Pl nta
Planta (1984)161:295-301
9 Springer-Verlag 1984
Potassium transport in salt-stressed barley roots Jonathan Lynch and Andr6 Lfiuchli Department of Land, Air and Water Resources, University of California, Davis, CA 95616, U S A
Abstract. Salinization of the medium inhibits both K + uptake by excised barley (Hordeum vulgare L.) roots and K + release from their stele, as measured by short-term 86Rb uptake and xylem exudation, respectively. Although inhibition was not specific to chloride, mannitol caused a different response from that of inorganic sodium salts, indicating that inhibition was at least partly the result of an ion effect. In roots previously exposed to low levels of NaC1, NaC1 stress directly affected stelar K + release, whereas in low-sodium roots stelar K § release was much less salt-sensitive than K § uptake. Key words: Hordeum- Potassium transport stress - Xylem exudation.
Salt
Introduction Two primary obstacles to plant growth in saline environments are high concentrations of potentially toxic ions and low external water potentials. Salt exclusion minimizes ion toxicity but exacerbates water stress, while salt accumulation facilitates osmotic adjustment but may lead to ion toxicity and nutritional imbalances (for recent reviews, see Greenway and Munns 1980; Wyn Jones 1981). Potassium nutrition is particularly important to salt-stressed nonhalophytes since considerable evidence indicates that metabolic damage results from high cytoplasmic Na § : K § ratios (Flowers et al. 1977; Winter and Preston 1982; Wyn Jones et al. 1979) and high Na § concentrations may interfere with K § acquisition (Greenway 1965; Orton 1980; Rains and Epstein 1967). In barley, which is a relatively salt-resistant nonhalophyte, it appears that under conditions of Abbreviation: chC1 = choline chloride
salt stress, favorable cytoplasmic Na + : K + ratios are maintained by a combination of K + : N a § uptake selectivity, Na § extrusion, vacuolar Na § compartmentation, and partitioning of Na § away from growing tissues at the expense of older organs (Greenway 1965; Nassery and Baker 1972; Orton 1980; Pitman et al. 1981; Wyn Jones and Storey 1978). In particular, Na + :K § ratios are typically higher in the roots than in the shoots of saltstressed barley plants (Greenway 1965; Lynch et al. 1982; Orton 1980; Wyn Jones and Storey 1978). This observation indicates that the salt sensitivity of K + transport into the xylem stream may be different from that of K + uptake by the root cortex. In this study we test this hypothesis by comparing the uptake and stelar release of K § in salttreated barley roots. We find the two processes to have differing relative sensitivities dependent upon the nutritional status of the root. A preliminary account of this work has been presented elsewhere (Lynch and Lfiuchli 1983).
Materials and methods Plant material Commercial quality barley (Hordeum vulgare L.) cv. Arivat was used in this investigation.
Exudation experiments Germination. Dry seed was rinsed for 30 s with deionized water. The moist seed was spread onto cheesecloth suspended over a solution of 0.5 m M CaSO4 by a stainless-steel grid inserted into a 4-1 plastic container. The solution was gently aerated with a single sintered-glass aeration tube supplied with filtered air. The germination container was covered with cellophane and placed in the dark for 94 h at 2 2 + 1 ~ C. At the end of this period, typical seedlings had 4 cm of coleoptile and five seminal roots 5-10 cm in length.
296
J. Lynch and A. L~iuchli : K + transport in salt-stressed barley roots 80
Greening. Following germination, nine seedlings were selected on the basis of vigor and uniformity, transferred to aerated 0.5 m M CaSO 4 solutions in opaque 4-1 containers, and placed in a growth chamber. The growth chamber was illuminated by fluorescent (" cool white"; General Electric Co., Cleveland, O., USA) and incandescent lamps, which supplied 70 gmol (photons) m -z s-1 of photosynthetically active radiation at plant level. During the 12-h light period, the temperature was maintained at 22 ~ C and the relative humidity at about 65% ; during the 12-h dark period the temperature was held at 19 ~ C and the relative humidity at about 90%. After 50 h in the growth chamber, the plants were moved to the laboratory for pretreatment.
