Acta Physiol Plant (2015) 37:1717 DOI 10.1007/s11738-014-1717-3

ORIGINAL PAPER

Freezing tolerance in Norway spruce, the potential role of pathogenesis-related proteins Lars Sandved Dalen • Øystein Johnsen • Anders Lo¨nneborg • Mahmoud W. Yaish

Received: 16 May 2014 / Revised: 4 November 2014 / Accepted: 4 November 2014 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2014

Abstract Cold-tolerant plants may endure subzero temperatures partially by inhibiting the development of ice crystals in the intercellular spaces and the xylem through the accumulation of antifreeze proteins (AFP) and the extra production of carbohydrates. Certain proteins associated with pathogen resistance in plants have the ability to bind and alter the growth of ice crystals. In this study, the accumulation of pathogenesis-related (PR) proteins and the development of freezing tolerance in seedlings of two latitudinal distinct Norway spruce (Picea abies L. Karst.) ecotypes were investigated. Despite freezing tolerance difference, timing of growth cessation and bud set variations, our results showed that there is no significant difference in the concentration of soluble carbohydrates between the two ecotypes. Immunoblots showed the presence of several b-1,3-glucanase and thaumatin PR proteins in the apoplastic fluid and the enzymatic assay

showed an extra accumulation of several isoforms of PR chitinases in cold-treated Norway spruce needles. In addition to PR proteins, a presence of de novo protein in coldtreated needles was noticed. In contrary to mature plants, total proteins isolated from freezing-tolerant Norway spruce seedling did not show antifreeze activity. Our results suggest that the activity of the PR proteins and the accumulation of soluble carbohydrates that increased during cold acclimation may have an indirect impact on the freezing tolerance in Norway spruce, however, deciphering the direct mechanism behind freezing tolerance in Norway spruce seedlings growing under controlled environmental conditions require further investigation. Keywords Antifreeze proteins  Norway spruce  Carbohydrates  Chitinases

Introduction Communicated by J. Gao.

Electronic supplementary material The online version of this article (doi:10.1007/s11738-014-1717-3) contains supplementary material, which is available to authorized users. L. S. Dalen ˚ s, Norway Norwegian Forest and Landscape Institute, 1431 A Ø. Johnsen Faculty of Environmental Science and Technology, ˚ s, Norway Norwegian University of Life Science, 1432 A A. Lo¨nneborg Sensilect Consulting AB, Spannma˚lsva¨gen 42, Dalby, Sweden M. W. Yaish (&) Department of Biology, College of Science, Sultan Qaboos University, Muscat, Oman e-mail: [email protected]

Freezing-tolerant perennial plants may survive subzero temperatures by inhibiting the formation of ice crystals in the extracellular spaces (Griffith and Antikainen 1996; Griffith and Yaish 2004). The ice crystals grow at the expense of water inside the cells, which dry out and experience supercooling. It is assumed that ice crystal formation inside cells is lethal due to mechanical damage to the membranes, and that the avoidance of intracellular freezing is dependent upon tolerance toward extracellular freeze dehydration (Levitt 1980). When extracellular ice is formed, the continued growth of ice crystals depends upon several factors that modify ice propagation, such as cell wall modifications, plasma membrane alterations (Steponkus 1984), arabinoxylanes (Olien 1965) and antifreeze proteins (AFPs) (Griffith and Antikainen 1996) accumulation.

