Introduction. Physiol. Plant. 28: XYLEM WATER POTENTIAL AND TRANSPIRATION 201

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1 Physiol. Plant. 28: XYLEM WATER POTENTIAL AND TRANSPIRATION 201 Changes in Transpiration, Net Carbon Dioxide Assimilation and Leaf Water Potential Resulting from Application of Hydrostatic Pressure to Roots of Intact Pepper Plants By B. E. JANES and G. W. GEE' Plant Science Department, College of Agriculture & Natural Resources, University of Connecticut, Storrs, Connecticut (Received August 25, 1972) Abstract Hydrostacic pressures varying from 0 to 6.0 bar were applied to roots of intact Capsicum annuum L. cv. California Wonder plants growing in nutrient solution and the rates of transpiration, and net COg assimilation, apparent compensation point and leaf is'ater potential measured. Increasing the pressure on the roots of plants with roots in solution with either -0.5 or -5.0 bar osmotic potential with 1 bar increments resulted in a decrease in transpiration. With the application of 1 or 2 bar pressure the rate of transpiration returned to near or above the original rate. An application of 3 or 4 bar pressure reduced the rare of transpiration of all plants. The transpiration of plants with roots in solution with -0.5 bar osmotic potential remained at the reduced rate for as long as these pressures were maintained. The transpiration of plants with roots in solution with -5.0 bar "was only temporarily suppressed at these pressures. Changing the applied pressure from 3 or 4 bar to 0 resuited in a rapid increase in transpiration which lasted approximately 15 minutes. Tliis was followed by a decrease im transpiration to a rate lower than before the pressure was applied. The pattern of response ^^^as similar for plants at low or high light intensity or at normal or low CO2 concentrations. When leaf diffusive resistance was 6.0 s cm"" or greater, changes in net CO2 assimilation were similar to those of transpiration. The apparent QO-y compensation point increased as pressure was applied and decreased with a release in pressure. Leaf water potential increased with an increase in pressure and decreased with a decrease in pressure. The changes ia leaf water potential were frequently but not always proportional to changes in pressure. It h postulated that the responses noted were due to changes in resistance to flow of water from xylem terminals through the mesophyll cells and stomatal cavities to the atmosphere. ' Present address: Institute of Natural & Environmental Resources, College of Life Sciences & Agriculture, University of New Hampshire, Durham, New Hampshire 03824, 'U.S.A. Possible control mechanisms associated with either a variable resistance in the mesophyll or the stomatal aperture are discussed. Introduction The rate at which water moves through an intact plant or a segment of the plant depends on the water potential gradient and the resistance encountered (Slatyer 1967). The water potential gradient in an intact plant can be altered by several different techniques, for example: altering the atmospheric stress, changing water potential of substrates, cooling roots, or applying pressure to roots. Many of the studies dealing with the relationship between water potential gradient and resistance to flow have employed techniques which decrease the water potential in the xylem of roots or stems or in the mesophyll of leaves. We felt it desirable to attempt to increase water potential in the water transport system by application of hydrostatic pressure to roots. Estimates of the effect of variations in the environment on resistance to fiow of water in roots have been made by measuring the rate of flow through decapitated roots by subjecting them to a range of applied pressures (Lopushinsky 1964, Mees and Weatherly 1957). We have been unable to find any reference to experiments dealing with changes in transpiration of an intact plant resulting from the application of hydrostatic pressure to the roots. In planning these experiments it was assumed that if plants were not transpiring at the maximum rate possible for the ambient environmental conditions because of a lack of water supply at the leaf surface, an increase in the water potential of the leaves would result in an increase in the rate of transpiration (Barrs 1971). Such an increase in leaf water potential should result from a corresponding increase in water potential in roots in-

