Two experiments, a split-root experiment and a root pressurizing experiment, were performed to test whether hydraulic signalling of soil drying plays a dominant role in controlling stomatal closure in herbaceous bell pepper plants. In the split-root experiment, when both root parts were dried, synchronous decreases in stomatal conductance (gs), leaf water potential (LWP) and stem sap flow (SFstem) were observed. The value of gs was found to be closely related to soil water potential (SWP) in both compartments. Tight relationships were observed between gs and stem sap flow under all conditions of water stress, indicating a complete stomatal adjustment of transpiration. When the half-root system has been dried to the extent that its water uptake dropped to almost zero, declines in gs of less than 20% were observed without obvious changes in LWP. The reduced plant hydraulic conductance resulting from decreased sap flow and unchanged LWP may be a hydraulic signal controlling stomatal closure; the results of root pressurizing supported this hypothesis. Both LWP and gs in water-stressed plants recovered completely within 25 min of the application of root pressurizing, and decreased significantly within 40 min after pressure release, indicating the hydraulic control of stomatal closure. Our results are in contrast to those of other studies on other herbaceous species, which suggested that chemical messengers from the roots bring about stomatal closure when plants are in water stress.
The control of stomatal conductance (gs) is the primary means by which plants regulate water flow through the soil–plant–atmosphere continuum (Saliendra, Sperry & Comstock 1995). When a partial root system of a plant is subjected to water stress, decreased gs may or may not be associated with decreased leaf water potential (LWP), depending on whether there is hydraulic or non-hydraulic control of stomatal closure.
Two techniques have been employed to test the two hypotheses. One is the pressurization of the whole root system (Gollan, Passioura & Munns 1986; Saliendra et al. 1995; Fuchs & Livingston 1996). This technique assumes that the pneumatic and hydraulic pressures in the soil are increased equally and do not affect the turgor of the roots, while only the hydraulic pressure is transmitted to the above-ground organs, which results in increases in turgor and cell volume. The other technique involves splitting the roots into two compartments (one above the other or the two adjacent and parallel) (Gowing et al. 1990; Zhang & Davies 1990; Khalil & Grace 1993), one kept wet and the other one dried.
The mechanism of stomata closure probably varies among species (Croker et al. 1998). Few reports on the mechanism in pepper plant have been published. It could be that the decline of hydraulic conductance resulting from partial root drying serves as a signal in controlling stomatal closure. The present experiments were undertaken to examine whether hydraulic signalling of soil drying plays a dominant role in controlling the stomatal behaviour in the herbaceous bell pepper plant.
Both of the techniques described above were used in the experiments. Following Sakuratani & Aoe (1997), we used the heat pulse method (Cohen et al. 1988) to measure the sap movements through the roots and through the stem separately, so that the changes in hydraulic conductance of the plant could be evaluated via measurements of LWP.
MATERIALS AND METHODS
Two experiments, a split-root experiment with water stress imposed on half or all of the root system, and a root pressurizing experiment, were conducted to examine the mechanism of stomatal closure of the bell pepper plant (Capsicum annuum L. vau. Maor). The experiments were performed in a temperature-controlled greenhouse.
Experiment 1: Split-root experiment
The plant roots were divided into two equal parts in two separate compartments formed by dividing 10 L pots with a waterproof, 2-mm-thick vertical plastic partition. The pots were perforated at the bottom to allow drainage. The growing medium was a mix of 60, 30 and 20%, by volume, of peat moss, plastic foam and chopped plastic sponge, respectively. Plants were irrigated and fertilized every 2 h during daytime by an automated drip irrigation system with one dripper per compartment that emitted 200 mL per irrigation.
In order to study the response of stomatal conductance to varied water stress, three water stress treatments were designed and applied separately in this experiment. In each treatment four plants with the split roots were subjected to water stress and another two were used as controls. The control plants were irrigated daily every 2 h as described above. In treatment 1 (dry/dry–dry/wet treatment), water was withheld from both compartments. When soil water content dropped to 0·4 of the maximum holding capacity, watering of one compartment was resumed. In treatment 2 (dry/wet–wet/wet treatment), water was withheld from one compartment and watering was resumed when the soil water content dropped to 0·4, as above. Treatment 3 (dry/wet–dry/dry–wet/wet treatment) was a combination of the previous two: water was withheld from the first compartment until soil moisture dropped to 0·4, and was then withheld from the second compartment also. When the soil water potential (SWP) in both compartments dropped to about − 90 kPa, watering of both compartments was resumed.
