Effects of Transpiration on Potassium and Sodium Fluxes in Root Cells and the Regulation of Ion Distribution Between Roots and Shoots of Barley Seedlings

Effects of Transpiration on Potassium and Sodium Fluxes in Root Cells and the Regulation of Ion Distribution Between Roots and Shoots of Barley Seedlings WOLF DIETER JESCHKE

Lehrstuhl fUr Botanik I, Universitat Wiirzburg, Mittlerer Dallenbergweg 64,8700 Wiirzburg, West Germany Received April 14, 1984· Accepted July 9, 1984

Summary The effects of transpiration on uptake and xylem transport of K and Na and on the individual fluxes of these ions in root cells of barley seedlings (Hordeum distichon L., age 4.5 d) were studied. Transpiration affected the fluxes of K even at low K concentrations (0.02 and 0.2 roM), provided that seedlings were used in which only one seminal root was left. In these seedlings transpiration induced substantially higher rates of water flow relative to the root weight than in seedlings with all (5-7) roots. Na fluxes were affected even in seedlings with all roots. Accelerated water flow promoted uptake and xylem transport of K to the shoot, but decreased the K concentration in the roots. Compartmental analysis showed that this was due to a decreased vacuolar K content. The cytoplasmic K content was unaffected or slightly increased. Many of the effects of transpiration on Na were similar, though more pronounced than those observed with K. As a consequence transpiration shifted the KINa selectivity in the roots and shoots in favour of Na. Na differed from K regarding effects on contents in the vacuole; transpiration either lowered or raised the vacuolar Na content, depending on the presence and concentration of K. Enhanced water flow promoted the plasmalemma influx of Na more than its xylem transport, and it led to an increase in the cytoplasmic Na content and in the plasmalemma efflux. These data suggest that increased water flow interacts with ion flow across membranes of root cells at more than one site. Moreover water flow interferes with the processes which regulate the balance between accumulation of ions in root cell vacuoles and transport to the shoot. The data are compatible with a cell to cell transport of water during its radial passage across the root.

Key words: Hordeum distichon L., transpiration, guttation, water flow, potassium, sodiUm, ion uptake, xylem transport, fluxes, compartmentation, recirculation, regulation of ion transport.

Introduction Transpiration can increase ion uptake by the whole plant (e.g. Broyer and Hoagland, 1943; Greenway and Klepper, 1968) and affect KINa selectivity (Pitman, 1965). This did not occur in low-salt plants (Broyer and Hoagland, 1943) or at very low external concentrations (Russell and Shorrocks, 1959; Bowling, 1968). Increased water flow due to transpiration was suggested to promote passive release of ions to the xylem vessels (Broyer and Hoagland, 1943; Bowling, 1976) or to increase a mass flow *) Dedicated to Prof. Dr. Wilhelm Simonis, Wiirzburg, on the occasion of his 75 th birthday.

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WOLF DIETER JESCHKE

of ions across the root (Hylmo, 1953). Clarkson (1974) suggested a sweeping away of ions through the root symplasm, particularly at higher concentrations (<
Materials and Methods a) Symbols used 1) Ion fluxes and contents, see Fig. 7
2) primary data of effiux analysis total tracer effiux across the plasmalemma cytoplasmic component of ~~ vacuolar, quasisteady component of ~~o, originating from exchange of vacuolar tracer half times of exchange of cytoplasmic and vacuolar tracer corresponding rate constants (h- I )

~~ ~~o(cyt) ~~o(vac)

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Potassium and sodium fluxes and transpiration in barley seedlings

269

3) water relation parameters JVr total water flow across the root (cm 3cm- 2s- 1), per cm2 of outer root surface, including water required for shoot growth J'Vr water flow due to either transpiration or guttation depending on the conditions.

b) Plant material Barley, Hordeum vulgare L., convar. distichon (Alef.) (= Hordeum distichon L.) cv. Villa was obtained from Mr. G. Herbolsheimer, Rottenbauer, Wiirzburg. Caryopses were germinated in the dark on filter paper moistened with 0.5 mM CaS04, seedlings were then grown for 4.5 d from sowing on aerated CaS04 solution in continuous light (AEG HQL lamp 400W, 4200 Lx or 12.5 Wm- 2) at 24°C day and 22°C night, humidity was high and there was ample guttation. In some cases the residual endosperm (caryopsis) was removed and/or all but one of the seminal roots were excised ca. 2 h before starting the experiments. The following tabulation gives the K and Na content [/Lmol gFW- 1 ±SEM(n)] in these low-salt plants: roots K+ 17.7 ± 0.6(47) Na + 3.7±0.2(39)

primary leaf coleoptile 37.4 ±0.6 (48) 33.2 ± 1(47) 0.56±0.06(37) 0.72±0.065(36)

c) Experimental solutions All solutions consisted of a basal medium containing 3 mM Ca(N03h and 0.5 mM MgS04. Na+ (1 mM) was added in the form of phosphate buffer (PH 5.8) and FeNa2EDTA (0.05mM). K + (0.2 mM) was given as KCl. If Na and hence phosphate was omitted, 1/6th (0.5 mM) of the Ca++ was given as phosphate buffer [H3P04 titrated with Ca(OHh to pH 5.8]. For tracer uptake and efflux measurements the solutions were labelled with 22Na or 42K.

