Planta (Berl.) 124, 99--103 (1975) 9 by Springer-Verlag 1975
Short Communication Effects of Magnesium, Calcium and Lanthanum Ions on Stomatal Oscillations in Avena sativa L. T. B r o g s
a n d A. J o h n s s o n
Department of Electrical Measurements, Lund Institute of Technology, P.O. Box 725, S-220 07 Lund, Sweden Received 27 November 1974; accepted 4 February 1975
Summary. Mg2+, Can+ and La 8+ caused an increased period time of the transpiratory oscillations when given to excised oscillating Arena plants (plants without root system). The effect was reversible, i.e. after withdrawal of the ions the period time returned to its original value. In order to achieve the same period lengthening as with 2.5 mM La a+, 20 mM Ca2+ and 40 mM Mg2+ was needed. The effects are discussed along two lines: (a) the ions interfere with ionic processes, central for the stomatal regulation, and (b) the ions decrease the water permeability of the guard cells. Simulations on a model, previously published in the literature, showed that an increased resistance against the water flow into the guard cells could explain the findings. The ions had no effect on the period length of the oscillations in intact plants, i.e. with root system. U n d e r certain e x p e r i m e n t a l conditions self-sustained oscillations c a n be f o u n d in t h e s t o m a t a l w a t e r r e g u l a t i o n of p l a n t s [1]. T h e oscillations, easily d e t e c t a b l e as r h y t h m i c t r a n s p i r a t i o n , p r o v i d e i m p o r t a n t clues to t h e d y n a m i c a l b e h a v i o u r of t h e s t o m a t a of i n t a c t p l a n t s a n d give v a l u a b l e i n f o r m a t i o n for t h e modelling of t h e s t o m a t a l w a t e r r e g u l a t i o n [7-10, 12]. T h e oscillations can arise in excised plants, i.e. p l a n t s w i t h o u t r o o t s y s t e m , n o r a bene if a sufficient resistance a g a i n s t w a t e r flow to t h e s t o m a t a l regions is present. A m e t h o d to increase t h e water-flow resistance b y p h y s i c a l compression of t h e leaf base has been used t o achieve oscillations of excised o a t p l a n t s [3, 5]. This m e t h o d allows ions to e n t e r t h e t r a n s p i r a t i o n s t r e a m a n d r e a c h t h e oscillating s t o m a t a w i t h o u t passing t h e n o r m a l ion r e g u l a t i o n of t h e r o o t s y s t e m . T h e a c t i o n of ions on s t o m a t a l oscillations, a n d hence on s t o m a t a l d y n a m i c s , can t h e r e f o r e be s t u d i e d in a convenient way. T h e a c t i o n of Li+ on t h e oscillatory t r a n s p i r a t i o n of o a t p l a n t s has been i n v e s t i g a t e d b y m e a n s of t h e m e t h o d m e n t i o n e d [5]. Some similarities b e t w e e n Li + a n d elements of group I I of t h e periodic s y s t e m [e.g. t h e r e l a t i v e l y large ionizing p o t e n t i a l ] p r o m p t e d t h e p r e s e n t s t u d y of t h e a c t i o n of Mg 2+ a n d Ca 2+. The seedlings used in the experiments (Avena sativa L., ev, Blenda; Sval6f, Sweden) were cultivated on tap water and under 12 h dark and 12 h of light daily [3]. In the experiments the first leaf of each plant (6 days old) was kept in a cuvette and stabilized carrier gas (air; 1.5 cm s-1, 20% relative humidity, 26~ passed the leaf. The relative humidity of the carrier gas leaving the cuvette was measured and could be calibrated to the transpiration rate of the plant (rama water h-l). The recordings were made in constant light irradiance (white light; Luma 250 W P8; 1.4 mW/cm 2) and temperature (26~ and the period length of the oscillations was then about 25 min. The number of experiments in each category to be discussed was about 10. 7*
100
T. Brog~rdh and A. JoMsson