Experiment 2: Root pressurizing experiment
Bell pepper plants were planted in 3 L pots and irrigated twice daily to field capacity. When the plants had grown to 1 m in height and 10 mm in stem diameter, water was withheld from one plant while the others were irrigated as before. When the SWP in the stressed plant dropped to − 0·037 MPa, the pot was put into a 10 L pressure chamber (as described by Fuchs & Livingston 1996) with the plant leaves and stem outside the chamber. A pressure of 0·25 MPa was applied to the plant root, and was released the next day. In order to prevent root damage from oxygen shortage, pressurized air was used for pressurizing the plant root. A rubber stopper was placed between plant stem and the cover of the pressure chamber, to protect the stem. The experiment was repeated twice. Pressure was applied and released at midday on clear days.
Sap flow in stem and in roots
The heat pulse method (Cohen et al. 1988) was used to measure sap flow in experiment 1. Three sensors were installed in each plant: one in the stem below the lowest leaves, and one in each root emerging from the two compartments. A data logger (Model CR7X; Campbell Scientific, Logan, UT, USA) triggered a heat pulse every 15 min throughout the day, and recorded the outputs of 18 probes. The calibration factor was unity (Y. Cohen and Y. Li, personal communication).
Soil water potential
Time-Domain Reflectometry (TDR) was used to measure the volumetric SWP. The TDR probe consisted of three parallel stainless steel rods, 250 mm long, 3 mm in diameter and 40 mm apart, connected to a detachable coaxial cable. One probe was embedded vertically in each compartment (experiment 1). Measurements were taken manually using a cable tester (Model 1502b; Tektronix, Beaverton, OR, USA). A preliminary test was carried out to determine the relationships between SWP (MPa) and soil water content. For details see Moreshet et al. (1999).
A steady-state porometer (Model LI1600; LI-COR, Lincoln, NE, USA) was used to measure gs. The porometer chamber was acclimated to ambient conditions for about 1 h before measurements began. Conductances of both abaxial and adaxial surfaces were measured, and the ratio between them was not affected by the drying treatments; the data presented are the sums of the two conductances. The sensor head was held to conform with the natural position and angle of the leaf.
Leaf water potential
The LWP was measured with a pressure chamber (Arimad 2; A.R.I., Kfar Charuv, Israel).
In experiment 1, the SWP and gs were measured at midday each day during the experiment, whereas LWP was measured only on the days before withholding water, the days before resumption of irrigation, and 2 d after resumption of irrigation.
In experiment 2, the LWP, SWP and gs of treated and control plants were measured before and after pressure application, and before and after pressure release. In addition, the gs of one leaf of the treated plant was measured continuously after pressure release at the intervals from 1 to 5 min until gs reached to an almost stable value.
The daily midday SWP in the three treatments are shown in Fig. 1a–c. In treatment 1, the SWP in both compartments dropped at the same rate after water was withheld (Fig. 1a). After resumption of irrigation of the first compartment, the fall of SWP in the second compartment became very slow. In treatment 2, only the first compartment was allowed to dry and its irrigation was resumed when its SWP dropped to about − 70 kPa (Fig. 1b). In treatment 3, water was withheld from the first compartment until its SWP dropped to − 80 kPa, and then the second compartment was also dried (Fig. 1c). When the SWP in both compartments had dropped to − 100 kPa, watering of both was resumed.
The LWP was measured less frequently, because only a limited number of leaves could be detached from the plants without affecting the transpiration. The results of all three experiments are presented in Fig. 1d–f. When both compartments were dried, the midday LWP dropped to − 1·62 MPa in treatment 1 (day 111, Fig. 1d) and to − 1·73 MPa in treatment 3 (day 155, Fig. 1f), respectively. The more negative value of LWP in treatment 3 can be attributed to the lower SWP (− 100 kPa) than that in treatment 1 (–90 kPa). Almost full recovery of LWP can be seen after irrigation was resumed in either one (day 113, Fig. 1d; day 132, Fig. 1e) or two compartments (day 157, Fig. 1f).
