Ionic and ph signalling from roots to shoots of flooded tomato plants in relation to stomatal closure

Transcription

1 Plant and Soil 253: , Kluwer Academic Publishers. Printed in the Netherlands. 103 Ionic and ph signalling from roots to shoots of flooded tomato plants in relation to stomatal closure Michael B. Jackson 1,4, Leslie R. Saker 1,CarolM.Crisp 2,MarkA.Else 2 & Franciszek Janowiak 3 1 School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, U.K. 2 Horticulture Research International-East Malling, West Malling ME19 6BJ, Kent, U.K. 3 Institute of Plant Physiology, Polish Academy of Sciences, Podluzna 3, Krakow, Poland 4 Corresponding author Key words: abscisic acid (ABA), flooding, mineral ions, roots, root to shoot communication, ph, signalling, tomato (Lycopersicon esculentum), xylem sap Abstract Soil flooding damages shoot systems by inhibiting root functioning. An example is the inhibition of water uptake brought about by decreased root hydraulic conductance. The extent of any resulting foliar dehydration this causes is limited by partial stomatal closure that begins within 4 h and is maintained for several days. Root to shoot signals that promote closure in flooded tomato plants have remained elusive but may include changes in solute delivery to the shoot by transpiration. Accordingly, we examined total osmolites and selected mineral ions in samples of xylem sap flowing at rates approximating whole plant transpiration. After 2.5 h flooding, delivery of total osmolites and of PO 3 4,SO 2 4,Ca 2+, K +,NO 3 and H + strongly decreased while Na + remained excluded. Several hours later, deliveries of osmolites, PO 3 4,SO 2 4,Ca 2+,andNa + rose above control values, suggesting that, after approximately 10 h, root integrity became degraded and solute uptake de-regulated. Deliveries of NO 3 remained below control values. Reducing or eliminating the supply of K + to detached leaves to test the potential of decreased K + delivery to close stomata proved negative. Decrease in H + delivery was associated with sap alkalisation. However, raising the ph of buffer from 6.0 or 6.5 to 7.0 did not close stomata when tested in the presence of abscisic acid (ABA) at a concentration (10 µmol m 3 ) typical of the transpiration stream of flooded plants. It is concluded that despite their rapidity and scale, negative messages in the form of increased ph and decreased solute delivery from roots to shoots are, themselves, unlikely initiators of stomatal closure in flooded tomato plants. Introduction In dryland species, flooding of the soil strongly depresses root metabolism and function, an outcome of impeded gas exchange that imposes oxygen shortage and accumulations of metabolically generated gases such as carbon dioxide and ethylene (Vartapetian and Jackson, 1997). However, the consequences are not restricted to roots. Damage soon extends to aerial parts as root functions upon which shoots depend become increasingly inadequate. One flooding-induced deprivation is impeded water supply. This is evid- FAX No: mike.jackson@bbsrc.ac.uk ent within a few hours of the start of flooding. It is the outcome of decreased root hydraulic conductance compared to well-drained plants (Else et al., 1995a, 2001) possibly caused by oxygen deficiency (Birner and Steudel, 1993; Everard and Drew, 1989) and increases in carbon dioxide (Kramer 1940). The intensity of any resultant loss of leaf hydration is tempered by counteractive responses such as stomatal closure that suppress water evaporation from the foliage. Accordingly, mutants of pea and tomato with impaired ability to close stomata suffer leaf water deficit more than the wild type when flooded (Jackson and Hall, 1987). Prompt stomatal closure by flooded plants is thus an adaptive feature. However, it is also one that is

