Planta (Berl.) 86, 1--9 (1969)
Phosphate Absorption by Air-Stressed Root Systems L ~ w I s D. D o w Department of Biology Tulane University, New Orleans, La. Received December 16, 1968
Summary. Root systems from plants grown in nutrient solution were exposed to air and either transferred to fresh nutrient solution containing 3sP-labeled phosphate or placed in a psyehrometer to determine their water potential. The amount of 32p absorbed by maize and soybean roots in the hour following their exposure to air was proportional to their water potential at the time they were transferred. Some cells, probably located in the stele, were more resistant to moisture stress than others. Absorption of 32p by all cells was severely inhibited by water potentials below -- 12 to -- 15 bars. Nearly normal amounts of the radioisotope and total phosphate were absorbed within 72 hr following root exposure of 4 of 5 species of detopped plants; some phosphorus was lost to the nutrient solution. Uptake of asp by passive processes was increased slightly by exposure of roots of intact maize plants to air, but the increase did not compensate for the substantial reduction in actively-absorbed asp.
Introduction R o o t surfaces s u d d e n l y e x p o s e d to air during t r a n s p l a n t i n g or t r a n s fer f r o m a solution culture are s u b j e c t e d to a v e r y steep g r a d i e n t in w a t e r p o t e n t i a l . E x t r e m e m o i s t u r e stress i r r e v o c a b l y d a m a g e s r o o t systems, b u t t h e effects of brief, s u b l e t h a l exposures to air h a v e n o t been i n v e s t i g a t e d in detail. This p a p e r e x a m i n e s t h e effects of a t m o s p h e r i c exposure on t h e active a b s o r p t i o n of ions b y roots. A b s o r p t i o n of ~2p. labeled p h o s p h a t e b y d e t o p p e d p l a n t s was used as a n i n d e x of d a m a g e t o anion a b s o r p t i o n processes. The m o i s t u r e stresses c r e a t e d b y t h e e x p o s u r e were m e a s u r e d on o t h e r sets of d e t o p p e d p l a n t s using a thermopile psyehrometer.
Materials and Methods a) Preparation o] Plant Material. Seeds of tomato (Lycopersicum esculentum M~L. ev. M-~ROLOBE),maize (Zea mays L., cv. DEKALB 441), barley (Hordeum vulgare L., cv. Co~PAI~.), sunflower (Helianthusannuus L., cv. Sum~isE), and soybean (Glycine max L., M~RR., cv. HAW~;E:CE)were soaked for 3--5 hr in aerated distilled water and planted in perlite moistened with distilled water. When the seedlings were 2---4 cm high, they were removed from the perlite, taking care to brush all particles from the root systems, and transferred to a continuously-aerated solution culture. The nutrient solution used to grow the plants had the following composition: l0 ~ME KH2P04, 500 ~M KNO3, 500 ~M C~(NO3)s, 200 ~zM MgSO4, l0 ~M FeC13, 1 Planta (Berl.),Bd. 86
2
L.D. Dew:
10 ~M ETDA, 1.20 t~M H3BOa, 0.20 ~M MuC12, 0.02 t~M ZnSO~, 0.007 EzM CuSO 4 and 0.0025 ~M Na2MoO4. The p H was adjusted to 6.0. The stems of 13 plants were supported by split plastic sponges while the roots extended into 6 L of nutrient solution through holes in the plastic top of a tray. The nutrient solution was renewed every 7 days. b) Procedure ]or Intact or Detopped Plants. 1--2 weeks after transplanting, 16 plants were selected for uniformity from 2 trays for an experiment. These plants were rinsed in fresh nutrient solution, and were either allowed to drain on a stainless-steel wire mesh, or were detepped and placed in a bottomless 50-ml centrifuge tube having a flange to support a retaining sponge which encircled the base of the cut stem. The centrifuge tube-plant unit was centrifuged for 2 rain at 50 • g to remove droplets of water and the root systems were immediately weighed. Plants which were exposed while intact were detopped and the root systems were weighed. Controls were set aside in an unlabeled nutrient solution. Other root systems were transferred to a closed glass chamber containing saturated CaC12 solution which maintained the atmosphere at 31% relative humidity at approximately 25 ~ The water potential of this atmosphere was approximately -- 1400 bars. Following 1 hr of exposure, the stressed root systems were reweighed and all root systems were either transferred to flasks containing 100 ml of nutrient solution labeled with 4 y.c.m1-1 of H~32p04, or placed in the chambers of a thermopfle psyehrometer for the measurement of water potential. This psychrometer uses a 9- junction thermopile, and is a modification of the thermoeouple psyehrometer of Ric~Am)s and OGATA (1958). 1-ml aliquots of the labeled nutrient solution were removed 30, 60, 90 minutes and 72 hr after the root transfer, or less often, depending upon the experiment. These aliqu0ts were added to 15 ml of scintillation fluid consisting of 60 g naphthalene, 4 g PFO (2,5-diphenyloxazole) and 100 ml of methanol dissolved in 1 L of dioxane. Samples were counted in a Beckman Liquid Scintillation System; quenching was negligible. The final p H of each nutrient solution was determined at the end of each experiment. Root systems were dried at i05 ~ and weighed. The fresh weight, dehydrated weight and dry weight were used to calculate the percent of the ~otal water content lost during exposure. Nutrient solutions used during the absorption period were evaporated to dryness and cleared of organic matter using a H202--H2SO ~ digestion when necessary. The residue was dissolved in distilled water and analyzed for phosphorus by a phosphomolybdate method. Total phosphorus uptake was calculated as the difference between the final and the original phosphorus content of the nutrient solution. c) Procedure ]or Root Segments. The primary and 3 adventitious roots were excised from 12 maize seedlings and placed in ridges in blotter paper moistened with nutrient solution. Holes, 1 or 2 em in diameter, exposed 1 or 2 cm portions of each root to air; the remainder of each root was in contact with the moist filter paper. The blotter paper and roots were covered with a plastic wrap except where holes exposed the roots to air. All blotters were hung in the CaC12 drying chamber for 24 hr. The exposed portions of the roots were excised, and 6 of these segments were transferred to each scintillation vial. Each vial contained 1 ml of 32P-labeled nutrient solution. 