Protoplasma (1999) 206:152-162 PROTOPLASMA �9 Springer-Verlag 1999 Printed in Austria

Diurnal changes in xylem pressure and mesophyll cell turgor pressure of the liana Tetrastigma voinierianum: the role of cell turgor in long-distance water transport

F. Thiirmer 1, J. J. Zhu 1, N. Gierlinger 2, H. Schneider 1, R. Benkert 1, P. Gegner 1, B. Herrmann 1, F.-W. Bentrup 2'*, and U. Zimmermann 1

~Lehrstuhl fiir Biotechnologie, Universitfit Wtirzburg, Wtirzburg and ZInstitut fiir Pflanzenphysiologie, Universit~it Salzburg, Salzburg

Received June 8, 1998 Accepted October 20, 1998

Summary. Long-term xylem pressure measurements were per- formed on the liana Tetrastigma voinierianum (grown in a tropical greenhouse) between heights of 1 m and 9.5 m during the summer and autumn seasons with the xylem pressure probe. Simultaneously, the light intensity, the temperature, and the relative humidity were recorded at the measuring points. Parallel to the xylem pressure mea- surements, the diurnal changes in the cell turgor and the osmotic pressure of leaf cells at heights of 1 m and 5 m (partly also at a height of 9.5 m) were recorded. The results showed that tensions (and height-varying tension gradients) developed during the day time in the vessels mainly due to an increase in the local light intensity (at a maximum 0.4 MPa). The decrease of the local xylem pressure from positive, subatmospheric or slightly above-atmospheric values (established during the night) to negative values after daybreak was associated with an almost ] : 1 decrease in the cell turgor pressure of the mesophyll cells (on average from about 0.4 to 0,5 MPa down to 0.08 MPa). Similarly, in the afternoon the increase of the xylem pressure towards more positive values correlated with an increase in the cell turgor pressure (ratio of about 1 : 1). The cell osmotic pres- sure remained nearly constant during the day and was about 0.75-0.85 MPa between 1 m and 9,5 m (within the limits of accura- cy). These findings indicate that the turgor pressure primarily deter- mines the corresponding pressure in the vessels (and vice versa) due to the tight hydraulic connection and thus due to the water equilibri- um between both compartments. An increase in the transpiration rate (due to an increase in light intensity) results in very rapid establish- ment of a new equilibrium state by an equivalent decrease in the xylem and cell turgor pressure. From the xylem, cell turgor, and cell osmotic pressure data the osmotic pressure (or more accurately the water activity) of the xylem sap was calculated to be about 0.35-0.45 MPa; this value was apparently not subject to diurnal changes. Con-

*Correspondence and reprints: Institut fiir Pflanzenphysiologie, Universit~t Salzburg, Hellbrunnerstral3e 34, A-5020 Salzburg, Aus- tria. E-mail: friedrich.bentrup @ sbg.ac.at

sidering that the xylem pressure is determined by the turgor pressure (and vice versa), the xylem pressure of the liana could not drop to - in agreement with the experimental results - less than -0.4 MPa, because this pressure corresponds to zero turgot pressure.

Keywords: Xylem pressure probe; Turgor pressure probe; Xylem osmotic pressure; Diurnal changes; Liana.

Introduction

Direct measurements of the xylem pressure in the liana Tetrastigma voinierianum with the xylem pres- sure probe have revealed (Benkert et al. 1995) that negative pressures develop during the day time. Their magnitude depended on the weather conditions. How- ever, the magnitude of the xylem pressure as well as the magnitude and direction of the pressure gradients were not always consistent with the predictions of the Cohesion Theory and with results obtained by indi- rect methods for determination of xylem pressure, respectively (see, e.g., Scholander et al. 1965, Turner et al. 1984, Passioura and Munns 1984). This finding, together with other results obtained with the pressure probe technique (Zimmermann et al. 1994a, b, 1995a, b), has reopened the debate about water ascent in higher plants (Canny 1995b, Pockman et al. 1995, Holbrook et al. 1995, Milburn 1996, Shackel 1996, Richter 1997). A conclusive answer to the question how water is lifted from the roots to the foliage of a tall tree can only be found through further measure- ments under various environmental conditions. The previous probe measurements on T. voinierianum

F. Thtirmer et al.: Diurnal xylem and turgor pressure variations 153

were performed in early spring under conditions of a tropical greenhouse at heights of 1 m and 5 m (Benkert et al. 1995). In the present communication, we report on long-term xylem pressure measurements recorded during summer and autumn up to heights of 9.5 m. The local light intensity, the temperature, and the relative humidity were measured simultaneously in order to identify the relevant environmental para- meter(s) responsible for the observed local changes in xylem pressure and to reveal the propagation of the latter in the xylem conduit. Additionally, we have recorded diurnal changes in turgor pressure and have determined the intracellular osmotic pressure of individual parenchyma cells, since changes in xylem pressure cannot be considered independent of changes in turgor pressure (and in osmotic pressure?). The xylem and cell compartments are hydraulically coupled (Andrews 1976, Malone 1993, Stahlberg and Cosgrove 1995, Schneider et al. 1997b). Therefore, it is well conceivable that the "water potential" of the parenchyma cells actually buffers the xylem pressure and changes of the latter in response to environmental factors (Balling and Zim- mermann 1990).

Material and methods Plant material

Experiments were performed on well-watered specimens of the liana Tetrastigma voinierianum, growing in the tropical greenhouses of the University of Salzburg, Austria. The plants had grown to a height of about 9.5 m, but the stem and the branches were considerably longer, because the plants had grown vertically upwards and then part of the way downwards. The data reported here were obtained during the summers of 1994, 1995, and 1997 as well as in the autumn of 1994. The greenhouses were illuminated after daybreak. The arti- ficial illumination was switched off automatically when the natural irradiance exceeded about 2500 lux (about 45/.tmol photons per m 2 . s). At full sunshine, the light intensity was appropriately limited by automatically operated blinds. The ambient relative humidity ranged from 60% to 95% and was controlled by means of an auto- matic befogging system. The ambient temperature varied between 17 ~ and 27 ~ Local temperature and relative humidity were contin- uously recorded by means of hygro-thermographs (Thies clima, Grt- tingen, Federal Republic of Germany) or by a sensor (Rotronic YA- 100, Rotonic AG, Bassersdorf, Switzerland) connected to a datalog- get (LI-1000 datalogger; LI-COR, Lincoln, Nebr., U.S.A.).