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ANTIFREEZE proteins isolated from plants exhibit low levels of hysteresis activity compared to those characterized in fish and insects. Therefore, plant’s AFPs can be considered as ice crystal modifiers rather than freezing inhibitor proteins (Griffith and Yaish 2004). Although ice recrystallization inhibition was detected in transgenic lines, overexpression of AFP-coding genes showed either no effect or a slight increase in the plant’s freezing tolerance (Ashworth et al. 1993). When freezing occurs, ice is formed in the xylem, in stomata, and in the extracellular spaces near the vascular tissue (Marentes et al. 1993). Several studies on freezing tolerance in herbaceous plants have shown that these plants secrete both AFPs and sugars to modify the growth of ice (Griffith and Yaish 2004; Hiilovaara-Teijo et al. 1999; Meyer et al. 1999; Worrall et al. 1998). The AFPs found in fish (DeVries 1970), insects (Duman et al. 1991) and plants (Doxey et al. 2006; Griffith and Yaish 2004) are structurally diverse, but they all interact with the surface of ice, depress the freezing temperatures of aqueous solutions, and inhibit ice crystal growth (Ashworth 1996). Some of these AFPs resemble pathogenesis-related (PR) proteins (Hon et al. 1995). PR proteins in plants normally accumulate in response to pathogen attack and are characterized as hydrolytic enzymes. For example in winter rye glucanases, chitinases, and thaumatins accumulate in the apoplastic fluid of the plant in response to pathogen attacks, as well as in response to low temperatures (Antikainen and Griffith 1997; Hon et al. 1995). This situation may provide a dual biotic and abiotic protection to the plant during early spring against mold infections and freezing temperatures (Griffith and Yaish 2004). Similar to other boreal coniferous species, Norway spruce (Picea abies L. Karst.) has a strong capacity to adapt to long-term periods of cold and freezing temperatures. It is therefore of special interest to investigate the putative role of PR proteins during development of freezing tolerance in these plants. In Norway spruce, soluble acidic PR proteins have been found in roots (Sharma et al. 1993), needles (Ka¨renlampi et al. 1994), buds (Baumann 1996), bark, and green cones (A. Lo¨nneborg, unpublished). Therefore, PR proteins which are produced in Norway spruce during cold acclimation may display antifreeze activity which likely has a fundamental function during the development of freezing tolerance in this plant at subzero temperatures. The goal of this project was to study the accumulation of PR proteins and carbohydrates during cold acclimation and to relate the PR protein activity with observed seedling survival after freezing. Because there are significant differences in freezing tolerance between latitudinal ecotypes of Norway spruce, we performed the experiments using seedlings from northern (66°N) and southern (60°N) Norway, and assayed for PR protein activity during cold acclimation.

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Materials and methods Plant material Norway spruce (Picea abies (L.) Karst.) seeds obtained from southern (lat. 60°110 N, long. 11°300 E) and northern (lat. 66°110 N, long. 11°300 E) ecotypes were germinated and grown in a growth chamber according to the previously described method (Dalen and Johnsen 2004; Dalen et al. 2001). Briefly, the seeds were planted in 50 ml pots containing peat with dolomite and perlite in a 3:1 ratio and initially incubated for 3 weeks with a 24 h day length and a photosynthetic photon flux density (PPFD) of 50–60 lmol m-2 s-1, 22 °C, and 0.8–1.0 kPa saturation vapor pressure deficit (VPD) before seedlings were thinned into one plant per pot. Subsequently, the seedlings were incubated for an additional 7 weeks in a continuous light at a fluorescence rate of 250 lmol m-2 s-1, 22–24 °C, and 0.3–0.6 kPa VPD. Growth cessation, bud set, and development of freezing tolerance were then gradually induced by reducing the photoperiod and the temperature. In Norway spruce, a day length below a certain critical value induces growth cessation, formation of terminal buds, and development of freezing tolerance (Baumann 1996). Lowered day and night temperatures further enhance the development of freezing tolerance. In this study, photoperiod was reduced gradually from 24 to 7 h, combined with a reduction in day/night temperatures from 22 °C/22 °C to 14 °C/7 °C. The seedlings were watered three times each week with a fertilizer solution containing 0.7 % red superba (Supra, Sweden) and 0.8 % (NH4)2SO4 (Johnsen 1989). Hourly average temperatures were measured using thermocouples and recorded with a data logger (Multi logger 21X, Campbell Scientific, UK). Light intensity and relative humidity were hourly recorded using a quantum sensor and a combined temperature and humidity sensor as previously described (Dalen et al. 2001). Growth measurements To characterize the cold acclimation process, we measured height growth and ranked the formation of terminal buds (Baumann 1996). Shoot height was measured every 2 weeks on four seedlings per ecotype and replicate (4 seedlings 9 2 ecotypes 9 12 replicates = 96 seedlings per sample date), for a total of 672 measurements following the previously described method (Dalen and Johnsen 2004). Carbohydrate analysis Needle samples obtained from two seedlings per ecotype were separately analyzed for the soluble sugar contents (Ashworth et al. 1993). 0.5 g of the frozen needles were