1 hr or 24 hr later the root segments were removed from the labeled solution, scintillation fluid was added to the vials and the latter were counted in the usual manner. The same portions of other root segments remained in contact with blotters in the drying chamber and served as controls. The percent water loss was calculated for each sample using mean fresh weights of the exposed segments, their weights after recovery, and their dry weights.
Phosphate Absorption by Air-Stressed Root Systems Results
Exposure of intact maize plants to air for 1 hr reduced their subsequent uptake of 32p; ~2p uptake by detopped plants was also reduced (Table 1). Passive transport of a2p, calculated as the difference between absorption by intact plants and absorption by detopped plants, was increased by the exposure. The net inhibition of 32p uptake, therefore, was due to reduced active uptake. The final p H of the nutrient solution in these and the following experiments ranged from 5,5 to 7.6 with no discernable trends. Table 1. Active and passive absorption o/32p by maize roots ]ollowing I hr exposure to air
Treatment
Stressed intact Stressed intact, detopped "Passive" transport under stress Control intact Control detopped "Passive" transport of controls
Hours after exposure 1
6
30
140 120 20 160 156 4
140 104 36 204 148 56
260 100 160 320 252 68
% Water lost 23 23
Uptake expressed in ~zc/gfresh wt. S.E. < 5 %, means of 4 plants each. Water loss calculated as % of total water capacity. Original solution contained 400 ~zc of 32p. 90% of the s2p label was absorbed by 3 g of detopped, unstressed maize plants within 30 rain (Fig. 1). There was negligible 32p uptake the following hour. Detopped and exposed plants absorbed 58 % of the 3~p in the first 0.5 hr and continued to absorb 32p the following hour, but at a reduced rate. Tomato root systems previously exposed to air absorbed 32p rapidly the first 0.5 hr but, unlike the controls, lost 32p to the nutrient solution the following hour (Fig. 2). 3 g of root systems were used in both of these experiments to assure substantial depletion of 32p; all other experiments used 1 g samples which usually depleted 50 % or less of the 1.0 ~ mole of total phosphate in each solution. Phosphate was lost by exposed tomato and soybean root systems in other experiments (Table 2). Smaller amounts were lost by other root systems, but the exact amount cannot be measured. Table 2 lists not only the ~ze of ,2p removed from solution by the root systems but also the ~zc of 3~p which should have been taken up on the basis of the phosphomolybdate phosphorus analysis and assuming no change in the original 32p/3~p ratio of the nutrient solution. Two factors operated to change the 32p/31p ratio, or specific activity of the nutrient solution: 1"
L. D. Dow:
4
Table 2. Changes in asp ancl total phosphate absorbed by detopped plants I h~ and 72 hr [ollowln9 1 hr o] exposure to air tze asp actually absorbed
~c asp theoretically absorbed
~tmoles total P actually absorbed
Maize unexposed controls 1 hr after exposure 72 hr after exposure
180 140 108
80 72 160
0.20 0.18 0.40
Barley unexposed controls 1 hr after exposure 72 hr after exposure
164 104 312
68 60 124
0.17 0.15 0.31
Sunflower unexposed controls 1 hr after exposure 72 hr after exposure
184 140 144
160 136 116
0.40 0.34 0.29
Tomato unexposed controls 1 hr after exposure
68 52
--36 --140
--0.09 --0.35
Soybean unexposed controls 1 hr after exposure 72 hr after exposure
346 88 376
140 --16 136
0.35 --0.04 0.34
Plant, treatment
Uptake expressed per gram fresh weight. S.E. < 5 %, means of 4 plants each. Negative values indicate loss of phosphate to the nutrient solution. Percent water loss calculated as percent of total water capacity. (1) phosphorus lost from " l e a k y " roots had a low specific activity and reduced the specific activity of the nutrient solution; (2) net uptake of a~P was greater than the net uptake of combined a~p and nip phosphate since an internal " p o o l " of phosphorus was labeled in all roots, including controls. Uptake of the radioisotope exceeded the uptake of 31p until an equilibrium was established between the specific activity of the internal " p o o l " and the external solution. Despite these disadvantages, a2p uptake remained a valuable parameter to assess relative degrees of damage to the anion absorption mechanism. I n most cases, total phosphate and a~P taken up by root systems in the 72 hr following exposure was almost as great or greater than uptake b y the controls. Soybean root systems lost water rapidly, and quickly developed low water potentials (Fig. 3). Uptake of 32p was inversely proportional to their water potentials at the end of the exposure period. A similar relationship existed between a~P uptake and the water potential of
Phosphate Absorption by Air-Stressed Root Systems maize
1o0
i
t-.