d) General procedure of uptake and efflux measurements Tracer ion uptake and efflux were measured in parallel using the same batch of plants and equal experimental conditions Qeschke and Jambor, 1981). Lids with 10 or 20 seedlings were placed on the experimental vessels Qeschke and Jambor, 1981) containing 38 ml of aerated, labelled solution and allowed to take up ions and tracer. In order to avoid depletion of nutrient ions the solution was circulated through a larger (up to 41) reservoir by means of a peristaltic pump. Particularly if low K concentrations were used, this ion was added at constant specific activity by a peristaltic pump regulated by an electronic timer. Ion concentrations were measured frequently and adjusted if necessary. Experiments were run at room temperature (2325°C) under continuous fluorescent light (3 Philips TL 65 W lamps, 7500 Lx). For conditions of low transpiration the vessels with the seedlings were enclosed in Perspex chambers with wet filter paper on the sides. Relative humidity was at the highest measurable level ( > 96 %) and the plants secreted large amounts of guttation fluid. High transpiration was achieved by exposing the plants to the ambient humidity, which varied between 20 and 40 % but was relatively stable throughout each experiment. Transpiration was promoted by gentle ventilation. Uptake of K and Na, or their tracers, was mesured by harvesting plants at intervals and by analysing the roots, coleoptiles, and leaves separately. All plant parts were first rinsed, twice shortly and 3 times 0.5 min in cold 0.5 mM CaS04 followed by weighing and extraction in HN0 3. Ion concentrations were expressed on a basis of fresh weight (d. Figs. 1 A, 2A). In order to determine the transport of K or Na to the shoots, the tracer ion content, or the increment in non-labelled ion content in the leaves and coleoptiles was expressed on the basis of fresh weight of roots (cp. Figs. IB, 2 B). Similarly, uptake of ions by the roots was obtained by expressing uptake by the plant as a whole on the basis of root weight.

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WOLF DIETEllJESCHKE

e} Measurement of individual fluxes ofJ(f" and Na + in root cells of whole barley seedlings The methods for determining ion fluxes using whole seedlings have been detailed recently Geschke and Jambor, 1981; Jeschke, 1982}. For measuring K fluxes the apical 1 mm of the root tips i.e. the meristematic tissue was removed from the roots. The reason was to avoid complex efflux kinetics due to the presence of tissues with high proportions of cytoplasm (root tip) and such with low cytoplasmic volume (differentiated root parts), see Behl and Jeschke (1981). This removal of the tip did not affect the interaction between transpiration and K fluxes. It also does not generate an «open root», in which external solution can freely enter the xylem vessels, as has been shown by measurements of the hydraulic conductivity of the root (Steudle and Jeschke, 1983). Flux measurements consisted of a loading and a washing-out period. During the loading period the roots - but not the entire seedlings - were brought to tracer flux equilibrium with the external, labelled solution. The duration of loading was 22 h for 22Na and ca. 16 h for 42K (depending on the time of delivery of this tracer) and well beyond the time required for equilibration. Labelled K was continuously added in order to ensure constant K concentration and specific activity; 22Na and Na were added as required. During the washing-out period the labelled solution was replaced by an unlabelled but otherwise identical one. This solution was exchanged at frequent intervals and the tracer washed out into these samples was measured by Cerenkov (42K) or -y-scintillation 2Na} counting. During the loading and washing-out periods the solutions were aerated and the environmental conditions (section d) were kept constant. For the graphical analysis of the efflux data and the evaluation of individual ion fluxes the reader is referred to Jeschke and Jambor (1981) and Jeschke (1982).

e

f) Rates ofguttation and transpiration Under conditions of high humidity guttation fluid was collected from the leaf and coleoptile tips with filter paper and weighed. A minor difficulty in this sampling was that sometimes droplets of the fluid were lost to the walls of the perspex covers. The weights of fluid were therefore standardized to a number of 10 (for plants having all roots) or 15 seedlings (for plants having 1 root). The amount of guttation fluid was related to the root fresh weight of 10 or 20 plants respectively. By this procedure the rate of guttation of the seedlings having one root was underestimated. The difference between the, higher, rate of guttation observed for the plants with one root compared to the plants having all roots (Table 5) could, therefore, be even greater than is apparent from this table. This procedure was adopted since some of the plants with one root apparently did not excrete guttation fluid. The rate of water loss and water uptake was measured by sealing seedlings into small vessels having a lateral capillary tube and filled with the nutrient solution. The plants were exposed to the same light, temperature, and low humidity conditions as in the uptake and flux experiments (section d). Water loss was determined by weighing, while water uptake by the roots was measured by following the movement of the meniscus of solution in the tube attached to the vessel. A time lag in the onset of water uptake and a resulting difference between loss from the leaves and uptake by the roots indicated a water deficit in the plants. The rate of water flow was related to the fresh weight and to the outer surface area of the roots; the water deficit was related to the fresh weight of the seedlings.

Results

a) Effect of transpiration on uptake of~ and Na + and their transport to the shoot In preliminary experiments with barley seedlings (age 4.5 d) having all seminal roots transpiration was found to increase uptake and xylem transport of Na but not J Plant. Physiol. Vol.