Pressure probe measurements

The construction, the principles and the possible pitfalls of the xylem pressure probe have been described in detail elsewhere (Balling and Zimmermann 1990, Benkert et al. 1995). The pressure transducer in the probe can measure negative pressures down to at least -1.4 MPa (Balling and Zimmermann 1990, Zimmermann et al. 1994a, Wei et al. 1998). This is also demonstrated by the very simple model

;~

:?0t ....................... Z 0 960 1920

Time (rain)

Fig. 1. Model experiment to demonstrate that the xylem pressure probe can read very negative pressure values. To this end, the tip of the glass capillary of the pressure probe was tightly sealed. Then, by appropriate displacement of the metal rod prior to time zero, the pressure in the perspex chamber of the probe was elevated above ambient pressure to start the experiment. Due to the water perme- ability of perspex, water slowly evaporates from the chamber caus- ing the depicted continuous pressure decrease. Cavitation occurred in this experiment when the negative pressure had dropped to about -0.95 MPa

experiment shown in Fig. 1. The tip of the glass capillary of a xylem pressure probe was tightly clogged and the pressure inside of the per- spex body was increased by appropriate displacement of the metal rod. Since perspex is slightly water-permeable, the pressure dropped during the following hours to about -0.95 MPa (due to slow water evaporation from the perspex chamber). At this value, cavitation occurred (Fig. 1). For probe measurements in single vessels of the liana, probes were mounted on portable, vibration-damping tables which were installed on the ground, on a metal platform which partly encircled the plants at 5 m height and on a movable (but not completely vibration-free) metal platform at about 9 m height. Branches were probed, if possi- ble simultaneously, at heights of 1 m, 5 m, and 9.5 m. The insertion sites of the probe at 1 m and 5 m height were about 17 m and 13 m away from the roots (due to the bending of the branches at about 9 m height). Frequently, the bark was partly removed before insertion in order to minimize clogging of the pressure probe tip during insertion through the tissue into a xylem vessel. Control experiments, without bark removal, demonstrated that this procedure did not influence the actual pressure in the xylem vessels. The probes were inserted slowly into about 1 cm thick twigs. Part of a typical cross-section of a twig is shown in Fig. 2. It is evident from Fig. 2 that the mature vessels of the liana were rather large (average diameter about 70 gin). Thus, probing of a vessel was rather easy. Insertion was stopped immediately when a rapid decrease of the pressure to positive, subatmospheric, or negative values was record- ed. The instantaneous drop from (above-)atmospheric to subatmo- spheric, pressures is a clear-cut indication that indeed a conducting vessel, rather than the apoplast or a nonfunctioning vessel was probed. Additionally, the rapid dissipation of pressure pulses applied via displacement of the metal rod of the probe (see below), as well as the movement of dyes injected into the transpiration stream (data not shown) provide further evidence that a conducting vessel has been probed (cf. Balling and Zimmermann 1990, Zimmermann et al. 1993, Benkert et al. 1995).

154 F. Thtirmer et al.: Diurnal xylem and turgor pressure variations

Fig. 2. Part of a light microscopy cross-section of a twig of the liana T. voinierianum showing the xylem. The conspicuously large size of the vessels (60-90 gm in diameter) considerably facilitated insertion of the pressure probe. Experiments with india ink added to the insert- ed microcapillary consistently demonstrated that in a successful recording indeed a xylem vessel had been hit by the probe. Bar: 75 btm

The construction and function of the cell turgor pressure probe has been described elsewhere (Zimmermann et al. 1969; Zimmermann 1978, 1989). Turgot pressure measurements were performed at heights of 1 m and 5 m. The microcapillary of the probe (filled with oil up to the very tip) was inserted into the upper leaf surface between the main vein and the leaf edge. Upon penetration of an epi- dermal cell, the turgor pressure pushed cell sap into the microcapil- lary tip. After lbrmation of an oil/sap meniscus, the microcapillary was inserted deeper into the leaf mesophyll and a constant cell turgor pressure was recorded. The oil/sap meniscus was kept at a constant position during the subsequent measurements by means of appropri- ate displacement of the metal rod (Zimmermann 1989, Zimmermann et al. 1992). The local temperature at the measuring points was determined by means of thermocouples (RS Components, MOrfelden-Walldorf, Federal Republic of Germany). The local photon flux density was determined by means of a planar quantum sensor (LI 189; LI-COR) or a luxmeter. All xylem pressure (tension) values are quoted as absolute pressures (tensions; atmospheric pressure =+0.1 MPa); according to these notations, 0.1 MPa tension corresponds to a xylem pressure of 0.0 MPa (vacuum). In contrast to the values of the xylem pressure, the turgor pressure (P~) of the ceils is defined relative to the ambient atmospheric pressure (Pare), i.e., Po = Pc* - Pare where P~* is the tur- gor pressure in absolute values.

Determination of the osmotic pressure of leaf cells

Extraction of sap from individual leaf cells and the measurement of its osmotic pressure were made as described elsewhere (Zimmer- mann et al. 1992).

Results

Xylem pressure and light intensity

Figure 3 shows xylem probe measurements at 1 m height recorded in the vessels of two plants of T. voinierianum grown in two neighbouring greenhous- es. Measurements were performed on a sunny and clear day in October 1994. A vessel of the plant grown in greenhouse A was probed at 06:35 p.m. At this time, the xylem pressure displayed a positive, subatmospheric value of +0.021 MPa. Shortly after vessel impalement, a leak occurred and the pressure returned to atmospheric. At 07:00 p.m. a second ves- sel was probed. The same pressure value was record- ed. Xylem pressure measurements on the plant grown in greenhouse B were started at 07:30 p.m. Analo- gously, long-term pressure recordings were only to be reached after a second insertion of the probe 30 rain later. In this plant, nearly identical xylem pressure values as in the specimen of greenhouse A were observed�9 Injection of a pressure pulse (via displace- ment of the metal rod of the probe) and its subsequent

13)

x

0.20

0.15 -

0.10

0.05 -

0 . 0 0 -

-0.05 -

-0.10 -

18:00

11i 1 i . . . . . . . . . .

