atmosphere and in condensed mist samples from the plant roots. Anaerobic conditions for this phase of the study were established by treatment with nitrogen gas in the mist chamber and ethanol concentrations were lower in the plants than where soil was used as the plant medium. Nevertheless, 59 the results show that root excretion provides a potentially important means of ethanol elimination by flooded tomato plants. Comparison of excretion data with transpiration re— sults also points to root excretion as being a more effective means of ethanol elimination than transpiration. Although the excretion of ethanol by plant roots may provide a means of alleviating toxicity within the plant the process may also induce pathogenicity. Weinhold (40) has shown that ethanol concentrations within the range established by this experi- ment are effective in initiating invasion of plant roots by certain soil pathogens. Ethanol in tomato plants subjected to 12 hours of flooding was observed to vary with the portion of the plant in which it was measured. The highest concentration of eth- anol was observed in the lower stem and in the top roots. Ethanol concentration decreased in the upper leaves and in the bottom roots. Lower ethanol concentration in the upper leaves is attributed to transpiration of the compound from the leaves and to higher oxygen concentration in the upper portions of the plant. Since ethanol removal was small the effect of oxygen due to photosynthesis and gaseous exchange is an important factor. Root excretion data indicate that ethanol concentration in the lower root system results from excretion of the compound and point to the lower root region as the most active zone of ethanol excretion. It is apparent that since ethanol increases in the plant with flood period that the means normally available for disposal or utiliza- tion of this compound are insufficient to offset the amounts 60 produced. The distribution study indicates, however, that root environment provides a significant sink for elimination of this constituent. Volume of soil, in which tomato plants were flooded, was shown to influence the time pattern of ethanol accu- mulation. Ethanol concentration was considerably higher on plants flooded in the smallest soil volume compared with concentration in plants flooded in volumes of double or quadruple magnitude. The rate of ethanol exudation was also higher in the smallest soil volume than in the larger volumes during early periods of flooding but this result was reversed at the longest flooding period. Potential oxygen availability in flooded treatments, measured as ox- ygen diffusion rate, was slightly higher on the largest soil volume than in the two smaller soil volumes, although all volumes provided values shown to be associated with the fermentation reaction. It is concluded that a greater sup— ply of oxygen relative to plant size was available in the form of entrapped air in the larger soil volumes. It is further concluded that the volume effect continued only un- til this supply of oxygen was consumed, which in this ex— periment was approximately 6 hours. Field flooding of tomatoes showed that field environ- mental conditions resulted in ethanol concentrations similar to those obtained in the growth chamber for identical flood- ing periods. It was necessary to use pot