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Freezing injury in sugar beet root cells: Sucrose leakage and modifications of tonoplast properties
Plant Science Letters, 26 (1982) 75--81 Elsevier Scientific Publishers Ireland Ltd.
75
FREEZING INJURY IN SUGAR BEET ROOT CELLS: SUCROSE LEAKAGE AND MODIFICATIONS OF TONOPLAST PROPERTIES
H. BARBIER a'b, F. NALIN c and J. GUERN a
aLaboratoire de Physiologic Cellulaire, C.N.R.S., 91190 Gif-sur~Yvette, blnstitut Teehique Franqais de la Betterave Industrielle, 45 rue de Naples, 75008 Paris and eGdndrale Sucri~re, Sucrerie de Cagny, 14630, Cagny (France) (Received November 9th, 1981) (Revision received December 22nd, 1981) (Accepted December 22nd, 1981)
SUMMARY
Freezing injury has been studied on sugar beet root cells. Sucrose effiux was monitored on disks prepared from control roots, or roots frozen at -5°C and thawed at both fast and slow rates. Results indicated that freezing affects the vacuolar membrane and increases its permeability for sucrose. The study of tonoplast properties on vacuole suspensions isolated from control or frozen roots showed that freezing increases membrane fragility and modifies the transtonoplast electrical potential difference. This confirmed the fact that tonoplast was the main site of freezing injury. Furthermore, all the tonoplast modifications induced by freezing are reversible, depending upon thawing temperature and duration.
INTRODUCTION
Freezing has multiple effects on higher plants and strongly disturbs the physiological equilibrium of affected cells. A rapid freezing induces ice formation inside the cells. During thawing, the small ice crystals grow and damage the membranes. A slow freezing produces extracellular ice formation, resulting in cell dehydration [ 1 ]. Degradation of physiological properties is progressive and depends upon freeze intensity. On onion bulbs, Palta and Li [2] showed that ions and sugar leakage precedes loss of cell turgot and water infiltration in tissues. These alterations are assumed to be due to a loss of membrane semi-permeability, or to membrane rupture [1]. However, Palta and coworkers [2,3] have recently shown that membrane semipermeability of frozen cells could remain intact. Furthermore, depending upon the degree of membrane damage, partial or complete recovery of frozen cells have been described [ 4--6] indicating that freezing injury may be reversible. 0304--4211/82/0000--0000l$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.
76 On sugar beet {Beta vulgaris L.) roots, the macroscopic consequences of freezing are well known. A decrease of sucrose c o n t e n t is followed by leakage of root sap and by the development of microorganisms on the root. Water infiltration induces the softening of root tissues, either with subsequent recovery to a normal state or development of definitive rotting. In sugar beet root cells, sucrose is stored in the large central vacuole and the vacuolar membrane is probably one of the regulatory sites of root sucrose accumulation. So we were particularly interested in the decrease of root sucrose content induced by freezing, and in the possible reversibility of freeze-induced symptoms. As freezing increases membrane permeability for solutes [2--4,7], sucrose loss in frozen beets may be due to tonoplast alterations. This paper shows the influence of freezing injury on the properties of the vacuolar membrane of root cells and the reversibility of induced alterations. Tonoplast properties were studied by sucrose efflux experiments and direct measurements on vacuoles isolated from normal or frozen roots. Results showed that freezing strongly increases tonoplast permeability for sucrose, reduces the yield of the preparation procedure for isolated vacuoles and modifies transtonoplast electrical potential difference (PD). MATERIALS AND METHODS
Plant material Roots of a tetraploid sugar beet variety (4675-31. CERES) were obtained from CERES (91660 Mdr~ville, France). Roots were harvested at m a t u r i t y (about 6 months after sowing} and stored in the cold (+4°C} for 3 months. Sucrose content of roots varies from 14% to 20% on a fresh weight basis.
Freezing and thawing procedures Roots were frozen at - 5 ° C for 64 h in a freezing chamber and then thawed in different conditions with either rapid thawing by direct passage from --5°C to +20°C, or slow thawing at +4°C, during various times (24, 48 and 96 h). Control roots, for comparison with frozen roots, were kept at +4°C. Temperature values were chosen as close as possible to climatic conditions encountered at the time of root harvesting. The freezing temperature was lower than the eutexia point for beet root cells (--2°C according to Oldfield et al. [8]).