Sustained transpiratory oscillations were obtained in excised plants by means of compression at the leaf base (Fig. 1 A). Distilled water then surrounded the stem at the site of the excision. When the distilled water was replaced by a 20 mM CaC4 solution, calcium ions entered the xylem. Fig. 1 B shows how 20 m ~ CaC12 solution increased the period length and the amplitude of the transpiratory oscillations. The effects were dependent on the concentration and in 40 m ~ CaC12 the oscillations were completely abolished. Treatments with MgC12 had the same general effects on the oscillations, but Fig. 1 C shows t h a t 40 mM MgC12 was required in order to obtain the same period lengthening as with 20 mM CaC12. The period lengthening caused by the divalent ions was mostly due to the longer time spent b y the stomata in the closed state (el. shape of curves in Fig. 1 A and 1 B). The ionic effects can be explained by changes of the stomatal parameters, but in principle they could be attributed to changes of the xylem resistance to water flow induced b y the ions at the site of the deformation. To study this possibility Ca 2+ was given to excised, non-compressed plants (Fig.1 D). The light was switched on, the transpiration rate increased and Ca 2+ was transported up into the leaf. After 15 rain the CaC12 solution around the plant stem was exchanged to distilled water, flushing the xylem some time before the xylem compression was applied. The oscillations now showed a typical Ca 2+ lengthening of the period which, therefore, was attributed to reactions in the stomatal regions. The Ca 2+ as well as the Mg 2+ effects were reversible as exemphfied in Fig. 1 E : treatment with 40 mM Mg C4 for 1 h caused a period lengthening and an amplitude increase, which disappeared one cycle after the treatment. These reversible effects point to the possibihty t h a t divalent ions can be continuously withdrawn from the sites, where they affect the stomatal dynamics, and transformed into an inactive form [el. 2]. The effects of lanthanum ions were more pronounced than those caused by the divalent ions: 2.5 mM LaC1a gave roughly the same period lenghtening as 20 mM CaC12 (Fig. 1 B). I t has previously been reported t h a t Li+ ions increase the period length of the transpiratory oscillations and t h a t K+ and Na + ions do not cause any period changes [5]. 80 mM LiC1 increased the period length by a factor of 1.6. The con-
Fig. 1A E. Sustained transpiratory oscillations in excised oat plants. (A) Transpiration rate of the primary leaf of one plant, kept in a euvette under constant environmental conditions. (B) Increased amplitude and period length of the oscillations under the influence of 20 mM CaCI~solution. (C) MgCI~solution, 40 raM, caused the same increase in amplitude and period length of the oscillations as 20 mM CaC12 solution; el. B. (D) A plant, treated with 20 m ~ CaC12prior to the compression of the leaf base (xylem deformation), showed an initial prolonged period length of the transpiratory oscillations. (E) Oscillations with normal amplitude and period length were found about one hour after a treatment with 40 mM MgClz. (F) Simulations were made on a model for plant water regulation in order to find a possible site of action of Ca~+ and )/Ig~+. The curve represents the leaf conductance per unit leaf area which in the simulation is directly proportional to the transpiration stream. An increased amplitude and period length was obtained when the resistance (R~) against water flow into the guard cells was increased by 10 percent from its original value Rg~ ~ should be compared with the results in B and C
| = % E
(~
9
1 hour
I"
20 mM CoCi 2
[
1 hour
J
I
~=:=I"(---'- 20 mM Ca C[2
~. 20
xyie m
on
0
~E 0
"
!
