(mag This is to certify that the dissertation entitled Growth, Respiration, Ion Uptake and Carbon Partitioning of Phaseolus vnlgaris L. Root Systems Exposed to Localized Anoxia presented by Thomas Edward Schumacher has been accepted towards fulfillment of the requirements for Ph.D. degreein Crop & Soil Sciences mm M {fir professor Date November 10, 1982 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 ‘bV1E;I_J RETURNING MATERIALS: Place in book drop to lJBRAfijfis remove this checkout from 11—. your record. FINES will be charged if book is returned after the date stamped below. GROWTH, RESPIRATION, ION UPTAKE AND CARBON PARTITIONING OF PHASEOLUS VULGARIS L. ROOT SYSTEMS EXPOSED TO LOCALIZED ANOXIA BY Thomas Edward Schumacher A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1982 ABSTRACT GROWTH, RESPIRATION, ION UPTAKE AND CARBON PARTITIONING OF PHASEOLUS VULGARIS L. ROOT SYSTEMS EXPOSED TO LOCALIZED ANOXIA BY Thomas Edward Schumacher Soil aeration may vary widely in space and time within compacted, poorly drained and even some well drained soil environments. The analysis of morphological and physiological responses of root systems to spatial and temporal heterogeneity of soil aeration could contribute to our understanding of root growth under field conditions. Split root cultures were used to simulate conditions of localized anoxia within the root systems of (Phaseolus vulgaris L.) genotypes. Aeration treatments, introduced to the solution cultures 14 days after germination, included an aerated control, a nonaerated control and a localized anoxia treatment where half of the root system was aerated and the remaining half nonaerated. A newly developed modified infrared gas analyzer method, sensitive to 3 x 10-12 moles of dissolved C02, was used to measure the respiration of root systems. Root growth during the 72 hour treatment period was determined using a neutral red staining technique. Thomas Edward Schumacher Root growth did not occur in the nonaerated control or the nonaerated portion of the localized anoxia treatment. Localized anoxia stimulated root growth in the aerated portion of the root system with values ranging from 150 to 185% of the aerated control. An experimental line partitioned the majority of new growth into roots which were present before the treatment period. A new stress resistant variety with improved upright architecture partitioned the new growth into lateral roots which emerged during the localized anoxia treatment. Compensatory root growth was primarily associated with an increase in the number of elongating lateral root tips which emerged during the treatment. Roots of one genotype also absorbed more K+ ions per unit of root weight in the aerated portion of the localized anoxia treatment when compared to the aerated control. Increased ion uptake was accompanied by a stimulation in oxygen respiration and xylem exudation rates. Aerated root systems accumulated between 40 to 60% of the l4C-sucrose translocated from the source leaf in a 2 h labeling period. Localized anoxia resulted in the greater translocation of l4C-sucrose to the elongating root tips of a stress line while the label accumulated in the nongrowing portions of the stress susceptible root system. Oxygen stress within a portion of the root system appeared to increase root growth, ion uptake, respiration and modified the carbon accumulation in the nonstressed Thomas Edward Schumacher portion of the root system. These varied responses of the genotypes to localized anoxia suggests that the adaptation of genotypes to specific soil environments combined with the development of soil management techniques designed to produce an improved root environment could contribute substantially to the utilization of marginal soils. To my grandmother Irene Stucky and my daughter Hannah Marie. ii ACKNOWLEDGEMENTS The advice and encouragement given to me by my committee members, Drs. M.W. Adams, A.E. Erickson, J.A. Flore, and B.D. Knezek have contributed greatly to the successful completion of my graduate program. I have appreciated their friendship and help over the years. Special acknowledgement is given to Dr. A.J.M. Smucker for the guidance and support given to me through the duration of my graduate program. I have especially appreciated his encouragement and the freedom given to me in the development of this project. The openness and free flow of ideas in our discussions has enabled me to grow both personally and professionally. Many people have provided me with encouragement, advice and help in carrying out the required tasks. I thank all of these people and wish to especially recognize the contributions of those individuals listed below. Dr. Ghassem Assar and Jim Reisen provided invaluable technical assistance in the use of the SMI liquid plasma emission spectrophotometer. The expert photographic advice and many hours of aid given by David Krauss are much appreciated. iii iv I would like to thank Tom Beckman, Mary Charley, Rod and Kim Friesen, Kathy Hammond, Jim Helm, Mary Hill, Teresa Howes, Joan Messner, Marianna Moore, Diane Moe, Dr. R.L. Perry, and Tracey Thomas for careful attention to the fine art of dissecting roots and for help given in the course of the experiments. Discussions, encouragement and critical help were provided by fellow graduate students, Hossein Assady and Bruce Riggle. Jean Aslakson and Cathy Orr provided all purpose tech- nical help and were instrumental in the successful completion of many tasks. I would like to thank Dr. M.L. Schumacher for the translation of a critical journal article. Dr. G.L. Hosfield kindly provided the use of several pieces of equipment needed for this study. Dr. J.C. Shickluna has provided me with encouragement and his interest in this project is much appreciated. The friendship and discussions with Shawn McBurney and Tim Falk greatly helped in the initial formation of ideas which stimulated this project. The flawless typing from my somewhat less than perfect writing was accomplished by Jodie Schonfelder. My undergraduate mentors from Bluffton College, Drs. Maurice Kaufmann, Richard Pannabecker and LaVerne Schirch deserve much of the credit for stimulating my interest in biology and the mechanisms involved in biological processes. The MSU Mennonite Fellowship has provided spiritual support and friendship these many years. My mother, father and brothers, Lucile, Vernon, Dan, Bill and Joe, and my in-laws Bob, Doris, Emily, Bobbie, Martha, Randy and Melinda have helped me to maintain a proper perspective on life and their encouragement gave me strength to continue on. If it weren't for my loving and faithful wife, Doris, I wouldn't have completed any of this dissertation. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . CHAPTER 1: LITERATURE REVIEW . . . . . . . Root Growth in Unfavorable Environments Compensatory Root Growth Under Localized Anoxia References . . . . . . . . . . . . . . CHAPTER 2: MEASUREMENT OF DISSOLVED C02 IN THE RHIZOSPHERE OF PLANT ROOT SYSTEMS BY A MODIFIED INFRARED GAS ANALYZER SYSTEM Abstract . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . Materials and Methods . . . . . . . . . Results and Discussion . . . . . . . . References . . . . . . . . . . . . . . CHAPTER 3: MEASUREMENT OF SHORT TERM ROOT GROWTH BY PRESTAINING WITH NEUTRAL RED Abstract 0 O O O O C O O O O O O O O I Introduction . . . . . . . . . . . . . Materials and Methods . . . . . . . . . vi Page ix xii 15 20 20 22 25 32 41 43 43 44 46 vii Results and Discussion . . . . . . References . . . . . . . . . . . . CHAPTER 4: LOCALIZED ANOXIA EFFECTS ON OF PHASEOLUS VULGARIS L. . . Abstract . . . . . . . . . . . . . Introduction . . . . . . . . . . . Materials and Methods . . . . . . . Results . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . References . . . . . . . . . . . . ROOT GROWTH CHAPTER 5: LOCALIZED ANOXIA EFFECTS ON RESPIRATION AND l4C-SUCROSE TRANSLOCATION or TWO DRY BEAN GENOTYPES . . . . . Abstract . . . . . . . . . . . . . Introduction . . . . . . . . . . . Materials and Methods . . . . . . . Results . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . References . . . . . . . . . . . . CHAPTER 6: ION UPTAKE, RESPIRATION AND TRANSPIRATION OF DRY BEAN ROOTS EXPOSED TO ANOXIA . . . . . . . . . . . Abstract . . . . . . . . . . . . . Introduction . . . . . . . . . . . Materials and Methods . . . . . . . LOCALIZED 48 54 55 55 57 59 64 74 80 83 83 85 87 92 106 113 117 117 119 121 viii Results and Discussion . . . . References . . . . . . . . . . CHAPTER 7: SUMMARY AND CONCLUSIONS LIST OF TABLES CHAPTER 2 1. Effect of varying the pH on XCOZ and [H2CO3*] under constant pCOz, ionic strength and temperature . . . . . . . . . 2. Comparison of four systems of measurement for calculating total C02 and carbonate components of Hoagland's solution (I = 3 x 10"2 M) at two pH levels and 20.0 i 0.1 C O O O O O O O O O I O I I O O O O O 3. Carbon exchange (CER), root reSpiration and calculated pH values of the nutrient solution as measured before and after the root chamber by the modified IRGA system after 18 h treatment . . . . . . . . . . . CHAPTER 3 1. Analyses of variance for shoot and root parameters of five dry bean genotypes three days after staining with neutral red O O O I O O O O O O O O O O I O O O O 2. Shoot and root parameters for five dry bean genotypes sixteen days after germination . . . . . . . . . . . . . . . CHAPTER 4 1. Effect of aeration treatments on shoot and root parameters for five dry bean genotypes O O O O O O O O O O O O O O O O 2. Main effect means for shoot parameters of the five dry bean genotypes averaged over all split root treatments . . . . . . . . ix CHAPTER 5 1. Comparisons of the number of growing root tips and root length accumulating during the three day treatment period for five dry bean genotypes . . . . . . . Comparison of the lengths of the zone of differentiation of branched roots for the aerated control (AC) and the aerated half of the localized anoxia treatment (ALA) for five dry bean genotypes . . . . . . . Modification of shoot and root growth of two genotypes of dry bean by 72 h aeration treatments of the root system . . . . . . Distribution of dry matter within the root systems of 31908 and Seafarer subjected to three aeration treatments for 72 h . . . . . . . . . . . . . . . . . Amount of l4C-sucrose recovered in the 80% ethanol wash and extraction,retained by the source leaf and translocated from the source leaf . . . . . . . . . . . . . Translocation of l4C-label from the source leaf to the shoot, root and root environment . . . . . . . . . . . . . . . Distribution of l4C-label among various shoot components . . . . . . . . . . . . . Proportion of 14C—label translocated from the source leaf to half the root system in four aeration environments . . . Activity of 14C per unit root dry weight for the root system and respiratory fraction of four root environment . . . . Distribution of 14C—label within root components during the treatment period . . C02 production and respiration by half root systems of Seafarer and 31908 . . . . Page 68 73 93 94 95 97 98 99 100 102 103 xi Page CHAPTER 6 1. Ion uptake by three dry bean genotypes treated with the following aeration treatments: aerated control (AC), nonaerated control (NC), and localized anoxia (LA) . . . . . . . . . . . . . . . . 127 2. Ion uptake per unit fresh weight of root for three dry bean genotypes . . . . . 128 3. Oxygen uptake rates per root system half per unit fresh weight and the change in oxygen uptake occurring during the treatment period (A02 uptake) for three dry bean genotypes . . . . 130 4. Xylem exudation rates for plants of three dry bean genotypes treated with the following aeration treatments: aerated control (AC), nonaerated control (NC), and localized anoxia treatment (LA) . . . . . . . . . . . . . . . 131 5. Comparison of K+ uptake with quarter and half strength Hoagland's nutrient solution for 31908 and Seafarer . . . . . . 133 6. Transpiration rates, leaf diffusive resistance values and leaf area for 31908 and Seafarer plants grown in half strength Hoagland's nutrient solution . . . . . . . . . . . . . . . . . . 138 CHAPTER 2 1. 2. 3. CHAPTER 1. CHAPTER 1. 5 LIST OF FIGURES Diagramatic representation of the system used to measure carbon dioxide in gas and liquid samples . . . . . . . . . . . Carbon dioxide standard curve based on ul injections of lmM Na2C03 . . . . . . Comparison of acidified and nonacidified sample peaks . . . . . . . . . . . . . . Diagram illustrating the root segment components measured after the three day treatment period . . . . . . . . . . Growth of three root components of five dry bean genotypes as affected by aera- tion treatments (aerated control and the aerated portion of the localized anoxia treatment) . . . . . . . . . . . . . . . Number of growing root tips during the treatment period for three root components and five dry bean genotypes as affected by aeration treatments (aerated control and the aerated portion of the localized anoxia treatment) . . . Accumulation of 14C02 respired by the root system and collected in ethanolamine during the labeling period . . . . . . . xii 26 27 35 63 70 71 104 CHAPTER 6 l. xiii Page Accumulative disappearance of potassium ion from half strength Hoagland's nutrient solution during 60 hours of aeration treatment for 31908 and Seafarer . . . . . . . . . . . . . . . . . . 135 Water losses occurring during the treat- ment period for Seafarer and line 31908 . . 140 INTRODUCTION The soil environment varies widely in physical, chemical and biological components over very short distances in the soil pedon. Most of these components can change quite rapidly with time and interact with each other to replace the former pattern of variation with a new pattern equally as complex as the former. Plant root systems, as part of the soil system, are continually exposed to spatial and temporal variation in concentrations of oxygen, water, nutrients, soil density and strength, microorganisms, toxic compounds, roots from other plants, etc. The heterogeneity of the soil system implies the presence of areas in the soil pedon which can support plant root growth better than other areas which are more detrimental to root growth. High crop productivity requires efficient exploration of the root system in the soil. Values as high as 50-60 percent of the plant's net photosynthate can be consumed by the root system (Pate et 31., 1979) while roots rarely occupy more than two percent of the soil volume (Barley, 1970). Man has attempted to improve the efficiency of the root-soil system primarily through energy inputs to the soil environment. The percentage of soil volume with high growth potential can be increased with fertilizer, irrigation, tillage methods, drainage, insecticides, herbicides, etc. Clearly, man- applied inputs to the soil, if correctly managed, can dramatically improve plant productivity. Man-applied inputs may be very high in soils where a large percentage of the soil volume is of low growth poten- tial. Even with extensive inputs the soil environment can not be controlled well enough to completely eliminate areas of stress to the plants. The high costs of oil and labor make it less economical to improve the production of marginal soils using man-applied inputs. The continuing scarcity of many of these inputs, particularly in the third world, makes it essential that man make the most efficient use of high energy inputs. These considerations give impetus to improve the ability of crop root systems to adapt to soil environments which are less than ideal. This approach until recently has been neglected as a tool for managing marginal soils. The use of man-applied inputs is not excluded by such an approach but could result in a decrease in the percentage of soil volume which must be changed and thus reduce expensive energy inputs. A combination of fitting the soil to the plant and fitting the plant to the soil should be more rewarding than pursuing either option alone. The objectives of this study were to examine the effects of localized anoxia, oxygen stress limited to a portion of the root system, on the growth, function and morphology of the aerated portion of the root system, to determine the effects of anoxia and localized anoxia on carbon (14C-sucrose) partitioning within the root system, to determine if differ- ences between dry bean genotypes exist and to develop methodology which could be used to quantify the response of the root system to varied environments. CHAPTER 1 LITERATURE REVIEW Root Growth in Unfavorable Environments The plant has evolved various strategies to increase efficiency of photosynthate utilization in a heterOgeneous soil environment. Efficient utilization of photosynthate by the root system requires that the root waste as little photosynthate as possible on unproductive soil environments. A root system which encounters an unfavorable environment has at least five strategies available to it. These strategies are given below with examples. 1) Change the unfavorable environment around the root to a more favorable environment. Rice roots have the ability to oxidize reduced iron in the rhizosphere thus precipitating the iron and preventing iron toxicity to the plant (Chen et 31., 1980; Green and Etherington, 1977). Iron efficient tomato cultivars excrete organic chelates and hydrogen ions into the rhiZOSphere in response to low concentrations of soluble iron. This action results in increased availability of iron to the tomato root (Brown and Ambler, 1974; Brown and Jones, 1974). Aluminum tolerance in wheat was related to the ability of the root to increase pH in the rhizosphere causing precipitation of the aluminum (Foy et 21., 1965). Pea roots exude substances favorable to the colonization of microorganisms which are antagonistic to the disease causing organism, Fusarium oxysporum t. pisi (Buxton, 1960). 2) Adapt to the unfavorable environment through changes in internal metabolism or structure. Roots from many species increase their solute potential in response to water deficits in the soil (Begg and Turner, 1976; Hsiao and Acevedo, 1974). Flood tolerant species produce diversified end products of glycolysis preventing the accumulation of large quantities of toxic ethanol (Crawford, 1978a). Some corn varieties have the potential of increasing the rate of potassium uptake under conditions of low potas- sium activity in the soil solution (Frick and Bauman, 1979). Phosphatase activity on the surface of wheat roots is increased under phosphorous deficiency (Gilliam, 1970). The iron efficient response described above is also an example of a change in the internal metabolism of the root (Brown gt gt., 1972). 3) Anticipate a future unfavorable environment in a particular volume of soil by a characteristic pattern of growth. One would expect to find this type of strategy in plant species which are particularly adapted to a specific environ- mental sequence. A period of favorable soil moisture followed by conditions of drought would give an advantage to a Species with an early formation of a deep root system. On the other hand, where water is in short supply throughout the growing season an early formation of a large root system would deplete the soil volume of water early and have disastrous effects later in the season (Hurd, 1974). Root systems of Atriplex confertifola appear to be genetically programmed to produce nonoverlapping peaks of activity at different depths in the soil in order to maintain root functions under favorable conditions and yet curtail excessive consumption of carbon (Holthausen and Caldwell, 1980). Various species of prairie plants have root systems which occupy different parts of the soil volume partially avoiding competition for water and nutrients (Weaver, 1926). 4) Continue characteristic growth through unfavorable environments and through chance encounter a more favorable environment. Root growth can continue into soils with a water potential as low as -40 bars (Portas and Taylor, 1976). Root growth can also occur through relatively infertile soil layers (Weaver, 1926). 