l
n
i
I I/ !.
50
I I
Ii
// I
~
!/ 0
30
60 min
90
Fig. 1. Absorption of asp by maize root systems following exposure to air. Exposure time: 1 hr. Each point mean of 4 plants. S.E. ~ 3%. Uptake expressed as % activity of soln./g fresh wt. of roots. Root systems 3 g each. o Exposed. 9 Unexposed
z
100 -
tomato
._~ /
s O'" .........
9 ...........
9
/ ! / /
a.
/ /
t
;/ ; .
/
9
~0
- ---~ .~.
-'-0
// // 7
..121
0
30
60 rain
90
Fig. 2. Absorption of a2p by tomato root systems following exposure to air. Exposure time: 1 hr. Each point mean of 4 plants. S.E. < 3 %. Uptake expressed as % activity of soln./g fresh wt. of roots. Root systems 3 g each. o Exposed. 9 Unexposed maize r o o t s y s t e m s (Fig. 4). I n b o t h eases t h e 82p/31p r a t i o p r o b a b l y c h a n g e d a t longer exposure times, due to leakage of sip from d a m a g e d roots. Thus, m o r e p h o s p h a t e was t a k e n u p a t higher stresses t h a n is i n d i c a t e d in Figs. 3 a n d 4. The error was g r e a t e r in t h e case of s o y b e a n t h a n maize. Since s o y b e a n was shown to be " l e a k i e r " t h a n maize in a n earlier e x p e r i m e n t (Table 2) ; t h e a b r u p t decline in u p t a k e in Fig. 3 was due in p a r t to this loss of p h o s p h a t e .
L. D. D o w : I
scybeon
-70
&
I
-6O
0.2
ID
~ -50
r~
.c
~q
.-_o -40 0_
I
o. -30
0.1 =t.
~ -20 /
-10
0
30 60 Minutes of exposure
90
Fig. 3. Changes in subsequent phosphate uptake and water potential developed during exposure of soybean root systems to air. S. E. ~ 5% for a2p uptake, S.E. 10% for water potential; points represent means of 4 plants each. 1)hosphate uptake calculated assuming no change in a2P/a11)ratio in nutrient solution; actual uptake greater than indicated for longer exposure times due to loss of a11)by roots
I
0.2
1
-70 -60
,,//
.m -5o 0
x~ -40
o.1
js
-30
_~ 13 O-
Q
~ -20
30 60 Minutes of exposure
90
Fig. 4. Changes in subsequent phosphate uptake and water potential developed during exposure of maize root systems to air. S.E. ~ 5 % for a21)uptake, S.E. ~ 10 % for water potential; points represent means of 4 plants each. 1)hosphate uptake calculated assuming no change in 321)/a11)ratio in nutrient solution; actual uptake greater than indicated for longer exposure times due to loss of 811)by roots
Phosphate Absorption by Air-Stressed Root Systems I n order to interpret the flat portions of the curves in Figs. 3 and 4, it was important to determine if changes in the phosphate absorption mechanism occnrred first at the root tips, or if the moisture stress was uniform along the root surfaces. Soybean roots showed a slight browning of the terminal and lateral root primordia after an hour of exposure to air. Maize roots, on the other hand, showed no browning and were efficient in translocating water to various 1- or 2-cm positions near or including the root apex. Damage to the phosphate absorption mechanism was slight; stressed portions absorbed as much as controls within 24 hr (Table 3). Isolated root apices did not recover completely within 24 hr since they were more dehydrated. Recovery was just as complete whether the terminal or the subterminal portions were exposed. Table 3. Changes in phosphate absorbed by portions o/maize root segments exposed to air ]or 24 hrs and their subsequent recovery Data expressed as 10-9 moles phosphate. Key: (t~-i-r)exposed . . . . removed, -in contact with blotter, ~ root apex. Water loss calculated as percent of total water capacity. Uptake calculated on basis of unchanged a2p/alp ratio in nutrient solution Exposure pattern
(..~
Exposed 24 hr
Water loss (%)
24 hr after exposure
Controls
3.5
48
5.4
5.1
70
2.5
4.1
::::::::::::~I~) 0.35
I,,,,,~ ~
4.2
20
6.0
5.8
!'"'~:::D
2.0
34
4.5
4.4
~,,,,,.,,,h ~
2.4
61
5.4
4.2
3.5
4.8
!lill|lllll~::::~ Discussion
Active uptake of asp by maize and soybean roots in the hour following exposure to air was proportional to the water potential of the roots at the time they were returned to a nutrient solution (Figs. 4 and 5). As they were exposed for longer periods of time, there were progressively fewer cells sufficiently recovered at the end of one hour to absorb phos. phate. The phosphate absorption mechanism of maize roots, however, continued to operate, probably at a slow rate (Fig. 2). Measurement of the remaining uptake was complicated by the loss of phosphate from the roots to the nutrient solution (Table 2). Appreciable phosphate uptake probably occurs only after cells regain turgor. FA.~X et al. (1955, 1956) found that 35S0~ uptake was
