Planta (Berl.) 91, 336--363 (1970)
Temperature Dependence of COg Assimilation and Stomatal Aperture in Leaf Sections of Zea mays KLAUS RASCHKE Botanisches Institut der Universit~t GieSen, Germany, and MSU/AEC Plant l~eseareh Laboratory, East Lansing, Michigan, U.S.A. Received January 31, 1970
Summary. CO2 exchange and air flow through the stomata were measured in leaf sections of Zea mays at temperatures between 7 and 52~ and under optimal water supply. The results were summarized in polynomials fitted to the data. In leaf samples brought from 16~ and darkness into different experimental temperatures and light, CO2 assimilation has a maximum near 30~ Above 37~ (in other experiments above 41~ net CO2 uptake stops abruptly and is replaced by CO2 evolution in light. If a 1-hr treatment with 25~ and light is inserted between darkness and the experimental temperatures, the threshold above which the assimilatory system collapses shifts 3 degrees upwards, to 40~ (or 44~ the decline of CO2 assimilation with high temperatures is less steep than without pretreatment; and the upper compensation point moves upscale by as much as 5 degrees. Stomata1 conductance for C02 does not, in general, follow an optimum curve with temperature. Between 15 and 35~ it is approximately proportional to net C02 assimilation, indicating control by C02; but above 35~ stomatal aperture increases further with temperature (and so does stomatal variability): the stomata escape the control by CO2 and above 40~ may be wide open even if CO2 is being evolved. Stomatal conductance for CO2 below 15~ may also be larger than would be proportional to CO2 assimilation. Net CO2 assimilation and stomatal conductance at 25~ were reduced if the leaf samples were pretreated with temperatures below approximately 20~ and above 30~ Stomata were more sensitive to past temperatures than was CO2 assimilation.
Introduction T h e g u a r d cells of a s t o m a respond to low CO 2 concentrations i n the leaf tissue b y opening the s t o m a t a l pore (Linsbauer, 1916; Scarth, 1932 ; F r e u d e n b e r g c r , 1941 ; H e a t h , 1948). W h e n air is forced t h r o u g h a maize leaf b y m e a n s of a flow porometer, the s t o m a t a respond to variations i n the CO 2 c o n t e n t of the air i n a q u a n t i t a t i v e m a n n e r , with the result t h a t s t o m a t a l c o n d u c t i v i t y , a t least u n d e r these e x p e r i m e n t a l conditions, is p r o p o r t i o n a l to the assimilatory power of the photos y n t h e t i c system, provided water s u p p l y does n o t become the factor l i m i t i n g s t o m a t a l a p e r t u r e (Raschke, 1965b, 1966).
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The present investigation was made to determine whether the effects of temperature on both CO~ fluxes in the leaf, the flux of CO 2 through the stomata on one hand and on net assimilation of CO 2 on the other are identical. If they are this would indicate that stomatal conductivity for CO 2 depends solely on C02 assimilation. If they are not, the C02 sensitivity of the guard cells would possess a temperature dependence of its own. A quantitative description of this dependence could help to identify the still unknown mechanism by which the guard cells sense CO 2. Knowledge of the temperature dependences of COs assimilation and stomatal conductance for C02 (and water vapor) is also required for designing models, specific for a given species, describing the complex relationships between energy and gas exchange of leaves and leaf canopies. For this purpose the results of temperature measurements should be expressed in the form of equations. In this investigation this was done by fitting second or third order polynomials to the data. Zea mays was chosen as experimental plant because stomatal responses to COs, fight, and changes in water loss and supply were already studied in this species (Karv6, 1961 ; Raschke, 1967 ; Raschke and Kiihl, 1969). In order to minimize interference by water stress all experiments were done with leaf sections supplied with water from their cut edges, and the humidity of the air was kept high. Net uptake of CO S and stomatal behavior vary considerable among leaves of the same individual, and pronounced gradients in activity exist along leaves. These variations can be eliminated, at least partially, by computing relative responses. Two methods are suitable. In the first one, half the number of the leaf sections is exposed to a reference tem. perature (in this investigation, 25 ~ and the other half to a temperature which varies from experiment to experiment. If a section treated with a certain temperature has two of its former neighbors exposed to 25 ~ its response can be compared to the arithmetic mean of the neighbors' responses. In the other method, leaf sections are subjected to temperature cycles between 25 ~ and another temperature, and responses to the two consecutive temperatures are compared. The latter method has the advantage of obtaining relative data with one and the same materiM. Unfortunately, however, the results are now affected by after-effects which past temperature treatments have on the physiological behavior of the samples. The present investigation was conducted in such a way that relative responses could be computed following either method. I t was also possible to describe quantitatively the effect past temperatures had on CO S assimilation and stornatM conductance measured subsequently at 25 ~. If it is true that stomatal conductance for COs is a function of the photosynthetic uptake of CO S a close coupling between the two variables
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should become most striking when assimilation and CO s flux through the stomata are measured simultaneoulsy, or almost simultaneously, in the same leaf sections. The present work included, therefore, a series of measurements of net uptake of COs which alternated with recordings of air flow through the same leaf samples. Flow was produced by periodic brief applications of pressure differences across the leaf sections. In this investigation, stomatal width does not appear to be very appropriate to characterize the physiological state of the guard cells. If stomata function as controllers of CO s exchange it is the flow of COs which should be the criterion. This flow, however, is not only a function of stomatal aperture but also of the CO2 concentration difference between the two ends of the stomatal pores. CO s flux through the stomata should also be the better parameter because it should help in recognizing a regulatory function of the guard cells if there is one. In the present investigation, stomatal responses were therefore recorded by means of flow porometers. KnoMng the CO s content of the air one can easily compute C02 fluxes through the leaf samples. One has, however, to realize that CO s fluxes into the leaves, as computed from porometer flows, almost certainly will be lower than the ones which would occur if the leaves could obtain COs from the surrounding air by diffusion. In a flow porometer the stomata through which the air enters the leaf will most probably be narrower than under diffusion conditions because they come into contact with air which has been only slightly depleted of COs by a diffusion process superimposed on the viscous flow. The stomata at the exit are exposed to air which has passed the assimilatory tissue and has lost a large proportion of its CO s. These stomata are most probably wider open than the entrance stomata; the entrance stomata, therefore, will limit air flow through and CO s supply to the leaf. In addition, it can be shown that at a given stomatal aperture and constant CO s gradient diffusion is more effective than viscous flow to supply the tissue with CO s. The results of the present investigation may, therefore, produce some information on the temperature dependence of stomatal COs regulation but they cannot be used to predict quantitatively stomatal behavior under field conditions without more experimentation to provide the additional functions necessary for such an extrapolation. Causal relationships between the magnitudes of net CO S assimilation and of C02 flux through the stomata can best be seen when both processes have reached their steady states. In order to recognize the establishment of equilibria time courses of assimilation and porometer flow had to be followed. Velocities of equilibration are themselves expected to be temperature dependent. The first part of the Results section of this paper contains information on the effect of temperature on the behavior with time of COz assimilation
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a n d COs flux t h r o u g h the s t o m a t a . The second p a r t describes how absolute COs fluxes d e p e n d o n t e m p e r a t u r e a n d o n past t e m p e r a t u r e , a n d the t h i r d a n d f o u r t h p a r t s s u m m a r i z e the c o m p u t a t i o n of relative t e m p e r a t u r e responses. The t h i r d p a r t also includes the results of quasis i m u l t a n e o u s m e a s u r e m e n t s of COs assimilation a n d porometer flow on the same leaf material. These m e a s u r e m e n t s were m a d e to d e m o n s t r a t e as directly as possible the presence or absence of a close coupling b e t w e e n CO S assimilation a n d s t o m a t a l c o n d u c t i v i t y for CO, over a wide temp e r a t u r e range.