5) Stop growth in the unfavorable environment and utilize the spared photosynthate in a more favorable environment for compensatory root growth. Root growth can shift easily from one part of the root system to another with more favorable growth conditions. The distribution of roots in the soil volume is determined to a large degree by local differences in soil conditions (deWit, 1978). Restriction of phOSphate and/or nitrate to a small part of the root system results in compensatory increases in growth of lateral roots (Drew and Saker, 1975; 1978). Compensatory root growth occurred in the crown root system of sorghum when most of the crown roots were removed (Jordan gt gt., 1979). Drying of the surface soil resulted in increased growth of roots in lower horizons of the soil con— taining moisture (Klepper gt gl., 1973). Barley responded to unfavorable temperatures by increasing root growth in soil with more favorable temperature (Crossett gt_gt., 1975). Half flooding of corn and sunflower root systems resulted in increased growth of roots in the nonflooded half and dry matter production of corn and sunflower was unaffected (Yu gt gt., 1969). These strategies are not exclusive of each other, several can take place at the same time or be included in the same process e.g. iron efficiency response by tomato. The first three strategies have been the most studied in terms of possible incorporation into plant breeding proqrams. Very little work has been done concerning the possible application of the strategy of compensatory root growth to plant adaptation to unfavorable soil environments. Compensatorngoot Growth Under Localized Anoxia Soil has a very heterOgeneous pore distribution, as a result, oxygen concentrations can vary considerably within the soil volume particularly at the higher moisture contents. Sinks and sources of oxygen can not be located in precise three dimensional coordinates. Their presence, densities and intensities vary tremendously in space and time depending on a large number of variables (Stolzy and Fluhler, 1978). Soils with a high percentage of clay aggregates may remain virtually waterlogged for extended periods after fissures between them have drained. Anaerobic processes are not limited to brief intervals when the whole profile is waterlogged (Smith, 1977). Facultative anaerobes capable of reducing oxidized forms of inorganic nitrogen, manganese and iron and true anaerobes such as sulfate reducers are found even in well aerated soils (Patrick, 1978). The high variability of oxygen diffusion rates after a rainfall or ponding of water was attributed to the heterogeneity of the aeration pattern within the soil (Fluhler gt 31., 1976). The formation of anaerobic zones in the soil results from a combination of restricted diffusion of oxygen and consumption of oxygen by microorganisms and roots. Microorganism growth is dependent generally on carbon compounds for energy. An increase in carbon compounds in the microenvironment results in an increase in the microorganism population and the rate of oxygen consumption. The rhizosphere is rich in carbon compounds and supports a dense microorganism population. This pOpulation can be expected to compete for oxygen with the root. At low oxygen concentrations the competition for oxygen increases since rhizosphere microorganisms can generally tolerate lower oxygen concentrations than the root (Griffin, 1968). Since microorganisms and plant roots require intimate contact with soil particles for uptake of nutrients and water they are predominately found in the finer pores of the soil (Currie, 1961). It is within these finer pores that the diffusion path of oxygen is partly or wholly through moisture and thus greatly reduced (Currie, 1965). According to Crawford (1978b) anoxia is a general condition in the root meristem of nonadventitious roots. The addition of sucrose to maize root tips tended to promote fermentation rather than aerobic reSpiration, supporting Crawford's hypothesis (Girton, 1979). Any competition for oxygen or restriction of oxygen diffusion will result in an increase in the extent of anoxia in the root system. A study by Silberbush gt gt. (1979) which examined the effect of trickle irrigation on wheat root growth found that root growth was confined to those parts of the soil profile with continuously high oxygen diffusion rates. The ability of a root system to exploit areas of relatively high oxygen concentrations by compensatory root growth could be advantageous under such conditions. Total root growth normally occurs in the root system at an exponential rate (Warncke and Barber, 1974). Individual root members, parts of the root associated with a single root tip, grow at an arithmetical rate characteristic of the type of root member i.e. primary, first order lateral etc. (Hackett and Rose, 1972a). For the root system to maintain exponential growth the initiation of new growing points is constantly required. Hackett and Rose (1972b) found that 10 when the cumulative growth of a particular class of root mem— ber became arithmetic the next order of root members began to develop to maintain overall exponential growth. Therefore, for compensatory root growth to occur, exponential root growth must be maintained by an increased amount of root branching, i.e. lateral root initiation and growth in the more favorable soil volume. Lateral root formation is also important for the exploration of a given volume of soil. Computer simulation demonstrates that the increased rate of elongation of an indi- vidual root member merely quickens the passage of the main absorbing portion of the root through the region. An increased rate of branching concentrates the absorbing regions of the root in that particular volume of soil (Lungley, 1973). For compensatory root growth to exploit a particular volume of soil an increase in lateral root formation is required in that localized volume. Compensatory root growth can be explained partially by a source-sink relationship. The stoppage of root growth in unfavorable soil environments increases photosynthate available for root growth in more favorable parts of the soil profile. Hormones have also been implicated in compensatory root growth (Russell, 1977). This is not surprising since there is much evidence to indicate hormonal control of lateral root initiation and growth as well as hormonal control of source- sink relationships. Lateral root formation and growth requires endogenous IAA (Feldman, 1980; Pilet gt gt., 1979). In the roots of 11 Egg mgyg lateral root production is preceded by an increase in IAA synthesizing capacity of the root segments (Feldman, 1980). Auxin is also apparently tranSported from the shoot and exerts a promoting effect on lateral root formation (Gersani gt gt., 1980). Cytokinins produced by the root tip appear to inhibit lateral root formation near the growing root apex (Goodwin and Morris, 1979; Wightman and Thimann, 1980). IAA synthesis is inhibited by the root tip as far back as 2 cm from the terminal millimeter of the root (Feldman, 1980). Lateral root emergence appears to be controlled by a balance between auxin promoting emergence and cytokinin inhibiting emergence in the pea root (Wightman gt gl., 1980). Auxin accumulation is also important in the development of adventitious roots from previously differen- tiated root primordia in the hypocotyl (Friedman gt gl., 1980). Sink activity may be controlled by positive hormonal feedback in bean seedlings. Auxin produced by the shoot enhances root activity and cytokinin produced by the root enhances shoot activity by controlling development of growth centers (Gersani gt gt., 1980). Metabolites then move in response to the sink effect this growth produces. The early development of lateral roots is probably not controlled directly by sink-source relationships between the root and shoot since vascular connections are not made with the developing lateral root until a later stage of development. Metabolites released by the breakdown of 12 cortical cells next to the lateral root primordia appear to provide the primordia with essential metabolites (Macleod and Francis, 1976). When the roots become longer than 2 mm in size they begin to increase rapidly in size which may indicate the connection of vascular tissue and the initiation of the sink-source relationship with the shoot. Plant hormones are also implicated in the continued growth of the root system. Endogenous auxin plays an essential role in the regulation of root elongation (Pilet gt gt., 1979). The maintenance of H+ currents between growing and nongrowing portions of the root also appear to be essential for continued root growth (Weisenseel, 1979). This also implicates auxin since it is involved in acid induced growth. The preceding discussion suggests that compensatory root growth may be characterized primarily by lateral root initia- tion and growth in more favorable environments of the soil. This process could occur in at least three steps: i) A change in hormonal relationships in the root growing in the favorable environment, triggering lateral root development. ii) Connection of the lateral root to the vascular system and ensuing competition for photosynthate. iii) Maintenance of conditions necessary for cell division and elongation. Critical to these general hypotheses of compensatory root growth is the presence of a signal from either the external environment or the stressed portion of the root to initiate increased lateral root formation. 13 Anoxia has a number of characteristic effects on root growth which suggests the possible occurrence of compensatory root growth. Two major modifications occurring to root growth under conditions of low oxygen concentrations are increased branching of roots and formation of adventitious roots (deWit, 1978). A higher number of lateral roots per unit of root length occurs under conditions of low oxygen concentration (Geisler, 1965). Adventitious root formation occurs near the surface of the soil where oxygen relations may be improved (Kramer, 1969). Adventitious roots formed under anaerobiosis are more porous and can serve to remove toxic anaerobic products from the root system (Crawford and Baines, 1977; deWit, 1978). Anoxia results in multiple changes in hormonal relation- ships within the plant. Gibberellin and cytokinin transport and production is reduced by anaerobiosis (Russell, 1977). Flooded plants contain three times the auxin content in their shoots than that of control plants (Phillips, 1964). Flood induced adventitious roots were formed primarily by an accumulation of auxins in the hypocotyls (Wample and Reid, 1979). The precursor of ethylene, l-aminocyclOpropane-l- carboxylic acid (ACC), is transported out of anaerobic roots (Bradford and Yang, 1980). ACC is prevented from forming ethylene by the absence of oxygen. When ACC enters a part of the plant under aerobic conditions the conversion to ethylene occurs (Branford and Yang, 1980). Thus ACC could 14 act as a possible signal to the aerated portion of the root system since ethylene is involved in the formation of lateral roots (McCully, 1975). Compensatory root growth discussed thus far has been of the "spatial" type, i.e. root growth stops in the unfavorable environment and growth is increased in a more favorable part of the soil volume. Another type of compensatory root growth should be possible with stresses which are of a transient nature such as soil waterlogging. This type of compensatory root growth is of a "temporal" type. Root growth stOps in the unfavorable environment. When the unfavorable environment becomes more favorable, root growth again resumes but at an accelerated rate to compensate for the loss of root growth during the period of stress. Under short term anoxia, temporal compensatory root growth may be as important as spatial compensatory root growth. REFERENCES Barley, K.P. 1970. The configuration of the root system in relation to nutrient uptake. Adv. Agron. 22:159-201. Begg, J.E. and N.C. Turner. 1976. Crop water deficits. Adv. Agron. 28:161-217. Bradford, K.J. and S.F. Yang. 1980. Xylem transport of l-aminocyclopropane-l-carboxylic acid, an ethylene precursor, in waterlogged tomato plants. Plant Physiol. 65:322-326. Brown, J.C. and J.E. Ambler. 1974. Iron-stress response in tomato (Lycopersicon esculentum) 1. Sites of Fe reduction, absorption and transport. Physiol. Plant. 31:221-224. Brown, J.C. and W.E. Jones. 1974. pH changes associated with iron-stress response. Physiol. Plant. 30:148-152. Brown, J.C., J.E. Ambler, R.L. Chaney and C.D. Foy. 1972. Differential responses of plant genotypes to micronu— trients. pp. 389-418. lg: Micronutrients in Agriculture, eds. J.J. Mortvedt, P.M. Giordano, and W.L. Lindsay. Madison, Wis. Buxton, E.W. 1960. Effects of pea root exudate on the antagonism of some rhizosphere microorganisms towards Fusarium oxygporum t. pisi. Gen. Microbiol. 22:678-689. Chen, C.C., J.E. Dixon and F.T. Turner. 1980. Iron coatings on rice roots: Mineralogy and quantity influencing factors. Soil Sci. Soc. Am. J. 44:635—639. Crawford, R.M.M. and M.A. Baines. 1977. Tolerance of anoxia and the metabolism of ethanol in tree roots. New Phytol. 79:519-526. Crawford, R.M.M. 1978. Metabolic adaptations to anoxia. pp. 119-154. £2: Plant Life in Anaerobic Environments, eds. D.D. Hook and R.M.M. Crawford. Ann Arbor Science, Ann Arbor, Mich. 15 16 Crawford, R.M.M. 1978. Critique - of "Measurement and pre- diction of anaerobiosis in soils." Metabolic indicators in the prediction of soil anaerobiosis. In: Nitrogen in the Environment. Nitrogen Behavior in—Field Soil Vol. 1, eds. D.R. Nielsen and J.G. MacDonald. Academic Press, New York. pp. 427-447. Crossett, R.N., D.J. Campbell and H.E. Stewart. 1975. Compensatory growth in cereal root systems. Plant Soil 42:673-683. Currie, J.A. 1961. Gaseous diffusion in the aeration of aggregated soils. Soil Sci. 92:40. Currie, J.A. 1965. Diffusion within soil microstructure, a structural parameter for soils. J. Soil Sci. 16:279- 289. deWit, M.C.J. 1978. Morphology and function of roots and shoot growth of crop plants under oxygen deficiency. pp. 333-350. £3: Plant Life in Anaerobic Environments, eds. D.D. Hook and R.M.M. Crawford. Ann Arbor Science Publ., Ann Arbor, Mich. Drew, M.C. and L.R. Saker. 1975. Nutrient supply and the growth of the seminal root system in barley. II. Localized, compensatory increases in lateral root growth and rates of nitrate uptake when nitrate supply is restricted to only part of the root system. J. Exp. Bot. 26:79-90. Drew, M.C. and L.R. Saker. 1978. Nutrient supply and the growth of the seminal root system in barley. III. Compensatory increases in growth of lateral roots, and in rates of phosphate uptake, in response to a localized supply of phosphate. J. Exp. Bot. 29:435-451. Feldman, L.J. 1980. Auxin biosynthesis and metabolism in isolated roots of Zea mays. Physiol. Plant. 49:145-151. Fluhler, H., M.S. Ardakani, T.E. Szuskiewicz and L.H. Stolzy. 1976. Field measured nitrous oxide concentrations, redox potentials, oxygen diffusion rates and oxygen partial pressures in relation to denitrification. Soil Sci. 122: 107-114. Foy, C.D., G.R. Burns, J.C. Brown and A.L. Fleming. 1965. Differential aluminium tolerance of two wheat varieties associated with plant-induced pH changes around their roots. Soil Sci. Soc. Am. Proc. 29:64-66. Frick, H. and L.F. Bauman. 1979. Heterosis in maize as measured by K uptake properties of seedling roots - 17 pedigree analyses of inbreds with high or low augmenta- tion potential. Crop Sci. 19:707-711. Friedman, R., A. Altman and E. Zamski. 1980. Adventitious root formation in bean hypocotyl cuttings in relation to IAA translocation and hypocotyl anatomy. J. Exp. Bot. 30:769-777. Geisler, G. 1965. The morphogenetic effect of oxygen on roots. Plant Physiol. 40:85-88. Gersani, M., S.H. Lips and T. Sachs. 1980. The influence of shoots, roots, and hormones on sucrose distribution. J. Exp. Bot. 31:177-185. Gilliam, J.W. 1970. Hydrolysis and uptake of pyrophosphate by plant roots. Soil Sci. Soc. Am. Proc. 34:83-86. Girton, R.E. 1979. Effects of oxygen concentration on the reSpiration of excised root-tip segments of maize and rice, and of germinating grains of rice and buckwheat. Physiol. Plant. 46:58-63. Goodwin, P.B. and S.C. Morris. 1979. Application of phyto- hormones to pea roots after removal of the apex: Effect on lateral root production. Aust. J. Plant Physiol. 6:195-200. Green, M.S. and J.R. Etherington. 1977. Oxidation of ferrous iron by rice (Otyza sativa L.) roots: A mechanism for waterlogging tolerance? J. Exp. Bot. 28:678-690. Griffin, D.M. 1968. A theoretical study relating the concentration and diffusion of oxygen to the biology of organisms in soil. New Phytol. 67:561-577. Hackett, C. and D.A. Rose. 1972. A model of the extension and branching of a seminal root of barley, and its use in studying relations between root dimensions. I. The model. Aust. J. Biol. Sci. 25:669-679. Hackett, C. and D.A. Rose. 1972. A model of the extension and branching of a seminal root of barley and its uses in studying relations between root dimensions. II. Results and inferences from manipulation of the model. Aust. J. Biol. Sci. 25:681-690. Holthausen, R.S. and M.M. Caldwell. 1980. Seasonal dynamics of root system respiration in Atriplex confertifolia. Plant Soil 55:307-321. Hsiao, T.C. and E. Acevedo. 1974. Plant responses to water deficits, water-use efficiency, and drought resistance. 18 Agric. Meteorol. 14:59-84. Hurd, E.A. 1974. Phenotype and drought tolerance in wheat. Agric. Meteorol. 14:39-55. Jordan, W.R., M. McCrary and F.R. Miller. 1979. Compensa- tory growth in the crown root system of sorghum. Agron. J. 71:803-807. Klepper, B., H.M. Taylor, M.C. Huck and E.L. Fiscus. 1973. Water relations and growth of cotton in drying soil. Agron. J. 65:307-310. Kramer, P.J. 1969. Plant and Soil Water Relationships: A Modern Synthesis. pp. 137-142. McGraw-Hill Book Co., New York. Lungley, D.R. 1973. The growth of root systems - a numerical computer simulation model. Plant Soil 38:145-159. Macleod, R.D. and D. Francis. 1976. Cortical cell breakdown and lateral root primordium development in Vicia faba L. J. Exp. Bot. 27:922-932. McCully, M.E. 1975. The development of lateral roots. pp. 105-121. lg: The Development and Function of Roots, eds. J.G. Torrey and D.T. Clarkson. Academic Press, New york. Pate, J.S., D.B. Layzell and C.A. Atkins. 1979. Economy of carbon and nitrogen in a nodulated and nonnodulated (N03 - grown) legume. Plant Physiol. 64:1083-1088. Patrick, W.H., Jr. 1978. Critique of: "Measurement and prediction of anaerobiosis in soils." In: Nitrogen in the Environment. Nitrogen Behavior IE Field Soil Vol. 1, eds. D.R. Nielsen and J.G. MacDonald. Academic Press, New York. pp. 449-457. Philips, I.D.J. 1964. Root-shoot hormone relations. II. Changes in endogenous auxin concentration produced by flooding of the root system in Helianthus annus. Ann. Bot. 28:37-45. Pilet, P.B., M.C. Elliott and M.M. Moloney. 1979. Endogenous and exogenous auxin in the control of root growth. Planta 146:405-409. Portas, C.A.M. and H.M. Taylor. 1976. Growth and survival of plant roots in dry soil. Soil Sci. 121:170-175. Russell, R.S. 1977. Plant root systems: Their function and interaction with the soil. pp. 298. McGraw-Hill 19 Book Co. (UK) Limited. Silberbush, M., B. Gornat and D. Goldberg. 1979. Effect of irrigation from a point source (trickling) on oxygen flux and on root extension in the soil. Plant Soil 52:507-515. Smith, K.A. 1977. Soil aeration. Soil Sci. 123:284-290. Stolzy, L.H. and H. Flfihler. 1978. Measurement and predic- tion of anaerobiosis in soils. In: Nitrogen in the Environment. Nitrogen Behavior i3 Field Soil Vol. 1, eds. D.R. Nielsen and J.G. MacDonald. Academic Press, New York. pp. 363-426. Wample, R.L. and D.M. Reid. 1979. The role of endogenous auxins and ethylene in the formation of adventitious roots and hypocotyl hypertrophy in flooded sunflower plants (Helianthus annuus). Physiol. Plant 45:219- 226. Warncke, D.D. and S.A. Barber. 1974. Root development and nutrient uptake by corn grown in solution culture. Agron. J. 66:514-516. Weaver, J.E. 1926. Root development of field crops. McGraw-Hill, New York. Weisenseel, M.H., A. Dorn and L.F. Jaffe. 1979. Natural H+ currents traverse growing roots and root hairs of barley (Hordeum vulgare L.). Plant Physiol. 64:512-518. Wightman, F. and K.V. Thimann. 1980. Hormonal factors controlling the initiation and development of lateral roots. 1. Sources of primordia-inducing substances in the primary root of pea seedlings. Physiol. Plant. 49:13-21. Wightman, F., E.A. Schneider and K.V. Thimann. 1980. Hormonal factors controlling the initiation and development of lateral roots. II. Effects of exogenous growth factors on lateral root formation in pea roots. Physiol. Plant. 49:304-314. Yu, P.T., L.H. Stolzy and J. Letey. 1969. Survival of plants under prolonged flooded conditions. Agron. J. 61:844-847. CHAPTER 2 MEASUREMENT OF DISSOLVED C02 IN THE RHIZOSPHERE OF PLANT ROOT SYSTEMS BY A MODIFIED INFRARED GAS ANALYZER SYSTEM ABSTRACT Total dissolved inorganic carbon (ECOZ) and aqueous carbon dioxide (HZCO3*, which includes C02 (aq) and H2CO3) in the solutions of root media may be measured by the injection of small gas or liquid samples (lul to 8m1) into a gas stripping column (GSC) connected in-line with an IRGA. The measurement of ZCOZ in solution requires sample acidifi- cation while HZCO3* and gaseous C02 are measured without the addition of lactic acid. The XCOZ standard curve was linear up to 300 nmol C02. Maximum sensitivity was approximately 300 pmol. H2C03* measurements were independent of pH. Consequently, ECOZ and HZCO3* could be used to calculate the pH, HCO3‘ and CO3= values of solutions in root media. Injec- tion and complete analyses required from 50 to 120 sec. The modified IRGA-GSC system was used to measure carbon exchange rates of shoots and ZCOZ respired by roots of plants subjected to anoxic stress. Carbon exchange rates of bean 20 21 (Phaseolus vulgaris, L.) shoots were unaffected while root respiration declined during 18 h of root anoxia. Gas or solution samples could be analyzed directly without modifying the IRGA-GSC system. INTRODUCTION Rates of C02 evolution from respiring plant roots have traditionally been determined by removing the C02 from rooting media with C02 free air (Barber and Martin, 1976; Pate gt gt., 1979). Typically in such systems the released C02 is trapped for a given period of time and the total quantity measured. Exact values for reSpiration are obtained only when gas equilibrium has been established under constant conditions of pH, temperature and flow rate (Jensen, 1957). C02 equilibration between the gas and liquid phases occurs most rapidly between pH 4 and 5. However, plant nutrient cultures are generally maintained between pH 6 and 7 and pH values may fluctuate in the root environment (Riley and Barber, 1969). Alternative methods for measuring root respiration rates involve closed or semi- closed systems in which the gas phase is excluded from the root media. One such method involves sealing the nutrient solution from the atmosphere for a short period of time and subsequently monitoring the release of total dissolved inorganic carbon (ZCOZ) into the nutrient solution (Jensen, 1957). Steady state respiration can be monitored using a semi-closed system in which the nutrient solution is 22 23 exposed to the atmosphere in an aeration reservoir and then pumped past the root system. Differential C02 can be monitored eliminating the problem of equilibration with a gas phase in the root medium. The measurement of ECG; in a closed system also eliminates the requirement for maintaining a constant pH. The measurement of C02 dissolved in aqueous solutions is complicated by the reaction of C02 with water to form carbonic acid (H2CO3) and the associated protolysis anions, bicarbonate (HCO3') and carbonate (CO3=). This reaction results in the interdependence of the aqueous solute components H2CO3* (representing the moieties of C02 (aq) and H2CO3 where only a small fraction of C02 (aq) occurs as H2CO3), HCO3', CO3=, H+, and OH' at equilibrium. H2C03* is related to the pC02 of the gaseous phase in equilibrium with the liquid phase by [H2CO3*] = a pCOz where a is the solubility coefficient for C02 in water at a specific temperature. Consequently measurements of dissolved C02 are a function of the pH, pCOZ of the equilibrium gas phase, carbonate alkalinity ([HCO3'] + 2[CO3=]), temperature and the ionic strength of the solution (Park, 1969; Stumm and Morgan, 1970). Any method used to measure C02 dissolved in solution must take into consideration the implications of the reaction of C02 with water. Numerous methods have been developed to measure ZCOZ (Birmingham and Colman, 1979; Park, 1969; Swinnerton gt gt., 1962). Indirect methods are based on the measurement of any 24 two of the following parameters, pCOZ, pH and carbonate alkalinity. Direct measurements of ZCOZ essentially require the acid conversion of bicarbonate and carbonate ions to C02, the separation of gas and liquid phases and the quantitative measurement of the released C02 gas. These methods require a considerable amount of time or relatively complex modifications of gas chromatographic equipment. This report describes an inexpensive modification of the Clegg approach (Clegg gt gt., 1978) which improves on other IRGA methods used to measure dissolved C02 (Gibbs, 1976; Kaschube and Reuter, 1978). A gas stripping column similar to that described by Swinnerton (1962) was used to remove C02 from solution. The described modification permitted the measurement of both carbon exchange rates and root respiration of plants by one IRGA system. The system was used to describe the effect of an 18 h period of anoxia on the carbon exchange rates and root respiration of Phaseolus vulgaris L. cv. Seafarer. MATERIALS AND METHODS The IRGA system described by Clegg gt gt. (1978) was modified by incorporating an integrating microprocessor and a gas stripping column (Fig. l). The integrating microprocessor was adjusted to measure the area under the peak and convert the area to ug or nmol of carbon dioxide. Flow rate of the N2 carrier gas was maintained at 0.6 l/min. Gas Stripping Column. The gas stripping column (GSC) used to remove dissolved carbon dioxide from sample solutions (Fig. 2), is similar to other systems reported for the GLC analyses of C02 (Swinnerton gt gl., 1962). The GSC consisted of a glass chromatographic tube 1 cm in diameter with a coarse glass frit 5 cm from the tip of the tube. A standard removable septum was attached to the chromatographic column at a 45° angle 1 cm above the glass frit. Tygon tubing connected the GSC to the IRGA sample line. Liquid or gas samples ranging from 1 to 8,000 ul were injected into the GSC using microsyringes (Hamilton type) or plastic surgical type syringes. Side injection near the glass frit resulted in an even exposure of the sample liquid to the stripping gas. The sample was removed through a three way stopcock at the bottom of the GSC connected to a vacuum trap. 25 26 .moamfimm pflsvfla paw mom ca opHxOHo conumo ousmmoe on poms Eoumwm onu mo cowumucommumou owumEmummHQ .H ousmflh copcuuuhz. Emufiwm... 