New Forests 13: 91–104, 1996. c 1996 Kluwer Academic Publishers. Printed in the Netherlands.
The effect of sub-zero temperatures in the light and dark on cold-hardened, dehardened and newly flushed white spruce (Picea glauca [Moench.] Voss) seedlings S.L. GILLIES1 and W.D. BINDER2
1 University College of the Fraser Valley, 33844 King Rd., Abbotsford, British Columbia, Canada; 2 British Columbia Ministry of Forests, Glyn Rd. Research Station, 1320 Glyn Road, Victoria, British Columbia, Canada V8W 3E7
Received 25 December 1994; accepted 1 December 1995
Key words: chlorophyll fluorescence (CF), cold hardiness, electrolyte leakage, freezing, frost hardiness, photodamage, photoinhibition Application. This study demonstrates that non-hardy, or newly flushed white spruce seedlings, in addition to suffering membrane damage, are subject to photoinhibition and/or photodamage if exposed to sub-zero temperatures in the presence of light. Both chlorophyll fluorescence and electrolyte leakage may be used to indicate freeze damage to white spruce needles. Abstract. Cold hardened, dehardened, and newly flushed foliage of one year old white spruce (Picea glauca [Moench.] Voss) seedlings were exposed to various sub-zero temperatures ( 2 to 22.5 C) either in the dark or light. The freezing treatment had no significant effect on the variable fluorescence to maximal fluorescence ratio (Fv /Fm ) of hardened seedlings, either in the light or dark. Also, no visible damage or increase in electrolyte leakage were evident in either the light or the dark treated seedlings. Both dehardened and newly flushed foliage were significantly affected by the freezing treatment, and light enhanced the effect. A decline in Fv /Fm increased electrolyte leakage and visible damage were observed at warmer temperatures in newly flushed needles than in dehardened needles. Seedlings exposed to subzero treatments in the light also had lower Fv /Fm , increased electrolyte leakage and showed more visible damage than seedlings exposed to the same sub-zero treatments in the dark. The temperature where 50% of the needles were damaged (LT50 ) as estimated from visible damage data was 10.8 C in the light and 12.1 C in the dark for dehardened, one year old needles. The LT50 in newly flushed needles was 4.8 C in the light and 6.2 C in the dark. Recovery of Fv /Fm values 3 days after freezing exposure was only evident in treatments where little visible damage was present. Both Fv /Fm and electrolyte leakage were strongly correlated with visible damage.
Introduction Conifer seedlings are often exposed to sub-zero temperatures, even during the growing season (Christersson et al. 1984). Before seedling hardening takes place sub-zero temperatures usually cause the free water in needles to freeze at about 4 C (Pisek 1973; Bauer et al. 1975). Such freezing can cause disruption of the plasma membrane and is considered a primary
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92 cause of freezing injury in plants (Steponkus 1984; Steponkus and Lynch 1989). For example, sub-zero temperatures caused an increase in efflux of cellular electrolytes in Norway spruce (Picea abies L.) needle tissue after ice crystallization injured cellular membranes (Pukacki and Pukacka 1987). Cold acclimation increases the cell membrane’s ability to resist injury by sub-zero temperatures (Levitt 1980). The developmental stage has been found to be an important factor in determining whether freezing injury will occur. In spruce, immature needles at the time of shoot elongation were found to be more sensitive to freezing temperatures than needles at other developmental stages, as indicated by decreased chlorophyll fluorescence (Welander et al. 1994). Furthermore, plants are subjected to sub-zero temperatures not only at night, but also during periods of moderate to high irradiance. Chlorophyll fluorescence (CF) is a non-destructive probe of photosynthetic function (Lichtenthaler et al. 1986; Krause and Weis 1991) and has been used extensively to investigate temperature effects on conifer foliage (Strand and Lundmark 1987; Smillie et al. 1988; Krause and Somersalo 1989; ¨ Bolhar-Nordenkampf et al. 1991; Ogren 1991; Gillies 1993). Several conifer species show a decrease in the ratio of variable fluorescence to maximal fluorescence (Fv /Fm ) in winter, indicating decreased electron flow (Hawkins ¨ and Lister 1985; Bolhar-Nordenkampf and Lechner 1988; Strand and Oquist 1988; Vidaver et al. 1989; Gillies 1993). Chlorophyll fluorescence has also been used to assess needle injury from freezing temperatures in Pinus contorta Dougl. ex Loud., Pinus sylvestris L. (Lindgren and Hallgren 1993) and Picea ssp. (Adams and Perkins 1993; Binder et al. 1993; Devisscher and Malek 1993; Binder and Fielder 1996). Several studies have examined the effects of freezing temperatures on conifer tissue in the dark (Pukacki and Pukacka 1987; Holopainen and Holopainen 1988; Rostad 1988), or followed by high light levels (Welander et al. 1994) or shade (Orlander 1993). Under natural conditions, conifers are often subjected to moderate to high light levels during the winter. However, the simultaneous application of freezing temperatures and moderate light levels has not been reported. This study investigated the susceptibility of white spruce (Picea glauca [Moench.] Voss) needles at different stages of development to sub-zero temperatures in both the light and dark and assessed the ability of chlorophyll fluorescence and electrolyte leakage to detect damage to needle tissue.