Material and Methods Plants. Zea mays L., or. "Asflo" (van der Have, Hamburg, Germany), identical with Pioneer variety 395, was grown in Knop's solution in growth cabinets, with a temperature of 26~ during the 16-hr day and 16~ during the night (more details in Raschke, 1966). Temperatures were controlled by contact thermometers which were exposed as test bodies to the radiation at the upper level of the plants. According to Bottl~nder (1966), in growth chambers growth and dry matter production is more significantly correlated with the temperature of an appropriate test body than with air temperature. As a rule, the experimental material was the fourth leaf, in the sequence of emergence, with fully developed auricles. Leaf length at this time was between 4~ and 55 cm; 23--27 days had elapsed since sowing. Air. Outside air was used (300--400 ppm C02, v/v, most frequently 350 ppm~ maximum daily drift 25 ppm). Humidity was kept constant at a level of 5 torr below saturation water-vapor pressure at leaf temperature. During the CO~assimilation experiments, water-vapor pressure in the air was 13 torr. Porometers and Assimilation Chambers (1%aschke, 1965a). Double chambers, made from Plexiglas, were submersed in groups of four in constant-temperature baths. The exposed leaf area in a chamber was 2.5 eme. Air at a pressure of 100 mm water was applied to the abaxial leaf side. The resulting porometer flow was measured by photoelectric transducers and recorded on a multipoint recorder with a scanning period of 28.8 see. Identical euvettes were used for the measurement of CO~ uptake. The lower and upper chamber were flushed with air at a rate of 301 hr -1. The readings of the flow meters used were corrected after comparison with a wet test meter. When CO2 assimilation and porometer flow were measured alternately, eight leaf chambers and one empty control chamber were connected to the gas analyzer in sequence, for 1 min each. Then, all chambers were switched automatically to operate as porometers for 1 rain and air flow through the stomata was scanned. Lea/ Temperatures were measured by running copper-constantan thermocouples (0.1 mm diameter) along the lower surfaces of the leaf sections and recording their output (with reference to ice) on the multipoint recorder. Temperatures were adjusted and maintained by constant temperature circulators (Meflgers Lauda, Germany). Light was produced by a water-cooled xenon arc lamp (Osram XBF 6000 W/I) with a 3 mm thick infra-red absorbing filter (KG 1; Schott, Mainz, Germany). The irradianee in the chambers was 28 mW cm-2 ~ 0.4 cal cm-2 min-1. For the lamp and filter used in this investigation this corresponds to a quantum-flux density of 130 nE cm-2 see-1. Gas Analyzers. Carbon-dioxide uptake was measured with a differential infrared gas analyzer (Uras 1, Hartmann & Braun, Frankfurt a. M., Germany). It was
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calibrated daffy by applying known pressure increments to gas of known composition. The results were corrected by a factor obtained from calibrations using mixing pumps (WSsthoff, Bochum, GelTaany) (for a discussion of the error introduced by pressure calibration see Legg and Parkinson, 1968). The absolute COz content of the air was recorded with a second infra-red gas analyzer, calibrated with test gases or mixing pumps. Execution o/the Experiments. At the end of the dark period, leaves were taken from the growth cabinets and cut under water into sections 3 to 4 cm long. Four sections, taken from two leaves, were brought to a temperature of 25~ another four from the same leaves to the temperature of the particular experiment. In some eases all eight sections were cut from the same leaf. Sections which had been adjacent in the leaf were always exposed to different temperature treatments. After switching on the light the circulators were adjusted so that the leaf sections attained the desired temperatures as closely as possible. When a more or less constant rate of C02 assimilation or a more or less constant porometer flow was reached and maintained for 20 or 30 rain (first equilibration), temperatures were changed by exchanging circulator connections. A new temperature equilibrium was reached within 10 to 30 rain, depending on the temperature step applied (second equilibration). The leaf sections were subjected to up to four cycles between an experimental temperature and the reference temperature of 25~ Fresh leaves were used for each new periodic comparison betwcer~ 25~ and another temperature. A temperature range from 7 to 52~ was covered by increments of approximately 5~. Evaluation. Absolute and relative data on C02 assimilation and CO2 flow through the stomata were obtained by following 860 equilibration processes in 250 sections cut from 50 leaves. These data, together with information on leaf temperatures and behavior with time, were processed at the Michigan State University Computer Laboratory, utilizing programs of the Michigan Agricultural Experiment Station for statistical analysis, curve fitting, and plotting.
Results
Time Courses o/COs Assimilation and Porometer Flow Through the Stomata and their Temperature Dependences COs Uptake reached a s t e a d y state faster a n d r e m a i n e d more stable with t i m e t h a n air flow t h r o u g h the s t o m a t a (Figs. 1, 2). After exposure to light, t h e assimilation process took I 0 - - 6 0 rain (most f r e q u e n t l y 30 rain) to become c o n s t a n t ; after a t e m p e r a t u r e change i t took 5 - - 6 0 rain (most f r e q u e n t l y 24 min). B e t w e e n 10 a n d 45 ~ e q u i l i b r a t i o n t i m e varied v e r y m u c h with the leaves used a n d showed no obvious v a r i a t i o n with t e m p e r a t u r e . Assimilation did n o t stabilize a t all a t 10 ~ a n d below, a n d a t 45 ~ a n d above. A t 50 ~ the leaves lost COs. This CO 2 evolution decreased with time. Air Flow Through the Stomata u s u a l l y reached a s t e a d y state 45 to 120 m i n after t h e light was switched on. T h e average e q u i l i b r a t i o n t i m e a t 25 ~ was 75 mln. W i t h increasing t e m p e r a t u r e i t increased to 250 rain a t 40 ~ b u t declined a g a i n with still higher t e m p e r a t u r e s (Fig. 3). Ad-
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341
justment of the stomata to a new temperature level took less time than an opening movement in response to light (Fig. 2). Equilibration after the first temperature change took on the average 40 rain, after the second only 25 rain. Again, equilibration time was particularly long at 40 ~.
Transient Stomatal Responses to Temperature Changes. I n many instances, stomata responded during the first 2 - - 4 rain after a temperature change in a direction opposite to the final one (Fig. 2). Stomatal aperture temporarily increased when the ultimate pore size was smaller than that before the change, and vice versa. Frequently, the transient movement was followed by a damped oscillation (Fig. 4). The amplitude of the transient response was large when air flow through the stomata was high ; it was smaller when the porometer reading was low. Transient responses were also correlated with the temperature interval applied, and with the time elapsed from the beginning of the experiment (Table 1). Table 1. CoeHicientso/determination (r2) /or the correlation betweentransient stomatal responses to sudden changes in temperature and several variables n ~ Number of observations; sign = sign of correlation coefficient r. Variable
Absolute COSflux through stomata Time from beginning of experiment ~iagnitude of temperature interval Temperature before change Temperature after change
Change in temperature downward (n = 83)
upward (n = 70)
sign
r2
sign
r~
(~-) (--) (+) (--) (+)
0.24 0.19 0.11 0.10 0.14
(-4-) (--) (--) (+) (--)
25 ~ Srt:5.72--0.676 T +0.0254 T 2 (4) 25~ Srt = 0.83 -- 0.042 T +0.0017 T 2
--0.000242 T a
n
r2
s
64 72
0.95 0.85
0.08 0.12
79 81
0.33 0.69
2.06 0.44
determination r ~ for the correlation between leaf temperature and CO2 flux t h r o u g h the s t o m a t a (Table 5). I f the past temperatures were variable, r~ was 0.33; if the past temperature was 25 ~ r 2 was 0.69. Of the three curves in the b o t t o m diagram in Fig. 11 the one for the change from a past t e m p e r a t u r e of 25 ~ to different following temperatures agreed best with the relative COs fluxes obtained b y comparing fluxes in neighboring leaf sections exposed to different temperatures (Fig. 8).
Discussion The Relation Between COs Assimilation and CO s F l u x Through the Stomata. I n the v a r i e t y of Zea mays investigated, net C02 assimilation and CO 2 flow t h r o u g h the s t o m a t a are proportional to each other between 15 and 35 ~ (Fig. 10). W i t h i n this temperature range, the COs-regulation hypothesis of stomatal function seems to hold. Regression equations c o m p u t e d for the two COs fluxes appear to agree less well (Fig. 8) b u t it should be kept in m i n d t h a t in a regression preconceived functions are fitted to the d a t a (in this case second and third order polynomials), with the result t h a t details are lost. This is particularly true if several experiments are combined in one statistical t r e a t m e n t as was the case here. Below 15 ~ and especially above 35 ~ the ratio between CO 2 flux t h r o u g h the s t o m a t a and CO 2 assimilation increases. Above 35 ~ Zea mays s t o m a t a m a y be wide open even if the leaves evolve COs. This m a y be the same p h e n o m e n o n as the s t o m a t a l opening in darkness observed in X a n t h i u m leaves when t h e y were b r o u g h t from 27 to 36 ~ (Mansfield, 1965). R e d u c e d or even absent stomata1 sensitivity to CO s at high temperatures m a y explain the m i n i m u m of CO s flux t h r o u g h the s t o m a t a which in Zea appeared near 35 ~ (Fig. 9, b o t t o m , 16 ~ -> Tx).
