THE EFFECT OF ABSCISIC ACID ON THESE LOOP GAIN OF THE FEEDBACK LOOP INVOLVING 002 AND STOMAI'A Thesis for the Degree of. M. S. MICE-“SAM STATE UNIVERSITY DEAN RICHARD :DUBBE 1977 lat/‘6» d6 Nil-’12; g {I ABSTRACT THE EFFECT OF ABSCISIC ACID ON THE LOOP GAIN OF THE FEEDBACK LOOP INVOLVING C02 AND STOMATA BY Dean Richard Dubbe Land plants are faced with a dilemma: assimilation of C02 from the atmosphere requires an intensive gas exchange; prevention of excessive water loss requires a minimal gas exchange. The stomata must balance these opposing priorities. In order to do this, the stomata make use of at least two major feedback loops, one involving C02 and the other involving H20. This thesis examines the importance of the feedback loop involving CO2 and stomata and the effect of abscisic acid (the plant hormone produced under conditions of stress) on this loop. In order to quantitatively examine the C02 feedback loop, several concepts from classical feedback theory have been used. Appropriate theory and techniques have been developed for measuring and interpreting the meaning of the individual gains within this feedback loop as well as the steady state loop gain of this loop. Because of its significance to the control of stomatal aperture, the feedback loop involving C02 Dean Richard Dubbe and assimilation has also been examined. Five plants (monocotyledon and dicotyledon; C3 and C4) were examined to see if any generalizations could be made about the feedback loops in different plants. These plants included Xanthium strumarium, Gossypium hirsutum, Avena sativa, Zea mays, and Amaranthus powellii. The effect of intercellular CO2 concentration on stomatal permeability and net assimilation in the absence and presence of abscisic acid was measured with a gas analysis system. These results were then used to calculate the physiological, physical, and loop gain of the feedback loops including C02 and stomata and C02 and net assimilation. The effect of abscisic acid on net assimilation rate, transpiration rate, and the transpiration ratio was also examined. It was found that the loop gains of the feedback loops involving C02 can have a large influence on the response of stomata to various environmental perturbations and on the regulation of intercellular C02 concentration. Abscisic acid significantly increased the magnitude of these loop gains in all cases except Egg mayg which had a high loop gain even in the absence of abscisic acid. An increase in abscisic acid also results in an increase in the water use efficiency of the plants tested. Not enough Dean Richard Dubbe plants were examined to draw any broad ecological conclusions, but it is shown that different plants do use different strategies to solve their dilemma. THE EFFECT OF ABSCISIC ACID ON THE LOOP GAIN OF THE FEEDBACK LOOP INVOLVING coz AND STOMATA BY Dean Richard Dubbe A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1977 ACKNOWLEDGEMENTS I would like to thank my advisor, Klaus Raschke, for all of his helpful advice and constructive criticism. I would also like to thank the other members of my committee, Ken Poff and Norm Good, for their suggestions. A very special acknowledgment goes to Graham Farquhar for having‘the patience to introduce a lowly botanist to the concepts of feedback theory. I am deeply indebted to him for all of his help and prodding. Graham has many good ideas to offer the field of plant physiology, and I am grateful that he shared several of them with me. I would also like to thank Tom Sharkey and Dennis Phillips for their technical assistance and for teaching me the fun and frustrations of computers. Lastly, a special thanks goes to Kathi for all of her moral support and understanding. ii TABLE OF CONTENTS Introduction The Plants Dilemma Stomatal Sensitivity to CO Effect of Abscisic Acid on Stomatal Sensitivity to C02 The Feedback Loops Statement of Purpose Theory Materials and Methods Results Net Assimilation Rate versus Intercellular [C02] Permeability versus Intercellular [C02] Ambient [C02] versus Intercellular [C02] The Effect of Ambient [C02] on the Loop Gains The Effect of Abscisic Acid on the Loop Gains The Effect of Abscisic Acid on the Transpiration Ratios Discussion The Effect of Abscisic Acid on Net Assimilation The Effect of Abscisic Acid on Stomatal Permeability The Effect of Ambient [C02] on the Loop Gains The Effect of Abscisic Acid on the Loop Gains . The Effect of Abscisic Acid on the Transpiration Ratio General Discussion of Gains Conclusions List of References iii ' mun (AH 11 19 27 27 33 39 39 43 43 47 47 47 48 SO 53 53 56 59 LI ST OF TABLES TABLE 1. Values of intercellular [C02], rate of assimilation, and epidermal permeability for three different ambient CO concentrations in Xanthium strumarium and Zea mays . . . . . . . . . . . . . . . TABLE 2. Values of the loop gains and their components, the physiological and physical gains, at three different C02 concentrations in Xanthium strumarium and £33 mays . . . . . . . . . . . . . . . TABLE 3. The gains of the feedback loops involving permeability, assimilation, and CO in the absence and presence of 10'5M abSClSiC acid in the transpiration stream . . . . . TABLE 4. The effect of abscisic acid (10-5 transpiration, assimilation, and transpiration ratio. . . . . . . . . . . . M) on iv Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Feedback loops involved in the control of stomatal aperture . . . . . . . . . . . The gains of the feedback loops involving assimilation and CO and stomatal permeability and C02 0 o o o o o o o o o o Photosynthetic response curves for Xanthium strumarium with respect to intercellular [C02] . . . . . . . . . . . Photosynthetic response curves for Gossypium hirsutum with respect to intercellular [C02] . . . . . . . . . . . Photosynthetic response curve for Avena sativa with respect to intercellular [C02] Photosynthetic response curve for Zea my with respect to intercellular [CO2 ] . Photosynthetic response curve for Amaranthus powelli with respect to intercellUlar [C02] 0 O O O O O O O O O O Permeability versus intercellular [C02] for Xanthium strumarium . . . . . . . . . Permeability versus intercellular [C02 ] for Gossypium hirsutum . . . . . . . . . . Permeability versus intercellular [C02] for Avena sativa . . . . . . . . . . . . . Permeability versus intercellular [CO2 ] for Zea mays . . . . . . . . . . . . . . Permeability versus intercellular [CO2 ] for Amaranthus powelli . . . . . . . . . . Ambient [C02] versus intercellular [C02] . Ambient _£CO2 ] versus intercellular [C02] with 10 M abscisic acid present in the transpiration stream V 8 13 28 29 31 32 34 35 36 37 38 4O . 41 Introduction The Plant's Dilemma ’In order to survive, plants must be able to carry on the energy capturing process of photosynthesis. For photosynthesis to occur, carbon dioxide must reach the chloroplasts. However, the epidermis of land plants is covered by a cuticle which is relatively impermeable to gasses. This cuticle is necessary in order to prevent excessive water loss, since under virtually all naturally occurring conditions, the water vapor pressure inside the leaf is greater than that of the surrounding air. To facilitate the assimilation of CO2 from the atmosphere, stomata are located throughout much of the epidermis. These turgor-operated valves have the ability to regulate gas exchange between plant and atmosphere by changing the aperture of the stomatal pore. Although the stomata are capable of regulating gas exchange, they are faced with a dilemma. The assimila- tion of CO2 from the atmosphere requires an intensive gas exchange and, hence, Open stomata. The prevention of excessive water loss demands that gas exchange be kept minimal, which requires closed stomata (29,30). Stomata must in some way balance these opposing priorities in order for the plant to survive. The question of how plants resolve