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homogenized in 10 ml 80 % ethanol, vortex for 30 min and then centrifuged as previously described (Dalen et al. 2001). An aliquot of 5 ml was dried out at 30 °C, dissolved in 5 ml H2O and purified using a Sep-pack CH and SAX filters. A 15 ll aliquot sample was injected into a Merck HPLC system (AS-4000 auto-sampler, L-6200 pump, L-5025 oven, RI-71 detector, all controlled using the D-6000 software), using a Waters NH2 column at 30 °C. Acetonitrile and water was used as an eluent at a 84:16 and 78:22 ratios for the mono- and disaccharides and for the raffinose and stachyose, respectively. Peak identity was determined using known carbohydrate reference standards. Total soluble sugars (fructose, glucose, inositol, raffinose, sucrose, and stachyose) were calculated on the basis of tissue water content and displayed in mg ml-1 unit as previously described (Dalen et al. 2001).

markers (Bio-Rad) were electroblotted onto a nitrocellulose membrane using the Mini Transblot as recommended by the supplier (Bio-Rad). Blots were blocked overnight in a buffer containing 1 % skim milk powder, followed by 4 h of incubation with antiserum against winter rye glucanase-like proteins (GLP), chitinase-like proteins (CLP), or thaumatin-like proteins (TLP), in a 1:2,000 dilution. The immunoreaction was detected by alkaline phosphatase conjugated with goat anti-rabbit secondary antibody (Sigma immunochemicals), using 5-bromo-4chloro-3-indolylphosphate-toluidine salt (BCIP; Sigma) and nitroblue tetrazolium (NBT; Sigma) as substrates. Apoplastic proteins from cold acclimated winter rye (Secale cereale L.) were used as positive controls on the immunoblots.

Protein extraction and visualization

Plant cold treatment and assessment of freezing damage

For extractions of proteins, whole and cut needles were harvested from northern (66°N) and southern (60°N) Norway spruce ecotypes and vacuum-infiltrated for 30 min at 0.8 kPa in a 0.1 M sodium citrate buffer (pH 5.0) with or without 0.3 M KBr and 1 % polyvinylpyrrolidone (PVP) 40. The needles were then carefully dried with paper towels and centrifuged at 2,0009g for 10 min. For the extracellular washing fluid (EWF), approximately 50 ll g-1 fresh weight, was collected and stored at– 20 °C. For the total extractions of protein, 1 g of needles was ground to a fine powder in liquid nitrogen along with a continuous addition of 100 mg caffeine (Sigma). The powder was transferred to a smaller mortar placed on ice, and 1 ml of 0.1 M sodium citrate buffer (pH 5.0) with 1 % PVP 40 (w/w), 1 % polyvinylpolypyrrolidone (PVPP) 360 (w/w) and 0.3 % Triton X-100 was added. Insoluble material was pelleted at 10,0009g for 15 min, and the supernatant collected and stored at -20 °C. Polyacrylamide Gel Electrophoresis–isoelectric focusing (PAGE-IEF) was performed essentially as previously described (Sharma 1995). Detection of chitinase activity was based on a method described by Pan et al. (Pan et al. 1991), using glycol chitin as a substrate in an overlay gel (Trudel and Asselin 1989) in the total protein and EWF using samples extracted from the northern (66°N) Norway spruce ecotype. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and immunoblotting was optimized and carried out using equal amounts (20 lg) of apoplastic proteins extracted from southern (60°N) Norway and local Norway spruce growing at the University of Waterloo campus. Protein samples were separated on 15 % SDS–polyacrylamide gels and stained with Coomassie Brilliant Blue (Laemmli 1970). Separated polypeptides and prestained low-molecular-weight

