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Frost hardiness of Norway spruce treated with acid mist. Evaluation of the electrolyte leakage rate technique
EnzlronmentalandExpetlmentalBotany,Vol. 35, No. 2, pp. 13~149, 1995 ElsevierScience Ltd Printed in Great Britain
Pergamon 0098-8472(94) 00041-7
FROST HARDINESS OF N O R W A Y SPRUCE TREATED WITH ACID MIST. EVALUATION OF THE ELECTROLYTE LEAKAGE RATE
TECHNIQUE LUCYJ. SHEPPARD, INGRID FRANSSEN andJ. NEIL CAPE Institute of Terrestrial Ecology, Bush Estate, Penicuik, Midiothian EH26 0QB, U.K.
(Received6June 1994; acceptedin revisedform 8 August 1994) Sheppard L.J., Franssen I. and Cape J. N. FrosthardinessofNorway sprucetreatedwith acidmist. Evaluation of the electrolye leakagerate technique. Environmental and Experimental Botany 35, 139-149, 1995.Solutions of acid mist at pH 2.5, 2.7 and 5.0 were applied at four misting frequencies to 2-year-old Norway spruce growing in pots in a glasshouse for 19 weeks, from July to November 1991. Frost hardiness assessments were made on detached shoots. Three methods for analysing ion leakage were compared: conductivity after 24 and 120 hr, and the rate of leakage derived from measurements at 1, 24 and 120 hr relative to the ion conductivity after autoclaving shoot segments in deionized water. The different methods of estimating the effects of acid mist on frost hardiness gave similar results, but the use of a single measurement after 24 hr was confounded by the inclusion of ions washed from the leaf surface. This bias was smaller for the single measurement after 120 hr, but only the electrolyte leakage rate determination could separate and restrict this potential source of bias. Estimates of leakage rate, which accommodates differences in hardening status and surfaceadsorbed ions, were found to be the most robust, sensitive method for determining acid mist effects on frost hardiness. Measurements of relative conductivity based on 100% fewer measurements are unlikely to save time since they will require exploratory studies throughout the hardening period to establish the optimum times for making the single, critical measurement.
K~ words: Norway spruce, acid mist, frequency, frost hardiness, ion leakage rate, relative conductivity.
INTRODUCTION
Norway spruce seedlings were exposed to simulated acid mist containing H +, N O s - , NH4 + and $ 0 4 2 A causal relationship between the deposition of a t three ionic concentrations and four frequencies of anthropogenic pollutants and reduced winter hardi- weekly exposure. Previous experiments 112'2°'22/had ness has been established for red spruce (Picea rubens also suggested that sensitivity could be further Sarg.). (7/In Europe, where Norway spruce [P. abies enhanced by increasing the exposure frequency for (L.) Kant] is the predominant species, forest decline a given dose, and this too was examined. has also been linked to increased frost sensitivity, The main objective of this study was concerned thought to reflect enhanced N inputs. (19)However, with assessing the relative merits of the ion leakage rigorous experiments examining the relationship rate technique/16) for evaluating the effects of acid between frost sensitivity, acid mist and frost hardi- mist on frost hardiness against ion leakage results ness of Norway spruce are lacking. This paper based on fewer measurements. O u r working reports a glasshouse experiment in which pot-grown hypothesis was that the taking of four measurements 139
140
L.J. SHEPPARD et al.
for the leakage rate method are justified and necessary, when examining the effects of acid mist, to overcome the bias introduced by accumulation and desorption.
Techniques current~ in use to detectfieezing injury Frost hardiness of plant tissue can be assessed under controlled conditions by subjecting the tissue to a range of sub-zero temperatures and a protocol for such tests is well established. <15/Likewise, many protocols for estimating resultant injury such as fluorescence diacetate, (27) impedance measurements, (28) visible injury(~'21/ chlorophyll fluorescence<26>and electrolyte leakage (2'6'14'16'27>are in current use. Unfortunately, the suitability of particular protocols for specific jobs is still the subject of intense debate, and many of the caveats are not widely recognized. Visible injury is probably most indicative of potential field damage, especially where whole seedlings are used. However, its development is slow (L. J. Sheppard, unpubl.) and the results rely on subjective scoring which is particularly inexact during midwinter. In assessing pollutant effects on frost hardiness, the ion leakage technique has been widely adopted, <3'5'8> but groups have utilized different measurement protocols. The timing of the measurements, e.g. 24 or 120 hr after freezing, is influenced by species, tissue type, season and tissue hardiness in relation to test temperatures. (4'24'27)Likewise, condltions of sample storage, temperature, the inclusion of wetting agents and the use of agitation will also influence the choice of timing (J. S. Jacobson, pers. comm.). Moreover, it is not possible to relate rates of ion leakage to biologically meaningful reference points for sample comparison, <17) since tissue death is difficult to determine, and the concept of 'death' may differ depending on the objective. Some groups have adopted the 'critical temperature' (Tc) criterion, corresponding to the point of earliest detectable freezing injury./61 The ability to meaningfully identify a threshold of ion leakage for death is dependent on the choice of test temperatures; ideally, the test intervals should be 2 or 3°C but, more often because of practical restrictions, intervals of up to 5°C are used. A single measurement of conductivity at a fixed time after immersion has often correlated poorly with visible damage. (25)Zhang and W i l l i s o n (27) found that the conductivity method systematically under-
estimated frost hardiness based on visible injury. This poor correlation appeared to result from the reading being taken too early, providing insufficient time for leakage to o c c u r . (24'27) Also, in some instances, freezing damage to membranes may not be permanent and can be repaired or compensated for, so that readings taken too soon after freezing, e.g. 24 hr, may overestimate the degree of damage. (l~/Temperature, agitation, surface area of material and other environmental conditions are all likely to influence leakage rate, thus necessitating preliminary tests to identify the most suitable conditions for the particular tissue under study? l> Where shoots have been treated with acid mist, a single measurement may be misleading because dissolution of ions deposited on leaf surfaces may contribute a large proportion of the total ions measured. The rate equation/161 which requires four readings, was developed to accommodate the effects of surface deposits and results correlate well with visible damage scoresJ 2~)In the ensuing experiment we evaluate the potential merits of relative conductivity measured at 24 hr (cfJ. S.Jacobson, pers. comm.) and 120 hr/4) against the rate of leakage. Measuring relative conductivity, as opposed to rate, reduces the number of measurements from four to two and may potentially reduce the time lapse between freezing and the end result. METHODS
Plant material and misting treatments One hundred and twenty 2-year-old Norway spruce seedlings, raised from seed in a glasshouse in their second flush of shoot growth, were arranged in 24 trays in July 1991. The plants were growing in 1.25 1 pots containing fertilized compost (peat, grit and loam, 40:40:20) + Vitax Q4 fertilizer (3.5% N, 7.5% P, 10% K). An additional 45 mg Vitax Q4 was added to the surface of each pot in July. Plants were watered with tap water (pH 8). The water was poured into trays fined with capillary matting on which the potted plants stood. Twelve trays of plants were randomly assigned to two replicate blocks per treatment. Spraying commenced on 11 July and continued for 19 weeks. The volume of spray applied to each replicate tray provided the rainfall equivalent of 4 mm week- 1. Four frequency treatments were included once a week (4 mm x week-1), twice a week (2 x 2 mm week-l), either
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE
141
Table 1. Concentrations (mM) of H +, NO3-, NH4+ and SO42- in the acid mist solutions and total deposition equivalent of N, S and H over 19 weeks pH
H÷
NO3-
NH4 +
8042-
mM 2.5 2.7 5.0
3.2 2.0 0.01
1.6 1.0 0.005
N
S
Deposition kg ha1.6 1.0 0.005