Sucrose efflux from root disks R o o t tissue cylinders were cut from the upper part of the root and sliced with a razor blade into disks (1.6 mm thick). Sucrose efflux was measured on eight disks (about 3 g o f fresh matter) immersed in distilled water, with magnetic stirring at room temperature. Total duration of each efflux was 120 min, including two successive steps. The first 15 min represented a
77 rinsing period, in 350 ml of di.qtilled water. Then disks were transferred to 250 ml of water to continue the efflux. To follow sucrose leakage from the disks, aliquots (1 ml) were taken at intervals from the efflux solution. At the end of efflux, the remaining sucrose was extracted from the disks with aqueous ethanol (80%). Sucrose concentration was measured by the resorcinol m e t h o d adapted from Foreman et al. [9].
Vacuole isolation Vacuolar suspensions were prepared from the same roots used in efflux experiments, according to a procedure previously described [10,11] and adapted from the general procedure described by Leigh and Branton [ 12]. Briefly, small cubes of r o o t tissues ( 0 . 5 - 0 . 8 g fresh wt.) were sliced with a razor blade in 1 M sorbitol, 50 mM Tris--HC1 (pH 8). The vacuole suspension was recovered by gentle pipetting. Population density of the vacuolar suspensions was a b o u t 500--1000 vacuoles/~l. All measurements were carried out on these crude vacuole preparations. Yield of vacuole preparation Vacuoles were prepared from calibrated cubes (0.3 g fresh wt.) in 0.1 ml of the buffered solution. Aliquots of 0.05 ml of vacuolar suspension were taken and placed in a Nageotte cell. Vacuoles were counted in suspensions from control or frozen roots and the amount of vacuoles obtained from treated samples was expressed as percentage of that corresponding t o c o n t r o l roots. Transtonoplast PD measurements A drop of the vacuolar suspension was placed on a microscope slide. Vacuoles were immobilized with a microholder. Potential difference was measured between a glass microelectrode filled with 3 M potassium chloride, inserted in the vacuole, and a reference electrode placed in the external solution [10,11,13]. RESULTS
Sucrose efflux from root tissue disks Sucrose efflux from r o o t disks into water is very rapid initially and slows down within the first 20--30 min (Fig. 1A). During the initial period, about 20% of the whole sucrose content leaked out. From 30 to 120 min sucrose leaked o u t from the disks with a much slower and constant rate. At the end of the efflux period, more than 75% of the initial sucrose content remained in the disks. A procedure of compartmental analysis may be applied to analyse sucrose leakage from root disks [14--16]. Three successive phases were characterized (Fig. 1B). Sucrose elimination from cells injured during disk cutting mainly accounts for the two first phases. Sucrose from the cytoplasm of intact cells is probably lost also at the end of this period [15].
78 :E 100,
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B
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2 70
2J
g
m 1"1,0
6`0 Time (rain)
120
O
1'0
6'0 Time Cmin)
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Fig. 1. Sucrose efflux from root disks prepared from a control, unfrozen root, and immersed in distilled water. Evolution of sucrose content, plotted against time as percentage of the initial content (1A) or as the s u c r o s e c o n c e n t r a t i o n (mg/g D.M.) on a logarithmic scale (1B).
So we used the percentage of sucrose lost during the first 15 min (rinsing period) as an indication of cell destruction intensity caused by disk cutting. According to Kholodova [14], the third phase was assumed to represent sucrose leakage from the vacuolar storage c o m p a r t m e n t of intact cells. The rate of sucrose leakage across the tonoplast is given by the slope of the curve within the period 30--120 min. This rate is directly related to tonoplast permeability for sucrose and was used in our study to estimate freezinginduced alterations. The rates of sucrose leakage from disks prepared from control roots (C) or roots frozen a t - 5 ° C for 64 h, with subsequent rapid (+20°C) or slow (+ 4°C) thawing were compared (Fig. 2). Results showed that freezing strongly increases the percentage of sucrose lost during the rinsing period and the rate of sucrose leakage from the vacuolar compartment. In the case of rapid thawing the disks have lost their whole sucrose content within the first 90 min. When frozen roots are thawed at + 4°C, and according to the duration of this thawing period (24--96 h), a gradual recovery towards the unfrozen values was observed. The fact that freezing increases tonoplast permeability for sucrose m a y account for a part of sucrose loss during rinsing. This permeabilization induces sucrose leakage from root cell vacuoles to the other cell compartments, cytoplasm and walls. These sucrose molecules diffuse into the external medium at the same time that sucrose from cells injured or destroyed by cutting, and raise the total a m o u n t of sucrose lost during the first efflux phases.