1
t
1 hour
J
1 hour
]
10 seconds Fig. 1
7a Planta (Berl.), Vol. 124
]
102
T. Brogtrdh and A. Johnsson
centrations of the investigated ions, necessary to cause a lengthening of this order, would indicate t h a t the ions are effective in the following order: L a a + > Ca 2+ > Mg2+> L i + > ( K + Na+, which cause no changes). The period lengthening could be explained along at least two different lines. (a) The ions could interfere with ionic transport processes of the stomata, e.g. the shuttling of K+ into and out from the guard cells, thus changing water transport processes across the g u a r d cell membranes. Changes in ion permeabilities caused b y divalent ions are c o m m o n l y k n o w n in biological systems [see e.g. 6] and b o t h La a+ and Ca 2+ are reported to inhibit K + absorption in corn roots [13]. (b) The ions could act on the water permeability of the guard-cell membranes. Ca 2+ has n a m e l y been found to decrease the water permeabilities of membranes [11, 14] and so the main action of the calcium ions could be on the guard-cell membranes, decreasing their water permeability and thus increasing the time constant involved in the oscillatory g u a r d cell movements. Simulations along this line were made b y means of a previously published model for oscillatory stomatal regulation [8]. Results from simulations with suddenly increased resistance towards water flow into the guard cells were in agreement with the experiments, see :Fig. 1 F. The increased time constant of the guard cells caused larger oscillations in the model. Because of non-linearities in the model, the time intervals between the transpiratory p e a k s - and consequently the period length of the oscillations then increased [cf. 4]. The period length of the oscillations in intact plants could not be changed b y a n y of the ions. This means t h a t the ions cannot penetrate the root tissues and reach the stomatal regions. P r o b a l y the Casparian strips form a barrier against the ion movements into the xylem vessels [el. 13]. We thank members of the staff of the Institute of Plant Physiology, Lurid University, for helpful comments and discussions. The work was financially supported by the Swedish Natural Science Research Council.
References 1. Barrs, H. D. : Cyclic variations in stomafal aperture, transpiration, and leaf water potential under constant environmental conditions. Ann. Rev. Plant Physiol. 22, 223-236 (1971) 2. Biddulph, 0., Nakayama, ~. S., Cory, R. : Transpiration stream and ascension of calcium. Plant Physiol. 36, 429-436 (1961) 3. Brogs T., Johnsson, A. : Oscillatory transpiration and water uptake of Arena plants. II. Effects of deformation of xylem vessels. Physiol. Plantarum (Cph.) 28, 341-345 (1973) 4. Brogs T., Johnsson, A. : Oscillatory transpiration and water uptake of Arena plants. III. Action of heavy water on the oscillation. Physiol. Plantarum (Cph.) 31, 112-118 (1974a) 5. Brog~rdh, T., Johnsson, A,: Effects of lithium on stomatal regulation. Z. Natufforsch. 29c, 298-300 (1974b) 6. BurstrSm, H. : Calcium and plant growth. Biol. Rev. 43, 287-316 (1968) 7. Claus, St. : Ein Modell ffir das Verhalten der SpaltSffnungen im Porometer. Studia biophys. 11, 110-117 (1968) 8. Cowan, I. R.: Oscillations in stomatal conductance and plant functioning associated with stomatal conductance: observations and a model. Planta (Berl.) 106, 185-219 (1972) 9. Hopmans, P. A. M. : Rhythms in stomatal opening of bean leaves. Meded. Landbouwhogeschool Wageningen 71, No. 3 (1971) 10. Karmanov, V. G., Meleschenko, S.N., Savin, V. N. : Study of the dynamics of the water metabolism of the plant and construction of an electrical analogue of the system of water exchange. Biofizika (Engl. Transl.) 11, 147-155 (1966)
Magnesium, Calcium and Lanthanum Ions in Avena sativa L.