2 202 B. E. JANES AND G. W. GEE Physiol. Plant duced by applying a hydrostatic pressure to the substrate. This hypothesis was incorrect. Hydrostatic pressure applied to roots of pepper plants did increase the water potential in the stem and leaves but did not consistently increase the rate of transpiration. The data presented here documents the changes in water relations of leaves of peppers and accompanying changes in transpiration and net CO^ assimilation as a result of changes in hydrostatic pressure applied to roots of plants growing in solution of different osmotic potentials and subjected to several different evaporative demands. Materials and Methods The experimental plants Capsicum annuum L. cv. California Wonder were grown in nutrient solution in a controlled environment. Plants were approxiamtely 4 weeks old at test maturity with a leaf surface of 4 to 5 dm^. The roots of the intact plants were sealed into a specially constructed Scholander-type pressure bomb and a continuous record of transpiration, COj assimilation and leaf temperature was obtained. The pressure bomb was approximately 15 cm in diameter containing a separate vessel to hold the nutrient solution. A split top was held down with a heavy threaded ring. The intact plant was placed in the solution with a split rubber stopper around the stem. The two halfs of the top were placed around the stopper and the plant and stopper sealed with silicone rubber. The rubber stopper Tvas held in place and compressed with two plates screwed to the top. The outer ring was placed over the plant and screwed tight to hold the whole assembly in place.. The techniques and equipment used for growing plants and making measurements were described in a previous paper by Janes (1970). Compressed air was used to apply the hydrostatic pressures. A stream of air was bubbled through the nutrient solution during periods of zero applied pressure. While it was impossible to aerate the roots during the time pressure,? were applied, at no time were there any visible detrimental effects due to a lack of aeration. In most instances the changes in pressure were rapid, being completed in less than one minute. The length of time the plant roots were subjected to a particular pressure and the sequence of pressure changes are indicated as the data is presented. The environmental conditions around the plant during measurement were varied as follows: CO2 270 xl 1"' with either (1) low light (3.5 x 10* erg cm"^ s~') or (2) high light ( ^ erg cm^' j-i). pj i^^ QQ^ and low light. The low COg concentration was obtained by shutting off the supply. The time for the plant deprived of a supply of CO2 to reduce the COg concentration in the 24 liters of air in the system to a constant low level (apparent COg compensation point) varied from 30 to 45 minutes depending on the size of the plant. Before pressure was applied, the plants were allowed to equilibrate to the test environment as indicated by a steady rate of transpiration and COg assimilation. The osmotic potential of the nutrient solution was maintained at either -0.5 or -5.0 bar with polyethylene glycol (PEG 400 or 1000). Leaf water potential was estimated from the balancing pressure values of detached leaves placed in a Scholander-type pressure bomb. The values reported are balance pressure values and are not corrected for osmotic potential of xylem sap. In a recent paper (Gee et al. 1972) we show that pepper plants exposed to similar environmental conditions have a near zero osmotic component and that satisfactory estimates of leaf water potential in the range -3 to -14 bar can be obtained from bomb readings. To avoid the brief discontinuity in records resulting from opening the system to sample leaves, the usual procedure was to measure the effect of pressure changes on transpiration and net COj assimilation of intact plants without sampling leaves. The treatments were repeated using the same or an other plant and the leaves were removed for estimation of changes in leaf water potential with each change in pressure. Leaf diffusive resistances were calculated using an analogy to Ohm's law (Slatyer 1967) r = -^: r is resistance in s cm"', AC is the vapor concentration difference between leaf and ambient air expressed as water