Study o f vacuolar suspensions isolated from root tissues Isolated vacuoles were prepared from the control and frozen roots used in efflux experiments. Vacuole diameter, preparation yield and transtonoplast PD were measured on the different suspensions. Vacuole diameter was not modified significantly by the various treatments
79 (~=
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Time (rain) Fig. 2. Sucrose efflux from root disks in distilled water. Evolution of sucrose content (sucrose concentration in mg/g of dry matter on a logarithmic scale) against time, from 30 to 120 rain of efflux. C, control roots (unfrozen). (1) freezing at --5°C, 64 h + thawing at +20°C; (2) freezing at--5°C, 64 h + thawing at +4°C, 24 h; (3) freezing at--5°C, 64 h + thawing at +4°C, 48 h; (4) freezing at - 5 ° C , 64 h + thawing at +4°C, 96 h. The rate of sucrose leakage from the vacuole is given by the slope(s) of the curve (x 10 3). Each curve is an average from 3 experiments on different roots. a p p l i e d t o t h e r o o t s : 1 4 . 3 + 1 . 2 a n d 1 3 . 2 + 1.3 p m , r e s p e c t i v e l y , f o r c o n t r o l and f r o z e n - r a p i d l y t h a w e d roots. T h e y i e l d o f vacuole p r e p a r a t i o n was s t r o n g l y decreased b y freezing (Fig. 3) t o 10% o f the c o n t r o l . T r a n s t o n o p l a s t PD was
small and positive, as already "described on vacuoles isolated from sugar beet
100
/
50
E
/~
I1.
0 u
0 - ' ! 64h 0
-5~C
24h
48h
96h
.4°C
Freezing- thawing treatment Fig. 3. Characteristics of vacuolar suspensions prepared from control or frozen roots. Vacuole preparation yield : average result from control roots, expressed in number of vacuoles per ul, was taken as a reference for 100% yield. Results from frozen roots were expressed in percentage of this reference value ± S.E. Transtonoplast PD was measured on the same vacuole preparations and expressed in mV ± S.E.
80
root cells [12,13]. Freezing induces a fall of PD from +10.5 mV for control roots to +4.0 mV for frozen roots. For these two last parameters, slow thawing allowed complete recovery. After 96 h at +4°C, the preparation yield was 92% of the control and PD value was +10.6 mV. Some qualitative observations, during vacuole micromanipulation, showed that vacuoles from frozen roots (especially in the case of rapid thawing) were more fragile. They badly endure sucking and fixation in the microholder, micropipette insertion, and they tear and burst very easily. This fragility, inducing vacuole bursting, could account for the observed decrease in the yield of vacuole preparation. DISCUSSION AND CONCLUSION
Freezing injury on plants has been studied by various methods, the most c o m m o n l y used being solute efflux measurements from frozen cells or tissues, in comparison with unfrozen ones. Analysing sugar and ion efflux from frozen onion bulbs, Palta et al. [3,4] have.shown that freezing increases solute efflux, and that the effusate originates from damaged, b u t living cells. In this way, they demonstrated that the efflux m e t h o d is a good tool to estimate freezing injury before any visual s y m p t o m s occur. With other plants, acid phosphatase release [17] or hydrogen cyanide release [7,18] were used as indicators of membrane damage following freezing or thawing. The results obtained from this efflux method show clearly that cellular membranes are the primary site of freezing injury in plants. In several cases, the vacuolar membrane seems to be the first, or even the sole target of frost injury. Yoshida et al. on Cormus stolonifera callus [19] have noted that lesion of the tonoplast is a primary step of chilling injury. Salt release from the vacuole is accompanied by ultrastructural changes in the tonoplast (invagination, unfolding) and precedes any change in the plasma membrane. On Amelanchier alnifolia, S t o u t has recently demonstrated [20] that the freeze-induced HCN release results specifically from tonoplast injury. In frozen tissues, semi-permeability of the cells remains intact [2], so the leakage o f solutes appears due to alterations in the cell membrane properties, rather than the membrane rupture. Nevertheless, the investigation of the initial injurious events in frozen cells is difficult, because of the lack of direct evidence at the membrane level. In sugar beet, this problem was resolved by the use of vacuolar suspensions. With these preparations, we could study directly tonoplast properties on vacuoles isolated from control (unfrozen) or frozen roots. Results showed that freezing increases membrane fragility, inducing more frequent vacuole bursting, and modifies significantly transtonoplast PD. The increase in sucrose efflux observed on disks provided other evidence that the tonoplast was the main site of freezing injury. Furthermore, all the tonoplast modifications induced by freezing: permeability for sucrose, fragility, lower PD, are reversible, depending upon thawing temperature and duration. Ion efflux following freezing has been attributed to a disruption of
81
active transport systems across the membrane [ 3,4,21 ], possibly related to protein alterations. Such an hypothesis o f protein structure modifications inside the tonoplast would account for the freeze-induced p h e n o m e n a observed on sugar beet. A repair of protein alterations would restore membrane properties and lead the tissue to recover from freezing injury. REFERENCES 1 J. Levitt, in: Responses of Plants to Environmental Stress, Academic Press, 1972, p. 188. 2 J.P. Palta and P.H. Li, Physiol. Plant., 50 (1980) 169. 3 J.P. Palta, J. Levitt and E.J. Stadelman, Plant Phyisol., 60 (1977) 393. 4 J.P, Palta, J. Levitt and E.J. Stadelman, Plant Physiol., 60 (1977) 398. 5 R.P. Creencia and W.J. Bramlage, Plant Physiol., 47 (1971) 389. 6 J.M. Lyons, J.K. Raison and P.L. Steponkus, The plant membrane in response to low temperature: an overview, in: J.M. Lyons, D. Grahams and J.K. Raison (Eds.), L o w Temperature Stress in Crop Plants. The Role of Membrane, Academic Press, 1979, p. 1. 7 D.G. Stout, B. Brooke, W, Majak and M. Reaney, Plant Physiol., 68 (1981) 248. 8 J.F.T. Oldfield, Sucrerie Beige, 89 (1970) 507. 9 D. Foreman, L. Gaylor, E. Evans and C. Trella, Anal. Biochem., 56 (1973) 584. 10 H. Barbier and J. Guern, C.R. Acad. Sci. Paris, 292 (1981) 785. 11 H. Barbier and J. Guern, Transmembrane potential of isolated vacuoles and sucrose accumulation by Beta vulgaris roots, in: D. M a r m e (Ed.), Higher Plant Membranes, Biomedical Press, 1982, in press. " 12 R.A. Leigh and D. Branton, Plant Physiol., 58 (1976) 656. 13 J.P. Rona, D. Cornel and R. Heller, in: M. TheUier, A. Monnier, M. Demarty and J. Dainty (Eds.), Transmembrane Ionic Exchange in Plant~Colloque International No. 258, CNRS, 1976, p. 349. 14 V.P. Kholodova, Fiziol. Rast., 14 (1967) 376. 15 Y. Signor, Th~se de 3° Cycle, Universith Pierre & Marie Curie, Paris, 1978. 16 R. Ehwald, D. Kowallick, A.B. Mescheryakov and V.P. Kholodova, J. Exp. Bot., 31 (1980) 607. 17 R.J. Bolduc, Plant Physiol., 67 (1981) 61. 18 D.G. Stout, W. Majak and M. Reaney, Plant Physiol., 66 (1980) 74. 19 S. Yoshida, T. Niki and A. Sakai, Possible involvement of the tonoplast lesion in chilling injury of cultured plant cells, in: J.M. Lyons, D. Grahams and J.K. Raison (Eds.), Low Temperature Stress in Crop Plants. The Role of Membrane, Academic Press, 1979, p. 275. 20 D.G. Stout, Plant Physiol., 67 (1981) 63. 21 J.P. Palta and J.K. Pohlman, Plant Physiol., 67 (1981) 62.