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Below 35 ~ stomatal conductivity is a function of the CO 2 content of the intercellular spaces and, therefore, follows the temperature curve of C02 assimilation which has a maximum near 30 ~ Above 35 ~ stomatal conductivity appears to b e determined by a process which accelerates rapidly with temperature. In conclusion, one can postulate that the magnitude of stomatal aperture indicates a balance between two oppossing processes, one of which is sensitive to COs. This hypothesis is similar to the hypothesis suggested by Williams (1954) who propounds that closure is an "active" process, or by Walker and Zelitch (1963) who propose that "biochemical reactions responsible for opening and closing occur simultaneously in the light". The uncoupling of stomatal conductivity from COs exchange at high temperatures may have ecological consequences, because it makes substantial transpirational cooling of leaves possible when net COs assimilation has stopped. Wide open stomata and leaf temperatures as much as 9 degrees below air temperature occurred in Xanthium at an air temperature of 43 ~ and an irradiation with 1.2 cal em -2 min -1 in a climatized wind tunnel, and at an air temperature of 36 ~ in the field (Drake et al., in press). Van Bavel and Ehrler (1968) also observed high transpirational cooling of leaves when air temperature was around 38~ in an irrigated sorghum crop with a radiation balance approaching 1 cal cm -2 rain -I leaf temperatures were several degrees below air temperature, and stomatal diffusion resistances were found to be very low. Temperature Dependence o/Net COs Assimilation. This investigation yielded several estimating equations describing net COs assimilation as a function of tissue temperature. They differ in their coefficients of determination, r 2, as well as in their standard errors of estimate, s (Tables 2, 4, 5). The highest correlation with temperature and the smallest errors resulted from the regression of relative COs fluxes measured while the leaf sections were subjected to changes from the experimental temperatures to 25 ~ (Table5, Eq. (1); r 2 ~ 0.95; s : 0.08). Nevertheless, this estimating equation is not suitable for representing the dependence of net-COs assimilation on temperature because low scatter and high correlation with temperature are due to the superimposition of very pronounced aftereffects of past temperatures on CO s assimilation at 25 ~ These aftereffects were not fully compensated by computing the geometric means of the responses to upward and downward temperature changes (p. 353 ; Fig. 11). On the other hand, relative fluxes computed from the responses to changes from 25 ~ to another temperature alone agreed very well with the relative values found by comparison of neighboring leaf sections. The curves in Figs. 8, 9 (top, 16 ~ --> Tx), and 11 (top, "present temp. ~- variable") are thought, therefore to be the best approximations to the true relationship between net-CO2 assimilation
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357
and leaf temperature which can be obtained from m y experimental data. They apply strictly only to the leaf material defined in the Methods section of this paper and for an irradiation with 28 m W cm -s of filtered xenon light. Nevertheless, some of the findings on COs assimilation in relation to temperature permit some generalizations. As is known to any farmer, net production of plant matter in maize is low at temperatures around 10 ~ I n m y experiments net uptake of COs at 10 ~ amounted to only between 20 and 30 % of t h a t measured at 30 ~ With increasing temperature the course of net-CO s assimilation b y maize appears to be determined by three processes (as in the response model of Itesketh and Baker, 1967): 1. an acceleration of CO2 uptake with temperature, 2. an antagonistic process which gradually increases with temperature, and 3. a rapid inactivation of the assimilatory apparatus which sets in abruptly above a certain threshold temperature. The combination of the first two processes results in the well-known optimum curve, with a m a x i m u m net uptake of CO s between 28 and 32 ~ (Figs. 6, 8, 9, top; 11 top, "present temp. ~-- variable"). The increase of net-COs assimilation between 15 and 25 ~ corresponds to a Q10 between 1.7 and 1.9 or an apparent activation energy of approximately 10.5 kcal tool -1 (computed from Tables 4, 5). These values shed some doubt on the validity of assimilation curves for maize which Gates (1965) derived from literature data and which Idso (1968) used for the computation of crop photosynthesis. Gates arrives at functions which for conditions comparable to the ones used in m y experiments predict a Q10 of 4.2 or an activation energy of 23 kcal tool -1. These values are twice as large as the ones found experimentally. The optimum curve of net-COs assimilation breaks off sharply at a critical temperature near 40 ~ CO s begins to be evolved at a temperature 3 or 4 degrees above this threshold; in individual leaves the transition from a high rate of CO s uptake to a negative balance m a y occur within 2 ~ At temperatures below 40 ~ COz evolution by maize leaves in the light is observed only if the leaves are seneseing. The abrupt change from a positive to a negative CO2 balance near 40 ~ m a y be typical for plants employing the diearboxylie acid p a t h w a y of carbon assimilation. This is snggested b y data of Murata et al. (1965) and Hofstra and Hesketh (1969) on the effects of temperature upon the COs exchange of plants with and without photorespiration. The authors presented smooth assimilation curves, but the data points which are shown in their figures indicate more or less pronounced breaks in the relation between net-CO s uptake and temperature near 40 ~ for Zea ~ays, Xndropogon sorghum, Echinochloa crus-galli and Xtriplex nummularia. An exposure of leaf sections to light and 25 ~ for about one hour increases the stabihty of the photosynthetic system against heat. The
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threshold temperature, above which the assimilatory system collapses, rises by about 3 degrees, the upper compensation point moves upscale b y about 5 degrees, and the decline of assimilation with high temperatures is less steep (p. 345). This conditioning is perhaps the result of an accumulation of photosynthetic products in the chloroplasts, similar to the protection of isolated chloroplasts against frost or drought by addition of sugars (Heber and Santarius, 1964; Santarius and Iteber, 1967). Temperature Dependence o/CO 2 Flux Through the Stomata. Several methods were used to arrive at a description of the relation between CO 2 flux through the stomata and temperature. Among these the comparison of fluxes through neighboring leaf sections exposed to different temperatures resulted in the highest coefficients of determination and in the least errors of estimate, although the latter ones still were considerable (Tables 4, 5). Relative fluxes computed from temperature changes from 25 ~ to other temperatures were very similar to the ones obtained from comparisons of leaf neighbors (Figs. 8, 11). The curves on the CO 2 flux through the stomata in Figs. 8--11 (" present temp. ~ variable" in the last one) are therefore, suitable for a discussion of the temperature dependence of stomatal aperture. Few data have been published on this relationship, and most of these have either not been obtained under sufficiently controlled conditions, or cover too small a temperature range. What Ketellapper wrote in 1963 still holds: "Much of the work with temperature is difficult to evaluate because the temperature effects on the stomata cannot always be separated from the effects of temperature o n . . . the water deficit of the leaf and on the relative humidity. This led to conflicting conclusions which for these reasons have not been discussed here". I, therefore, refer only to Sts (1962) microscopic measurements of the stomatal aperture in leaves of Vicia/aba. According to Sts stomatal opening increases steeply and almost linearly between 5 and 35 ~ in C02-containing air and light, and between 5 and 40 ~ in C02-free air and darkness. Above these temperatures stomata undergo slight closure. From the curves St~lfelt has provided, a Qlo of 1.5 between 20 and 30 ~ for stomatal apertures in Vieia can be computed. My data on Zea result in a Q10 of 1.8 between 20 and 30 ~ and of 1.6 between 30 and 40 ~ for the COs flux through the stomata which corresponds to an "activation energy" of 10.5 keal/mol. This approximate agreement is somewhat surprising because stomatal width and stomatal conductivity for viscous flow are not linearly related. The agreement cannot be a result of stomatal regulation of CO S exchange because StMfelt found a similar temperature dependence of stomatal aperture in COs-free air and darkness. The relationship between temperature and stomatal aperture (and CO2 flux
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359
through the stomata) resembles the temperature dependence of the water conductivity in roots of transpiring maize plants, as reported by Brouwer (1965). The Q10 of this process is 1.8 between 20 and 30 ~ Both functions, stomatal aperture and water conductivity of roots, rise with temperature more steeply than the inverse of the viscosity of water. The latter has an activation energy of 4.1 kcal reel -1 and cannot, therefore, be solely responsible for the temperature dependence of the two physiological processes compared. Another physical parameter which could cause the temperature dependence of stomatal aperture is the solubility of COs in water (Levitt, 1967), but again the inverse of this does not increase as rapidly with temperature (activation energy =- 5.7 kcal reel -1) as does stomatal aperture. If water supply is not limiting stomatal aperture increases with temperature to as high as 50 ~. This finding confirms Moss' (1963) observation of wide-open stomata in maize leaves at 40 ~ although Moss himself doubted his own results because transpiration and photosynthesis were less at 40 ~ than at lower temperatures. I t appears that occurrence of wide-open stomata at high temperatures in leaves well supplied with water can be generally expected, for Drake et al. (in press) found maximum apertures in leaves of X a n t h i u m strumarium above 40 ~ and Heath and Meidner (1957) established that high temperature caused stomatal opening in onion leaves, provided accumulation of a high CO S concentration in the leaves was prevented. A further peculiarity of the stomatal temperature response is the minimum of aperture occurring between 13 and 18 ~ (Fig. 6, second equilibration; Figs. 8--11). A similar increase in stomatal conductivity was also observed below 10 ~ in X a n t h i u m if the moisture content of the air was high (Drake et al., in press). This phenomenon could be explained on the basis of a stomatal model involving two opposing mechanisms, as was already suggested in the discussion of the coupling between COs assimilation and stomatal conductivity for COe. I t could be that below 15 ~ the closing force is more affected by decreasing temperatures than the opening force; the turgor in the epidermal cells could be the force antagonizing opening. Temperatures above 35 ~ increase the lability of the stomatal mechanism, and repeated changes between high temperatures and 25 ~ fatigue the stomatal apparatus to such an extent that the original steep increase of stomatal aperture with temperature no longer occurs but an optimum curve is established similar to that of CO S assimilation, with a maximum stomatal aperture between 25 and 30 ~ (Fig. 6, third equilibration). Stomata responded to sudden temperature increases with a transient closure, and to temperature drops with a temporary opening movement