117. pp. 267-285 {1984}

Potassium and sodium fluxes and transpiration in barley seedlings

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Fig. 1: A) Accumulation of K+e2K) in the root .0, coleoptile • ¢ or primary leaf ~ of 4.5 d old barley seedlings having one seminal root, at high (> 96 %) • • • and low (25 %) relative humidity 0 ¢ L, T = 23 - 25 °e,continuous light, external solution 0.2 rnM K + and 1 rnM Na + • B) Uptake .0 and xylem transport "'\7 of K+ (42K) in the same seedlings and under the same conditions as in A). Open symbols and dashed lines: with transpiration, as in A). Seedlings with caryopsis.

of K at the low concentrations used (0.2 mM K and 1 mM Na)I), cpo the results of Pitman (1965) at higher concentrations. However, transpiration clearly stimulated K uptake in seedlings with one root (e.g. Fig. 1). This was not due to K uptake through the root stumps, which were above the experimental vessels. In subsequent experiments with K, therefore, seedlings with a single seminal root were used. Experiments were carried out with seedlings having their residual endosperm (caryopsis) (Figs. 1-3) or with seedlings from which the endosperm had been removed (Tables 1, 2). The response of ion fluxes to transpiration was similar in seedlings with and without the residual endosperm, but the competition between shoot and root for K, to be referred to below, was aggravated by removal of the endosperm. In detail, there were various interactions between increased water flow (transpiration) and K and Na uptake, as is seen from Figs. 1-4 and Tables 1 and 2. 1) Transpiration increased the rates of tracer K accumulation in the primary leaf (Fig. 1 A) and correspondingly accelerated the xylem K transport (Fig. 1 B). Transpiration also increased the rate of net K accumulation. This occurred at different K

1) With older seedlings (5.5 d) having a higher shoot to root ratio and higher rates of transpiration relative to the root weight transpiration increased K uptake.

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WOLF DIETER JESCHKE

A

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Fig. 2: A) Time-dependent change in the sodium content in roots . 0 and the primary leaf AL, of 4.5 d old barley seedlings with all seminal roots at low (26 %, open symbols) or high (> 96 %, closed symbols) relative humidity; T = 22 - 24°C, continuous light, external solution: 0.2 mM K + + 1 mM Na +. B) Uptake and xylem transport ~'V of Na + in the same seedlings as in A). Open symbols and dashed lines: with transpiration. Seedlings with caryopsis.

.0

concentrations and in presence or absence of Na (Tables 1 and 2). Even though the rates of Na accumulation in leaves and xylem transport of Na were low in the presence of K, they were both strongly stimulated by transpiration (Fig. 2). 2) Increased water flow did not, or only slightly, affect the rate of K or Na accumulation in the root. The initial rate of K or Na uptake by the seedlings between 0 and 8 h (Figs. 1-3), which is governed by accumulation within the root was, therefore, essentially unaffected by transpiration (Table 1). 3) In contrast, transpiration promoted the steady rate of K or Na uptake by the seedlings between 10 and 30 or 50 h, which is dominated by transport to the shoot (Figs. 1-3 and Table 1). 4) The final level of K accumulation in the root was clearly decreased by transpiration under all our conditions (Figs. 1,3, Table2). With Na transpiration induced either a decrease in the final level of accumulation in the root (when K was low or absent in the external solution: Fig.3, Table 2 and Fig.5: Qv) or an increase (in the presence of 0.2 mM K, Fig. 2). These data suggest that under conditions of transpiration K in the root is in part replaced by Na and that higher rates of water flow across the root interfere sensitively with the delicate balance of ion flow either to the vacuoles of the root cells or to the shoot. The apparent replacement of K by Na was much more evident at a low K concentration (0.02 mM K and 1 mM Na, Fig. 3): Here the K level in the root decreased after reaching a transient maximum even in the

J. Plant. Physiol. Vol.

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Potassium and sodium fluxes and transpiration in barley seedlings

A

B

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Fig. 3: A} Time-dependent change in the potassium content in the root Ai'::" and primary leaf . 0 of 4.5 d old barley seedlings having one root, with transpiration O/::,. or without ...., conditions as in Fig. 1, but 0.02 mM K + and 1 mM Na + . B} Corresponding to A}, but change in the Na + content. Seedlings with caryopsis.

Table 1: Rates (pMol gFW- 1 h- 1) of net uptake Joe and net transport to the shoot JRS (= Jex) of potassium and sodium in barley seedlings having one seminal root. Uptake was measured at low (25-35% R.H., «+transp.») or at high humidity (>96% R.H., «-transp.»); seedlings without their residual endosperm were used. Joe(init} is the net rate of uptake during the first 8 -10 hand Joc{steady} is the rate between 10 and 30 h, cpo Figs. 1 Band 2 B. External conc.:

0.02mM K+

0.02mM K+, ImMNa+

0.2mM K+, ImM Na+

net K+ fluxes -transp. Joe(init} +transp. J oe(steady} -transp. +transp. -transp. JRS +transp.

3 4.1 1.7 2.6 1.5 2.6

3 3 1.6 2.5 2.0

5 5 7.4 10.2 7.3

3.4

lOA

6.9 6.9 4.5 5.5 3.9 5.3

4.5 4.5 1.5 2.5

net Na+ fluxes -transp. Joe(init) +transp. Joe(steady) -transp. +transp. -transp. JRS +transp.