8
L.D. D o w :
inhibited at incipient plasmolysis; minimum uptake occurred when the concentration of mannitol surrounding the maize roots became slightly hypertonic. Uptake then became independent of the degree of plasmolysis. On the other hand, comparable studies of air-stressed roots have not been made, so these data using hypertonie solutions may not apply to air-stressed roots. Anions, including phosphate and sulfate, are absorbed by processes which depend upon the metabolism of ATP. The ATP-turnover rate of maize roots exposed to KC1 solutions, for example, is higher than the turnover rate of non-accumulating roots while the total high energy phosphate contents of these roots are the same (WEIGL, 1963). Any treatment which affects phosphate absorption by roots is likely to influence anion absorption generally, by controlling the amount of phosphate available for ATP synthesis. Root systems resisted further water loss more effectively within a range of water potentials from --12 to --15 bars for maize (Fig. 3) and -- 8 to -- 12 bars for soybean (Fig. 4). A group of cells which account for 50--75 % of the absorbed 32p must be unusually resistant to water loss. Rapid and efficient water transport took place in the maize root system. Roots which were partly in contact with nutrient solution translocated considerable water to exposed portions. These findings are similar to those of SLAVlKOVA(1967 a, b) who found that root systems of Fraxinus excelsior developed overall water potentials (suction forces) which were intermediate between high and low potentials of the soil to which they were exposed. These moisture-stress-resistant cells of maize roots are therefore well supplied with water as long as it is available anywhere in the root system. Their ready access to water and resistance to steep water potentials at the root surface indicate that they are located along the central axis of the root in the stele where gradients m water potential are least and there is ready access to water from the xylem. The final phase of the dehydration process is marked by sharp decreases in water potential and a2P absorption. These changes are accompanied by a wholesale destruction of cells and a loss of phosphate to the nutrient medium. The active absorption process was the predominant component of 3~p uptake by intact maize plants. Exposure of roots to air reduced total asp uptake by decreasing active absorption. Some increase in root permeability increased passive uptake slightly. A barrier exists in roots which retards water loss more effectively than the root surface. This barrier is believed to be the endodermis and associated cells of the stele. At high water potentials, atmospheric exposure exerts a reversible effect on the phosphate absorption mechanism; at low water potentials, there is massive cellular destruction.
Phosphate Absorption by Air-Stressed Root Systems
9
Future work will compare these results with those obtained by increasing the osmotic potential of the nutrient solution. The responses of roots to these two types of treatment should indicate t o what extent data obtained with hypertonic media can be used to explain effects on root systems due to direct water loss. The author wishes to thank Dr. STERLING B. HENDRICKS for making available the facilities of the Mineral Nutrition Laboratory, U.S.D.A., for one summer. He is also indebted to Dr. JAMES E. LEGGETT and the other members of the Laboratory for their valuable suggestions and criticism. This study was supported by a National Science Foundation grant (GB-6919).
References FALK,H., U. LUTTGE,U. J. WEIGL: Untersuchungen zur Physiologie plasmolysierter Zellen. I. Markierung des Plasmolysevorraumes mit Isotopen. Z. Pflanzenphysiol. 53, 19-31 (1965). - Untersuchungen zur Physiologie plasmolysierter Zellen. 11. Ionenaufnahme, 0,-Wechsel, Transport. Z. Pflanzenphysiol. 54, 4 4 6 4 6 2 (1966). RICHARDS, L. A., and G. OGATA:Thermocouple for vapor pressure measurements in biological and soil systems a t high humidity. Science 128, 1089 (1958). SLAVIKOYA, J.: Compensation of root suction force within a single root system. Biol. Plantarum (Kbh.) 9, 20-27 (1967). - Dependence of the root suction force on the soil water content. Biol. Plantarum (Kbh.) 9, 149-156 (1967). WEIGL,J.: Die Bedeutung der energiereichen Phosphate bei der Ionenaufnahme durch Wurzeln. Planta (Berl.) 60, 307-321 (1963).