ooq �9 ioo4~ - 08i00 09[00 10i00 11100 "

Time of day

'21100' 'oo;o' '0~;0' "061o0' 'o8100' '12;0 Time of day

Fig. 3. Parallel xylem pressure recordings on twigs of two specimens of T. voinierianum growing in neighbouring greenhouses A (solid line) and B (dashed line). The measurements were performed at 1 m height on a sunny and cloudless day in October 1994. Greenhouse conditions during the day: ambient temperature 21 ~ to 24 ~ ambient relative humidity 64% to 67%. The ambient relative humid- ity in greenhouse B was by 3% to 8% higher than in greenhouse A. The vessels were probed in the late afternoon of the first day. Because of the occurrence of leakages shortly after puncturing the vessels, probing was repeated (arrows pointing upwards). Both mea- surements were ended by cavitations occurring around noon. The arrow pointing downwards indicates the injection of a pressure pulse (by appropriate displacement of the metal rod within the probe) in order to exclude clogging of the microcapillary tip of the xylem pres- sure probe. Note that the xylem pressure in plant B was always sig- nificantly higher than in plant A. Inset Plot of the differences in xylem pressure measured in the morning hours in plants A and B. The circles indicate the difference in local light intensities between the two pressure probing points

F. Thtirmer et al.: Diurnal xylem and turgor pressure variations

0.20

0 . 1 5 -

0.10 -

v 0.05 - O

0.00 -

o.. -0.05 -

~ , -0.10 -

X -o.15 -

18:00' 00100' 06100 12100 'lAO' 00100 '06100 12:00 '18100' Time of day

Fig. 4. A two-day-and-night recording of the xylem pressure at 1 m height of plant A. Measurements were performed during November 1994. The days were sunny, but partly clouded in the afternoon. Greenhouse conditions during the days: the ambient temperature var- ied between 20 ~ and 22 ~ the ambient relative humidity ranged from 53% to 89%. Note the reproducibility of the diurnal changes in xylem pressure

I55

0.10-

0.05-

0.00-

~:z -o.o5- E e

-0.10-

-0.15-

i J i i

: / ,,, \

10100 12':00 ' ' 14:00 16:00

Time of day

0.8

0.6

g 0.4 ('P

2

0.2 " ~ d

X

0,0

Fig. 5. Plots of the changes in xylem pressure (observed on the sec-

ond day of the experiment shown in Fig. 4 on a larger time scale; sol-

id line) and of changes in the light intensity ( 0 ) measured simulta- neously close to the probing point. Similar data were obtained on the

first day of measurement (data not shown). Greenhouse conditions

during the day: the ambient temperature varied between 20 ~ and

22 ~ the ambient relative humidity ranged from 76% to 88%

rapid dissipation indicated that the capillary tip insert- ed in plant B was not blocked. Overnight, both plants showed a further continuous increase in pressure whereby the pressure in plant B was always by about 0.01 MPa higher than that of plant A. Both plants reached atmospheric pressure at 10:20 p.m. and 11:05 p.m., respectively. The maxi- mum above-atmospheric values were reached between 04:00 a.m. and 06:00 a.m. with +0.12 MPa for plant A and +0.135 MPa for plant B. Above- atmospheric pressures were accompanied by visible guttation. With the onset of illumination and conse- quently of transpiration at 07:00 a.m., tensions devel- oped in the vessels of both plants. At noon (when neg- ative pressure values were reached) both experiments ended in cavitation. The developing tension in the xylem of plant A was significantly higher than in the xylem of plant B. This is clearly concluded from the plot of the xylem pres- sure differences observed between both plants in the morning hours (Fig. 3 inset). Note the corresponding differences in light intensity measured at both loca- tions and selected times. Although the day was sunny and clear, significant differences in the local light intensities were measured. It is obvious that both parameters showed the same trend as the day pro- ceeded, suggesting that the local differences in light intensity provide a plausible explanation for the observed differences in the xylem pressures of both plants. Temperature effects could be excluded since the local changes in temperature at both measuring

points were very similar (data not shown). The tem- perature ranged from 21 ~ to 24 ~ However, a small influence of the relative humidity on the xylem pressure difference between the two plants seemed possible, since this parameter was always slightly higher in greenhouse B compared to greenhouse A (by 3% to a maximum of 8%). A long-term xylem pressure recording on plant A over 52 h during November 1994 at 1 m height (Fig. 4) provided convincing evidence that the local light intensity was the relevant parameter for the develop- ment of tension (Fig. 5). A vessel was probed at 06:00 p.m. on the first day with the light intensity gradually decreasing and the xylem pressure showing positive, subatmospheric values (Fig. 4). The maximum above- atmospheric pressure (+0.137MPa) was reached again at about 05:00 a.m. Parallel with the increase in the local light intensity (i.e., exposure to direct sun- shine; at about 10:00 a.m.), xylem tension developed in the vessels until at about 03:30 p.m. a maximum was reached. Similar changes in xylem pressure (ten- sion) were recorded during the following night and day which conspicuously arose slightly delayed upon changes in the local light intensity (compare Figs. 4 and 5). At about 02:20 p.m. the local light intensity reached its maximum (0.7 klux; Fig. 5). The maxi- mum lfght intensity coincided with the recording of the most negative pressure in the vessel (-0.131 MPa; Figs. 4 and 5). Afterwards, the sky turned cloudy, resulting in an immediate drop of light intensity, fol- lowed by a continuous decrease to very low values at

156

0.10 -

(3_ 0 .05 -

0~ 0.00

~.. -0 .05

E (11

, - 0 . 1 0

-0 .15

� 8 4

" - " ' . 1 ' , [

' I ' ' I ' '

18:00 00:00 06100 I ' ' I ' I ' ' i ' I '

12:00 18:00 00:00 06 O0 12:00

Time of day

Fig. 6. Parallel long-term xylem pressure measurements at heights of 1 m (solid line), 5 m (dashed line), and 9.5 m (dotted line). The mea- surements were performed on very cloudy days. Greenhouse condi- tions during the days: the ambient temperature varied between 17 ~ and 23.5 ~ the ambient relative humidity between 60% and 84%. The artificial illumination was switched on during day time; the maximum light intensity was 35 gmol/m 2 �9 s

05:00 p.m. Correspondingly, xylem tension again decreased with a delay of about 20 rain. Similar mea- surements performed during summer confirmed the above findings.