their dilemma is an important one. Plants must maintain an adequate rate of photosynthesis to enable them to grow and reproduce during an often limited growing season. Growth is also necessary if plants are to be successful in outcompeting their neighbors for sunlight. However, in both natural and agricultural situations water is often available in limited amounts, so the plant must wisely use its supply of water. Thus it would seem that a plant with a high water use efficiency (CO2 assimilated/ H20 transpired) would be more successful than a plant with a low water use efficiency. This may be the case in certain situations, but a high water use efficiency is not always a desirable characteristic of a plant. For example, plants utilizing Crassulacean acid metabolism have water use efficiencies five to twenty times greater than those of other plants (1), but their extremely SIOW' rate of growth makes them impractical from an agricul- tural point of view. Because assimilation follows approximately saturation kinetics with respect to stomatal permeability while transpiration is nearly linearly related to permeability, the stomatal aperture for maximum photosynthesis is normally greater than that for optimum water use efficiency (30). Therefore a successful plant might be one which is capable of maximizing either photosynthesis or water use efficiency depending on various environmental factors such as water supply, evaporative demand, light intensity, and temperature. Stomatal Sensitivity to CO2 In order to maximize their efficiency of water use, plants must adjust stomatal opening to the CO2 require- ment of the assimilatory tissue (29). One means of achieving this is to have the stomata sense the CO2 depletion of the intercellular spaces. The question which then arises is whether or not stomatal sensitivity to CO2 is a common feature of all land plants. As early as 1898, Darwin (5) performed experiments examining the effects of COZ-free air and pure CO2 on stomata. He found that stomata in light remain Open in an atmosphere free of carbon dioxide, and that they close slowly in an atmosphere of pure C02. In 1916, Linsbauer (19) reported that in both light and darkness, the removal of CO2 from the atmosphere resulted in stomatal opening. Using physiological concentrations of C02, Freudenberger (8) clearly demonstrated that increases in CO2 concentration caused stomatal closure and decreases caused opening. She found that these results applied to leaves in the dark as well as those in the light and also to etiolated leaves. In 1948, Heath (10) reported similar findings. However, he found that if the stomata had been completely closed, they would not reopen in COZ—free air. He interpreted this as meaning that the effect of carbon dioxide is due to its concentration in the sub-stomatal cavities and not the concentration at the outer surface of the guard cells. From this and other evidence (26,16, 22) it can be concluded that sensitivity to CO2 is a common feature of stomata, and that stomata probably sense the CO2 concentration in the intercellular spaces within the leaf. Can this sensitivity actually function in such a way as to make stomatal opening proportional to the photosynthetic demand for C02? Using Zea mays and an airflow parameter, Raschke (26) was able to demonstrate that stomata are capable of maintaining a relatively constant intercellular CO2 concentration in an environ- ment with a fluctuating ambient CO2 concentration. He " went on to describe how the stomatal sensitivity to CO2 could function as a regulatory mechanism making stomatal aperture proportional to the CO2 requirement of the assimilatory tissue. Effect of Abscisic Acid on Stomatal Sensitivity to CO2 Although stomatal sensitivity to CO2 appears to be fairly common among species, it is not universal. Stomata of well watered, greenhouse-grown plants of Xanthium strumarium appear to be insensitive to changes in ambient CO2 concentration (27). However, they can be sensitized to CO2 by raising the abscisic acid (ABA) content of the leaves (27,28). This is an important observation in view of the fact that the ABA concentration in leaves has been shown to increase dramatically under conditions of water stress (13,33,34,35) or chilling (31). Wright and Hiron (34) showed that the concentration of ABA in wilted wheat leaves may be forty—fold greater than that in fully turgid leaves. It has recently been shown that not only does ABA sensitize the stomates to C02, but CO2 sensitizes the stomates to ABA (28). In COZ-free air, stomata of 5 Xanthium strumarium will not respond to ABA (if <10- M) in the transpiration stream. Increased stomatal sensitivity to CO2 with increased ABA levels has only been shown with Xanthium strumarium~' (27,28). However, there are indications that this sensitization may occur in other species as well. Heath and Meidner (ll) speculated that stomatal sensitivity to CO2 might be greatly enhanced by water stress. One year later, Heath and Mansfield (12) showed in Xanthium pennsylvanicum and probably in Triticum aestivam and Taraxacum officinale that stomatal sensitivity to CO2 is greatly increased when plants are subjected to water Stress . It has been known for almost ten years that increased ABA concentrations in the leaf result in a reduction in transpiration and stomatal aperture (3,13, l4,l7,20,23,24). However, it now appears that ABA may do more than simply cause stomatal closure. The simultaneous requirement for ABA and C02 provides a mechanism which can allow the plant to either maximize photosynthesis or improve water use efficiency. For example, under conditions of adequate water supply and low evaporative demand, stomata are insensitive to CO2 and Open widely to allow in as much CO2 as possible for photosynthesis. If water loss becomes too great, some ABA is formed causing the stomata to become sensitized to C02 and close slightly which reduces water loss. Thus, the plant shifts from maximizing photosynthesis to improving water use efficiency since at wide stomatal apertures assimilation is affected less by stomatal movement than is transpiration (30). If too much water is still being lost, more ABA is formed which further closes the stomata. The degree of stomatal closure will be proportional to the need for CO2 by the assimilatory tissue since assimilation rate is one of the factors determining intercellular CO2 concentration which in turn affects stomatal aperture. If the assimilation rate is low, the intercellular CO2 concentration will be high thus causing stomata to close more than if the assimilation rate was high and the intercellular CO2 concentration was low. The Feedback Loops We have seen that a change in the intercellular CO2 concentration can result in a change in stomatal permeability, P. However, if stomatal permeability changes, this will also change the intercellular CO2 concentration, Ci‘ This is a feedback loop because a causal chain exists which may be summarized by Ci-—-" P -——. ci (where -———> is read as "affects"). As seen in Figure 1, there are several such feed- back loops involved directly or indirectly in the control of stomatal aperture. The two major feedback loops affecting stomatal permeability are the ones involving CO2 and H20 (29). Because of the number of interacting feedback loops, it is difficult to predict what the final stomatal aperture will be if one of the parameters in the 100ps is disturbed. For example, a change in light intensity will result in a change in assimilation which will, in turn, directly or indirectly affect intercellular C02 concentration, stomatal aperture, evaporation rate, and leaf temperature. Thus, the results of many experi- ments involving stomata are ambiguous. In order to assess the importance of each of the various interacting feedback loops and the biological ...muommm.m= mm coon mum Ail-V , L mzouum