LEWISD. DOVE Department of Biology Tulane University New Orleans, Louisiana 70118, USA
Phosphate Absorption by Air-Stressed Root Systems L ~ w I s D. D o w Department of Biology Tulane University, New Orleans, La. Received December 16, 1968
Summary. Root systems from plants grown in nutrient solution were exposed to air and either transferred to fresh nutrient solution containing 3sP-labeled phosphate or placed in a psyehrometer to determine their water potential. The amount of 32p absorbed by maize and soybean roots in the hour following their exposure to air was proportional to their water potential at the time they were transferred. Some cells, probably located in the stele, were more resistant to moisture stress than others. Absorption of 32p by all cells was severely inhibited by water potentials below -- 12 to -- 15 bars. Nearly normal amounts of the radioisotope and total phosphate were absorbed within 72 hr following root exposure of 4 of 5 species of detopped plants; some phosphorus was lost to the nutrient solution. Uptake of asp by passive processes was increased slightly by exposure of roots of intact maize plants to air, but the increase did not compensate for the substantial reduction in actively-absorbed asp.
Introduction R o o t surfaces s u d d e n l y e x p o s e d to air during t r a n s p l a n t i n g or t r a n s fer f r o m a solution culture are s u b j e c t e d to a v e r y steep g r a d i e n t in w a t e r p o t e n t i a l . E x t r e m e m o i s t u r e stress i r r e v o c a b l y d a m a g e s r o o t systems, b u t t h e effects of brief, s u b l e t h a l exposures to air h a v e n o t been i n v e s t i g a t e d in detail. This p a p e r e x a m i n e s t h e effects of a t m o s p h e r i c exposure on t h e active a b s o r p t i o n of ions b y roots. A b s o r p t i o n of ~2p. labeled p h o s p h a t e b y d e t o p p e d p l a n t s was used as a n i n d e x of d a m a g e t o anion a b s o r p t i o n processes. The m o i s t u r e stresses c r e a t e d b y t h e e x p o s u r e were m e a s u r e d on o t h e r sets of d e t o p p e d p l a n t s using a thermopile psyehrometer.
Materials and Methods a) Preparation o] Plant Material. Seeds of tomato (Lycopersicum esculentum M~L. ev. M-~ROLOBE),maize (Zea mays L., cv. DEKALB 441), barley (Hordeum vulgare L., cv. Co~PAI~.), sunflower (Helianthusannuus L., cv. Sum~isE), and soybean (Glycine max L., M~RR., cv. HAW~;E:CE)were soaked for 3--5 hr in aerated distilled water and planted in perlite moistened with distilled water. When the seedlings were 2---4 cm high, they were removed from the perlite, taking care to brush all particles from the root systems, and transferred to a continuously-aerated solution culture. The nutrient solution used to grow the plants had the following composition: l0 ~ME KH2P04, 500 ~M KNO3, 500 ~M C~(NO3)s, 200 ~zM MgSO4, l0 ~M FeC13, 1 Planta (Berl.),Bd. 86
2
L.D. Dew:
10 ~M ETDA, 1.20 t~M H3BOa, 0.20 ~M MuC12, 0.02 t~M ZnSO~, 0.007 EzM CuSO 4 and 0.0025 ~M Na2MoO4. The p H was adjusted to 6.0. The stems of 13 plants were supported by split plastic sponges while the roots extended into 6 L of nutrient solution through holes in the plastic top of a tray. The nutrient solution was renewed every 7 days. b) Procedure ]or Intact or Detopped Plants. 1--2 weeks after transplanting, 16 plants were selected for uniformity from 2 trays for an experiment. These plants were rinsed in fresh nutrient solution, and were either allowed to drain on a stainless-steel wire mesh, or were detepped and placed in a bottomless 50-ml centrifuge tube having a flange to support a retaining sponge which encircled the base of the cut stem. The centrifuge tube-plant unit was centrifuged for 2 rain at 50 • g to remove droplets of water and the root systems were immediately weighed. Plants which were exposed while intact were detopped and the root systems were weighed. Controls were set aside in an unlabeled nutrient solution. Other root systems were transferred to a closed glass chamber containing saturated CaC12 solution which maintained the atmosphere at 31% relative humidity at approximately 25 ~ The water potential of this atmosphere was approximately -- 1400 bars. Following 1 hr of exposure, the stressed root systems were reweighed and all root systems were either transferred to flasks containing 100 ml of nutrient solution labeled with 4 y.c.m1-1 of H~32p04, or placed in the chambers of a thermopfle psyehrometer for the measurement of water potential. This psychrometer uses a 9- junction thermopile, and is a modification of the thermoeouple psyehrometer of Ric~Am)s and OGATA (1958). 1-ml aliquots of the labeled nutrient solution were removed 30, 60, 90 minutes and 72 hr after the root transfer, or less often, depending upon the experiment. These aliqu0ts were added to 15 ml of scintillation fluid consisting of 60 g naphthalene, 4 g PFO (2,5-diphenyloxazole) and 100 ml of methanol dissolved in 1 L of dioxane. Samples were counted in a Beckman Liquid Scintillation System; quenching was negligible. The final p H of each nutrient solution was determined at the end of each experiment. Root systems were dried at i05 ~ and weighed. The fresh weight, dehydrated weight and dry weight were used to calculate the percent of the ~otal water content lost during exposure. Nutrient solutions used during the absorption period were evaporated to dryness and cleared of organic matter using a H202--H2SO ~ digestion when necessary. The residue was dissolved in distilled water and analyzed for phosphorus by a phosphomolybdate method. Total phosphorus uptake was calculated as the difference between the final and the original phosphorus content of the nutrient solution. c) Procedure ]or Root Segments. The primary and 3 adventitious roots were excised from 12 maize seedlings and placed in ridges in blotter paper moistened with nutrient solution. Holes, 1 or 2 em in diameter, exposed 1 or 2 cm portions of each root to air; the remainder of each root was in contact with the moist filter paper. The blotter paper and roots were covered with a plastic wrap except where holes exposed the roots to air. All blotters were hung in the CaC12 drying chamber for 24 hr. The exposed portions of the roots were excised, and 6 of these segments were transferred to each scintillation vial. Each vial contained 1 ml of 32P-labeled nutrient solution. 