The midday value of gs and the sap flows in the stem (SFstem) and root (SFroot) were normalized to the respective conductance and sap flows on the first day of the experiment, in order to cancel out biological differences between plants. The normalized gs, SFstem and SFroot of the stressed plants were normalized again to those of the control plants to cancel out the environmental variations between days (see also Ray & Sinclair 1997); it was assumed that irrigated and water-stressed plants responded similarly to the changing environmental conditions. The results are presented in Fig. 1g–i. Withholding water from only one compartment affected the value of gs only slightly and a decrease in gs of less than 20% was observed (treatment 2, Fig. 1h; treatment 3 before day 152, Fig. 1i). When water was withheld from both compartments gs declined drastically to less than 10% of that of control plants (treatment 1, Fig. 1g; treatment 3, Fig. 1i after day 152). Stomatal conductance was not completely recovered, following resumption of watering, either in plants stressed in only one part of the root (treatment 2, Fig. 1h) or in plants stressed in both parts of the root (treatment 3, Fig. 1i).
The response of SFstem to the variation of SWP was very similar to that of gs (Figs 1j–l). The relative root water uptake (or ratio of root water uptake to stem sap flow, Fig. 1m–o) depended on the difference in SWP between the compartments. In treatment 1, the stem sap flow dropped gradually, as the SWP decreased in both compartments, and fell to 0·36 of that in the control plants (Fig. 1j) on day 111 when the lowest SWP was reached. After resumption of watering in one compartment the SFstem recovered to about 0·9 of that of the control plants. The water uptake by the re-watered root increased almost four-fold whereas that in the dry compartment dropped almost to nearly zero (Fig. 1m). In treatment 2, the SFstem diminished gradually after one compartment was dried, and dropped to 0·72 of that of the control plants by day 129 (Fig. 1k). The fraction of the total uptake taken by the dried root fell gradually from 0·42 to 0·13 whereas that by the well-watered root increased gradually from 0·58 to 0·87 (Fig. 1n). The fractions taken in by both roots recovered almost completely as the SWP difference between the compartments was removed by the resumption of watering (Fig. 1n). In treatment 3, when the first compartment was dried to − 80 kPa (day 151) the midday SFstem dropped to 0·65 of that in the control plants (Fig. 1l). The fraction of the total water uptake taken by the first root fell to almost zero, which was lower than that found in experiment 2, because the soil was drier, and the second root, which was well watered, absorbed almost all of the water needed to meet the requirements of leaf transpiration (Fig. 1o). Drying of the second compartment caused SFstem to drop at an increased rate to 0·20 of that of the control plants (day 155, Fig. 1l). The fractions of the total water uptake increased to 0·39 in the first root and dropped from almost 1·00 to 0·61 in the second root, respectively, compared with the control plants (Fig. 1o). Resumption of watering in both compartments resulted in recovery of stem sap flow to 0·8 of that in the control, but did not change the fraction of water uptake by each root.
Figure 2 illustrates the sap flow patterns in treatment 3, in the control plant (Fig. 2b) with both well-watered compartments, and in the treated plant (Fig. 2a) with two split-root compartments allowed to dry one after the other (see Fig. 1c), and then both watered again. Flows on the last day of the drying cycle (Fig. 2a) were low in both roots and stem until early in the afternoon, when irrigation of both compartments was resumed, and flows in the stem and in the roots increased within 15–30 min. The flow was lower in the root that had been dried for a longer period.
On the basis of the data from the three treatments, quite close relationships were found between normalized stomatal conductance (gs) and SWP (Fig. 3). When water was withheld from both compartments the relationship between gs and SWP in the dry compartment was very similar to that in the wet compartment (Fig. 3a & b). When only one root was dried, the SWP had only a slight influence on gs, represented by the much lower slope (Fig. 3a) than that obtained by withholding water from both compartments.
The relationships between gs and SFstem are presented in Fig. 4, in which Fig. 4(a–c) represent treatments 1, 2 and 3, respectively, and Fig. 4d includes data from all three treatments. It can be seen that stomatal conductance was closely related to SFstem when water was withheld from either one compartment (Fig. 4b) or both compartments (Fig. 4a & c), as well as after resumption of watering (Fig. 4d).