103
11. Kuiper, P. J. C.: Water transport across membranes. Ann. Rev. Plant Physiol. 23, 157-172 (1972) 12. Lang, A. R. G., Klepper, B., Cumming, M. J. : Leaf water balance during oscillation of stomatal aperture. Plant Physiol. 44, 826-830 (1969) 13. Nagahashi, G., Thomson, W. W., Leonard, I~. T. : The Casparian strip as a barrier to the movement of lanthanum in corn roots. Science 188, 670-671 (1974) 14. Whittembury, G., Sugino, N., Solomon, A. K. : Effect of antidiuretic hormone and calcium on the equivalent pore radius of kidney slices from Necturus. Nature (Lond.) 187, 699-701 (1960)
Short Communication Effects of Magnesium, Calcium and Lanthanum Ions on Stomatal Oscillations in Avena sativa L. T. B r o g s
a n d A. J o h n s s o n
Department of Electrical Measurements, Lund Institute of Technology, P.O. Box 725, S-220 07 Lund, Sweden Received 27 November 1974; accepted 4 February 1975
Summary. Mg2+, Can+ and La 8+ caused an increased period time of the transpiratory oscillations when given to excised oscillating Arena plants (plants without root system). The effect was reversible, i.e. after withdrawal of the ions the period time returned to its original value. In order to achieve the same period lengthening as with 2.5 mM La a+, 20 mM Ca2+ and 40 mM Mg2+ was needed. The effects are discussed along two lines: (a) the ions interfere with ionic processes, central for the stomatal regulation, and (b) the ions decrease the water permeability of the guard cells. Simulations on a model, previously published in the literature, showed that an increased resistance against the water flow into the guard cells could explain the findings. The ions had no effect on the period length of the oscillations in intact plants, i.e. with root system. U n d e r certain e x p e r i m e n t a l conditions self-sustained oscillations c a n be f o u n d in t h e s t o m a t a l w a t e r r e g u l a t i o n of p l a n t s [1]. T h e oscillations, easily d e t e c t a b l e as r h y t h m i c t r a n s p i r a t i o n , p r o v i d e i m p o r t a n t clues to t h e d y n a m i c a l b e h a v i o u r of t h e s t o m a t a of i n t a c t p l a n t s a n d give v a l u a b l e i n f o r m a t i o n for t h e modelling of t h e s t o m a t a l w a t e r r e g u l a t i o n [7-10, 12]. T h e oscillations can arise in excised plants, i.e. p l a n t s w i t h o u t r o o t s y s t e m , n o r a bene if a sufficient resistance a g a i n s t w a t e r flow to t h e s t o m a t a l regions is present. A m e t h o d to increase t h e water-flow resistance b y p h y s i c a l compression of t h e leaf base has been used t o achieve oscillations of excised o a t p l a n t s [3, 5]. This m e t h o d allows ions to e n t e r t h e t r a n s p i r a t i o n s t r e a m a n d r e a c h t h e oscillating s t o m a t a w i t h o u t passing t h e n o r m a l ion r e g u l a t i o n of t h e r o o t s y s t e m . T h e a c t i o n of ions on s t o m a t a l oscillations, a n d hence on s t o m a t a l d y n a m i c s , can t h e r e f o r e be s t u d i e d in a convenient way. T h e a c t i o n of Li+ on t h e oscillatory t r a n s p i r a t i o n of o a t p l a n t s has been i n v e s t i g a t e d b y m e a n s of t h e m e t h o d m e n t i o n e d [5]. Some similarities b e t w e e n Li + a n d elements of group I I of t h e periodic s y s t e m [e.g. t h e r e l a t i v e l y large ionizing p o t e n t i a l ] p r o m p t e d t h e p r e s e n t s t u d y of t h e a c t i o n of Mg 2+ a n d Ca 2+. The seedlings used in the experiments (Avena sativa L., ev, Blenda; Sval6f, Sweden) were cultivated on tap water and under 12 h dark and 12 h of light daily [3]. In the experiments the first leaf of each plant (6 days old) was kept in a cuvette and stabilized carrier gas (air; 1.5 cm s-1, 20% relative humidity, 26~ passed the leaf. The relative humidity of the carrier gas leaving the cuvette was measured and could be calibrated to the transpiration rate of the plant (rama water h-l). The recordings were made in constant light irradiance (white light; Luma 250 W P8; 1.4 mW/cm 2) and temperature (26~ and the period length of the oscillations was then about 25 min. The number of experiments in each category to be discussed was about 10. 7*