on consecutive days (2 x c) or with a 3-day interval (2 x ), and four times a week (4 x 1 m m week-'). Mist treatments made up at p H 2.5, 2.7 and 5.0 from NH4NO3 and H2SO4 (Table 1) were delivered for 5 min by hand using a pump action sprayer which provided droplets up to 10 ttm. No supplementary heat or light was supplied. Frost hardiness Twelve current year lateral shoots were removed on 26 November from each tree and stored in black polythene bags at 2°C. Each day, 2 shoots tree-1 were subjected to a simulated night, air frost at different test temperatures in a programmable freezing cabinet. (3'81Temperatures were lowered at 5°C hr-1, the test temperature was held for 3 hr then temperatures were raised at 10°C hr-1 to 2°C. Due to the expected treatment effects on frost hardiness, shoots were exposed to two temperature ranges: - 13, - 17, - 22, - 27, - 32 and - 37°C for p H 2.5; and - 27, - 32, - 37, - 44 and - 49°C for p H 2.7 and p H 5. Shoots were not pre-immersed to remove surface salts. A 1 cm section was excised from the middle portion of each frozen shoot and placed in a 20 ml vial. Fifteen ml of deionized water were added to each vial and the conductivity of the bathing solution was measured using a platinum electrode with built-in temperature correction, at 1 hr and 120 hr, autoclaved at 1050C for 5 min and measured 24 hr later./3/Ion leakage rates were calculated for each frozen shoot by fitting the data to the equation C, = Co+(C .... -Co)(1-e-~a). 06) Relative conductivities were also calculated by taking the value for 24 or 120 hr and dividing by the autoclaved value which represents the sum total of leakable ions. After log-transformation, the electrolyte leakage rate (k) and relative conductivities were analysed
1.6 1.0 0.005
36 22.5 0.005
39 24.3 0.1
H l
25 15 --
using two-way A N O V A . (18/ Separate analyses were performed at each test temperature to test for effects o f p H and frequency on leakage rates. The analyses were performed with blocks as the unit of replication and (pH * frequency * block) as the error term. Contrast analyses were constructed to test the effect of frequency on leakage rates (k) and relative conductivities at all temperatures and p H combinations. Temperatures equivalent to 50% shoot death were calculated by Probit Analysis/18~ from the numbers of dead shoots per treatment at each test temperature. Shoots were designated dead or alive depending on whether their leakage rate exceeded a 'critical' rate. This critical leakage rate was determined as being around the separation point between the population of leakage rates from unfrozen shoots and those frozen at the coldest test temperature - 4 9 ° C . The population of leakage rates is composed from shoots of all treatments. RESULTS
Frost hardiness Salt accumulation on the needle suoCace. The ratio of ion conductivity values after 1 hr in deionized water to the autoclaved values were 0.05, 0.04 and 0.03 for p H 2.5, 2.7 and 5.0, respectively. Ion accumulation (data not shown) was significantly greater (9) (P < 0.001) for the p H 2.5 treated needles which received the highest ion concentrations. Treatment with p H 2.7 also significantly enhanced surface ion accumulation. There was no significant (P > 0.05) frequency effect on surface ion accumulation. Effects of treatment on log transformed leakage rates (k) and the lethal temperature causing 50% shoot death (LTs0). Treatment with p H 2.5 mist significandy increased leakage rates from unfrozen control shoots (pH 5) but p H 2.7 mist significantly reduced leakage from
142
L.J. SHEPPARD et aL
Table 2. Geometric dai~ rates of ion leakage ~ day-')3%rn Norway spruce shoots treated with mist at pH 5.0, 2.7 and 2.5, at a range offreezing temperatures. Results represent the leakage rates averagedover thefour fiequency treatments. (The overalleffect of)qequenqy was not sign~qcant P > 0.05.) Means in the same rowfollowed by the same letter are not sign}qcant~ different
Table 3. Temperatures (°C) required to kill 50% (LTs0) of Norway spruce shoots treated with acid mist at differentpHs and with different application J~equencies, on 26 November 1991. LTs0s determinedfrom a critical ion leakage rate of 2.7% day -I
pH k values (% day- 1) Temperature (°C) pH 5.0 pH 2.7 pH 2.5 -49 -44 - 37 -32 -27 2
14.6a 9.7a 4.0a 1.9a 1.1a 0.6a
17.9a 14.5a 7.2b 3.1b 1.7b 0.3b
NA NA 9.6b 5.4c 2.7c 1.0c
P value treatment effect 0.18 0.08 0.0007 0.0001 0.003 0.0001
* NA = not available.
unfrozen shoots (Table 2). Frequency effects measured over all three treatment p H s were significant (P < 0.05) only at - 1 7 ° C but patterns were consistent across the test temperatures, so that the more often spraying occurred, the greater the ion leakage rate post-freezing stress. D a m a g e and high leakage rates followed the order 4 x > 2 x c > 2 x > 1 x . Increasing spraying frequency from once or twice a week to 4 x w e e k - 1 caused a significant increase in ion leakage from shoots treated with p H 2.5 mist at - 17, - 2 2 and - 2 7 ° C . T h e frequency distribution of ion leakage rates from all treatments and test temperatures (Fig. 1, lower) was used to determine a critical leakage rate. Values for the unfrozen shoots and those frozen at - 4 9 ° C fall into a bimodal distribution; unfrozen shoots had values log k < - 6 . 8 while shoots exposed to - 4 9 ° C had values k > - 6 . 6 . A value of loge k corresponding to a leakage rate of 2.7% day-~ was chosen as the leakage rate which best discriminated between 'live' and 'dead' shoots. There was a significant effect of p H but not frequency on the LTs0 (Table 3). T h e LT50 for the p H 2.5 mist treatment was 8.5°C warmer than that for p H 5.0. Shoots treated with p H 2.5 mist 4 x week-1 were significantly less hardy (P = 0.05) by 4°C than those receiving the same dose once a week. W h e r e the ion concentration/dose was reduced by 33% (pH = 2.7), the only significant differences in LTs0 relating to frequency were between the twice weekly application on con-
LTs0
2.5
5
ANOVA P value
-34.6
<0.05
2.7
--26.1a - 3 1 . 5 b
Frequency
4x
2Xc
2x
lx
LTs0
29.4a
pH* frequency
LTs0
95% confidence limits - 2 5 . 7 to 22.3 --32.0 to 22.7 No confidence limits - 2 9 . 9 to 25.7
30.6a 31.5a
31.5a >0.05
2.5 2.5 2.5 2.5
4x 2×c 2x 1x
-23.7a --26.9a -- 26.3a -27.7b
2.7 2.7 2.7 2.7
4x 2x c 2x lx
-30.8a -29.2a -33.3a - 32.7b
-32.2 -31.2 -37.8 -34.4
to to to to
29.3 26.0 27.3 31.7
5.0 4 x 5.0 2 x ¢ 5.02x 5.0 1 x
- 33.8a - 35.8a -34.9a -34.1a
- 35.6 - 37.4 --36.7 -36.1
to to to to
32.0 34.5 33.3 32.2
Means followed by different letters within a pH treatment are significantlydifferent. (95% C.I. do not overlap.)
secutive days (2 x c w e e k - l ) and the once weekly application (1 x week-l). No frequency effects were detected in shoots treated with mist at p H 5.0. Treatment effects based on ion leakage, relative conductivity after 24 hr (Cr24). Cr24 was significantly higher for shoots treated with p H 2.5 than with p H 2.7 mist and c o m p a r e d with the controls at all temperatures (Table 4). Frequency effects, averaged over p H 2 . 5 5 were not significant (P > 0.05) at any of the test temperatures. Ranking the Cr24 values for different frequencies of treatment at each individual temperature showed higher relative conductivities from shoots treated more frequently (4 x w e e k - l) or with less recovery time between treatments (2 x c w e e k - 1) (data not shown). Contrast tests (SAS, G L M procedure), however, showed only three instances when shoots from the 4 x week-~ treatment were
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE
143
100 0
,--,
5
10
.
.
15 20
.
25
...-., r ~ tT"~7"/AI V A , . , ~ , ~ . . . .
.
30
35
40
Frequency
o
60 r-
0
45
50
55
60
65
70
75
10
11
12
13
14
Cr120 %
- - - - - -
0
1
2
3
4
5
6
7
8
9
15
16
17
18
19 20
Cr~ %
~ r"n ~:~ ~ ~:m 1223 ~ ~2~ °'~ 0.3 0.4 0.4 0.5 0.7 0.8 1.5 1.8 2.2 2.7 3.3 4 4.9 5.9 7.3 8.9 11 13 K %day-I
16 20 24
29
36 44
Midpoint
Fig. 1. Frequency distribution of (lower) k values, mean geometric daily leakage rates (% day-1), (middle) Cr24 , relative ion conductivity values after 24 hr, and (upper) % Crt20, ion conductivity values after 120 hr (%) in relation to autoclaved values from different acid mist/frequency appfication treatments. Open boxes [] represent unfrozen, live shoots and slashed boxes [] represent dead shoots frozen at -49°C.
significantly more damaged than shoots from the once weekly treatment. Relative conductivity Cr24 values following freezing at - 4 9 ° C ranged from 5 to 20% while values for unfrozen shoots ranged from 3 to 11% (Fig. 1, middle). The absence of a clearly defined bimodal distribution, and the large overlap between the two temperature extremes make the choice of a 'critical' relative conductivity rather arbitrary. For comparison with the analyses based on leakage rates, a value of 12% was chosen to separate dead from live shoots, it being the largest value above which there were no live shoots in the unfrozen population. The resultant mean LTs0s (Fig. 2) were significantly affected by mist acidity but not frequency. Differences between the p H treatments were 19°C between the p H 2.5 and 5.0, and 7.7°C between the p H 2.5 and 2.7 treatments (Table 5).
Table 4. Relative conductivities (Cr24) of solutions measured after 24 hr in relation to the ion conductivi~ (Cr~4/C~u~o)of Norway spruce shoots treated with mist at pH 5.0, 2.7 and 2.5 at a range offieezing temperatures
Mean relative conductivities Cr24 Temperature (°C) pH5.0 --49 -44 -37 - 32 -27 2
0.12a 0.1 la 0.09a 0.08a 0.06a 0.04a
pH 2.7
Probabilities (treatment) one-way pH 2.5 ANOVA
0.16b 0.18b 0.14b
--0.17c
0.03 0.0002 0.0001
0.1 l b
0.14c
0.0001
0.09b 0.07b
0.11c 0.08c
0.0001 0.0001
Means followed by the same letter in a row are not significandy different.
L.J. SHEPPARD et al.