Pretreatment. During the pretreatment period, the air temperature was 22_+ 1~ C, the relative humidity 60+ 10%, and fluorescent lamps ("cool white") supplied 70 gmol m -z s-1 of photosynthetically active radiation of plant level. The pretreatment solutions contained 0.5 m M KNOa, 0.5 m M CaSOr and in some cases 1 m M NaC1; K + and Na + were radiolabeled with S6Rb and 22Na as necessary. Seedlings were pretreated in this manner for 24 h, at which point K + accumulation in the roots had equilibrated (Fig. 1).
Xylem exudation measurement. For the measurement of ion transport through excised roots we used a slightly modified version of the technique developed by Pitman (1971). Following pretreatment, the seedling roots were rinsed in 0.5 m M CaSO 4, inserted into plexiglass transport chambers as shown in Fig. 2, and the shoots removed. The roots were inserted into the chambers horizontally so that the terminal 8 cm of the root system extended into the uptake compartment. The upper portions of the two vertical partitions were then fitted down on top of the roots and sealed with silicone grease (high-vacuum grease; Dow Coming Corp., Midland, Mich., USA), forming three water-tight compartments. Then the shoot was excised, leaving about 5 mm of each root protruding into the collection compartment. Leakage between the compartments was determined by assaying the radionuclide in the guard compartment at the end of each experiment. Data from leaky chambers were discarded. During the 60 rain following shoot excision, the roots were in 0.5 mM CaSO 4 at 22+0.1 ~ C. Each compartment of each chamber was aerated with filtered air passed through a hypodermic needle. Sixty min after excision, the uptake-compartment solution was replaced with the pretreatment solution (" time zero") and samples were collected at intervals by withdrawing 10-ml aliquots from the collection compartment. The remaining 8 ml of solution were removed and discarded, and the compartment refilled with 18 ml of 0.5 mM CaSO 4. After equilibrating for 3 h, the uptake-compartment solution was removed and replaced with the treatment solution, which contained 0.5 mM CaSO,, 0.5 mM K N O 3 (radiolabeled where appropriate) and varying amounts of the applied solute (radiolabeled where appropriate). Sample collection was continued for 2 h following treatment application, at either 15- or 30-min intervals. After collection of the final xylem-exudate sample, the portion of the root system in the uptake compartment was removed from the chamber, blotted dry, and weighed. Thus, a total of 6 h elapsed from the time of root excision to the termination of the experiment. In one set of experiments the treatment solution applied at 3 h did not contain K § In these experiments K § (S6Rb+) efflux from the roots was measured by periodically sampling the solution in the uptake compartment.
Radionuclide assay. Samples were made up to a final volume of 20 ml with a mixture of 8 g Omnifluor (New England Nucle-
0 0 Roots [] [ ] Shoots - 0 - - - - . 4 Roots + N a C I u~ 60 - - J ' - - " - m Shoots + N a C I 70
To
"
~
50 -
+
~
~
/..11
2o
I0 j
/
;="
~O~ M ~lr ~
0
I0
20
I
30
TIME (hours)
Fig. 1. Uptake of K + in roots and shoots of intact barley seedlings during a 24-h treatment with 86Rb-labeled 0.5 mM K N O 3 and 0.5 mM CaSO~. Salt treatment (closed symbols) applied as 15 m M NaCL Two replicates per determination
Col lect ion Absorpt ion Compartment Compartment I Cut end I Guard Root of root J Compartment tip
i :
Fig. 2. Plexiglass transport chamber used for xylem-exudation measurement (after Pitman 1971)
ar, Boston, Mass., USA) in 1 I dioxane prior to liquid scintillation counting (300 C; Packard, Downers Grove, Ill., USA). Samples containing [laC]choline (Sigma Chemical Co., St. Louis, Mo., USA) were evaporated to dryness in polyethylene scintillation vials, then taken up with I ml of H 2 0 and 9 ml of the dioxane-Omnifluor mixture prior to counting.