Cold treatment and freezing damage assessment was carried out using the previously described method with some minor modifications (Dalen et al. 2001). Freezing tests were carried out on whole seedlings three times during the cold acclimation period. A range of temperature from -6 °C to 47 °C was used during this test in which six different freezing temperatures were applied for each of the three freezing tests. Since the two latitudinal ecotypes differ significantly in freezing tolerance during the early parts of cold acclimation, the seedlings of the northern ecotype were freeze-tested at an earlier date, and at lower temperatures than the seedlings of the southern ecotype. Twelve seedlings from each ecotype were randomly selected for each freezing test temperature. In this experiment, a total of 432 seedlings were used in which 12 seedlings of each ecotype were tested at six different freezing temperatures within three freezing dates. The freezing tests were carried out in six different programmable freezing chambers (Weiss Umwelttechnik, Reiskirchen, Germany). The freezing test program was initiated by exposing the root-insulated seedlings to 5 °C for 1 h. To reach the suitable test temperature, the chamber temperature was gradually lowered by 2 °C h-1 and maintained at test temperature for 4 h. Subsequently, the temperature was gradually increased at 2 °C h-1 to 5 °C. To enhance ice formation at 0 °C, plants were misted for 10 s with tap water. The freezing test temperatures employed for the southern ecotype were -6, -8, -10, -12, -14, and -16 °C for the freeze test on day 101 after germination; -15, -17.5, -20, -22, -24, and -26 °C for the freeze test on day 116; and -29, -32, -35, -38, -41, and -44 °C for the freeze test on Day 129. For the northern ecotype, the freezing test temperatures employed were -11, -13, -15, -17, and -19 °C for the freeze test on day 99 after germination; -18, -20.5, -23, -25, -27, and -29.5 °C for the

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Experimental strategy and data analysis Norway spruce seedlings were placed in trays and incubated in two different phytotron rooms. A split-plot design was used, with six replicates (complete blocks) experiment in each phytotron room, as previously described (Dalen and Johnsen 2004). Each replica was composed of two latitudinal ecotypes, with 44 seedlings per ecotype. ANOVA was carried out using the general linear model in SAS (Institute 1987). Yijk ¼ l þ si þ bj þ dkð jÞ þ eijkl where Yijk was the mean of all tested plants for each combination of the experimental factors considered, l was the total mean, si was the fixed influence of ecotype, bj was the fixed influence of phytotron room, dk(j) was the random influence of replicate within room, and eijk was the experimental error. When data were analyzing for height growth, bud set, and freezing tolerance, additional terms for sampling date and/or temperature were inserted to the model. LT50-values were computed depending on the freezing damage scores. A damage index (DI), ranging from 0 to 1, was calculated based on needle damage score (ND), utilizing the formula DI = ND/11. The test temperatures utilized on a test date i, were converted to relative values ranging from 0 to 1 by the following formula. Ti ¼ ðXmax  Xi Þ=ðXmax  Xmin Þ where Xmax was the maximum test temperature on a test occasion; Xmin was the minimum test temperature on a test occasion; and Xi representing the test temperatures used on a test occasion. DI was fitted to a logistic model.
DIi ¼ 1= 1 þ aebTi by employing the NLIN procedure in SAS. The model was denoted by the linear equation.

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logð1=DIi  1Þ ¼ bTi þ logðaÞ where the constants a and b determined where the T value DI started to increase (a) and the rate of increase in DI associated with a decrease in Ti (b). The LT50 values were computed as the freezing temperatures where DI achieved the value 0.5 by the following expression. LT50 ¼  ð½logðaÞ=b  ½Xmax  Xmin  þ Xmax Þ Antifreeze assay Extracted proteins where assayed for their antifreeze activity following the previously described method (Hon et al. 1994). The antifreeze activity assay is based on the ability of the proteins to alter the morphology of ice crystals during a slow-warm and cool cycle of the microscope stage. As an experimental negative control, the antifreeze assays were performed using water and the protein buffer.