144
Table 5. The temperature (°C) required to kill 50% (LTso) of Norway spruce shoots treated with mist at different pHs and frequencies, on 26 November. LTsos determined from critical log k, Cr24 and Crl2o values LT5o pH frequency
k
mean
Cr24
mean
pH 2.5 -22.2 -26.6
Crl2o
4x 2x c
-23.7a -26.9
2x 1x
--26.3 - 27.7b
4x 2x c
- 30.8a - 29.2a
2x 1x
- 33.3b - 32.7b
4x 2xc
- 33.8 - 35.8
2x 1 x
- 34.9 -34.1
- 47.3 -43.5
- 38.6 -38.6
Overall mean
-- 30.7
-- 35.8
-- 35.0
-26.1
--27.1a --31.4 -26.7
-28.2 - 29.7 pH 2.7 - 32.0 -- 36.2 -31.5
-30.7 -30.8 - 33.3b - 34.3a -- 33.2a
--34.5 -31.9 - 38.0 pH 5 - 49.7 - 44.0
-34.6
mean
-35.1 -38.1b - 35.0ab - 37.6 -- 41.9
--46.1
-39.2
Means followed by different letters in the same column within a pH treatment are significantly different (P < 0.05) (95% C.I. did not overlap).
Treatment effects based on relative conductivi~ ajqer 120 hr (Crl2o). A significant effect of mist acidity was found at all but the coldest temperatures (Table 6). As with leakage rate (k) and Cr24 , frequency effects were present at each temperature with the greatest values for Crl2 0 from the 4 x and 2 x c treatments (data not shown). Contrast analysis indicated significant differences (P < 0.04) between 4 x and 1 x week -~ treatments at p H 2.5 for the less severe (cold) test temperatures. In the p H 2.7 treatment, twice weekly applications on consecutive days (2 x c week -~) were significantly more damaging than twice weekly applications (2 x week-1) with recovery time in between. The establishment of a critical Cr120 value from the frequency distribution was less subjective than for Cr24. The population of Crl20 values from unfrozen shoots and shoots frozen at - - 4 9 ° C did not overlap, and a value midway between the populations of 25% was chosen as the critical Crl20 value
for shoot death (Fig. 1, upper). The resulting LTs0s, based on this critical value had relatively small confidence intervals about the predicted values. The mean LTs0 for the p H 2.5 treatment was 8.3°C warmer than that for the m e a n of the p H 5 treatment, and 4.2°C warmer than the mean for the p H 2.7 treatments. For the p H 2.5 treatments, misting four times a week significantly (P < 0.05) reduced the LTs0 compared with once a week; for p H 2.7, the twice weekly treatment on consecutive days proved as detrimental as 4 x week-~ treatment. The twice weekly treatment, allowing time for recovery, increased the frost resistance (Table 5). Comparison ofLTsos derived ~om leakage rate k, Cr24 andCrl2o. M e a n LTso values based on kwere always warmer than those calculated from Cr24 or Crl2o (Table 5). M e a n values derived from k and Crl20 estimates separated the acid effects by similar amounts (Table 5). The Cr~2o results provided the most sensitive test for effects of frequency of acid
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE
145
--40
~,
-3s
ffJ
/Jl I1/ Ill Ill
~-3~ E,..1
III
~
ri"~
/iZ fii
-25t~IL~~ II ~//~/~/#'#~/IJ "/li.1l/i."1t .7, i.lII1#11
-20
4x 2xc 2x
I
Ix
4x 2xc 2x
Ix
/-/-/-
4x 2xc 2x
Ix
Treatment frequency
Fig. 2. Effect of the pH/ion concentration of acid mist and frequency of exposure on the frost hardiness (LTs0°C) of Norway spruce seedlings. Legend [] pH 2.5; [] pH 2.7, [] pH 5.
Table 6. Relative conductivities (Crl20) of solutions containing assessment of acid mist effects on frost hardiness is Norway spruce shoots treated with mist at pH 5.0, 2.7 and 2.5 justified despite the large investment in labour costs. at a range offreezing temperatures The data highlight the huge influence of the time Mean relative conductivities (Crl20) Temperature (°C) pH 5.0 -49 -44 -37 -32 -27 2
0.54a 0.43a 0.24a 0.15a 0.09a 0.06a
pH 2.7 0.60a 0.55b 0.37b 0.21a 0.14b 0.08b
Probability from one-way pH 2.5 ANOVA --0.44b 0.31b 0.20c 0.10c
0.12 0.05 0.0006 0.0002 0.0002 0.0001
Means followed by the same letter in a row are not significantly different P > 0.05. mist exposure on frost hardiness (LTs0). LT50 values based on 24 hr relative conductivity values were the most variable having the least precision. Where significant effects of frequency were observed, both leakage rates and Crl20 values gave similar results. DISCUSSION
The data presented here support the working hypothesis that the use of four measurements in the
interval between immersion, post-freezing, and making the single conductivity measurement. However, as will become clear in the ensuing discussion, because our understanding of the ion leakage process is incomplete, much of our hypothesis is still based on untested assumptions some of which may yet prove to be misconceived. O u r ignorance of the source of ions leaking into the bathing solution, whether they are of apoplastic or symplastic origin, may prove critical in the choice of a protocol for testing the effects of acid mist on frost hardiness.
Does the method of analysis influence the result/interpretation of acid mist effects onfrost hardiness? A single reading, when taken within 24 hr may not provide sufficient time for all ions to leak into the bathing solution from damaged cells. (4) Cr24 may thus underestimate freezing damage, unlike a reading taken after 120 hr, Crl20./4) In this study, all three estimates of damage, relative conductivity after 24 hr (Cr24), after 120 hr (Cri20) and leakage rate (k) indicated a significant effect of mist p H on ion leakage post-freezing. The three methods all ranked the treatments with respect to increasing ion leakage
146
L.J. SHEPPARD et aL
p H 2.5 > p H 2.7 > p H 5. Cr24 values indicated significant differences between all p H treatments at all test temperatures but for Cr120 and k values some combinations of p H and test temperatures, not necessarily the same ones, were not significantly different. A large effect of treatment on ion leakage was necessary to detect frequency effects which were only observed at the lowest pH. A high frequency of treatment (4 x week-1) or twice weekly treatments on consecutive days (2 x c week -1) caused the greatest increases in ion leakage for a given pH. Comparisons of frequency effects for a given p H identified differences in apparent sensitivity between the three methods. Six significant temperature contrasts were identified at pH 2.5 and 1 at p H 2.7 based on k values, 1 at p H 2.5 and 2 at p H 2.7 with Cr24, and 5 at p H 2.5 and 3 at p H 2.7 using Crl20 values. Leakage rates post-freezing showed that, at a few temperatures, p H 2.5 mist increased ion leakage most when it was applied 4 x week -l, significantly > 2 x or 1 x week -1. Relative conductivity after 24 hr showed that the 4 x week- ~cause significantly more ion leakage than 1 x week- 1but was unable to discriminate between the twice weekly application. Interestingly, while similar contrasts were shown by Crl20 and k for the same temperatures, Cr120 also identified differences between the twice weekly treatments (2 x c and 2 x week -I) at p H 2.7. To summarize, Cr24 is quick, but the potential for misrepresentation of the effects of acid mist is high due to (1) interference from surface ions and (2) incomplete ion leakage. It is possible that agitation as used by Jacobson e t a / . (13) may minimize the effects of (2). Our analysis has also revealed that Cr24 is the least sensitive for spruce of the methods used although it did show the greatest number of significant temperature-treatment combinations. This greater apparent power of Cr24 probably reflects the interaction with surface residues, 59% of the C24 value reflects surface residue/rapidly desorbed ions compared to 47% in the C 120reading with respect to calculating k. There were no real differences in sensitivity between results for relative conductivity after 120 hr and those for ion leakage rate, but since the workload was halved, Crl20 appears to offer a promising alternative to the rate method. Whilst other workers have experienced microbial contamination after 5 days, no such prob-
lems were observed here where specimens were stored in the refrigerator to restrict microbial activity. Ion leakage - - what does it tell us?