Xylem and cell turgor pressure

In the following set of experiments the xylem pres- sure and/or the cell turgor pressure was recorded in parallel at different heights of plant A. Figure 6 shows simultaneous long-term measurements of the xylem pressure at 1 m, 5 m, and 9.5 m height. The experi- ment was performed on very cloudy days during November 1994; the greenhouse was illuminated arti- ficially during day time. The recordings expanded over nearly two days. Probing of the vessels occurred during noon and at 04:00 p.m. As can be seen f rom Fig. 6, the max imum pressure during the nights exceeded the atmospheric value at 1 m height only by about 0.01 MPa; the corresponding values recorded by the probes at 5 m and 9.5 m heights remained in the positive, subatmospheric range. Consistent with this, guttation was only observed at ground level. Maximum tension in the vessels was recorded at 02:00 p.m. the following day. At this time, the pres- sure in the 1 m and 5 m vessels assumed values around -0.05 MPa; slightly more negative values (about -0 .12 MPa) were measured at 9.5 m height. The changes in xylem pressure at the various heights measured in the following night were nearly identical to those recorded in the first night, although the 1 m

F. Thtirmer et al.: Diurnal xylem and turgor pressure variations

0.2 , I . , �9 , ' , �9 , . �9 �9 , , �9 ' n 0 .5

-0.1 0.2 ~

-0 .2 0.1 "13

0 3 u 0.0

24 :00 04 :00 08 :00 12:00 16:00

Time of day

Fig. 7. Parallel xylem pressure recordings in plant A at heights of 1 m (solid line), 5 m (dashed line), and 9.5 m (dotted line). Measure- ments were performed during a very sunny week in July 1995. The vessels were probed in tile very late evening of the first day shown. Parallel to the continuous xylem pressure recordings, values of the cell turgor pressure were taken of mesophyll cells of a leaf at a height of 1 m (0). Inset Plot of the difference in xylem pressure (APx) against the corresponding difference in turgor pressure (APe) between 10:00 a.m. and 05:00 p.m. at 1 m height reveals that both showed a nearly 1 : 1 relationship (dashed line)

and 9.5 m probes became slightly clogged. Clogging occurred presumably due to small amounts of mucilaginous substances within the vessels detected via staining with alcian blue (data not shown; see Zimmermann et al. 1994b). It is interesting to note that the pressure difference between the 1 m and 9.5 m measuring points was 0.09 MPa at maximum. The pressure difference between the 1 m and 5 m probes varied between 0.03 MPa dur- ing the nights and 0.01 MPa during day-time. Figure 7 shows simultaneous recordings of the xylem pressure at the three measuring points and of the cell turgor pressure at a height of 1 m. The measurements were performed during a very sunny week in July 1995. The vessels were probed in the night between 10:30 p.m. and 11:30 p.m. Contrary to the results obtained in late autumn (see above), negative pres- sures could still be recorded during the night in the vessels at 9.5 m height and partly at 5 m height at this t ime of the year. At 1 m height the xylem tension was also larger than during autumn; the pressure reached just the positive, subatmospheric range. Up to day- break at 05:45 a.m., the pressure continuously increased in all vessels, not exceeding the atmospher- ic value. In the 5 m and 9.5 m vessels the pressure remained in the positive, subatmospheric and negative pressure range, respectively. The difference in pres- sure between 9.5 m and 1 m amounted to about

F. Thfirmer et al.: Diurnal xylem and turgor pressure variations 157

0.5-

D D ~" 0.4- n

0 . 3 -

if) ii1 o.2- Q..

S ~o.1

0.0

00:00

~9

D D

D IDc~D ~7

r i

04:00 08:00 1210O 16:001 2010O 24100

Time of day

Fig. 8. Cumulative plot of turgor pressure values measured in indi-

vidual leaves of plant A at the same time of the year as in Fig. 7 at

1 m (O) and 5 m ([7) height. Greenhouse conditions during the days:

the ambient temperature varied between 22 ~ and 28 ~ the ambi-

ent relative humidity between 67% and 87%; the max imum local light intensity was 800 gmol /m 2 �9 s

0.1 MPa. With the onset of illumination (i.e., transpi- ration), the tension increased in all vessels, but differ- ently. Around 11:00 a.m., the pressure difference between the 9.5 m and the 5 m vessel was consider- ably larger than during the night, whereas the pressure difference between the 5 m and the 1 m vessel was significantly smaller. At this time, heavy clogging of the 5 m probe was observed and cavitation occurred in the 9.5 m probe due to vibrations of the recording site (metal platform) and/or because of small move- ments of the leaves and twigs due to water loss and the concomitant changes in stem and twig diameter (see below). In contrast, the 1 m recording continued until late afternoon. As can be seen from Fig. 7, the xylem pressure at this height approached a value of -0.225 MPa around 05:00 p.m., as apposed for the 9.5 m probing recorded at about 11:00 a.m. Similar results were obtained from many other recordings in which only the xylem pressure in two vessels at different heights could be measured in par- allel or the xylem pressure at one measuring point together with the cell turgor pressure. The maximum negative pressures recorded on sunny days during the summer were about -0.4 MPa. Below this value always cavitation occurred. Furthermore, it has to be noted that between 01:00 p.m. and 03:00 p.m. in the summer negative pressures could only occasionally be read at 5 m and in particular at the height of 9.5 m. Repeated insertion of the probe showed the occur- rence of cavitation in the majority of the vessels, because only subatmospheric, positive values were recorded.