Uptake experiments Seeds were germinated as described above, but greened without thinning in order to generate large amounts of root material. Greening and pretreatment conditions were as described above, with the exception that the pretreatment solution did not contain radionuctides. After excision, groups of 15-20 roots were enclosed in cheesecloth "teabags" (Epstein et al. 1963) that permitted immersion of the root material in experimental solutions for specified time periods. As in the xylem exudation experiments, the roots were first exposed to 0.5 mM CaSO 4 for 1 h followed by exposure to the pretreatment solution (0.5mM CaSO4, 0.5 mM K N O 3 and either 0 or 1 m M NaC1) for 180 min. At 180 min the roots were transferred to treatment solutions, containing 0.5 m M CaSO4~ 0.5 m M KNO3, and variable amounts of NaC1. As in the exudation experiments, the treatment continued for 2 h.
J. Lynch and A. L/iuchli: K + transport in salt-stressed barley roots Experimental solutions were maintained at 22_+ 0.5 ~ C. The ratio of solution volume to root mass was approx. 800 ml/g, as in the exudation experiments: with this ratio the solutions were not detectably depleted during the course of the experiment. All solutions were gently aerated with filtered air by means of sintered-glass rods. Ion uptake was determined by transferring some roots from the pretreatment or treatment solutions for 10 rain to identical solutions containing trace amounts of either 86RbC1 or 22NaC1. Series of uptake measurements were taken over time, so that the midpoints of uptake periods coincided with the midpoints of xylem-exudate collection periods in the exudation experiments. On the basis of previous work in this laboratory (Welch and Epstein 1969) and Cram's work with barley roots (Cram 1969; Cram and Laties 1971), this 10-min uptake period was taken as an estimate of influx across the plasmalemma. Uptake was terminated by immersing the roots in 0.5 mM CaSO4 at 5~ followed by free-space desorption in aerated 0.5 m M CaSO~ at 5 ~ C for i0 min. The roots were then blotted dry, weighed, and dry-ashed in glass scintillation vials at 575 ~ C for 3 h. The ash was dissolved in 1 ml deionized H 2 0 and taken up to 10 ml with dioxane and Omnifluor prior to counting as described above.
297 20 - - influx
a 1 5 m M NaCI
16 _ - . . . . . 30rnM NaCI - - - - - - 45rnM NaCI 12 L
8
_
L.--.X.. n
--
[d- --~}~ .
Exudation data were analyzed for equality of treatment means with multivariate linear regression (Timm 1975). Uptake data from intact plant and excised root experiments were analyzed using repeated measures analysis of variance (Dixon 1981).
Results
To interpret the effects of applied solutes on K + appearance in the collection compartment of our transport chambers, we first had to determine the lag time between events occurring in the uptake compartment and consequent events detected in the collection compartment. This lag time was estimated by applying a pulse of 86Rb to the uptake compartment at the time of salinization (180 min) of standard roots (i.e. pretreated with 0.5 m M K N O 3 and 0.5 m M CaSO4). Rubidium-86 was detected in the collection compartment within 5 rain of the start of the pulse in both control (0.5mM CaSO 4 and 0.5raM K N O 3 ) and salinized (0.5 m M CaSO4, 0.5 m M KNO3, and 45 m M NaC1) treatments, indicating that the lag time between the uptake of K + and its subsequent appearance in the collection compartment was small relative to the sample collection intervals. The magnitude of K + exudation was ra:ther variable in our system, possibly because o f seed variability and size differences among root sys-. terns. Approximately 80% of the root systems were: composed of five individual roots, the remainder having either four or five individual roots. However, when the exudation data were normalized by converting fluxes (gmol g - ~ fresh weight h-1) to percentages of the flux preceding treatment appli-
.
.
.