Ion leakage from unfrozen shoots provides information about treatment effects on membrane integrity. All three methods indicated significant effects of p H on ion leakage ( P < 0.001) but whereas k values suggested treatment with pH 2.7 enhanced membrane integrity (reduced ion leakage), the wash-off bias inherent in the conductivity methods inferred increased ion leakage with increasing acidity. Reductions in ion leakage have previously been observed in N deficient plants in response to the additional N supplied in the acidic mist. (22) The large number of treatment-temperature combinations makes it almost impossible to establish treatment effects and compare the three methods. Clearly, there is a requirement for reducing the freezing temperature data into a single value for each treatment. We have calculated a lethal temperature at which a given proportion of shoots is killed (LTs0 = 50% kill). Shoots are first categorized as 'alive' or 'dead', which means establishing a means of distinguishing dead and live shoots from measured leakage rates or relative conductivities. In other studies utilizing relative conductivities, an arbitrary percentage value has been used, often > 20 or > 50%. (~°'l1)In this experiment, the maximum relative conductivity Cr24 measured was 20% in contrast to 80% for Cr120 thus showing how critical the choice of end time is for spruce shoots. In the initial development of the rate conductivity method, (16~the rate of ion leakage from untreated red spruce shoots was found to be linear over 120 hr but after that the curve flattened off. Similar evaluations need to be conducted to determine the optimum time for measuring Cr. Deans et al. (41 found that oak stems required at least 168 hr to maximize ion leakage post-freezing. These workers also observed that for oak stem tissue the ion concentration, post-autoclaving for short periods, even at 121°C and high pressure, was influenced by the freezing temperature. Where membranes suffered little freezing perturbation, a longer time interval between autoclaving and ion conductivity measurement was required to ensure that the measurement represented the total ion balance. Observations by Sutinen et al. (23~ suggest that
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE the hardening status of the shoots also influences the length of time required to maximize the concentration of leaked ions in the bathing solution. Whilst the relative conductivity method may save on the number of measurements required, the time elapsing between freezing and estimating Cr must first be assessed to take into account species and hardiness effects. The time saved over the rate method is unlikely to exceed that used in determining the optimum leakage times, over the winter. (4/ The determination of a critical Cr by a more objective method such as the one described for leakage rates (2/was not satisfactory for Cr24 because of the significant overlap of values for unfrozen tissue and that frozen at - 4 9 ° C , which reflected the over-riding influence of wash-off. The choice of 12% as the critical value over-estimated shoot hardiness by implying 30% of the shoots frozen to - 4 9 ° C were alive. Visual assessments of shoots frozen at this temperature (data not shown) indicated all shoots had been killed (shoots turned brown) at - 49°C. In contrast, separation was good for Cr120 so that a critical value could be chosen with confidence. The degree of separation probably reflected (i) that all potential ions had had time to move into the bathing solution and (ii) that effects of wash-off residues were comparatively small. However, if we compare estimates of LTs0 based on leakage rate with those based on relative conductivity after 120 hr, agreement is poor. Much better agreement was obtained between treatment means for Cry20 and Cr24 , despite the latter being based on such an ill-defined choice of critical Cr. The discrepancies between LTsos calculated by the three methods arise from the choice of a threshold value. Perkins et a/. (~7/ also identified this stage as the weak link and these observations support their conclusion. DeHayes et a/. (6) do not make comparisons of frost hardiness based on LT50 but prefer to identify a critical freezing temperature, from their range of test temperatures, which causes a significant increase in relative conductivity. In this experiment, each 4°C fall in temperature resulted in a significant increase in k, Cr2~ and Cry20 values, invalidating this method. For the purpose of making comparisons between acid mist treatments within a particular study, the absolute value of the LT~0 in °C is of secondary importance to identifying treatment differences and
147
rankings. However, when extrapolating to the field or between studies, the absolute value is of paramount importance and so the assessment of LT50 must be based on a protocol that can deliver on both accounts. Close similarities between rankings for frequency at pH 2.5 based on k and Cr120 w e r e observed. Both methods showed that increasing the frequency of mist application exacerbates the detrimental effect of acid mist on the ability to withstand low temperature-induced damage to the plasmalemma. Likewise, both methods detected significant differences between treatment 1 x week -1 vs 4 x week -1 at p H 2.7 not surpringly no significant effects of frequency on LTs0 were detected by either method at pH 5, but both methods ranked the frequency means the same. On the basis of the LT50 rankings and the similarities in sensitivity rankings for the frequency effects at the various test temperatures, we recommend both the leakage rate and relative conductivity (Crl20) as suitable methods of evaluating acid mist effects on frost hardiness. Relative conductivity has the advantage of 100% fewer measurements but may require a large time investment on development since it is strongly influenced by hardening status. (24i By contrast, the rate method requires more measurements but these make it more robust and less dependent on wash off and hardening status. One final cause for concern which must be levelled at all these methods which utilize ion leakage is the observation that ion leakage is itself affected by acid mist, cfunfrozen tissue. However, the effect is so small by comparison with the effects of freezing that it is unlikely to exert a strong influence on our interpretation of the results./23/No correlation has been found between the leakage rate from unfrozen shoots and the LTs0 (L. J. Sheppard, unpubl.). Recommendations
Based on observations from this experiment with acid mist treated spruce shoots and a study undertaken by Deans et a/. (4) utilizing leafless stem sections, we recommend the ion leakage rate method as the most robust and sensitive for distinguishing phenoand genotypic differences in frost hardiness. In the absence of preliminary tests to determine the appropriate time at which to make the single measurement for determination of relative conductivity its usefulness is questionable./4/The apparent time sav-
148
L.J. SHEPPARD et al.
ing in making only two compared with four measurements is thus rarely realized and often at the expense of'accuracy'. Unfortunately, in the case of freezing studies, there are no short cuts, and estimations of frost hardiness are only as good as the data, which should include a comprehensive range of test temperatures with intervals of 2-4°C maximum. Confidence in the selection of threshold leakage rates can be greatly improved by including visual assessments of damage to a small sample of shoots covering the range of test temperatures.
REFERENCES 1. Cannell M. G. R. and Sheppard L.J. (1982) Seasonal changes in the frost hardiness of provenances of Picea sitchensis in Scotland. Forestry55, 137-153. 2. Cape J. N., Fowler D., Eamus D., Murray M. B., Sheppard L.J. and Leith I. D. (1989) Effects of acid mist and ozone on frost hardiness of Norway spruce seedlings. Pages 331-335 in H. D. Payer, P. Firrman and P. Mathy, eds. Environmental research with plants in closed chambers. Proc. Int. Symp. Munchen 1989. Report 26 - - Air Pollution Report Series CEC. 3. Cape J. N., Leith I. D., Fowler D., Murray M. B., Sheppard L. J., Eamus D. and Wilson, R. H. F. (1991) Sulphate and ammonium in mist impair the frost hardening of red spruce seedlings. New Phytol. 118, 119-126. 4. DeansJ. D., Billington H. F. and Harvey F. (1994) The assessment of frost damage to leafless stem tissues of Quercuspetraea: a reappraisal of the method of relative conductivity. Forestry (in press). 5. DeHayes D. H. (1992) Winter injury and developmental cold tolerance of Red spruce. Pages 295338 in C. Eagar and M.B. Adams, eds. The ecologyand declineof red spruce in the Eastern United States. SpringerVerlag, New York. 6. DeHayes D. H. and Williams M. W. (1989) Critical temperature: A quantitative method of assessing cold tolerance. Gen. Tech. Rep. NE 134, USDA, 6 pp. 7. Eagar C. and Adams M. B. (1992) Ecology anddecline of red sprucein the Eastern United States. Springer-Verlag, New York. 8. Fowler D., Cape J. N., Deans J. D., Leith I. D., Murray M. B., Smith R. I., Sheppard L. J. and Unsworth M. H. (1989) Effects of acid mist on the frost hardiness of red spruce seedlings. New Phytol. 113, 321-335. 9. Genstat (1990) Genstat 5, Release 2. Reference manual. Oxford Science Publications. 10. Green L. M. and Warrington I.J. (1978) Assessment of frost damage in radiata pine seedlings using the
different electro-conductivity technique. N.z. 07. For. Sd. 8~ 344-350. 11. HaUam P. M. and Tibbits W. N. (1988) Determination of the frost hardiness of Eucalyptus using the electrical conductivity of the diffusate in conjunction with a freezing chamber. Can. jT. For. Res. 18, 595600. 12. JacobsonJ. S., Bethard T., Heller L. I. and Lassoie J. P. (1990) Response of Picearubensseedlings to intermittent mist varying in acidity, and in concentrations of sulphur and nitrogen-containing pollutants. Physiol. Pl. 78, 595-601. 13. JacobsonJ. S., Heller L. I., L'Hirondelle S.J. and LassoieJ. P. (1992) Phenology and cold tolerance of Picea rubens Sarg. seedling exposed to sulphuric and nitric acid mist. Scand.J. For. Res. 7, 331-344. 14. Leith I. D., CapeJ. N., Murray M. B., Sheppard L. J., DeansJ. D. and Fowler D. (1989) Frost hardiness and visible injury of spruce seedlings subjected to simulated acid mist. Pages 175-180 inJ. B. Bucher and I. Bucher-Wallen, eds. Air pollution and forest decline, IUFRO Switzerland. 15. LevittJ. (1980) Responses of plants to environmental stresses. Vol. 1, 2nd Edition, Chillin~freezing and high temperaturestresses. Academic Press, London, 471 pp. 16. Murray M. B., Cape J. N. and Fowler D. (1989) Quantification of frost damage in plant tissue by rates of electrolyte leakage. New Phytol. 113, 307311. 17. Perkins T., Adams G. T., Lawson S. and Hemmerlein M. T. (1993) Cold tolerance and water content of current year red spruce foliage over two winter seasons. Tree Physiol. 13, 119-130. 18. SAS (1985) SAS User's Guide."Statistics, Version 5. SAS Institute Inc., Cary, NC, U.S.A. 19. Schulze E. D. and Freer-Smith P. H. (1991) An evaluation of forest decline based on field observations focussed on Norway spruce, Picea abies. Proc. Roy. Soc. Edinburgh, 97B, 155-168. 20. Sheppard L.J., Cape J. N. and Leith I. D. (1992) Effects of acid mist on visible injury, nutrients and frost hardiness in Sitka, Norway and red spruce. Pages 115-119 in M. Tesche and S. Feiler, eds. Air pollution and interactions between organisms in forest ecoo,stems. Proc. 15th Int. IUFRO meeting, Dresden, Germany. 21. Sheppard L.J., Cape J. N. and Leith I. D. (1993) Acid mist affects dehardening, budburst and shoot growth in red spruce? For. ScL 39, 680-691. 22. Sheppard L.J., Cape J. N. and Leith I. D. (1993) Influence of acidic mist on frost hardiness and nutrient concentrations in red spruce seedlings Part II. Effects of misting frequency and dose. New PhytoL 124) 607-615. 23. Sheppard L.J., Leith I. D. and CapeJ. N. (1994)
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE Effects of acid mist on mature grafts of Sitka spruce. Part 1. Frost hardiness and foliar nutrient concentrations. Env. PoUut. 85, 229-238. 24. Sutinen M. L., Palta J. P. and Reich P. B. (1992) Seasonal differences in freezing stress resistance of needles of Pinus n~ra and Pinus resinosa: evaluation of the electrolyte leakage method. Tree Physiol. 11, 241254. 25. Van den Driessche R. (1976) Prediction of cold hardiness of Douglas fir seedlings by index of injury and conductivity methods. Can. 7. For. Res. 6, 511515.