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WOLF DIETER JESCHKE

Table2: K+ and Na+ content (JLMol gFW- 1) in the root and the primary leaf of barley seedlings with one seminal root after 30 h ion uptake from solutions containing different concentrations of K+ and Na+; uptake occurred at low (25-35% R.H. «+transp.») or at high humidity ( > 96 % R.H. «- transp.»); seedlings without their residual endosperm were used. Each value was taken from uptake curves obtained from averages of two independent experiments similar to those of Figs. 2 and 3. External conc.: ion cont. in root K+ -transp. +transp. Na+ -transp. +transp.

0.02mM K+

0.02mMK+, 1mMNa+

0.2mM K+, 1mM Na+

34 23

15 10 75 65

50 31 57 62

57 62 20 30

76 95 4.5 8

ion cant. in prim. leaf K+ -transp. 45 +transp. 55 Na+ -transp. +transp.

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Fig. 4: Selectivity ratio SK/Na = (K +i*Na +0)/ (K +0 *Na +i) in the primary leaf (above) or the root (below) of barley seedlings after 30 h K + and Na + uptake from solutions containing 1 mM Na + and 0.02 mM (left) or 0.2 mM K + (right). The selectivity ratios relate to the external solution and are given for transpiring (hatched) or nontranspiring seedlings (open) without the residual endosperm (caryopsis). Seedlings with one seminal root.

absence of transpiration which, when present intensified this pattern (Fig. 3 A). Under these conditions the accumulation of Na in the root was remarkably high (Fig.3B). 5) Under all our conditions the KINa selectivity of accumulation in roots and shoots was decreased by transpiration, i.e. shifted in favour of Na (Fig. 4). J Plant. Physiol. Vol. 117. pp. 267-285 (1984)

Potassium and sodium fluxes and transpiration in barley seedlings

275

Table 3: Primary data used for the calculation of sodium and potassium fluxes (Fig. 5 A, B), as obtained from 22Na and 42K uptake and efflux experiments with barley seedlings (age ca. 4.5 d at start of loading with tracer, 5.5 d at the time of efflux measurements). For sodium: seedlings with all (5 -7) roots; 1 mM Na. For potassium: seedlings with one seminal root; 0.2 mM K+ and 1 mM Na+. 23-25 °e,continuous light, humidity 25-35% or 96%. Averages of n experiments ±s.e. mean. Fluxes in pMol gFW- I h- I ; the tracer fluxes 4>* were divided by the specific activity So in the external solution. K+ (42K) Na+ 2Na) IOn:

e

humidity: n:

high 5

low 5

high 4

Jex Jey 4>Mcyt)/so 4>Mvac)/so ke (h- I ) tll2c (min) ky *102 (h- I ) tl12y (h)

2.5 ±0.3 0.19 1.21±0.32 0.17±0.01 l.S7±0.16 22.2 0.93±0.03 74.2

3.S ±0.3 0.21 2.94±0.63 0.27±0.02 l.S1±0.09 23 1.17±0.07 59.3

4.9 ±0.7 0.13 5.7 ± 1.1 0.60±0.OS 1.25±0.06 33.3 2.06±0.25 33.5

low 4 6.7 ±OA

0.2 5.5 ±0.9 0.61±0.05 1.4S±0.13 2S.1 2.81±0.22 24.7

b) Effects of increased water flow on individual ion fluxes and compartmentation 1) Sodium fluxes Na fluxes were measured with seedlings having all (5-7) roots, since effects of transpiration could be observed here. Fluxes were determined at 1 mM Na in the absence of K. Transpiration increased both the fast, cytoplasmic, and the slow, vacuolar component of 22Na efflux [4>~o(cyt) and ~o(vac)] and accelerated the vacuolar turnover of tracer as shown by a higher rate constant kv (Table 3). The rate constant of cytoplasmic tracer exchange ke was almost unaffected, though there was a trend to lower values for the transpiring seedlings. Transpiration increased the rate of xylem transport CPex and the plasmalemma influx CPoe (Fig. 5 A) in agreement with the changes in the net fluxes (Table 1) and it accelerated also the plasmalemma efflux 4>eo of Na (Fig. 5 A). The vacuolar Na content Qv (Fig. 5 A) was decreased (1 mM N a +), this is similar to the effect of transpiration on Na levels in roots at low external K (Fig. 3 A). Remarkably, however, the cytoplasmic Na content Qc was increased by transpiration (Fig. 5 A), and this occurred similarly in the presence of 0.02 mM and 0.2 mM K (data not shown). This appears important, as it implies that interactions between water flow and Na fluxes are not confined to the site of xylem secretion. If increased water flow would promote only the secretion ofNa to the xylem sap, then a sweeping away ofNa from the symplasm would result in a lower cytoplasmic Na content (Clarkson, 1974).

2) Potassium fluxes Measurements were made at 0.2 mM K in the presence of 1 mM Na using 42K as a tracer. For the reasons given in Section a) seedlings with one seminal root only were

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WOLF DIETER JESCHKE

Na· fluxes ~ (I.Jmol gFW-'h-') , Na· contents Q(l.Jmol gFW-')

K+fluxes f1 (pmol gFW-'h-') or K+ contents (j-Imol gFW-')

0.