~
/f/l- --
4
7 o E X / i,
+ (3
0 5
I --exudation
I -
I -
-
I
I b
-
4
~---~ . . . . . . . . ~ . . . . . . . 5
-1
F
I
-
-
2-I -o
Data analysis
/
/
150
180
I I I 210 240 270 TIME (minutesl
I 300
Fig. 3a, b. Influx of Na + (a) and xylem exudation (b) in excised barley roots following NaC1 application. NaC1 applied at 180 min. a Five replicates per determination; salt effect highly significant (P
Planta (1984)161:295-301
9 Springer-Verlag 1984
Potassium transport in salt-stressed barley roots Jonathan Lynch and Andr6 Lfiuchli Department of Land, Air and Water Resources, University of California, Davis, CA 95616, U S A
Abstract. Salinization of the medium inhibits both K + uptake by excised barley (Hordeum vulgare L.) roots and K + release from their stele, as measured by short-term 86Rb uptake and xylem exudation, respectively. Although inhibition was not specific to chloride, mannitol caused a different response from that of inorganic sodium salts, indicating that inhibition was at least partly the result of an ion effect. In roots previously exposed to low levels of NaC1, NaC1 stress directly affected stelar K + release, whereas in low-sodium roots stelar K § release was much less salt-sensitive than K § uptake. Key words: Hordeum- Potassium transport stress - Xylem exudation.
Salt
Introduction Two primary obstacles to plant growth in saline environments are high concentrations of potentially toxic ions and low external water potentials. Salt exclusion minimizes ion toxicity but exacerbates water stress, while salt accumulation facilitates osmotic adjustment but may lead to ion toxicity and nutritional imbalances (for recent reviews, see Greenway and Munns 1980; Wyn Jones 1981). Potassium nutrition is particularly important to salt-stressed nonhalophytes since considerable evidence indicates that metabolic damage results from high cytoplasmic Na § : K § ratios (Flowers et al. 1977; Winter and Preston 1982; Wyn Jones et al. 1979) and high Na § concentrations may interfere with K § acquisition (Greenway 1965; Orton 1980; Rains and Epstein 1967). In barley, which is a relatively salt-resistant nonhalophyte, it appears that under conditions of Abbreviation: chC1 = choline chloride
salt stress, favorable cytoplasmic Na + : K + ratios are maintained by a combination of K + : N a § uptake selectivity, Na § extrusion, vacuolar Na § compartmentation, and partitioning of Na § away from growing tissues at the expense of older organs (Greenway 1965; Nassery and Baker 1972; Orton 1980; Pitman et al. 1981; Wyn Jones and Storey 1978). In particular, Na + :K § ratios are typically higher in the roots than in the shoots of saltstressed barley plants (Greenway 1965; Lynch et al. 1982; Orton 1980; Wyn Jones and Storey 1978). This observation indicates that the salt sensitivity of K + transport into the xylem stream may be different from that of K + uptake by the root cortex. In this study we test this hypothesis by comparing the uptake and stelar release of K § in salttreated barley roots. We find the two processes to have differing relative sensitivities dependent upon the nutritional status of the root. A preliminary account of this work has been presented elsewhere (Lynch and Lfiuchli 1983).
Materials and methods Plant material Commercial quality barley (Hordeum vulgare L.) cv. Arivat was used in this investigation.
Exudation experiments Germination. Dry seed was rinsed for 30 s with deionized water. The moist seed was spread onto cheesecloth suspended over a solution of 0.5 m M CaSO4 by a stainless-steel grid inserted into a 4-1 plastic container. The solution was gently aerated with a single sintered-glass aeration tube supplied with filtered air. The germination container was covered with cellophane and placed in the dark for 94 h at 2 2 + 1 ~ C. At the end of this period, typical seedlings had 4 cm of coleoptile and five seminal roots 5-10 cm in length.
296
J. Lynch and A. L~iuchli : K + transport in salt-stressed barley roots 80
Greening. Following germination, nine seedlings were selected on the basis of vigor and uniformity, transferred to aerated 0.5 m M CaSO 4 solutions in opaque 4-1 containers, and placed in a growth chamber. The growth chamber was illuminated by fluorescent (" cool white"; General Electric Co., Cleveland, O., USA) and incandescent lamps, which supplied 70 gmol (photons) m -z s-1 of photosynthetically active radiation at plant level. During the 12-h light period, the temperature was maintained at 22 ~ C and the relative humidity at about 65% ; during the 12-h dark period the temperature was held at 19 ~ C and the relative humidity at about 90%. After 50 h in the growth chamber, the plants were moved to the laboratory for pretreatment.