containers for the plants to ensure accurate flooded conditions in the 61 field experiment and the size of container, 3.8 liters in this experiment, could have influenced the actual concentra— tion of ethanol produced. Nevertheless the results showed that similar effects for flooding occurred for both envi- ronments and it is concluded that measurements in the growth chamber have valid application to field conditions. Considerable variation occurred between ethanol values within each treatment throughout the experiments in this investigation. It is believed that these differences were due to variability inherent in the plant materials. Regardless of this variability sufficient consistency was obtained to establish statistical differences described between treatments and it is concluded that the averages fairly represent treatment effects. The investigations reported here Show that measure— ment of ethanol concentration in tomato plants is a use- ful means of determining the degree of damage resulting from environmental conditions during short periods of flooding. The studies conducted by Kenefick and also by Grineva indicated that alcohol measurement served as an index of insufficient oxygen supply. Fulton determined ethanol qualitatively and showed a quantitative relation— ship between this compound in xylem exudate of tomato_plants and soil oxygen diffusion rate. The present study is in agreement with these results and further shows that en- vironmental conditions during a short term flooding period can be assessed on the basis of ethanol measurement. 62 One worker, Letey (2), was unable to obtain a rela- tionship between ethanol concentration, in tomato plants, and different oxygen concentrations in nutrient solutions. However, in Letey's investigation oxygen levels were es- tablished by bubbling oxygen through the solution medium at 21, 10, 3.5 and 1.5 percent concentrations. Russell (33) has questioned the applicability of dynamic conditions of this nature in studying oxygen requirements for plants. In addition the treatments were continued for periods extended to three weeks. The question is also raised con- cerning adaptability of the plants to these low oxygen conditions through development of aerenchyma tissue as has been shown (36, 37) for certain plants. The data shown by Letey for tomato plants are in close agreement with those reported by Fulton and Erickson where similar condi- tions were provided during treatment. It is considered that ethanol determinations consti- tute a very suitable tissue test method for evaluating the conditions involved in soil anaerobic stress for plants. Undoubtedly other plant metabolites have potential usefule ness in this area. In addition the metabolic processes may only be an initial indicator of anaerobic effects since structural changes could also follow, especially with prolonged periods of oxygen deficiency. It is concluded that the plant fermentation reaction, as evaluated by eth~ anol measurements, can serve a useful purpose in answering 63 questions regarding flooding effects on plants and in seek- ing means to alleviate the disastrous results that can oc— cur where plants are subjected to flooding. APPENDICES 64 55 Hm mm mm Hm m: mm NH mm on Om mm mm we mH mH NH NH OH OH O a mm mH Ow em mm we : om HH mm mH om OH meoz OH 5 HO O m mm meoz ustq 