2 104 currently without a satisfactory explanation in tomato. Our objective has been to rectify this deficiency. In tomato plants, the negative hydraulic message created by decreased root hydraulic conductance does not itself instigate closure (Else et al., 1995a). Furthermore, unlike droughted plants (Zhang and Davies 1989), closing of stomata cannot be ascribed to increased ABA in xylem sap. On the contrary, delivery rates of ABA from roots to shoots in the transpiration stream are strongly suppressed by flooding (Else et al., 1996, 2001). Thus, other signals must be sought. Our previous studies have shown that 24 h flooding results in many quantitative changes to solute concentration and delivery rate (Else et al., 1995b; Jackson et al., 1996). Prominent amongst the changes were much altered deliveries of nutrient cations and anions. Each of these changes is a potential signal that could influence shoot behaviour and be implicated in stomatal regulation. Two pre-requisites for identifying active signals are that changes in delivery precede or accompany stomatal closure and that the strength of the signal is demonstrably sufficient to promote closure. Accordingly, the present paper provides, for the first time, detailed time courses of changes in the delivery of various anions and cations, concentrating on the first few hours of flooding. We show that alkalisation and a sharp decrease in K + delivery are two of several rapid changes in xylem sap that are associated closely with stomatal closure. The efficacy of these potential signals is tested in detached-leaf transpiration assays. Materials and methods Plant Material Tomato plants (Lycopersicon esculentum Mill. cv. Ailsa Craig) were grown from the two-leaf stage in 95- mm diameter pots containing a general-purpose peatbased compost ( Levington, Fisons, Ipswich, UK) to which 2 kg m 3 of a slow release fertilizer (Osmocote, Grace Sierra UK, Nottingham, UK) was added. The controlled environment room was set to a 15 h photoperiod (10:00 01:00 h) with PAR 210 µmol m 2 s 1, 25/20 C light/dark temperature and 50 60% relative humidity. Plants were sub-irrigated automatically via capillary matting, and any side shoots removed daily. Plants at the seven- to eight-leaf stage were flooded at 10:00 h, immediately following the start of the photoperiod, by submerging their pots individually in plastic containers holding m 3 tap waterwarmedto25 C. The water level was maintained approximately 10 mm above the soil. Water relations Plant transpiration was determined by loss of weight over known periods of time and after correcting for evaporation from the soil. A diffusion porometer (Delta-T, Mark 4, Delta-T Devices, Cambridge, UK) was used to estimate stomatal closure on the abaxial surface of the fifth oldest leaf (mmol H 2 Om 2 s 1 ). Leaf areas were measured destructively using a Delta T leaf area planimeter after plants were decapitated for xylem sap collection. Collection of xylem sap Shoots were excised just below the cotyledonary node using a sharp blade and a 20-mm long rubber sleeve attached to the hypocotyl stump. The assembly was then placed in a pressure chamber designed to express xylem into the rubber sleeve and out to glass scintillation vials for collection (Else et al., 1994). Compressed air (for controls) or oxygen-free nitrogen (for flooded plants) was used to pressurize root systems of welldrained and flooded plants over the range MPa until xylem sap flow approximated that of whole plant transpiration. Between 2 and m 3 of sap were collected for solute analysis once an initial 200 mm 3 from each root system was discarded to avoid contamination from wounding. After collection, sap samples were weighed and then frozen in liquid nitrogen before storing at 20 C. Solute analysis Potassium, calcium and sodium concentrations in xylem sap were assayed by atomic absorption spectrophotometery (1100B, Perkin-Elmer, Buckinghamshire, UK) after the addition of lanthanum chloride. Phosphate, NO 3 and SO 4 2 were measured by anion-exchange chromatography using a Dionex Ion- Pac AS4 analytical column (250 4 mm i.d.), a Dionex AG4-SC guard column (100 4mmi.d.) and a Dionex Conductivity Detector-3 (Dionex UK Ltd). An Anion Self-Regenerating Suppressor-1 (Dionex UK Ltd) was used in-line to suppress background conductivity. Anions were eluted with a buffer containing 2.0 mol m 3 sodium carbonate and 0.75 mol m 3 sodium hydrogen carbonate (BDH Chemicals Ltd, Poole, UK) at a flow rate of m 3