0.

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Fig. 5: Effect of transpiration on the unidirectional fluxes cf> and the cytoplasmic Qc and vacuolar Qv content of Nae2Na) (A) and K(42K) (B) in root cells of barley seedlings (without endosperm). A: seedlings having all roots; averages of 3 or 5 experiments ± s.e. mean (bars); 1 mM Na +. B: seedlings with one seminal root; averages of 4 experiments ± s.e. mean (bars); 0.2 mM K+ and lmMNa+. A and B: continuous light, 25-35%R.H.

used. Transpiration increased the cytoplasmic and vacuolar components of 42K tracer exchange [~to(cyt) and ~to(vac)] respectively but these changes in 42K effluxes were proportionately less than with 22Na (Table3). As with 22Na, transpiration increased the rate constant kv of vacuolar exchange, but also the rate constant of cytoplasmic exchange kc of 42K was somewhat increased (Table 3). The prominent effects of transpiration on K fluxes and compartmentation in the root cells were faster rates of xylem transport CPcx and of plasmalemma influx CPoc and a lower vacuolar K content Qv (Fig. 5 B). These changes confirm the data of uptake measurements (Section a), and show the decrease in root K content (e.g. Table 2) to be due to lower vacuolar K content as had been suggested above. In keeping with this suggestion the cytoplasmic K content was not decreased (Fig. 5 B). By contrast to Na, the plasmalemma efflux CPco of K was not increased in response to transpiration (Fig.SB). The proportionate increases in xylem transport and the plasmalemma influx were lower with K than with Na (Figs. 5 A, B), although seedlings with a single or all roots were used for K and Na fluxes respectively. The difference in the promotion of K and Na fluxes may, therefore, be even greater than is evident from the data of Figs. 5 A, B, because transpiration accelerates water flow more in seedlings with one than with all roots (Table 4).

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c) Water flows under conditions ofguttation and transpiration

Rates of water flow across the root were evaluated by measuring guttation and transpiration (Table 4). Water uptake was either estimated from the sum of water loss and the water needed for expansion growth of the shoot (metabolic water use was neglected) or under conditions of transpiration it was also measured by photometry. The latter value agreed well with the estimated one (Table 4). The volume flows J'Vr (cm3cm- 2s- 1) due to transpiration or guttation and the total flow Jvr were estimated by relating rates of water loss or uptake to the root surface using a specific surface area of 80 cm2gFW- 1 (Pitman et al. 1976). In seedlings with all roots, and more so in those with one seminal root, both J'Vr and ]V" were greatly increased by transpiration (Table 4). In addition, transpiration induced a water deficit of 0.05 or 0.16 gl gFW in seedlings with all or one of the roots respectively.

Table 4: Rates of water flow in barley seedlings having one or all seminal root(s) under conditions favouring guttation (high humidity, >96%) or transpiration (low humidity, 25% R.H.). T = 23-25 °C, external solution 0.2mM K+ and 1 mM Na+; age of seedlings was the same as in the efflux measurements: 4.5 d at time of exposing to experimental solution, 5.5 d at measurement. low

humidity: number of roots:

high

water loss (mg ~-I h- ) ' J'Vra) (cm 3 cm- s-I)*107 1'v/Lprb) (bar) required xylem sap osmolarityC) (mosM)

102± 12(6) 3.54 2.72

[K+ + Na+Jxd) calculated xylem sap osmolaritye) (mosM) water uptakef ) (mg gFW- 1 h- ) ' water deficit (mg gFW-I) total water flowg) (mg gFW-I h- 1) Jv/) (cm 3 cm- 2 s-I)*107

1

5-7 75±10(6) 2.6 2.0

123

94

36

69

1

5-7

600±90(4) 20.8 (16)

253±40(3) 8.6 (6.6)

12

n.d.

72

138

(24)

n.d.

n.d.

630±79(4) 163± 9(4)

257±39(3) 49± 5(3)

633 22

n.d.

157 5.4

87 3.0

a) Volume flow across root relative to outer surface area.

b) Using a mean value of 1.3*10-7 cm bar-I s-I for Lpr (Steudle and Jeschke, 1983). Calculated from 11" = cRT, accounting for external osmolarity. ) Estimated from cf>cx (K+ + Na+) and the total volume flow lvr. e) Assuming a univalent counteranion. f) Estimated from water loss and the water needed for shoot growth, assuming 0.85 g water per gFW. g) see Methods.

j,

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Under conditions of high humidity water flow is driven osmotically and the osmolarity required for the observed flow rates can be estimated. Assuming purely osmotic water movement the flow rate is