1 hr or 24 hr later the root segments were removed from the labeled solution, scintillation fluid was added to the vials and the latter were counted in the usual manner. The same portions of other root segments remained in contact with blotters in the drying chamber and served as controls. The percent water loss was calculated for each sample using mean fresh weights of the exposed segments, their weights after recovery, and their dry weights.
Phosphate Absorption by Air-Stressed Root Systems Results
Exposure of intact maize plants to air for 1 hr reduced their subsequent uptake of 32p; ~2p uptake by detopped plants was also reduced (Table 1). Passive transport of a2p, calculated as the difference between absorption by intact plants and absorption by detopped plants, was increased by the exposure. The net inhibition of 32p uptake, therefore, was due to reduced active uptake. The final p H of the nutrient solution in these and the following experiments ranged from 5,5 to 7.6 with no discernable trends. Table 1. Active and passive absorption o/32p by maize roots ]ollowing I hr exposure to air
Treatment
Stressed intact Stressed intact, detopped "Passive" transport under stress Control intact Control detopped "Passive" transport of controls
Hours after exposure 1
6
30
140 120 20 160 156 4
140 104 36 204 148 56
260 100 160 320 252 68
% Water lost 23 23
Uptake expressed in ~zc/gfresh wt. S.E. < 5 %, means of 4 plants each. Water loss calculated as % of total water capacity. Original solution contained 400 ~zc of 32p. 90% of the s2p label was absorbed by 3 g of detopped, unstressed maize plants within 30 rain (Fig. 1). There was negligible 32p uptake the following hour. Detopped and exposed plants absorbed 58 % of the 3~p in the first 0.5 hr and continued to absorb 32p the following hour, but at a reduced rate. Tomato root systems previously exposed to air absorbed 32p rapidly the first 0.5 hr but, unlike the controls, lost 32p to the nutrient solution the following hour (Fig. 2). 3 g of root systems were used in both of these experiments to assure substantial depletion of 32p; all other experiments used 1 g samples which usually depleted 50 % or less of the 1.0 ~ mole of total phosphate in each solution. Phosphate was lost by exposed tomato and soybean root systems in other experiments (Table 2). Smaller amounts were lost by other root systems, but the exact amount cannot be measured. Table 2 lists not only the ~ze of ,2p removed from solution by the root systems but also the ~zc of 3~p which should have been taken up on the basis of the phosphomolybdate phosphorus analysis and assuming no change in the original 32p/3~p ratio of the nutrient solution. Two factors operated to change the 32p/31p ratio, or specific activity of the nutrient solution: 1"
L. D. Dow:
4
Table 2. Changes in asp ancl total phosphate absorbed by detopped plants I h~ and 72 hr [ollowln9 1 hr o] exposure to air tze asp actually absorbed
~c asp theoretically absorbed
~tmoles total P actually absorbed
Maize unexposed controls 1 hr after exposure 72 hr after exposure
180 140 108
80 72 160
0.20 0.18 0.40
Barley unexposed controls 1 hr after exposure 72 hr after exposure
164 104 312
68 60 124
0.17 0.15 0.31
Sunflower unexposed controls 1 hr after exposure 72 hr after exposure
184 140 144
160 136 116
0.40 0.34 0.29
Tomato unexposed controls 1 hr after exposure
68 52
--36 --140
--0.09 --0.35
Soybean unexposed controls 1 hr after exposure 72 hr after exposure
346 88 376
140 --16 136
0.35 --0.04 0.34
Plant, treatment
Uptake expressed per gram fresh weight. S.E. < 5 %, means of 4 plants each. Negative values indicate loss of phosphate to the nutrient solution. Percent water loss calculated as percent of total water capacity. (1) phosphorus lost from " l e a k y " roots had a low specific activity and reduced the specific activity of the nutrient solution; (2) net uptake of a~P was greater than the net uptake of combined a~p and nip phosphate since an internal " p o o l " of phosphorus was labeled in all roots, including controls. Uptake of the radioisotope exceeded the uptake of 31p until an equilibrium was established between the specific activity of the internal " p o o l " and the external solution. Despite these disadvantages, a2p uptake remained a valuable parameter to assess relative degrees of damage to the anion absorption mechanism. I n most cases, total phosphate and a~P taken up by root systems in the 72 hr following exposure was almost as great or greater than uptake b y the controls. Soybean root systems lost water rapidly, and quickly developed low water potentials (Fig. 3). Uptake of 32p was inversely proportional to their water potentials at the end of the exposure period. A similar relationship existed between a~P uptake and the water potential of
Phosphate Absorption by Air-Stressed Root Systems maize
1o0
i
t-.