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26. Wttlff A., Sheppard L. J. and Leith I. D. (1994) Evaluation of electrolyte leakage, chlorophyll fluorescence and ultrastructural techniques for detecting effects of acid mist on the frost hardiness of Sitka spruce shoots. Env. Exp. Bot. 35, 261-273. 27. Zhang M. I. N. and Willison J. H. M. (1987) An improved conductivity method for the measurement of frost hardiness. Can. 07. Bot. 65, 710-715. 28. Zhang M. I. N., Stout D. G. and WillisonJ. H. M. (1992) Plant tissue impedance and cold acclimation: A reanalysis. 07. Exp. Bot. 43, 263-266.
Pergamon 0098-8472(94) 00041-7
FROST HARDINESS OF N O R W A Y SPRUCE TREATED WITH ACID MIST. EVALUATION OF THE ELECTROLYTE LEAKAGE RATE
TECHNIQUE LUCYJ. SHEPPARD, INGRID FRANSSEN andJ. NEIL CAPE Institute of Terrestrial Ecology, Bush Estate, Penicuik, Midiothian EH26 0QB, U.K.
(Received6June 1994; acceptedin revisedform 8 August 1994) Sheppard L.J., Franssen I. and Cape J. N. FrosthardinessofNorway sprucetreatedwith acidmist. Evaluation of the electrolye leakagerate technique. Environmental and Experimental Botany 35, 139-149, 1995.Solutions of acid mist at pH 2.5, 2.7 and 5.0 were applied at four misting frequencies to 2-year-old Norway spruce growing in pots in a glasshouse for 19 weeks, from July to November 1991. Frost hardiness assessments were made on detached shoots. Three methods for analysing ion leakage were compared: conductivity after 24 and 120 hr, and the rate of leakage derived from measurements at 1, 24 and 120 hr relative to the ion conductivity after autoclaving shoot segments in deionized water. The different methods of estimating the effects of acid mist on frost hardiness gave similar results, but the use of a single measurement after 24 hr was confounded by the inclusion of ions washed from the leaf surface. This bias was smaller for the single measurement after 120 hr, but only the electrolyte leakage rate determination could separate and restrict this potential source of bias. Estimates of leakage rate, which accommodates differences in hardening status and surfaceadsorbed ions, were found to be the most robust, sensitive method for determining acid mist effects on frost hardiness. Measurements of relative conductivity based on 100% fewer measurements are unlikely to save time since they will require exploratory studies throughout the hardening period to establish the optimum times for making the single, critical measurement.
K~ words: Norway spruce, acid mist, frequency, frost hardiness, ion leakage rate, relative conductivity.
INTRODUCTION
Norway spruce seedlings were exposed to simulated acid mist containing H +, N O s - , NH4 + and $ 0 4 2 A causal relationship between the deposition of a t three ionic concentrations and four frequencies of anthropogenic pollutants and reduced winter hardi- weekly exposure. Previous experiments 112'2°'22/had ness has been established for red spruce (Picea rubens also suggested that sensitivity could be further Sarg.). (7/In Europe, where Norway spruce [P. abies enhanced by increasing the exposure frequency for (L.) Kant] is the predominant species, forest decline a given dose, and this too was examined. has also been linked to increased frost sensitivity, The main objective of this study was concerned thought to reflect enhanced N inputs. (19)However, with assessing the relative merits of the ion leakage rigorous experiments examining the relationship rate technique/16) for evaluating the effects of acid between frost sensitivity, acid mist and frost hardi- mist on frost hardiness against ion leakage results ness of Norway spruce are lacking. This paper based on fewer measurements. O u r working reports a glasshouse experiment in which pot-grown hypothesis was that the taking of four measurements 139
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L.J. SHEPPARD et al.
for the leakage rate method are justified and necessary, when examining the effects of acid mist, to overcome the bias introduced by accumulation and desorption.
Techniques current~ in use to detectfieezing injury Frost hardiness of plant tissue can be assessed under controlled conditions by subjecting the tissue to a range of sub-zero temperatures and a protocol for such tests is well established. <15/Likewise, many protocols for estimating resultant injury such as fluorescence diacetate, (27) impedance measurements, (28) visible injury(~'21/ chlorophyll fluorescence<26>and electrolyte leakage (2'6'14'16'27>are in current use. Unfortunately, the suitability of particular protocols for specific jobs is still the subject of intense debate, and many of the caveats are not widely recognized. Visible injury is probably most indicative of potential field damage, especially where whole seedlings are used. However, its development is slow (L. J. Sheppard, unpubl.) and the results rely on subjective scoring which is particularly inexact during midwinter. In assessing pollutant effects on frost hardiness, the ion leakage technique has been widely adopted, <3'5'8> but groups have utilized different measurement protocols. The timing of the measurements, e.g. 24 or 120 hr after freezing, is influenced by species, tissue type, season and tissue hardiness in relation to test temperatures. (4'24'27)Likewise, condltions of sample storage, temperature, the inclusion of wetting agents and the use of agitation will also influence the choice of timing (J. S. Jacobson, pers. comm.). Moreover, it is not possible to relate rates of ion leakage to biologically meaningful reference points for sample comparison, <17) since tissue death is difficult to determine, and the concept of 'death' may differ depending on the objective. Some groups have adopted the 'critical temperature' (Tc) criterion, corresponding to the point of earliest detectable freezing injury./61 The ability to meaningfully identify a threshold of ion leakage for death is dependent on the choice of test temperatures; ideally, the test intervals should be 2 or 3°C but, more often because of practical restrictions, intervals of up to 5°C are used. A single measurement of conductivity at a fixed time after immersion has often correlated poorly with visible damage. (25)Zhang and W i l l i s o n (27) found that the conductivity method systematically under-
estimated frost hardiness based on visible injury. This poor correlation appeared to result from the reading being taken too early, providing insufficient time for leakage to o c c u r . (24'27) Also, in some instances, freezing damage to membranes may not be permanent and can be repaired or compensated for, so that readings taken too soon after freezing, e.g. 24 hr, may overestimate the degree of damage. (l~/Temperature, agitation, surface area of material and other environmental conditions are all likely to influence leakage rate, thus necessitating preliminary tests to identify the most suitable conditions for the particular tissue under study? l> Where shoots have been treated with acid mist, a single measurement may be misleading because dissolution of ions deposited on leaf surfaces may contribute a large proportion of the total ions measured. The rate equation/161 which requires four readings, was developed to accommodate the effects of surface deposits and results correlate well with visible damage scoresJ 2~)In the ensuing experiment we evaluate the potential merits of relative conductivity measured at 24 hr (cfJ. S.Jacobson, pers. comm.) and 120 hr/4) against the rate of leakage. Measuring relative conductivity, as opposed to rate, reduces the number of measurements from four to two and may potentially reduce the time lapse between freezing and the end result. METHODS
Plant material and misting treatments One hundred and twenty 2-year-old Norway spruce seedlings, raised from seed in a glasshouse in their second flush of shoot growth, were arranged in 24 trays in July 1991. The plants were growing in 1.25 1 pots containing fertilized compost (peat, grit and loam, 40:40:20) + Vitax Q4 fertilizer (3.5% N, 7.5% P, 10% K). An additional 45 mg Vitax Q4 was added to the surface of each pot in July. Plants were watered with tap water (pH 8). The water was poured into trays fined with capillary matting on which the potted plants stood. Twelve trays of plants were randomly assigned to two replicate blocks per treatment. Spraying commenced on 11 July and continued for 19 weeks. The volume of spray applied to each replicate tray provided the rainfall equivalent of 4 mm week- 1. Four frequency treatments were included once a week (4 mm x week-1), twice a week (2 x 2 mm week-l), either
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE
141
Table 1. Concentrations (mM) of H +, NO3-, NH4+ and SO42- in the acid mist solutions and total deposition equivalent of N, S and H over 19 weeks pH
H÷
NO3-
NH4 +
8042-
mM 2.5 2.7 5.0
3.2 2.0 0.01
1.6 1.0 0.005
N
S
Deposition kg ha1.6 1.0 0.005