2a.: “£04... 0... 2.93.. :23 ¢ 5.. ,-I||.H-.IE . u D 20:85 on noooaoha .:. «we. 1... _ . - mac Iv . - 222...; 3.523 nlv can i wanna m¢o.|v xii-mac .uz. .Iv n 35.8 3.32... 8553: v.4... “8:20 I CN.C¢CU 55.. H A... was» $3» .I to... n 2:350:33: i n .lvu 3:23. u «up»: o ’9: n r- _ 20:83. - .--- Hate-(v. 27 IOOOO 8000’ 5000* 2000 » / IOOOP //// 800 - 600 » / 400* I V 200* ‘ IOO' //// 80* 1 60' u 40» . INTEGRATOR RESPONSE -§ (75030 t—o—a LA; 1 4.11141 _L JJJIJIJAI A A 4414' 0 l 2 5 IO 20 50 ICC 200 500 '000 2000 5000(0000 C02 (“"10” Figure 2. Carbon dioxide standard curve based on ul injections of lmM NaZCO3. 28 ZCOZ and HZCO3* Measurement. Prior to a series of measurements the IRGA system was calibrated by the injec- tions of standards. Measurement of ZCOZ involved an initial injection of 0.5 m1 of 0.1 N lactic acid into the GSC. The amount and concentration of the acid could be varied according to the alkalinity and volume of the sample. Generally, multiple measurements could be made using one injection of acid. Sample injection was made after CO2 dissolved in the lactic acid was removed by the C02 free N2 stripping gas flowing at a rate of 0.4 l/min. Sample volume could be varied based on the approximate ZCOZ in the sample. The surfactant qualities of lactic acid also improved the efficiency of separating gases from the solution by reducing the size of gas bubbles in the GSC. Total time between injections required from 1 to 2 min and was reduced if reacidification was not required. Standards were prepared from ul injections of a 1 mM Na2CO3 solution (Birmingham and Colman, 1979). The procedure for H2C03* measurement involved initial rinsing of the GSC with distilled water to remove residual acidity. Sample manipulation was similar to that described for the ZCOZ measurement except there was no acidification. Standards were prepared by equilibrating aqueous solutions with gas from commercially prepared standards of 225, 339 and 635 ul/l pC02 (Matheson) at known temperatures. Standard curves were constructed for H2C03* using the relationship: H2CO3* = a pC02 where a is the solubility coefficient of 29 C02 in distilled water. Solutions of known pCOz, pH and temperature were prepared and analyzed. Calculations were based on the appropriate equations used to calculate the various XCOZ components. Comparisons were made with measurements made by the modified IRGA system. Respiration and Photosynthesis Measurements. Dry bean seeds, Phaseolus vulgaris L. (cv. Seafarer), were surface sterilized in 1% sodium hypochlorite and rinsed throughly with distilled water. Seeds were germinated on paper toweling in the dark, at a constant temperature of 25° C. Seedlings were transferred to glass root chambers (Schumacher and Smucker, 1981) when the hypocotyls were approximately 2 to 3 cm in length. Plants were grown in a growth chamber having a photoperiod of 13 h, a relative humidity of 40 to 60%, with day/night temperatures of 27° and 20° C, and irradiance of 425 uEinsteins m‘zsec”l at the top of the plant chambers. Modified half-strength Hoagland's solutions (containing 0.92 pH Mn) equilibrated with air or N2 were circulated past the root system at an average rate of 15 ml min-1. Sampling ports were inserted into the rubber circulation lines at the inlet and outlet of the root chamber for sampling. Nutrient solutions were replaced as needed and immediately before initiating the gas treatments. Duplicate l to 3 ml samples of the nutrient solutions were taken at the inlet and outlet of the root chambers. Samples were injected directly into the GSC of the modified IRGA system for the direct measurement 30 of ZCOZ and [H2C03*]. Root reSpiration rates could be calculated from the AZCOZ concentrations and the nutrient solution flow rate. Thirteen days after seed incubation the shoots were sealed into 2 liter cylindrical acrylic chambers equipped with entrance and exit ports for measuring ACOZ of the shoot system. Compressed air (350-400 ul/l C02) flowed through the chamber at the rate of 8.0 1 min-l. This flow rate was rapid enough to minimize flow rate dependent changes in carbon exchange rate (CER) of the shoot. Duplicate gas samples were collected at the inlet and outlet and injected into the modified IRGA system (Clegg gt gt., 1978). The AC02 in conjunction with the flow rate measurements were used to estimate the CER. Periodic measurements were made of root respiration and shoot CER until the prebloom stage of growth, 26 days after the incubation period. At the prebloom stage of growth, half the experimental units were randomly assigned to an anoxia stress root treatment and the remainder to an aerated control treatment. Nutrient solution reservoirs were separated into the control, which was aerated with compressed air, and the anoxia stress treatment, which was equilibrated with nitrogen gas to an oxygen level of <<0.l% 02 (measured by a YSI Model 53 oxygen monitor). Shoot CER and root respiration measurements were taken before and after the 18 h anoxic treatment period. At the conclusion of the experiment, leaf area, fresh weight and 31 dry weight were measured. The experimental design was a completely randomized block design with three replications. One chamber without roots was used to estimate the background respiration of the nutrient solution due to microbial respiration. RESULTS AND DISCUSSION Injection of ul quantities of Na2C03 for the ZCOZ standard curve resulted in a linear response up to 300 nmol C02 (Fig. 2). Quantities greater than 300 nmol C02 over- loaded the IRGA detection system. The minimum limit of detection achieved was 300 pmol C02. This sensitivity approximates the detection limits achieved by the GLC- methanation technique (Birmingham and Colman, 1979) and is at least three orders of magnitude more sensitive than the detection limits of thermal conductivity or previous infrared analyzer methods for measuring total C02 dissolved in solution. The coefficient of variation at 2 nmol of C02 was 7.1% (n = 10). Coefficients of variation for larger quantities of C02 were generally in the range of 0.5 to 3.0% (n = 6). The H2C03* standard curve was similar to the ZCOZ standard curve. Chemical equilibrium analysis of the rela- tionship between pCOz and H2C03* indicates that for an open system, the [H2CO3*] should be independent of ZCOZ and pH. This was verified by the experiment summarized in Table l in which the pH values of the test solutions were varied. A comparison of IRGA responses from acidified samples (ECOZ) 32 33 Table 1. Effect of varying the pH on ECOZ and [H2CO3*] under constant pCOz, ionic strength and temperature. (pCOz = 225 ul/l, I = 3.0 x 10'2 M, T = 23° C). pH [H2c03*] zcoz mg ml'1 5.09 .340 .346 5.91 .364 .513 6.95 .335 1.892 7.72 .395 10.114 34 and nonacidified samples (HZCO3*) indicated a symmetrical peak for the acidified sample (Fig. 3). The assymetric tailing of the nonacidified sample indicated that a slower conversion of HCO3' to C02 occurred at near neutral pH values (Kaschube and Reuter, 1978; Kern, 1960). Nutrient solution concentrations had essentially no affect on the measurement of ZCOZ and H2C03* concentrations in Hoagland's solutions for concentrations of 1.5 >< 10'2, 3.7 x 10"2 and 5.9 x 10'2 M (one quarter, one half and full strength modified Hoagland's solutions, respectively). The ZCOZ measured by our modified IRGA system correlated well with ZCOZ values calculated from aqueous solutions of known pH, temperature and pCOz at constant ionic strength (Table 2). In addition, relative standard deviations (5) calculated according to Waser (1964) were lower when measuring the ZCOZ by our modified IRGA system (s = 0.02) than were the calculated values for ZCOZ based on measured values for pH, temperature and reported pCO2 values (3 = 0.05). The carbon dioxide - carbonate system is described at equilibrium by a series of equations based on concentrations of the appropriate solute components balanced for electro- neutrality and on equilibrium constants for specific temperatures and ionic strengths (Park, 1969; Stumm and Morgan, 1970). The appropriate equations may be solved for equilibrium concentrations of all components by utilizing two or more of the four measurable concentration parameters (pH, carbonate alkalinity, pC02 and ZCOZ) with apprOpriate Figure 3. 35 Integrator Response 0 ms!— 02468 Time (min) Comparison of acidified and nonacidified sample peaks. All conditions are similar except for the acidification of the sample. a) Sample injection into 0.1 N lactic acid in the gas stripping column. b) Sample injection into acid free gas stripping column. 36 Table 2. Comparison of four systems of measurement for calculating total C02 and carbonate components of Hoagland's solution (I = 3 x 10'"2 M) at two pH levels and 20.0 0.1 C. Measured Parameters pH H2C03* Hc03‘ co3= 2c02 ug ml'l pH, pCOza 6.08 1.062 0.614 10‘5 1.676 H2C03*, zeozb 6.16 1.046 0.737 10‘5 1.783 pH, zcoz 6.08 1.128 0.655 10‘5 1.783 pH, H2C03* 6.08 1.046 0.603 10‘5 1.649 pH, pC02 6.82 1.062 3.49 10‘3 4.557 H2C03*, zco2 6.91 0.987 3.83 10‘3 4.817 pH, 2c02 6.82 1.157 3.66 10'3 4.817 pH, H2C03* 6.82 0.987 3.20 10’3 4.190 apCO2 - Matheson standard (635 01/1). bH2C03* and ZCOZ measured by the modified IRGA-GSC system. 37 equilibrium constants. Table 2 compares four systems of measurements used to calculate the concentrations of all carbon dioxide - carbonate solute components from Hoagland's nutrient solutions having two pH levels. These results indicate the interchangeability of pH, H2C03* and XCOZ for use in determining carbonate solute components. An analysis of relative errors using the present techniques for C02 and pH measurement indicate that the smallest 5 value for all component calculations would be from the pH and ZCOZ combina- tion. The H2CO3* and ZCOZ combination could be used to calculate pH values in situations where this might be difficult such as in samples of low volume. The application of the modified IRGA system was tested by measuring the effect of an 18 h period of anoxia on the root respiration and net photosynthesis of dry bean plants. Gas samples taken from the shoot environment for photosynthesis and the solution samples taken from the rhizosphere solution could be interspersed with no adjustments to the system. In addition, the standard curve for ZCOZ and the gas samples were found to be similar facilitating the use of one standard curve for both sample types. Measurements of CER and root respiration before the start of the anoxia treatment indicated no significance between treatments. After an 18 h period of anoxia the CER was unchanged while root respiration rates declined significantly (Table 3). Low root respiration rates after 18 h of anoxia indicates a possible depletion of substrate I?“ 38 .wuflaflnmnoum mo Ho>oH mo.o man no oocmoHMHcmflm moumoflccfl « n .HonEmno noon onu Houmm can oHOMoQ «mOUNm pan NOON mo mucoEoHsmmoE oCHHCH Eoum powmasoamo mosam> mm comm .ucoEumouu n ma Houmm Eopmwm «UmH pmamaUOE on» >3 Honfimno Doom may Houmm can ouowon pouswmoa mm cowusaom usoflnuss on» mo mosam> mm Goudasoamo can coflumuflmmou noon .Ammuv omcmnoxo conuwu .m magma 39 for glycolysis (Vartapetian, 1978). This reduction in substrate available to the roots appears to indicate that root anoxia either inhibits the transport of photoassimilates to the root system, promotes the loss of photoassimilates by roots or partitions the photoassimilates into a cellular pool unavailable for glycolysis. Nutrient solutions also became more alkaline during the 18 h treatment period. Solution pH increased from an initial 6.0 to 6.7 and 7.0 for the anoxic and control treatments. This ApH suggested that roots either excreted bicarbonate and hydroxyl ions or absorbed hydrogen ions (Riley and Barber, 1969). Table 3 indicates that pH of the nutrient solution was lowered by the reSpiring root system before being returned to the reservoir. This contradiction may result from the reactions between carbonic acid and hydroxyl ions which accumulate during anion uptake. Large quantities of ZCOZ accumulating in the nutrient solution surrounding plant roots could mask a pH increase until the ECOZ is driven from the bulk nutrient solution by equilibrating the solution with air or N2 gases. In conclusion, the modified IRGA system provides a rapid method for the measurement of CER (gas samples) and root respiration rates (solution samples) with a minimal amount of time and effort. The method is nondestructive and additional gases removed by the stripping system could be analyzed by connecting other analytical systems to the IRGA. The system also permits the trapping of labeled l4C02 to determine the 40 specific activity of root respiration and plant photo- synthesis. The IRGA-GSC approach is routinely used in our carbon transport studies of plant responses to stress environments. REFERENCES Barber, D.A. and J.K. Martin. 1976. The release of organic substances by cereal roots into soil. New Phytologist 76:69-80. Birmingham, B.C. and B. Colman. 1979. Measurement of carbon dioxide compensation points of freshwater algae. Plant Physiol. 64:892-895. Clegg, M.D., C.Y. Sullivan and J.D. Eastin. 1978. A sensi- tive technique for the rapid measurement of carbon dioxide concentrations. Plant Physiol. 62:924-926. Gibbs, C.F. 1976. A continuously recording polarographic respirometer and its use in oil biodegradation studies. Water Res. 10:443-451. Jensen, G. 1957. Application of the tonometer principle for root respiration measurements. Physiol. Plant. 10:967-983. Kaschube, K. and W. Reuter. 1973. Quantitative determina- tion of free carbon dioxide in water. Deutsche Gewaesserkunliche Mitt 22:30-31. Kern, D.M. 1960. The hydration of carbon dioxide. J. Chem. Educ. 37:14-22. Park, P.K. 1969. Oceanic C02 system: An evaluation of ten methods of investigation. Limnology and Oceanography 14:179-186. Pate, J.S., D.B. Layzell and D.L. McNeil. 1979. Modeling the transport and utilization of carbon and nitroqen in a nodulated legume. Plant Physiol. 63:730-737. Riley, D. and S.A. Barber. 1969. Bicarbonate accumulation and pH changes at the soybean (Glycine max (L.) Merr.) root-soil interface. Soil Sci. Soc. Am. J. 33:905-908. Schumacher, T.E. and A.J.M. Smucker. 1981. Mechanical inpedance effects on oxygen uptake and porosity of drybean roots. Agron. J. 73:51-55. 41 42 Stumm, W. and J.J. Morgan. 1970. Dissolved carbon dioxide. In Aquatic chemistry: An introduction emphasizing EHemical equilibria in natural waters. John Wiley and Sons, New York, pp 118-159. Swinnerton, J.W., V.J. Linnebom and C.H. Cheek. 1962. Determination of dissolved gases in aqueous solutions by gas chromatography. Anal. Chem. 34:483-485. Vartapetian, B.B., I.N. Andreeva and N. Nuritdov. 1978. Plant cells under oxygen stress. In D.D. Hook and R.M.M. Crawford, eds, Plant Life iH_Anaerobic Environments. Ann Arbor Science Publ, Ann Arbor, Michigan. waser, J. 1964. Quantitative chemistry - a laboratory text. W.A. Benjamin, Inc., New York, pp 361-381. CHAPTER 3 MEASUREMENT OF SHORT TERM ROOT GROWTH BY PRESTAINING WITH NEUTRAL RED ABSTRACT A root staining procedure is described for measuring root growth which occurs during short term treatment periods. A vital stain, neutral red chloride, was used to stain roots £2 tttg. Root growth after the staining period appeared white and was easily differentiated from the stained portion of the pretreated root system. The staining treatment did not significantly alter plant growth or root respiration rates. The neutral red was primarily confined to cell walls of the cortex for the short term studies reported. Use of this or similar staining techniques combined with image processing equipment promises to provide a suitable method for rapid, inexpensive and quantitative measurement of root responses to short term treatments. 43 INTRODUCTION The measurement of tg gttg plant growth is often complicated by the difficulty of collecting quantitative data without destroying the root system. Nondestructive measurements of tg gttg root growth generally involve measuring individual roots at a glass-soil interface (Bma, 1979). These methods measure only a fraction of the total root growth occurring in the soil volume. In addition it is difficult to quantify the root environment at the glass- soil interface which is certainly modified during the installation procedure and may be quite different from the bulk soil (Dyer and Brown, 1981). Root measurements may also be made on solution grown roots by marking the root tip with india ink or by measuring root length before and after each treatment. Both these methods involve handling individual roots which can be tedious and may damage the roots. Identification of established roots by staining appears to be one method for determining root responses to specific plant or root environment treatments. A root staining technique was developed which could be applied to the entire root system of solution cultures before treatment. Following treatment, several morphological characteristics of new 44 45 (white) and previous (red) root growth were measured. Neutral red chloride, a basic dye of the azine group, was used to stain the root system (Gurr, 1960). Objectives of this study were to determine the suitability of neutral red chloride for measuring the effects of short term treatments on root growth of dry bean genotypes and to determine whether staining adversely affected the growth or metabolism of plant root systems. MATERIALS AND METHODS Five genotypes of Phaseolus vulgaris L. (cv., Domino, Pinto 111, Seafarer, Swan Valley, and MSU experimental line 31908) were germinated on moist paper toweling in an incuba- tor at 23 C. Seedlings were subsequently transferred to containers filled with aerated quarter strength Hoagland's nutrient solution. Root systems were stained by removing them from the nutrient solution, carefully washing with distilled water and placing the roots in a container filled with 0.03% neutral red chloride for a predetermined time. Roots were carefully rinsed four times in containers of distilled water and returned to the nutrient solution. Since neutral red chloride is less soluble in water at a pH greater than 7.0 and the nutrient solution pH was 6.5, the pH of the staining solution was adjusted to 6.5 with additions of acetic acid (Gurr, 1960). Staining intensity of plant roots was examined by exposing roots to neutral red chloride concentrations of 0.50, 0.25, 0.13, 0.06, and 0.03 g/l for periods of 5 or 10 minutes. Root staining intensity was evaluated for plants ranging from the primary leaf to the flowering stage. Color intensity was evaluated by visual comparisons. 46 47 Short term root respiration rates were determined for Phaseolus vulggris L. roots by monitoring the oxygen con- sumption by the root system in the nutrient solutions immediately before and after staining. Long term effects of the staining procedure on the oxygen consumption rates of root systems were made using the YSI oxygen monitor, model 53, three days after the staining treatment. Leaf area was measured using the LICOR leaf area meter, model 3000. Shoot and root fresh weights and root length measure- ments were made three days after staining the root systems. Nitrate concentrations before and after the treatment period were determined potentiometrically by an Orion, model 92-07, selective ion electrode. Root branching, length, and other morphological and physiological characteristics were measured in experiments employing this staining technique. Cross sections of both roots and shoots were examined to determine the localization or translocation of the stain within the plant. RESULTS AND DISCUSSION A continuous gradation in the visual intensity of color occurred from 0.50 to 0.03 g/l of neutral red chloride. Stain concentrations greater than 0.25 g/l gave acceptable intensities with a 5 minute exposure. Increasing the exposure period to 10 minutes increased the color to an intensity similar to color resulting from a doubling of the stain concentration. Staining conditions of 0.30 g/l for 5 minutes were chosen to minimize exposure to an uncontrolled environment and reduce the potential adverse effects of a high stain concentration. Microscopic examination of the Phaseolus vulgaris L. root system immediately after staining indicated that the dye was primarily localized to the cell walls of the cortical cells, with the increased color intensities observed up to the endodermis. No stain was observed in the stele region of the root. Newly emerged lateral root tips and elongation of previously emerged roots could easily be differentiated one day after staining. The color differentiation between stained and unstained root segments could be observed for periods of time greater than six days. This indicates that 48 49 the stain was permanently fixed to the cell walls and this technique may be used for long-term measurements of differ- ential root growth and losses of root cell debris during root senescence. It was also observed that nodules could be stained by neutral red and that nodules which formed after the staining period could be differentiated from those formed before the staining period. Oxygen uptake rates of dry bean root systems were similar before and immediately after staining. Oxygen respiration of stained and unstained root systems were also very similar three days after staining. A summary of the analyses of variance for the two-factor experiment indicated no significant effect on root respiration rates, leaf area, nitrate ion uptake, or plant weight (Table l). The neutral red staining approach to measuring differential root growth had no detrimental influence on plant growth. Although there were no significant effects from staining, there were significant differences between genotypes for leaf area, nitrate ion uptake, and shoot and root weights (Table 2). The small seed genotypes (Seafarer, Swan Valley, and Domino) produced smaller shoots and roots at the third trifoliate stage of growth compared to the large seed geno- types (Pinto 111 and line 31908). Swan Valley, a white seed type, and Domino, a black seed type, are progeny of the cross between Black Turtle Soup, a black seed type, and Nep-2, a mutated white seed type derived from irradiated San Fernando seed, a black seed type. Within the small seeded genotypes, 50 .mm mm3 oxmum: :ofl oumnuflc mom mp Houum + .suhaflnanoua no Hm>ma Ho.o 6:0 06 suaaananona Hmohumapaum on mummmm44 +ooo.oma Nmm.