30H new casemeomEme 30A ucqu anm new modemsmosoe 30H ee mmH om as Hm mmH em emH. mOH omH ON OO omm em Om Om mm ms em Hm mH mmH mOH mm OHH meH ONH mH mH MH OH OH mH mH : H: mm e: em em em s OH OH om s :H em meoz mH mH s mm Hm O eeoz ocmHH 30H new osspmeoosos EsHooz uanq cme Och oesbmomosoe EsHooz mHH mmH msH OOH we mm em ewm mmm mOH mam mom ooe em ONH mmm me OOH :wH me mH sOH mzH eeH OOH mOH Oem mH mm mm mm em Om em 3 mm em we mm OH OH s OH mm Hz 2H OH m meoz OH OH NH Om mH mm eeoz ucwHH 30H new OHOOOLOQEoB anm sag coHummucoocoo Hocmnpm OewHH Ome see magpmooQEoe cme .>< m a r m H mpsom mom OoHomm weHOOOHm Lfl .>< H mssom gum OoHpmm wcHUOOHm .moHuHmcmocH ustH ozu um pom onsumpoQEmu HHm mo newsmp moss» smog: OOOoOHm mbcmHQ oOmEou no mumpsxo anmx OH coHOmmOc.O:OO HocmzomII.H mqmqe 66 Summary_of Tablel. Source of Variance Flood Period Air Temperature Light Intensity Flood Period x Air Temperature Flood Period x Light Intensity Air Temperature x Light Intensity l'ij 94.72 24.14 8.47 15.62 1.38 2.27 67 TABLE 2.--Ethanol concentration in xylem exudate of tomato plants flooded at three soil temperatures in greenhouse. Soil Flooding Period Rep Hours Ee$p° 1 2 3 4 5 6 Av. Ethanol Concentration ppm 10.0 22 l6 19 26 22 24 22 None 18.2 30 36 l8 17 16 18 23 26.7 17 7 21 23 16 17 17 10.0 68 67 28 45 36 27 45 4 18.2 51 81 24 27 25 37 41 26.7 48 79 26 22 23 15 36 10.0 16 74 29 28 69 53 45 12 18.2 49 73 33 34 78 57 54 26.7 61 72 27 18 63 100 57 Summapy of Table 2. Source of Variance §_ Nec. F 0.05 0.01 Flood Period 16.43 3.23 5.18 Soil Temperature .12 3.23 5.18 Flood Period x Soil Temperature .71 2.61 3.83 68 TABLE 3.——Rate of ethanol exudation by tomato plants at three soil temperatures in the greenhouse. Ll Soil Flooding Period Rep Hours Temp. 1 2 3 4 5 6 Av. Ethanol, ugm hr-l 10.0 .7 2.2 6.5 18.9 .7 4.3 5.6 None 18.2 5.4 8.3 18.0 5.6 1.4 4.0 7.1 26.7 3.9 5.1 9.9 9.7 2.4 2.6 5.6 10.0 6.1 6.7 20.4 6.0 3.6 1.1 7.3 4 18.2 12.8 11.3 21.6 46.7 1.0 30.3 20.6 26.7 13.4 20.5 24.1 24.9 32.4 9.0 20.7 10.0 2.0 2.0 29.9 28.0 21.4 13.3 . 16.1 12 18.2 16.3 20.3 17.5 17.5 27.3 20.0 19.8 26.7 13.4 16.6 25.1 20.3 65.5 82.0 37.2 Spmmary of Table 3. Source of Variance F. Nec. F _- —i 0.05 0.01 Flood Period 9.54 3.23 5.18 Soil Temperature 3.78 3.23 5.18 Flood Period x Soil Temperature 1.66 2.61 3.83 69 TABLE 4.-—Ethanol concentration in xylem exudate of tomato plants flooded at two soil temperatures under medium air tem- perature and high light intensity. Flooding Period Rep Hours 1 2 3 4 5 Av. Ethanol ConCentration ppm Soil Temperature 9.1 - 30° C None 0 21 33 7 18 16 4 26 54 57 47 23 41 12 170 175 110 23 165 129 24 220 60 70 120 165 127 fi' Soil Temperature 18.2° C None 4 10 9 4 10 7 4 52 10 48 14 40 32 12 140 24 55 49 79 69 24 100 132 130 144 94 120 Summagy of Table 4. Source of Variance F_ Nec. F ' 0.05 0.01 Flood Period 18.70 2.95 4.57 Soil Temperature 2.97 4.20 7.64 Flood Period x Soil Temperature 1.13 2.95 4.57 70 TABLE 5.—-Transpiration loss of ethanol from flooded tobacco plants. ‘— Flooding Period - Hours 24-Hour Plant Xylem None 4 12 24 Exudate Ethanol Concentration, ppm in Transpiration Condensate at 26.70 C in Laboratory 1 1 2 4 6 362 2 3 4 10 4 120 Average 2.0 3.0 7.0 5.0 241 Ethanol Concentration, ppm in Transpiration Condensate at 14.4 - 35.6° C in Growth Chamber 1 5 4 2 20 212 2 2 2 7 7 25 Average 3.5 3.0 4.5 13.5 119 TABLE 6.