one .onsuummm Hmumaoum mo Honucoo wnu a“ Um>ao>cw mmooH xomncmmm "a musmwm ovum coflumuucmocoo onsuummm mumm moo mnsumuwmswe “MadaamoumucH HmumEoum coflumuomm>m :oflumHflEHmmm consequences of the regulation exerted by each of the loops, it is useful to measure the degree of regulation (=gain) that occurs. We need to know not only that stomata are sensitive to C02, but also quantitatively how sensitive the stomata are to changes in C02 concentration and how sensitive CO2 concentration is to changes in stomatal permeability. In classical feedback theory, the gain of the feedback loop, which is the amplification that a signal receives moving around the loop, is an appropriate measure of the amount of regulation that occurs (2,6). Statement of Purpose The purpose of this thesis will be to examine the feedback loop involving CO2 and stomata. Appropriate theory and methods for measuring the gain of this loop will be developed. This thesis will examine 1) how the“ gain affects stomatal responses to various environmental perturbations, 2) how the magnitude of the gain can give us an indication of how well intercellular CO2 concen- tration is being controlled, and 3) how the magnitude of the gain can change when the concentration of ABA in the leaf increases. It will also examine the effects of ABA on photosynthesis and water use efficiency. Finally, several different species (C3 and C4, monocotyledon and dicotyledon) will be examined to see if any generaliza- tions can be made regarding strategies that different i 10 plants use to solve their dilemma. Theory Let us consider the steady-state relationships between the intercellular CO2 concentration, ci, and the permeability of stomata to diffusion of water vapor, P. We know that c. depends on the ambient CO 1 2 concentration, c, and the stomatal permeability, P, and the net rate of assimilation, A. The following partial differential equation describes Changes in Ci' dci = (aci/ac)A'Pdc + (Sci/3P)CIAdP + (sci/3A)c,PdA (1) We also know that the stomatal permeability and the net assimilation are dependent on certain metabolic and environmental factors. Permeability is assumed to be a function of Ci' light intensity, I, concentration of abscisic acid [ABA], and perhaps other factors which we will call np. Assimilation rate is a function of Ci' I, temperature, T, and other factors which we will call nA. Both P and A can now be expressed in terms of the following two partial differential equations: dP = (aP/aCi)I,ABA,n dci + (SP/aI)Ci,ABA,nde P + (3P/3ABA)C. I n dABA + (aP/an ) d 1, p P ci,I,ABA np (2) ll 12 dA = (BA/aci) dc. + (BA/BI) dI I,T,nA l Ci’T’na + (BA/3T)c. dT + (BA/anA)c dn (3). ll I’D. i'I'T A A Initially, for the sake of simplicity, let us assume that all factors except intercellular CO2 concentration, stomatal permeability, assimilation rate, and ambient CO2 concentration are held constant. The partial differen- tials aP/aci and EBA/3ci become total differentials and equations (2) and (3) simplify to: dP dA (dA/dci)dci (5) The two feedback loops of interest to us here are indicated in Figure 2, which is a diagrammatic represen- tation of equations (l),(4), and (5). One loop involves permeability and the other assimilation rate and both affect the intercellular [C02]. We define dP/dci and dA/dci as the physiological gains of the permeability loop and the assimilatory loop, respectively. These gains are so named because they are dependent on metabolic processes in the guard cells and assimilatory tissues. moi/BF)!“c and (Sci/amp,c will be defined as the physical gains of the permeability loop and the assimilatory loop, respectively. The magnitudes of these gains depend on physical characteristics of the stomatal pore as 13 deJt :11: dci C 3c I 1 I 3c. .95 ------1-- dCi 3A P,c Figure 2: The gains of the feedback loops involving Assimilation and CO2 and Stomatal Permeability and CO 2 This is a diagrammatic representation of Equations (1), (4) and (5). 14 expressed in the following Ohm's Law type of relationship. ci a c - 1.6A/P (6) The factor 1.6 arises because of the difference in diffusivities of water vapor and CO2 in air. We now define the open loop gains of the feedback loOpS involving stomatal permeability and assimilation as the products of their respective physiological and physical gains. In both loops this product is usually negative in the steady state. The open 100p gain is so called because if a change, dci, were imposed causing a change, (dP/dci)dci (=dP), in permeability, and if it were possible to artificially open the loop and examine the change in Ci' (aci/BP)C'AdP, resulting from the change in permeability, under conditions not affecting the original perturbation of c that change would be it (aci/BP)c A(dP/dci)dci. Thus, the amplification that the I small perturbation has undergone after one transit around the feedback loop 15 (aci/BP)C'A(dP/dci) = Gp. An analogous example can be worked through for the assimi- latory loop, and here the amplification would be (Sci/3A)CIP(dA/dci) = GA' It is 1mportant to note that 1t does not matter which factor is initially perturbed within the loop. The amplification after one transit around the 100p will always be equal to the loop gain. 15 Apart from being independent of the way that the loop is broken up, the measure of loop gain is also usefully independent of the choice of units of the components. For example, if instead of permeability we chose stomatal resistance as our measure of stomatal opening, the new measures of the physiological and physical gains would have different magnitudes and opposite signs, but the product of the two gains would have the same magnitude and sign as that obtained using permeability. We now examine how the open loop gain comes into play in the normal closed loop situation. Returning to equation (1), we see that dP and dA can be replaced by using equations (4) and (5). This yields the following equation: dci = (sci/3c)P'Adc + (aci/aP)C’A(dP/dci)dci + (aci/aA)c,p(dA/dci)dci (7) Equation (7) can then be rearranged to obtain: (aci/ac)P’Adc l-(Bci/BP)C'A(dP/dci) - (Sci/3A)C'P(dA/dci) doi = (3c./8c) dc = 1 P,A (8) l-GP-GA We see that the open loop gains of both the permeability 100p and the assimilation loop appear in the denominator of this equation. Thus, in effect, a change in 16 intercellular CO2 concentration that one might expect to see if one perturbed the ambient [C02] without modifying permeability or assimilation will be modified by l/l—GP-GA. Since both loops are normally negative in the steady state, the denominator becomes greater than one, thus reducing the expected change in ci. Therefore, the greater that the abSolute value of the loop gain becomes, the more "resistant" Ci is to change. We can also look at the effect of the open loop gain on the response of permeability. If we take equation (4) and substitute for dci using equation (8) we obtain the following: (dP/dci)(3ci/3c)P'Adc dP = (9) l-GP-GA A change in P that one would expect from a change in ambient CO2 concentration is also modified by l/l-G From equations (8) and (9) one can see that P-GA. both permeability and intercellular CO2 concentration are under the influence of feedback. The degree to which this occurs depends on the magnitudes of the loop gains. Thus far, we have considered only the influence of a change in ambient CO2 concentration on permeability or intercellular CO2 concentration. We now examine the effects of a change in light intensity, dI, that may involve direct effects on both permeability and 17 assimilation. Although it has not been conclusively shown that light directly effects stomatal permeability, for the purposes of this discussion we will assume that there are direct effects. We rewrite equations (4) and (5) as: dP (ap/aci)Idci + (3P/3I)C_dI (10) 1 . and dA (BA/aci)Idci + (BA/BI)CidI (11) These two equations can then be substituted for dP and dA in equation (1) and the change in C1 is: dci = (sci/3P)C’A(3P/31)cidl + (3°1/31)cp(3A/31)cifi (12) l-GP-GA It can be seen that light directly affects ci by changing the permeability and assimilation rate. This effect is modified by the loop gains of both the permeability and assimilation 100ps. For example, if GP = -l.5 and GA = -O.5 the denominator becomes 3 and the effect of a change of light intensity on Ci would be 33% of what one would expect if there were no feedback. Similarly, we can look at the effect of light intensity on permeability. Using equations (l),(10), and (11), one can derive the following: 18 (3.)