3 105 min 1. A standard solution of anions was chromatographed to establish retention times and calibration, and the data output was collected and processed by a Grams/386 Data Handling System (Galactic Industries Corp. USA). Osmolality of 50 mm 3 samples was determined by freezing-point depression using a Roebling cryo-milliosmometer (Camlab, Cambridge, UK). Acidity of xylem sap was measured in 15-mm 3 samples with a Camlab ph Boy meter (Camlab Ltd., Cambridge, UK) and ph values converted to [H + ]. Assays of signal activity Detached leaflet experiments Twelve leaflets with petioles attached were excised from the third and fourth oldest leaves of well-drained plants under de-ionised water. These were re-cut under de-ionised water and inserted through holes in the lids of scintillation vials containing solutions that varied according to the experiment. The ph was varied using 1 mol m 3 potassium-phosphate buffer (KH 2 PO4 and K 2 HPO 4 in ratios generating the desired ph). At hourly intervals, weight loss by transpiration was recorded. Weight loss continued to be recorded at hourly intervals for a further 5 h before lights were switched off and water loss assessed for a further 1 h to check stomatal functioning. At the end of the experiment, leaflets were photocopied, scanned and areas determined electronically with image analysis software (Delta-T Devices). The assay was housed in a small growth chamber (CH 500 VL, Angelantoni Industrie, Italy) set to provide a temperature of 25 Cand a light intensity of 190 µmol m 2 s 1 PAR. Figure 1. Effect of flooding 1-month-old tomato plants for up to 36 h on (A) stomatal conductance of the abaxial surface of the fifth oldest leaf and (B) whole plant transpiration rates. Vertical bars represent the least significant difference (LSD) at p 5%. Each plant had seven or eight visible leaves. says of xylem sap, a new batch of plants was analysed at each time point. Numerical analyses Delivery rates of solutes in the transpiration stream were calculated by multiplying the concentration of each solute by the flow rate of the sap being analysed and dividing by the leaf area to give a specific delivery rate (mol s 1 m 2 ). Before sap was collected, the flow rate from each plant root system was adjusted to approximate whole plant transpiration rate appropriate for the time of day and treatment. This was necessary because transpiration varied with time and duration of flooding (Figure 1). Fully randomised experimental designs were used. Least significant differences (LSD) were calculated (p 0.05) on untransformed data. For conductance and transpiration measurements, the same plants or leaves were sampled each time. For as- Results Within 4 h of the start of soil flooding, stomatal conductances were already substantially smaller than in well-drained plants; flooding reversing the normal morning rise in stomatal opening (Figure 1A). The effect was maximal in the middle of the photoperiod when conductances of flooded plants were 66% smaller than in controls. During the second half of the photoperiod, stomatal conductances declined in control plants. Conductance also continued to decrease in flooded plants except for a reproducible temporary partial recovery after approximately 8 h treatment. During the second photoperiod, controls again showed a mid-day peak in conductance; in flooded plants conductance values remained small and declined further

4 106 Figure 2. Effect of flooding 1-month-old tomato plants for up to 36 h on estimated delivery in the transpiration stream of total osmolites from roots to shoots expressed on a leaf area basis. Vertical bar represents the least significant difference (LSD) at p 5%. over time (Figure 1A). Generally, changes in conductance measured in leaf 5 were mirrored by changes to whole plant transpiration rate (Figure 1B), demonstrating the limiting effect on overall water use brought about by a general closing of stomata. The impact of flooding on transpiration stream solutes, measured in terms of osmolality, is shown in a time-course of delivery rates (Figure 2). A large (59%) decrease occurred within 2.5 h flooding compared to a statistically insignificant fall of only 5% in controls. Later, the position was reversed since, by the end of the first photoperiod, solute transport to the shoots of flooded plants increased sharply and came to equal or even exceed that in well-drained controls. By then, solute delivery in controls had fallen away naturally to less than 16% of the starting rate. During the second photoperiod, the reversal became more marked, with delivery in flooded plants consistently exceeding that of well-drained plants especially during the early afternoon (Figure 2). Three major nutrients (NO 3,PO 4 3 and SO 4 2 ) were chosen as anion components of the total solute load. Each suffered marked decreases in delivery to the shoot during the first 2.5 h of flooding that were sustained for several more hours. The decrease in NO 3 delivery was especially large at this time and amounted to a 63% decrease from the