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(Figs. 2, 4). Both responses were often followed by a damped oscillation, very similar to the stomatal responses observed after sudden changes in water supply (Rasehke, in press). One possible explanation is that this effect is due to a decrease with temperature of the viscosity of water (roughly 20% per 10~ at physiological temperatures). A rise in temperature would be equivalent to an improvement in water supply. This effect should increase with increasing transpiration; there was indeed a correlation of the transient stomatal response with stomatal aperture (Table 1). The first (often oscillatory) phase of stomatal adjustment to a new temperature level could represent an adjustment to the new water supply situation. In the experiment described in Fig. 4 this phase took about 7 min if the first approach to an equilibration is taken as end point, or about 20 rain if the subsequent oscillation is included. Conclusion Net CO~ assimilation and stomatal conductance for COs are proportional to each other between 15 and 35 ~ (Fig. 10); the hypothesis of stomatal regulation of the intercellular C02 concentration seems to hold within this temperature range and for the variety of maize used in this investigation. The CO~ sensitivity of the stomata itself depends on temperature: The temperature curves of net-CO s assimilation and CO s flux through the stomata diverge below 15 ~ and particularly above 35 ~. The regression equations for net-C02 assimilation and CO S flux through the stomata (Tables 4, 5) can be used to estimate the relative temperature dependence of the CO~ sensitivity of the stomata. The divergence between COs assimilation and stomatal condnetance for CO S below 15 and above 35 ~ adds to already available evidence (Heath, 1959; Ketcllapper, 1963) against a requirement of newly synthesized assimilates for the operation of the stomatal mechanism. Net CO 2 assimilation of Zea mays as a function of temperature approximates an optimum curve which abruptly breaks at a temperature near 40 ~ This effect is so pronounced that a different estimating equation is to be used to describe net-COs assimilation above the threshold temperature (Table 4, Fig. 8). Stomatal conductance for COa (and other gases) increases with temperature. Q10's between 1.5 and 1.8 have been found (p. 358). The temperature dependence of the stomatal diffusion resistance must, therefore, be included in the design of leaf or crop models. Idso (1969) as well as Waggoner (1969) omitted the temperature dependence of the stomatal diffusion resistances from their leaf and canopy models, presumably because pertinent information was not available to them. The present investigation provides estimating equations on which more realistic models can be based. An attempt to include the temperature
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sensitivity of the stomata into a theoretical treatment of the energy and gas exchange of a leaf has recently been made by Raschke (1968). Although the estimating equations given in Tables 2, 4 and 5 were derived from measurements with flow porometers they appear to be generally representative for the relative course of stomatal aperture with temperature because continuous air flow through the stomata yielded functions which agreed with the ones obtained by extracting cube roots from the data of intermittent porometer readings. The functions found agree also quite satisfactorily with Sts (1962) microscopical measurements of stomatal aperture on Vicia /aba at different temperatures. The " a c t i v a t i o n energy" of stomatal aperture is approximately 10.5 kcal mo1-1 between 15 and 35 ~ A value of about this magnitude is to be expected for a physiological mechanism under metabolic control ; it is equal to the aetiviation energy found for net-COs assimilation between 15 and 25 ~ The computation of an activation energy of stomatal opening m a y not, however, be meaningful at all if stomatal aperture is the result of at least two opposing processes with differing temperature dependences. Determinations of stomatal opening and closing velocities as functions of temperature appear, therefore, to be desirable. They could also help to test the conclusion t h a t transient stomatal responses to temperature changes (Figs. 2, 4; p. 360) can in p a r t be explained as resulting from changes in the viscosity of water. Net CO~. assimilation and CO S flux through the stomata depend strongly on previous temperature treatments. For some well-defined situations these dependences can be described quantitatively (Tables 3, 5; Figs. 7, ll). The range of past temperatures which cause no negative aftereffects on COs assimilation and stomatal aperture is surprisingly small. I t extents about from 20 to 30 ~ Pretreatment of leaf tissue with light at 25 ~ made the photosynthetic system more resistant to high temperatures (p. 345). This conditioning effect appears to be worth a further study. The experimental part of this work was supported by the Deutsche Forschungsgemeinschaft, and the evaluation of the data by the U. S. Atomic Energy Commission under contract AT(11-1)-1338. Use of the Michigan State University computing facilities was made with support, in part, from the National Science Foundation. I thank Miss WMtraud Bonke for careful assistance during the performance and the evaluation of the experiments, and Miss Erika Hartwieg, Miss Freia Schulz-BMdes and Mrs. Sandra Watson for help in processing the data.
References Bottl~nder, W. : Technische Hilfsmittel zur Erzeugung und Ermittlung optimaler Lebensbedingungen fiir Pflanzen. Bodenkultur ][7, 195--236 (1966). Brouwer, R. : Water movement across the root. Syrup. Soc. exp. Biol. 19, 131--149 (1965).
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Drake, B.G., Raschke, K., Salisbury, F . B . : Temperatures and transpiration resistances of X a n t h i u m leaves as affected by air temperature, humidity, and wind speed. Plant Physiol. (in press). Freudenberger, H. : Die Reaktionen der SchlieBzellen auf Kohlens~ure und Sauerstoffentzug. Protoplasma (Vienna) 35, 15--54 (1941). Gates, D.M.: Energy, plants, and ecology. Ecology 46, 1--13 (1965). Heath, O . V . S . : Control of stomatal movement by a reduction in the normal carbon dioxide content of the air. l~ature (Lond.) 161, 179--181 (1948). Light and carbon dioxide in stomatal movements. Handbuch der Pflanzenphysiologic (W. Rnhland, ed.), vol. XVII/1, p. 415--464. Berlin-G5ttingenHeidelberg: Springer 1959. - - Meidner, H. : Effects of carbon dioxide and temperature oa stomata of A l l u r e ce2a L. Nature (Lond.) 180, 180--182 (1957). Heber, U. W., Santarius, K. A.: Loss of adenosine triphosphate synthesis caused by freezing and its relationship to frost hardiness problems. Plant Physiol. 89, 712--719 (1964). ttesketh, J., Baker, D.: Light and carbon assimilation by plant communities. Crop Sei. 7, 285--293 (1967). Hofstra, G., Hesketh, J. D.: Effects of temperature on the gas exchange of leaves in the light and dark. Planta (Berl.) 85, 228--237 (1969). Idso, S.B." A holoeoenotie analysis of environment-plant relationships. Teehn. Bull. No 264, Agrie. Expt. star., Univ. of Minnesota, St. Paul (1968). A theoretical framework for the photosynthetic modeling of plant communities. Advancing Frontiers of Plant Sciences 23, 91--118 (1969). Karv6, A.: Die Wirkung verschiedener Lichtqualiti~ten auf die Offnungsbewegung der Stomata. Z. Bot. 49, 47--72 (1961). Ketellapper, H. J.: Stomatal physiology. Ann. Rev. Plant Physiol. 14, 249--267 (1963). Legg, B. J., Parkinson, K. J.: Calibration of infra-red gas analysers for use with carbon dioxide. J. Sci. Instr. (J. Phys. E) Ser. II, 1, 1003--1006 (1968). Levitt, J. : The mechanism of stomatal action. Planta (Berl.) 74, 101--118 (1967). Linsbauer, K.: BeitrEge zur Kenntnis der SpaltSffnungsbewegungen. Flora (Jena) 9, 100--143 (1916). Mansfield, T. A. : Studies in stomatal behaviour X I L Opening in high temperature in darkness. J. exp. Bot. 16, 721--731 (1965). Meidner, H., Spanner, D. C.: The differential transpiration porometer. J. exp. Bot. 10, 190--205 (1959). Moss, D. lq.: The effect of environment on gas exchange of leaves. Conn. Agrie. Exp. Star., Bull. No 664, 86--100 (1963). Murata, Y., Iyama, J., ttonma, T.: Studies on the photosynthesis of forage crops IV. Influence of air-temperature upon the photosynthesis and respiration of alfalfa and several southern type forage crops. Proe. Crop Sci. Soc. Jap. 84, 154--158 (1965). Raschke, K. : Eignung und Konstruktion registrierender Porometer fiir das Studium der Schliel3zellenphysiologie. Planta (Berl.) 67, 225--241 (1965a). - - Die Stomata als Glieder eines schwingungsf~higen C02-Regelsystems. Experimenteller !~achweis an Zea mays L. Z. Naturforsch. 26b, 1261--1270 (1965b). Die Reaktionen des COs-l~egelsystems in den SehlieBzellen yon Zea mays auf weifles Licht. Planta (Berl.) 68, 111--140 (1966). M Zur Steuernng der Transpiration durch die Photosynthese. Ber. dtseh, bot. Ges. 80, 138--144 (1967).
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Raschke, K. : Ein Modell zur Besehreibung des Energie- und Gasaustausehes eines Maisblattes. Studia biophys. 11, 41---46 (1968). Stomatal responses to pressure changes and to interruptions in the water supply of detached leaves of Zea mays L. Plant Physiol., in press. Santarius, K.A., Heber, U. : Das Verhalten yon Hill-Reaktion und Photophosphorylierung isolierter Chloroplasten in AbhEngigkeit yore Wassergehalt. II. Wasserentzug fiber CaCle. Planta (Berl.) 73, 109--137 (1967). Scarth, G. W. : l~Iechanism of the action of light and other factors on stomatal movement. Plant Physiol. 7, 481--504 (1932). Sts M. G. : The effect of temperature on opening of the stomatal cells. Physiol. Plantarum (Cph.) 15, 772--779 (1962). Van Bavel, C. H. M., Ehrler, W. L.: Water loss from a sorghum field and s~omatal control. Agron. J. 69, 84---86 (1968). Waggoner, P. E. : Predicting the effect upon net photosynthesis of changes in leaf metabolism and physics. Crop Sci. 9, 315--321 (1969). Walker, D. A., Zelitch, I. : Some effects of metabolic inhibitors, temperature and anaerobic conditions on stomatal movement. Plant Physiol. 88, 390--396 (1963). Williams, W. T. : A new theory of the mechanism of stomatal movement. J. exp. Bot. 5, 343--352 (1954). -
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K. Rasehke MSU/AEC Plant Research Laboratory East Lansing, Michigan 48823, U.S.A.