75
FREEZING INJURY IN SUGAR BEET ROOT CELLS: SUCROSE LEAKAGE AND MODIFICATIONS OF TONOPLAST PROPERTIES
H. BARBIER a'b, F. NALIN c and J. GUERN a
aLaboratoire de Physiologic Cellulaire, C.N.R.S., 91190 Gif-sur~Yvette, blnstitut Teehique Franqais de la Betterave Industrielle, 45 rue de Naples, 75008 Paris and eGdndrale Sucri~re, Sucrerie de Cagny, 14630, Cagny (France) (Received November 9th, 1981) (Revision received December 22nd, 1981) (Accepted December 22nd, 1981)
SUMMARY
Freezing injury has been studied on sugar beet root cells. Sucrose effiux was monitored on disks prepared from control roots, or roots frozen at -5°C and thawed at both fast and slow rates. Results indicated that freezing affects the vacuolar membrane and increases its permeability for sucrose. The study of tonoplast properties on vacuole suspensions isolated from control or frozen roots showed that freezing increases membrane fragility and modifies the transtonoplast electrical potential difference. This confirmed the fact that tonoplast was the main site of freezing injury. Furthermore, all the tonoplast modifications induced by freezing are reversible, depending upon thawing temperature and duration.
INTRODUCTION
Freezing has multiple effects on higher plants and strongly disturbs the physiological equilibrium of affected cells. A rapid freezing induces ice formation inside the cells. During thawing, the small ice crystals grow and damage the membranes. A slow freezing produces extracellular ice formation, resulting in cell dehydration [ 1 ]. Degradation of physiological properties is progressive and depends upon freeze intensity. On onion bulbs, Palta and Li [2] showed that ions and sugar leakage precedes loss of cell turgot and water infiltration in tissues. These alterations are assumed to be due to a loss of membrane semi-permeability, or to membrane rupture [1]. However, Palta and coworkers [2,3] have recently shown that membrane semipermeability of frozen cells could remain intact. Furthermore, depending upon the degree of membrane damage, partial or complete recovery of frozen cells have been described [ 4--6] indicating that freezing injury may be reversible. 0304--4211/82/0000--0000l$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.
76 On sugar beet {Beta vulgaris L.) roots, the macroscopic consequences of freezing are well known. A decrease of sucrose c o n t e n t is followed by leakage of root sap and by the development of microorganisms on the root. Water infiltration induces the softening of root tissues, either with subsequent recovery to a normal state or development of definitive rotting. In sugar beet root cells, sucrose is stored in the large central vacuole and the vacuolar membrane is probably one of the regulatory sites of root sucrose accumulation. So we were particularly interested in the decrease of root sucrose content induced by freezing, and in the possible reversibility of freeze-induced symptoms. As freezing increases membrane permeability for solutes [2--4,7], sucrose loss in frozen beets may be due to tonoplast alterations. This paper shows the influence of freezing injury on the properties of the vacuolar membrane of root cells and the reversibility of induced alterations. Tonoplast properties were studied by sucrose efflux experiments and direct measurements on vacuoles isolated from normal or frozen roots. Results showed that freezing strongly increases tonoplast permeability for sucrose, reduces the yield of the preparation procedure for isolated vacuoles and modifies transtonoplast electrical potential difference (PD). MATERIALS AND METHODS
Plant material Roots of a tetraploid sugar beet variety (4675-31. CERES) were obtained from CERES (91660 Mdr~ville, France). Roots were harvested at m a t u r i t y (about 6 months after sowing} and stored in the cold (+4°C} for 3 months. Sucrose content of roots varies from 14% to 20% on a fresh weight basis.
Freezing and thawing procedures Roots were frozen at - 5 ° C for 64 h in a freezing chamber and then thawed in different conditions with either rapid thawing by direct passage from --5°C to +20°C, or slow thawing at +4°C, during various times (24, 48 and 96 h). Control roots, for comparison with frozen roots, were kept at +4°C. Temperature values were chosen as close as possible to climatic conditions encountered at the time of root harvesting. The freezing temperature was lower than the eutexia point for beet root cells (--2°C according to Oldfield et al. [8]).