vapor density H2O ^ig cm""', and E is the rate of transpiration expressed as H^O ^ig cm"* s"*. A constant water vapor content of 8.9 (xg cm"^ was maintained in the ambient air by controlling air temperature at 25.5 C and relative humidity at 37.5 percent. Vapor content of the leaf atmosphere was estimated by assuming the air in the stomatal cavity was saturated at the leaf temperature. The values for leaf temperature were obtained by averaging the values from three, AWG no. 40 copper constantan thermocouples pressed to the underside of a leaf fully exposed to light. The rate of transpiration from this leaf was assumed to he the same as for the whole plant. Results During the course of the experimentation, pressure was applied to the roots of 40 pepper plants. The following results for individual plants are representative of the observed responses. 1. Transpiration (a) Light and osmotic potential. The changes in transpiration of plants in solutions of either -0.5 or -5,0 bar osmotic potential and exposed to either high or low light are illustrated in Figures 1 to 4. At no time were there any signs of wilting in these plants. Plants in solution with -5.0 bar osmotic potential were adjusted to increase stress over a period of 18 hours. While there

3 PhysioL Plant XYLEM WATER POTENTIAL AND TRANSPIRATION 203 Figure 1. Changes in transpiration and net assimilation rate of leaves on a pepper plant in -0.5 bar osmotic potential solution subjected to varying hydrostatic pressures on the root. Measurements made at low light. Numbers on transpiration trace indicate leaf resistance s cm~*, at the time indicated. were some variations between individual plants, there was a more or less consistent pattern or response. A sudden increase in pressure applied to roots in increments of 1 bar resulted in a temporary decrease in transpiration. With an application of I or 2 bar pressure the rate of transpiration returned to near or above the original level (except 2 bar Figure 2). An application of 3 or 4 bar pressure to roots of plants in -0.5 bar solution reduced the rate of transpiration and it remained below the rate recorded at lower pressures for as long as these pressures were maintained (Figures 1, 2 and 6). The equilibrated rate of transpiration after application of 3 or 4 bar pressure to roots of plants in solution with -5.0 bar osmotic potential was either the same or slightly higher than the rate with 2 bar of applied pressure (Figures 3, 4, 5 and 7). 7 JC 0.8 f 0.6 "a c x" OA h -TRi>HS PI RATION 1 1 AP'PUED PRessm. E BARS 5 IT.Z v 2 H 0 U R 5 ASSIMILATION X'' ji^ "^ ^ / A - ' % i 0 t US. 7 1 Figure 3. Changes in transpiration and net assimilation of leaves on a pepper plant equilibrated in -J.O bar osmotic potential solution for IS hours then subjeeted to varying hydrostatic pressures. Measurements made at low light. The numbers on transpiration trace indicate leaf resistances, s cm""', at times indicated. Changing the pressure from 3 or 4 bar to 0 resulted in a rapid increase in transpiration which lasted approximately 15 minutes. This was followed by a decrease in transpiration to a rate lower than before pressure was first applied. With the exception of plants shown in Figure 2 there was no visible indication of guttation at applied pressures of 4 bars. The apparent increase in transpiration which occurred at time of leaf sampling (break in curve Figure 2) most likely resulted from evaporation of drops on margins of the leaves. Pressures of 3 or more bar frequently resulted in guttation from plants in S r NtT ASSIMDLUTION T i I O X w * "5(1,7 ^j - n 1 1 J 1, \ (SI / "^ 1-1 V^ Z tt.pplie:d' PRESSURE ajurs 1 'll \ E" "'"^ - 0 Figure 2, Changes in transpiration and net assimilation rate of leaves on a pepper plant in -0.$ b.ar osmotic potential solution subjected to varying hydrostatic pres.sures on the root. Measurements made at high light. Numbers on transpiration trace indicate leaf resistancej, s cm"^, at the time indicated. Figure 4. Changes in transpiration and net assimilation of leaves on a pepper plant equilibrated in -5.0 bar osmotic potential solution for 42 hours then subjected to uarying hydrostatic pressures,. Measurements made at high light on the same plant as Figure 3, 24 hours later. The numbers on transpiration trace indicate leaf resistance, s cm"-'', at tim.es indicated.