“ <:;>1<::>C,A<:>.+<2—:->C,P<—:%u f dP dI —— 1 -a- (13) L l-GP-G A J From this equation, one can see that light can affect permeability in three ways. There is the direct effect and the two indirect effects which operate via the CO2 feedback . loops. The 100p gains can have a large influencecnxthe2two indirect effects. Because of the complexity of the light response, it is apparent why direct effects of light on permeability are difficult to measure. The feedback loops involving CO2 would have to be broken (i.e., ci must be held constant) before one could actually measure direct stomatal responses to light. Other responses to environmental perturbations are also affected by these feedback loops and can be evaluated in the same way as the light response. 19 Materials and Methods Plant Materials Plants of Xanthium strumarium L., a C3 species, were grown in a soil mixture in a greenhouse at East Lansing, Michigan. The strain used, a strict short-day plant, was originally collected near Chicago, Illinois and has been propagated in California and later in Michigan. The natural light period was extended to 20 h 2 from day.1 by supplementary illumination with 0.3 W m- Sylvania Grolux fluorescent lamps, so that the plants were kept in a vegetative state. Temperature maxima in the greenhouse were between 23 and 29°C; the relative humidity was between 70 and 80%. Fully expanded leaves from 2 month old plants were used. The fifth leaf from the apex was detached under water after submerging the h tops of the plants for about 2 minutes (to produce temporary hydropassive stomatal closure); the cut end of the petiole was kept under water throughout the experiment. Plants of Zea mays L. (cv. Michigan 500; seeds from Michigan State University Crop and Soil Science Department), a C4 species, were cultivated in a sand- vermiculite mixture in a high light intensity growth 20 1 from chamber. The plants were illuminated for 20 h day- a combination of General Electric lamps H400Dx33-l and LU 400. The intensity varied in three steps through the day with a peak irradiance of 240 W m-2 of photosynthe- tically usable light (defined as the light passing through an infrared-absorbing glass (Corning 4600)). The day temperature was 31°C and the night temperature was 20°C; the relative humidity was 70%. The sixth leaf from the base of 1 month old plants was used. Plants of Amaranthus powellii S. Wats. (seeds were collected from the Michigan State University campus and were a gift from Dr. Stephenson), a C4 species, were cultivated in a soil mixture containing 1/3 part perlite in a high light intensity growth chamber. Conditions were the same as those described for Zea mays. The‘ sixth leaf from the apex of 1 month old plants was used.. Plants of Gossypium hirsutum L. (cv. Acala SJ-l; seeds from Dr. C. A. Beasley, University of California, Riverside), a C3 species, were cultivated in a growth chamber. The plants were illuminated for 12 h day-1 (60 W m-2 of photosynthetically usable light) from fluorescent lamps followed by l h from incandescent lamps. The temperatures were 32°C during the day and 22°C during the night. The relative humidity was 60%. The fifth leaf from the apex of 2 month old plants was used. The petiole was cut under water and remained under 21 water throughout the experiment. Plants of Avena sativa L. (cv. Rodney), a C3 species, were cultivated in a vermiculite-gravel mixture in a growth chamber. Plants were illuminated for 12 h -1 (80 W 111-2 day of photosynthetically usable light) from fluorescent lamps. The temperatures were 21°C during the day and 18°C during the night. The relative humidity was 60%. A fully expanded leaf section 20-25 cm from the tip was used in the experiment. Plants were 1.5 months old. Measurement of Gas Exchange Water-jacketed chambers were attached to the upper and lower surface of each detached leaf, and water of 22.S°C was circulated through the jackets. Four pairs of chambers were available for simultaneous measurements on four leaves. Air of known water vapor (dew point = 18.S°C) and CO2 content was passed at 50 l h.1 through these chambers. The exposed leaf surface was 2.44 cm2 in each chamber. The petiole of dicotyledonous plants or the leaf blade and midrib of monocotyledonous plants were kept in a beaker containing 10 ml of distilled water. In 4 experiments using ABA, 1 ml of 10- M (i) ABA was added to 9 ml of distilled water in the beaker to produce a 5 concentration of 10- M (1) ABA. Since only the (+) enantiomer of the cis, trans isomer of ABA is effective 22 . in causing stomatal closure (4), the effective concentra- 6 M. With tion of ABA in the irrigation water was 5 x 10- respect to ABA, stomata respond in normal air with slightly more than half their maximal amplitude at this concentration (28). Formation of endogenous ABA in the leaf tissue in response to low water potentials was kept low by trimming all parts of the leaf not covered by the gas-exchange chambers, thereby reducing the transpiring area (27). Changes in gas composition were measured with differential infrared gas analyzers (Uras 2; Hartmann and Braun, Frankfurt a.M., Germany). The absolute CO2 content of the air was monitored with an additional Uras analyzer. Thermocouples were used to measure leaf temperatures and the temperature of the condensor setting the dew point of the air supplied to the chambers. The leaves of Xanthium strumarium, Zea mays, and 2 Amaranthus powellii were illuminated with 340 W m7 of photosynthetically usable light (400-700 nm wavelength). This corresponds to 1.53 mE m-Zs-l. The leaves of Gossypium hirsutum and Avena sativa were illuminated 2 with 240 W m- of photosynthetically usable light. This corresponds to 1.08 mE m-zs-1. The light source was a water cooled 6 kw xenon-arc lamp (Osram XBF 6000) behind an infrared-absorbing glass (Corning 4600) and neutral 23 density filters (Plexiglas No. 800; Rohm und Haas, Dormstadt, Germany). Voltages from the thermocouples and the gas analyzers were amplified and fed into a Hewlett-Packard 21008 minicomputer which computed the time courses of the exchange of CO2 and water vapor through the upper and lower epidermis, epidermal per- meabilities to water vapor exchange, and intercellular CO2 concentrations (=CO2 concentration at the sites of evaporation). The intercellular‘CO2 concentrations pre— sented in this paper were calculated using equation (6). Values for assimilation and permeability are combined values from the upper and lower epidermis. Results are from a 20 minute time span after the stomata had reached a steady state and are the average of four separate leaves. Readings for each leaf were taken once every five minutes. Measurements of the Various Gains The basic experiment consisted of varying the ambient CO2 concentration, c, from 0 to 550 ul-l+ over a time span of 5 h and measuring the steady state values of the permeability, P, net assimilation rate, A, and intercellular CO2 concentration, Ci’ at each value of the ambient [C02]. From these values, plots of permeability and net assimilation versus intercellular [C02] were made. Using these plots and a rearranged 24 equation (6), plots showing the relationship between intercellular [C02] and ambient [C02] were drawn. An