100
T. Brog~rdh and A. JoMsson
Sustained transpiratory oscillations were obtained in excised plants by means of compression at the leaf base (Fig. 1 A). Distilled water then surrounded the stem at the site of the excision. When the distilled water was replaced by a 20 mM CaC4 solution, calcium ions entered the xylem. Fig. 1 B shows how 20 m ~ CaC12 solution increased the period length and the amplitude of the transpiratory oscillations. The effects were dependent on the concentration and in 40 m ~ CaC12 the oscillations were completely abolished. Treatments with MgC12 had the same general effects on the oscillations, but Fig. 1 C shows t h a t 40 mM MgC12 was required in order to obtain the same period lengthening as with 20 mM CaC12. The period lengthening caused by the divalent ions was mostly due to the longer time spent b y the stomata in the closed state (el. shape of curves in Fig. 1 A and 1 B). The ionic effects can be explained by changes of the stomatal parameters, but in principle they could be attributed to changes of the xylem resistance to water flow induced b y the ions at the site of the deformation. To study this possibility Ca 2+ was given to excised, non-compressed plants (Fig.1 D). The light was switched on, the transpiration rate increased and Ca 2+ was transported up into the leaf. After 15 rain the CaC12 solution around the plant stem was exchanged to distilled water, flushing the xylem some time before the xylem compression was applied. The oscillations now showed a typical Ca 2+ lengthening of the period which, therefore, was attributed to reactions in the stomatal regions. The Ca 2+ as well as the Mg 2+ effects were reversible as exemphfied in Fig. 1 E : treatment with 40 mM Mg C4 for 1 h caused a period lengthening and an amplitude increase, which disappeared one cycle after the treatment. These reversible effects point to the possibihty t h a t divalent ions can be continuously withdrawn from the sites, where they affect the stomatal dynamics, and transformed into an inactive form [el. 2]. The effects of lanthanum ions were more pronounced than those caused by the divalent ions: 2.5 mM LaC1a gave roughly the same period lenghtening as 20 mM CaC12 (Fig. 1 B). I t has previously been reported t h a t Li+ ions increase the period length of the transpiratory oscillations and t h a t K+ and Na + ions do not cause any period changes [5]. 80 mM LiC1 increased the period length by a factor of 1.6. The con-
Fig. 1A E. Sustained transpiratory oscillations in excised oat plants. (A) Transpiration rate of the primary leaf of one plant, kept in a euvette under constant environmental conditions. (B) Increased amplitude and period length of the oscillations under the influence of 20 mM CaCI~solution. (C) MgCI~solution, 40 raM, caused the same increase in amplitude and period length of the oscillations as 20 mM CaC12 solution; el. B. (D) A plant, treated with 20 m ~ CaC12prior to the compression of the leaf base (xylem deformation), showed an initial prolonged period length of the transpiratory oscillations. (E) Oscillations with normal amplitude and period length were found about one hour after a treatment with 40 mM MgClz. (F) Simulations were made on a model for plant water regulation in order to find a possible site of action of Ca~+ and )/Ig~+. The curve represents the leaf conductance per unit leaf area which in the simulation is directly proportional to the transpiration stream. An increased amplitude and period length was obtained when the resistance (R~) against water flow into the guard cells was increased by 10 percent from its original value Rg~ ~ should be compared with the results in B and C
| = % E
(~
9
1 hour
I"
20 mM CoCi 2
[
1 hour
J
I
~=:=I"(---'- 20 mM Ca C[2
~. 20
xyie m
on
0
~E 0
"
!