> m.o n.m voa.v mv Houum ooo.~m omm.a H.o 5.6 mms.a a mamuocmo x camum 44ooo.omm.a onm.m 4.6.m .4N.mam ..mmm.mma a mmsuocmo ooo.sam aom.m m.o H.m AGG.~ H :Amum mxmud: 006m ucwamz named: amps L6 mousom coH oxmumb cmoum smouh mood oumuufiz commxo Doom Doozm .Amucmam NV muflcs Hmucoeflnomxo Mom mosam> usomoumou moumsgm coo: .poH Hmuuso: QDHB msflcflmum Houmm whom mossy mom>uosom soon who o>Hm mo muouofimumm uoou mam poonm HOW moumsqm cmoE oosmflum> mo momwamc< .H manna 51 NNN wz v.0 o.H mm Amo.ov omq who omm m.~ m.oa omm momam how 0mm m.H H.m mom HHH oucflm wmm oom o.a n.m omH ocflEoo com omm m.H m.s mmm smaam> casm mom 0mm N.H v.v moa monommom alucmam Hun: m: HI3mmHIHn m: IIIIIIIII alucmHm m IIIIIIIII HIDCMHQ mEo oxmums mumuuflz oumm oxmumb unmfloz nmoum unmfloz nmoum comm mmmwuocow comwxo uoom poonm wood .coflumcflEuom nouwm mmmc coouxflm momwuocoo coon xuo o>Hm MOM muouoEmumm uoou paw poocm .N manna 52 Domino had larger shoots and roots compared to Seafarer, a white seed type. Swan Valley produced shoots and roots of intermediate weight but was not significantly different from Domino. A comparison of stained and nonstained root systems at similar stages of growth indicated no observable changes in root morphology. However, one needs to exercise care in transferring the root systems during the staining procedure to prevent damage to the root tips. The neutral red staining technique appears to be an efficient and effective method of determining differential root growth among dry bean, soybean, and perhaps many other crOp species. Root length of the unstained and stained portions of the root system can be measured using a number of methods (Bfihm, 1979). Perhaps the most promising approach involves the use of the improved color image pro- cessing equipment similar to that reported by Voorhees gt gt. (1980) for differentiating white and red portions of stained root systems (unpublished data). Using this approach one could measure multiple changes in the morphological characteristics of root growth after a treatment period, including the growth which occurred before staining and the total length and branching of the root system. A recent report (Carman, 1981) indicates that multiple color stains may be added to sand cultures throughout a treatment period. The differential staining of plant root systems appears to be a method for selecting plant cultivars 53 having superior root systems, as well as measuring root responses to abiotic and biotic soil stresses during the different stages of root growth. This method is much easier to perform than other standard methods, especially when working with well developed root systems. When combined with color photo- graphy it does not involve handling of the individual roots and it is suitable for collection of a large number of short term or long term root growth data. REFERENCES B6hm, W. 1979. Methods of studying root systems. Springer- Verlag, Berlin, pp. 188. Carman, J.G. 1981. A nondestructive stain technique for investigating root systems. Agron. Abst. 73rd Annual Meeting, p. 81. Dyer, Dan and D.A. Brown. 1981. Computer modeling of crop root systems. Agron. Abst. 73rd Annual Meeting, p. 174. Gurr, Edward. 1960. Encyclopedia of microsc0pic stains. The Williams and Wilkins Co., Baltimore, pp. 290-292. Voorhees, W.B., V.A. Carlson and E.A. Hallauer. 1980. Root length measurements with a computer-controlled digital scanning microdensitometer. Agron. J. 72:847-851. 54 CHAPTER 4 LOCALIZED ANOXIA EFFECTS ON ROOT GROWTH OF PHASEOLUS VULGARIS L. ABSTRACT Compensatory root growth in response to nonuniform aeration within a root-soil system can occur in heterogeneous media such as the soil. This study was undertaken to more fully evaluate this response in five genotypes of dry bean, Phaseolus vulggris L. Three aeration treatments were applied to split root systems, an aerated control, a nonaerated control and a localized anoxia treatment in which half the root system was aerated and the remaining half nonaerated. Shoot and root growth was reduced in the nonaerated control but not in the localized anoxia treatment. Contributions of the root components differed depending on the genotype examined. MSU experimental line 31908 partitioned the added growth into branched and lateral roots which were present before the treatment period. Swan Valley partitioned the extra growth into lateral roots which emerged during the treatment period. The other genotypes responded in an intermediate manner. The Pinto cultivar did not significantly 55 56 increase the number of growing root tips in response to the localized anoxia treatment. These differences are discussed in terms of the long term effects on root system growth. INTRODUCTION The annual Phaseolus vulgaris L. sp. has many charac- teristics of the competitive-ruderal growth strategy, including a relatively rapid growth rate and the capacity to produce a high leaf area index. During early stages of growth, these plants exhibit a competitive growth strategy resulting in rapid responses to environmental conditions. This strategy is characterized by rapid adjustments to environmental changes in photosynthate partitioning within the plant, accompanied by changes in the size, morphology and distribution of individual leaves and roots (Grime, 1979). One dynamic resource of the soil environment is the supply of molecular oxygen. Water excludes air from soil pores resulting in the formation of an anoxic environment. Phaseolus vulgaris L. cvs. are very susceptible to water- logging. Flooding for periods less than twelve hours caused reductions in bean yields of greater than 50% (Forsythe gt gt., 1979). Oxygen deficits may also occur in the absence of complete submergence of the soil profile. Soils with a high percentage of clay aggregates may remain virtually waterlogged for extended periods after fissures between 57 58 them have drained (Smith, 1977). The high variability of oxygen diffusion rates after a rainfall has been attributed to the heterogeneity of the aeration patterns within the soil (Flfihler gt gt., 1976). Strong evidence for limited soil and root aeration is the widespread distribution of anaerobes and facultative anaerobes in the profiles of most soil types (Patrick, 1978). Root systems growing in a heterogeneous soil environment may be exposed to both aerobic and anaerobic conditions. The ability of root systems to exploit the aerated zones of the soil profile should provide a mechanism which enables plants to avoid the adverse effects of anoxia. Compensatory growth in response to stress, localized within a part of the root system, has been observed for many types of adverse conditions (Brouwer, 1981). Although the effects of anoxia localized to a part of the root system have been reported (Brouwer, 1981; Yu gt gt., 1969) most studies examining compensatory root responses are limited to root weights. A more detailed analysis of root morphological changes within stressed root systems is needed. Objectives of this study were to examine the extent of compensatory root growth responses to localized anoxia for five genotypes of Phaseolus vulgaris L.; to provide a more detailed analysis of root components contributing to these responses; and to determine the extent to which these con— tributions might differ among the genotypes examined. MATERIALS AND METHODS The five genotypes of dry bean (Phaseolus vulgaris L.) used in this experiment were Pinto lll, Seafarer (a small white seed-type), Swan Valley (a small white seed-type), Domino (a small black seed-type) and MSU experimental line 31908 (a white kidney seed-type). Seeds of these plants were surface sterilized with 1% sodium hypochlorite solution for 10 minutes, rinsed with distilled water and germinated in the dark at 23 : 0.5C on trays containing wet cheesecloth covered with moist paper toweling. The radical root tip was removed at 24-48 hours after germination by cutting directly below the zone of basal root formation. This produced a split root system composed of basal roots which developed laterally from the main axis of the plant. Seedlings were grown in the dark for an additional period of 96 h, followed by a 24 h exposure to high humidity and light before being transferred to the greenhouse environment. Two seedlings of the appropriate genotype were selected for uniform hypocotyles (>2 cm) and root systems and transplanted into each experimental unit. Each half of the split root system was placed into a separate compartment within the container. Compartments consisted of two 59 60 polyethylene bags lined with saran to prevent the diffusion of gases into the nutrient solution. Polyvinyl inserts were used to provide support for the bags and maintain constant volume. Each compartment, one liter in volume, was filled with a modified quarter-strength Hoagland's nutrient solution adjusted to a pH of 6.0. Compressed air flowing through fritted glass aerators maintained nutrient solution oxygen concentrations above 0.19 atm p02. The tOp and outer surfaces of the container were covered with aluminum foil to exclude light and reduce temperature fluctuations in the nutrient solution. Supplemental cool white fluorescent lighting was used to maintain a photoperiod of 14 h light and 8 h dark. Photosynthetically active radiation ranged from 200 to 1,900 uE m-2 sec’l during the course of the experiment. Three aeration treatments were randomly allocated to the root systems of each genotype after a period of eight days of growth in a constantly aerated root environment. Aeration treatments consisted of two split root controls (both halves of the root system aerated; both halves of the root system subjected to N2 gas) and a localized anoxia treatment (one half the split root system aerated and the second half nonaerated). Oxygen partial pressures (YSI Model 53 Biological Oxygen Monitor) in the nonaerated control and the anoxia portion of the localized anoxia treatment were <0.005 atm p02. Gas flow rates through the nutrient solution were >150 cm3 min-l. Aeration treatments 61 were imposed for a period of 72 h before harvest. A staining technique (Schumacher and Smucker, 1982) was used to measure the amount of root growth taking place during the treatment period. The plant roots were stained prior to the start of the aeration treatments. The nutrient solution in the split root container was replaced with fresh quarter strength Hoagland's nutrient solution before return- ing the stained root system to the container. Root growth occurring during the treatment period was unstained and appeared white. Root staining had no detrimental effects on plant growth or root respiration rates (Schumacher and Smucker, 1982). Color transparencies were taken of the root systems at the conclusion of the treatment period. An opsiometer was used to measure the unstained portion of the roots (Reicosky et 31., 1970). The root system was divided into several components which were actively growing at the time of length measurement. Root categories included a) branched roots (basal and lateral roots bearing other laterals); b) nonbranched lateral roots; and c) lateral roots which emerged (i.e. completely white) during the treat- ment period, Figure 1. The number of root tips in each category was determined concurrently with the length. Root fresh weight was determined by immediately weighing the roots after aSpirating excess water and blotting with tissue paper. Leaf area was measured using a LiCor Model 3100 leaf area meter. The data was analyzed as a split plot randomized 62 complete block design. Genotypes were the main treatments and aeration the subtreatments. Orthogonal comparisons were made between the appropriate control and aeration treatments. Comparisons between genotypes were conducted using an F restricted Least Significant Difference test. Figure l. 63 DJ . - . I I I I I I I III Diagram illustrating the root segment components measured after the three day treatment period. Solid lines represent stained roots present before the treatment. Dash lines represent root growth occurring during the treatment period. Root components examined included the branched parent roots (a), nonbranched lateral roots (b), and unbranched lateral roots which emerged during the treatment period (c). RESULTS Leaf area, dry weight, dry matter percentage, root and plant fresh weight and shoot to root ratios were similar for the aerated control and localized anoxia treatments (Table 1). Total root fresh weight declined as a result of the localized anoxia treatment in the Pinto 111 cultivar in contrast to the other genotypes which showed no change in weight. Root anoxia decreased several shoot parameters during the 72 h treatment (Table 1). The greater shoot to root ratios of the nonaerated control appeared to be a function of decreased root growth of plants subjected to oxygen stress. Wilting of the primary leaves occurred for the nonaerated control in two replications for Domino and 31908, and in one replication for Pinto 111. Wilting began immediately after flushing with N2 gas. Shoots regained turgidity within two to four hours after the onset of anoxia. Bpinastic curvature of the petiole and hyponastic curvature of the primary leaf blades were observed more frequently and to a greater degree in nonaerated controls compared to the other treatments. Plant size (i.e. leaf area and weight) was greater for the large seed genotypes Pinto 111 and 31908 (Table 2). 64 65 .Hm>ma mo.o on» um unmoflmwcmflm <4 .m> Um cmm3umn confluwQEou+ .>Hm>fluommwmu .Hm>mH Ho.o Ho mc.o man an ucmoflmwcmflm dd .0« .m> oz cmwzumn GOmMHmQEOO*«.* >.m m.ma m.~ mmh com «A «ym.m *«o.m ««m.H *«mmm *«mwa Oz w.m m.~H o.m can omm Um woman m.m v.m +m.H omm 05H ma «to.m «*o.m ««o.H mmm «omH UZ h.v H.oH m.H mmm mma Ufl HHH oucwm h.m m.m m.H owe mma flu *rm.m *zm.v zrh.o xtmhm *oaa UZ w.m m.m m.H mvv ova Ué OCHEOQ H.m H.o m.H mmm oaa ma ««M.m ttv.v «*w.o *mmm «om UZ a.m m.o e.H mam mma o< smaam> cmzm ~.m m.m m.a ovm OHH dq «*m.¢ «¥V.v *tm.o mom om UZ m.m m.m m.a omm mHH 04 Hmummmmm HIDCMHQ m alucmam mE HIHCMHQ NED oflumm Doom unmflmz :mwum ucmflwz cmwum acmflmz who mmud uswEumeB mmmuocmw ou uoonm pamHm Doom wood mama coflumuwd .Aaqv mflxocm UmNHHmooH cam .AOZV Houucoo pwumummcoc .AU¢V Honucoo pmumumm wumB mucmEpmwuu coflumumd .mcoflumoflamwu me mo mammE mum mosam> .mmmwuocmm coma >Hp w>flm How mumqumumm Doom cam poocm co mucwEummuu coflumuwm m0 powwmm .H magma 66 Table 2. Main effect means for shoot parameters of the five dry bean genotypes averaged over all split root treatments. Leaf Leaf Shoot to Genotype Area Dry Weight Root Ratio cm2 mg Seafarer 105a+ 330a 3.9a Swan Valley 110a 375ab 4.0a Domino 130b 425b 4.7a Pinto 111 170c 535C 6.lb 31908 215d 685d 4.7a +Means within columns followed by the same letter do not differ significantly at the 0.05 probability level. 67 Domino tended to be larger compared to Seafarer and Swan Valley. The Pinto lll cultivar had larger shoot to root ratios than the other genotypes indicating a possible difference in carbon allocation compared to the other geno- types at this stage of growth. Root length measurements were conducted only on growth occurring during the treatment period to provide a more detailed analysis of the treatment effects. Anoxia severely inhibited both the length and number of roots growing during the treatment period (Table 3). Compensation for the lack of growth in the nonaerated portion of the localized anoxia treatment was demonstrated by the highly significant increase in length and number of growing roots in the aerated half of the localized anoxia treatment compared to the aerated control. Pinto lll appeared to respond less dramatically to localized anoxia in terms of numbers of growing root tips (Table 3). An analysis of the contribution of numbers of growing roots and the mean length for each root component indicated that the larger number of growing root tips in the localized anoxia treatment accounted for 52% (Pinto) to 83% (31908) of the increased root growth. The remaining root growth presumably occurred as a result of increased root elongation rates. Total new growth was positively correlated with the total number of growing root tips in the aerated treatment for the Pinto lll cultivar but not in the localized anoxia treatment indicating that the compensatory root growth of 68 . 04 .ummuucoo Hocmmonuuo may Scum msam> m may no comma Hw>ma muflaflnmnoum Ho.o on» an mocmoHMHcmflmee oo oo om oH ow om om oH oH oH dqz «*omm oaw «zoom *«omo «*ono «zoom ¥«omo «aoom «*oom «zoom flafi ow om oa oa om oH OH m OH m 02 can omv ohv ovv omv omm omv ovm own cum Ué HIAucmam mo .ozo HIADCMHQ mo Eoo momam HHH oafleoo smaam> umumummm moaam Add ocfleoo smaam> “mummmmm unashamue OHCHQ Em3m O#C..nnw Cm3m fiOHHMHO/N ucwEummHB mcfluso coflumHuHcH uocm ucmEumeB ocflusa nuBouw uoom .Emummm uoou mcu mam: How mum mmsHm> .Aéqzo ucmfiummuu mflxocm owuflamooa 03» mo cofluuom omumummcoc way can .AHm new poflumm ucwEummuu woo mmucu may mcfluso mcflumHDEsoom cuocma Doom pom mmflu boon mafi3oum mo Hogans ocu mo mcomflummEoo .m magma 69 Pinto 111 was not closely related to the number of growing root tips. An examination of the partitioning of root growth occurring during the treatment period indicates that the root components differed in the degree of compensatory growth (Figure 2). The distribution of root growth in the aerated control varied only slightly between genotypes. This pattern changed dramatically in the localized anoxia treatment particularly in the distribution of length between the three root components. The contribution of the root components appeared to depend on the genotype examined. The branched roots and lateral roots which emerged before the treatment period contributed significantly to the extra growth in the line 31908. Most of the new root growth for the cultivar Swan Valley appeared to occur in the lateral roots which emerged during the treatment period. Compensatory root growth responses to anoxia increased the emergence of roots in the aerated portion of the roots during the treatment period for all cultivars with the exception of Pinto 111 (Figure 3). There was a significant portion of the lateral root system which did not elongate during the treatment period. These lateral roots tended to be nonbranched and at a distance from the root tip of the parent root compared to actively growing roots. The length of the branched root segment from which roots emerged during the treatment period (zone of differentiation) was increased by the localized anoxia treatment for most of ‘58 482 F 288 156 ROOT LENGTH (cm) 58 359 322 252 282 152 ROOT LENGTH (cm) 182 50 Figure 350 ’ 388 ’ 256 > 70 458 one > 31988 3523 1 no 1 250 > 5 I1] ROOT LENGTH (cm) [ A A A l__‘_____‘_.. A _ A J A « L DOMINO PINTO 111 p C II .. i k ' J L l I 1 > . D 1 p 1 D I ‘ r J } SwSN VQ_LEY SERVRRER 11m i_.__l.___.L-—_L_—_‘_ _. A.._._‘. Y Y Y ifllbilcll Growth of three root components of five dry bean genotypes as affected by aeration treatments (aerated control and the aerated portion of the localized anoxia treatment). Black bars represent means of one half the root system in the completely aerated control. White bars represent the root growth for aerated half of the localized anoxia treatment. Root system components examined included the branched parent roots (a), nonbranched lateral roots present before the treatment period (b), and unbranched lateral roots which emerged during the treatment period (c). The single and double asterisks represent significance at the 0.05 and 0.01 levels respectively for the orthog- onal contrast of aerated control vs. aerated half of the localized anoxia treatment within genotypes. 71 an 539 m E; .50 > - '- wa - .— 8 350 » (x no 1 u E, m > . z o 202 > ‘1. ‘-’ use > 6 we > 2 se 1 552 e. ' see . 1 U‘ 1 E: 452 4, _ daet novzwo ‘ > PINTO 111 1 8 359 .. D: ‘ sea '— y Li Z 252 1 + 3 L o 222, a I ‘5 user 6 we . z ; . 52 If 1 r E ' I :52 , r . 1' see . 1 1 U1 1 f; ase‘. _____ J . ,_ 1 Stwqxti’ ! SNRN VRLLEY Q22 5 b g l 1 « O‘ 35? , —— .; , ., ‘ a l ‘ 1 388 It . . L7 ' 1 E 252{ 1 . . z ' ' 0 we . . a ‘ U ISEL . . . g G 182 L 4 . z ' 1 5e f i 4 L . e r 1 _: 1 a c a b c Figure 3. Number of growing root tips during the treatment period for three root components and five dry bean genotypes as affected by aeration treatments (aerated control and the aerated portion of the localized anoxia treatment). Black bars represent means of one half the root system in the completely aerated control. White bars represent root numbers for the aerated half of the localized anoxia treat- ment. Root system components examined included the branched parent roots (a), nonbranched lateral roots present before the treatment period (b), and unbranched lateral roots which emerged during the treatment period (c). The single and double asterisks represent significance at the 0.05 and 0.01 levels respectively for the orthogonal contrast of aerated control vs. aerated half of the local- ized anoxia treatment within genotypes. 72 the genotypes (Table 4). The Pinto cultivar showed no response from the localized anoxia treatment. Mean spacing of lateral roots emerging during the treatment period showed very little difference between the two aerated treatments for all genotypes. The aerated control had a mean lateral root spacing of 4.2 roots emerged per cm of parent root compared to 4.6 for the aerated portion of the localized anoxia treatment averaged over all genotypes. 