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93 Materials and methods White spruce seedlings were provided by Hybrid Nursery, Pitt Meadows, British Columbia. Seed origin was 56 070 N 124 490 W. Seed was sown in March into PSB313a containers (2.7 cm diameter, 13.2 cm depth, 60 cc volume; Styroblocks, Beaver Plastics, Edmonton, AB, Canada) containing 3:1 (v:v) peat:vermiculite. The stock was fertilized and watered with the commercial crop. Seedlings were brought to Simon Fraser University (Burnaby, Canada) in November and placed in an unheated glass greenhouse. Seedlings were fertilized (150 ppm Plant Prod 20:20:20) once a week and watered 3 times weekly or when styro block weight declined to 7.5 kg. The experiment was a full 2 3 7 factorial for light developmental stage freezing temperature. Seedlings were exposed to sub-zero temperature treatments (described below) in either the light, or dark at three different stages of development. The stages of development were: 1) one year old needles, hardened under natural winter conditions, 2) one year old needles, dehardened for a minimum of 35 days (described below), and 3) shoot elongation phase, after transfer into growth conditions. The shoots at that point had reached about 75% of their final length. Seedlings were transferred into growth conditions in February. Three hundred and Eighty seedlings were placed in a growth chamber at 22/16 C, 16 h photoperiod, photosynthetic photon flux density (PPFD) of 375 mol m 2 s 1 , and 70–85% relative humidity (RH). Seedlings were watered frequently and grown for a minimum of 35 days. Seedlings were considered completely dehardened after 35 days. One half of the seedlings had all new foliage removed prior to application of freezing treatments, and only dehardened one year old needles remained. All new foliage was left on the rest of the seedlings, and when shoot elongation reached 75% of the final length they were used in the experiment. Freezing treatments were done in a modified chest freezer with a minimum possible temperature of 22.5 C. The freezer lid was removed and replaced with an insulating foam lid with a 40 30 cm double pane glass window. A plexiglass water bath with 9 cm of water was placed over the window. A light source external to the freezer was supplied by quartz-halide lamps. The maximal PPFD at foliage height was 400 mol m 2 s 1 as measured by a LICOR 185A quantum sensor (LI-COR, Lincoln, NE, USA). To ensure uniform air temperature, air circulation was provided by two fans in the bottom of the freezer. Seedlings were exposed to 6 sub-zero temperatures ( 2, 4.1, 6.2, 10, 13.2, 22.5 C) with an external light source, control temperature was 25 C. Samples were placed horizontally in the freezer to ensure all foliage received a relatively uniform flux density. Samples to be frozen in the dark were placed in a light proof black box with air vents on the bottom to allow
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94 air circulation. Temperatures inside the box were the same as in the rest of the freezer compartment. Temperature in the freezer was lowered at 6 C h 1 from the initial temperature of 18 C and held at the lowest intended temperature for 1 h. Five samples (seedlings) were used at each temperature, each temperature treatment was repeated three times. Each treatment group was exposed to 4 h of light, beginning 3 hours before the desired temperature was reached, including the control group which was left in the freezer for 4 h. After freezing treatment was complete, the light was turned off and the temperature increased at 6 C h 1 to room temperature (22 2 C) in the dark. Seedlings were given 16 h dark recovery at room temperature and then exposed to a PPFD of 150 mol m 2 s 1 for 2 h. Control seedlings received the same conditions. Seedlings were then placed in an incubator at 22 2 C, 16 h daylength, PPFD of 170 mol m 2 s 1 , and 75–85 % RH for two weeks. It took approximately 7 days to complete the freezing treatments. The 3 replications for hardened needles were done between December 7th and January 4th. Freezing treatments for dehardened and newly flushed needles