operating point was chosen at some particular c, and, from the above mentioned graphs, values were obtained for P, A, and ci at this point. It is necessary to choose an operating point because the physical gains, (Bolt/3P)“A and (aci/BA)C'P are defined at some particular constant ambient [C02]. As will be seen, choosing different operating points results in different values for the various gains. From the plots of P and A versus c the physio- i' logical gains of the permeability (dP/dci) and assimilation (dA/dci) loops, respectively, were determined by measuring the slope of these curves at the operating point. I The physical gains at the operating point were obtained from the following equations which were derived from equation (6). _ 2 (BCi/BP)C,A — 1.6 A/P (14) (sci/amp,C = -1.6/P (15) (Sci/3c)P'A = 1 ' (16) The Open loop of each loop is then the product of the physical and physiological gains of that loop. 25 Example: Calculation of the physiological, physical, and loop gains for the feedback loops involving permeability, assimilation, and intercellular [C02]. We 5 will use Xanthium strumarium with 10- M (i) ABA in the transpiration stream. We will choose our Operating ' —1 point to be [C02] ambient = 300ul l . Using Figures 3, 8, and 14, we determine that: ci = 210 ull-l = 24.5 c mole mmzs"l A = 12.4 u mole m-Zs"l c = 300 pl 1-1 The calculations of the physiological gains are shown in Figures 3 and 8. (dP/dci) = 34.44 X 102 mole m-ZS-l (1 1-1) (dA/dci) = 5.00 x 10"2 mole mmzs"l (1 1-1) The physical gains would be: (sci/3p)A C = (1.6)(12.4x10-6 mole m‘zs‘1)/ (0.245 mole m-Zs'l) = 3.31 x 10.4 mzs mole"1 (1 1'1) _ _ -2 -1 (BCi/BA)P'C - 1.6/0.245 mole m s = -6.53 mzs mole-1 (1 1'1) The loop gains would be: (dP/dci)(aCi/3P)A,c = -1.14 (dA/dci)(3ci/3A)P c = 26 -0. 33 Results Net Assimilation versus Intercellular [C02] Figures 3,4,5,6, and 7 show the photosynthetic response curves for Xanthium strumarium, Gossypium hirsutum, Avena sativa, Zea mays, and Amaranthus powellii, respectively. The solid lines (._____,) represent the response of net rate of assimilation to a changing inter- cellular [C02] in the absence of any ABA in the transpira- tion stream. The dashed lines (+ ----- +) represent the 5 M (i) ABA was present in the response when 10- transpiration stream. The arrows indicate the assimilation rate and intercellular [C02] at normal ambient [C02] (=3oo u11‘1). The shape of the photosynthetic response curves is typical for each species. Net assimilation rates of the two C4 species, Zea mays and Amaranthus powellii, saturate with respect to [C02] at low values of the intercellular [C02] (~100 ul 1-1) at the light intensity used. The net assimilation rates of the three C3 species do not saturate at CO2 concentrations less than or equal to the normal ambient [C02]. The relatively low maximum rates of photosynthesis for the two C4 species are unexplained. An experiment designed to test 27 28 _~oo_ Haasaawoumucfi Op uoommmu nuw3 Esaumsnuum annucmx How w>usu Omcommwu ofiuonuc>mouonm "m musmwm AH...” .13 HNOUH HMHOHHOOHOucH oom cow com oom - r n p . ¢m¢+ " al.l.|+ dmdl u . . . 1 and H omH n8 H-H.H: omH n E O OE x u m . woo .cwmo Hmomeaowmanm m .III 1 «U S7u n1 IOH ma ON mm m atomn) uorqetrmrssv gen 5 I- Z- ( 29 A08 unasaawoumucfl Ou powmmmu saw: savanna: sawmmmmow Mom O>uoo wmcommmu owumnucwmouonm HNOOH HMHOHHOouOucH com 00% Con OON OOH flmfll "v wusmwm OH .N mm Iomn) uorqetrmrssv gen 6 ma T"Z’ 30 108 “wasaaoonwucw ou nommmwu gaw3 m>aumm mcw>¢ How O>uao mucommmn vauwnuaamouonm "m whamflm AHIH.H:v ”NoU_ MMHDHHOOHOHCH oom OOG OOm OON OOH ¢m<1 u OH ma ON mm m 310mm) uorqetrmrssv gen 3 I- Z- 31 _NOUH “OHSHHOOMOUGH ou uowmmwu nuH3 mNmE MON How w>uao oncommwu OOm fig... mmfll OO¢ A H 1H.H:O .mOOO unasaamouwucH OOm OON oflumnucamouonm OOH no OHDmHh OH mH ON mm m atomn) uorqetrmrssv nan S 2- I— 32 amoul umHsHHmoumucw ou uommmmu suHS HHHHmson manucmumsd “Ow O>uso oncommwu OHuwnucamouosm "h muzmwm AH H.H1O ”Nova HmHsHHmouOucH dm¢+ dfldl + + ‘ .+“‘ m atomn) UOIJPIImISSV new 9 (I- Z- 33 whether or-not these low rates were the result of sub- optimal light and temperature conditions used in the experiments indicated that this was not the cause. Possibly light quality in the high light intensity growth chamber was unsuitable for the growth of these species. Permeability versus Intercellular [C02] Figures 8,9,10,11, and 12 show the relationship between epidermal permeability for water vapor and intercellular [C02] for Xanthium strumarium, Gossypium hirsutum, Avena sativa, Zea mays, and Amaranthus powellii, respectively. Again, the solid line represents the response in the absence of ABA, and the dashed line represents the response in the presence Of ABA. The arrows pointing to the curves show permeability and intercellular [C02] at normal ambient [C02]. Arrows on the curves of Figures 8,10, and 11 indicate in which direction the ambient [C02] was changing in the experiment. Permeability depended on the direction of change in [C02] at relatively low intercellular CO2 concentrations. This hysteresis has been observed in several experiments and in several unrelated species. Although the reason for the two different curves is unclear, I have chosen to use the lower curve for this discussion. As will be seen, this 34 ESHumasuum Eanucmx new ”Nova HMHDHHOOHOHOH msmuw> quHHnmmEumm A IH.H:v HNOU_ HmHsHHmoumucH um OMDOHm H oom oov com com OOH o om ¢m¢+ " + I.I.I¥ gm? 1 . . IE IT.. 11.. 773 om m m E OHOE OHwi.OMI u I, I. - I I N m E m 080 I o H N HI NI H Hm my; LI ON m I .MWW .camw moaoo 0am» b mo . H . H . :m s. I I.om r.ov / + \ / \ II \ ITn7.l.l.lik.\.\ 1.! H-om 1gIIO> o O 0 III. 0 (I_Sz_m atoms) Anrtrqeemzad 35 .EsuOman Bowmxmmow now ”NOO_ HMHOHHOOHODOH msmuo> muHHHnmmsfiom "m onsmHm OOm ¢m¢+ aumm wombafnow Hmoou HMHDHHOUHOHGH m5mum> >uHHHQMOEHOm oov A . 3 TH: OOm HNOUH umHzHHooumucH OON “OH mustm OOH OH ON Om ow Om OO m atoms) Aqrtrqeemled s (I- Z- mNmE MON How ”moo. HmHsHHmouwucH msmno> muHHHnmmauom “HH wuzmHm A IH.H:V Hmoo_ uaHsHHmoumucH H 00m 00v 00m CON OCH 0 O ¢m¢+ " +IZI.J+ dmdl " . . o + I 37 Iowa) Kntttqeemlad S 1118 (1' Z- .lOm Ow HHHHw3om.msnu:mumE¢ Mom ”moo” HmHsHHOOHOucH mswum> >uHHHnmmfiumm "NH ousmHm AHIH H1O HNOO. HmHoHHooumucH 00m 00m OON OOH OH 38 / 0v lOm m atoms) 53111q99m13d s I- Z- ( 39 rather arbitrary decision will not affect the interpre- tation except in certain special cases. Ambient [C02] versus Intercellular [C02] Figure 13 shows the relationship between inter- cellular [C02] and ambient [C02] in the absence of ABA for the five species examined. With the exception of Zea mays, the relationship is nearly linear with the intercellular [C02] being slightly less than the ambient [C02]. There is virtually no regulation of intercellular [C02] in the sense of maintenance at a constant level. Figure 14 shows this relationship when ABA is present in the transpiration stream. With ABA present, the change is intercellular [C02] resulting from a change in ambient [C02] is much smaller than that when ABA is absent as evidenced by the change in slope. The Effect of Ambient [COO] on the LOOp Gains In this section the effects of ambient [C02] on the gains of the feedback loops involving net assimila- tion, permeability, and [C02] in the absence of ABA will be presented. To avoid repetition, one species with stomata insensitive to C02, Xanthium strumarium, will be compared with another species, Zea mays, having sensitive stomata. Three Operating points for each species have been chosen for examination, and these are listed in Table 1. Two of these operating points, A and C, are (ul-l’l) [C02] intercellular 600 500 400 300 200 100 40 A o o 100 200 300 400 500 600 -1 [C02] ambient ((11 1 ) Figure 13: Ambient [C02] versus intercellular [C02]. O -Xanthium strumarium, [5 - Gossypium hirsutum, x - Avena sativa, o -Zea mays, + - Amaranthus powellii (pl-1‘1) Intercellular [C02] 600 500 400 300 200 100 41 //M .