initial value while delivery in control plants remained unchanged. After 12 h flooding, NO 3 delivery was almost completely extinguished (Figure 3A). A small recovery was seen during the first half of the following day but, values remained substantially below those of well-drained plants. After 36 h flooding, almost no NO 3 was delivered in the xylem sap since the concentration (2 Figure 3. Effect of flooding 1-month-old tomato plants for up to 36 h on estimated delivery from roots to shoots of (A) NO 3,(B) PO 4 3 and (C) SO 4 2 expressed on a leaf area basis. Vertical bars represent the least significant difference (LSD) at p 5%. mol m 3 ) and sap flow per plant (1 mm 3 s 1 )were both extremely small by this time. Phosphate delivery was also decreased by 2.5 h of flooding compared to well-drained plants. The effect was most marked after 6 h when deliveries of PO 4 3 were only 67% of controls (Figure 3B). Nevertheless, unlike NO 3,deliveries of PO 4 3 began to rise towards the end of the first photoperiod and remained larger for much of the following day before collapsing after 33 h flooding. A similar pattern was obtained for SO 4 2 (Figure 3C) except that the final downturn was absent. Delivery of Ca 2+ was depressed within 2.5 h to rates that were substantially below those of control

5 107 plants (Figure 4A). However, within 12 h, a clear reversal was evident as deliveries rose sharply to exceed well-drained values. During the following day, this abnormally fast delivery of Ca 2+ to the shoots of flooded plants was sustained. Potassium delivery was also inhibited strongly by 2.5 h flooding resulting in values that remained below control values for 10 h. However, by 12 h, values for well-drained plants had decreased to similarly low levels (Figure 4B). During the second photoperiod, delivery in well-drained plants increased again but the rise was not fully matched by flooded plants. In contrast to the other cations, Na + delivery was very slow during most of the first day. Rates were similar for both treatments until 12h when the values for flooded plants rose suddenly to exceed those of well-drained plants many fold (Figure 4C). During the following day, Na + transport in controls remained close to zero but in flooded plants it rose to maximum deliveries of nmol s 1 m 2. The delivery of H + ions was decreased 75% by 2.5 h flooding while in control plants, delivery increased slightly over this time. Although delivery rates of controls then subsided steadily throughout the photoperiod, they always exceeded rates for flooded plants (Figure 5A). During the following day, values for welldrained plants continued to exceed those for flooded plants although differences were not statistically significant. Measurements of transpiration stream ph (Figure 5B) show a temporary increase that was evident after 2.5 h flooding and, for the most part, retained until at least 12 h. In the second photoperiod, ph was similar for both treatments. The possible impact of strongly decreased deliveries of K + on stomatal behaviour was tested in a detached leaf bioassay. Explants comprising three leaflets from a fully expanded leaf were initially provided with potassium phosphate buffer (ph 6.5) at a concentration of K + of 5 mol m 3. This is similar to that estimated for the transpiration stream of well-drained plants. After 1 h, half the leaves were switched to a much smaller K + concentration (1 mol m 3 ) typical of xylem sap from flooded plants at the time of first stomatal closure. A more extreme treatment, that of supplying only deionised water and thus withdrawing K + supply completely, was also included. Neither treatment slowed the rate of water loss from the explants compared to 5 mol m 3 K + (Figure 6) indicating no promotion of stomatal closure. However, stomata remained responsive to closing stimuli since darkness strongly suppressed water loss. The following morning, illumination raised rates of water Figure 4. Effect of flooding for up to 36 h on estimated delivery from roots to shoots of (A) Ca 2+, (B) K + and (C) Na + in 1-month-old tomato plants expressed on a leaf area basis. Vertical bars represent the least significant difference (LSD) at p 5%. loss, but the response (interpreted as stomatal reopening) was not inhibited by lowering or eliminating K + supply (Figure 6). The impact on stomatal closure of raising the ph of xylem sap to 7 was tested using potassium phosphate buffer containing 1 mol m 3 K +, a concentration similar to that of the transpiration stream of flooded plants at the time stomata started to close. This upward ph shift had no statistically discernible effect compared to buffer set at ph 6.5, a value typical of well-drained plants (result not shown). When the test was repeated