Pretreatment. During the pretreatment period, the air temperature was 22_+ 1~ C, the relative humidity 60+ 10%, and fluorescent lamps ("cool white") supplied 70 gmol m -z s-1 of photosynthetically active radiation of plant level. The pretreatment solutions contained 0.5 m M KNOa, 0.5 m M CaSOr and in some cases 1 m M NaC1; K + and Na + were radiolabeled with S6Rb and 22Na as necessary. Seedlings were pretreated in this manner for 24 h, at which point K + accumulation in the roots had equilibrated (Fig. 1).
Xylem exudation measurement. For the measurement of ion transport through excised roots we used a slightly modified version of the technique developed by Pitman (1971). Following pretreatment, the seedling roots were rinsed in 0.5 m M CaSO 4, inserted into plexiglass transport chambers as shown in Fig. 2, and the shoots removed. The roots were inserted into the chambers horizontally so that the terminal 8 cm of the root system extended into the uptake compartment. The upper portions of the two vertical partitions were then fitted down on top of the roots and sealed with silicone grease (high-vacuum grease; Dow Coming Corp., Midland, Mich., USA), forming three water-tight compartments. Then the shoot was excised, leaving about 5 mm of each root protruding into the collection compartment. Leakage between the compartments was determined by assaying the radionuclide in the guard compartment at the end of each experiment. Data from leaky chambers were discarded. During the 60 rain following shoot excision, the roots were in 0.5 mM CaSO 4 at 22+0.1 ~ C. Each compartment of each chamber was aerated with filtered air passed through a hypodermic needle. Sixty min after excision, the uptake-compartment solution was replaced with the pretreatment solution (" time zero") and samples were collected at intervals by withdrawing 10-ml aliquots from the collection compartment. The remaining 8 ml of solution were removed and discarded, and the compartment refilled with 18 ml of 0.5 mM CaSO 4. After equilibrating for 3 h, the uptake-compartment solution was removed and replaced with the treatment solution, which contained 0.5 mM CaSO,, 0.5 mM K N O 3 (radiolabeled where appropriate) and varying amounts of the applied solute (radiolabeled where appropriate). Sample collection was continued for 2 h following treatment application, at either 15- or 30-min intervals. After collection of the final xylem-exudate sample, the portion of the root system in the uptake compartment was removed from the chamber, blotted dry, and weighed. Thus, a total of 6 h elapsed from the time of root excision to the termination of the experiment. In one set of experiments the treatment solution applied at 3 h did not contain K § In these experiments K § (S6Rb+) efflux from the roots was measured by periodically sampling the solution in the uptake compartment.
Radionuclide assay. Samples were made up to a final volume of 20 ml with a mixture of 8 g Omnifluor (New England Nucle-
0 0 Roots [] [ ] Shoots - 0 - - - - . 4 Roots + N a C I u~ 60 - - J ' - - " - m Shoots + N a C I 70
To
"
~
50 -
+
~
~
/..11
2o
I0 j
/
;="
~O~ M ~lr ~
0
I0
20
I
30
TIME (hours)
Fig. 1. Uptake of K + in roots and shoots of intact barley seedlings during a 24-h treatment with 86Rb-labeled 0.5 mM K N O 3 and 0.5 mM CaSO~. Salt treatment (closed symbols) applied as 15 m M NaCL Two replicates per determination
Col lect ion Absorpt ion Compartment Compartment I Cut end I Guard Root of root J Compartment tip
i :
Fig. 2. Plexiglass transport chamber used for xylem-exudation measurement (after Pitman 1971)
ar, Boston, Mass., USA) in 1 I dioxane prior to liquid scintillation counting (300 C; Packard, Downers Grove, Ill., USA). Samples containing [laC]choline (Sigma Chemical Co., St. Louis, Mo., USA) were evaporated to dryness in polyethylene scintillation vials, then taken up with I ml of H 2 0 and 9 ml of the dioxane-Omnifluor mixture prior to counting.