were conducted between March 15th and April 28th. Chlorophyll fluorescence was measured immediately after seedlings were warmed to room temperature and again after three days using an integrating fluorometer constructed in the laboratory (Toivonen and Vidaver 1984; Vidaver et al. 1989). Before measurement seedlings were dark pretreated for 30 min at room temperature. The variable chlorophyll fluorescence of the foliage was monitored, and recorded for 10 seconds. The light level in the integrating sphere had a PPFD of 115 mol m 2 s 1 provided by a quartz-iodine projection lamp. To measure chlorophyll fluorescence of the new foliage separately from one year old foliage in the integrating sphere, foliage not measured was either left external to the sphere or covered by aluminum foil. The foil had no effect on chlorophyll fluorescence measurement values. Measurement of relative electrolyte leakage was modified from the procedure of Colombo et al. (1984) (S. L’Hirondelle, B.C. Ministry of Forests pers. comm.). Needle samples were removed from seedlings after freezing treatment and cut into 1 cm lengths and placed into polypropylene vials with 3.5 ml deionized water (3 needles per vial, 3 vials per seedling). A blank containing 3.5 ml deionized water was prepared. Samples and blank were placed at 4 C for 24 h then electrolyte leakage was measured using a Digital Conductivity Meter Model 1481–90,82 (Cole-Parmer Inst. Co., Chicago, IL, USA). Samples were then boiled for 1 h at 100 C in a boiling water bath,
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95 then placed at 4 C for 24 h and conductivity remeasured. Percent electrolyte leakage was calculated using the formula: [(conductivityfrozen
blank) * (conductivityboil
blank) 1 ] *100 %
Visible damage to foliage was assessed after two weeks. Damage to needles was defined as the percentage (in 5 % increment categories i.e. 0–5, 6–10, 11–15% etc) of discolored, or dead needles to total number of needles on the sample. Needle water content (WC) as a percentage of fresh mass was determined by measuring the mass of fresh needles from well watered seedlings (FWT) and oven dry mass (DWT) of needles (48 h at 70 o C) using the formula: WC = [(FWT
DWT)*FWT
1 ]*100.
Statistical analysis was performed using SAS/STAT (1988). Analysis of variance (PROC GLM) with developmental stage, temperature and light as main factors was used to analyze electrolyte leakage, fluorescence and damage data. The Bonferroni test of differences was used to determine significance between individual means. Linear regression was used to determine the relationship between Fv /Fm and needle damage on the linear portion of the curve, over a range of 5 to 95 % damage. Visible needle damage data was subjected to an arcsine square root transformation (Binder and Fielder 1995). However, means of the transformed data did not change the r2 of the regression against electrolyte leakage or the distribution of the residuals so the untransformed results are presented.
Results Chlorophyll fluorescence The freezing treatments had no significant effect on Fv /Fm of hardened seedlings measured immediately following thawing, either in the light or the dark (Figure 1). The Fv /Fm parameter value was higher for all treatments, including the control, after 3 days in the incubator, and no significant treatment effect was evident. The Fv /Fm of dehardened needles was significantly affected by the freeze temperature treatments. The Fv /Fm decreased with temperatures below 6.2 C. There was no significant difference between the control and treatment temperatures of 6.2 C and above. The Fv /Fm declined significantly at 10 C in the dark, and was even lower in the light treatment group. After
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Figure 1. A). The variable fluorescence to maximum fluorescence ratio (Fv /Fm ) of hardened (H), dehardened (O) and newly flushed (N) white spruce needles after sub-zero temperature exposure in the dark (D – dotted lines) or in the light (L – solid lines) (400 mol mol m 2 s 1 ). B). The same seedlings as in A) but after three days in low light (120 mol mol m 2 s 1 ).