In the experiment of Fig. 7 the cell turgor pressure was also determined at a height of 1 m. Obviously, this parameter showed similar diurnal changes as experienced with the xylem pressure (Fig. 7). At the time of probing of the 1 m vessel, the cell turgor pres- sure was about 0.37 MPa (10:11 p.m.). A similar val- ue (0.34 MPa) was also recorded at 10:00 a.m. of the following day. Subsequent cell turgor pressure mea- surements performed in intervals of about 30 min showed a continuous decrease to 0.07 MPa at 04:55 p.m. The decrease in turgor pressure correlated well with the decrease in xylem pressure at this height (ratio about 1 : 1; see Fig. 7 inset); evidently, the total change in xylem pressure of 0.22 MPa corresponded well (within the range of accuracy) with the total change in turgor pressure of about 0.27 MPa. [Note that the cell turgor pressure values represent discrete measurements in different cells which were located in the same tissue layer (see also Fig. 8). Continuous cell turgor pressure recordings from a given cell were not feasible.] In Fig. 8 a cumulative plot of the turgor pressure val- ues measured in the mesophyll cells of leaves of T. voinierianum at 1 m and 5 m height during a sunny week in July 1995 is given. Due to the scatter of the data it is not possible to decide whether a turgor pres- sure difference between cells at 1 m and 5 m height existed. (Cell turgor pressure recordings at 9.5 m height were not feasible because of inevitable vibra- tions of the mobile metal platform.) However, there were some evidences that the cell turgor pressures at 5 m height were on average somewhat smaller than those at 1 m height (see Fig. 8). Despite this uncer- tainty, the data show clearly that the diurnal changes in turgor pressure were consistent with the diurnal changes in xylem pressure measured in Fig. 7. The maximum values of the turgor pressure (ranging between 0.35 MPa and 0.48 MPa) were reached dur- ing the night (10:00 p.m. to 08:00 a.m.). Turgor pres- sure dropped immediately upon illumination and reached the lowest values (ranging between 0.05 MPa and 0.08 MPa) between 04:00 p.m. and 06:00 p.m. (during the summer maximal daily light intensity in the greenhouse occurred considerably later than in late autumn because of the operation of automatic blinds which strongly dimmed the light at noon, while in late afternoon horizontally impinging light fully reached the plants; see Material and methods). The subsequent continuous increase in cell turgor pressure exactly correlated with the first occurrence of nega- tive pressures in the vessels (data not shown). The

158 F. Thtirmer et al.: Diurnal xylem and turgor pressure variations

average maximum change in turgor pressure and xylem pressure (data not completely shown, but see Figs. 7 and 8) during a day-and-night period was 0.4-0.5 MPa. In some experiments during July 1995, plant A was watered after determination of the cell turgor pressure around noon, when turgor pressure values of 0.1 to 0.2 MPa were recorded. Measurements of the cell tur- gor about 30 min later showed that the turgor pressure had increased by about 10% to 50%. Consistently, a similar change in xylem pressure towards less nega- tive values was also observed (data not shown). Measurements of the diurnal changes in the osmotic pressure within the mesophyll cells showed that this parameter was nearly independent of day time and height. The osmotic pressure measured at heights between 1 m and 9.5 m showed an average of 0.82 +_ 0.06 MPa (n = 101).

Discussion

The data presented here have demonstrated that the local light intensity, and thus the local transpiration rate, is the relevant parameter for the diurnal changes in the xylem pressure of the liana T. voinierianum

(provided that the plant was well-watered). Local dif- ferences in temperature and relative humidity certain- ly played a role, but apparently only a minor one under greenhouse conditions. This result is consistent with similar findings for maize plants grown in a greenhouse in subtropical latitudes (Schneider et al. 1997a). The delayed response of xylem pressure to changes in the local light intensity can also easily be explained by the lag time of stomata opening and clo- sure which is usually in the range of 20 min (K. Raschke pers. commun.), The development of (more) positive pressures as well as the tension gradients measured during the night between 1 m and 9.5 m height of about 0.08 MPa to 0.1 MPa were just high enough to overcome the gravitational force. Some- times, however, slightly smaller gradients were obtained. This is particularly obvious if the tension gradients between 1 m and 5 m are considered. Root pressure together with stem pressure (Kramer 1983, Braun 1984) can explain these findings (for a more detailed discussion, see Benkert et al. 1995). It is important to note that any positive pressure at 1 m height never exceeded +0.14 MPa. This is consistent with the occurrence of guttation which was only observed at ground level (and in some cases at 5 m; data not shown). Guttation and the water-leaky walls

of the xylem (Canny 1991) apparently prevent the generation of higher pressures, i.e., the plant repre- sents an open system, allowing a rapid dissipation of pressure. (Therefore, it is very unlikely that root pres- sure is involved in the water ascent to the foliage of very tall trees!) As the day proceeded, xylem tensions developed locally and essentially independent of each other, pre- sumably due to the differences in the local light inten- sity at the different heights (see also Benkert et al. 1995; Zimmermann et al. 1995a, b). Clearly, local changes in tension did not immediately propagate, and thus led to a temporary reduction or increase, respectively, of the gradient between each of the two measuring points (see Figs. 6 and 7). This is an important finding, because in a continuous hydraulic system immediate propagation of a pressure signal would be expected. Note also the Cohesion Theory requires that the tension gradients are at least linear, or even steeper due to the known hydraulic resis- tances. Gradients less than linear, therefore, are not consistent with the assumption that tension is the sole driving force for water ascent.

However, another possible explanation is that the local pressure in the xylem is "buffered" by the turgor pressure of the adjacent cells for some time, depend- ing on the water supply by the roots relative to tran- spirational loss of water. The latter explanation is sup- ported by parallel measurements of xylem pressure and turgor pressure in mesophyll cells. These experi- ments have demonstrated convincingly that the xylem (x) and the surrounding tissue cells (c) behave like a Hepp-type osmometer (Mauro 1965, 1981; Balling et al. 1988; Balling and Zimmermann 1990; Zimmer- mann et al. 1995b; see also Malone 1993). Due to the rapid water exchange times of tissue cells (in the order of 1 s to some 10 s; Zimmermann 1989; Steudle 1989, 1992; Moore and Cosgrove 1991; Malone 1993) one can always assume water equilibrium or, more correctly, a quasi-stationary state between the two compartments (for respective considerations in thermodynamics, see Dainty 1963). Thus, the chemi- cal water potentials (y) of both compartments must be equal at a given height h:

#x(h) = #c(h) (1)

and correspondingly (Pickard 1981, Benkert et al. 1995)