Sucrose efflux from root disks R o o t tissue cylinders were cut from the upper part of the root and sliced with a razor blade into disks (1.6 mm thick). Sucrose efflux was measured on eight disks (about 3 g o f fresh matter) immersed in distilled water, with magnetic stirring at room temperature. Total duration of each efflux was 120 min, including two successive steps. The first 15 min represented a
77 rinsing period, in 350 ml of di.qtilled water. Then disks were transferred to 250 ml of water to continue the efflux. To follow sucrose leakage from the disks, aliquots (1 ml) were taken at intervals from the efflux solution. At the end of efflux, the remaining sucrose was extracted from the disks with aqueous ethanol (80%). Sucrose concentration was measured by the resorcinol m e t h o d adapted from Foreman et al. [9].
Vacuole isolation Vacuolar suspensions were prepared from the same roots used in efflux experiments, according to a procedure previously described [10,11] and adapted from the general procedure described by Leigh and Branton [ 12]. Briefly, small cubes of r o o t tissues ( 0 . 5 - 0 . 8 g fresh wt.) were sliced with a razor blade in 1 M sorbitol, 50 mM Tris--HC1 (pH 8). The vacuole suspension was recovered by gentle pipetting. Population density of the vacuolar suspensions was a b o u t 500--1000 vacuoles/~l. All measurements were carried out on these crude vacuole preparations. Yield of vacuole preparation Vacuoles were prepared from calibrated cubes (0.3 g fresh wt.) in 0.1 ml of the buffered solution. Aliquots of 0.05 ml of vacuolar suspension were taken and placed in a Nageotte cell. Vacuoles were counted in suspensions from control or frozen roots and the amount of vacuoles obtained from treated samples was expressed as percentage of that corresponding t o c o n t r o l roots. Transtonoplast PD measurements A drop of the vacuolar suspension was placed on a microscope slide. Vacuoles were immobilized with a microholder. Potential difference was measured between a glass microelectrode filled with 3 M potassium chloride, inserted in the vacuole, and a reference electrode placed in the external solution [10,11,13]. RESULTS
Sucrose efflux from root tissue disks Sucrose efflux from r o o t disks into water is very rapid initially and slows down within the first 20--30 min (Fig. 1A). During the initial period, about 20% of the whole sucrose content leaked out. From 30 to 120 min sucrose leaked o u t from the disks with a much slower and constant rate. At the end of the efflux period, more than 75% of the initial sucrose content remained in the disks. A procedure of compartmental analysis may be applied to analyse sucrose leakage from root disks [14--16]. Three successive phases were characterized (Fig. 1B). Sucrose elimination from cells injured during disk cutting mainly accounts for the two first phases. Sucrose from the cytoplasm of intact cells is probably lost also at the end of this period [15].
78 :E 100,
c:l
A
B
II U
_
--
=
-
.
•
L¢I
2 70
2J
g
m 1"1,0
6`0 Time (rain)
120
O
1'0
6'0 Time Cmin)
120
Fig. 1. Sucrose efflux from root disks prepared from a control, unfrozen root, and immersed in distilled water. Evolution of sucrose content, plotted against time as percentage of the initial content (1A) or as the s u c r o s e c o n c e n t r a t i o n (mg/g D.M.) on a logarithmic scale (1B).
So we used the percentage of sucrose lost during the first 15 min (rinsing period) as an indication of cell destruction intensity caused by disk cutting. According to Kholodova [14], the third phase was assumed to represent sucrose leakage from the vacuolar storage c o m p a r t m e n t of intact cells. The rate of sucrose leakage across the tonoplast is given by the slope of the curve within the period 30--120 min. This rate is directly related to tonoplast permeability for sucrose and was used in our study to estimate freezinginduced alterations. The rates of sucrose leakage from disks prepared from control roots (C) or roots frozen a t - 5 ° C for 64 h, with subsequent rapid (+20°C) or slow (+ 4°C) thawing were compared (Fig. 2). Results showed that freezing strongly increases the percentage of sucrose lost during the rinsing period and the rate of sucrose leakage from the vacuolar compartment. In the case of rapid thawing the disks have lost their whole sucrose content within the first 90 min. When frozen roots are thawed at + 4°C, and according to the duration of this thawing period (24--96 h), a gradual recovery towards the unfrozen values was observed. The fact that freezing increases tonoplast permeability for sucrose m a y account for a part of sucrose loss during rinsing. This permeabilization induces sucrose leakage from root cell vacuoles to the other cell compartments, cytoplasm and walls. These sucrose molecules diffuse into the external medium at the same time that sucrose from cells injured or destroyed by cutting, and raise the total a m o u n t of sucrose lost during the first efflux phases.