4 204 B. E. JANES AND G. W. GEE Pbysiol. Plant CONCENT-nttTKM APPLIIECl PRE55URE BAAS APPLIED PnES?URE BARS Figure 5. Effect of changes in applied hydrostatic pressure on transpiration and leaf water potential of pepper plants. Solution osmotic potential changed from -0.5 to -5.0 bar osmotic potential at start of measurements. Measurements made at low light. Break in curve represents disruption due to leaf sanapling. Leaf resistances s cm~^, for critical points are indicated by figures. Leaf water potentials at 0 and 3 bars applied pressure obtained from a similarly treated plant. Tbe leaves were wilted during tbe period from 2.0 to 3.5 hours. bar solution. Higher pressures were required to produce guttation in plants in -5.0 bar solution. (b) Wilting. The effect of pressure changes on the rate of transpiration from plants which were wilted by a sudden change in osmotic potential of the nutrient solution from -0.5 to -5.0 bar while the plants were exposed to light are shown in Figure 5. The decrease in osmotic potential caused a rapid increase in rate of transpiration which reached a maximum 15 minutes Figure 7. Changes in transpiration and apparent CO2 compensation point of pepper leaves on a plant equilibrated in 5.0 bars osmotic potential nutrient solution jar 18 hours then subjected to changes in hydrostatic pressures applied to roots. Numbers on transpiration trace indicate leaf resistance, s cm"', at time indicated. MeasBremerats made at low ligbt. after the change. This was followed by a rapid decrease in rate to a value much lower than the original level. This response has been reported in some detail by Falk (1966). Approximately 1.7 hours after change of the nutrient solution osmotic potential the rate of transpiration was approximately half the rate before treatment and the leaves all showed signs of wilting. Pressure on the roots was then increased in 1 bar increments at one half hour intervals. Each increment of pressure resulted in a sudden drop in the rate of transpiration followed by an increase in rate to a slightly higher value than before the pressure was applied. The leaves gradually regained turgidity and all signs of wilting had disappeared approximately 1.7 hours after pressure was first applied. There were no signs of guttation or bleed- Figure 6. Changes in transpiration and apparent CO2 compensation point of pepper lea'ves on a plant in OJ bar osmotic potential solution subjected to varying hydrostatic pressures on the roots. Numbers on transpiration trace are leaf resistances, s cm"', at the time indicated. Measurements made at low light. Figure 8. Relationship between apparent CO2 compensation point and rate of transpiration of pepper plants. Data obtained from plants in either -0.5 or -5.0 bars osmotic potential nutrient solution, at high or low ligbt and witb varying pressures applied to roots.

5 Physiol. Plant XYLEM WATER POTENTIAL AND TRANSPIRATION 205 ing from petiole stubs even with 6 bar pressure on the roots. On release of pressure there was an abrupt rise in transpiration followed by a decrease to a very low level. (c) COg concentration. The changes in rate of transpiration of plants in an atmosphere low in COj and subjected to different hydrostatic pressures were similar to those in air with 270 [il 1"' COg. A pressure of 2 bar applied to roots of a plant in solution of -0.5 bar osmotic potential resulted in only a very brief reduction in rate of transpiration followed by an increase in rate (Figure 6). Changing the applied pressure from 4 to 0 bar resulted in a rapid increase in transpiration. This was the only instance in which the initial increase in rate of transpiration following a change in applied pressure from 3 or more bar to 0 bar was not followed by a rapid decrease. A plant in solution of -5.0 bar osmotic potential at low light and low COg (Figure 7), responded in a manner consistent with previous, results, except for a drop in transpiration for a brief period with a change in pressure from 2 to 0 bar. Changing the applied pressure from 4 to 0 bar resulted in a rapid increase in transpiration with a subsequent reduction in rate. Meidner and Mansfield (1968) have reviewed in some detail the relationship between internal COo concentration and stomatal aperture and concluded that in many instances the effect of an environmental factor on stomatal aperture was a reflection of the influence of the variable factor on internal COg concentrations. Data on the relationship between internal CO, concentration and stomatai aperture as indicated by rate of transpiration (Figure 8) were obtained from plants in an atmosphere with the COg concentration at the apparent compensation point and exposed to the different environments used in this study (high or low light, roots in nutrient solution with an osmotic potential of -0.5 or -5.0 bar and varying pressure applied to the roots). It is difficult to determine what mechanism was responsible for changes in the CO., equilibrium but these data do demonstrate the close correlation between internal CO., concentration in the range 40 to 100 fil 1"' COj and rate of transpiration over an appreciable part of the range encountered in present experiments. 