73 Table 4. Comparison of the lengths of the zone of differen- tiation of branched roots for the aerated control (AC) and the aerated half of the localized anoxia treatment (ALA) for five dry bean genotypes. Genotypes AC ALA cm Seafarer 50 70ns Swan Valley 50 80** Domino 50 75** Pinto 111 55 60ns 31908 65 100** **Significant at the 0.01 level of probability as determined by the F test of the orthoganol contrast between the aerated treatments within genotypes. DISCUSSION Flooding of the plant root system has been reported to cause a wide range of effects on the entire plant (Cannell and Jackson, 1981). These have included the wilting, epi- nasty, and slow rates of extension and dry matter accumulation observed in this study. The reduction in leaf area found in the nonaerated control could be the result of reductions in leaf cell expansion, net photosynthesis, and in growth hormones exported from the root. Wenkert 33 31. (1981) found that leaf elongation rates slowed within 20 to 40 minutes of flushing the root environment of EEE.EEX§ L. with N2. This supports the initial role of flooding in reducing leaf cell expansion presumably by affecting leaf water potential through increased root resistance to water absorption. Increases in the stomatal resistance of £33 gays L. and Phaseolus vulgaris L. 20 to 50 hours after the initiation of root anoxia, suggests a delay in the effect of reduced net photosynthesis on the accumulation of dry matter (Moldau, 1973; Wenkert et 21., 1981). A decline in net photosynthesis in Phaseolus vulgaris L. was attributed to a reduction of both stomatal and meSOphyll conductances as 74 75 Opposed to only a reduction in stomatal conductance under water deficits (Moldau, 1973). In contrast, Brouwer (1963) presented results indicating that the decrease in dry matter production in Phaseolus vulgaris L. is a result of the reduction in net photosynthesis per plant due to the smaller leaf area occurring from an extended reduction in leaf water potential. The possible shortage of growth hormones needed for shoot growth provides an alternate explanation for the reduced leaf elongation of the nonaerated control of our study. Carmi and Heuer (1981) concluded that the reduction of growth hormones supplied to the shoot from a restricted root system caused starch to accumulate in the leaves of Phaseolus vulgaris, L. concurrently with reduced leaf elongation even though the leaf was found not to have reduced water potential relative to a control. The lack of a flooding effect on Pinto lll leaf dry weight may be due to a smaller effect of flooding on net photosynthesis or different requirements for transport of plant growth hormones. Another possible interpretation is that the Pinto lll cultivar accumulates carbon products within the leaf under flooding stress rather than translocating the carbon compounds to other parts of the plant. The transient and infrequent occurrence of wilting in the nonaerated controls may have resulted from a combination of plants with large leaf areas (the smaller sized cultivars did not wilt) and greenhouse conditions which promoted high transpiration rates. Wilting was only observed on bright 76 sunny days when the relative humidity was low. Plants tended to regain full turgor when transpirational demands were reduced in the late afternoon or early evening. Shoot growth was not substantially affected when compen- satory root growth occurred. This is in agreement with the observations of others (Brouwer, 1981; Yu 33 31., 1969). The aerated half of the root system evidently was able to supply the metabolites (e.g. cytokinins, nutrients, etc.) needed for continued high rates of photosynthesis and the water needed for leaf expansion. The removal of oxygen from the nutrient solution caused essentially complete inhibition of root growth. Root elonga- tion of several plant species are inhibited by the removal of molecular oxygen (Huck, 1970). Compensatory root growth in response to localized anoxia was qualitatively similar to studies conducted with Zea mays L. and Triticum aestivum L. (Brouwer, 1981; Yu SE 31., 1969). The observed increase in the number of growing root tips of the aerated half of the localized anoxia treatment may be quite crucial to the development and maintenance of compensatory growth within the root system. Total root growth normally occurs in the root system at an exponential rate (Hackett and Rose, 1972). Individual root members grow at an arithmetic rate charac- teristic of the type of root member i.e. primary, first order lateral, etc. The initiation of new growing points is constantly required for the root system to maintain exponential growth. The failure of the Pinto 111 cultivar 77 to respond to localized anoxia by increasing the number of growing root tips in the aerated portion of the root system could result in a future slowing of the compensatory response since individual root members may not be able to sustain the increased rates of growth needed for exponential growth of the total root system. The rate of growth of the parent root is a major factor governing the rate of production of new lateral roots (May 33 31., 1967). The ability of line 31908 to stimulate growth of the parent roots when treated with localized anoxia suggests potential for future lateral root initiation. Computer simulations indicate the importance of branching on exploiting localized soil environments (Lungley, 1973). Phaseolus vulgaris L. has approximately a three day period between lateral root initiation and emergence (Thompson and Macleod, 1981). This implies that the majority of roots observed to emerge during the treatment period were from primordia developed before the start of the aeration treatments. The measured mean spacing of lateral roots compared favorably with the findings of Thompson and Macleod (1981). Because of the delay between lateral root initiation and emergence, our measurements primarily reflected the pattern of lateral root initiation under completely aerated conditions. Growth potential, the timing and duration of elongation, of secondary roots are also partly determined at the time of root initiation (Yorke and Sagar, 1970). Localized anoxia 78 within the root system may have had an effect on mean spacing and growth potential of lateral roots which we could not detect. An extended examination of root growth after the treatment period could provide information on this possibility. The increased emergence of lateral roots in the local- ized anoxia treatment relative to the control could be attributed to the release from inhibition of primordia already formed. Close proximity of primordia to the parent root tip causes inhibition of lateral root development presumably by an inhibitor formed in the parent root tip (Goodwin and Morris, 1979; Wightman and Thimann, 1980). An increase in the elongation rate of the parent root could stimulate lateral root emergence by affecting the balance between auxin transported from the shoot and inhibitor moving basipetally from the parent root tip (Pilet 31 31., 1979). Alternatively there could be either an increase in auxin transport from the shoot or a decrease in inhibitor from the parent root tip as a result of the localized anoxia treatment. Root systems of several plant species have significant genotypic variation (McIntosh and Miller, 1980; Jenison 33 31., 1981; Stoffella 33 31., 1979). The root system characteristics in these species were relatively stable over environments. In contrast, the root system growth of the five dry bean genotypes measured in this study were relatively similar under optimum growing conditions. This relative similarity changed under conditions in which half 79 the root system was exposed to less than optimal conditions. This situation is somewhat analogous to induced differentia- tion found in Sinapis albus under water stress conditions in that stress caused a change in root morphology which was not expressed in the absence of the stress (Vartanian, 1981). More complete interpretation of the effects of the differences observed between genotypes in this study will depend on longer term measurements of root growth after the treatment period and on more complete modeling of root growth in heterogeneous media. In summary, compensatory root growth occurred in all five genotypes of Phaseolus vulgaris L. in response to localized anoxia. The expression of compensation varied between genotypes with some root components compensating more in particular genotypes than in others. These results could help explain the nonlinear effect of some soil conditions such as soil compaction on plant growth and yield. Soil compaction promotes soil heterogeneity and poor aeration (Cannell and Jackson, 1981). Unless the soil is uniformly and severely compacted, and nonswelling there will be pockets of relatively better aeration which the root system can exploit. This could help ameliorate the otherwise severe damage to the shoot. REFERENCES Brouwer, R. 1963. Some physiological aspects of the influ- ence of growth factors in the root medium on growth and dry matter production. Jaarb. I.B.S. 212:11-30. Brouwer, R. 1981. Co-ordination of growth phenomena within a root system of intact maize plants. Plant Soil 63:65-73. Cannell, R.Q. and M.E. Jackson. 1981. Alleviating aeration stresses. p. 141-192. 13: Modifying the root environ- ment to reduce crop stress, eds. G.F. Arkin and H.M. Taylor. ASAE, St. Joseph, Michigan. Carmi, A. and B. Heuer. 1981. The role of roots in control of bean shoot growth. Ann. Bot. 48:519-529. Flfihler, H., M.S. Ardakani, T.E. Szuskiewicz and L.H. Stolzy. 1976. Field-measured nitrous oxide concentrations, redox potentials, oxygen diffusion rates, and oxygen partial pressures in relation to denitrification. Soil Sci. 122:107-114. Forsythe, W.M., A. Victor and M. Gomez. 1979. Flood tolerance and surface drainage requirements of Phaseolus vulgaris L. p. 205-214. 13: Soil physical conditions and crop production in the tropics, eds. R. Lal and D.J. Greenland. J. Wiley and Sons, Chichester. Goodwin, P.B. and S.C. Morris. 1979. Application of phytohormones to pea roots after removal of the apex: Effect on lateral root production. Aust. J. Plant Physiol. 6:195-200. Grime, J.P. 1979. Plant strategies and vegetation processes. pp. 222. John Wiley and Sons, New York. Hackett, C. and D.A. Rose. 1972. A model of the extension and branching of a seminal root of barley and its uses in studying relations between root dimensions. II. Results and inferences from manipulation of the model. Aust. J. Biol. Sci. 25:681-690. 80 81 Huck, M.G. 1970. Variation in taproot elongation rate as influenced by composition of the soil air. Agron. J. 62:815-818. Jenison, J.R., D.B. Shank and L.H. Penny. 1981. Root characteristics of 44 maize inbreds evaluated in 4 environments. Crop Sci. 21:233-237. Lungley, D.R. 1973. The growth of root systems - a numeri- cal computer simulation model. Plant Soil 38:145-159. May, L.H., R.E. Randles, D. Aspinall and L.C. Paleg. 1967. Quantitative studies of root growth. II. Growth in the early stages of development. Aust. J. Biol. Sci. 20:273-283. McIntosh, M.S. and D.A. Miller. 1980. Development of root- branching in three alfalfa cultivars. Agron. J. 20: 807-809. Moldau, H. 1973. Effects of various water regimes on stomatal and mesophyll conductances of bean leaves. Photosynthetica 7:1-7. Patrick, W.H., Jr. 1978. Critique of: "Measurement and prediction of anaerobiosis in soils." p. 449-457. In: Nitrogen in the environment. NitrOgen behavior in _— field soil. Vol. 1, eds. D.R. Nielsen and J.G. MacDonald. Academic Press, New York. Pilet, P.B., M.C. Elliott and M.M. Moloney. 1979. Endogenous and exogenous auxin in the control of root growth. Planta 146:405-409. Reicosky, D.C., R.J. Millington and D.B. Peters. 1970. A comparison of three methods for estimating root length. Schumacher, T.E. and A.J.M. Smucker. 1982. A staining technique for measurement of short term root growth. In Press. Smith, K.A. 1977. Soil aeration. Soil Sci. 123:284-290. Stoffella, P.J., R.E. Sandsted, R.W. Zobel and W.L. Hymes. 1979. Root characteristics of black beans. II. Morphological differences among genotypes. CrOp Science 19:826-830. Thompson, A. and R.D. Macleod. 1981. Lateral root anlage development in excised roots of Vicia faba L., Pisum sativum L., 333 mays L. and Phaseolus vulgaris L. Ann. Bot. 47:583-595. 82 Vartanian, N. 1981. Some aspects of structural and functional modifications induced by drought in root systems. Plant Soil 63:83-93. Wenkert, W., N.R. Fausey and H.D. Watters. 1981. Flooding responses in Zea mays L. Plant Soil 62:351-367. Wightman, F. and K.V. Thimann. 1980. Hormonal factors controlling the initiation and development of lateral roots. I. Sources of primordia-inducing substances in the primary root of pea seedlings. Physiol. Plant. 49:13-21. Yorke, J.S. and G.R. Sagar. 1970. Distribution of secondary root growth potential in the root system of Pisum sativum. Can. J. Bot. 48:699-704. Yu, P.T., L.H. Stolzy and J. Letey. 1969. Survival of plants under prolonged flooded conditions. Agron. J. 61:844-847. CHAPTER 5 LOCALIZED ANOXIA EFFECTS ON RESPIRATION AND l4C-SUCROSE TRANSLOCATION OF TWO DRY BEAN GENOTYPES ABSTRACT Short term 14 C-labeling experiments can provide informa- tion on the initial partitioning of carbon within plants. U-14C-sucrose was used to determine translocation patterns in root systems of two dry bean genotypes after a 3 day exposure to aerated and nonaerated environments. A split aeration treatment which exposed half the root system to anoxic conditions was used to simulate localized anoxia within the root system. Controls consisted of completely aerated and nonaerated split root systems. Complete anoxia within the root system for 72 h reduced the movement of l4C-label into the root system and resulted in an accumulation of label in the hypocotyl region. The 14C-label translocated to anoxic root systems appeared to be excluded from respiratory metabolism during the 3 h pulse/chase period. The non- aerated portion of the localized anoxia treated root system responded similarly to the nonaerated control in terms of 14C translocation. The aerated portion of the localized 83 84 anoxia treatment increased the proportion of label to the root system relative to the aerated control. Partitioning appeared to occur differently in the two dry bean genotypes. MSU experimental line 31908 partitioned an increased percentage of l4C-compounds to the actively growing fraction of the root system while Seafarer tended to increase the relative 14C content of the nongrowing region of the root system. INTRODUCTION Root systems growing in heterogeneous soil environments may be exposed to both aerobic and anaerobic conditions (Cannell and Jackson, 1981). Several studies have demon- strated the occurrence of compensatory growth in aerated portions of the root system when the remaining part of the root system is exposed to anoxic conditions (Brouwer, 1981; Trought and Drew, 1981; Schumacher and Smucker, 1982b). Root length and weight were used in these studies to provide an indication of the effects of localized anoxia. Although these measurements are an integration of the partitioning response of the plant during the treatment period they do not necessarily reflect translocation patterns at the conclu- sion of the treatment period. Short term l4C-labeling experiments provide information on the pattern of carbon translocation within the plant at a particular point in time. A number of short term l4C-labeling studies have been conducted on flooded plants (Grineva and Nechiporenko, 1977; Nagao and Wada, 1970; Nechiporenko and Grineva, 1976; Nuritdinov and Vartapetyan, 1980; Starck 3E 31., 1975). 14 These studies, however, did not account for losses of C to the root environment through exudation or respiration 85 86 during the labeling period. Objectives of this study were: 1) to determine the translocation patterns for two dry bean genotypes whose root systems were subjected to aerated, nonaerated and localized anoxia treatments for 72 h, and 2) to account for losses of 14C within the root environment during the labeling period. MATERIALS AND METHODS Seed from two dry bean (Phaseolus vulgaris L.) geno- types, cultivar Seafarer and MSU experimental line 31908, were sterilized with 0.5% sodium hypochlorite solution for 3 minutes and rinsed with distilled water. Germination occurred in the dark on trays containing moist paper toweling and cheesecloth. An incubator maintained temperatures at 23°C for germination and initial seedling growth. Radical root tips were removed 24 to 48 hours after radicals emerged. This produced a split root system composed of 6 to 8 basal roots forming laterally to the main axis of the plant. Seedlings were given a 24 h exposure to light when the hypocotyls were >2 cm in length. Seedlings were transferred to acrylic chambers designed to allow each half of the root system to be sealed in separate compartments. Two seedlings of the appropriate genotype were transplanted into each chamber. Root system halves were grown in compartments containing approximately 1 liter of modified half-strength Hoagland's nutrient solution adjusted to a pH of 6. Filtered compressed 1 air flowed through fritted glass tubes at >150 cm3 min" , maintaining nutrient solution oxygen partial pressures >0.l9 87 88 atm oxygen (YSI model 53 Biological Oxygen Monitor). Light was excluded from the split root systems by placing the acrylic chambers into opaque PVC cylinders. Chambers were randomly assigned positions on the greenhouse bench. Supple- mental cool white fluorescent light was used to provide a photoperiod of 16 h light and 8 h dark. Photosynthetically active radiation at midday ranged from 200 (cloudy) to 1,900 zsec"l (clear) uE m' at the primary leaf surface. Three aeration treatments were randomly allocated to the root systems of each cultivar after a period of eight days of growth in a well aerated root environment. Aeration treatments consisted of two controls, both halves of the root system aerated (AC); both halves of the root systems non- aerated (NC); and a localized anoxia treatment in which the gaseous treatments were split, with half the root system aerated (ALA) and the second half nonaerated (NLA). Oxygen partial pressures in the nonaerated treatments were maintained below 0.005 atm oxygen by continually equilibrating the nutrient solution with nitrogen gas (>150 cm3 min-1). The neutral red staining technique (Schumacher 3E 31., 1982) was used to differentiate between root growth occurring before and during the treatment period. Roots were stained immediately before the aeration treatments were established. Carbon translocation was monitored by applying 15 uCi U-l4C-sucrose (sp. act. 673 mCi/mmol; Source New England Nuclear; 90% ethanol solution) to abraded areas of the middle leaflet of the oldest trifoliate. Phosphate buffer, 89 (5 mM adjusted to pH 6), was periodically added to the leaf to maintain a wet surface. Source leaflets were removed from plants two hours after labeling and rinsed with 5 ml of 80% ethanol to determine the amount of unabsorbed [14C] sucrose. Plants were dissected into various components after a one hour chase period. Shoots were dissected into six components identified as the middle trifoliate source leaf, two trifoliate leaves adjacent to the source plus primary leaves, immature rapidly eXpanding trifoliates, stem above source petiole, stem below source petiole to cotyledonary node and hypocotyl. Roots were dissected into five components depending on position and color. White unstained root segments indicated growth which occurred during the treat- ment period. Root components included white segments of: 1) parent roots bearing lateral roots; 2) lateral roots which emerged before staining; and 3) lateral roots which emerged after staining. The nongrowing part of the root system (red portion) was divided into sections of the parent root: 4) originating lateral roots which emerged before staining; and 5) originating lateral roots which emerged after staining. Root systems which did not grow during the treatment period were not dissected but were otherwise treated similarly to the dissected roots. Component parts of roots and shoots were frozen on dry ice and stored at -20°C. Ethanol soluble l4C-compounds were extracted from plant tissue by hot (55°C) 80% ethanol for one hour. Extracts were filtered using preweighed dry filter paper. Residues were dried at 70°C 90 and weighed. Filtrate was cooled to -120°F in Erlenmeyer (125 ml) flasks fitted with funnels and cheesecloth to prevent loss, then flash evaporated in a Virtis Freezemobile II freeze dryer. The dried samples were resolubilized with 20 m1 of hot 80% ethanol and subsequently divided into two equal volume aliquots. One aliquot was reserved for additional analyses. Aqueous counting scintillant (ACS, Amersham) was added to the samples for determination of radioactivity. Samples with counting efficiencies less than 30% or giving inconsistent duplicate readings were diluted to reduce color quenching. Counting efficiency was generally in the range of 60-90%. Each sample was counted twice on a LS-8100 Beckman Scintilla- tion Counter. Radioactivity was adjusted for background and quench based on a standard quench curve develOped using similar quenching agents to those in the samples. Control samples with no radioactivity were periodically subjected to the entire extraction procedure to determine background radiation and contamination. Root respiration of 14C02 was monitored by bubbling the exhaust gases from the split root chambers into separate vials containing 20 ml of ethanolamine. Aliquotes (1 ml) were withdrawn from the vials every half hour. Methanol (2 ml) was added to each sample to increase ethanolamine solubility in the scintillation cocktail. Absorbant volumes were adjusted for the time of sampling. Root exudation of l4C-materials were measured at harvest by acidifying a 10 ml 91 sample of nutrient solution, to a pH 4.0-5.0, to remove the HCO3’, freeze drying, and determining radioactivity as stated previously. These values were adjusted for the total volume of nutrient solution in each split root compartment. Root respiration (C02 production) was evaluated 1 h before labeling the plants with l4C-sucrose. The flow rate of treatment gases were measured and gas samples were taken in triplicate using a 1 ml tuberculin syringe. Syringe needles were stOppered and the C02 concentrations immediately determined by injection into a modified Beckman model 865 infrared C02 analyzer (Schumacher and Smucker, 1982c). This procedure was repeated for all treatments and background samples. The experiment was designed and analyzed as a randomized complete block design. Orthogonal comparisons were made between the aerated control and aerated portion of the localized anoxia treatments. Percentage and dpm data were analyzed as arcsine and log (x-l) transformations, respec- tively. RESULTS Nonaeration of the complete root system significantly reduced leaf area, the growth of roots and increased shoot to root ratios in both genotypes (Table l). Localized anoxia treatments had shoot dry weights and leaf areas which were in general similar to the aerated control (Table 1). Roots in the aerated half of the localized anoxia treatments showed little increase in dry weight for either genotype when compared to the aerated control (Table 2). There were significant interactions between genotypes and aeration treatments in dry matter allocated to the previously emerged lateral root growth. Localized anoxia increased the allocation of dry weight to this component in Seafarer but not in the 31908 line. Table 2 indicates that the total dry weights of the nonaerated controls were similar to the nonaerated portions of the localized anoxia treatments. The amount of 14C-label recovered by hot ethanol extraction of plant components and by washing the source leaf with 80% ethanol was not affected by aeration treat- ment (Table 3). There was a greater amount of 14C recovered from Seafarer than from 31908. The amount of l4C-sucrose 92 93 .qu .mocmummmwo ucmoflmacowm ummmq on» ocfim: muflafinmnoum mo Hw>mH mm mzu um ucmHmMMHU >Hucmoflmecoflm no: mum Hmpuwa mEmm mnu an owBOHHOM GEDHOU 50mm cw mmsam>+ DH.v comma Doom nomm «a Uo.h nmoa MHHH nmmw oz Qm.m ooam Comm nova Ufl momam Qm.m 6mm nmvma mmom «A 0o.m mom omm wmwv oz Mm.m mmm onooa +Mmmv U4 Hmumwmmm Alucmam NED HlucmHQ o8 ucowmz who mmud ucmflwz >MQ ucmflwz xuo wucmaumona mmwuocmo poomuuoonm mama uoom uoocm coflumumd .Adqo mflxocm omuflamooa ocm .Aozv Honucoo omumuwmcoc .Avflv Honucoo omumumm mumB mucmEummHu cofiumnm< .Emum>m uoou map mo mucmfiumwug coflumumm a N5 >3 smog who mo mwmwuocmm o3u mo nu3ouo Doom pom poonm mo GOHDMUHMHGOS .H manme 94 .Qma .mocmummwao mammamacoam ummma mzu ocams wuaaanmnoum mo am>ma wm mnu um ucmummmao >aucmoawacoam uoc mum Hmuuma mEmm may >9 ©m30aa0m cEsaoo comm ca wmsam>+ I I I I 6mm «dz Mom mma QmNa maa vaa find I I I I Mom oz omm maa nqa moa omma U4 momam I I I I Mom daz 6mm on Qva an boo mum muoom unoamz mun ucmEummHB mm>vocmw Bmz muoom amumumq cocmum Doom COaDMHmd mucmcomeou poem oca3ouw wam>auo¢ .Aquo ucmEummHu maxocw pmmaaMUOa may mo mam: Umumummcoc 6cm .Amado ucmEummuu maxocm pmwaaMUOa mnu mo mam: omumumm .Aozv aouucom omumummco: .A0¢o aouucoo pmumumm mHmB mucmEummuu amaumumm .n mm HOM mucmEpmmuu coaumumm mmucu on omuommnsm HmHMwmmm pom mooam wo mEmum>m uoou mcu casua3 umuumE wuo mo scausnauumao .m manme 95 Table 3. Amount of l4C-sucrose recovered in the 80% ethanol wash and extraction, and translocated from the source leaf. treatments were aerated control (AC), control (NC), retained by the source leaf Aeration nonaerated and localized anoxia (LA). Source leaf was washed and removed after 2 h and the plant harvested after an additional hour. Aeration Genotype Treatment l4C-Label Applied to Source Leaf Recovered Retained Translocated Seafarer AC NC LA 31908 AC NC LA l982a+ 2742a 1975a 1340b 1035b 1066b dpm x 10"4 plant'lI 483ab 1278a 860ab 305ab 300ab 225b 232a 107b 202ab l37ab 37c 112ab IValues in each column followed by the same letter are not significantly different at the 5% level of probability using the Least Significant Difference, LSD. +The power of 10 represents the amount by which the actual quantity was multiplied to give the reported quantity, e.g. 232 represents 2,320,000. 