6 108 but with a larger ph difference (ph 6.0 increased to 7.0) there was still no discernible effect (Figure 7A). This was so even when the buffer also contained a small amount of ABA (Figure 7A). This amount was the concentration (10 µmol m 3 ) estimated to be present in the transpiration stream of flooded plants during the first few hours of flooding. Despite this lack or response to K + or ph, detached leaves were found to retain sensitivity to ABA supplied at 50 µmol m 3 (Figure 7B) This concentration exceeded by 40% the approximately 30 µmol m 3 present in xylem sap of unstressed tomato plants. Figure 7B also indicates that transpiration by leaves fed with only deionised water remained stable for several hours indicating no effect on stomatal apertures. Discussion Figure 5. Effect of flooding for up to 36 h on (A) estimated delivery of H + ions from roots to shoots of 1-month-old tomato plants expressed on a leaf area basis, and (B) changes in the ph of xylem sap. Vertical bars represent the least significant difference (LSD) at p 5%. Figure 6. Bioassay of stomatal closure estimated from changes in the rate of transpiration by detached leaflets taken from fully expanded tomato leaves and inserted into vials of treatment solution. Effect of transfer from 5 mol m 3 potassium phosphate buffer to1molm 3 buffer or to deionised water after 1 h. The impact of switching to dark conditions and a return to light is also shown. Vertical bar represents the least significant difference (LSD) at p 5%. We measured statistically detectable decreases in stomatal apertures of tomato plants (deduced from smaller leaf conductances) within 4hofthestartofsoil flooding, compared to controls. Despite a temporary partial recovery at about mid-day, the extent of closure intensified with time and extended to the second day. The closure was sufficiently widespread to decrease the rate of water loss by the entire tomato plant, an effect offsetting the potentially dehydrating influence that loss of root hydraulic conductance brings about in the first few hours of flooding (Else et al., 1995a; Jackson et al., 1996). Plants that are deficient in the hormone abscisic acid (ABA), and thus have an impaired ability to close their stomata when flooded, suffer more leaf damage than the wild type (Jackson and Hall, 1987). Thus, the stomatal response to flooding in tomato is a rapid, persistent and effective acclimation step. Our aim is to understand how it is initiated. The effect is, necessarily, a response to one or more signals from the roots. The transpiration stream is a likely conduit for either positive or negative messages from flooded roots that may promote closing of stomata (Jackson, 2002). These are conceived as increases or decreases, respectively, in the carriage of physiologically active solutes from roots to the shoot. In the present paper, we examine the possible role that changes in ion transport to the shoot may have in effecting stomatal closure. Our strategy was to characterize changes in delivery of major mineral ions that take place before or simultaneously with the start of stomatal closure and to test experimentally the efficacy

7 109 Figure 7. (A) Comparison of the effect of increasing ph of 1 mol m 3 potassium phosphate buffer from 6.0 or 7.0 in the presence and absence of 10 µmol m 3 ABA on apparent stomatal conductances estimated as transpiration rates of detached leaflets taken from fully expanded tomato leaves. (B) Effect of adding 50 µmol m 3 ABA to deionised water on apparent stomatal conductances measured as water loss from excised leaflets taking up the treatment solutions by transpiration. Vertical bars represent the least significant difference (LSD) at p 5%. of promising signals as a possible effectors of stomatal closure. transfer to the xylem across membranes (Marshner, 1995; Miller, 1985; Scheurwater et al., 1998). Total solutes It had been shown previously (Jackson et al., 1996) that solute delivery increases on the second day of flooding and this was confirmed in the present work. One source of this flush of solutes could be root cells dying from prolonged oxygen