2. Net CO2 assimilation Changes in the rate of net COg assimilation in plants in solution with -5.0 bar osmotic potential were similar to those for the rate of transpiration (Figures 3 and 4) indicating that stomatal control of COg diffusion was the limiting factor in these plants. The imposition of an osmotic stress resulted in partial closure of stomates reducing the rate of COg diffusion to the point where CO., concentration at the site of photochemical reaction and not light became the limiting factor. The rate of net assimilation in the plants in -0.5 bar solution and low light (Figure 1) showed a response to pressure treatments, but in contrast to plants in -5.0 bar solution the magnitude was small. There was some indication that application of 1 and 2 bar pressure increased net assimilation slightly. Stomatal aperture did not limit COg assimilation until a pressure of 3 bar was applied and leaf resistance was 6.0 s cm"'. There were changes in assimilation associated with changes in diffusive resistance when pressure was reduced from 3 to 0 bar. The indications were that the low diffusive resistance of leaves of plants in solution with -0.5 bar and at high light was at no time limiting net assimilation (Figure 2). There is no obvious explanation for reduction in net assimilation with application of 1 bar pressure to roots of plants under high light. 3. CO2 concentration The changes in CO., concentration of the air in equilibrium with plants deprived of a supply of CO, (Figures 6 and 7) were a result of variations in the equilibrium between respiration and photosynthesis of the entire top of the plants as effected by changes in hydrostatic pressure applied to roots and reflect differences in the compensation point. It is possible that the compensation point of an individual leaf would have been different from these values for the stems and leaves combined (Bravdo 1971). The slope of the COg curve (Figures 6 and 7) represents the rate at which the plant is removing CO^ from approximately 24 liters of the system. An abrupt alteration in the rate of change in CO, concentration as pressure was applied indicates that the balance between respiration and COg assinailation varied with a change of pressure on the roots. The imposition of pressure to roots of pepper plants deprived of CO.J supply increased the CO2 equilibrium concentration. On release of pressure on the roots the CO, concentration returned to approximately the pretreatment level. Plants with a low water potential in the substrate (Figure 7) had a higher equilibrium value at zero pressure than those at higher water potential (Figure 6). There was some indication that the greater the applied pressure the higher the COg concentration at equilibrium, but this was not as marked as was the change in transpiration. 4. Leaf water potential The water potential values (iljj presented in Table 1 represent the equilibrium pressure in the xylem of the leaves without a correction for the osmotic potential of the xylem sap and are typical of those obtained. Leaves for IJIL measurements were cut from the plant Va to 1 V2 hours after change in pressure. The measurements were made on plants treated similarly to those

6 206 B. E. JANES AND G. W. GEE Physiol. Plant Table 1. Effect of hydrostatic pressure applied to roots on leaf and stem water severed at root shoot junction at end of experiment. potential. Stems Plant No. Nutrient solution osmotic potential bar Water potential, bar Leaf hydrostatic pressure applied to roots, bar 0 start j 6 0 end Stem 0 end = ' > ^ ' " = ' Measurements made at high light. 