Temperature Dependence of COg Assimilation and Stomatal Aperture in Leaf Sections of Zea mays KLAUS RASCHKE Botanisches Institut der Universit~t GieSen, Germany, and MSU/AEC Plant l~eseareh Laboratory, East Lansing, Michigan, U.S.A. Received January 31, 1970
Summary. CO2 exchange and air flow through the stomata were measured in leaf sections of Zea mays at temperatures between 7 and 52~ and under optimal water supply. The results were summarized in polynomials fitted to the data. In leaf samples brought from 16~ and darkness into different experimental temperatures and light, CO2 assimilation has a maximum near 30~ Above 37~ (in other experiments above 41~ net CO2 uptake stops abruptly and is replaced by CO2 evolution in light. If a 1-hr treatment with 25~ and light is inserted between darkness and the experimental temperatures, the threshold above which the assimilatory system collapses shifts 3 degrees upwards, to 40~ (or 44~ the decline of CO2 assimilation with high temperatures is less steep than without pretreatment; and the upper compensation point moves upscale by as much as 5 degrees. Stomata1 conductance for C02 does not, in general, follow an optimum curve with temperature. Between 15 and 35~ it is approximately proportional to net C02 assimilation, indicating control by C02; but above 35~ stomatal aperture increases further with temperature (and so does stomatal variability): the stomata escape the control by CO2 and above 40~ may be wide open even if CO2 is being evolved. Stomatal conductance for CO2 below 15~ may also be larger than would be proportional to CO2 assimilation. Net CO2 assimilation and stomatal conductance at 25~ were reduced if the leaf samples were pretreated with temperatures below approximately 20~ and above 30~ Stomata were more sensitive to past temperatures than was CO2 assimilation.
Introduction T h e g u a r d cells of a s t o m a respond to low CO 2 concentrations i n the leaf tissue b y opening the s t o m a t a l pore (Linsbauer, 1916; Scarth, 1932 ; F r e u d e n b e r g c r , 1941 ; H e a t h , 1948). W h e n air is forced t h r o u g h a maize leaf b y m e a n s of a flow porometer, the s t o m a t a respond to variations i n the CO 2 c o n t e n t of the air i n a q u a n t i t a t i v e m a n n e r , with the result t h a t s t o m a t a l c o n d u c t i v i t y , a t least u n d e r these e x p e r i m e n t a l conditions, is p r o p o r t i o n a l to the assimilatory power of the photos y n t h e t i c system, provided water s u p p l y does n o t become the factor l i m i t i n g s t o m a t a l a p e r t u r e (Raschke, 1965b, 1966).
Temperature Dependence of 002 Assimilation and Stomata] Aperture
337
The present investigation was made to determine whether the effects of temperature on both CO~ fluxes in the leaf, the flux of CO 2 through the stomata on one hand and on net assimilation of CO 2 on the other are identical. If they are this would indicate that stomatal conductivity for CO 2 depends solely on C02 assimilation. If they are not, the C02 sensitivity of the guard cells would possess a temperature dependence of its own. A quantitative description of this dependence could help to identify the still unknown mechanism by which the guard cells sense CO 2. Knowledge of the temperature dependences of COs assimilation and stomatal conductance for C02 (and water vapor) is also required for designing models, specific for a given species, describing the complex relationships between energy and gas exchange of leaves and leaf canopies. For this purpose the results of temperature measurements should be expressed in the form of equations. In this investigation this was done by fitting second or third order polynomials to the data. Zea mays was chosen as experimental plant because stomatal responses to COs, fight, and changes in water loss and supply were already studied in this species (Karv6, 1961 ; Raschke, 1967 ; Raschke and Kiihl, 1969). In order to minimize interference by water stress all experiments were done with leaf sections supplied with water from their cut edges, and the humidity of the air was kept high. Net uptake of CO S and stomatal behavior vary considerable among leaves of the same individual, and pronounced gradients in activity exist along leaves. These variations can be eliminated, at least partially, by computing relative responses. Two methods are suitable. In the first one, half the number of the leaf sections is exposed to a reference tem. perature (in this investigation, 25 ~ and the other half to a temperature which varies from experiment to experiment. If a section treated with a certain temperature has two of its former neighbors exposed to 25 ~ its response can be compared to the arithmetic mean of the neighbors' responses. In the other method, leaf sections are subjected to temperature cycles between 25 ~ and another temperature, and responses to the two consecutive temperatures are compared. The latter method has the advantage of obtaining relative data with one and the same materiM. Unfortunately, however, the results are now affected by after-effects which past temperature treatments have on the physiological behavior of the samples. The present investigation was conducted in such a way that relative responses could be computed following either method. I t was also possible to describe quantitatively the effect past temperatures had on CO S assimilation and stornatM conductance measured subsequently at 25 ~. If it is true that stomatal conductance for COs is a function of the photosynthetic uptake of CO S a close coupling between the two variables
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should become most striking when assimilation and CO s flux through the stomata are measured simultaneoulsy, or almost simultaneously, in the same leaf sections. The present work included, therefore, a series of measurements of net uptake of COs which alternated with recordings of air flow through the same leaf samples. Flow was produced by periodic brief applications of pressure differences across the leaf sections. In this investigation, stomatal width does not appear to be very appropriate to characterize the physiological state of the guard cells. If stomata function as controllers of CO s exchange it is the flow of COs which should be the criterion. This flow, however, is not only a function of stomatal aperture but also of the CO2 concentration difference between the two ends of the stomatal pores. CO s flux through the stomata should also be the better parameter because it should help in recognizing a regulatory function of the guard cells if there is one. In the present investigation, stomatal responses were therefore recorded by means of flow porometers. KnoMng the CO s content of the air one can easily compute C02 fluxes through the leaf samples. One has, however, to realize that CO s fluxes into the leaves, as computed from porometer flows, almost certainly will be lower than the ones which would occur if the leaves could obtain COs from the surrounding air by diffusion. In a flow porometer the stomata through which the air enters the leaf will most probably be narrower than under diffusion conditions because they come into contact with air which has been only slightly depleted of COs by a diffusion process superimposed on the viscous flow. The stomata at the exit are exposed to air which has passed the assimilatory tissue and has lost a large proportion of its CO s. These stomata are most probably wider open than the entrance stomata; the entrance stomata, therefore, will limit air flow through and CO s supply to the leaf. In addition, it can be shown that at a given stomatal aperture and constant CO s gradient diffusion is more effective than viscous flow to supply the tissue with CO s. The results of the present investigation may, therefore, produce some information on the temperature dependence of stomatal COs regulation but they cannot be used to predict quantitatively stomatal behavior under field conditions without more experimentation to provide the additional functions necessary for such an extrapolation. Causal relationships between the magnitudes of net CO S assimilation and of C02 flux through the stomata can best be seen when both processes have reached their steady states. In order to recognize the establishment of equilibria time courses of assimilation and porometer flow had to be followed. Velocities of equilibration are themselves expected to be temperature dependent. The first part of the Results section of this paper contains information on the effect of temperature on the behavior with time of COz assimilation
Temperature Dependence of CO2 Assimilation and Stomatal Aperture
339
a n d COs flux t h r o u g h the s t o m a t a . The second p a r t describes how absolute COs fluxes d e p e n d o n t e m p e r a t u r e a n d o n past t e m p e r a t u r e , a n d the t h i r d a n d f o u r t h p a r t s s u m m a r i z e the c o m p u t a t i o n of relative t e m p e r a t u r e responses. The t h i r d p a r t also includes the results of quasis i m u l t a n e o u s m e a s u r e m e n t s of COs assimilation a n d porometer flow on the same leaf material. These m e a s u r e m e n t s were m a d e to d e m o n s t r a t e as directly as possible the presence or absence of a close coupling b e t w e e n CO S assimilation a n d s t o m a t a l c o n d u c t i v i t y for CO, over a wide temp e r a t u r e range.