-~Effect of flooding on rate of ethanol transpiration from tobacco plants. Flooding Period - Hours Plant None I“ 4 12 24 Ethanol ugm hr-l Rate of Transpiration of Ethanol at 26.70 C in Laboratory 1 1.6 2.1 1.6 3.7 2 .7 1.3 8.3 .8 Average 1.2 1.7 5.0 2.3 Rate of Transpiration of Ethanol at 14.4 - 35.6° C in Growth Chamber l 1.6 3.8 .3 6.4 2 1.5 2.1 1.4 5.1 Average 1.6 3.0 .9 5.8 71 TABLE 7.--Transpiration loss of ethanol from flooded tomato plants. Flooding Period — Hours 24-Hour Plant Xylem None 4 12 24 Exudate Ethanolpppm Ethanol Concentration in Transpiration Condensate at 26 7° c in Laboratory 1 2 4 11 7 2oo 2 3 6 9 8 138 Average 2.5 5.0 10.0 7.5 179 Ethanol Concentration in Transpiration Condensate at 14.4 - 35.6° C in Growth Chamber 1 5 7 12 10 212 2 4 2 4 7 105 Average 4.5 4.5 8.0 8.5 159 TABLE 8.--Effect of flooding on rate of ethanol transpiration from tomato plants. Flooding Period ~ Hours Plant None 4 12 24 Ethanol; ugm hr“l Rate of Transpiration of Ethanol at 26.70 C in Laboratory l 3.4 7.0 13.5 2.9 2 2. 8. 8.5 5.6 Average 3.0 7.7 11.0 4.3 Rate of Transpiration of Ethanol at 14.4 - 35.6° C in Growth Chamber 1 3.6 26.3 16.0 16.8 2 .6 2.3 .9 3.7 Average 2 1 14.3 8.5 10.3 72 TABLE 9.--Ethanol concentration in xylem exudate of tomato plants flooded in three soil volumes at medium temperature and high light intensity. Soil Rep Flooding Period — Hours Volume ‘ ' (Liters) NO‘ None 3 6 9 Ethanol, ppm l 12 60 305 330 2 40 105 420 500 1.9 3 42 135 378 525 4 16 38 425 600 Av. 28 85 382 489 1 22 45 140 245 2 32 33 160 250 3.8 3 50 70 150 300 4 10 125 195 260 Av. 29 68 161 264 1 8 27 75 185 2 37 39 142 212 7.6 3 20 65 320 145 4 23 70 80 70 Av. 22 50 154 153 Summary of Table 9. Source of Variance F. Nec,~E 0.05 0.01 Flood Period 74.08 2.90 4.46 Soil Volume 35.54 3.30 5.34 Flood Period x Soil Volume 10.24 2.40 3.42 73 TABLE 10.—-Rate of etharml exudation from tomato plants flooded in three soil volumes at medium temperature and high light intensity. Soil R Flooding Period - Hours ep. Volume No (Liters) ‘ None 3 6 9 Ethanol ugm hr-1 1 24 33 154 78 2 5 63 33 229 1.9 3 6 96 97 79 4 28 119 235 142 Av. 16 78 130 132 1 19 10 44 149 2 10 28 54 237 3.8 3 22 75 189 118 4 16 94 148 166 Av. 17 52 109 168 l 21 32 76 161 2 24 42 113 267 7.6 3 17 64 86 195 4 6 66 122 9 Av. 17 51 99 158 Summarypof Table 10. Source of Variance F. Nec. F 0.05 0.01 Flood Period 15.15 2.90 4.46 Soil Volume .08 3.30 5.34 Flood Period x Soil Volume .54 2.40 3.42 74 TABLE ll.-—0xygen diffusion rates in three soil volumes flooded at medium temperature and high light intensity. Soil Flooding (81:22:) 52:13? 2 3 I Oxygen, gm x 10.8 cm.2 min-l None 43.8 38.2 32.6 28.8 35.9 3 17.0 15.1 11.3 19.8 15.8 1.9 6 13.2 1443 16.5 10x7 13.7 9 13.5 9.0 11.0 11.2 11.2 None 38.4 45.5 40.1 32.6 39.2 3 8 3 16.5 15.0 13.2 13.5 14.6 6 14.7 13.3 16.2 13.2 14.4 9 12.5 11.0 13.6 12.4 12.4 None 41.4 28.4 27.0 24.1 30.2 3 13.8 17.6 17.3 17.0 16.4 7'2 6 18.2 16.9 13.8 16.2 16.3 9 20.1 15.9 11.6 15.9 15.9 Summary of Table 11. Source of Variance F. Nec. F Flood Period 101.22 2:88 4:46 Soil Volume .30 3.30 5.34 Flood Period x Soil Volume 2.96 2.40 3.42 75 TABLE 12.