96 translocated from the source leaf was reduced in the nonaerated control for both genotypes. Absence of aeration to the entire root system reduced the translocation of l4C-label to the root system and as a result ethanol soluble labeled metabolites accumulated in the shoot (Table 4). The hypocotyl and adjacent stem below 14C_ the source leaf were the primary sites of increased label (Table 5). The translocation of l4C-label to the nonaerated half of the localized anoxia treatment was similar to the nonaerated control (Table 6). Most of the label translocated to the anoxic root system remained in the root and was not lost by roots as C02 or soluble exudates. Up to one third of the label translocated to the root system was respired and exuded by roots of the aerated treatments while less than 10% was lost by anoxic roots. Activity per unit dry weight of the roots indicated that the reduced label in the anoxic root system of the nonaerated control resulted from less translocation and was not simply due to a reduction in the size of the root system (Table 7). Similar allocations of l4C-label occurred between shoots and roots of the aerated control and localized anoxia treat- ments (Table 4). However, when a portion of the root was subjected to anoxia more 14C was transported to the aerated half of the root system relative to the aerated control (Table 6). The orthogonal comparison between the aerated control and the aerated half of the localized anoxia treatment was significant at the 5% level of probability 97 Table 4. Translocation of l4C-label from the source leaf to the shoot, root and root environment. Root losses include both exudation and respiration. Values for the root system include both halves of the root environment. Aeration treatments were aerated control (AC), nonaerated control (NC), and localized anoxia (LA). Aeration Root Genotype Treatment Shoot Root Loss % Seafarer AC 39a+ 40a 20a NC 84b 15bc ma wm map um ucmumwmao waucmoamacmam uoc mum umuuma mEmm mnu an ©m30aaom cadaoo numm ca mmsam>+ maa on nama we mma «a Dom moa mam av mma Oz nova mm Dana we mma U< momam noma moa nama am do da Dam om comm maa ova Oz soma av maa mm +mn Om umummmmm w a>uooom>m mousom ahuooomwm mmumaaowaue mmumaa0mau9 ucmEummuB mmwuocmw m>on¢ pom mousom musumEEH ummpao 0cm c0apmum¢ Emum cmmBumm Emum mm>mma mumEaum .Amao maxocm ommaaMOOa pom .Auzo aouucoo omumummcoc .AUc Am “macaumm mUHDOm m>onm Emum Av “macaumm mousom ocm moo: >umcooma>uoo cmm3umn Emum Am “mmumaa0mauu mcaocmmxm maoammu aamEm AN “mm>mma mumEauQ ocm umammma mousom may ou uxmc mumawmma Aa "mumz ommwamcm mmSmmau ucmcomEoo Doonm mJOaum> macaw amQMaIUva mo cOausnauumao .m magma 99 Table 6. Proportion of l4C-label translocated from the source leaf to half the root system in four aeration environments. Aeration treatments were aerated control (AC), nonaerated control (NC), localized anoxia-aerated portion (ALA), localized anoxia-nonaerated portion (NLA). Aeration Root C02 Genotype Treatments System Exudates Respiration Seafarer AC 20.2b+ 0.28a 9.4ab NC 7.6a 0.02a 0.1c ALA 27.7b 0.33a 12.8a NLA 4.6a 0.02a 0.4c 31908 AC 15.6b 0.14a 5.5b NC 3.0a 0.15a 0.2c ALA 25.4b 0.29a 10.1ab NLA 7.7a 0.24a 0.3c 1'Values in each column followed by the same letter are not significantly different at the 5% level of probability using the Least Significant Difference, LSD. 100 Table 7. 14Carbon activity per unit root dry weight for the root system and respiratory fraction of four root environments. Aeration treatments were aerated control (AC), nonaerated control (NC), aerated half of the localized anoxia treatment (ALA), and nonaerated half of the localized anoxia treatment (NLA). Aeration Root System l4C02 Genotype Treatment Activity Respiration dpm x 10'4 gdw"l dpm x 10'4 gdw'lI Seafarer AC 430ab+ 199a NC l35bc lde ALA 550a 247a NLA 238abc l7bc 31908 AC l45abc 52ab NC 14d le ALA 156abc 55ab NLA 108c Scd +Values in each column followed by the same letter are not significantly different at the 5% level of probability using the Least Significant Difference, LSD. IThe power of 10 represents the amount by which the actual quantity was multiplied to give the reported quantity, e.g. 1 represents 10,000. 101 for the percentage of label translocated to the root systems of the combined genotypes. Allocation of the extra l4C- label appeared to be controlled by genotype. The prOportion of l4C-label translocated to the actively growing roots was increased by localized anoxia for the 31908 line but not for Seafarer (Table 8). Seafarer tended to increase the propor- tion of 14C allocated to the segment of parent root bearing previously emerged laterals from 9.5% in the aerated control to 15.5% in the aerated portion of the localized anoxia l4C treatment. A comparison of the relative amounts of translocated to the actively growing roots (Table 8) and to the total root system (Table 6) indicates that the majority of label was found in the nongrowing portions of the root system in both genotypes. Carbon dioxide production by the entire root system was greater for the 31908 line, but when adjusted for root system size the respiration rates for the two genotypes were similar (Table 9). Respiration rates of the aerated half of the localized anoxia treatments tended to be larger than the aerated controls. Very little of the total carbon dioxide respired during the 3 h pulse/chase period was represented by 14 C02 I Nonaerated treatments responded with uniformly low l4CO2 respiratory loss over the 3 h treatment period (Figure 1). Approximately 1 to 1.5 h were required before significant amounts of 14C were respired by the root system. Seafarer respired significantly more l4C02 than did the 31908 genotype 102 .mocmumwmao m mo wuaaanmnoum manumumo on Umms mum3 cowaummfioo amcomosuno mumaumoummm may :0 pmmmn mummu m .mum>auaso casuaB mucmEummuu smaumumm :mm3umn mcomauwQEoo new muaaanmnoum mo am>ma mm man no mocmoamacmam mucmmmummma «ma N m m ¢a< o a m m Um momam m m m v find aa m m o Dd Hmummmmm amuoa mDoom amumuma omoumem >am50a>mum muoom ucmEummHB mmwuocmw 3mz muoom amumumq omnmcmum ceaumumd mucmcomaou uoom oca3ouw xam>auo< .Aam mum mmSam> .pmammma mousom may Eonm ceauMUOamcmuu usmoumm co ommmn mum paw Emumwm moon mnu mam: How mum mmsam> .ooaumm ucmEummnu mnu mcauso mucmcomfioo Doom Casua3 amQMaIUva mo coausnauumaa .m magma 103 .oma .mocmummmaa ucmoamacmam ummma map ocams mgaaanmnoum mo am>ma mm map um ucmnmmmao waucmoamacmam uoc mum Hmuuma mEmm mcp ma ©m30aa0m canaoo comm ca wmsaw>+ ma.o woma.m comvm .m ¢a< ma.ov mmo6.6 comma oz mm.a awomm.m nomm.m Om momam mv.o emmm.m 6mme «qz mm.a nmmm.ma naem.m age ma.o ammoa.n oommm 02 no.3 nmome.m +onmam.a om umammmmm w an: Hum Noo ma HI: anxuemae xv on Nov omuammmm amuoe mumm ceauosooum wucmEDmmHB mmwuocmo mo mmmucmoumm Ova coaumuammmm moo omumumd .Aquo ucmEummuu maxocm omwaaMUOa may mo mamn omumummcoc ocm .A¢Aacs mcafidmmm cam mmouosm omamnma mo mua>auom mamaommm map so Ummmn mmaOE ou Eon moauum>coo >3 omCaEHmumo mum3 Umuamwmu moo mo mmmucmonmm Ova .munmamz who moon co ommmn mum mmumu coaumuammmm .momam cam umumwmmm mo mEmmem Doom mam: an ceaumuammmw mam ceauosooum NOD .m manna 104 .uamoawacmamaoa mm3 mum>auasm auoa Mom muamEummuu maxoam ommaaMUOa map mo amaauom omumumm cam aouuaoo omumumm may mo aOmaHmQEoo amaoooauuo a< .>am>aaommmmu .wuaaaamaoum wo mam>ma ao.o oam mo.o man an pamoawacoam mam3 mucmEummuu coauwumm ammBuma paw mum>auaso cmmSuma maomaummeoo “Om ummu m .UOaumm oaaamaMa map maauso maaEMaoamaum ca omuomaaoo Uam Emummm noon map wa omuammmu NOUva mo aOHHMasadooa nay Uta... mHZUthcumh awhtmuxzoz .C .4 awhccuc Goa—n Jomhzou Dahcmuc cam—n r .c .J awhcmuc mumcuxum Jouhzou awhcmuc muccucum doIIx mm <,-aI x wdp) AllAIlDU 3,, .a madmam 105 even though the Seafarer root system was smaller than the 31908 root system. After the removal of the source leaf, at 2 h, the respiration of 14C02 became linear indicating a steady state in 14C metabolism for both genotypes (Figure l). DISCUSSION A reduction in plant mass as the result of exposing the total root system to anoxia has been well documented (Cannell and Jackson, 1981; Meek and Stolzy, 1978; Brouwer, 1963). An anoxic root system essentially stOps growing while the attached shoot continues growth but at a reduced rate. This results in an increase in the shoot to root ratio (Brouwer, 1963; Meek and Stolzy, 1978). Shoot growth appears unaffected when anoxic conditions are limited to only part of the root system (Brouwer, 1981; Schumacher and Smucker, 1982b; Yu 3E 31., 1969). Trought and Drew (1981) found that the aeration of a single seminal root would prevent shoot injury in young wheat plants if a complete range of nutrients were provided in the aerated nutrient solution. Root growth in the aerated portions of root systems exposed to localized anoxia tends to compensate for the inactive anoxic roots (Brouwer, 1981; Schumacher and Smucker, 1982b). Although there was a trend for greater root dry weight in the aerated portion of the localized anoxia treated root system the same degree of compensation was not as apparent as in previous studies (Brouwer, 1981; Schumacher and Smucker, 1982b). Corn roots subjected to localized anoxia 106 107 in the Brouwer (1981) study apparently were grown for a longer period of time than in this study. Lengthening the treatment period would increase the weight differences observed. The measurement of root dry weight is likely to be less sensitive than length measurements reported in a previous study (Schumacher and Smucker, 1982b). Root dry weight analysis could be biased toward roots with larger diameters particularly since large diameter roots comprise the smallest proportion of the total root system (Fiscus, 1981). The nutrient solution concentration of this study was double that in a previous study (Schumacher and Smucker, 1982b) which may have altered compensatory responses to localized anoxia. Local concentrations of ions in the rhizosphere can significantly affect root system growth and morpholOgy (Hackett, 1972; Drew and Saker, 1978; Drew _3 _1., 1973). Accumulation of label in the basal region of the stem of anoxia stressed plants has been observed in studies with cotton (Nuritdinov and Vartapetyan, 1980), dwarf bean (Starck 3E 31., 1975) and corn (Grineva and Nechiporenko, 1977; Nechiporenko and Grineva, 1976). The hypocotyl of the stem may become a greater sink for photoassimilates in anoxia stressed plants due to the initial inhibition of auxin movement to an anoxic root system and subsequent accumulation in the hypocotyl (Phillips, 1964). Auxin build up in the hypocotyl can induce the development of preformed root initials (Friedman 3E 31., 1980) and could result in the 108 hypertrophy of the hypocotyl observed in flooded plants (Wample and Reid, 1978). The development of adventitious roots in the hypocotyl could stimulate assimilate transport (Turvey and Patrick, 1979) by producing significant amounts of cytokinins (Forsyth and Vanstaden, 1981). Differences in the formation of adventitious roots were not apparent in our study. The short duration of the treatment period may have been a contributing factor to this observation since there is a lag period between initial activity in the root primordia and emergence of roots (Thompson and Macleod, 1981). Other short term l4C-1abeling studies of flooded plants indicate an inhibition of phloem translocation to anoxic root systems (Nagao and Wada, 1970; Nechiporenko and Grineva, 1976; Grineva and Nechiporenko, 1977; Starck 3E 31,, 1975). When the time between labeling and harvest is lengthened to several hours or days the amount of ethanol soluble label found in the root system appears to be similar or even increased relative to the control (Fulton 3E 31., 1964; Wiedenroth and Poskuta, 1981). These observations may result from an increased incorporation of label into structural components (Fulton 3E 31., 1964) or greater respiratory loss within the aerated control root system. When considering the fate of carbon in long term studies one must integrate the initial translocation with subsequent carbon metabolism and possible retranslocation of newly formed carbon compounds from the root system. 109 Label in the root exudate fraction of this 3 h pulse/ chase study was relatively low compared to the longer labeling period of Wiedenroth and Poskuta (1981). Short term labeling apparently limits the incorporation of label into compounds which are exuded and may be too short for leakage of labeled assimilates from the root stele. The reduced proportion of 14C-label in the respiratory fraction l4C translo- of the nonaerated root systems indicates that cated to the anoxic root system may be separate from the respiratory pool. Although there have been observations of elevated sugar levels in flooded roots (Spek, 1981; Benjamin and Greenway, 1979; Limpinuntana and Greenway, 1979), starch grains are consumed from amyloplasts during flooding (Papenhuijzen and R005, 1979). Additionally soluble carbohydrates are observed to increase only in flooded roots attached to the shoot, anaerobic excised roots have reduced levels of sugars (Saglio and Pradet, 1980). These observations could be interpreted to support either passive unloading or leakage of sucrose from the phloem under anaerobic conditions. Phloem unloading initially occurs in the apOplast and from there sucrose is either taken up directly (Chin 3E 31., 1981) by the cells in the stele or hydrolyzed into fructose and glucose by an acid invertase (Eschrich, 1980). Entry into living cells either as sucrose or hexoses requires active uptake (Cronshaw, 1981) which is greatly reduced under energy poor anoxic conditions. Movement of sugars out of the stele and 110 into the cortex predominately occurs in the symplasm, an energy requiring process (Dick and apRees, 1975). Soluble carbohydrates in the apoplast of the stele may also leak out at the point of secondary root emergence, a passive process (Peterson 33 31., 1981). The appearance of label in the anoxic root system but not in the respired C02 could indicate that the majority of l4C-label was initially confined to the apoplast of the stele in the anoxic root and unavailable or very slowly available for metabolism. This could provide an alternative explanation for the high sugar content in anoxic roots and disagrees with the conclusions by Limpinuntana and Greenway (1979) that there is no lack of available substrate in anaerobic root systems. The similarity of the translocation of 14C-label to the nonaerated control roots and the nonaerated roots in the localized anoxia treatment indicated that the activity of the nonaerated root system was primarily controlled by the immediate anoxic environment surrounding the root. The alleviation of shoot injury in the localized anoxia treatment did not appear to be a result of an alteration in the response of root cells to anoxia. Length, oxygen uptake and ion uptake were previously found to increase in the aerated roots of localized anoxia treated dry beans (Schumacher and Smucker, 1982b; 1982c). The increase in C02 production and 14C translocation to aerated roots of the localized anoxia treatment support these findings. The 31908 line appeared to be partitioning 111 additional l4C-labeled compounds into actively growing roots while Seafarer appears to have partitioned these compounds into portions of the root system which were not white, i.e. not actively growing. The response by Seafarer could indicate storage of the assimilate within the parent roots or it may indicate an allocation of carbohydrate to newly developing lateral roots (cv. discussion concerning hypocotyls). Lateral roots in Phaseolus vulgaris L. require at least three days between initiation and emergence from the parent root (Thompson and Macleod, 1981). Increased translocation of assimilate within the root when growth in a portion of the root system stops has a number of possible explanations. One hypothesis is that there is strong competition within the root system for limited amounts of carbohydrate and as a result the total root system is growing at a less than maximal rate (Brouwer, 1981). A second hypothesis proposes hormonal control of assimilate partitioning either by affecting phloem unloading at the sieve tubes or by stimulating the growth of a particular tissue (Turvey and Patrick, 1979). Phloem loading and unloading under nonstress conditions is dependent on active membrane transport processes (Lfittge and Higinbotham, 1979). Local control of energy compounds (ATP, etc.) needed for membrane transport could be a possible mechanism for accomplishing assimilate partitioning. Respiration rates based on the dry weight of the roots tended to be higher in the aerated portion of the localized 112 anoxia treatment. This could reflect an increase in any one or all of three reSpiratory processes, root growth, uptake and transport of ions and maintenance processes (Veen, 1981). The higher respiration rates could reflect an increased supply of carbohydrate within the root. However, a recent study by Farrar (1981) with shaded plants and excised roots indicates that respiration rates are not a simple function of carbohydrate supply from the shoot and that other factors are involved. Farrar's observations brings in to question much of the correlative evidence used to support the strong competition hypothesis for sink activity within the root system. In summary, the translocation of l4C-sucrose was affected by aeration treatment. Completely anoxic root systems inhibited the movement of l4C-labeled compounds into the root system. The labeled compounds which entered the anoxic root system appeared to be separate or only slowly available to the respiratory pool. Anoxic root activity is primarily determined by the immediate environment around the roots. Localized anoxia stimulates the movement of assimilates to the aerated portion of the root system. After 72 hours of treatment the 31908 line appears to allocate relatively more assimilate to actively growing regions as compared to Seafarer. This study in combination with other studies indicates that the various dry bean genotypes studied differ in their response to localized anoxia primarily through different assimilate allocation patterns. REFERENCES Benjamin, L.R. and H. Greenway. 1979. Effects of a range of 02 concentrations on porosity of barley roots and on their sugar and protein concentrations. Ann. Bot. 43:383-393. Brouwer, R. 1963. Some physiological aspects of the influence of growth factors in the root medium on growth and dry matter production. Jaarb. I.B.S. 212:11-30. Brouwer, R. 1981. Co-ordination of growth phenomena within a root system of intact maize plants. Plant Soil Cannell, R.Q. and M.B. Jackson. 1981. Alleviating aeration stresses. 13: Modifying the root environment to reduce crop stress, ed. G.F. Arkin and H.M. Taylor. pp. 141- 192. ASAE, St. Joseph, Michigan. Chin, C., M. Lee and M. Weinstein. 1981. Some characteristics of the sucrose uptake system of excised tomato roots. Can. J. Bot. 59:1159-1164. Cronshaw, J. 1981. Phloem structure and function. Ann. Rev. Plant Physiol. 32:465-485. Dick, P.A. and T. apRees. 1975. The pathway of sugar transport in roots of Pisum sativum. J. Exp. Bot. 26:305-314. Drew, M.C. and L.R. Saker. 1978. Nutrient supply and the growth of the seminal root system in barley. III. Compensatory increases in growth of lateral roots, and in rates of phosphate uptake, in response to a localized supply of phosphate. J. Exp. Bot. 29:435-451. Drew, M.C., L.R. Saker and T.W. Ashley. 1973. Nutrient supply and the growth of the seminal root system in barley. I. The effect of nitrate concentration on the growth of axes and laterals. J. Exp. Bot. 24: 1189-1202. 