2 Polyethylene giycole 1000 added at start of measurements. There was no equilibrium time. ^ Ambient CO2 concentration at CO2 compensation point. ' Petiole stubs bled when petiole cut off. ^ At! cut petioles on these plants bled when 3 bar pressure was applied. reported in Figures 1-7. Application of pressure to the roots resulted in an increase in iji^. There was some indication that there was a greater increase In IJJL per increment of applied pressure in leaves of plants with roots in nutrient solution with -5.0 bar osmotic potention than in leaves of plants io nutrient solution with -0.5 bar osmotic potential. However, it was difficult to determine any consistent pattern in the change in IJJL. The increases were sometimes greater and sometimes less than the applied pressure. The data on plant 6 (Table 1) showing the relationships between the hydrostatic pressure in the xylem of the petiole and ijj[^ were of particular interest. This plant was growing in a solution of -0.5 bar osmotic potential, and doring measurement of leaf water potentials the light was at low intensity and the CO^ concentration was kept at the apparent compensation point by removal of external COg supply. The application of 2 bar pressure resulted in an increase in leaf water potential of 3.1 bar (from -5.8 to -2.7). When a leaf was cut off after approximately one half hour at 2 bar pressure, water exuded from the stub of the petiole attached to the plant. A pressure of 3 bar on the roots caused 3 cut petiole stubs to bleed. Despite this positive pressure in the petiole the leaf water potential was -2.7 bar with 2 bar applied pressure and -1.7 bar with 4 bar applied pressure. Transpiration from a similarly treated plant is indicated in Figure 6. The existence of a negative leaf water potential despite a positive xylem poteotial is also illustrated in Figure 2. Drops of water appeared on the leaf margins of a plant to which 4 bar pressure in increments of 1 bar per hour were applied. When a leaf was sampled at this time the petiole stub left on the plant bled but no water appeared at the cut surface of the petiole attached to the leaf until 7.2 kg pressure was applied. Similar data have been obtained from plants guttating in a dark humid atmosphere. 5. Leaf diffusive resistances The calculated resistance to diffusion of water from leaf to air (leaf resistance) at critical points are indicated on the graphs:. These values are the sum of the leaf and boundary layer resistances (r, 4- rj. The boundary layer, r^, estimated by substituting pieces of wet blotters of similar size and orientation as pepper leaves, was approximately 1.3 s cm"'. Changes in pressure applied to roots did influence leaf diffusive resistance. However, the changes were small in comparison to the changes in i )j^.. For example, when the leaves were wilted the resistance was 14.7 s cm"' with a leaf water potential of bar (Figure 5). Applying 6 bar pressure to roots increased 1(1^ to -2.7 bar but only reduced diffusive resistance to 12.1 s cm"'. The indications were that the leaf diffusive resistance was to a large extent determined by the root and top environment and only to a slight extent by the ^L. Thus, plants in -0.5 bar solution had resistances varying from 2.5 to 7.8 s cm"', plants in -5.0 bar solution had resistances varying from 6.1 to 87.6 s cm"'. Stomatal resistances of plants in -0.5 bar were lowest at low concentrations of COg (Figure 6) and highest at low light and in air with 270 ^1 1"' COg (Figure 1). The highest stomatal resistances were in plants in solution of -5.0 bar with 270 (il 1"' COg air and low light (Figure 3). Discussion The presence of a negative water potential of -2 bar or lower in the leaves when the water potential in the petioles was 0 or positive would indicate that the re-

7 Physiol. Plant XYLEM WATER POTENTIAL AND TRANSPIRATION 207 sponses reported here were primarily associated with the passage of water from xylem terminals to the atmosphere and not in the root, stem or petiole. Flow of water from the xylem terminals to the atmosphere would encounter resistance within the leaf as well as stomatal resistance at the leaf air interface. Any control mechanism responding to pressure changes would have to be located either in the movement of liquid water in the mesophyll or in the diffusion of water vapor through the stomates. There is evidence to support control at both locations, and it is probable that both were involved. 