-—Ethanol concentration in xylem exudate of tomato plants flooded in the field at a medium-high air temperature and high light intensity. Flooding Period Rep. Hours 1 2 3 4 5 Average Ethanol, ppm None 14 O 5 32 7 l2 4 39 70 120 47 33 62 12 65 125 145 200 150 137 24 85 200 245 285 345 232 Nec. F 0.05 0.01 Flood Period 17.53 3.49 5.95 Summary of Table 12. [’13 BI BLI OGRA PHY 76 11. BIBLIOGRAPHY Aubertin, G. M. and L. T. Kardos. Root growth through porous media under controlled conditions. II. Effect of aeration levels and rigidity._ Soil Sci. Soc. Amer. Proc. 29:363-365. 1965. Aubertin, G. M., R. W. Rickman and J. Letey. Plant ethanol content as an index of soil—oxygen status. Agron. J. 58:305-307. 1966. Bartholomew, W. V. and F. E. Clark. Soil,fitrogep. Monograph No. 10. Madison, Wisconson: Amer. Soc. Agron., 1965. Baten, W. D. and A. H. Eichmeier. A comparison of weather conditions at Monroe, East Lansing and South Haven, Michigan: Michigan State University, 1958. Baver, L. D. Soil Physics. New York: John Wiley and Sons, 1956. Baver, L. D. and R. B. Farnsworth. Soil structure ef- fects in the growth of sugar beets. Soil Sci. Soc. Amer. Proc. 5:45-48, 1940. Bergman, H. F. Oxygen deficiency as a cause of disease in plants. Botan. Rev. 25:417-485, 1959. Bonner, James and J. E. Varner. Plant Biochemistry. New York: Academic Press, 1965. Brown, N. J., E. R. Fountaine and M. R. Holden. The oxygen requirement of crop roots and soils under near field conditions. J. Agr. Sci. 64:195-203, 1965. Epstein, E. Effect of soil temperature at different growth stages on growth and development of potato plants. Agron. J. 58:169—172, 1966. Erickson, A. E. and D. M. Van Doren. The relation of plant growth and yield to soil oxygen availability, Trans. 7 Int. Cong. Soil Sci. Vol. III (1960), 428:434. 77 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 78 Fulton, J. M. The relation between soil aeration and the accumulation of ethanol and certain other metabolites in tomato plants. Doctors disser- tation, Michigan State University, 1963. Grineva, G. M. Alcohol formation and excretion by plant roots under anaerobic conditions. Trans- lated from Fiziol. East. 10:361-369, 1963. Gudjunsdottir, Sigrun and H. Burstrom. Growth promo— ting effect of alcohol on excised wheat roots. Physiolog. Plantarum. 15:498—504, 1962. Haagen-Smit, A. J., J. G. Kirchner, A. N. Prater and C. L. Deasy. Chemical studies of pineapple (Ananas sativus Lindl) 1. The volatile flavor and odor constituents of pineapple. J. Am. Chem. .599. 67:1646-1650, 1945. Hageman, R. H. and D. Flesher. The effect of an aerobic environment on the activity of alcohol dehydrogenase and other enzymes of corn seedlings. Arch. Biochem. Biophysics. 87:203-209, 1960. Handbook of Chemistry and Physics. 44th ed. Cleve— land: The Chemical Rubber Pub. Co, 1963. Huck, M. G., R. H. Hageman and J. B. Hanson. Diurnal variation in root respiration. Plant Phys. 37: 371-379: 1962. Jackson, L. P. Effect Of soil water content and oxygen diffusion rate on growth of potato sets. M. S. Thesis, Michigan State University, 1952. Kenefick, D. G. Formation and elimination of ethanol in sugar beet roots. Plant Phys. 37:434-439, 1962. Kramer, P. J. Transpiration and the water economy of plants. In: Plant Physiology. F. C. Steward, ed. New York: Academic Press, 11:607-626, 1959. Kristensen, K. J. and E. R. Lemon. Soil aeratibn and plant root relations. III. Physical aspects of Oxygen diffusion in the liquid phase of the soil. Agron. J. 56:295-301, 1964. Laing, H. E. ReSpiration of the rhizomes of Nuphar advenum and other water plants. Amer. J. Bot. 27:574-581, 1940. 