Study o f vacuolar suspensions isolated from root tissues Isolated vacuoles were prepared from the control and frozen roots used in efflux experiments. Vacuole diameter, preparation yield and transtonoplast PD were measured on the different suspensions. Vacuole diameter was not modified significantly by the various treatments
79 (~=
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~ - - - -.-- . _,,_ --~
~E
~2.5
-
g
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~,---._,,_._.,.__
L_
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. ,,__ . _.=_ S = . 1 . ~
•
d
gz0 E u
•
-'-~_
s =-4.12
• T~ \
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Time (rain) Fig. 2. Sucrose efflux from root disks in distilled water. Evolution of sucrose content (sucrose concentration in mg/g of dry matter on a logarithmic scale) against time, from 30 to 120 rain of efflux. C, control roots (unfrozen). (1) freezing at --5°C, 64 h + thawing at +20°C; (2) freezing at--5°C, 64 h + thawing at +4°C, 24 h; (3) freezing at--5°C, 64 h + thawing at +4°C, 48 h; (4) freezing at - 5 ° C , 64 h + thawing at +4°C, 96 h. The rate of sucrose leakage from the vacuole is given by the slope(s) of the curve (x 10 3). Each curve is an average from 3 experiments on different roots. a p p l i e d t o t h e r o o t s : 1 4 . 3 + 1 . 2 a n d 1 3 . 2 + 1.3 p m , r e s p e c t i v e l y , f o r c o n t r o l and f r o z e n - r a p i d l y t h a w e d roots. T h e y i e l d o f vacuole p r e p a r a t i o n was s t r o n g l y decreased b y freezing (Fig. 3) t o 10% o f the c o n t r o l . T r a n s t o n o p l a s t PD was
small and positive, as already "described on vacuoles isolated from sugar beet
100
/
50
E
/~
I1.
0 u
0 - ' ! 64h 0
-5~C
24h
48h
96h
.4°C
Freezing- thawing treatment Fig. 3. Characteristics of vacuolar suspensions prepared from control or frozen roots. Vacuole preparation yield : average result from control roots, expressed in number of vacuoles per ul, was taken as a reference for 100% yield. Results from frozen roots were expressed in percentage of this reference value ± S.E. Transtonoplast PD was measured on the same vacuole preparations and expressed in mV ± S.E.
80
root cells [12,13]. Freezing induces a fall of PD from +10.5 mV for control roots to +4.0 mV for frozen roots. For these two last parameters, slow thawing allowed complete recovery. After 96 h at +4°C, the preparation yield was 92% of the control and PD value was +10.6 mV. Some qualitative observations, during vacuole micromanipulation, showed that vacuoles from frozen roots (especially in the case of rapid thawing) were more fragile. They badly endure sucking and fixation in the microholder, micropipette insertion, and they tear and burst very easily. This fragility, inducing vacuole bursting, could account for the observed decrease in the yield of vacuole preparation. DISCUSSION AND CONCLUSION
Freezing injury on plants has been studied by various methods, the most c o m m o n l y used being solute efflux measurements from frozen cells or tissues, in comparison with unfrozen ones. Analysing sugar and ion efflux from frozen onion bulbs, Palta et al. [3,4] have.shown that freezing increases solute efflux, and that the effusate originates from damaged, b u t living cells. In this way, they demonstrated that the efflux m e t h o d is a good tool to estimate freezing injury before any visual s y m p t o m s occur. With other plants, acid phosphatase release [17] or hydrogen cyanide release [7,18] were used as indicators of membrane damage following freezing or thawing. The results obtained from this efflux method show clearly that cellular membranes are the primary site of freezing injury in plants. In several cases, the vacuolar membrane seems to be the first, or even the sole target of frost injury. Yoshida et al. on Cormus stolonifera callus [19] have noted that lesion