Root pressurizing experiment
Pressurizing the root induced complete recoveries of gs and LWP in the plant with water stress. When the SWP dropped to − 0·037 MPa, the values of gs and LWP were 476 mmol m−2 s−1 and − 1·34 MPa, i.e. 26 and 23%, respectively, lower than those of the control plant (Fig. 5a–c). When the plant root was pressurized at 0·25 MPa, the gs and LWP of the stressed plant increased to values very close to those of the control plant within 25 min: 572 mmol m−2 s−1 and − 0·98 MPa, respectively (Fig. 5a–c), whereas gs and LWP of the control plant shown almost no change during the process. After pressure release on the next day, the gs and LWP of the treated plant decreased from 758 mmol m−2 s−1 and − 0·89 MPa, respectively, to 322 mmol m−2 s−1 and − 1·26 MPa, respectively, within 40 min (Fig. 5e, f), and the SWP declined to − 0·047 MPa (Fig. 5d). Figure 6 presents the decline of gs of one leaf in the stressed plant after pressure release; it decreased from about 800 mmol m−2 s−1 before pressure release to 282 mmol m−2 s−1, within 34 min after pressure release. The fluctuation in gs at the beginning represents the response to a change in solar radiation caused by a passing cloud.
Drying one half of a root system of a bell pepper plant reduced stomatal conductance by less than 20% (Fig. 1h & i) without a decrease in LWP (Fig. 1e & f), much less than the reductions by 50% or more reported in maize (Zhang & Davies 1989, 1990), the 74% found in sycamore (Khalil & Grace 1993), the 40–60% found in six temperate, deciduous tree species, the 25–35% reported for sorghum (Auge et al. 1995), the 47% obtained in sunflower (Neales et al. 1989) or the 50% found in rice (Bano et al. 1993). This indicates that the bell pepper plant with a well-established root system can utilize localized supplies of soil water to maintain stomatal opening. Similar results were reported for lupins by Gallardo, Turner & Ludwig (1994) who found that drying part of the root system for 36 d did not influence gs, the photosynthesis rate or plant growth, when adequate water was provided to the wet portion of the root system. A decline in gs of less than 20% may have been only the expression of biological variation and not a real non-hydraulically regulated decrease (Croker et al. 1998). It is possible also that the observed reductions in gs of half-root-dried plants, compared with well-watered plants could have been due to undetectable changes in leaf water status (Croker et al. 1998). It is also possible that the slight decline in gs was a response to a reduction in plant hydraulic conductance, serving as a hydraulic signal initiated by the combination of decreased sap flow and unchanged LWP.
The result of root pressurizing supported our hypothesis. The fact that LWP and gs recovered almost to those of the control plant within 25 min after pressure was applied, decreased significantly within 40 min after pressure release, in the water-stressed bell pepper plant eliminates the possibility of non-hydraulic control of stomatal closure, and strongly suggests that leaf water status does indeed have a dominant influence on gs. Our result is consistent with that obtained by Fuchs & Livingston (1996) who found very rapid stomatal responses to both increased and decreased root chamber pressure in Douglas fir and alder seedlings, and strongly suggested that the hydraulic signals overwhelmed any chemical signals transmitted from roots to shoots. Saliendra et al. (1995) presented similar results for the woody plant Betula occidentalis. Furthermore, the sap flow recovered within 30 min of irrigation resumption, as shown in Fig. 2. This is the time required for stomata to open (Petersen, Moreshet & Fuchs 1991), and this finding may be an indication of a hydraulic signal.
A number of experiments with herbaceous species (Gollan et al. 1986; in wheat and sunflower; Schurr, Gollan & Schulze 1992; in sunflower) employed a root pressure chamber to provide compelling evidence that gs is not controlled exclusively at the leaf level. In these experiments, pressurizing the soil did not bring about any significant increase in gs when plants were subjected to soil drought. Fuchs & Livingston (1996) interpreted this difference in stomatal response (to root pressurization), between woody and herbaceous species, as indicating that woody plants, by virtue of their larger size, are less reliant on relatively slow-moving root signals for short-term stomatal control. Schulze (1991) suggested that large woody species would lack a chemical root signal, because the long transport time would make root-signalling ineffective for short-term stomatal regulation. However, these arguments cannot account for the results we obtained with the herbaceous bell pepper plant. A possible explanation is that stomata respond much more strongly to hydraulic signals than to any non-hydraulic signal in bell pepper plants.