l
n
i
I I/ !.
50
I I
Ii
// I
~
!/ 0
30
60 min
90
Fig. 1. Absorption of asp by maize root systems following exposure to air. Exposure time: 1 hr. Each point mean of 4 plants. S.E. ~ 3%. Uptake expressed as % activity of soln./g fresh wt. of roots. Root systems 3 g each. o Exposed. 9 Unexposed
z
100 -
tomato
._~ /
s O'" .........
9 ...........
9
/ ! / /
a.
/ /
t
;/ ; .
/
9
~0
- ---~ .~.
-'-0
// // 7
..121
0
30
60 rain
90
Fig. 2. Absorption of a2p by tomato root systems following exposure to air. Exposure time: 1 hr. Each point mean of 4 plants. S.E. < 3 %. Uptake expressed as % activity of soln./g fresh wt. of roots. Root systems 3 g each. o Exposed. 9 Unexposed maize r o o t s y s t e m s (Fig. 4). I n b o t h eases t h e 82p/31p r a t i o p r o b a b l y c h a n g e d a t longer exposure times, due to leakage of sip from d a m a g e d roots. Thus, m o r e p h o s p h a t e was t a k e n u p a t higher stresses t h a n is i n d i c a t e d in Figs. 3 a n d 4. The error was g r e a t e r in t h e case of s o y b e a n t h a n maize. Since s o y b e a n was shown to be " l e a k i e r " t h a n maize in a n earlier e x p e r i m e n t (Table 2) ; t h e a b r u p t decline in u p t a k e in Fig. 3 was due in p a r t to this loss of p h o s p h a t e .
L. D. D o w : I
scybeon
-70
&
I
-6O
0.2
ID
~ -50
r~
.c
~q
.-_o -40 0_
I
o. -30
0.1 =t.
~ -20 /
-10
0
30 60 Minutes of exposure
90
Fig. 3. Changes in subsequent phosphate uptake and water potential developed during exposure of soybean root systems to air. S. E. ~ 5% for a2p uptake, S.E. 10% for water potential; points represent means of 4 plants each. 1)hosphate uptake calculated assuming no change in a2P/a11)ratio in nutrient solution; actual uptake greater than indicated for longer exposure times due to loss of a11)by roots
I
0.2
1
-70 -60
,,//
.m -5o 0
x~ -40
o.1
js
-30
_~ 13 O-
Q
~ -20
30 60 Minutes of exposure
90
Fig. 4. Changes in subsequent phosphate uptake and water potential developed during exposure of maize root systems to air. S.E. ~ 5 % for a21)uptake, S.E. ~ 10 % for water potential; points represent means of 4 plants each. 1)hosphate uptake calculated assuming no change in 321)/a11)ratio in nutrient solution; actual uptake greater than indicated for longer exposure times due to loss of 811)by roots
Phosphate Absorption by Air-Stressed Root Systems I n order to interpret the flat portions of the curves in Figs. 3 and 4, it was important to determine if changes in the phosphate absorption mechanism occnrred first at the root tips, or if the moisture stress was uniform along the root surfaces. Soybean roots showed a slight browning of the terminal and lateral root primordia after an hour of exposure to air. Maize roots, on the other hand, showed no browning and were efficient in translocating water to various 1- or 2-cm positions near or including the root apex. Damage to the phosphate absorption mechanism was slight; stressed portions absorbed as much as controls within 24 hr (Table 3). Isolated root apices did not recover completely within 24 hr since they were more dehydrated. Recovery was just as complete whether the terminal or the subterminal portions were exposed. Table 3. Changes in phosphate absorbed by portions o/maize root segments exposed to air ]or 24 hrs and their subsequent recovery Data expressed as 10-9 moles phosphate. Key: (t~-i-r)exposed . . . . removed, -in contact with blotter, ~ root apex. Water loss calculated as percent of total water capacity. Uptake calculated on basis of unchanged a2p/alp ratio in nutrient solution Exposure pattern
(..~
Exposed 24 hr
Water loss (%)
24 hr after exposure
Controls
3.5
48
5.4
5.1
70
2.5
4.1
::::::::::::~I~) 0.35
I,,,,,~ ~
4.2
20
6.0
5.8
!'"'~:::D
2.0
34
4.5
4.4
~,,,,,.,,,h ~
2.4
61
5.4
4.2
3.5
4.8
!lill|lllll~::::~ Discussion
Active uptake of asp by maize and soybean roots in the hour following exposure to air was proportional to the water potential of the roots at the time they were returned to a nutrient solution (Figs. 4 and 5). As they were exposed for longer periods of time, there were progressively fewer cells sufficiently recovered at the end of one hour to absorb phos. phate. The phosphate absorption mechanism of maize roots, however, continued to operate, probably at a slow rate (Fig. 2). Measurement of the remaining uptake was complicated by the loss of phosphate from the roots to the nutrient solution (Table 2). Appreciable phosphate uptake probably occurs only after cells regain turgor. FA.~X et al. (1955, 1956) found that 35S0~ uptake was