//+ 0 A I l I O 100 200 300 400 500 600 Ambient [C02] (pl-1'1) Figure 14: Ambient [C02] versus intercellular [C02] with 10'5 M ABA present in the transpiration stream. 0 - Xanthium strumarium; A - Gossypium hirsutum + - Amaranthus powellii; o - Zea mays 42 m.o O.¢H OOH com 0 «H m.MH an 00m m m>ms «MN m.m~ mb.m Om OOH 4 mm O.HN Omv com o . m EdHuMEduum Nb 0 @H hmm 00m 3 m E 020 m E OE: . 1 3 HI NI H Nb HA NI H m c HIH H mm HIH H OOH d huaHanMOEuOm OOHuMHaEamma HMHSHHOoumuca HNOOH ucoanEM HNOUH uCHom HMaumuMz uCMHm . . . . . . . OOHHMNMQO . mmME MON OGM EzHuMssuum ESHCOCMX :H meoHuMuucmocoo N on uannEM uCMHMMMHM mmucu MOM muHHHQMMEHMQ HMEHMOHOM OCM :oHuMHHEHMMM mo MuMu .HNOOO EMHsHHMOHMOCH mo mmzHM> .H MHnMa 43 unnatural cases since the ambient [C02] at these points is not normally encountered in a natural environment. Operating point B is representative of a natural environ- ment. The values of the physiological, physical, and loop gains at each Operating point are given in Table 2. The Effect of Abscisic Acid on the Loop_Gains Table 3 lists values for the various gains of the feedback loops involving permeability, net assimilation, and intercellular [C02] for the five species examined.’ The ambient [C02] is 300 ul 1"1 in all cases. For all species except Avena sativa, gains in the absence and presence of ABA are given. For Avena sativa in the presence of ABA, no steady state values of permeability were achieved making the calculation of the various gains impossible. The results for Avena sativa in the absence of ABA were included because they are a good example of stomatal insensitivity to C02. The Effect of Abscisic Acid on the Transpiration Ratios Table 4 lists values for the transpiration rate, net assimilation rate, and transpiration ratio (mole H20 transpired/mole CO2 assimilated) for all the species examined. Values in the presence and absence of ABA are given. The ambient [C02] = 300 pl l-l. The percent reduction is calculated as percent of the original value in the absence of ABA. ‘44 OH.OI HO.MHI Ov.mml Ov.Hmv mh.o mO.hNI U H~.oa mm.mu oe.HHI mm.soH vm.H cm.MMI m msme MON hm.oI vo.o+ ~m.v- Hm.mfl om.HH oo.~+ a m0.OI 00.0I mv.mI 00.0 mm.H mm.hI U . . . . . . . EsHuMEduum m0 Ol 00 O HN NI . Hm v mt m 00 O m 3 - hH.0I 00.0 Hm.~I mm.H Hm.h mm.0+ 0 III III 0E m E 0E m E m E OE m E 0E HIH N HIH N mIOH HI NI H NIOH HI NI H «OH HO HO 0. . Ho Ho Ham Ilh Hmm .IMW m MMW U MAMMW Ilm. .Ilm ucHom MHuOuM CM .om <0 .Om b .0m .0m zg mcHMJ HMOHOOHOmenm mmmE MON 02M anumsanum Ezwcuan :H meoHuMHOCOocoo woo u:OHoEM uCOutmep OOgc» uM .mcwmm HMonch rcM HMoHOrH0wm30Q Ocu .mtcOcOQEou HHOcu hem MCHMO QOOH OCu Mo OOaHM> .HH OHQME 45 0H.0I H0.~I 0m.vHI 0v.~0H mn.H 00.HHI mO» HHHHO3om nszucMuME< 00.0 0v.0I «0.0 I No.0m 00.0 00.0 I 02 0v.0I 00.0I 0H.~HI 00.0vH mmHm 00.m I MO» mxms MON Hm.0I mm.MI 0v.HHI 00.00H v0.H 0m.nnI 02 I I I cOcHMuuM OOMHM> mcMOum 02 II mO» M>HuMm MCO>< 00.0I 00.0 mm.~ I ”v.5 mm.m 00.0 oz mv.0I Hm.0I m0.HHI O.vn v0.m 5H.v I OOH Esuaman 85H mmoo 00.0I ~0.0I FO.MI mv.w 0~.~ om.~ I 02 nm.0I vH.H mm.0I H.nm 00.m vv.va OO> anHuMazuum Echuch EH.0I 00.0 H~.~I mm.H mh.m 00.0 oz HOMOHOOHOOOEHE IHOEm~E IHoE a «a HIONIE HOE ~I0H m IE HoE N0H moon 003 06 a :6 A me 234 13.83: 230 53305.34 afidfioaumm mm .8 MGHMO HMOHO>£0 ncHMo HMonoHonusm .EMOqu coHuMuHmucduu 0:» 0H 40¢ zmIOH uo OocOmoum 00M OocOmnM Onu 0H N00 000 .cOHuMHHEHmmM .>9HHHAMOEHOQ mcH>Ho>cH OmooH x010600u on» no OEHMO 0:9 .HHH OHnMB 46 up ob.nH oo.a no» HHHHmzmm mv «I On mosucouosa mMH .mo.~H HF.H oz ms oo.~H mm.o no» mums mm ma Om non OHH mm.MH mm.H 02 I I I OOH M>HuMm I I I MCO>¢ mod Mm.mH oH.m oz mmz mo.m mH.H mo» sousmuzn mm «H cm 22H mmoo mom op.m om.~ oz aha hm.HH oH.~ mo» eozuosouum mm mm om sozsucmx omN mm.mH oo.¢ oz cowuosoom ~ooa on: :oHuoooom HImNIa oaoa cowuoooom HImNIa macs mama ucmHo ucOouOm OHUMM coHUMuHmmcMuB UGOOHOO :oHuMHHEHmO¢ ucOouOm COHUMuHmmcMuB .H¢m¢ unonuH3 OaHM> On» 00 uaOouOm OH OH cO>Hm :oHuochu Onav .coHuMu coHuMuHmmcMuu ucM .coHvMHHEHOOM .coHuMuHmmcMuu :0 Hz 0H. mac no “OOqu Oak .>H OHnMa m {I I‘ll"! I’ll" 47 Discussion The Effect of Abscisic Acid on Net Assimilation It is apparent that ABA had no effect on photosynthesis during the time span of these experi- ments (~5 hours). The photosynthetic response curves with respect to intercellular [C02] are virtually identical with and without ABA in all cases. It is possible that under longer time spans, increased ABA levels might indirectly cause a reduction in photosynthesis via its oxidation product, phaseic acid (9). Phaseic acid has recently been shown to strongly inhibit photosynthesis ((18), T. Sharkey, personal communication). The Effect of Abscisic Acid on Stomatal Permeability It can be seen that in the absence of ABA the stomata of Zea mays are quite sensitive to changes in intercellular [C02], whereas those of Xanthium strumarium and Avena sativa are virtually insensitive to changes in intercellular [C02]. The stomata of Gossypium hirsutum and Amaranthus powellii might be classified as intermediate in their sensitivity to C02. Comparing the stomatal responses to CO2 in the presence and absence of ABA, one can see that ABA results in a general reduction in stomatal permeability 48 as well as an increased sensitivity to CO2 in all species except Avena sativa. These responses are most strikingly demonstrated in the case of Xanthium strumarium. In the case of Avena sativa, ABA caused a gradual decline in stomatal permeability during the course of the experiment which was independent of changes in [C02]. This indicates that the effective concentration of ABA that the stomata respond to was changing during the experiment, or, possibly, that the ABA concentration used was higher than that normally found in the stressed plant. It may be that Avena cannot sequester or metabolize ABA as effectively as the other species. The Effect of Ambient [C02] on the Loop Gains The loop gain of the assimilation loop is most significant at low CO2 concentrations. This loop performs less of a regulatory role at high CO2 concentrations than at low concentrations. This is due to the saturation characteristics of the photosynthetic response curves. On the other hand, the absolute value of the loop gain of the permeability 100p increases with increasing [C02]. At operating point A for Zea mays, the permeability loop gain is positive which means that positive feedback is present. However, its effect is overridden by a larger negative loop gain for the assimilation lOOp.. Let us now examine the loop gains at operating point B and the significance that they have to the plant. lill.11llcllll 1)!) I) JIIIIIIlfIIIllI-Il 49 The absolute values of the loop gains of both the permeability and assimilation loops for Zea mays are greater than those for Xanthium strumarium. Returning to equation (8), we see that there is a one to one correspondence between a change in ambient [C02] and the resulting change in intercellular [C02] if no feedback occurs (i.e., if the loop gains are zero). (If we now substitute the values of the loop gains for Xanthium strumarium into this equation, we see that a change in c would result in a change in Ci of 93% of that which would be expected without any feedback occurring. For - would be 21% of that which Zea mays, the change 1n cl would be expected without any feedback. Clearly, there is much greater regulation of C1 in Zea mays than there is in Xanthium strumarium. From equation (9), it can be seen that the loop gains also influence the response of stomata to changes. in ambient [C02]. Again, in this case, the larger loop gains of Zea mays would reduce the change in permeability that one would expect if there were no feedback occurring. It should be noted that this does not mean that the stomata of Zea mays are less sensitive to changes in ambient [C02] than those of Xanthium strumarium, since the physiological gain, (dP/dci), in the numerator is much larger for Zea mays than for Xanthium strumarium. 