of the tonoplast is a primary step of chilling injury. Salt release from the vacuole is accompanied by ultrastructural changes in the tonoplast (invagination, unfolding) and precedes any change in the plasma membrane. On Amelanchier alnifolia, S t o u t has recently demonstrated [20] that the freeze-induced HCN release results specifically from tonoplast injury. In frozen tissues, semi-permeability of the cells remains intact [2], so the leakage o f solutes appears due to alterations in the cell membrane properties, rather than the membrane rupture. Nevertheless, the investigation of the initial injurious events in frozen cells is difficult, because of the lack of direct evidence at the membrane level. In sugar beet, this problem was resolved by the use of vacuolar suspensions. With these preparations, we could study directly tonoplast properties on vacuoles isolated from control (unfrozen) or frozen roots. Results showed that freezing increases membrane fragility, inducing more frequent vacuole bursting, and modifies significantly transtonoplast PD. The increase in sucrose efflux observed on disks provided other evidence that the tonoplast was the main site of freezing injury. Furthermore, all the tonoplast modifications induced by freezing: permeability for sucrose, fragility, lower PD, are reversible, depending upon thawing temperature and duration. Ion efflux following freezing has been attributed to a disruption of
81
active transport systems across the membrane [ 3,4,21 ], possibly related to protein alterations. Such an hypothesis o f protein structure modifications inside the tonoplast would account for the freeze-induced p h e n o m e n a observed on sugar beet. A repair of protein alterations would restore membrane properties and lead the tissue to recover from freezing injury. REFERENCES 1 J. Levitt, in: Responses of Plants to Environmental Stress, Academic Press, 1972, p. 188. 2 J.P. Palta and P.H. Li, Physiol. Plant., 50 (1980) 169. 3 J.P. Palta, J. Levitt and E.J. Stadelman, Plant Phyisol., 60 (1977) 393. 4 J.P, Palta, J. Levitt and E.J. Stadelman, Plant Physiol., 60 (1977) 398. 5 R.P. Creencia and W.J. Bramlage, Plant Physiol., 47 (1971) 389. 6 J.M. Lyons, J.K. Raison and P.L. Steponkus, The plant membrane in response to low temperature: an overview, in: J.M. Lyons, D. Grahams and J.K. Raison (Eds.), L o w Temperature Stress in Crop Plants. The Role of Membrane, Academic Press, 1979, p. 1. 7 D.G. Stout, B. Brooke, W, Majak and M. Reaney, Plant Physiol., 68 (1981) 248. 8 J.F.T. Oldfield, Sucrerie Beige, 89 (1970) 507. 9 D. Foreman, L. Gaylor, E. Evans and C. Trella, Anal. Biochem., 56 (1973) 584. 10 H. Barbier and J. Guern, C.R. Acad. Sci. Paris, 292 (1981) 785. 11 H. Barbier and J. Guern, Transmembrane potential of isolated vacuoles and sucrose accumulation by Beta vulgaris roots, in: D. M a r m e (Ed.), Higher Plant Membranes, Biomedical Press, 1982, in press. " 12 R.A. Leigh and D. Branton, Plant Physiol., 58 (1976) 656. 13 J.P. Rona, D. Cornel and R. Heller, in: M. TheUier, A. Monnier, M. Demarty and J. Dainty (Eds.), Transmembrane Ionic Exchange in Plant~Colloque International No. 258, CNRS, 1976, p. 349. 14 V.P. Kholodova, Fiziol. Rast., 14 (1967) 376. 15 Y. Signor, Th~se de 3° Cycle, Universith Pierre & Marie Curie, Paris, 1978. 16 R. Ehwald, D. Kowallick, A.B. Mescheryakov and V.P. Kholodova, J. Exp. Bot., 31 (1980) 607. 17 R.J. Bolduc, Plant Physiol., 67 (1981) 61. 18 D.G. Stout, W. Majak and M. Reaney, Plant Physiol., 66 (1980) 74. 19 S. Yoshida, T. Niki and A. Sakai, Possible involvement of the tonoplast lesion in chilling injury of cultured plant cells, in: J.M. Lyons, D. Grahams and J.K. Raison (Eds.), Low Temperature Stress in Crop Plants. The Role of Membrane, Academic Press, 1979, p. 275. 20 D.G. Stout, Plant Physiol., 67 (1981) 63. 21 J.P. Palta and J.K. Pohlman, Plant Physiol., 67 (1981) 62.