(1) with Lpr the hydraulic conductivity of the entire root and the osmotic pressures in the xylem sap 11"x and external medium 11"0' Before applying equ. (1) it must be asked, whether the total water flow JVr or only the guttational component J'Vr is relevant. JVr contains the water needed for expansion growth of the leaf. Since cell expansion occurs by water uptake in the leaf itself, it removes water from the leaf apoplast and the xylem sap: In this respect it resembles transpiration (and will act in parallel to transpiration at low humidity). Expansion growth in the leaf will, therefore, exert a differential drag on the xylem fluid and should not be included in the calculation of the osmolarity required to drive xylem sap across the root at low humidity. With Lpr = 1.3 * 10- 7 (Steudle and Jeschke, 1983) the 1I"x required for J/ Vr was 2.7 or 2.0 bar for seedlings with one or all roots. The corresponding osmolarities of the xylem sap (Table4) may be compared to estimated ones obtained from the (K + Na) concentration in the xylem sap - assuming an univalent counteranion (Table 4). These estimated osmolarities were in the order of the required ones. However, for seedlings with one root the estimated concentration (72 mM) was considerably lower than required (123 mM) for J'Vr. The implications of this difference will be referred to in the Discussion. Discussion The various observed interactions between transpiration and K and Na uptake (items 1- 5, p. 8), confirm and extend earlier observations. Enhanced K uptake in transpiring barley seedlings has been found by Broyer and Hoagland (1943), Hooymans (1969) (0.2 and 10mM K) but not by Pitman (1965) (15mM K and 45mM Na). The differences could be due to the K concentrations, to the presence of Na, and to the use of low-salt (Pitman, 1965) or high-salt plants (Hooymans, 1969). As early as 1943 Broyer and Hoagland observed that transpiration accelerated K uptake in highsalt but not in low-salt plants and this was confirmed by our finding that net K and N a uptake was not affected by transpiration during the initial 10 h (Figs. 1-3) but accelerated later on when the roots already were at a high-salt state. In our experiments K uptake was promoted by transpiration at low external K concentrations in the presence or absence of Na (Table 1). Remarkably, however, this was restricted to seedlings having one seminal root and hence a high shoot to root ratio and correspondingly high rates of transpiration-induced water flows (Table 4) or else to older seedlings with all roots which had a higher shoot to root ratio too. The greater effects of transpiration on Na compared to K fluxes (Figs. 5 A, B) combined with a shift of the KINa selectivity in the shoots in favour of Na (Fig. 4) confirms results of Pitman (1965) at much higher concentrations. But under our condi-

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tions transpiration also shifted the KINa selectivity in the roots towards Na (Fig. 4). The closer interaction between water flow and Na compared to K influx is strengthened by the response of Na fluxes to transpiration in seedlings with all roots. The greater ease of an interaction with Na could be due to a larger passive component in Na uptake (Pitman and Saddler, 1967; Cheeseman, 1982). The transpiration-induced decrease in the final K content of the roots (Fig. 3 and Table2) confirms observations by Hooymans, 1969. She attributed this to decreased accumulation of K in the cytoplasm rather than in vacuoles. As is clearly shown by compartmental analysis, however, transpiration decreased the vacuolar and not the cytoplasmic content in the roots. Under the same conditions (0.2 mM K and 1 mM Na) the Na content in the roots was increased. This indicates that accelerated water flow across the root shifts the vacuolar ion accumulation from K to Na. Thus water flow appears to interfere with the processes that regulate the balance of ions, in particular of K, between root cells or their vacuoles, and the shoot. So far no data on responses of individual ion fluxes to transpiration are available. Jensen and Kylin (1980) found an increase in the efflux of 86Rb from roots of cucumber, oat, and wheat and suggested that a «drought signal» by the shoot led to increased leakage of ions from the root and thereby also to improved transport of ions with the water stream to the xylem vessels. In barley seedlings transpiration also increased the cytoplasmic and vacuolar components of 42K and 22Na efflux (Table 3). As shown by an evaluation of these efflux data (Fig. 5 A, B), however, the increased rates of tracer exchange mainly reflect increased rates of K and Na influx and net uptake (Figs. 1- 3). There was no transpiration-induced leakage of ions. However, under certain conditions (low external K) K ions absorbed in the roots may be released for transport to the shoot (Fig. 3 A), K being replaced by Na, if this cation is present externally. This process was accelerated by transpiration (Fig. 3 A). The extent of this replacement of K is indicated by the K content in the roots after 30 h in 0.02 mM K and 1 mM Na solution (Table2). Even in the absence of transpiration the final K level (15 /LMol gFW-l) was below the endogenous content (17.7 /LMol gFW-1, see Methods) but in transpiring seedlings it dropped to as low as 10 ~ol gFW- 1 (Table2). These changes were due to a replacement of vacuolar K with Na within the root, as follows from estimates of the K concentration in vacuoles. Using 110 mM for the cytoplasmic K concentration, which is little affected by external conditions eschke and Stelter, 1976; Jeschke, 1977), and 5 % for the cytoplasmic and 85 % for the vacuolar volume fractions the following vacuolar K concentration were calculated for transpiring seedlings from the data of Table 2: 5.3 mM at 0.02 mM K and 1 mM Na and 21 mM at 0.02 mM K compared to 14.3 mM in low salt roots. According to these data, in the presence of N a part of the vacuolar K had been replaced by Na and then transported to the shoot, since no leakage of K occurred. In nontranspiring seedlings the vacuolar K concentrations were considerably higher (cp. the K content in the roots, Table2).