three days, Fv /Fm values remained relatively constant for seedlings receiving temperature treatments higher than 6.2 C. Mean Fv /Fm of seedlings receiving temperatures of 10 C and lower did not recover to the level of seedlings exposed to the higher temperatures (Figure 1). The Fv /Fm value of newly flushed needles measured immediately after thawing was significantly affected by both the temperature and light treatments. The Fv /Fm values declined with exposure to lower temperatures. The Fv /Fm of new foliage exposed to sub-zero temperatures in the dark declined significantly at 6.2 C, whereas this occurred at 4.1 C for seedlings in
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97 the light. The Fv /Fm values after 3 days recovery in low light were not significantly different from the control seedlings, either in the dark or the light, for temperatures 4.1 o C and above. The Fv /Fm values after 3 days recovery for new foliage exposed to 6.2 C in the dark remained low, but declined further for new foliage exposed to these temperatures in the light (Figure 1). For new foliage, freezing to 4.1 C in the light resulted in a reversible decline of Fv /Fm , the Fv /Fm values returning to control levels after three days recovery at low light level. Visible damage There was no visible damage to the foliage of hardened seedlings after two weeks for all treatment groups (Figure 2a). Visible damage was observed in dehardened one year old needles, the estimated LT50 was 10.5 C in the light and 12.4 C in the dark and were significantly different at P = 0.01. Newly flushed needles showed visible damage at higher temperatures than dehardened needles. Newly flushed needles had an LT50 of 4.8 C in the light and 6.2 C in the dark and were significantly different at P = 0.05. Electrolyte leakage There was no significant change in electrolyte leakage for hardened needles over the freezing temperature range used, in either the light or dark treatment groups (Figure 2b). Electrolyte leakage of dehardened needles was significantly affected by the both the temperature and light, and electrolyte leakage increased at lower temperatures. A small amount of electrolyte leakage was observed in the dark treatment group at 6.2 C and increased to near 100 % at 13.2 C. Greater leakage was observed in light, compared to dark treated seedlings. Electrolyte leakage was observed at 4.1 C in newly flushed needles, and increased at a greater rate in the light treated seedlings, compared to the dark treatment groups. Relationship of Fv /Fm and electrolyte leakage to visible needle damage after freezing The amount of visible damage observed in needles after freezing is highly correlated with both Fv /Fm (Figure 3a) and electrolyte leakage resulting from membrane rupture (Figure 3b) if values above 5 and below 95 % damage are used to calculate the fit.
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98
Figure 2. A). Visible damage to foliage of hardened (H), dehardened (O) and newly flushed (N) white spruce seedlings after two weeks recovery from sub-zero temperature treatments in the dark (D – dotted lines) or light (L – solid lines), and B). Electrolyte leakage of the needles of hardened (H), dehardened (O) and newly flushed (N) white spruce needles after sub-zero temperature treatments in the dark (D – dotted lines) or light (L – solid lines).
Needle water content Newly flushed needles had more (%) water content (90.3), than either dehardened needles (83.5) or hardened needles (71.2).
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99
Figure 3. A). The variable fluorescence to maximum fluorescence ratio (Fv /Fm ) related to visible needle damage (using data between 5 and 95 % damage), and B). The electrolyte leakage related to visible needle damage (using data between 5 and 95 % damage).
Discussion Since seedlings with hardened needles were not affected by the freezing treatments used, in either the light or dark, such seedlings are either unaffected by the presence of light during freezing, or lower temperatures and/or higher light levels are necessary to cause a measurable effect. In either case, hardened seedlings apparently are resistant to the freeze temperatures at the light level tested in this study and seedling quality is not affected.