1. Mesophyll resistance. The resistance to movement of water from the xylem terminals to' the stomatal cavity can be appreciable as evidenced by the presense of a negative leaf water potential when the xylem potential was positive. The fact that during periods when conditions favored guttation root pressure forced water through the hydathodes rather than through the stomates also suggests an appreciable mesophyll resistance. It appears that there is some mechanism in the leaf which prevents the positive xylem pressure produced by application of pressure to roots, from completely saturating the pepper leaves. This is illustrated best in data of Figure 2. An application of 4 bar pressure produced drops of water on the leaf margins. When a leaf was cut the petiole stub attached to the stem bled but the meniscus retreated into the petiole attached to the leaf. A pressure of 1.1 bar was required to return the meniscus to the cut surface. This would indicate that the resistance to flow of water from the xylem terminals through the mesophyll cells to the stomatal cavity was appreciably greater than the resistance to flow from the xylem to the hydathodes. Rawlins (1963) observed that shaded leaves of tobacco commonly remain turgid, while adjacent leaves on the same plant exposed to the sun wilt. He concluded that since the adjacent leaves remained turgid, indicating that the water potential in the xylem was not low enough to cause wilting, a major part of the supply resistance to the wilted leaves occurred between the xylem and leaf cells. The data of Hoffman and Splinter (1968) demonstrating that the water potential gradient between two sections of a leaf may be greater than the water potential gradient between the leaf and soil would suggest significant mesophyll resistance. Weatherly (1963) has shown that the bulk of the water moves from the xylem terminals to the stomatal cavity in the mesophyll cell walls and intercellular spaces and that this resistance is much less than the resistance to movement through cellular membranes into mesophyll cells. Two possible explanations for the occurrence of a negative water potential in the leaf and positve potential in the petioie xylem occurred to us. A. There may be a pressure sensitive resistance in the pathway between the xylem and the stom.atal cavity which acts as a check valve, increasing the resistance to flow when xylem pressure is. positive and decreasing the resistance to flow when the pressure in the xylem is negative. Raschke (1970 b) postulates two resistances in this pathway. We visualize the first resistance (rj) as being located in the passage between the xylem terminals and mesophyll cells and rg in the passage through mesophyll cells to the stomatal cavity. It is probable that a membrane of some sort is associated with r^. Essau (1953) states that the bundle sheath that surrounds: the xylem terminals in leaves has some features that are comparable to the endodermis of the root. Brouwer (1954) has presented data indicating that the conductivity of Vicia faba roots increased as the suction tension in the xylem increased and attributed this to either an increase in number or size of channels functioning. The existence of a similar pressure-sensitive variable resistance located in the pathway between the xylem terminals and mesophyll cells (r ) functioning as a check valve could at least in part explain the responses to change in hydrostatic pressures. The persistent reduction in rate of transpiration of plants in -0.5 bar osmotic potential with applied pressure of 3 or 4 bar could be explained by such a pressuresensitive resistance. B. It is possible that with the increase in hydrostatic pressure in the xylem the larger elements in the leaves swelled and partly closed off or in some way compressed the small pliable terminal xylem vessels. This could have prevented these small vessels from completely expanding for as long as the pressure was maintained. A sudden release ol the pressure would allow them to expand and draw water from the larger elements, producing the negative xylem pressure in the leaf. 2. Stomatal resistance. While there was some evidence of the existence of a variable mesophyll resistance there was more convincing evidence that at least some of the changes in transpiration and net COj assimilation were brought about by the response of stomates to stimuli originating from both the external environment and from within the plant. These stimuli could have been physical or chemical. The short periods of change in rate of transpiration associated with changes in pressure were in all probability a response to hydropassive movements of the stomates (Meidner and Mansfield 196S, Raschke 1970 a). In addition to this response to change in volume and shape of cells a6 pressure in the xylem was changed, it is probable that there were some much more complex controls operating. In a recent review of cycling of stomatal aperture Barrs (1971) presented evidence of both a water control system and COj control system. The lack of correlation between leaf water potential and transpiration or photosynthesis would indicate that the water control system was not important in these studies. There is, however, evidence to support the functioning of a system controlled by the COj concentration within the leaf.