a

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Our finding that transpiration did not affect on uptake during the first 10 hours (Figs. 1-3), when root accumulation governed ion uptake by the seedlings, asks for an explanation. Firstly, the primary leaf of low-salt barley seedlings does not unfold before ion uptake to the shoot commences. Transpiration of seedlings on 0.5 mM CaS04 was, therefore, much lower than after 20 h on 0.2 mM K and 1 mM Na (as in Table4). Secondly the lack of an effect might be due to the accompanying anion, the uptake of which may be less affected by water flow than that of K and Na. This possibility needs to be checked experimentally, however. Also the stimulation of the plasmalemma Na efflux by transpiration (Fig. 5 A) is not easily explained by water and ion flow interactions. It may either be due to the increased cytoplasmic Na content (Fig. 5 A) or to a contribution of exchange transport to the plasmalemma Na efflux Geschke, 1982).

b) Causes and sites of the interaction between water and ion flows The net flow of ions across the root to the shoot (see Fig. 6) can be described by the equation (Pitman, 1977, see also Anderson, 1976)

(2) Jact and Jpass are ion transport against or along the electrochemical gradient, while the first two terms account for interactions between water flow across the symplast membranes GYm) or the apoplast GYb) with ion flows. (Jm and (Jb are the reflection coefficients of the membranes or the apoplastic pathway respectively, Cm is the ion concentration at the relevant membrane(s). (Jb was suggested to be near one, the second term becomes then}vb . Co and is hence non-discriminating; this term may be significant at high ionic concentrations (Pitman, 1977). In Fig. 6 the pathway of water includes passage across vacuoles.}vm is then the cell to cell water flow and consists of a symplastic and a transcellular component. Equ. (2) together with Fig. 6 allow one to predict the properties of coupling between water and ion flows: a) Since (Jm is a property of the membrane(s) as well as the solute and since Cm depends on the specific solute, the coupling term J ym( 1- (Jm)Cm will differ between solutes (ions) and hence be selective. b) Jvm passes at least two membranes, the plasmalemma of the root cortex and that of the xylem parenchyma cells (Fig.6). Consequently, interactions between water and solute flows can occur at both membranes. c) Along the pathway of JYm water may either cross vacuoles or move along the symplasm only (Fig. 6). In the first case water flow can interact with ion fluxes across the tonoplast. In the second case a «sweeping away» of ions bypassing the vacuoles together with water can be envisaged, and this could interfere indirectly with vacuolar ion accumulation. Our data clearly agree with the above properties a-c):

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caspanan band

symplasm (cytoplasm) Qc vacuole(s) Qv

Pvc ¢cv

> xylem vessel

=>

Fig. 6: Schematic representation of the ion fluxes t/> and the possible pathways for the radial water movement across the root, shown in a simplified longitudinal section of a root. The single cell layer stands for the series of layers extending from the rhizodermis to the xylem parenchyma.hm is the water flow from cell to cell, occurring either along the symplasm or transcellularly, hb is the apoplastic volume flow of water, bypassing the cells. - unidirectional, -? net ion fluxes. Modified after Pitman (1977). a) Transpiration affected ion uptake selectively relative to K and Na uptake (Table 2 and Fig. 4). b) At least for Na water flow promoted the influx c/Joc more than and independently of the transport to the xylem, see also the increase in the cytoplasmic Na content (Fig. 5 A). c) Transpiration interfered with the accumulation of K and N a in root cell vacuoles (Figs. 1- 3 and 5 A, B). It is suggested, therefore, that water and ion flows interact according to the term J vm{1- Um)Cm and that a considerable proportion of the water flow across the root during high transpiration occurs by a cell to cell pathway. This pathway is indicated also by measurements of the hydraulic conductivity Lp of root cells and that of the entire root Lpr (Steudle and Jeschke, 1983; Jones et aI., 1983). Undoubtedly a radial transport of ions across the root via a transcellular pathway is highly improbable. However, there is no need to assume identical pathways for

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water and solutes. If water moves across vacuoles, interactions can occur either during entry or exit from vacuoles depending on the facility of ion movement in either direction. In barley roots the tonoplast efflux of K was preferred relative to that of Na, while for Na uptake into the vacuole was favoured Geschke, 1977, 1979). Increased water flow could, therefore, promote K release from vacuoles and thereby favour its transport to the shoot as is indicated by our results (Fig. 3 A). With Na, the response depended on the K concentration. At 0.2 mM K higher water flows promoted vacuolar Na accumulation, possibly in exchange for K (Fig. 2). This agrees with the preference of tonoplast influx for Na compared to K Geschke, 1977, also 0.2 mM Na). At low external K or in its absence a decrease in the vacuolar Na accumulation was found (Figs. 3 Band 5 A). Under these conditions Na appears to mimic the behaviour of K. In transpiring seedlings neither K nor Na showed a lower cytoplasmic content than in non-transpiring ones (Figs. 5 A, B). This argues against a «sweeping away» of ions by water flow in the symplasm, see c), above, as was envisaged by Clarkson, 1974.