Results The results showed some differences in critical photoperiod and freezing tolerance between the two ecotypes seedlings employed in this study. The growth measurements indicated that the northern ecotype had terminated shoot growth 2 weeks before the southern one (Fig. 1) and developed terminal buds 2 weeks earlier than the southern ecotype (data not shown). The concentration of soluble sugars, a factor which sometimes correlate with the

100

80

Height (mm)

freeze test on day 114; and -30, -34, -37, -40, -43, and -47 °C for the freeze test on day 126. After each freeze test, the seedlings were kept in a greenhouse for 3 weeks at 22 °C, with a 24-h day length and a PPFD of 100 lmol m-2 s-1. The seedlings were irrigated three times a week with a fertilizer solution as mentioned earlier. Freezing damage on whole shoots (browning and discoloration) was visually evaluated on a scale from 0 (no visible damage in needles) to 11(all needles are completely brown) according to the previously published method (Dalen et al. 2001; Johnsen et al. 1995). In this freezing test, LT50 was defined as the temperature where 50 % of the frozen needles were either brown or discolored. The LT50 was visually estimated based on the average damage scores for each replicate.

Acta Physiol Plant (2015) 37:1717

60

40

20

0 60

70

80

90

100

110

120

140

160

Days After Germination Fig. 1 Mean shoot height in Norway spruce seedlings obtained from northern (66°N) or southern (60°N) Norway. Each data point stands for the average of 12 replicates, i.e., 48 seedlings ± the standard deviation of the mean

Acta Physiol Plant (2015) 37:1717

Chitinolytic activity during cold acclimation To investigate possible accumulation of chitinases in Norway spruce needles during cold acclimation, we examined the presence of chitinolytic activity during cold treatments using isoelectric focusing combined with a chitin overlay gel. The results showed that there were several chitinolytic isoforms both in the extracellular fluid and in the total protein of Norway spruce needles regardless of the treatment temperature (Table 1; Fig. 5); however, there was a slight increase in the enzymatic activity during cold acclimation. In the apoplastic extracts, the chitinolytic isoforms were either basic or acidic, with no neutral isoforms appearing (Fig. 5a). In the total extracts, the majority of the chitinolytic enzymes were either basic or acidic, but a few enzymes with pIs values &8.0 appeared toward the end of cold acclimation when night temperature was lowered (Fig. 5b). The similarity in chitinolytic enzyme patterns between apoplastic and total extracts indicates an extracellular location of most of these proteins. Some variations in the chitinolytic protein accumulation pattern were noticed between the northern (66°N) and southern (60°N) ecotype (Fig. S1). This variation was probably due to the difference in their freezing tolerance

500

mM of Carbohydrate

400

300

200

100

0 60

70

80

90

100

110

120

Days After Germination Fig. 2 Carbohydrate concentration in Norway spruce needles during cold acclimation. The values correspond to each data point are the average amount of soluble sugars displayed in molar units and computed on the basis of tissue water content, ± the standard deviation

–40 Ecotype 60°N

–35

Temperature (°C)

development of freezing tolerance, increased with the reduced photoperiod and temperature, but there was no statistically significant difference in the concentration of soluble carbohydrates between the two ecotypes (Fig. 2). Freezing tests performed during cold acclimation showed that freezing tolerance developed faster in the northern ecotype, and there were some variations in freezing tolerance between the northern and the southern ecotype (Fig. 3). The estimated LT50 values show that the northern ecotype is more freezing tolerant than the southern ecotype as the average difference in LT50 was 4.2 °C during freeze test I; 8.8 °C during freeze test II; and 6.5 °C during freeze test III. Apoplastic polypeptides from cold-treated Norway spruce needles were separated by SDS-PAGE and tested with antibodies raised against PR proteins of antifreeze activity isolated from winter rye. SDS-PAGE analysis showed the existence of various proteins in the apoplastic fluid. Based on the Coomassie staining, the estimated molecular mass of these proteins ranges from 18 to 100 kDa (Fig. 4a). In the immunoblot, we have detected one protein from spruce reacting to the anti-GLP antibody (Fig. 4b), and several proteins reacting against the anti-TLP antibody (Fig. 4c). These proteins had apparent molecular weights of 32 kD and 16–25 kD, respectively. We did not detect any proteins from Norway spruce reacting against the antiCLP antibody (Fig. 4d).