8 208 B. E. JANES AND G. W. GEE PhysioJ. Plant The data of Figure 8 clearly indicates that at low concentrations of CO^ there is a relationship between internal CO^ concentration and stomatal resistance. In the studies reported here there was no means of determining the internal COg concentration of leaves of plants growing in air with 270 \il 1"' COg. However, the indications were that the internal CO5, concentration, and thus stomatal resistance, was regulated to a large extent by factors which influence the rate of photosynthesis (light intensity, previous history of the plant, COg and water vapor concentration of the atmosphere and osmotic potential of the nutrient solution) and not by changes in ijij^. 3. Hormonal control. It is recognized that factors such as the concentration of kinetins, abscisic acid (Mansfield and Jones 1971, Tal, Imber and Itai 1970, and Tal and Imber 1970) and potassium {Graham and Ulrich 1972) influence the stonaatal mechanism and that they may be involved in the responses reported here. No attempt was made to assay the activity of these compounds. This research w^as supported in part by funds from the National Science Foundation; from the University of Connecticut Institute of Water Resources through the U. S. Department of the Interior and authorized in the Water Resources Research Act of 1964, PL ; and by the Storrs Agricultural Experiment Station as a part of Northeastern Regional Research Project NE-48. This is Scientific Contribution No. 532 of the Experiment Station. Betercnees Barrs, H. D Cyclic variations in stomatal aperture, transpiration, and leaf water potential under constant environmental conditions. Annu. Rev. Plant Physiol. 22: Bravdo, B Carbon dioxide compensation points of leaves and stems and their relation to net photosynthesis. Plant Physiol. 48: Brouwer, R The regulating influence of transpiration and suction tension on the water and salt uptake by the roots of intact Vicia faba plants. Acta Bot. Neerl. 3: Essau, K Plant Anatomy. John Wiley & Sons, New York. Falk, S. O Effect on transpiration and water uptake by rapid changes in the osmotic potential of the nutrient solution. Physio!. Plant. 19: Gee, G. W., Liu, W., Olvang, H. & Janes, B. E Measurement and control of water potential in a soilplant system. Soil Science 115. In press. Graham, R. D. & Ulrich, A Potassium deficiencyinduced changes in stomatal behavior, leaf water potentials, and root system permeability in Beta vulgaris L. Plant Physiol. 49: Hoffman, G. J. & Splinter, W. E Water potential measurements of an intact plant-soil system. Agron. J. 60: Janes, B. E Effect of carbon dioxide, osmotic potential of nutrient solution, and light intensity on transpiration and resistance to flow of water in pepper plants. Plant Physiol. 45: Lopushinsky, W Effect of water movement on ion movement into xylem of tomato roots. Ihid. 39: Mansfield, T. A. & Jones, R. J Effects of abscisic acid on potassium uptake and starch content of stomatal gcard cells. Pianta (Berl.) 101: Mees, G. C. & Weatherly, P. E The mechanism of water absorption by roots. 11. The role of hydrostatic pressure gradients: across the cortex. Proc. R. Sec. (Lond.) B 147: Meidner, H. &c Mansfield, T. A Physiology of Stomata. McGraw-Hill, New York. Raschke, K a. Leaf hydraulic system: Rapid epidermal and stomatal responses to changes in water supply. Science 167: b. Stomatal responses to pressure changes and interruptions in the water supply of detached leaves of 2ea Mays L. Plant PhysioL 45: 415^23. Rawlins, S. L Resistance to water flow in the transpiration stream. Conn. Agric. Exp. Stn. Bull. No. 664: Slatyer, R. O Plant-water Relationships. Academic Press, New York. Tal, M., Imber, D. & Itai,, C Abnormal stomatal behavior and hormonal imbalance in flacca,, a wilty mutant of tomato. I. Root effect and kioetin-like activity. Plant. Physiol. 46: Abnormal stomatal behavior and hormonal imbalance in flacca, a wilty mutant of tomato. II. Auxin and abscisic acid like activity. Ihid. 46: Weatherley, P. E The pathway of water movement across the root cortex and leaf mesophyll of transpiring plants. In The Water Relations of Plants (Rutter and Whitehead eds.), pp John Wiley and Sons, New York.

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