Mgh + V[Px - Pam] + RTlnax = VPc + RTlnac (2)

where M is the molecular mass, g is the gravitational

F. Thtirmer et aI.: Diurnal xylem and turgor pressure variations

constant, h is the distance from the ground, V is the molar volume, Px is the xylem pressure, Pare is the ambient atmospheric pressure (+0.1 MPa), Pc is the turgor pressure (relative to atmospheric pressure), ax is the activity of water in the xylem, ac is the activity of water in the cells, R is the gas constant, and Tis the absolute temperature. Reduction of the water activity term can be envisaged by osmotically active (low-molecular-weight) solutes and/or by gel-like structures (Plumb and Bridgman 1972, Zimmermann et al. 1994b, Benkert et al. 1995). In the first case, Eq. (3) holds:

pgh + (Px - Pare) - arx : Pc - arc (3a)

or, if the hydrostatic pressure within the cells is expressed in absolute values:

pgh + (Px - Pare) - 7gx = (Pc* - Pam) -- arc, (3b) pgh + Px - arx = Pc* - 7re

where p is the density of the xylem sap and r is the osmotic pressure of the respective compartment. [Equation (3b) shows that the absolute values of the hydrostatic pressure in the xylem and the surrounding cells are independent of the ambient pressure! This was exactly found when the equilibrium xylem pres- sure was measured in tobacco plants exposed to ele- vated ambient pressures in a hyperbaric chamber (Balling and Zimmermann 1990). Passioura (1991) termed this phenomenon "bizarre".] Equation (3b) shows that the xylem pressure Px at a certain height is determined exclusively by the water potential of the cells, but not a priori by the transpira- tion rate (Balling and Zimmermann 1990, Stahlberg and Cosgrove 1995). Transpiration will not disturb the water equilibrium state as long as sufficient water from the roots is available. If water uptake through the roots becomes rate-limiting in response to an increase in transpiration rate, a new water equilibrium state will be established very quickly by an appropriate decrease of Px and Po. The change in both parameters should occur strictly linearly (as has been observed experimentally here) provided that the osmotic pres- sure of the parenchymal cells remains constant. According to the measurements of the osmotic pres- sure of the cell sap of Z voinierianum, this seems to be the case. Similar results were also obtained for sugarcane (Zhu et al. unpubl, data). A nearly constant cell osmotic pressure is not surpris- ing, because the time constants of solute exchange are usually one to two magnitudes larger than those of the water exchange and the volumetric elastic moduli of the cell wails of higher plants are fairly high (Katchal-

159

sky and Curran 1965; Zimmermann 1978; Steudle 1989, 1992). Therefore, two important conclusions can be drawn from the inferred tight hydraulic connection between the xylem and tissue cell compartments. First, if we assume an average value of 0.8 MPa for the cell osmotic pressure at 1 m to 5 m height and if we use the Px and Pc values recorded after daybreak (at 10:00 a.m.; Fig. 7 and data not shown), the osmotic pressure of the xylem sap is estimated to be about 0.37-0.39 MPa (assuming that the gravitational term is 0.01 MPa and 0.05 MPa, respectively). This value is rather high compared to those in the bulk of litera- ture on xylem sap composition. However, there are reports suggesting values (at least in some higher plants and trees) which well compare to the above estimate (e.g., Ferguson etal. 1981; Kramer 1983; Canny 1993, 1995a; Barker and Becker 1995; Schill etal. 1996; Marienfeld etal. 1996). Furthermore, mucilaginous substances, i.e., gel-like structures as found in mangroves (Zimmermann et al. 1994b), were apparently present in the xylem (see above), which can also lower the water activity (Plumb and Bridgman 1972). This would correspondingly de- crease the osmotic pressure value. The osmotic pressure of the xylem sap did not show substantial diurnal changes as found for other higher plants and tall trees (e.g., poplar, Siebrecht and Tisch- ner pers. commun.). Using the Px and Pc values from Fig. 7 recorded during the daytime, it can easily be shown that the osmotic pressure of the xylem sap did not significantly change with the increasing light intensity (i.e., with increasing transpiration and water flow). The reason for the absence of substantial diur- nal changes in the osmotic pressure of the xylem ves- sels may be the relatively low transpiration rate of the liana and/or the limited accuracy of the estimates. Secondly, along with the lines of the arguments pre- sented above, a stable xylem pressure of less than about -0.4MPa (corresponding to a value of -0.5 MPa relative to the atmospheric pressure, Para) is very unlikely according to Eq. (3b) because the turgor pressure did not reach values lower than 0.05 MPa (at least up to a height of 5 m). This, in addition to the bulk of other evidence (e.g., Balling et al. 1988; Schneider et al. 1997a, b), demonstrates that the probe indeed reads the actual xylem pressure. The important question remains whether in the liana at noon tensions higher than about 0.5 MPa develop, but are not read by the pressure probe, because instan- taneous cavitation is induced by puncturing the ves-

160 F. Thtirmer et al.: Diurnal xylem and turgor pressure variations

sel. In order to answer this question we have to dis- cuss how water equilibrium between the xylem and (turgorless) parenchyma cells is achieved. Theoretically, in the turgorless state a new water equi- librium can only be established by an increase of the cell osmotic pressure and/or xylem osmotic pressure. An increase in xylem osmotic pressure could only be achieved by shrinkage of the xylem volume, provided that the delivery of osmotically active solutes by the roots was constant during the day. There are reports that contraction of the xylem and of individual vessels indeed occurs (Bode 1923, MacDougal 1924). How- ever, contraction of the xylem volume is not large enough to account for significant changes in xylem osmotic pressure. Furthermore, if the increase of the xylem osmotic pressure were high enough (e.g., by shifting of osmotically active substances from the xylem parenchyma cells into the vessels; Braun 1984), the tension should be kept nearly constant; however, this was not observed. A rapid increase in cell osmotic pressure could be achieved by plasmolysis and subsequent shrinkage of the turgorless cells, or by synthesis of osmotically active (compatible) solutes. Plasmolysis was not found in the liana (data not shown), and so far has not been reported in the literature, whereas diurnal changes in stem and root diameter are well-known (Klepper et al. 1971, Huck et al. 1970, M. Zimmer- mann 1983). However, these changes mainly correlate with water loss from the walls and the bark. Produc- tion of osmotically active substances is conceivable, but the cell osmotic pressure measurements have giv- en no indication for changes in the order of a few 0.1 MPa, clearly required for the establishment of higher stable negative pressures than recorded here or by Benkert et al. (1995) and Zimmermann et al. (1995a). In fact, pressure probe measurements on many plants have shown (see the above references) that the xylem pressure, when approaching negative values of about -0.4 to -0.5 MPa, rapidly decreases further until cav- itation occurs. This observation suggests that, as soon as the hydraulically coupled parenchyma cells be- come turgorless, the plant fails to maintain the water equilibrium between the xylem and the adjacent tis- sues by osmotic adjustments in the vessels and/or parenchyma cells. The assumption that primarily the "water potential" of turgescent tissue cells rather than the transpiration rate determines the xylem pressure, was supported by recent analogous measurements on sugarcane (un-