on consecutive days (2 x c) or with a 3-day interval (2 x ), and four times a week (4 x 1 m m week-'). Mist treatments made up at p H 2.5, 2.7 and 5.0 from NH4NO3 and H2SO4 (Table 1) were delivered for 5 min by hand using a pump action sprayer which provided droplets up to 10 ttm. No supplementary heat or light was supplied. Frost hardiness Twelve current year lateral shoots were removed on 26 November from each tree and stored in black polythene bags at 2°C. Each day, 2 shoots tree-1 were subjected to a simulated night, air frost at different test temperatures in a programmable freezing cabinet. (3'81Temperatures were lowered at 5°C hr-1, the test temperature was held for 3 hr then temperatures were raised at 10°C hr-1 to 2°C. Due to the expected treatment effects on frost hardiness, shoots were exposed to two temperature ranges: - 13, - 17, - 22, - 27, - 32 and - 37°C for p H 2.5; and - 27, - 32, - 37, - 44 and - 49°C for p H 2.7 and p H 5. Shoots were not pre-immersed to remove surface salts. A 1 cm section was excised from the middle portion of each frozen shoot and placed in a 20 ml vial. Fifteen ml of deionized water were added to each vial and the conductivity of the bathing solution was measured using a platinum electrode with built-in temperature correction, at 1 hr and 120 hr, autoclaved at 1050C for 5 min and measured 24 hr later./3/Ion leakage rates were calculated for each frozen shoot by fitting the data to the equation C, = Co+(C .... -Co)(1-e-~a). 06) Relative conductivities were also calculated by taking the value for 24 or 120 hr and dividing by the autoclaved value which represents the sum total of leakable ions. After log-transformation, the electrolyte leakage rate (k) and relative conductivities were analysed
1.6 1.0 0.005
36 22.5 0.005
39 24.3 0.1
H l
25 15 --
using two-way A N O V A . (18/ Separate analyses were performed at each test temperature to test for effects o f p H and frequency on leakage rates. The analyses were performed with blocks as the unit of replication and (pH * frequency * block) as the error term. Contrast analyses were constructed to test the effect of frequency on leakage rates (k) and relative conductivities at all temperatures and p H combinations. Temperatures equivalent to 50% shoot death were calculated by Probit Analysis/18~ from the numbers of dead shoots per treatment at each test temperature. Shoots were designated dead or alive depending on whether their leakage rate exceeded a 'critical' rate. This critical leakage rate was determined as being around the separation point between the population of leakage rates from unfrozen shoots and those frozen at the coldest test temperature - 4 9 ° C . The population of leakage rates is composed from shoots of all treatments. RESULTS
Frost hardiness Salt accumulation on the needle suoCace. The ratio of ion conductivity values after 1 hr in deionized water to the autoclaved values were 0.05, 0.04 and 0.03 for p H 2.5, 2.7 and 5.0, respectively. Ion accumulation (data not shown) was significantly greater (9) (P < 0.001) for the p H 2.5 treated needles which received the highest ion concentrations. Treatment with p H 2.7 also significantly enhanced surface ion accumulation. There was no significant (P > 0.05) frequency effect on surface ion accumulation. Effects of treatment on log transformed leakage rates (k) and the lethal temperature causing 50% shoot death (LTs0). Treatment with p H 2.5 mist significandy increased leakage rates from unfrozen control shoots (pH 5) but p H 2.7 mist significantly reduced leakage from
142
L.J. SHEPPARD et aL
Table 2. Geometric dai~ rates of ion leakage ~ day-')3%rn Norway spruce shoots treated with mist at pH 5.0, 2.7 and 2.5, at a range offreezing temperatures. Results represent the leakage rates averagedover thefour fiequency treatments. (The overalleffect of)qequenqy was not sign~qcant P > 0.05.) Means in the same rowfollowed by the same letter are not sign}qcant~ different
Table 3. Temperatures (°C) required to kill 50% (LTs0) of Norway spruce shoots treated with acid mist at differentpHs and with different application J~equencies, on 26 November 1991. LTs0s determinedfrom a critical ion leakage rate of 2.7% day -I
pH k values (% day- 1) Temperature (°C) pH 5.0 pH 2.7 pH 2.5 -49 -44 - 37 -32 -27 2
14.6a 9.7a 4.0a 1.9a 1.1a 0.6a
17.9a 14.5a 7.2b 3.1b 1.7b 0.3b
NA NA 9.6b 5.4c 2.7c 1.0c
P value treatment effect 0.18 0.08 0.0007 0.0001 0.003 0.0001
* NA = not available.
unfrozen shoots (Table 2). Frequency effects measured over all three treatment p H s were significant (P < 0.05) only at - 1 7 ° C but patterns were consistent across the test temperatures, so that the more often spraying occurred, the greater the ion leakage rate post-freezing stress. D a m a g e and high leakage rates followed the order 4 x > 2 x c > 2 x > 1 x . Increasing spraying frequency from once or twice a week to 4 x w e e k - 1 caused a significant increase in ion leakage from shoots treated with p H 2.5 mist at - 17, - 2 2 and - 2 7 ° C . T h e frequency distribution of ion leakage rates from all treatments and test temperatures (Fig. 1, lower) was used to determine a critical leakage rate. Values for the unfrozen shoots and those frozen at - 4 9 ° C fall into a bimodal distribution; unfrozen shoots had values log k < - 6 . 8 while shoots exposed to - 4 9 ° C had values k > - 6 . 6 . A value of loge k corresponding to a leakage rate of 2.7% day-~ was chosen as the leakage rate which best discriminated between 'live' and 'dead' shoots. There was a significant effect of p H but not frequency on the LTs0 (Table 3). T h e LT50 for the p H 2.5 mist treatment was 8.5°C warmer than that for p H 5.0. Shoots treated with p H 2.5 mist 4 x week-1 were significantly less hardy (P = 0.05) by 4°C than those receiving the same dose once a week. W h e r e the ion concentration/dose was reduced by 33% (pH = 2.7), the only significant differences in LTs0 relating to frequency were between the twice weekly application on con-
LTs0
2.5
5
ANOVA P value
-34.6
<0.05
2.7
--26.1a - 3 1 . 5 b
Frequency
4x
2Xc
2x
lx
LTs0
29.4a
pH* frequency
LTs0
95% confidence limits - 2 5 . 7 to 22.3 --32.0 to 22.7 No confidence limits - 2 9 . 9 to 25.7
30.6a 31.5a
31.5a >0.05
2.5 2.5 2.5 2.5
4x 2×c 2x 1x
-23.7a --26.9a -- 26.3a -27.7b
2.7 2.7 2.7 2.7
4x 2x c 2x lx
-30.8a -29.2a -33.3a - 32.7b
-32.2 -31.2 -37.8 -34.4
to to to to
29.3 26.0 27.3 31.7
5.0 4 x 5.0 2 x ¢ 5.02x 5.0 1 x
- 33.8a - 35.8a -34.9a -34.1a
- 35.6 - 37.4 --36.7 -36.1
to to to to
32.0 34.5 33.3 32.2
Means followed by different letters within a pH treatment are significantlydifferent. (95% C.I. do not overlap.)
secutive days (2 x c w e e k - l ) and the once weekly application (1 x week-l). No frequency effects were detected in shoots treated with mist at p H 5.0. Treatment effects based on ion leakage, relative conductivity after 24 hr (Cr24). Cr24 was significantly higher for shoots treated with p H 2.5 than with p H 2.7 mist and c o m p a r e d with the controls at all temperatures (Table 4). Frequency effects, averaged over p H 2 . 5 5 were not significant (P > 0.05) at any of the test temperatures. Ranking the Cr24 values for different frequencies of treatment at each individual temperature showed higher relative conductivities from shoots treated more frequently (4 x w e e k - l) or with less recovery time between treatments (2 x c w e e k - 1) (data not shown). Contrast tests (SAS, G L M procedure), however, showed only three instances when shoots from the 4 x week-~ treatment were
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE
143
100 0
,--,
5
10
.
.
15 20
.
25
...-., r ~ tT"~7"/AI V A , . , ~ , ~ . . . .
.
30
35
40
Frequency
o
60 r-
0
45
50
55
60
65
70
75
10
11
12
13
14
Cr120 %
- - - - - -
0
1
2
3
4
5
6
7
8
9
15
16
17
18
19 20
Cr~ %
~ r"n ~:~ ~ ~:m 1223 ~ ~2~ °'~ 0.3 0.4 0.4 0.5 0.7 0.8 1.5 1.8 2.2 2.7 3.3 4 4.9 5.9 7.3 8.9 11 13 K %day-I
16 20 24
29
36 44
Midpoint
Fig. 1. Frequency distribution of (lower) k values, mean geometric daily leakage rates (% day-1), (middle) Cr24 , relative ion conductivity values after 24 hr, and (upper) % Crt20, ion conductivity values after 120 hr (%) in relation to autoclaved values from different acid mist/frequency appfication treatments. Open boxes [] represent unfrozen, live shoots and slashed boxes [] represent dead shoots frozen at -49°C.
significantly more damaged than shoots from the once weekly treatment. Relative conductivity Cr24 values following freezing at - 4 9 ° C ranged from 5 to 20% while values for unfrozen shoots ranged from 3 to 11% (Fig. 1, middle). The absence of a clearly defined bimodal distribution, and the large overlap between the two temperature extremes make the choice of a 'critical' relative conductivity rather arbitrary. For comparison with the analyses based on leakage rates, a value of 12% was chosen to separate dead from live shoots, it being the largest value above which there were no live shoots in the unfrozen population. The resultant mean LTs0s (Fig. 2) were significantly affected by mist acidity but not frequency. Differences between the p H treatments were 19°C between the p H 2.5 and 5.0, and 7.7°C between the p H 2.5 and 2.7 treatments (Table 5).