24. R) U] [\1 O \ H; N 28. 29. 30. 31. DU I‘O 79 Lemon, E. R. and A. E. Erickson. Principle of the platinum micro-electrode as a method of charac- terizing soil aeration. Soil Sci. 79:383—392, 1955. The measurement of oxygen diffusion in the soil with a platinum micro-electrode. Spil Sci. Soc. Amer. Proc. 16:160—163, 1952. Lemon, E. R. Soil aeration and plant root relations. 1. Theory. Aggpgp_;. 54 167-170, 1962. Lemon, E. R. and C. L. Wiegand. Soil aeration and plant root relations. II. Root respiration. Agron. J. 54:171-175, 1962. Letey, J., L. H. Stolzy, M. Valoras and T. E. Szuszkiewics. Influence of oxygen diffusion rate on sunflower growth at various soil and aig temperatures. Agron. J. 54:316-317, 19 2. Meyer, B. S., D. B. Anderson and R. H. Bohning. Introduction to Plant Physiology. New York: D. Van Nostrand Co., 1964. McCree, K. J. Light measurements in plant growth investigations. Nature. 206:527, 1965. Richards, S. J., R. M. Hagan and T. M. McCalla. Soil temperature and plant growth. Soil Physical Conditions and Plant Growth. figppp. Monograph No. 2. pp. 303-480, New York: Academic Press, Co., 1952. Riviere. J. Etude de la rhizosphere du ble. App. Agronomique. 11:397-440, 1960. Russell, M. B. Soil aeration and plant growth. Soil Physical Conditions and Plant Growth. Agron. Monograph No. 2. pp. 253-301, New York: Academic Press, Inc., 1952. Turner, J. S. Respiration (the Pasteur effect in plants). Ann. Rev. Plant Physiol. 2:145- 168, 1951. Vaadia, Y. Autonomic diurnal fluctuations in the rate of exudation and root pressure of decapitated sunflower plants. Physiol. Plantarum. 13:701- 717, 1960. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 80 Vallance, K. B. and D. A. Coult. Observations on the gaseous exchanges which take place between Menyanthes trifoliata L. and its environment. I. The composition of the internal gas of the plant. J. Exp. Bot. 2:212—222, 1951. Van Der Heide, H., Berendina M. De Boer—Bolt and M. H. Van Raalte. The effect of a low oxygen content of the medium on the roots of barley seedlings. Acta Bot. Neerlandica. 12:231-247, 1963. Van Doren, D. M. Relationship between oxygen diffusion rates, as measured with the platinum micro- electrode, and plant growth. Doctors disserta- tion, Michigan State University, 1958. Vartapetyan, B. B. Polarographic investigation of oxygen transport in plants. Translated from Fiziol. Rast. 11:659-665, 1964. Weinhold, A. R. Rhizomorph production by Armillaria mellea induced by ethanol and related compounds, Science. 142:1065-1066, 1963. Went, F. W. Plant growth under controlled conditions. II. Correlation between various physiological processes and growth in the tomato plant. Am, J. Botany. 31:597-618, 1944. White, A., P. Handler and E. L. Smith. Principles of Biochemistry. New York: McGraw—Hill Book Co., 1964. Williamson, R. E. The effect of root aeration on plant growth. Soil Spi. Soc. Amer. Proc. 28:86-90, 1964. Yocum, C. S., L. H. Allen and E. R. Lemon. Photosyn— thesis under field conditions. VI. Solar radiation balance and photosynthetic efficiency. Agronpi. 56: 249—253, 1964. Yoder, R. E. The significance of soil structure in relation to the tilth problem. Soil Sci. Soc. Amer. Proc. 2:21-23, 1937. I ll . ill-il- IIII. I.