publ. results, but see the review article of Zimmer- mann et al. 1995b) and particularly on maize roots (Schneider et al. 1997b). Maize roots allowed long- term cell turgot pressure measurements. Thus, manip- ulation of the environmental conditions or short-term exposure to salt stress has been followed instanta- neously by both turgot and xylem probe. The results provided clear evidence that both parameters indeed are well correlated. In summary, we conclude that the turgot pressure of the parenchyma cells in a conducting stem critically affects and thus effectively buffers the xylem pressure and vice versa. While we now have presented clear- cut experimental evidence for this conclusion, the idea is not new. Otto Rennet (1911), reviewing the work of several authors, wrote: "As said by Leclerc du Sabon, it is hardly possible other than that the turgor pressure of the xylem parenchyma cells attains equi- librium with the pressure of the adjacent vessels. The parenchyma of the xylem thus should be able to wilt just as does that of the leaves" (p. 237, our transla- tion). Finally, we have shown that the osmotic pressure (or, more precisely, the water activity) of the xylem sap can be determined from concomitant, height-depen- dent xylem pressure, cell turgot, and cell osmotic pressure measurements, provided that the cell osmot- ic pressure can be accurately measured, i.e., that the extraction rate of the cell sap is high enough in order to avoid dilution of the cell sap by water influx from the surroundings (Malone et al. 1989). If the relation between the turgor and the xylem pressure is found to be nearly 1 : 1 (see Fig. 7 inset) for a set of experi- mental conditions, the measurement of the cell osmotic pressure for one Px or Pc data point is suffi- cient, because ~c can be assumed to be constant. Oth- erwise, if Too changes diurnally (i.e., the relation between the two parameters is less than 1 : 1), the osmotic pressure of the cell sap must be determined for each data point. It is evident that calculations of the osmotic pressure of the xylem sap and its height dependence from probe measurements could consid- erably facilitate the elucidation of long-distance water transport: this process (particularly its height depen- dence) is difficult to assess accurately by current methods, i.e., by analysis of sap expressed from tissue organs (Jachetta et al. 1986, Canny 1993), or of sap withdrawn from single xylem vessels of intact plants by the xylem pressure probe upon pressurization of the roots (Marienfeld et al. 1996). These sap collec- tion methods can be subject to errors due to progres-

F. Thtirmer et al.: Diurnal xylem and turgor pressure variations

sive dilution of the sap with water forced out of the tissue cells during the extraction process. Only the compression/decompression method recently intro- duced by Schillet al. (1996) seems to circumvent this problem. However, the latter method is applicable only to rigid stem pieces.

Acknowledgement This work was supported by grants from the BMBF (DARA 50WB 9643) to UZ and from the DFG (1782/1-1) to RB.

References Andrews FC (1976) Colligative properties of simple solutions. Sci-

ence 194: 567-57l B alling A, Zimmermann U (l 990) Comparative measurements of the

xylem pressure of Nicotiana plants by means of the pressure bomb and pressure probe. Planta 182:325-338

- - Btichner K-H, Lange OL (1988) Direct measurement of nega- tive pressure in artificial-biological systems. Naturwis- senschaften 75:409-411

Barker M, Becker P (1995) Sap flow rate and sap nutrient content of a tropical rain forest canopy species, Dryobalanops aromatica, in Brunei. Selbyana 16:201-211

Benkert R, Zhu J-J, Zimmermann G, Ttirk R, Bentrup F-W, Zimmer- mann U (1995) Long-term xylem pressure measurements in the liana Tetrasfigma voinierianum by means of the xylem pressure probe. Planta 196:804-813

Bode HR (1923) Beitr~ige zur Dynamik der Wasserbewegung in den Gef~iBpflanzen. Jahrb Wiss Bot 62:92-127

Braun HJ (1984) The significance of the accessory tissues of the hydrosystem for osmotic water shifting as the second principle of water ascent, with some thoughts concerning the evolution of trees. IAWA Bull 5:275-294

Canny MJ (1991) The xylem wedge as a functional unit: speculations on the consequences of flow in leaky tubes. New Phytol 118: 367-374

- (1993) The transpiration stream in the leaf apoplast: water and solutes. Philos Trans R Soc Lond Biol Sci 341:87-100

- (1995a) Potassium cycling in Helianthus: ions of the xylem sap and secondary vessel formation. Philos Trans R Soc Lond Biol Sci 348:457-469

- (1995b) A new theory for the ascent of sap: cohesion supported by tissue pressure. Ann Bot 75:343-357

Dainty J (1963) Water relations of plant cells. Adv Bot Res 1: 279-326

Ferguson AR, Eiseman JA, Dale JR (1981) Xylem sap from Actini- dia chinensis: gradients in sap composition. Ann Bot 48:75-80

Holbrook NM, Burns M J, Field CB (1995) Negative xylem pressures in plants: a test of the balancing pressure technique. Science 270: 1 1 9 3 - 1 1 9 4

Huck MG, Klepper B, Taylor HM (1970) Diurnal variations in root diameter. Plant Physiol 45:529-530

Jachetta J J, Appleby AP, Boersma L (1986) Use of the pressure ves- sel to measure concentrations of solutes in apoplastic and mem- brane-filtered symplastic sap in sunflower leaves. Plant Physiol 82:995-999

Katchalsky A, Curtan PF (1965) Nonequilibrium thermodynamics in biophysics. Havard University Press, Cambridge, Mass

161

Klepper B, Browning VD, Taylor HM (1971) Stem diameter in rela- tion to plant water status. Plant Physiol 48:683-685