113 114 Eschrich, W. 1980. Free space invertase, its possible role in phloem unloading. Ber. Dtsch. Bot. Ges. 93:363-378. Farrar, J.F. 1981. ReSpiration rate of barley roots: Its relation to growth, substrate supply and the illumination of the shoot. Ann. Bot. 48:53-65. Fiscus, E.L. 1981. Analysis of the components of area growth of bean root systems. Crop Sci. 21:909-913. Forsyth, C. and J. Vanstaden. 1981. The effects of root decapitation on lateral root formation and cytokinin production in Pisum-sativum. Physiol. Plant. 51:375- 380. Friedman, R., A. Altman and E. Zamski. 1980. Adventitious root formation in bean hypocotyl cuttings in relation to IAA translocation and hypocotyl anatomy. J. Exp. Bot. 30:769-777. Fulton, J.M., A.E. Erickson and N.E. Tolbert. 1964. Distribution of 14C among metabolites of flooded and aerobically grown tomato plants. Agron. J. 56:527-529. Grineva, G.M. and G.A. Nechiporenko. 1977. Distribution and transformation of sucrose-14C in maize plants in conditions of innundation. Sov. Pl. Physiol. 24:32-37. Hackett, C. 1972. A method of applying nutrients locally to roots under controlled conditions, and some morphological effects of locally applied nitrate on the branching of wheat roots. Aust. J. Biol. Sci. 25:1169-1180. Limpinuntana, V. and H. Greenway. 1979. Sugar accumulation in barley and rice grown in solutions with low concentrations of oxygen. Ann. Bot. 43:373-383. Lfittge, V. and N. Higinbotham. 1979. Transport in plants. Springer-Verlag, New York. Meek, B.D. and L.H. Stolzy. 1978. Short-term flooding. pp. 351-373. 13: Plant life in anaerobic environments, eds. Donal D. Hook and R.M.M. Crawford. Ann Arbor Science Publ. Inc. Ann Arbor, Michigan. Nagao, T. and Y. Wada. 1970. Studies on the growth of tobacco roots. IV. Effect of oxygen concentration of media on the translocation of 4C assimilated into root. Proc. Crop Sci. Soc. Japan 39:21-24. Nechiporenko, G.A. and G.M. Grineva. 1976. Effect of 115 duration of innundation on distribution of 14C-sucrose in maize. Sov. Pl. Physiol. 23:826-830. Nuritdinov, N. and B.B. Vartapetyan. 1980. Translocation of l4C-sucrose in cotton under conditions of root anaerobiosis. Sov. Pl. Physiol. 27:616-621. Papenhuijzen, C. and M.B. Roos. 1979. Some changes in the subcellular structure of root cells of Phaseolus vulgaris as a result of cessation of aeration in the root medium. Acta Botanica Neerlandica 28:491-497. Peterson, C.A., M.E. Emanuel and G.B. Humphreys. 1981. Pathway of movement of apoplastic fluorescent dye tracers through the endodermis at the site of secondary root formation in corn (£33 mays) and broad bean (Vicia faba). Can. J. Bot. 59:618-637. Phillips, I.D.J. 1964. Root-shoot hormone relations. II. Changes in endogenous auxin concentration produced by flooding of the root system in Helianthus annuus. Ann. Bot. 28:37—45. Saglio, P.H. and A. Pradet. 1980. Soluble sugars, respira- tion and energy charge during aging of excised maize root tips. Plant Physiol. 66:516-519. Schumacher, T.E. and A.J.M. Smucker. 1982. Measurement of dissolved C02 in the rhizosphere of plant root systems by a modified IRGA system. Plant Physiol. (Accepted). Schumacher, T.E. and A.J.M. Smucker. 1982. Localized anoxia effects on root growth of Phaseolus vulgaris L. In prep. Schumacher, T.E. and A.J.M. Smucker. 1982. Localized anoxia effects on ion uptake, respiration and transpiration of drybean roots. In prep. Schumacher, T.E., A.J.M. Smucker, A. Eshel and B. Currie. 1982. A staining technique for measurement of short term root growth. In prep. Spek, L.Y. 1981. Influence of nitrate and aeration on growth and chemical composition of Zea mays L. Plant Soil 63:115-119. Starck, Z., R. Karwowska and E. Kraszewska. 1975. The effect of several stress conditions and growth regulators on photosynthesis and translocation of assimilates in bean plant. Acta Soc. Bot. Pol. 44:567-588. Thompson, A. and R.D. Macleod. 1981. Lateral root anlage 116 development in excised roots of Vicia faba L., Pisum sativum L., Zea mays L. and Phaseolus vulgaris L. Ann. Bot. 47:583-595. Trought, M.C.T. and M.C. Drew. 1981. Alleviation of injury to young wheat plants in anaerobic solution cultures in relation to the supply of nitrate and other inorganic nutrients. J. Exp. Bot. 32:509-523. Turvey, P.M. and J.W. Patrick. 1979. Kinetin-promoted transport of assimilates in stems of Phaseolus vulgaris L. Localized versus remote site(s) of action. Planta 147:151-155. Veen, B.W. 1981. Relation between root respiration and root activity. Plant Soil 63:73-77. Wample, R.L. and D.M. Reid. 1978. Control of adventitious root production and hypocotyl hypertrophy of sunflower (Helianthus annuus) in response to flooding. Physiol. Plant. 44:351-359. Wiedenroth, E. and J. Poskuta. 1981. The influence of oxygen deficiency in roots on C02 exchange rates of shoots and distribution of C-l4-photossimilates of wheat seedlings. Z. Pflan. Physiol. 103:459-469. Yu P.T., L.H. Stolzy and J. Letey. 1969. Survival of plants under prolonged flooded conditions. Agron. J. 61:844- 847. CHAPTER 6 ION UPTAKE, RESPIRATION AND TRANSPIRATION OF DRY BEAN ROOTS EXPOSED TO LOCALIZED ANOXIA ABSTRACT Ion uptake by dry bean root systems was examined by measuring the disappearance of ions from the nutrient solution during a three day treatment period. Aeration treatments consisted of split root systems with both halves aerated (aerated control); both halves nonaerated (nonaerated control); and one half aerated and the remaining half non- aerated (localized anoxia). Ion absorption by each plant was similar in the aerated control and localized anoxia treatments. Anoxic root systems absorbed 2, 40, and 60 percent of the aerated control for K+, Ca++, and NO3‘, respectively. Ion absorption appeared to increase directly with root growth in the aerated portion of the localized anoxia treatment. Localized anoxia increased potassium ion uptake per unit root weight, xylem exudation and root respiration rates for the Pinto lll cultivar. Transpiration rates of Seafarer were stimulated by localized anoxia to 135% of the aerated control and may have contributed to the 117 118 additional ion uptake by mass flow in the nonaerated portion of the localized anoxia treatment. A doubling of the nutrient solution concentration appeared to greatly reduce compensatory ion uptake in line 31908. Nutrient solution of the nonaerated control became more alkaline during the stress than did the nonaerated portion of the localized anoxia treatment, indicating a possible direct or indirect effect of the aerated portion of the localized anoxia treatment on the corresponding nonaerated half. An eXamination of the response of root systems to localized stresses could be valuable in the determination of root system efficiency. Compensation in ion uptake by dry bean roots subjected to localized anoxia appeared to be the result of increased root growth, greater transpiration rates and an increase in the ion uptake rate per unit root weight of one genotype. The differences observed between genotypes indicate that there is a considerable amount of variation in plant response to localized stress. Efficient uptake of water and nutrients by the root system may help overcome localized stress within the soil environment. INTRODUCTION Anaerobic zones may be distributed throughout the root- soil profile and can change with time (Stolzy and Flfihler, 1978). Anoxia reduces the uptake of ions by the root system presumably through an inactivation of the active transport system and a depolarization of cell membranes (Lfittge and Higinbotham, 1979). Localization of anaerobic zones to parts of the root system could reduce the root surface area available for ion uptake. Under conditions of partial exposure of the root system to anaerobiosis (localized anoxia) it is conceivable that the aerated portion of the root system could compensate for the reduced ion uptake in the anaerobic zone. Compensatory increases in ion uptake and stimulation of root growth have been shown to occur when localized portions of root systems from nutrient depleted plants are exposed to high nutrient concentrations (Drew and Saker, 1975; 1978; Drew, Saker and Ashley, 1973; Hackett, 1972). Trought and Drew (1981) found a similar response in wheat seedlings when one seminal root was exposed to an aerated complete nutrient solution while the remainder of the roots were anoxic. This study examines ion uptake responses of a number of 119 120 genotypes of Phaseolus vulgaris L. to conditions of localized anoxia. Compensatory uptake occurred in all genotypes examined, however, there appeared to be different mechanisms for achieving this compensation. MATERIALS AND METHODS Two experiments were conducted using five and two dry bean genotypes, respectively. Cultivars Seafarer, Swan Valley, Domino, Pinto 111 and MSU experimental line 31908 were represented in the first experiment. Results for Swan Valley and Domino were qualitatively similar to Seafarer and are not reported here. The second experiment examined in more detail the effects of localized anoxia on Seafarer and line 31908. Both experiments utilized similar initial procedures and aeration treatments (Schumacher and Smucker, 1982b; 1982c). Seeds were surface sterilized and germinated in the dark on trays containing moist paper toweling and cheese- cloth. An incubator maintained temperatures at 23°C for germination and initial seedling growth. Radical root tips were removed 24 to 48 hours after radicals emerged. This produced a split root system composed of basal roots forming laterally to the main axis of the plant. Seedlings were given a 24 h exposure to light when the hypocotyls were >2 cm in length. Seedlings were transferred to split root containers which contained aerated Hoagland's nutrient solution. The composition of the nutrient solution in the 121 122 first experiment consisted of quarter strength Hoagland's nutrient solution with 1.50 mM K+, 0.25 mM H2PO4‘, 3.75 mM NO3', 0.5 mM Mg++, 0.5 mM 504:, 1.25 mM Ca++ and 2.25 x 10"2 mM Fe as Fe EDDHA solution. Trace elements were provided according to Hoagland and Arnon (1950) with a 20% reduction of manganese to reduce the possibility of Mn toxicity. Plants in the second experiment were initially grown in quarter strength Hoagland's nutrient solution for four days and then replaced with half strength Hoagland's solution. Both nutrient solutions were initially adjusted to a pH of 6.0. Chambers in the first experiment were constructed from plastic containers lined with saran coated polyethylene bags. The second experiment utilized acrylic chambers which could be sealed to collect rhizosphere gases and inhibit the loss of water through evaporation. The compartments of both types of chambers contained approximately 1 liter of the appropriate nutrient solution. Compressed air flowed (>150 cm3 min-1) through fritted glass tubes to maintain oxygen partial pressures in the nutrient solution above 0.19 atm oxygen (YSI Model 53 Biological Oxygen Monitor). Chambers were randomly assigned positions on the greenhouse bench. Supplemental cool white fluorescent light was used to provide a photoperiod of 16 h light and 8 h dark. Photosynthetically active radiation at midday ranged from 200 (cloudy) to 1,900 (clear) uEinsteins m'2 sec"1 at the primary leaf surface. Three aeration treatments were randomly allocated to 123 the root systems of each genotype after a period of eight days of growth in a well aerated root environment. Aeration treatments consisted of split root systems with an aerated control (AC) where both halves of the root system were aerated, a nonaerated control (NC) where both halves of the root system were nonaerated and a localized anoxia treatment (LA) with half the root system aerated (ALA) and half the root system nonaerated (NLA). Oxygen partial pressures in the nonaerated treatments were maintained below 0.005 atm oxygen by continually equilibrating the nutrient solution with nitrogen gas. The neutral red staining technique (Schumacher 33 31., 1982) was used to differentiate between root growth occurring before and during the treatment period. The first experiment included measurements of oxygen uptake, ion uptake and xylem exudation rates. Root growth measurements were also measured and are reported elsewhere (Schumacher and Smucker 1982b). Oxygen uptake was measured for each treatment combination before the imposition of the aeration treatments and after a 72 hour treatment period. Measurements were made by flowing nutrient solution from solution surrounding the root system past a clark electrode (YSI Model 53 Biological Oxygen Monitor) maintained at a constant temperature of 20°C. Oxygen uptake rates were calculated from the decrease in oxygen content of the solution with time and the volume of the nutrient solution. Xylem exudation rates were determined by measuring the volume of exudate collected in latex tubing placed over 124 the ends of the cut stem during a specified time period. Nitrate ion content was measured in solution samples, taken immediately before and after the treatment period, by an Orion NO3' selective ion electrode standardized against known concentrations of NO3’ in a matrix of Hoagland's solution. Measurements were made of the pH of samples using a Corning Model 130 pH meter. The disappearance of H+ ions from the nutrient solution was estimated by reference to a titration curve for 0.25 mM H2P04'. Concentrations of Ca++, Mg++, K+ and P in the solution were determined by measuring ions in the solution before and after the treatment periods with an SMI liquid plasma emission multi-ion spectrophotometer. The second experiment measured ion uptake measurements with time from samples taken every 12 hours beginning with replacement of the nutrient solution at 2-4 hrs after the initiation of the aeration treatments. A control with no plants was also sampled during each time period for back— ground losses. Measurements were made of the volume of containers from gradations on the sides of the acrylic chambers at the same time as sampling for ion uptake. Background losses from the chambers with no plants were subtracted from these values to provide estimates of water loss. Distilled water was added when appropriate to maintain solution volume. Leaf area was measured at the conclusion of the experiment using a LiCor 3000 leaf area meter. Diffusive resistance measurements were made 24 and 48 hours after the initiation of the aeration treatments during the 125 early afternoon using a LiCor LI 60 diffusive resistance meter and leaf clamp. Photosynthetically active radiation at the time of measurement varied depending on cloud cover but was nearly constant within replications. The first experiment was analyzed as a randomized complete block design with split, the main plots were the cultivars and the subplots the aeration treatments. The second experiment was a randomized complete block design. Both experiments used time as the blocking variable. RESULTS AND DISCUSSION There was a decrease in total ion uptake in plants with completely anoxic root systems during the treatment period (Table 1). In contrast when one half the root system was exposed to anoxia, total ion uptake was similar to the aerated control. Trought and Drew (1981) found a similar response in wheat seedlings when a single seminal root was grown in aerated nutrient solution. The increase in root growth as a result of localized anoxia (Schumacher and Smucker, 1982b) accounted for most of the compensation in ion uptake as there was no change in ion uptake rates per unit root weight for four of the five genotypes. Pinto 111 had an increase in the ion uptake per unit root weight (Table 2). These results are similar to reports of the response of barley root systems to localized concentrations of nutrients (Claassen and Barber, 1977; Drew and Saker, 1975; 1978; Hackett, 1972; Drew 33 31., 1973). Drew and Saker (1975) found that a localized treatment of nitrate within barley root systems growing in low nitrate nutrient solution caused an increase in ion uptake both from a localized increase in root growth and an increase in ion uptake rate per unit 126 127 mm.o mm.m mm.o ao.o mo.o aa.a mo.omq am.o oa.oa mo.o me.o mm.~ hm.o do ao.o ee.m mm.o oo.o oa.a oa.o oz om.a Ne.m mm.o oh.o am.m ao.o o< HHH oosam mm.o om.sa mo.o ao.o om.m mm.o ama auoa mosaoaa oam macaumoaaamu xam wO mammE may mum mmsam> .Adao maxoam omnaaMOOa cam .AUZV aouucoo Omumummaoc .Aomv aonuaoo Omumumm ”mucmEummHu aOapmumm oaaBOaaOm map aua3 omummuu mmmwoocmo coma xuo mmuau wa mamums COH .a maame 128 Hm.o om.m Hm.o ao.o oe.o oa.o mo.omq m~.o mo.a mm.o ao.o aa.a sm.~ qu mm.o am.m as.o ma.o sm.m mo.m mam ao.o e~.m om.a oo.o so.a oo.o oz ma.o ao.o mm.o mm.o mm.a an.m om aaa oosam ma.o mm.m em.o ao.o mm.o mo.a «oz oa.o ao.o sm.o ma.o oo.a mo.m mam mm.o ma.m ao.a ao.o aa.a oo.o oz mm.o mo.m mm.o sm.o mo.a mm.m om momam aa.o mm.v am.o ao.o am.a mm.a «oz mm.o ~m.m mm.o om.o Hm.a Ha.m mam ao.o mm.m om.a ao.o mo.a em.o oz am.o ma.a mm.o mm.o ea.a mo.m om nosoooom aIzmo aIa aOE: III neommm Imoz +: ++oz ++oo +z ososuooue moaoosoo COHHMHOAN .auoamuum umuumsq um mmS aOausaOm acmauusc m.ocmammom .mcoaamoaaomu xam mo mcmmE map ucmmmummu mama .Aama Emammm aoou How cm>ao mum mmsam> .mmmmuoamm cmma >HO mmuau HOw uoou mo Damamz ammuw pas: umm mamums aOa .N maame 129 weight of root. Trought and Drew (1981) concluded that shoot injury (i.e. reduced growth, chlorosis, etc.) in partially flooded wheat plants was prevented by a compensation in ion uptake by aerated portions of the root system supplied with a complete compliment of nutrients. They also observed a stimulation of lateral root growth in the aerated portion of the root system similar to that found in this study (Schumacher and Smucker, 1982b). Differences in ion uptake rates between cultivars have been demonstrated in several crop species and appear to be genetically controlled (Glass 33 31., 1981; Baligar and Barber, 1979; Lindgren 33 31., 1977). Maize lines have varying genetic potentials to alter K+ uptake rates in response to environmental stress (Frick and Bauman, 1979). Dry bean genotypes differed primarily in the mechanisms of achieving compensatory ion uptake. Pinto 111 was the only genotype to respond to localized anoxia with an increase in ion uptake rate per unit root weight (Table 2). Pinto 111 also had higher oxygen uptake rates per unit root weight (Table 3) as well as an increase in the xylem exudation rate (Table 4) as a result of the localized anoxia treatment. The increased oxygen uptake rates per unit root weight in Pinto 111 were probably a reflection of increased respiratory requirements for the increased uptake of ions. Root respiration is a reflection of energy needs for root growth, ion uptake and transport, and maintenance processes. Veen (1981) estimates that 130 Table 3. Oxygen uptake rates per root system half per unit fresh weight and the change in oxygen uptake occurring during the treatment period (A02 uptake) for three dry bean genotypes. Measurements are given for the following aeration treatments: aerated control (AC) and aerated half of the localized anoxia treatment (ALA). Values repre- sent the means Of six replications. Hoagland's nutrient solution was at quarter strength. Aeration A 02 02 Uptake:Root Genotype Treatment 02 Uptake Uptake Fresh Weight nmol h'l (% plant)’l nmol h"l gfw"l Seafarer AC 4.5 1.0 7.1 ALA 8.2* 5.6* 9.4 Pinto 111 AC 5.8 2.2 6.3 ALA 12.3** 8.5** 12 5** 31908 AC 7.4 3.1 5.8 ALA 12.9** 8.5** 7.6 *,**Indicates significance at the .05 and .01 levels of probability, reSpectively, for the orthogonal contrast of AC vs. ALA. 131 Table 4. Xylem exudation rates for plants of three dry bean genotypes treated with the following aeration treatments: aerated control (AC), nonaerated control (NC), and localized anoxia treatment (LA). Values represent the means of six replications. Hoagland's nutrient solution at quarter strength. Genotype Aeration Treatment Seafarer Pinto 111 31908 ul h'l AC 120a“r 250a 250b NC 2b 80b 60a LA 90ab 380c 270b +Values within the same column with the same letter are not significantly different at the .05 level of probability. Using the Least Significant Difference, LSD. 132 respiration for ion uptake and transport processes represents approximately 60 percent of total root respiration. Xylem exudation is proportional to the gradient in osmotic potential between the external solution and the xylem sap (Bowling, 1976). Stimulation of ion transport by abscisic acid resulted in increased xylem exudation rates in Phaseolus vulgaris (Karmoker and Steveninck, 1978). Butz and Long (1979) found that glutamine translocation and xylem exudation rates were closely correlated and proposed that transport of uncharged glutamine into the xylem could provide the driving force for the exudation process. The increase in ion uptake may stimulate the rate of xylem exudation (Table 4) by increasing the osmotic potential of the xylem sap through an increase in the ionic and/or reduced nitrogen concentration of the xylem exudate. A second experiment suggested differences in the response of 31908 and Seafarer to localized anoxia when nutrient solu- tion concentrations were doubled. An increase in K+ ion uptake per half root system occurred as a result of doubling the ion concentration of the nutrient solution for all treatments except the aerated portion of the localized anoxia treatment for line 31908 (Table 5). The increase in rate of potassium ion uptake was similar to that indicated by the Langmuir adsorption isotherm which appears to adequately describe the relationship between external ion concentration and ion uptake (Bowling, 1976; Michalik, 1982). Potassium ion uptake of excised barley roots did not become 133 Table 5. Comparison of K+ uptake with quarter and half strength Hoagland's nutrient solution for 31908 and Seafarer. Aeration treatments are aerated control (AC), nonaerated control (NC), aerated half of the localized anoxia treatment (ALA) and nonaerated half of the localized anoxia treatments (NLA). Values are the means of six and four replications for the experiments with quarter and half strength Hoagland's nutrient solution, respectively. H. Sol. % H. Sol. Aeration K+ Uptake Per Genotype Treatment K+ Uptake K+ Uptake Root Dry Weight pmol h"l (% plant)"1 pmol h'l gdw"l Seafarer AC 1.99 3.66 23.19 NC 0.10 0.43 4.48 ALA 3.14 4.63 24.44 NLA 0.74 1.79 23.14 31908 AC 3.63 7.14 27.41 NC 0.00 2.05 13.61 ALA 5.50 6.73 25.64 NLA 0.79 1.74 14.50 LSD.05 0.54 1.65 3.01 134 independent of external K+ concentrations until external K+ ion concentrations were >5 mM (Bowling, 1976). An examina- tion of ion uptake with time illustrates the difference in response between 31908 and Seafarer (Figure 1). The aerated portion of the localized anoxia treatment and the aerated control for 31908 were similar in K+ uptake throughout the duration of the experiment. Seafarer tended to increase K+ uptake in response to localized anoxia relative to the aerated control after approximately 36 hours of treatment. This lag time could be indicative of the time necessary for additional growth of the root system to have an effect on ion uptake. There appeared to be a shift in the proportion of cations removed from the nutrient solution under conditions of anoxia (Tables 1 and 2). This was due to an inhibition of K+ uptake rather than an increase in the uptake of selected divalent cations. Ca++ and Mg++ are primarily absorbed by the root passively while K+ uptake has been shown to have an active component (Bowling, 1976). The lack of oxygen in the root media limits the amount of energy available for active membrane transport and rapidly depolarizes the cell membrane (Lfittge and Higinbotham, 1979). Anoxic roots have a K+ influx which is similar to that predicted for cells with constant passive permeability (Cheeseman and Hanson, 1979). Table 2 indicates a greater rate of loss of H+ from the nutrient solution for the nonaerated control compared 135 . Swazo pamEummuu maxocm OmmaamOOa map mo mama omumummaoa mam Adamo ucmEammnu maxoam omwaamOOa map mo mama mmumumm .Auzo aouucoo omumummcoc .Aumo aouucoo omamumm mums muamEummuu aOaumumm .umummmmm mam mooam MOM ucmEummuu ceaumamm mo musoa om usausm aOauDaOm ucmauasa m.ocmaomom aumamnum mama Eoum COa Esammmuom mo mocmummmmmwao m>aumasesood may Utah .3 on 2. on v~ w. o W ......... ... ........