Saliendra et al. (1995) argued that to refer to a root signal transported at the relatively sluggish velocity of the transpiration stream as a feedforward response to soil water status is misleading, because a chemical signal will necessarily arrive at the leaf after the hydraulic one has influenced leaf water status. They suggested that the hydraulic signal is a simple and rapid form of root-to-shoot communication that can initiate stomatal responses or other leaf-level changes. We do not rule out the possibility of some chemical signal such as abscisic acid leading to partial stomatal closure. Certainly, the results of other split-root experiments (e.g. Zhang & Davies 1990; Gowing et al. 1990; Khalil & Grace 1993) provide strong evidence that chemical signals from the roots can play a dominant role in influencing gs. However, a chemical signal produced from the root in dry soil may influence gs only when it is transported to the leaf by upward sap flow, in which the magnitudes of water uptake of the roots, both in dry and in well-irrigated soil play an important role. When water is adequately supplied to one half of the root system while water uptake by the other half is very small because of its low SWP, any chemical signal sent from roots in dry soil could be diluted sufficiently to be ineffective in closing stomata and acting as a root signal (Gallardo et al. 1994). Khalil & Grace (1993) found that stomatal conductance of sycamore seedlings decreased progressively to 26% of the control as the soil water content decreased in one half of the root part while the other root part was well irrigated; when the soil water content in the drying root part dropped below 0·13 g g−1, stomatal conductance recovered sharply to 70% of that of the control plants. They attributed this phenomenon to decreased xylem sap abscisic acid concentration, caused by reduced water uptake from the root in dry soil. However, our results showed that after its slight fall gs never recovered (Fig. 1h & i) even if the water uptake of the root in dry soil decreased to almost zero (Fig. 1o), indicating that the influence, if any, of non-hydraulic signals on stomatal behaviour in the bell pepper plant was slight.
When water was withheld from both compartments, declines in gs, LWP and SFstem were observed. Stomatal conductance showed close relationships with SFstem when one half of the root was dried (Fig. 4b), when the whole root system was dried (Fig. 4a & c), and after the water supply to the dried root(s) was resumed (Fig. 4d), indicating a complete stomatal adjustment of transpiration. In addition, very tight relationships between gs and SWP in both compartments (Fig. 3) were observed, suggesting a hydraulic control of stomatal conductance when water was withheld from the whole root system.
The incomplete recovery of stomatal conductance when watering was resumed after several days of water stress, and the slight decline in gs without changes in LWP when half the root system was dried, may be attributed to the decrease of hydraulic conductance, since a lower sap flow with the same LWP indicates reduced hydraulic conductance. Meinzer & Grantz (1990) observed a close relationship between stomatal and hydraulic conductance over a wide range of plant sizes, growth conditions, seasons and manipulations of leaf and root area in sugarcane. In a split-root system, the roots in fully watered soil will continue to grow, whereas those in dry soil would not be expected to grow as much, and might even die under severe soil drying (Croker et al. 1998). Thus, in the present study, when the water to the dried root was resumed, root hydraulic conductance would not be expected to recover at once to the level in the root in fully watered soil, resulting, in turn, in incomplete recovery of stomatal conductance (Fig. 1g–i) so that root water uptake never returned to its original level (Fig. 1j–l).
Our experiments were conducted under the conditions in which SWP was above −0·1 MPa because of the soil medium. Zhang & Davies (1989) found that when SWP dropped to between −0·2 and −0·3 MPa, abscisic acid content in the roots of maize plants increased substantially. However, Croker et al. (1998) observed that the SWP required to trigger non-hydraulic signals to cause declines in gs of half-root-dried plants varied from −0·044 to −0·013 MPa in six deciduous tree species. In other experiments, non-hydraulic signals was found to be triggered in roots as decline of SWP without controlling the stomatal behaviour (Saab & Sharp 1989; Gallardo et al. 1994; Fort et al. 1997). It is apparent that the value of SWP at which hormonal signals are triggered in roots is different among plants, and should therefore not be a factor changing the mechanism of stomatal closure.
This project was supported in part by the Dutch-Israeli Agricultural Research Program (DIAPR 95/11) ‘Optimization of fruit quality of greenhouse pepper and tomato by manipulating transpiration and solute content’. The assistance of Yefet Cohen is greatly appreciated.