Kramer PJ (1983) Water relations of plants. Academic Press, New York

MacDougal DT (1924) Dendrographic measurements. Carnegie Inst Washington Publ 350:1-88

Malone M (1993) Hydraulic signals. Philos Trans R Soc Lond Biol Sci 341:33-39

- Leigh RA, Tomos AD (1989) Extraction and analysis of sap from individual wheat leaf cells: the effect of sampling speed on the osmotic pressure of extracted sap. Plant Cell Environ 12: 919-926

Marienfeld S, Zhu JJ, Koyro H-W, Schr6der WH, Zimmermann U (1996) Direkte Probenentnahme aus dem Xylem: Vergleich mit konventionell gewonnenen Proben. In: Abstracts of the Botanikertagung, Dtisseldorf, p 325

Mauro A (1965) Osmotic flow in a rigid porous membrane. Science 149:867-869

- (1981) The role of negative pressure in osmotic equilibrium and osmotic flow. In: Ussing HH, Bindslev N, Lassen NA, Sten- Knudsen O (eds) Water transport across epithelia. Alfred Ben- zon Symp. 15. Munksgaard, Copenhagen, pp 107-119

Milburn JA (1996) Sap ascent in vascular plants: challengers to the cohesion theory ignore the significance of immature xylem and the recycling of Mtinch water. Ann Bot 78:399407

Moore PH, Cosgrove DJ (1991) Developmental changes in cell and tissue water relations parameters in storage parenchyma of sug- arcane. Plant Physiol 96:794-801

Passioura JB (1991) An impasse in plant water relations? Bot Acta 104:405-411

- Munns R (1984) Hydraulic resistance of plants II: effects of rooting medium, and time of day, in barley and lupin. Aust J Plant Physiol 11:341-350

Pickard WF (1981) The ascent of sap in plants. Prog Biophys Mol Biol 37:181-229

Plumb RC, Bridgman WB (1972) Ascent of sap in trees. Science 176:1129-1131

Pockman WT, Sperry JS, O'Leary JW (1995) Sustained and signifi- cant negative water pressure in xylem. Nature 378:715-716

Renner O (1911) Experimentelle Beitr~ige zur Kennmis der Wasser- bewegung. Flora 113:171-247

Richter H (1997) Water relations of plants in the field: some com- ments on the measurement of selected parameters. J Exp Bot 48: 1-7

Schill V, Hartung W, Orthen B, Weisenseel MH (1996) The xylem sap of maple (Acer platanoides) trees: sap obtained by a novel method shows changes with season and height. J Exp Bot 47: 123-133

Schneider H, Wistuba N, Miller B, Gegner P, Thtirmer F, Melcher P, Meinzer F, Zimmermann U (1997a) Diurnal variation in the radial reflection coefficient of intact maize roots determined with the xylem pressure probe. J Exp Bot 48:2045-2053

- Zhu JJ, Zimmermann U (1997b) Xylem and cell turgor pressure probe measurements in intact roots of glycophytes: transpiration induces a change in the radial and cellular reflection coefficients. Plant Cell Environ 20:221-229

Scholander PF, Hammel HT, Bradstreet ED, Hemmingsen EA (1965) Sap pressure in vascular plants. Science 148:339-346

Shackel K (1996) To tense, or not too tense: reopening the debate about water ascent in plants. Trends Plant Sci 1:105-106

162

Stahlberg R, Cosgrove DJ (1995) Comparison of electric and growth responses to excision in cucumber and pea seedlings II: long-dis- tance effects are caused by the release of xylem pressure. Plant Cell Environ 18:33-41

Steudle E (1989) Water flow in plants and its coupling to other processes: an overview. Methods Enzymol 174:183-225

- (1992) The biophysics of plant water: compartmentation, cou- pling with metabolic processes and flow of water in plant roots. In: Somero GN, Osmond CB, Bolis CL (eds) Water and life: comparative analysis of water relationships at the organismic, cellular, and molecular levels. Springer, Berlin Heidelberg New York Tokyo, pp 173-204

Turner NC, Spurway RA, Schulze E-D (1984) Comparison of water potentials measured by in situ psychrometry and pressure cham- ber in morphologically different species. Plant Physiol 74: 316-319

Wei C, Steudle E, Tyree MT (1998) Xylem pressure in maize leaves measured with a cell pressure probe: a test of the Cohesion-Ten- sion theory. J Exp Bot 49 Suppl: 10

Zimmermann MH (1983) Xylem structure and the ascent of sap. Springer, Berlin Heidelberg New York

Zimmermann U (1978) Physics of turgor- and osmoregulation. Annu Rev Plant Physiol 29:121-148

- (1989) Water relations of plant cells: pressure probe technique. Methods Enzymol 174:338-366

F. Thiirmer et al.: Diurnal xylem and turgor pressure variations

- R~ide H, Steudle E (1969) Kontinuierliche Druckmessung in Pflanzenzellen. Namrwissenschaften 56:634

- Rygol J, Balling A, K16ck G, Metzler A, Haase A (1992) Radial turgor and osmotic pressure profiles in intact and excised roots of Aster tripolium. Plant Physiol 99:186-196

- Haase A, Langbein D, Meinzer F (1993) Mechanisms of long- distance water transport in plants: a re-examination of some pm- adigms in the light of new evidence. Philos Trans R Soc Lond Biol Sci 341:19-31

- Meinzer FC, Benkert R, Zhu J J, Schneider H, Goldstein G, Kuchenbrod E, Haase A (1994a) Xylem water transport: is the available evidence consistent with the cohesion theory? Plant Cell Environ 17:1169-1181

- Zhu JJ, Meinzer FC, Goldstein G, Schneider H, Zimmermann G, Benkert R, Thtirmer F, Melcher P, Webb D, Haase A (1994b) High molecular weight organic compounds in the xylem sap of mangroves: implications for long-distance water transport. Bot Acta 107:218-229

- Bentrup F-W, Haase A (1995a) Xylem pressure, flow, and sap composition of trees determined by means of the xylem pressure probe. In: Terazawa M, McLeod CA, Tamai Y (eds) Tree sap. Hokkaido University Press, Sapporo-shi, pp 47-58

- Meinzer F, Bentrup F-W (1995b) How does water ascend in tall trees and other vascular plants? Ann Bot 76:545-551