50 What significance do these differences in gains have to each plant? For Zea mays, the value of the intercellular [C02] at the normal ambient [C02] corresponds approximately to that found at the break point ((2), Figure 6) of the photosynthetic curve. Further increases in intercellular [C02] caused by increased permeability would not increase net assimila- tion appreciably and would only lead to greater water loss. In this case, control of intercellular [C02] would probably be beneficial to the plant. It can maintain a maximum rate of photosynthesis and at the same time have a high water use efficiency. In the case of non-stressed Xanthium strumarium, there is virtually no regulation of c Because of the shape of its 1' photosynthetic response curve, increase in ci result in increases in net rate of assimilation. If the plant is not suffering from water stress, it is beneficial to the. plant, from a photosynthetic standpoint, to have stomates open as wide as possible even though water loss is quite high. In the next section, we shall examine what happens when water loss becomes too great and ABA is formed. The Effect of Abscisic Acid on the Loop Gains It can be seen that ABA significantly increased the absolute value of the lOOp gain of the assimilation loop in all species examined. ABA also increased the lOOp gain of the permeability loop in all species except 51 §33_m§y_. The loop gain declined in Zea mays when ABA was present, but still remained relatively large compared with the other species.’ This decline was due to a decrease in the physiological gain (dP/dci). The area of high physiological gain for Zea mays shifted to a lower intercellular [C02] in the presence of ABA. This shift may be of advantage to the plant if, for example, light intensity increased resulting in an increase in assimilation and a decrease in intercellular [C02]. The high gain at the lower intercellular [C02] would help the plant to maintain a certain minimum [C02] in the leaf. In the previous section we saw that for Xanthium strumarium a change in c would result in a change in ci of 93% of that which would be expected without any feedback occurring. Using values for the loop gains from Table 3, we see that when ABA is present in the transpiration stream, a change in c would result in a change in ci of 40% of that which would be expected without any feedback occurring. There is much greater regulation of intercellular [C02] when ABA is present. Similar examples can be worked through for the other species. The measurement of the loop gain allows us to directly measure the stomatal sensitivity to C02. It can be seen that ABA is capable of significantly Ir III'III'I I'll-ll“ 52 increasing the stomatal sensitivity to C02. Using a factorial experiment, Mansfield (21) stated that there was no interaction between CO2 and ABA. He concluded that the effects of ABA and CO2 were purely additive. It is clear from the results presented here that this is not the case. There is a definite interaction between CO2 and ABA providing for communication between the CO2 feedback loop and the H20 feedback loop. It is possible that the plants which Mansfield used were stressed prior to his experiments which resulted in stomatal sensitivity to CO2 in both his control leaves and his leaves which were fed ABA. The additional ABA given to leaves with high levels of endogenous ABA may have led to results which would erroneously indicate that the effects of ABA and C02 on stomata are purely additive. ‘It should be noted that the loop gains reported here are dependent on the environmental conditions and the condition of the plants used. The measurements of the magnitudes of the loop gains probably underestimate the actual values because other feedback loops involving stomata remained intact when measurements Of the physiological components were made. The most important of these is the loop involving evaporation from the leaves. When the ambient [C02] was increased and stomata closed, the rate of evaporation, E, declined, and the water status of the leaf may have improved, reducing the stomatal closure. This loop has two 53 components, an environmental one with gain (dE/dP) (7) and a physiological one with gain (aP/BE)ci. The former was minimized by using only a moderate humidity gradient and the latter was minimized by using detached leaves, removing the drop in water potential through the rest of the plant. The Effect of Abscisic Acid on the Transpiration Ratio All species examined show a large decrease in the transpiration ratio when ABA is present. Most of this reduction is due to a decrease in transpiration; net assimilation rate is affected only slightly. In both the presence and absence of ABA, the C4 species have lower transpiration ratios than the C3 species. General Discussion of Gains How do the open lOOp gains reported in this paper compare with those of other biological systems? The open loop gain for the pupillary reflex arc has been measured as -0.16, but may be greater; for the semicircular control of balance, -0.1; for eye tracking, -l.l; for eye movement control, -4 (32). Open loop gains of -9 (which may be an overestimate) and -16 have been measured for body temperature control and for the respiratory chemostats (C02), respectively. It would appear that the measurements presented in this paper for stomata are in the range of those previously reported 54 for physiological systems. However, when compared with those found in electronic circuitry and other technical systems (open loop gains of the order of 105), it would appear that the physiological Open loop gains are quite small. Why do the open loop gains in physiological systems appear to be low in magnitude and what signifi- cance do these low gains have in terms of physiological ' processes?~ If we return to the general equation, AControlled Parameter = Disturbance of the Controlled Parameter 1-Loop Gain which is applicable to the system described in this report as well as other physiological systems, it can be seen that a low value fOr the lOOp gain means that the system acts to moderate disturbances rather than eliminate them. If the loOp gain had a value of minus infinity, then the system would eliminate any distur- bances of the controlled parameter. In the case of the C02 system described herein, a fixed "reference" intercellular C02 concentration with which stomata compare the actual intercellular CO2 concentration probably does not exist. There is little or no evidence for the usefulness of the error concept in biology (15), and in this respect physiological and technological control systems appear quite different. 55 Why are physiological systems moderators rather than governors? It is possible that they use less energy in relation to the amount of information that they process. In all control systems, not only does the. regulator, in a sense, control the flow of energy, but it must use energy in the control process. Also, there are Opposing priorities in biology. For example, in the case of stomata, precise control of the rate of assimilation would mean imprecise control of water use and vice versa. In a natural environment it is unlikely that steady state conditions ever occur. The light intensity, temperature, humidity, and other environmental variables are constantly changing. The stomata must continually respond to the plant's changing environment. Although the steady state loop gain which is obtained under artificially constant conditions may never occur naturally, it does give an indication of the speed of system response and the dynamic behavior in general. The steady state loop gain also allows one to order in magnitude the relative importance of interacting feedback loops. It is hOped that this paper will lay some of the groundwork necessary to the understanding of dynamic stomatal behavior. 