+ IlII-I‘IIII .. ‘11“... O W . \. . ....-I\.\ I 1 On I. \. U I a 1 S t 0‘ \ 8— s ..n \ M . .......\.\.. . ...... u . .. \ . ...“\..\.\ 52 32a 0 25 M . ..... . . 5c can.» I . 03 N I. \. oz com; o m _. \. oz 32.” o . 3n 1 . \ 52 cuccucum a U . \. . . can on K. \ Sc mumEcum I 3 \ . oz muccucum d J .. no a 00? Q. ”_H 93 o: cuccucum d w a . 02. o . . . P . can ( .a musoam 136 to the other aeration treatments. The loss of H+ from solution could be due to either H+ influx from the solution or neutralization of the H+ from an efflux of base from the root system. Anion uptake particularly NO3‘ uptake appears to occur in aerated root systems with an efflux of OH’ or HCO3‘ ions. Keltjens (1981) provided evidence for nitrate reduction as the driving for OH" efflux. However, it is difficult to apply this observation to an anoxic root system since both active ion uptake and nitrate reduction require aerobic respiration for energy. A previous study examining an 18 h period of anoxia did not show an increase in alkalinity compared to the aerated control (Schumacher and Smucker, 1982a). This may indicate that the loss of H+ ions may be associated with longer exposure to anoxia and increased damage to the root system. A comparison with the nonaerated portion of the localized anoxia treatment indicates that the effect of H+ can not be explained simply in terms of the immediate anaerobic environment. The aerated portion of the localized anoxia treatment appeared to inhibit the loss of H+ from the nutrient solution in the nonaerated half (Table 2). This could be a direct influence by transfer of some component to the anoxic portion of the root system from the aerated portion Of the root system or more likely an indirect influence by the transfer or removal of a component by the shoot to or from the root system. Nitrogen deficiency as a result of an inhibition of nitrate uptake under flooded conditions appears to contribute 137 in a large part to flooding induced chlorosis of the shoot (Cannell and Jackson, 1981). In the present study shoot chlorosis or other overt symptoms of nitrogen deficiency were not observed in any of the treatments during the duration of the experiments. Pretreatment or treatment with high concentrations of NO3' (5.0 vs. 0.1 mM) prevented the appearance of shoot injury and promoted the growth of wheat seedling shoots with anoxic root systems (Trought and Drew, 1981). This could partly explain the relatively greater reduction in leaf area when plants were grown in the less concentrated nutrient solution (Table 6). Nitrate ion disappearance from the nutrient solution was substantially reduced by total anoxia but not completely inhibited (Table 1). There appeared to be an increase in nitrate uptake per unit weight of root in the nonaerated control of 31908 (Table 2). This was possibly a result of a greatly reduced root system and relatively high tranSpiration rates in the 31908 nonaerated control (Table 6). Tables 1 and 2 indicates that substantial amounts of ions were removed from the nutrient solution by anoxic root systems. These results agree with other studies which examined the absorption of ions by anoxic root systems of intact plants (Jackson, 1979; Woodford and Gregory, 1948; Loughman, 1981; Trought and Drew, 1981; Hammond 33 31., 1955). Studies of ion uptake on excised roots exposed to anoxic conditions indicate almost complete inhibition of ion uptake under anoxic condi- tions (Hopkins 33 31., 1950; Steward 33 31., 1936; Lee, 1978). 138 mo om a.m m.HH mo.omq omN mma m.m h.mm ¢Q mwa owa ©.m ®.¢N OZ omm oaN m.m h.mN Ufl momam oaa mm a.m H.0m 4Q om mm v.h m.ma OZ maH om m.m m.mm Dd Hmummmmm Illalaamam NEOIII aIEO 0mm anmm NIE me mmum mmua mocmumawmm mumm pamEummHB mmwuoamw wmma mmmq m>am5mmaa mmma coaumuammamne aOaumumm .aOm .m w .aOm .m x .mmnm mmma amaam cam oOaHmm pamEammHu >m© mmuau man ocauso mmOa HmumB co Ommma mum mmumu coaumuammamua .muamEamme map mo aOaumauaca umumm a mo .wmooae um cmamu mHmB muamE ImusmmmE mocmumammu m>am5mmam mmma .Amqo maxocm mmuaamOOa cam .aozv aouucoo mmumummcoa .Aumv aouuaoo omumumm Mo mmumamaoo muamEammHu COaumHm< .aoausaOm ucmauusc m.oamammom aumamuum Hmuumsv ca :3Oum muamam How am>ao Omam mum mmSam> mmum mmmq .aoausaOm aamauusa m.©:mammom auoamuum mama ca GBOHm muamam umumwmmw ocm momam How mmum mmma mam mmSam> mocmumammu m>amsmmam mmma .mmamu coaumuammamue .o maamB 139 Trought and Drew (1981) attributed the differences in the effect of anoxia on ion uptake of excised roots and intact plants to passive accumulation of ions by mass flow through damaged roots in the intact plant. An examination of the average transpiration rates (Table 6) and the accumulative water uptake during the course of the second experiment (Figure 2) support this possibility. Calculation of the amount of ions which could be absorbed through mass flow based on the amount of water absorbed by the nonaerated treatments and the concentration of the nutrients in solution indicated that mass flow could account for the amount of ions removed from the nutrient solution during the treatment period. Figure 1 indicates that significant amounts of K+ were not absorbed until after approximately 36 h Of anoxia. This may indicate the time at which root injury became so severe as to allow absorbtion of ions primarily by mass flow. Transpiration rates were reduced by the nonaerated control in Seafarer but not in 31908 (Table 6). The low amounts of water lost initially by the nonaerated control of Seafarer (Figure 2) could be a result of increased root and/or stomatal resistance to water movement. Later responses in water loss observed in Figure 2 reflect a reduction in absorbing surfaces (smaller root system), reductions in transpiring surfaces (smaller leaf area) and reductions in stomatal resistance for the nonaerated control (Table 6). Transpiration rates were greater in the localized Figure 2. HRTER UPTRKE (ml) 140 7° 1 1 TI fl 1 an L 4 3190: so 0 RCRRTCO CONTROL 9 NONaCRaTCO CONTROL “50.65 1 o RCRRTCO L.R. . «I o NONRCRRTCO L.R. « an . an « IO . O 7. I fi 1 1 so a . ... sgarnRCR s so _ A RCRRTCO CONTROL L's!)J35 1 u A NONRCRRTEO CONTROL U I RCRRTCO L.R. E «I » O NONRCRRTCO L.R. I .- (L . D 30 - - tr E 20 A 1 E ,/ _ 1° ’ // - '1 j.- ‘ + J n a 0 I2 24 as «a CO 72 TIME (h) Water losses occurring during the treatment period for Seafarer and line 31908. Plants were grown in half strength Hoagland's nutrient solution. Aera- tion treatments were aerated control, nonaerated control, aerated half of the localized anoxia treatment (aerated LA) and nonaerated half of the localized anoxia treatment (nonaerated LA). 141 anoxia treatment for the Seafarer cultivar compared to the aerated control (Table 6 and Figure 2). These high transpiration rates were reflected in the relatively high absorption of K+ in the nonaerated portion of the localized anoxia treatment for Seafarer (Figure 1). In summary, localized anoxia treated plants had similar ion uptake rates compared to the aerated control plants. Compensatory ion uptake in the localized anoxia treatment was a function of increased root growth in the aerated portion of the root system, increased ion uptake rate per unit root weight in the Pinto lll cultivar and to a small, but significant contribution from the non- aerated portion of the root system. The transpiration rates probably contributed to ion removal from the solution by the anoxic root systems through mass flow. As a result, the nonaerated portion of the localized anoxia treatment tended to remove more ions than the nonaerated control because of the reduced resistance in the leaves of the localized anoxia treatment compared to the nonaerated control. The apparent differences observed between the limited number of genotypes examined in this study indicate that there is a considerable amount of variation in responses to localized stress in the rhizosphere. The extent and types of responses by the plant tO a particular soil environment could have bearing on the subsequent efficiency of the root system in terms of water utilization, ion 142 uptake, etc. In addition to the numerous factors cited by Nielsen (1979) which determine the efficiency of ion uptake one might add the compensatory response of the root system to localized stress. REFERENCES Baligar, V.C. and S.A. Barber. 1979. Genotypic differences of corn for ion uptake. Agron. J. 71:870-873. Bowling, D.J.F. 1976. Uptake of ions by plant roots. pp. 207. Chapman and Hall, London. Butz, R.G. and R.C. Long. 1979. L-Malate as an essential component of the xylem fluid of corn seedling roots. Plant Physiol. 64:684-690. Cannell, R.Q. and M.B. Jackson. 1981. Alleviating aeration stresses. pp. 141-192. In: Modifying the root environment to reduce crop—stress, ed. G.F. Arkin and H.M. Taylor. ASAE, St. Joseph, Michigan. Cheeseman, J.M. and J.B. Hanson. 1979. Energy-linked potassium influx as related to cell potential in corn roots. Plant Physiol. 64:842-846. Claassen, N. and S.A. Barber. 1977. Potassium influx characteristics Of corn roots and interaction with N, P, Ca, and Mg influx. Agron. J. 69:860-864. Drew, M.C. and L.R. Saker. 1975. Nutrient supply and the growth of the seminal root system in barley. II. Localized, compensatory increases in lateral root growth and rates of nitrate uptake when nitrate supply is restricted to only part of the root system. J. Exp. Bot. 26:79-90. Drew, M.C. and L.R. Saker. 1978. Nutrient supply and the growth of the seminal root system in barley. III. Compensatory increases in growth of lateral roots, and in rates of phosphate uptake, in response to a localized supply of phosphate. J. Exp. Bot. 29:435- 451. Drew, M.C., L.R. Saker and T.W. Ashley. 1973. Nutrient supply and the growth of the seminal root system in barley. I. The effect of nitrate concentration on the growth of axes and laterals. J. Exp. Bot. 24:1189-1202. 143 144 Frick, H. and L.F. Bauman. 1979. Heterosis in maize as measured by K uptake properties of seedling roots - pedigree analyses of inbreds with high or low augmentation potential. CrOp Sci. 19:707-711. Glass, A.D.M., M. Yaeesh Siddiqi and K.I. Giles. 1981. Correlations between potassium uptake and hydrOgen efflux in barley varieties. A potential screening method for the isolation of nutrient efficient lines. Plant Physiol. 68:457-459. Hackett, C. 1972. A method of applying nutrients locally to roots under controlled conditions and some morphological effects of locally applied nitrate on the branching of wheat roots. Aust. J. Biol. Sci. 25:1169-1180. Hammond, L.C., W.H. Allaway and W.E. Loomis. 1955. Effects of oxygen and carbon dioxide levels upon absorption of potassium by plants. Plant Physiol. 30:155-161. Hoagland, D.R. and D.I. Arnon. 1950. The water-culture method for growing plants without soil. Calif. Agr. Expt. Sta. Circular, 347 (Revised). Hopkins, H.T., A.W. Specht and S.B. Hendricks. 1950. Growth and nutrient accumulation as controlled by oxygen supply to plant roots. Plant Physiol. 25:193-209. Jackson, M.B. 1979. Rapid injury to peas by soil water- logging. J. Sci. Food Agric. 30:143-152. Karmoker, J.L. and R.F.M. Steveninck. 1978. Stimulation of volume flow and ion flux by abscisic acid in excised root systems of Phaseolus vulgaris L. cv. Redland Pioneer. Planta 141:37-43. Keltjens, W.G. 1981. Absorption and transport of nutrient cations and anions in maize roots. Plant Soil 63:39-47. Lee, R.E. 1978. Inorganic nitrogen metabolism in barley roots under poorly aerated conditions. J. Exp. Bot. 29:693-708. Lindgren, D.T., W.H. Gabelman and G.C. Gerloff. 1977. Variability of P uptake and translocation in Phaseolus vulgaris L. under P stress. J. Am. Soc. Hort. Sci. 102:674-677. Loughman, B.C. 1981. Metabolic aspects of the transport of ions by cells and tissue of roots. Plant Soil 63:47-57. Lfittge, U. and N. Higinbotham. 1979. Transport in plants. pp. 204-234. Springer-Verlag, Berlin. 145 Michalik, I. 1982. The influence of phosphate concentration on the kinetics of uptake by maize roots. Biol. Plant 24:161-170. Nielsen, E.N. 1979. Plant factors determining the efficiency of nutrient uptake from soils. Acta Agr. Scand. 29:81- 85. Schumacher, T.E. and A.J.M. Smucker. 1982. Measurement of dissolved C02 in the rhizosphere of plant root systems by a modified IRGA system. Plant Physiol. (Accepted). Schumacher, T.E. and A.J.M. Smucker. 1982. Localized anoxia effects on root growth of Phaseolus vulgaris L. (In prep.) Schumacher, T.E. and A.J.M. Smucker. 1982. Localized anoxia effects on reSpiration and l4C-sucrose translocation of two drybean cultivars. (In prep.) Schumacher, T.E., A.J.M. Smucker, A. Eshel and B. Currie. 1982. A staining technique for measurement of short term root growth. (In prep.) Steward, F.C., W.H. Berry and T.C. Broyer. 1936. The absorbtion and accumulation of solutes by living cells. VIII. The effect of oxygen upon respiration and salt accumulation. Ann. Bot. 50:345-366. Stolzy, L.H. and H. Flfihler. 1978. Measurement and prediction of anaerobiosis in soils. PP. 363-426. In: Nitrogen in the environment. Nitrogen behavior IE field soil Vol. 1, eds. D.R. Nielsen and J.G. MacDonald. Academic Press, New York. Trought, M.C.T. and M.C. Drew. 1981. Alleviation of injury to young wheat plants in anaerobic solution cultures in relation to the supply of nitrate and other inorganic nutrients. J. Exp. Bot. 32:509-523. Veen, B.W. 1981. Relation between root respiration and root activity. Plant Soil 63:73-77. Woodford, E.K. and F.G. Gregory. 1948. Preliminary results obtained with an apparatus for the study of salt uptake and root respiration of whole plants. Ann. Bot. 12:335-370. CHAPTER 7 SUMMARY AND CONCLUSIONS The heterOgeneity of field soils is generally absent from most artificially controlled studies. The absence of heterogeneity in experimental systems can result in root activity quite different than that observed in the field. This greenhouse study was conducted to determine the possible effects of spatial heterogeneity in oxygen concen- trations on dry bean root growth and physiology. Split root methodology was used to test the general hypothesis that the growth and functioning of the aerated portion of the root system is modified when the remaining portion of the root system is subjected to anoxia. Two methods are reported for measuring root responses to environmental variations within the rhizosphere. Neutral red stain was used to determine the growth of roots during discrete periods of time. A modification of an infrared gas analyzer was developed to measure dissolved carbon dioxide and the pH of ul amounts of nutrient solution. Localized anoxia had a significant effect on the aerobic portion of dry bean root systems. In general, localized anoxia resulted in an increase in root growth 146 147 rates, the number of growing root tips, the uptake of ions, and the prOportion of carbon translocated to the aerated half of the root system. Shoot growth rates of localized anoxia treated plants were similar to the aerated control. Anoxic conditions throughout the root system stOpped root growth. Ion absorption, transpiration, root respira- tion and carbon translocation continued during anoxia but at greatly reduced rates. The reSponses of anoxic roots were similar whether the entire or only part of the root system was exposed to anoxia. There were two exceptions to this observation. There was a greater movement of water through anoxic roots of the localized anoxia treatment compared to the nonaerated control. This was primarily due to larger transpiration rates in the localized anoxia treated plants. Localized anoxia treatments reduced the loss of H+ from the anaerobic environment relative to the nonaerated control. Dry bean genotypes tended to respond differently to localized anoxia. The distribution of root growth among various root components after treatment with localized anoxia depended on the genotype examined. Line 31908 tended to increase the growth of roots which were present before the treatment period in response to localized anoxia. While Swan Valley appeared to increase the growth rate of roots which emerged during the treatment period. The other three genotypes responded with intermediate patterns. Genotypic differences were also observed between 148 Seafarer and 31908 in a more detailed study. Although both genotypes tended to increase the percentage of carbon translocated to the aerated portion of the localized anoxia treated root system, 31908 appeared to partition a higher percentage of 14C to the growing root tips than Seafarer. Indicating a potential difference in the future growth of the respective root systems. Line 31908 appeared to respond differently than Seafarer when the nutrient solution concentration was doubled. Seafarer tended to have higher ion uptake rates in the aerated portion of the localized anoxia treatment at both nutrient solution concentrations. Line 31908 strongly responded to localized anoxia by increasing ion uptake in quarter strength Hoagland's solution but this did not occur when the ion concentrations were doubled. Localized anoxia stimulated transpiration rates in Seafarer but not in line 31908. Pinto 111 responded differently to localized anoxia in several respects compared to the other genotypes. The number of growing root tips and the length of parent root initiating new roots was not increased by localized anoxia to the same extent as that found for other genotypes. Pinto 111 appeared to increase the ion uptake rates per unit root weight, oxygen uptake rates per unit root weight and xylem exudation rates to a much greater extent than in other genotypes. The responses to localized anoxia observed in this 149 study indicate that spatial heterogeneity of oxygen concen- trations in the root environment can have a broad range of effects on root system growth and physiology. The response to localized anoxia appears to depend on both the environment surrounding roots and the genetic composition of the plant species grown. There are a number of implications and questions arising from these results which need further exploration. The short term effects on the pattern of root growth found in this study implicate long term changes in root system morphology. A study examining root growth during a recovery period following anoxia treatments could indicate whether the change in growth patterns would continue, as well as providing more information on lateral root primordia initiated during the treatment period. Plant responses to localized anoxia are likely to vary depending on the stage of growth. An examination of the effects of localized anoxia during reproductive stages of develOpment could help determine the significance of localized anoxia on plant activities (carbon partitioning, floral deve10pment, pod abortion, seed fill, etc.) which directly influence plant yield. Responses observed in a biphasic system such as the split root system used in this study may be considerably modified in a multi-phase system such as the soil. Aerobic and anaerobic zones in the soil are not divided evenly within the root system but are more likely to occur as a 150 mosaic of zones within the soil volume. Additionally, oxygen concentrations will be much more variable in the soil. Vary- ing portions of root systems growing under field conditions may be exposed to normoxia, hypoxia and anoxia. The effect of nutrient solution concentrations on the reSponse of 31908 to localized anoxia indicate that interactions with soil parameters such as soil temperature, fertility, rhiZOSphere population, etc. may also have modifying effects on the results observed in this study. An examination of root growth in well characterized heterogeneous media and the more complete characterization of prOperties affecting root growth under field conditions could be helpful in the prediction of the effects of soil modification on root growth. The modeling of root growth in heterogeneous media will require more information on the effects of various soil environments on root growth than is presently available. Tillage methodology and management could benefit by the determination of the minimum "ideal" rhizosphere volume needed under various climatic conditions and soils. Another question concerns root systems needed for field vs. greenhouse conditions. Plants exposed to nearly homogeneous "ideal" root environments may not need the same capabilities in their root systems as plants designed to grow in field environments. The minimum functioning root system size needed for plants well supplied with nutrients, water and oxygen remains an unanswered question. 151 The ability of root systems to respond differentially to their immediate environment implicates an underlying system in the control of root system growth, perhaps involving plant growth regulators, plant nutrient status and balance, etc. The effects of growth regulator trans- port, shoot nutrient status and the compartmentation of various metabolites all deserve further inquiry in terms of a role in the localized anoxia responses observed in this study. These are a few of the questions confronting the field of plant and soil sciences. I would encourage the reader to heed the advice of Cardinal Newman and not be overwhelmed by the amount of the unknown but to continue the sometimes seemingly slow advancement of knowledge. "A man would do nothing if he waited until he could do it so well that no one would find fault with what he has done." - Cardinal Newman