c) Implications for the regulation of ion transport in the whole plant In the whole plant ion uptake by the root and transport to the shoot are tightly regulated in relation to growth and demand of the shoot (Pitman, 1972, 1975; Clarkson, 1974). Increased shoot growth promoted the uptake and xylem transport of K + (Pitman, 1972) and an artificially increased shoot to root ratio accelerated the guttational water flow and strongly affected the individual fluxes of K and Na in root cells of barley seedlings Geschke, 1982). Though at first sight unrelated, a comparison between effects of transpiration and of an increased shoot to root ratio yields remarkable parallels. Similar to transpiration a high shoot to root ratio promoted the plasmalemma influx and the xylem transport of K and had little effect on the plasmalemma efflux. The vacuolar K content was decreased, the cytoplasmic content was less affected but tended to be increased at a high shoot to root ratio Geschke, 1982) as well as by transpiration (Fig. 5 B). Similar parallels were found for Na fluxes, which responded more strongly than K fluxes in both cases (cp. Figs. SA, B). Other parallels were increases in plasmalemma efflux or the cytoplasmic and vacuolar Na content in presence of 0.2 mM K Geschke, 1982, Fig. 5 and this paper, Fig. 2)2). Clearly these parallel changes argue for a common basis. It is suggested that increased water flow is this common basis and that water flow is one of the ways by which the shoot regulates ion fluxes in roots even in the absence of transpiration. This suggestion receives support from the large difference between the xylem sap osmolarity (123 mM) required to drive the observed rates of water flow across the 2) Fig. 2 shows an increased root content of Na Considering the distribution of Na between cytoplasm and vacuoles Geschke and Stelter, 1976), however, most of the Na will be in vacuoles.

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root in non-transpiring seedlings and the osmolarity (72 mM) calculated from the rates of ion and water flow (Table 4, seedlings with one root). The calculated osmolarities, however, do not include any contribution of ions recirculated from the shoot. Retranslocation of ions from barley leaves has been measured (Greenway and Pitman, 1965) and there is no doubt that K together with malate (Dijkshoorn et al., 1968) or inorganic anions is recirculated from shoots to the roots (Pate, 1975; Pitman, 1975). Recently Armstrong and Kirkby (1979) and Jeschke et al. 1984 found that recirculated K contributed by 20 % to the xylem transport in tomatoes or lupins. This contribution probably is considerably higher in seedlings with one root, since in these very high rates of phloem transport have been observed (Passioura and Ashford, 1974). It is suggested, therefore, that recirculated K, together with anions, accounts for the difference between the osmolarity of the xylem sap observed and that required to drive water at the rate of J'Vr (see below) in seedlings with one root (Table4). This implies that here a considerable part of the xylem sap osmolarity originates from phloem solutes, and is thus linked to the photosynthetic activity of the shoot. This shoot-dependent component 3) of osmotic water flow due to recirculated ions can explain the similarities in the responses of ion fluxes in roots to transpiration on the one hand and to an increased shoot to root ratio in non-transpiring seedlings on the other. Further experiments are needed to substantiate this suggestion. In particular, information on the in situ composition of the xylem sap is needed. Analyses of the root exudate would be insufficient here since the phloem-dependent component is interrupted by excision. There is yet another way in which shoot growth or a high shoot to root ratio can interact with ion fluxes in the root via water flow. As was outlined above, only the guttational component of water flow must be driven osmotically across the root, whereas the flow of water needed for expansion growth in the shoot is driven by osmotic gradients set up in that very organ. As can be seen from Table 4, the water flow due to expansion growth (JVr-J'Vr) was 1.9 or OAcm3cm-2 s- 1 in seedlings with one or all roots respectively. Thus shoot expansion by itself drives a considerable water flow across the root and this can interact with ion flows in a similar way as shoot-dependent water flow due to recirculation (see above) or as transpiration. Conclusions In the intact barley plant water and ion flows seem to interact at more than one membrane. Thus Na influx and secretion to the xylem were promoted independently by transpiration. For both K and Na also the vacuolar accumulation in roots was af3) That a shoot-dependent water flow an occur is conspicuously demonstrated by the ample guttation of low-salt barley seedlings growing on CaS04 solution (see methods). Undoubtedly this guttational water flow is virtually independent of ion uptake, and must mainly be driven by phloemborne solutes derived from the endosperm and the primary leaf.

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fected by transpiration suggesting that water flow interferes with the delicate controls, which regulate the allocation of ions to root cell vacuoles and to the shoot. Moreover transpiration shifted the KINa selectivity in the root and more so in the shoot in favour of Na. The data can neither be ascribed to an apoplastic interaction in the root nor can they be attributed to a dilution of the symplastic ion content in roots of the transpiring plant. They are consistent with interactions of ions and water as they move across the plasmalemma and the tonoplast, from cell to cell across the root, and into the xylem vessels (Fig. 6). Similarities in the responses of K and Na fluxes to transpiration and to an increased shoot to root ratio point to a common basis. It is suggested that the shoot may regulate ion uptake by the root a) by contributing to osmotic water flow across the root though ions recirculated from the shoot via the phloem, b) by controling the rate of water flow needed for expansion growth and c) by the rate of transpiration. The common basis is the interaction between water and ion flows. This type of regulation via water flow does not exclude other types of shoot-root interactions such as by the supply of photosynthates or by hormonal control. Acknowledgements This investigation was supported by the Deutsche Forschungsgemeinschaft. Thanks are extended to Miss Andrea Schniering for untiring technical assistence, to Mr. Ulrich Schliwa for constructing an electronic timer and to Mr. G. Herbolsheimer for the barley seed. Thanks are also expressed to Dr. H. Greenway, Perth, for comments and helpful suggestions to the manuscript.

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