Material and Methods Plants. Zea mays L., or. "Asflo" (van der Have, Hamburg, Germany), identical with Pioneer variety 395, was grown in Knop's solution in growth cabinets, with a temperature of 26~ during the 16-hr day and 16~ during the night (more details in Raschke, 1966). Temperatures were controlled by contact thermometers which were exposed as test bodies to the radiation at the upper level of the plants. According to Bottl~nder (1966), in growth chambers growth and dry matter production is more significantly correlated with the temperature of an appropriate test body than with air temperature. As a rule, the experimental material was the fourth leaf, in the sequence of emergence, with fully developed auricles. Leaf length at this time was between 4~ and 55 cm; 23--27 days had elapsed since sowing. Air. Outside air was used (300--400 ppm C02, v/v, most frequently 350 ppm~ maximum daily drift 25 ppm). Humidity was kept constant at a level of 5 torr below saturation water-vapor pressure at leaf temperature. During the CO~assimilation experiments, water-vapor pressure in the air was 13 torr. Porometers and Assimilation Chambers (1%aschke, 1965a). Double chambers, made from Plexiglas, were submersed in groups of four in constant-temperature baths. The exposed leaf area in a chamber was 2.5 eme. Air at a pressure of 100 mm water was applied to the abaxial leaf side. The resulting porometer flow was measured by photoelectric transducers and recorded on a multipoint recorder with a scanning period of 28.8 see. Identical euvettes were used for the measurement of CO~ uptake. The lower and upper chamber were flushed with air at a rate of 301 hr -1. The readings of the flow meters used were corrected after comparison with a wet test meter. When CO2 assimilation and porometer flow were measured alternately, eight leaf chambers and one empty control chamber were connected to the gas analyzer in sequence, for 1 min each. Then, all chambers were switched automatically to operate as porometers for 1 rain and air flow through the stomata was scanned. Lea/ Temperatures were measured by running copper-constantan thermocouples (0.1 mm diameter) along the lower surfaces of the leaf sections and recording their output (with reference to ice) on the multipoint recorder. Temperatures were adjusted and maintained by constant temperature circulators (Meflgers Lauda, Germany). Light was produced by a water-cooled xenon arc lamp (Osram XBF 6000 W/I) with a 3 mm thick infra-red absorbing filter (KG 1; Schott, Mainz, Germany). The irradianee in the chambers was 28 mW cm-2 ~ 0.4 cal cm-2 min-1. For the lamp and filter used in this investigation this corresponds to a quantum-flux density of 130 nE cm-2 see-1. Gas Analyzers. Carbon-dioxide uptake was measured with a differential infrared gas analyzer (Uras 1, Hartmann & Braun, Frankfurt a. M., Germany). It was
340
K. Rasehke:
calibrated daffy by applying known pressure increments to gas of known composition. The results were corrected by a factor obtained from calibrations using mixing pumps (WSsthoff, Bochum, GelTaany) (for a discussion of the error introduced by pressure calibration see Legg and Parkinson, 1968). The absolute COz content of the air was recorded with a second infra-red gas analyzer, calibrated with test gases or mixing pumps. Execution o/the Experiments. At the end of the dark period, leaves were taken from the growth cabinets and cut under water into sections 3 to 4 cm long. Four sections, taken from two leaves, were brought to a temperature of 25~ another four from the same leaves to the temperature of the particular experiment. In some eases all eight sections were cut from the same leaf. Sections which had been adjacent in the leaf were always exposed to different temperature treatments. After switching on the light the circulators were adjusted so that the leaf sections attained the desired temperatures as closely as possible. When a more or less constant rate of C02 assimilation or a more or less constant porometer flow was reached and maintained for 20 or 30 rain (first equilibration), temperatures were changed by exchanging circulator connections. A new temperature equilibrium was reached within 10 to 30 rain, depending on the temperature step applied (second equilibration). The leaf sections were subjected to up to four cycles between an experimental temperature and the reference temperature of 25~ Fresh leaves were used for each new periodic comparison betwcer~ 25~ and another temperature. A temperature range from 7 to 52~ was covered by increments of approximately 5~. Evaluation. Absolute and relative data on C02 assimilation and CO2 flow through the stomata were obtained by following 860 equilibration processes in 250 sections cut from 50 leaves. These data, together with information on leaf temperatures and behavior with time, were processed at the Michigan State University Computer Laboratory, utilizing programs of the Michigan Agricultural Experiment Station for statistical analysis, curve fitting, and plotting.
Results
Time Courses o/COs Assimilation and Porometer Flow Through the Stomata and their Temperature Dependences COs Uptake reached a s t e a d y state faster a n d r e m a i n e d more stable with t i m e t h a n air flow t h r o u g h the s t o m a t a (Figs. 1, 2). After exposure to light, t h e assimilation process took I 0 - - 6 0 rain (most f r e q u e n t l y 30 rain) to become c o n s t a n t ; after a t e m p e r a t u r e change i t took 5 - - 6 0 rain (most f r e q u e n t l y 24 min). B e t w e e n 10 a n d 45 ~ e q u i l i b r a t i o n t i m e varied v e r y m u c h with the leaves used a n d showed no obvious v a r i a t i o n with t e m p e r a t u r e . Assimilation did n o t stabilize a t all a t 10 ~ a n d below, a n d a t 45 ~ a n d above. A t 50 ~ the leaves lost COs. This CO 2 evolution decreased with time. Air Flow Through the Stomata u s u a l l y reached a s t e a d y state 45 to 120 m i n after t h e light was switched on. T h e average e q u i l i b r a t i o n t i m e a t 25 ~ was 75 mln. W i t h increasing t e m p e r a t u r e i t increased to 250 rain a t 40 ~ b u t declined a g a i n with still higher t e m p e r a t u r e s (Fig. 3). Ad-
Temperature Dependence of CO2 Assimilation and Stomatal Aperture
341
justment of the stomata to a new temperature level took less time than an opening movement in response to light (Fig. 2). Equilibration after the first temperature change took on the average 40 rain, after the second only 25 rain. Again, equilibration time was particularly long at 40 ~.
Transient Stomatal Responses to Temperature Changes. I n many instances, stomata responded during the first 2 - - 4 rain after a temperature change in a direction opposite to the final one (Fig. 2). Stomatal aperture temporarily increased when the ultimate pore size was smaller than that before the change, and vice versa. Frequently, the transient movement was followed by a damped oscillation (Fig. 4). The amplitude of the transient response was large when air flow through the stomata was high ; it was smaller when the porometer reading was low. Transient responses were also correlated with the temperature interval applied, and with the time elapsed from the beginning of the experiment (Table 1). Table 1. CoeHicientso/determination (r2) /or the correlation betweentransient stomatal responses to sudden changes in temperature and several variables n ~ Number of observations; sign = sign of correlation coefficient r. Variable
Absolute COSflux through stomata Time from beginning of experiment ~iagnitude of temperature interval Temperature before change Temperature after change
Change in temperature downward (n = 83)
upward (n = 70)
sign
r2
sign
r~
(~-) (--) (+) (--) (+)
0.24 0.19 0.11 0.10 0.14
(-4-) (--) (--) (+) (--)
25 ~ Srt:5.72--0.676 T +0.0254 T 2 (4) 25~ Srt = 0.83 -- 0.042 T +0.0017 T 2
--0.000242 T a
n
r2
s
64 72
0.95 0.85
0.08 0.12
79 81
0.33 0.69
2.06 0.44
determination r ~ for the correlation between leaf temperature and CO2 flux t h r o u g h the s t o m a t a (Table 5). I f the past temperatures were variable, r~ was 0.33; if the past temperature was 25 ~ r 2 was 0.69. Of the three curves in the b o t t o m diagram in Fig. 11 the one for the change from a past t e m p e r a t u r e of 25 ~ to different following temperatures agreed best with the relative COs fluxes obtained b y comparing fluxes in neighboring leaf sections exposed to different temperatures (Fig. 8).
Discussion The Relation Between COs Assimilation and CO s F l u x Through the Stomata. I n the v a r i e t y of Zea mays investigated, net C02 assimilation and CO 2 flow t h r o u g h the s t o m a t a are proportional to each other between 15 and 35 ~ (Fig. 10). W i t h i n this temperature range, the COs-regulation hypothesis of stomatal function seems to hold. Regression equations c o m p u t e d for the two COs fluxes appear to agree less well (Fig. 8) b u t it should be kept in m i n d t h a t in a regression preconceived functions are fitted to the d a t a (in this case second and third order polynomials), with the result t h a t details are lost. This is particularly true if several experiments are combined in one statistical t r e a t m e n t as was the case here. Below 15 ~ and especially above 35 ~ the ratio between CO 2 flux t h r o u g h the s t o m a t a and CO 2 assimilation increases. Above 35 ~ Zea mays s t o m a t a m a y be wide open even if the leaves evolve COs. This m a y be the same p h e n o m e n o n as the s t o m a t a l opening in darkness observed in X a n t h i u m leaves when t h e y were b r o u g h t from 27 to 36 ~ (Mansfield, 1965). R e d u c e d or even absent stomata1 sensitivity to CO s at high temperatures m a y explain the m i n i m u m of CO s flux t h r o u g h the s t o m a t a which in Zea appeared near 35 ~ (Fig. 9, b o t t o m , 16 ~ -> Tx).