Uptake experiments Seeds were germinated as described above, but greened without thinning in order to generate large amounts of root material. Greening and pretreatment conditions were as described above, with the exception that the pretreatment solution did not contain radionuctides. After excision, groups of 15-20 roots were enclosed in cheesecloth "teabags" (Epstein et al. 1963) that permitted immersion of the root material in experimental solutions for specified time periods. As in the xylem exudation experiments, the roots were first exposed to 0.5 mM CaSO 4 for 1 h followed by exposure to the pretreatment solution (0.5mM CaSO4, 0.5 mM K N O 3 and either 0 or 1 m M NaC1) for 180 min. At 180 min the roots were transferred to treatment solutions, containing 0.5 m M CaSO4~ 0.5 m M KNO3, and variable amounts of NaC1. As in the exudation experiments, the treatment continued for 2 h.
J. Lynch and A. L/iuchli: K + transport in salt-stressed barley roots Experimental solutions were maintained at 22_+ 0.5 ~ C. The ratio of solution volume to root mass was approx. 800 ml/g, as in the exudation experiments: with this ratio the solutions were not detectably depleted during the course of the experiment. All solutions were gently aerated with filtered air by means of sintered-glass rods. Ion uptake was determined by transferring some roots from the pretreatment or treatment solutions for 10 rain to identical solutions containing trace amounts of either 86RbC1 or 22NaC1. Series of uptake measurements were taken over time, so that the midpoints of uptake periods coincided with the midpoints of xylem-exudate collection periods in the exudation experiments. On the basis of previous work in this laboratory (Welch and Epstein 1969) and Cram's work with barley roots (Cram 1969; Cram and Laties 1971), this 10-min uptake period was taken as an estimate of influx across the plasmalemma. Uptake was terminated by immersing the roots in 0.5 mM CaSO4 at 5~ followed by free-space desorption in aerated 0.5 m M CaSO~ at 5 ~ C for i0 min. The roots were then blotted dry, weighed, and dry-ashed in glass scintillation vials at 575 ~ C for 3 h. The ash was dissolved in 1 ml deionized H 2 0 and taken up to 10 ml with dioxane and Omnifluor prior to counting as described above.
297 20 - - influx
a 1 5 m M NaCI
16 _ - . . . . . 30rnM NaCI - - - - - - 45rnM NaCI 12 L
8
_
L.--.X.. n
--
[d- --~}~ .
Exudation data were analyzed for equality of treatment means with multivariate linear regression (Timm 1975). Uptake data from intact plant and excised root experiments were analyzed using repeated measures analysis of variance (Dixon 1981).
Results
To interpret the effects of applied solutes on K + appearance in the collection compartment of our transport chambers, we first had to determine the lag time between events occurring in the uptake compartment and consequent events detected in the collection compartment. This lag time was estimated by applying a pulse of 86Rb to the uptake compartment at the time of salinization (180 min) of standard roots (i.e. pretreated with 0.5 m M K N O 3 and 0.5 m M CaSO4). Rubidium-86 was detected in the collection compartment within 5 rain of the start of the pulse in both control (0.5mM CaSO 4 and 0.5raM K N O 3 ) and salinized (0.5 m M CaSO4, 0.5 m M KNO3, and 45 m M NaC1) treatments, indicating that the lag time between the uptake of K + and its subsequent appearance in the collection compartment was small relative to the sample collection intervals. The magnitude of K + exudation was ra:ther variable in our system, possibly because o f seed variability and size differences among root sys-. terns. Approximately 80% of the root systems were: composed of five individual roots, the remainder having either four or five individual roots. However, when the exudation data were normalized by converting fluxes (gmol g - ~ fresh weight h-1) to percentages of the flux preceding treatment appli-
.
.
.
~
/f/l- --
4
7 o E X / i,
+ (3
0 5
I --exudation
I -
I -
-
I
I b
-
4
~---~ . . . . . . . . ~ . . . . . . . 5
-1
F
I
-
-
2-I -o
Data analysis
/
/
150
180
I I I 210 240 270 TIME (minutesl
I 300
Fig. 3a, b. Influx of Na + (a) and xylem exudation (b) in excised barley roots following NaC1 application. NaC1 applied at 180 min. a Five replicates per determination; salt effect highly significant (P