1
t
1 hour
J
1 hour
]
10 seconds Fig. 1
7a Planta (Berl.), Vol. 124
]
102
T. Brogtrdh and A. Johnsson
centrations of the investigated ions, necessary to cause a lengthening of this order, would indicate t h a t the ions are effective in the following order: L a a + > Ca 2+ > Mg2+> L i + > ( K + Na+, which cause no changes). The period lengthening could be explained along at least two different lines. (a) The ions could interfere with ionic transport processes of the stomata, e.g. the shuttling of K+ into and out from the guard cells, thus changing water transport processes across the g u a r d cell membranes. Changes in ion permeabilities caused b y divalent ions are c o m m o n l y k n o w n in biological systems [see e.g. 6] and b o t h La a+ and Ca 2+ are reported to inhibit K + absorption in corn roots [13]. (b) The ions could act on the water permeability of the guard-cell membranes. Ca 2+ has n a m e l y been found to decrease the water permeabilities of membranes [11, 14] and so the main action of the calcium ions could be on the guard-cell membranes, decreasing their water permeability and thus increasing the time constant involved in the oscillatory g u a r d cell movements. Simulations along this line were made b y means of a previously published model for oscillatory stomatal regulation [8]. Results from simulations with suddenly increased resistance towards water flow into the guard cells were in agreement with the experiments, see :Fig. 1 F. The increased time constant of the guard cells caused larger oscillations in the model. Because of non-linearities in the model, the time intervals between the transpiratory p e a k s - and consequently the period length of the oscillations then increased [cf. 4]. The period length of the oscillations in intact plants could not be changed b y a n y of the ions. This means t h a t the ions cannot penetrate the root tissues and reach the stomatal regions. P r o b a l y the Casparian strips form a barrier against the ion movements into the xylem vessels [el. 13]. We thank members of the staff of the Institute of Plant Physiology, Lurid University, for helpful comments and discussions. The work was financially supported by the Swedish Natural Science Research Council.
References 1. Barrs, H. D. : Cyclic variations in stomafal aperture, transpiration, and leaf water potential under constant environmental conditions. Ann. Rev. Plant Physiol. 22, 223-236 (1971) 2. Biddulph, 0., Nakayama, ~. S., Cory, R. : Transpiration stream and ascension of calcium. Plant Physiol. 36, 429-436 (1961) 3. Brogs T., Johnsson, A. : Oscillatory transpiration and water uptake of Arena plants. II. Effects of deformation of xylem vessels. Physiol. Plantarum (Cph.) 28, 341-345 (1973) 4. Brogs T., Johnsson, A. : Oscillatory transpiration and water uptake of Arena plants. III. Action of heavy water on the oscillation. Physiol. Plantarum (Cph.) 31, 112-118 (1974a) 5. Brog~rdh, T., Johnsson, A,: Effects of lithium on stomatal regulation. Z. Natufforsch. 29c, 298-300 (1974b) 6. BurstrSm, H. : Calcium and plant growth. Biol. Rev. 43, 287-316 (1968) 7. Claus, St. : Ein Modell ffir das Verhalten der SpaltSffnungen im Porometer. Studia biophys. 11, 110-117 (1968) 8. Cowan, I. R.: Oscillations in stomatal conductance and plant functioning associated with stomatal conductance: observations and a model. Planta (Berl.) 106, 185-219 (1972) 9. Hopmans, P. A. M. : Rhythms in stomatal opening of bean leaves. Meded. Landbouwhogeschool Wageningen 71, No. 3 (1971) 10. Karmanov, V. G., Meleschenko, S.N., Savin, V. N. : Study of the dynamics of the water metabolism of the plant and construction of an electrical analogue of the system of water exchange. Biofizika (Engl. Transl.) 11, 147-155 (1966)
Magnesium, Calcium and Lanthanum Ions in Avena sativa L.
103
11. Kuiper, P. J. C.: Water transport across membranes. Ann. Rev. Plant Physiol. 23, 157-172 (1972) 12. Lang, A. R. G., Klepper, B., Cumming, M. J. : Leaf water balance during oscillation of stomatal aperture. Plant Physiol. 44, 826-830 (1969) 13. Nagahashi, G., Thomson, W. W., Leonard, I~. T. : The Casparian strip as a barrier to the movement of lanthanum in corn roots. Science 188, 670-671 (1974) 14. Whittembury, G., Sugino, N., Solomon, A. K. : Effect of antidiuretic hormone and calcium on the equivalent pore radius of kidney slices from Necturus. Nature (Lond.) 187, 699-701 (1960)