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Below 35 ~ stomatal conductivity is a function of the CO 2 content of the intercellular spaces and, therefore, follows the temperature curve of C02 assimilation which has a maximum near 30 ~ Above 35 ~ stomatal conductivity appears to b e determined by a process which accelerates rapidly with temperature. In conclusion, one can postulate that the magnitude of stomatal aperture indicates a balance between two oppossing processes, one of which is sensitive to COs. This hypothesis is similar to the hypothesis suggested by Williams (1954) who propounds that closure is an "active" process, or by Walker and Zelitch (1963) who propose that "biochemical reactions responsible for opening and closing occur simultaneously in the light". The uncoupling of stomatal conductivity from COs exchange at high temperatures may have ecological consequences, because it makes substantial transpirational cooling of leaves possible when net COs assimilation has stopped. Wide open stomata and leaf temperatures as much as 9 degrees below air temperature occurred in Xanthium at an air temperature of 43 ~ and an irradiation with 1.2 cal em -2 min -1 in a climatized wind tunnel, and at an air temperature of 36 ~ in the field (Drake et al., in press). Van Bavel and Ehrler (1968) also observed high transpirational cooling of leaves when air temperature was around 38~ in an irrigated sorghum crop with a radiation balance approaching 1 cal cm -2 rain -I leaf temperatures were several degrees below air temperature, and stomatal diffusion resistances were found to be very low. Temperature Dependence o/Net COs Assimilation. This investigation yielded several estimating equations describing net COs assimilation as a function of tissue temperature. They differ in their coefficients of determination, r 2, as well as in their standard errors of estimate, s (Tables 2, 4, 5). The highest correlation with temperature and the smallest errors resulted from the regression of relative COs fluxes measured while the leaf sections were subjected to changes from the experimental temperatures to 25 ~ (Table5, Eq. (1); r 2 ~ 0.95; s : 0.08). Nevertheless, this estimating equation is not suitable for representing the dependence of net-COs assimilation on temperature because low scatter and high correlation with temperature are due to the superimposition of very pronounced aftereffects of past temperatures on CO s assimilation at 25 ~ These aftereffects were not fully compensated by computing the geometric means of the responses to upward and downward temperature changes (p. 353 ; Fig. 11). On the other hand, relative fluxes computed from the responses to changes from 25 ~ to another temperature alone agreed very well with the relative values found by comparison of neighboring leaf sections. The curves in Figs. 8, 9 (top, 16 ~ --> Tx), and 11 (top, "present temp. ~- variable") are thought, therefore to be the best approximations to the true relationship between net-CO2 assimilation
Temperature Dependence of COs Assimilation and Stomatal Aperture
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and leaf temperature which can be obtained from m y experimental data. They apply strictly only to the leaf material defined in the Methods section of this paper and for an irradiation with 28 m W cm -s of filtered xenon light. Nevertheless, some of the findings on COs assimilation in relation to temperature permit some generalizations. As is known to any farmer, net production of plant matter in maize is low at temperatures around 10 ~ I n m y experiments net uptake of COs at 10 ~ amounted to only between 20 and 30 % of t h a t measured at 30 ~ With increasing temperature the course of net-CO s assimilation b y maize appears to be determined by three processes (as in the response model of Itesketh and Baker, 1967): 1. an acceleration of CO2 uptake with temperature, 2. an antagonistic process which gradually increases with temperature, and 3. a rapid inactivation of the assimilatory apparatus which sets in abruptly above a certain threshold temperature. The combination of the first two processes results in the well-known optimum curve, with a m a x i m u m net uptake of CO s between 28 and 32 ~ (Figs. 6, 8, 9, top; 11 top, "present temp. ~-- variable"). The increase of net-COs assimilation between 15 and 25 ~ corresponds to a Q10 between 1.7 and 1.9 or an apparent activation energy of approximately 10.5 kcal tool -1 (computed from Tables 4, 5). These values shed some doubt on the validity of assimilation curves for maize which Gates (1965) derived from literature data and which Idso (1968) used for the computation of crop photosynthesis. Gates arrives at functions which for conditions comparable to the ones used in m y experiments predict a Q10 of 4.2 or an activation energy of 23 kcal tool -1. These values are twice as large as the ones found experimentally. The optimum curve of net-COs assimilation breaks off sharply at a critical temperature near 40 ~ CO s begins to be evolved at a temperature 3 or 4 degrees above this threshold; in individual leaves the transition from a high rate of CO s uptake to a negative balance m a y occur within 2 ~ At temperatures below 40 ~ COz evolution by maize leaves in the light is observed only if the leaves are seneseing. The abrupt change from a positive to a negative CO2 balance near 40 ~ m a y be typical for plants employing the diearboxylie acid p a t h w a y of carbon assimilation. This is snggested b y data of Murata et al. (1965) and Hofstra and Hesketh (1969) on the effects of temperature upon the COs exchange of plants with and without photorespiration. The authors presented smooth assimilation curves, but the data points which are shown in their figures indicate more or less pronounced breaks in the relation between net-CO s uptake and temperature near 40 ~ for Zea ~ays, Xndropogon sorghum, Echinochloa crus-galli and Xtriplex nummularia. An exposure of leaf sections to light and 25 ~ for about one hour increases the stabihty of the photosynthetic system against heat. The
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threshold temperature, above which the assimilatory system collapses, rises by about 3 degrees, the upper compensation point moves upscale b y about 5 degrees, and the decline of assimilation with high temperatures is less steep (p. 345). This conditioning is perhaps the result of an accumulation of photosynthetic products in the chloroplasts, similar to the protection of isolated chloroplasts against frost or drought by addition of sugars (Heber and Santarius, 1964; Santarius and Iteber, 1967). Temperature Dependence o/CO 2 Flux Through the Stomata. Several methods were used to arrive at a description of the relation between CO 2 flux through the stomata and temperature. Among these the comparison of fluxes through neighboring leaf sections exposed to different temperatures resulted in the highest coefficients of determination and in the least errors of estimate, although the latter ones still were considerable (Tables 4, 5). Relative fluxes computed from temperature changes from 25 ~ to other temperatures were very similar to the ones obtained from comparisons of leaf neighbors (Figs. 8, 11). The curves on the CO 2 flux through the stomata in Figs. 8--11 (" present temp. ~ variable" in the last one) are therefore, suitable for a discussion of the temperature dependence of stomatal aperture. Few data have been published on this relationship, and most of these have either not been obtained under sufficiently controlled conditions, or cover too small a temperature range. What Ketellapper wrote in 1963 still holds: "Much of the work with temperature is difficult to evaluate because the temperature effects on the stomata cannot always be separated from the effects of temperature o n . . . the water deficit of the leaf and on the relative humidity. This led to conflicting conclusions which for these reasons have not been discussed here". I, therefore, refer only to Sts (1962) microscopic measurements of the stomatal aperture in leaves of Vicia/aba. According to Sts stomatal opening increases steeply and almost linearly between 5 and 35 ~ in C02-containing air and light, and between 5 and 40 ~ in C02-free air and darkness. Above these temperatures stomata undergo slight closure. From the curves St~lfelt has provided, a Qlo of 1.5 between 20 and 30 ~ for stomatal apertures in Vieia can be computed. My data on Zea result in a Q10 of 1.8 between 20 and 30 ~ and of 1.6 between 30 and 40 ~ for the COs flux through the stomata which corresponds to an "activation energy" of 10.5 keal/mol. This approximate agreement is somewhat surprising because stomatal width and stomatal conductivity for viscous flow are not linearly related. The agreement cannot be a result of stomatal regulation of CO S exchange because StMfelt found a similar temperature dependence of stomatal aperture in COs-free air and darkness. The relationship between temperature and stomatal aperture (and CO2 flux
Temperature Dependence of C02 Assimilation and Stomatal Aperture
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through the stomata) resembles the temperature dependence of the water conductivity in roots of transpiring maize plants, as reported by Brouwer (1965). The Q10 of this process is 1.8 between 20 and 30 ~ Both functions, stomatal aperture and water conductivity of roots, rise with temperature more steeply than the inverse of the viscosity of water. The latter has an activation energy of 4.1 kcal reel -1 and cannot, therefore, be solely responsible for the temperature dependence of the two physiological processes compared. Another physical parameter which could cause the temperature dependence of stomatal aperture is the solubility of COs in water (Levitt, 1967), but again the inverse of this does not increase as rapidly with temperature (activation energy =- 5.7 kcal reel -1) as does stomatal aperture. If water supply is not limiting stomatal aperture increases with temperature to as high as 50 ~. This finding confirms Moss' (1963) observation of wide-open stomata in maize leaves at 40 ~ although Moss himself doubted his own results because transpiration and photosynthesis were less at 40 ~ than at lower temperatures. I t appears that occurrence of wide-open stomata at high temperatures in leaves well supplied with water can be generally expected, for Drake et al. (in press) found maximum apertures in leaves of X a n t h i u m strumarium above 40 ~ and Heath and Meidner (1957) established that high temperature caused stomatal opening in onion leaves, provided accumulation of a high CO S concentration in the leaves was prevented. A further peculiarity of the stomatal temperature response is the minimum of aperture occurring between 13 and 18 ~ (Fig. 6, second equilibration; Figs. 8--11). A similar increase in stomatal conductivity was also observed below 10 ~ in X a n t h i u m if the moisture content of the air was high (Drake et al., in press). This phenomenon could be explained on the basis of a stomatal model involving two opposing mechanisms, as was already suggested in the discussion of the coupling between COs assimilation and stomatal conductivity for COe. I t could be that below 15 ~ the closing force is more affected by decreasing temperatures than the opening force; the turgor in the epidermal cells could be the force antagonizing opening. Temperatures above 35 ~ increase the lability of the stomatal mechanism, and repeated changes between high temperatures and 25 ~ fatigue the stomatal apparatus to such an extent that the original steep increase of stomatal aperture with temperature no longer occurs but an optimum curve is established similar to that of CO S assimilation, with a maximum stomatal aperture between 25 and 30 ~ (Fig. 6, third equilibration). Stomata responded to sudden temperature increases with a transient closure, and to temperature drops with a temporary opening movement