starvation. A second, possibly dominant source, may be an increasingly unrestricted entry of minerals from the soil solution as dead or dying cells fail to regulate selective ion uptake and create increasingly open apoplastic pathways for the entry of soil solution (Everard and Drew, 1989). Above normal solute deliveries were confirmed in the present study and commenced after 12 h of flooding stress. However, the situation after shorter flooding times when stomata first started to close was found to be very different. Instead of increases in delivery, marked reductions in overall solute delivery were observed. These commenced no later than 2.5 h after flooding began, thus anticipating or accompanying stomatal closure. Since, in tomato, solute deliveries are partially or wholly independent of sap flow rates (Else et al., 1995b) this early decrease in solute delivery is probably an outcome of inhibited energydependent uptake of ions from the soil and of their Mineral anion signalling Phosphate, NO 3 and SO 4 2 were assayed since they are important mineral nutrients and major contributors to the total solute content of xylem sap (Schurr and Schulze, 1995). In addition, PO 4 3 shortage is a potential message that closes stomata since Radin (1984) showed that withholding PO 4 3 promotes ABA-induced closure of stomata. Nitrate also has a promising role in root to shoot signalling since decreasing its supply in cotton raised the apparent sensitivity of stomata to ABA in tests with detached leaves (Radin et al., 1982). Furthermore, NO 3 delivery in xylem sap also reflects its concentration in root tissues, depression of which may decrease root hydraulic conductance (Hoarau et al., 1996) and the synthesis of cytokinins (Takei et al., 2001). Our ion measurements showed a slowing of NO 3, PO 4 3 and SO 4 2 delivery that preceded the beginning of stomatal closure. Decline in NO 3 delivery was especially marked, with a decrease of approximately 64% evident in 2.5 h. It was also the most sustained of the three. In contrast to PO 4 3 and SO 4 2, there was no reversal to above-normal deliveries towards the end of the first day of flooding and during the second day. This was presumably the outcome of

8 110 anaerobic soil bacteria utilizing NO 3 as an alternative terminal acceptor of electrons (Gambrell et al., 1991). These effects of depleted NO 3 correlate to known effects of flooding on root hydraulic conductance (e.g., Else at al. 1995a) and xylem sap cytokinin activity when measured by bioassay (Carr and Reid, 1969). In contrast to those of NO 3, concentrations of PO 4 3 available for unregulated entry can increase as a consequence of increased solubilization of iron phosphates (Rubio et al., 1998) in the more chemically reducing conditions of flooded soil. This may contribute to increases in foliar phosphorus sometimes reported after a few days flooding (e.g., Jackson, 1979) and to the raised delivery rates in xylem sap when flooding lasted for 12 h or more. Mineral cation signalling We chose to measure K + because it is the most abundant mineral nutrient cation in xylem sap (Schurr and Schulze, 1995) and because of its central role in the expansion and contraction of stomatal guard cells (Wilmer and Fricker, 1996). We chose to measure Ca 2+ because of its intracellular role in the signal transduction pathway of ABA-mediated stomatal closure. Elevated cytoplasmic Ca 2+ is required for both stomatal opening and for closure. Its action is linked to activation or de-activation, respectively, of plasma membrane H + /ATPaseandaK + -H + symport (Netting, 2000). Sodium was chosen as a cation for which there is a particularly active exclusion mechanism in roots that is vulnerable to oxygen shortage (Drew and Läuchli, 1985). Our analyses of changes in K + delivery were reminiscent of changes in NO 3. Within 2.5 h of flooding, transport to the shoot was depressed by 73%, making it a promising candidate signal for closing stomata. The inhibition of uptake from the soil into the xylem was such that K + decreased from approximately 6 to almost 1 mol m 3 in sap flowing at rates close to those of whole plant transpiration. Inhibition of K + uptake by flooded plants is well established (e.g., Jackson, 1979; Zhang and Davies, 1986). As with NO 3, the negative K + message persisted into the second day and thus may also play a part in sustaining closure in addition to its initiation. Guard cells of initially open stomata