Table 4. Relative conductivities (Cr24) of solutions measured after 24 hr in relation to the ion conductivi~ (Cr~4/C~u~o)of Norway spruce shoots treated with mist at pH 5.0, 2.7 and 2.5 at a range offieezing temperatures
Mean relative conductivities Cr24 Temperature (°C) pH5.0 --49 -44 -37 - 32 -27 2
0.12a 0.1 la 0.09a 0.08a 0.06a 0.04a
pH 2.7
Probabilities (treatment) one-way pH 2.5 ANOVA
0.16b 0.18b 0.14b
--0.17c
0.03 0.0002 0.0001
0.1 l b
0.14c
0.0001
0.09b 0.07b
0.11c 0.08c
0.0001 0.0001
Means followed by the same letter in a row are not significandy different.
L.J. SHEPPARD et al.
144
Table 5. The temperature (°C) required to kill 50% (LTso) of Norway spruce shoots treated with mist at different pHs and frequencies, on 26 November. LTsos determined from critical log k, Cr24 and Crl2o values LT5o pH frequency
k
mean
Cr24
mean
pH 2.5 -22.2 -26.6
Crl2o
4x 2x c
-23.7a -26.9
2x 1x
--26.3 - 27.7b
4x 2x c
- 30.8a - 29.2a
2x 1x
- 33.3b - 32.7b
4x 2xc
- 33.8 - 35.8
2x 1 x
- 34.9 -34.1
- 47.3 -43.5
- 38.6 -38.6
Overall mean
-- 30.7
-- 35.8
-- 35.0
-26.1
--27.1a --31.4 -26.7
-28.2 - 29.7 pH 2.7 - 32.0 -- 36.2 -31.5
-30.7 -30.8 - 33.3b - 34.3a -- 33.2a
--34.5 -31.9 - 38.0 pH 5 - 49.7 - 44.0
-34.6
mean
-35.1 -38.1b - 35.0ab - 37.6 -- 41.9
--46.1
-39.2
Means followed by different letters in the same column within a pH treatment are significantly different (P < 0.05) (95% C.I. did not overlap).
Treatment effects based on relative conductivi~ ajqer 120 hr (Crl2o). A significant effect of mist acidity was found at all but the coldest temperatures (Table 6). As with leakage rate (k) and Cr24 , frequency effects were present at each temperature with the greatest values for Crl2 0 from the 4 x and 2 x c treatments (data not shown). Contrast analysis indicated significant differences (P < 0.04) between 4 x and 1 x week -~ treatments at p H 2.5 for the less severe (cold) test temperatures. In the p H 2.7 treatment, twice weekly applications on consecutive days (2 x c week -~) were significantly more damaging than twice weekly applications (2 x week-1) with recovery time in between. The establishment of a critical Cr120 value from the frequency distribution was less subjective than for Cr24. The population of Crl20 values from unfrozen shoots and shoots frozen at - - 4 9 ° C did not overlap, and a value midway between the populations of 25% was chosen as the critical Crl20 value
for shoot death (Fig. 1, upper). The resulting LTs0s, based on this critical value had relatively small confidence intervals about the predicted values. The mean LTs0 for the p H 2.5 treatment was 8.3°C warmer than that for the m e a n of the p H 5 treatment, and 4.2°C warmer than the mean for the p H 2.7 treatments. For the p H 2.5 treatments, misting four times a week significantly (P < 0.05) reduced the LTs0 compared with once a week; for p H 2.7, the twice weekly treatment on consecutive days proved as detrimental as 4 x week-~ treatment. The twice weekly treatment, allowing time for recovery, increased the frost resistance (Table 5). Comparison ofLTsos derived ~om leakage rate k, Cr24 andCrl2o. M e a n LTso values based on kwere always warmer than those calculated from Cr24 or Crl2o (Table 5). M e a n values derived from k and Crl20 estimates separated the acid effects by similar amounts (Table 5). The Cr~2o results provided the most sensitive test for effects of frequency of acid
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE
145
--40
~,
-3s
ffJ
/Jl I1/ Ill Ill
~-3~ E,..1
III
~
ri"~
/iZ fii
-25t~IL~~ II ~//~/~/#'#~/IJ "/li.1l/i."1t .7, i.lII1#11
-20
4x 2xc 2x
I
Ix
4x 2xc 2x
Ix
/-/-/-
4x 2xc 2x
Ix
Treatment frequency
Fig. 2. Effect of the pH/ion concentration of acid mist and frequency of exposure on the frost hardiness (LTs0°C) of Norway spruce seedlings. Legend [] pH 2.5; [] pH 2.7, [] pH 5.
Table 6. Relative conductivities (Crl20) of solutions containing assessment of acid mist effects on frost hardiness is Norway spruce shoots treated with mist at pH 5.0, 2.7 and 2.5 justified despite the large investment in labour costs. at a range offreezing temperatures The data highlight the huge influence of the time Mean relative conductivities (Crl20) Temperature (°C) pH 5.0 -49 -44 -37 -32 -27 2
0.54a 0.43a 0.24a 0.15a 0.09a 0.06a
pH 2.7 0.60a 0.55b 0.37b 0.21a 0.14b 0.08b
Probability from one-way pH 2.5 ANOVA --0.44b 0.31b 0.20c 0.10c
0.12 0.05 0.0006 0.0002 0.0002 0.0001
Means followed by the same letter in a row are not significantly different P > 0.05. mist exposure on frost hardiness (LTs0). LT50 values based on 24 hr relative conductivity values were the most variable having the least precision. Where significant effects of frequency were observed, both leakage rates and Crl20 values gave similar results. DISCUSSION
The data presented here support the working hypothesis that the use of four measurements in the
interval between immersion, post-freezing, and making the single conductivity measurement. However, as will become clear in the ensuing discussion, because our understanding of the ion leakage process is incomplete, much of our hypothesis is still based on untested assumptions some of which may yet prove to be misconceived. O u r ignorance of the source of ions leaking into the bathing solution, whether they are of apoplastic or symplastic origin, may prove critical in the choice of a protocol for testing the effects of acid mist on frost hardiness.
Does the method of analysis influence the result/interpretation of acid mist effects onfrost hardiness? A single reading, when taken within 24 hr may not provide sufficient time for all ions to leak into the bathing solution from damaged cells. (4) Cr24 may thus underestimate freezing damage, unlike a reading taken after 120 hr, Crl20./4) In this study, all three estimates of damage, relative conductivity after 24 hr (Cr24), after 120 hr (Cri20) and leakage rate (k) indicated a significant effect of mist p H on ion leakage post-freezing. The three methods all ranked the treatments with respect to increasing ion leakage
146
L.J. SHEPPARD et aL
p H 2.5 > p H 2.7 > p H 5. Cr24 values indicated significant differences between all p H treatments at all test temperatures but for Cr120 and k values some combinations of p H and test temperatures, not necessarily the same ones, were not significantly different. A large effect of treatment on ion leakage was necessary to detect frequency effects which were only observed at the lowest pH. A high frequency of treatment (4 x week-1) or twice weekly treatments on consecutive days (2 x c week -1) caused the greatest increases in ion leakage for a given pH. Comparisons of frequency effects for a given p H identified differences in apparent sensitivity between the three methods. Six significant temperature contrasts were identified at pH 2.5 and 1 at p H 2.7 based on k values, 1 at p H 2.5 and 2 at p H 2.7 with Cr24, and 5 at p H 2.5 and 3 at p H 2.7 using Crl20 values. Leakage rates post-freezing showed that, at a few temperatures, p H 2.5 mist increased ion leakage most when it was applied 4 x week -l, significantly > 2 x or 1 x week -1. Relative conductivity after 24 hr showed that the 4 x week- ~cause significantly more ion leakage than 1 x week- 1but was unable to discriminate between the twice weekly application. Interestingly, while similar contrasts were shown by Crl20 and k for the same temperatures, Cr120 also identified differences between the twice weekly treatments (2 x c and 2 x week -I) at p H 2.7. To summarize, Cr24 is quick, but the potential for misrepresentation of the effects of acid mist is high due to (1) interference from surface ions and (2) incomplete ion leakage. It is possible that agitation as used by Jacobson e t a / . (13) may minimize the effects of (2). Our analysis has also revealed that Cr24 is the least sensitive for spruce of the methods used although it did show the greatest number of significant temperature-treatment combinations. This greater apparent power of Cr24 probably reflects the interaction with surface residues, 59% of the C24 value reflects surface residue/rapidly desorbed ions compared to 47% in the C 120reading with respect to calculating k. There were no real differences in sensitivity between results for relative conductivity after 120 hr and those for ion leakage rate, but since the workload was halved, Crl20 appears to offer a promising alternative to the rate method. Whilst other workers have experienced microbial contamination after 5 days, no such prob-
lems were observed here where specimens were stored in the refrigerator to restrict microbial activity. Ion leakage - - what does it tell us?