56 Conclusions From the results presented here, we can conclude that the loop gains of the feedback loops involving C02 can have a large influence on the response of stomata to various environmental perturbations, and on the regulation of intercellular CO2 concentration. Abscisic acid, the plant hormone produced under condi- tions of stress, can significantly increase the magnitude Of these loop gains. An increase in abscisic acid also results in an increase in the water use efficiency of the plants tested. Although many more plants would have to be examined before any broad ecological conclusions can be drawn, we can begin to see that different plants do use different strategies to solve their dilemma. The three C3 species examined had relatively insensitive stomata in the absence of ABA and sensitive stomata in the presence of ABA. This would enable them to maximize photosynthesis at the expense of water loss when water was not limiting. However, when water becomes limiting and the ABA concentration in the leaf increases, the ‘ stomata become sensitive to the intercellular [C02], and their aperture will depend on the photosynthetic demand for C02. In the case of Zea mays, the stomata are always sensitive to C02, and, in the experiments described here, 57 they are able to keep the intercellular [C02] at or near the break point of the photosynthetic curve. Photo- synthesis appears tO be maximized and the rate of water loss is low. Amaranthus powellii is an interesting case in that the stomata open much wider than is necessary to maximize photosynthesis when ABA is not present. It would seem that this would be disadvantageous to the plant since a large amount of water is being lost. When ABA is present, [C02] is controlled near the break point of the photosynthetic curve which greatly improves the water use efficiency without decreasing the net rate of assimilation. Although this strategy (if indeed it is a strategy) is puzzling, it appears to work since Amaranthus powellii is a very successful weed. Other strategies will no doubt be discovered in the future. For example, it has recently been found (25) that a C3 winter annual Camissonia claviformis, found in the deserts Of the southwestern United States has exceptionally high rates of photosynthesis (higher than any reported C4 species). In order to allow enough C02 into the mesophyll of the leaf, it has very 2s-l) meaning that wide stomatal apertures (102 cmole- a great deal of water is being lost. However, the extremely high rate of photosynthesis allows the plant to complete its entire life within 6 to 10 weeks during 58 the time of the year when water is available. Thus, the plant appears to maximize photosynthetic rate at the expense of large water losses. It would be interesting to examine what would happen if the water supply ran out before the plant was able to complete its life cycle (i.e., is ABA formed and are the stomata sensitized to €02?). l) 2) 3) 4) 5) 6) 7) 8) 9) 10) LI ST OF REFERENCES Black, C., Photosynthetic carbon fixation in relation to net C02 uptake. Ann. Rev. Plant Physiol. 24, 253-286 (1973) Cowan, I.R., Stomatal behavior and environment. Advances in Botanical Research (in press)(l977) Cummins, W.R., Kende, H., and Raschke, K., Specificity and reversibility of the rapid stomatal response to abscisic acid. Planta 99, 347-351 (1971) Cummins, W.R. and Sondheimer, E., Activity of the Asymmetric Isomers of abscisic acid in a rapid bioassay. — Planta 111, 365-369 (1973) Darwin, R., Observation on stomata Roy. Soc. London, Phil. Trans. Ser B 190, 531- 608 (1898) Farquhar, G.D., A study of the responses of stomata to perturbations of environment. Ph.D. thesis, Australian National University, Canberra (1973) Farquhar, G.D. and Cowan, I.R., Oscillations in stomatal conductance. The influence of environmental gain. Plant Physiol. 54, 769-772 (1974) Freudenberger, H., Die Reaktion der Schliesszellen auf Kohlensaure und Sauerstoff-Entzug. Protoplasma 35, 15-54 (1940) Harrison, M.A. and Walton, C.D., Abscisic acid metabolism in water-stressed bean leaves. Plant Physiol. 56, 250-254 (1975) Heath, O.V.S., Control of stomatal movement by a reduction in the normal carbon dioxide content of the air. Nature 161, 179-181 (1948) 59 ll) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 60 Heath, O.V.S. and Meidner, H., The influence of water strain on the minimum intercellular space carbon dioxide concentration and stomatal movement in wheat leaves. J. Expt. Bot. 12, 226-242 (1961) Heath, O.V.S. and Mansfield, T.A., A recording porometer with detachable cups operating on four separate leaves. Proc. Roy. Soc. B. 156, l-13 (1962) Hiron, R.W.P. and Wright, S.T.C., The role of endogenous abscisic acid in the response of plants to stress. J. Expt. Bot. 24, 769-781 (1973) Jones, R.J. and Mansfield, T.A., Suppression of stomatal Opening in leaves treated with abscisic acid. J. Expt. Bot. 21, 714-719 (1970) Jones, R.W., Principles of biological regulation; an introduction to feedback systems. Academic Press, New York (1973) Ketellapper, H.J., Stomatal physiology. Ann, Rev. Plant Physiol. 14, 249-270 (1963) Kriedemann, P.E., Loveys, B.R., Fuller, G.L., and Leopold, A.C., Abscisic acid and stomatal regulation Plant Physiol. 49, 842-847 (1972) Kriedemann, P.E., Loveys, B.R., and Downton W.J.S., Internal control of stomatal physiology and photosynthesis. II. Photosynthetic responses to phaseic acid. AustJ J. Plant Physiol. 2, 553-67 (1975) Linsbauer, K., Beitrage zur Kenntnis der Spaltoffnungs- bewegungen. Flora (Jena) N.F. 9, 100-143 (1916) Little, C.H.A. and Eidt, D.C., Effect of abscisic acid on budbreak and transpiration in woody species. Nature 220, 498-499 (1968) Mansfield, T.A., Delay in the response of stomata to abscisic acid in COz-free air. J. Expt. Botany 27, 559-564 (1976)' 22) 23) 24) 25) 26) 27) 28) 29) 30) 31) 32) 61 Meidner, H., and Mansfield, T.A., Physiology of stomata. New York: McGraw-Hill (1968) Mittelheuser, C.J. and Van Steveninck, R.F.M., Stomatal closure and inhibition of transpiration induced by (RS)-abscisic acid. Nature 221, 281-282 (1969) Mizrahi, Y., Blumenfeld, A., and Richmond, A., Abscisic acid and transpiration in leaves in relation to osmotic root stress. Plant Physiol. 46, 169-171 (1970) Mooney, H.A., Ehleringer, J. and Berry, J.A., High photosynthetic capacity of a winter annual in Death Valley. Science 194, 322-323 (1976) Raschke, K., Die Stomata als Glieder eines schwin- gungsfahigen COz-Regelsystems. Experimenteller Nachweis an Zea mays L. Z. F. Naturforsch. 20b, 1261-1270 (1965) Raschke, K., Abscisic acid sensitizes stomata to C02 in leaves of Xanthium strumerium L. Proc. VIII Intern. Conf. on Plant Growth Substances. Tokyo (1973) - Raschke, K., Simultaneous requirement of carbon dioxide and abscisic acid for stomatal closing in Xanthium strumerium L. Planta 125, 243-259 (1975) Raschke, K., Stomatal action. Ann. Rev. Plant Physiol. 26, 309-339 (1975) Raschke, K., How stomata resolve the dilemma of opposing priorities. Phil. Trans. R. Soc. Lond. B., 273, 551-560 (1976) Raschke, K., Pierce, M., and Popiela, C.C., Abscisic acid content and stomatal sensitivity to C02 in leaves of Xanthium strumarium L. after pretreatments in warm and cold growth chambers. Plant Physiol. 57, 115-121 (1976) Riggs, D.S., Control theory and physiological feedback mechanisms. Williams and Wilkins Press, Baltimore (1970) 62 33) Wright, S.T.C., An increase in the "inhibitor-B" content of detached wheat leaves following a period of wilting. Planta 86, 10-20 (1969) 34) Wright, S.T.C. and Hiron, R.W.P., (+) Abscisic acid, the growth inhibitor induced in detached leaves by a period of wilting. Nature 224, 719-720 (1969) 35) Zeevaart, J.A.D., (+)-Abscisic acid content of spinach in relation to photoperiod and waterstress. Plant Physiol. 48, 86-90 (1971)