are rich in cytoplasmic K + and thus independent of external supplies until stomata attempt to re-open at the start of the second photoperiod when light activates K + uptake (Wilmer and Fricker, 1996). At least 1 mol m 3 apoplastic K + is needed to support inward movement via the K + inward rectifier channel and thus underpin guard cell expansion (Blatt, 1985). The concentration of K + in xylem sap at the base of the shoot and flowing at rates similar to the plant s transpiration rate was mol m 3 in our flooded tomato plants and thus close to this threshold value. Potassium application was shown by Zhang and Davies (1986) to re-opened closed stomata of leaves taken from flooded pea plants, implying that a low external supply of K + may indeed have been insufficient to support opening on the morning of the second photoperiod. However, our tests with detached leaves showed that reducing the supply of K + by 80% or eliminating it did not promote stomatal closure or inhibit light-driven re-opening This indicates that over the short term, there are sufficient K + reserves in the leaf apoplast for stomatal adjustments to be made independently of xylem K +. Calcium is undoubtedly a key component of intracellular signalling during ABA-induced closure at warm temperatures and increasing external Ca 2+ can enter guard cells via calcium channels and enhance ABA activity (De Silva et al., 1985). However, it is believed that Ca 2+ levels in the guard cell apoplast are protected in some way from changes in xylem Ca 2+ (Atkinson et al., 1990). In the light of this, and our finding that Ca 2+ delivery from flooded roots decreased rather than increased at the time stomata start to close, Ca 2+ changes alone are unlikely to instigate stomatal closure in flooded tomato plants. However, after 10 h flooding, when Ca 2+ fluxes reverse as rootcell integrity collapses, any consequential addition of this cation to the guard cell apoplast may help to sustain closure. The behaviour of Na + illustrates the changing ability of roots to operate as selective ion absorbers as flooding proceeds. Sodium exclusion processes are complex but, in roots of non-halophytes, they probably include an energy-dependent outwardly directed H + /Na + antiport. In maize, the Na + exclusion process is susceptible to a >25% fall in oxygen from the 20.8 kpa found in well-aerated water (Drew and Läuchli, 1985). Our delivery results indicate that roots of tomato remain sufficiently energized and structurally intact to exclude Na + successfully for about 10 h. At this time the equilibrium partial pressure of dissolved soil oxygen is known to have decreased from 20.8 to approximately 4 kpa (Else et al., 2001). Thereafter, the exclusion process appeared to degenerate. As a consequence, transport of Na + to the shoot in the second photoperiod came to rival that of K +.The

9 111 impact of this Na + if it reaches the guard cell apoplast is unknown and worthy of testing. Proton and ph signalling The significance of changes in delivery of protons out of the root system in the xylem centres on its impact on the ph of xylem sap and its ABA concentration. The central tenant (Wilkinson, 1999) is that if root stress alkalinises xylem sap from just below neutral values of unstressed plants (from approx. 6.4 to 7.3), ABA concentrations rise in response to a steepened concentration gradient of undissociated and thus uncharged abscisic acid (ABAH). This occurs because ABAH dissociates more completely in alkaline xylem sap thus maintaining a steep concentration gradient of undissociated ABAH from symplast to sap The net effect is a promotion of diffusion of ABAH from cells surrounding the vasculature into the alkalinised sap of water-conducting vessels. If the resulting increase in the ABA titre raises ABA around guard cell to more than approximately 30 µmol m 3, the plasma membrane H + /ATPase is activated and stomata begin to close in minutes. This effect is strong enough to override a stomatal opening effect that alkaline xylem sap can bring about in the absence of ABA (Wilkinson et al., 1998). While such a system has been shown to operate in species, such as tomato exposed to drying soil, the position has been less clear for flooded plants. This is because evidence of xylem sap alkalisation has been minimal and contradictory (cf. Else et al., 1995b; Jackson et al., 1996). In addition, because flooding strongly depresses ABA concentrations in xylem