Ion leakage from unfrozen shoots provides information about treatment effects on membrane integrity. All three methods indicated significant effects of p H on ion leakage ( P < 0.001) but whereas k values suggested treatment with pH 2.7 enhanced membrane integrity (reduced ion leakage), the wash-off bias inherent in the conductivity methods inferred increased ion leakage with increasing acidity. Reductions in ion leakage have previously been observed in N deficient plants in response to the additional N supplied in the acidic mist. (22) The large number of treatment-temperature combinations makes it almost impossible to establish treatment effects and compare the three methods. Clearly, there is a requirement for reducing the freezing temperature data into a single value for each treatment. We have calculated a lethal temperature at which a given proportion of shoots is killed (LTs0 = 50% kill). Shoots are first categorized as 'alive' or 'dead', which means establishing a means of distinguishing dead and live shoots from measured leakage rates or relative conductivities. In other studies utilizing relative conductivities, an arbitrary percentage value has been used, often > 20 or > 50%. (~°'l1)In this experiment, the maximum relative conductivity Cr24 measured was 20% in contrast to 80% for Cr120 thus showing how critical the choice of end time is for spruce shoots. In the initial development of the rate conductivity method, (16~the rate of ion leakage from untreated red spruce shoots was found to be linear over 120 hr but after that the curve flattened off. Similar evaluations need to be conducted to determine the optimum time for measuring Cr. Deans et al. (41 found that oak stems required at least 168 hr to maximize ion leakage post-freezing. These workers also observed that for oak stem tissue the ion concentration, post-autoclaving for short periods, even at 121°C and high pressure, was influenced by the freezing temperature. Where membranes suffered little freezing perturbation, a longer time interval between autoclaving and ion conductivity measurement was required to ensure that the measurement represented the total ion balance. Observations by Sutinen et al. (23~ suggest that
EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE the hardening status of the shoots also influences the length of time required to maximize the concentration of leaked ions in the bathing solution. Whilst the relative conductivity method may save on the number of measurements required, the time elapsing between freezing and estimating Cr must first be assessed to take into account species and hardiness effects. The time saved over the rate method is unlikely to exceed that used in determining the optimum leakage times, over the winter. (4/ The determination of a critical Cr by a more objective method such as the one described for leakage rates (2/was not satisfactory for Cr24 because of the significant overlap of values for unfrozen tissue and that frozen at - 4 9 ° C , which reflected the over-riding influence of wash-off. The choice of 12% as the critical value over-estimated shoot hardiness by implying 30% of the shoots frozen to - 4 9 ° C were alive. Visual assessments of shoots frozen at this temperature (data not shown) indicated all shoots had been killed (shoots turned brown) at - 49°C. In contrast, separation was good for Cr120 so that a critical value could be chosen with confidence. The degree of separation probably reflected (i) that all potential ions had had time to move into the bathing solution and (ii) that effects of wash-off residues were comparatively small. However, if we compare estimates of LTs0 based on leakage rate with those based on relative conductivity after 120 hr, agreement is poor. Much better agreement was obtained between treatment means for Cry20 and Cr24 , despite the latter being based on such an ill-defined choice of critical Cr. The discrepancies between LTsos calculated by the three methods arise from the choice of a threshold value. Perkins et a/. (~7/ also identified this stage as the weak link and these observations support their conclusion. DeHayes et a/. (6) do not make comparisons of frost hardiness based on LT50 but prefer to identify a critical freezing temperature, from their range of test temperatures, which causes a significant increase in relative conductivity. In this experiment, each 4°C fall in temperature resulted in a significant increase in k, Cr2~ and Cry20 values, invalidating this method. For the purpose of making comparisons between acid mist treatments within a particular study, the absolute value of the LT~0 in °C is of secondary importance to identifying treatment differences and
147
rankings. However, when extrapolating to the field or between studies, the absolute value is of paramount importance and so the assessment of LT50 must be based on a protocol that can deliver on both accounts. Close similarities between rankings for frequency at pH 2.5 based on k and Cr120 w e r e observed. Both methods showed that increasing the frequency of mist application exacerbates the detrimental effect of acid mist on the ability to withstand low temperature-induced damage to the plasmalemma. Likewise, both methods detected significant differences between treatment 1 x week -1 vs 4 x week -1 at p H 2.7 not surpringly no significant effects of frequency on LTs0 were detected by either method at pH 5, but both methods ranked the frequency means the same. On the basis of the LT50 rankings and the similarities in sensitivity rankings for the frequency effects at the various test temperatures, we recommend both the leakage rate and relative conductivity (Crl20) as suitable methods of evaluating acid mist effects on frost hardiness. Relative conductivity has the advantage of 100% fewer measurements but may require a large time investment on development since it is strongly influenced by hardening status. (24i By contrast, the rate method requires more measurements but these make it more robust and less dependent on wash off and hardening status. One final cause for concern which must be levelled at all these methods which utilize ion leakage is the observation that ion leakage is itself affected by acid mist, cfunfrozen tissue. However, the effect is so small by comparison with the effects of freezing that it is unlikely to exert a strong influence on our interpretation of the results./23/No correlation has been found between the leakage rate from unfrozen shoots and the LTs0 (L. J. Sheppard, unpubl.). Recommendations
Based on observations from this experiment with acid mist treated spruce shoots and a study undertaken by Deans et a/. (4) utilizing leafless stem sections, we recommend the ion leakage rate method as the most robust and sensitive for distinguishing phenoand genotypic differences in frost hardiness. In the absence of preliminary tests to determine the appropriate time at which to make the single measurement for determination of relative conductivity its usefulness is questionable./4/The apparent time sav-
148
L.J. SHEPPARD et al.
ing in making only two compared with four measurements is thus rarely realized and often at the expense of'accuracy'. Unfortunately, in the case of freezing studies, there are no short cuts, and estimations of frost hardiness are only as good as the data, which should include a comprehensive range of test temperatures with intervals of 2-4°C maximum. Confidence in the selection of threshold leakage rates can be greatly improved by including visual assessments of damage to a small sample of shoots covering the range of test temperatures.
REFERENCES 1. Cannell M. G. R. and Sheppard L.J. (1982) Seasonal changes in the frost hardiness of provenances of Picea sitchensis in Scotland. Forestry55, 137-153. 2. Cape J. N., Fowler D., Eamus D., Murray M. B., Sheppard L.J. and Leith I. D. (1989) Effects of acid mist and ozone on frost hardiness of Norway spruce seedlings. Pages 331-335 in H. D. Payer, P. Firrman and P. Mathy, eds. Environmental research with plants in closed chambers. Proc. Int. Symp. Munchen 1989. Report 26 - - Air Pollution Report Series CEC. 3. Cape J. N., Leith I. D., Fowler D., Murray M. B., Sheppard L. J., Eamus D. and Wilson, R. H. F. (1991) Sulphate and ammonium in mist impair the frost hardening of red spruce seedlings. New Phytol. 118, 119-126. 4. DeansJ. D., Billington H. F. and Harvey F. (1994) The assessment of frost damage to leafless stem tissues of Quercuspetraea: a reappraisal of the method of relative conductivity. Forestry (in press). 5. DeHayes D. H. (1992) Winter injury and developmental cold tolerance of Red spruce. Pages 295338 in C. Eagar and M.B. Adams, eds. The ecologyand declineof red spruce in the Eastern United States. SpringerVerlag, New York. 6. DeHayes D. H. and Williams M. W. (1989) Critical temperature: A quantitative method of assessing cold tolerance. Gen. Tech. Rep. NE 134, USDA, 6 pp. 7. Eagar C. and Adams M. B. (1992) Ecology anddecline of red sprucein the Eastern United States. Springer-Verlag, New York. 8. Fowler D., Cape J. N., Deans J. D., Leith I. D., Murray M. B., Smith R. I., Sheppard L. J. and Unsworth M. H. (1989) Effects of acid mist on the frost hardiness of red spruce seedlings. New Phytol. 113, 321-335. 9. Genstat (1990) Genstat 5, Release 2. Reference manual. Oxford Science Publications. 10. Green L. M. and Warrington I.J. (1978) Assessment of frost damage in radiata pine seedlings using the
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EVALUATION OF ELECTROLYTE LEAKAGE TECHNIQUE Effects of acid mist on mature grafts of Sitka spruce. Part 1. Frost hardiness and foliar nutrient concentrations. Env. PoUut. 85, 229-238. 24. Sutinen M. L., Palta J. P. and Reich P. B. (1992) Seasonal differences in freezing stress resistance of needles of Pinus n~ra and Pinus resinosa: evaluation of the electrolyte leakage method. Tree Physiol. 11, 241254. 25. Van den Driessche R. (1976) Prediction of cold hardiness of Douglas fir seedlings by index of injury and conductivity methods. Can. 7. For. Res. 6, 511515.
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26. Wttlff A., Sheppard L. J. and Leith I. D. (1994) Evaluation of electrolyte leakage, chlorophyll fluorescence and ultrastructural techniques for detecting effects of acid mist on the frost hardiness of Sitka spruce shoots. Env. Exp. Bot. 35, 261-273. 27. Zhang M. I. N. and Willison J. H. M. (1987) An improved conductivity method for the measurement of frost hardiness. Can. 07. Bot. 65, 710-715. 28. Zhang M. I. N., Stout D. G. and WillisonJ. H. M. (1992) Plant tissue impedance and cold acclimation: A reanalysis. 07. Exp. Bot. 43, 263-266.