8
L.D. D o w :
inhibited at incipient plasmolysis; minimum uptake occurred when the concentration of mannitol surrounding the maize roots became slightly hypertonic. Uptake then became independent of the degree of plasmolysis. On the other hand, comparable studies of air-stressed roots have not been made, so these data using hypertonie solutions may not apply to air-stressed roots. Anions, including phosphate and sulfate, are absorbed by processes which depend upon the metabolism of ATP. The ATP-turnover rate of maize roots exposed to KC1 solutions, for example, is higher than the turnover rate of non-accumulating roots while the total high energy phosphate contents of these roots are the same (WEIGL, 1963). Any treatment which affects phosphate absorption by roots is likely to influence anion absorption generally, by controlling the amount of phosphate available for ATP synthesis. Root systems resisted further water loss more effectively within a range of water potentials from --12 to --15 bars for maize (Fig. 3) and -- 8 to -- 12 bars for soybean (Fig. 4). A group of cells which account for 50--75 % of the absorbed 32p must be unusually resistant to water loss. Rapid and efficient water transport took place in the maize root system. Roots which were partly in contact with nutrient solution translocated considerable water to exposed portions. These findings are similar to those of SLAVlKOVA(1967 a, b) who found that root systems of Fraxinus excelsior developed overall water potentials (suction forces) which were intermediate between high and low potentials of the soil to which they were exposed. These moisture-stress-resistant cells of maize roots are therefore well supplied with water as long as it is available anywhere in the root system. Their ready access to water and resistance to steep water potentials at the root surface indicate that they are located along the central axis of the root in the stele where gradients m water potential are least and there is ready access to water from the xylem. The final phase of the dehydration process is marked by sharp decreases in water potential and a2P absorption. These changes are accompanied by a wholesale destruction of cells and a loss of phosphate to the nutrient medium. The active absorption process was the predominant component of 3~p uptake by intact maize plants. Exposure of roots to air reduced total asp uptake by decreasing active absorption. Some increase in root permeability increased passive uptake slightly. A barrier exists in roots which retards water loss more effectively than the root surface. This barrier is believed to be the endodermis and associated cells of the stele. At high water potentials, atmospheric exposure exerts a reversible effect on the phosphate absorption mechanism; at low water potentials, there is massive cellular destruction.
Phosphate Absorption by Air-Stressed Root Systems
9
Future work will compare these results with those obtained by increasing the osmotic potential of the nutrient solution. The responses of roots to these two types of treatment should indicate t o what extent data obtained with hypertonic media can be used to explain effects on root systems due to direct water loss. The author wishes to thank Dr. STERLING B. HENDRICKS for making available the facilities of the Mineral Nutrition Laboratory, U.S.D.A., for one summer. He is also indebted to Dr. JAMES E. LEGGETT and the other members of the Laboratory for their valuable suggestions and criticism. This study was supported by a National Science Foundation grant (GB-6919).
References FALK,H., U. LUTTGE,U. J. WEIGL: Untersuchungen zur Physiologie plasmolysierter Zellen. I. Markierung des Plasmolysevorraumes mit Isotopen. Z. Pflanzenphysiol. 53, 19-31 (1965). - Untersuchungen zur Physiologie plasmolysierter Zellen. 11. Ionenaufnahme, 0,-Wechsel, Transport. Z. Pflanzenphysiol. 54, 4 4 6 4 6 2 (1966). RICHARDS, L. A., and G. OGATA:Thermocouple for vapor pressure measurements in biological and soil systems a t high humidity. Science 128, 1089 (1958). SLAVIKOYA, J.: Compensation of root suction force within a single root system. Biol. Plantarum (Kbh.) 9, 20-27 (1967). - Dependence of the root suction force on the soil water content. Biol. Plantarum (Kbh.) 9, 149-156 (1967). WEIGL,J.: Die Bedeutung der energiereichen Phosphate bei der Ionenaufnahme durch Wurzeln. Planta (Berl.) 60, 307-321 (1963).
LEWISD. DOVE Department of Biology Tulane University New Orleans, Louisiana 70118, USA