sap as it exits from the roots (e.g., Else et al., 1995b) this will raise substantially the amount of enrichment by alkali trapping before stomatal-closing concentrations can be attained in the transpiration stream as it passes up the plant to target guard cells. Our present results, show, for the first time, a large decrease in proton delivery out of flooded roots in the transpiration stream. The effect started within 2.5 h and lasted for the duration of the first photoperiod. Although the biochemical explanation may be more complex than simple arrest of plasma membrane H + - ATPase activity (Gerendás and Schurr, 1999), there is no doubt about its impact on sap ph. This showed alkalisation up to ph 7.0 while well-drained control values usually held at about 6.5. The change could be enough to close stomata via ABA redistribution to xylem sap. However, our tests with excised leaves did not encourage the view. Switching from a ph 6.5 or 6.0 buffer to a ph 7.0 buffer feed did not effect stomatal closure in detached tomato leaves. This was true even when 10 µmol m 3 ABA was added to the uptake buffer to simulate the background level of ABA found in xylem sap of flooded plants at the time stomata close. Presumably, switching to ph 7.0 did not raise xylem sap ABA sufficiently to reach active concentrations. The required level lies between 30 µmol m 3 (typical of well drained plants) and 50 µmol m 3 (the concentration we found to promote closure). Such values are substantially above the 10 µmol m 3 present in sap of flooded plants as it exits from the root system. Despite this result, the impact of ph change deserves further study for two reasons. Firstly, the target ph values we used may not have been the most appropriate. Our values were based on those of sap as it enters the base of the shoot. However, sap ph may be subject to change before its evaporation in the leaf mesophyll. In future work, it will be important to ascertain the ph gradient from root to target leaf. Values from these positions will be more appropriate guides for testing the impact of sap alkalisation on stomatal regulation. A second reason for re-examining the ph signal could be that the path length along which more alkaline sap travels may be inadequate in our excised leaves. Presumably over greater distances, more ABA can be re-distributed into the transpiration stream thereby increasing the probability of enrichment to physiologically active levels. There is also the possibility that the conditions of the assay, particularly light intensity (Wilkinson and Davies, 2002) may be critical, although in our work, light intensity of the transpiration assay and of the flooding experiment with whole plants were comparable and low enough to ensure high ABA sensitivity. Summary Sharply decreasing delivery of total osmolites, protons and several mineral nutrients typified the impact of the first few hours of flooding on solutes of the transpiration stream as it entered the shoot from the root system. These changes preceded and accompanied partial closing of stomata that slowed overall transpiration. This contrasts with responses to effects of soil drying when solute levels decrease only after several days and by relatively small amounts often long after stomatal closing begins (Bahrun et al., 2002). Several hours later, the situation reversed as deliveries increased above control values, indicating a breakdown in selectivity of ion uptake as root cells degenerated

10 112 in response to soil anaerobiosis. Early decreases in delivery of NO 3, K + and protons (increased ph) were particularly marked and prompt and held promise as negative messages involved in closing stomata. However, negative K + and ph signals were ineffective when tested using an isolated-leaf transpiration assay. Nitrate remains to be tested. However, since halting all solute input into a leaf also failed to close stomata we conclude that negative messages alone are unlikely instigators of stomatal closure. Further work on negative messages carried in the transpiration stream should concentrate on possible interactions with positive or accumulation messages once these become identified more certainly. Acknowledgements This work was supported by the Royal Society of London under its Joint Projects with Central & Eastern Europe scheme. We thank Mr Richard Parkinson and Mrs Kazimiera Hajduk for growing the plants. 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