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(Figs. 2, 4). Both responses were often followed by a damped oscillation, very similar to the stomatal responses observed after sudden changes in water supply (Rasehke, in press). One possible explanation is that this effect is due to a decrease with temperature of the viscosity of water (roughly 20% per 10~ at physiological temperatures). A rise in temperature would be equivalent to an improvement in water supply. This effect should increase with increasing transpiration; there was indeed a correlation of the transient stomatal response with stomatal aperture (Table 1). The first (often oscillatory) phase of stomatal adjustment to a new temperature level could represent an adjustment to the new water supply situation. In the experiment described in Fig. 4 this phase took about 7 min if the first approach to an equilibration is taken as end point, or about 20 rain if the subsequent oscillation is included. Conclusion Net CO~ assimilation and stomatal conductance for COs are proportional to each other between 15 and 35 ~ (Fig. 10); the hypothesis of stomatal regulation of the intercellular C02 concentration seems to hold within this temperature range and for the variety of maize used in this investigation. The CO~ sensitivity of the stomata itself depends on temperature: The temperature curves of net-CO s assimilation and CO s flux through the stomata diverge below 15 ~ and particularly above 35 ~. The regression equations for net-C02 assimilation and CO S flux through the stomata (Tables 4, 5) can be used to estimate the relative temperature dependence of the CO~ sensitivity of the stomata. The divergence between COs assimilation and stomatal condnetance for CO S below 15 and above 35 ~ adds to already available evidence (Heath, 1959; Ketcllapper, 1963) against a requirement of newly synthesized assimilates for the operation of the stomatal mechanism. Net CO 2 assimilation of Zea mays as a function of temperature approximates an optimum curve which abruptly breaks at a temperature near 40 ~ This effect is so pronounced that a different estimating equation is to be used to describe net-COs assimilation above the threshold temperature (Table 4, Fig. 8). Stomatal conductance for COa (and other gases) increases with temperature. Q10's between 1.5 and 1.8 have been found (p. 358). The temperature dependence of the stomatal diffusion resistance must, therefore, be included in the design of leaf or crop models. Idso (1969) as well as Waggoner (1969) omitted the temperature dependence of the stomatal diffusion resistances from their leaf and canopy models, presumably because pertinent information was not available to them. The present investigation provides estimating equations on which more realistic models can be based. An attempt to include the temperature
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sensitivity of the stomata into a theoretical treatment of the energy and gas exchange of a leaf has recently been made by Raschke (1968). Although the estimating equations given in Tables 2, 4 and 5 were derived from measurements with flow porometers they appear to be generally representative for the relative course of stomatal aperture with temperature because continuous air flow through the stomata yielded functions which agreed with the ones obtained by extracting cube roots from the data of intermittent porometer readings. The functions found agree also quite satisfactorily with Sts (1962) microscopical measurements of stomatal aperture on Vicia /aba at different temperatures. The " a c t i v a t i o n energy" of stomatal aperture is approximately 10.5 kcal mo1-1 between 15 and 35 ~ A value of about this magnitude is to be expected for a physiological mechanism under metabolic control ; it is equal to the aetiviation energy found for net-COs assimilation between 15 and 25 ~ The computation of an activation energy of stomatal opening m a y not, however, be meaningful at all if stomatal aperture is the result of at least two opposing processes with differing temperature dependences. Determinations of stomatal opening and closing velocities as functions of temperature appear, therefore, to be desirable. They could also help to test the conclusion t h a t transient stomatal responses to temperature changes (Figs. 2, 4; p. 360) can in p a r t be explained as resulting from changes in the viscosity of water. Net CO~. assimilation and CO S flux through the stomata depend strongly on previous temperature treatments. For some well-defined situations these dependences can be described quantitatively (Tables 3, 5; Figs. 7, ll). The range of past temperatures which cause no negative aftereffects on COs assimilation and stomatal aperture is surprisingly small. I t extents about from 20 to 30 ~ Pretreatment of leaf tissue with light at 25 ~ made the photosynthetic system more resistant to high temperatures (p. 345). This conditioning effect appears to be worth a further study. The experimental part of this work was supported by the Deutsche Forschungsgemeinschaft, and the evaluation of the data by the U. S. Atomic Energy Commission under contract AT(11-1)-1338. Use of the Michigan State University computing facilities was made with support, in part, from the National Science Foundation. I thank Miss WMtraud Bonke for careful assistance during the performance and the evaluation of the experiments, and Miss Erika Hartwieg, Miss Freia Schulz-BMdes and Mrs. Sandra Watson for help in processing the data.
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Drake, B.G., Raschke, K., Salisbury, F . B . : Temperatures and transpiration resistances of X a n t h i u m leaves as affected by air temperature, humidity, and wind speed. Plant Physiol. (in press). Freudenberger, H. : Die Reaktionen der SchlieBzellen auf Kohlens~ure und Sauerstoffentzug. Protoplasma (Vienna) 35, 15--54 (1941). Gates, D.M.: Energy, plants, and ecology. Ecology 46, 1--13 (1965). Heath, O . V . S . : Control of stomatal movement by a reduction in the normal carbon dioxide content of the air. l~ature (Lond.) 161, 179--181 (1948). Light and carbon dioxide in stomatal movements. Handbuch der Pflanzenphysiologic (W. Rnhland, ed.), vol. XVII/1, p. 415--464. Berlin-G5ttingenHeidelberg: Springer 1959. - - Meidner, H. : Effects of carbon dioxide and temperature oa stomata of A l l u r e ce2a L. Nature (Lond.) 180, 180--182 (1957). Heber, U. W., Santarius, K. A.: Loss of adenosine triphosphate synthesis caused by freezing and its relationship to frost hardiness problems. Plant Physiol. 89, 712--719 (1964). ttesketh, J., Baker, D.: Light and carbon assimilation by plant communities. Crop Sei. 7, 285--293 (1967). Hofstra, G., Hesketh, J. D.: Effects of temperature on the gas exchange of leaves in the light and dark. Planta (Berl.) 85, 228--237 (1969). Idso, S.B." A holoeoenotie analysis of environment-plant relationships. Teehn. Bull. No 264, Agrie. Expt. star., Univ. of Minnesota, St. Paul (1968). A theoretical framework for the photosynthetic modeling of plant communities. Advancing Frontiers of Plant Sciences 23, 91--118 (1969). Karv6, A.: Die Wirkung verschiedener Lichtqualiti~ten auf die Offnungsbewegung der Stomata. Z. Bot. 49, 47--72 (1961). Ketellapper, H. J.: Stomatal physiology. Ann. Rev. Plant Physiol. 14, 249--267 (1963). Legg, B. J., Parkinson, K. J.: Calibration of infra-red gas analysers for use with carbon dioxide. J. Sci. Instr. (J. Phys. E) Ser. II, 1, 1003--1006 (1968). Levitt, J. : The mechanism of stomatal action. Planta (Berl.) 74, 101--118 (1967). Linsbauer, K.: BeitrEge zur Kenntnis der SpaltSffnungsbewegungen. Flora (Jena) 9, 100--143 (1916). Mansfield, T. A. : Studies in stomatal behaviour X I L Opening in high temperature in darkness. J. exp. Bot. 16, 721--731 (1965). Meidner, H., Spanner, D. C.: The differential transpiration porometer. J. exp. Bot. 10, 190--205 (1959). Moss, D. lq.: The effect of environment on gas exchange of leaves. Conn. Agrie. Exp. Star., Bull. No 664, 86--100 (1963). Murata, Y., Iyama, J., ttonma, T.: Studies on the photosynthesis of forage crops IV. Influence of air-temperature upon the photosynthesis and respiration of alfalfa and several southern type forage crops. Proe. Crop Sci. Soc. Jap. 84, 154--158 (1965). Raschke, K. : Eignung und Konstruktion registrierender Porometer fiir das Studium der Schliel3zellenphysiologie. Planta (Berl.) 67, 225--241 (1965a). - - Die Stomata als Glieder eines schwingungsf~higen C02-Regelsystems. Experimenteller !~achweis an Zea mays L. Z. Naturforsch. 26b, 1261--1270 (1965b). Die Reaktionen des COs-l~egelsystems in den SehlieBzellen yon Zea mays auf weifles Licht. Planta (Berl.) 68, 111--140 (1966). M Zur Steuernng der Transpiration durch die Photosynthese. Ber. dtseh, bot. Ges. 80, 138--144 (1967).
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Raschke, K. : Ein Modell zur Besehreibung des Energie- und Gasaustausehes eines Maisblattes. Studia biophys. 11, 41---46 (1968). Stomatal responses to pressure changes and to interruptions in the water supply of detached leaves of Zea mays L. Plant Physiol., in press. Santarius, K.A., Heber, U. : Das Verhalten yon Hill-Reaktion und Photophosphorylierung isolierter Chloroplasten in AbhEngigkeit yore Wassergehalt. II. Wasserentzug fiber CaCle. Planta (Berl.) 73, 109--137 (1967). Scarth, G. W. : l~Iechanism of the action of light and other factors on stomatal movement. Plant Physiol. 7, 481--504 (1932). Sts M. G. : The effect of temperature on opening of the stomatal cells. Physiol. Plantarum (Cph.) 15, 772--779 (1962). Van Bavel, C. H. M., Ehrler, W. L.: Water loss from a sorghum field and s~omatal control. Agron. J. 69, 84---86 (1968). Waggoner, P. E. : Predicting the effect upon net photosynthesis of changes in leaf metabolism and physics. Crop Sci. 9, 315--321 (1969). Walker, D. A., Zelitch, I. : Some effects of metabolic inhibitors, temperature and anaerobic conditions on stomatal movement. Plant Physiol. 88, 390--396 (1963). Williams, W. T. : A new theory of the mechanism of stomatal movement. J. exp. Bot. 5, 343--352 (1954). -
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K. Rasehke MSU/AEC Plant Research Laboratory East Lansing, Michigan 48823, U.S.A.