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100 Needle tissue damage in dehardened and newly flushed foliage occurred at higher freezing (warmer) temperatures in the presence of light than if exposed to these temperatures in darkness. These results are similar to those of Welander et al. (1994), who found that freezing temperatures followed by high irradiance increased damage in newly flushed Picea abies seedlings. The reversible decreases in Fv /Fm values observed in this study are attributed to photoinhibition due to a decrease in Fv (variable fluorescence) and not ¨ to increases in Fo (background or initial fluorescence) (see also Oquist and Malmberg 1989). Photoinhibition occurs when the photosynthetic antennae absorb light in excess of the capacity of photosynthesis to process light energy ¨ (Oquist et al. 1987). The primary site of photoinhibition is still uncertain but most reports suggest it is the D1 protein of the reaction centre of PSII (Kyle et al. 1984; Arnzt and Trebst 1986; L¨onneborg et al. 1988). Godde and Buchhold (1992) suggest that increased turnover of the D1 protein in trees explains the synergistic effects of stress conditions and high light intensities often observed in the field. Where photoinhibition is involved the recovery of Fv /Fm is quite fast. Sharma and Singhal (1992) reported recovery of Fv /Fm in Triticum aestivum was almost complete within 12 h after photoinhibition treatments of 650 mol m 2 s 1 at either 10 or 30 C. Alternately, in either the light or dark, a substantial and irreversible decrease in Fv /Fm is indicative of severe and permanent tissue damage. In such cases the combination of low temperature and moderate light results in photodamage, and is caused by photooxidation (light and oxygen dependent bleaching) (Wise and Naylor 1987; Gillies and Vidaver 1990, Vidaver et al. 1991). Damage results from the production of toxic oxygen compounds which can inactivate photosystem reaction centres (Vidaver et al. 1991), cause the photodestruction of pigments (Anderson 1986), and produce peroxidation of lipid membranes and proteins (Salin 1987). Damage to needles exposed to sub-zero temperatures in darkness cannot, of course, be explained by light dependent photodamage. In the dark, damage to needles most likely is due to freezing injury, caused by ice crystal formation and dehydration (Schmitt et al. 1985), and appears to be related to the percent water content of seedlings. Seedlings with higher percent water content showed greater injury at warmer temperatures than seedlings of lower percent water content. The increased electrolyte leakage observed in these seedlings indicates that membranes had been damaged. Irreversible decreases in Fv /Fm observed in seedlings in the dark are attributed to freezing injury to chloroplast membranes. Any increase in damage observed in the light treatment groups we attribute to photodamage since decreases in Fv as well as large increases in Fo were seen after three days (data not shown).
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101 Similar to others (D¨uring et al. 1990; Sundblad et al. 1990; Lindgren and H¨allgren 1993; Adams and Perkins 1993, Binder and Fielder 1996), we found a strong correlation between Fv /Fm and visible damage (Figure 3a). Also, as reported by others, (Simpson 1989; Sutinen et al. 1992; L’Hirondelle et al 1992; Binder et al. 1993; Binder and Fielder 1996) we have shown a strong linear relationship between electrolyte leakage and visible damage (Figure 3b). We conclude that chlorophyll fluorescence and electrolyte leakage are both useful techniques to estimate freezing injury to needles of white spruce, and presumably other conifer species. Conclusions Susceptibility to photoinhibition, photodamage, and damage to membranes after freezing depends upon the freezing temperature, the developmental stage of the needles, and is enhanced by light. Hardened needles were not affected by temperature and light treatments, but both dehardened and newly flushed needles were affected. Newly flushed needles were significantly more susceptible to freezing in both the light and dark than dehardened needles. Both chlorophyll fluorescence and electrolyte leakage may be used to assess freezing injury in white spruce needles. In addition, chlorophyll fluorescence is a diagnostic tool that can be used to assess light enhanced freezing damage to the photosynthetic system. Acknowledgements We thank Drs. P. Puttonen, S. L’Hirondelle and Mr. P. Fielder for reviewing the original manuscript. Thanks go to Kevin Conley for reproduction of the graphics. Funding for this work was provided to one of us (SLG.) by the Natural Sciences and Engineering Council of Canada (Post Graduate Scholarships) and Simon Fraser University Doctoral Fellowships. We also thank the Ministry of Forests, Research Branch for support, and Hybrid Nursery, Pitt Meadows, B.C. for supplying the seedlings. References Adams, G. T. and Perkins, T. D. 1993. Assessing cold tolerance in Picea using chlorophyll fluorescence. Env. and Exp. Bot. 33: 377–382. Anderson, J. M. 1986. Photoregulation of the composition, function, and structure of thylakoid membranes. Annu. Rev. Plant Physiol. 37: 93–136. Arntz, B. and Trebst, A. 1986. On the role of the QB-protein of PSII in photoinhibition. FEBS Letters. 194: 43–49.
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