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Ecotype 66°N

–30 –25

–20 –15 –10 –5 0

I

II

III

Freezing Test Fig. 3 Freezing tolerance in Norway spruce seedlings obtained from northern (66°N) and southern (60°N). Average LT50 values ± the standard error of the mean was estimated from assessment of visual damage after freezing whole seedlings at temperatures ranging from -6 °C to -47 °C. See ‘‘Materials and methods’’ for a description of the freezing procedure

phenotype; however, more experiments are needed to prove this hypothesis. Antifreeze activity of the EWF proteins Antifreeze proteins have the capacity to slow down and to alter the growth of ice crystals under freezing conditions (Griffith et al. 1992). The antifreeze assay results showed that proteins extracted from cold-treated plants, hardy down to -47 °C, did not exhibit antifreeze activity.

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Fig. 4 Specificity of antisera produced against purified AFPs from cold-treated winter rye leaves used against proteins from Norway spruce EWF. 15 % SDS-PAGE was stained with Coomassie Brilliant Blue (a). The protein standard is shown in lane M, followed by the samples of extracellular washing fluid from cold-treated Norway spruce (Spruce) needles and cold-treated winter rye leaves (Rye). Thaumatin (b), b1,3-glucanase (c), and Chitinase (d) immunoblotting assay using antibodies raised against the respective proteins from winter rye

A

B

Chinase Spruce Rye

97.4 66.2 45.0 31.0 21.5 14.5

SDS-PAGE

Table 1 Photoperiod and day/night temperature (°C) during cold acclimation of Norway spruce seedlings Days

Photoperiod (h)

Temperature (°C) (day/night)

42–51

24

20

51–57

21

20/18

57–63

18

20/18

63–70

17

20/13

70–77

16

19/13

77–84

15

18/12

84–91

14

19/8

91–98

13

18/7

98–05

12

17/7

105–112

11

15/7

112–119

10

14/7

119–126

9

14/7

The light intensity was 250 lmol m-2 s-1 photosynthetic active radiation (PAR) at wavelength between 400 and 700 nm

However, our results showed that EWF samples prepared from mature Norway spruce trees in Waterloo, Canada, during the winter time were found to exhibit antifreeze activity. This antifreeze activity was observed in EWF prepared from whole and cut needles (Fig. 6).

Discussion Pathogenesis-related proteins such as chitinases and b-1,3glucanases accumulate in roots of Norway spruce in

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D

C

MW EWF Protein β -1,3-Glucanase Thauman (kD) M Spruce Rye Spruce Rye Spruce Rye

Immunoblot-Assay

response to fungal attack (Sharma et al. 1993). In cold acclimated winter rye, similar PR proteins have been found to display an antifreeze activity (Hon et al. 1995; Marentes et al. 1993). Recent experiments showed that overexpression of two PR-glucanase and one chitinase gene enhances freezing tolerance in Arabidopsis (Cabello et al. 2012). Photoperiod is the main factor regulating cold acclimation in perennial trees such as Norway spruce (Heide 1974). As the critical photoperiod is reached, shoot growth is reduced and the plant develops terminal buds. Subsequently, freezing tolerance increases as photoperiod and temperature is reduced. During cold acclimation, there is an increase in dry mass and in the concentrations of the soluble sugars (Ashworth et al. 1993). We have shown that freezing tolerance increased with decreasing photoperiod and temperature, and that it developed quicker and to a greater extent in the northern ecotype, and that there were clear dissimilarities in freezing tolerance between the northern and the southern ecotype (Fig. 3). The concentration of soluble sugars such as sucrose and raffinose, which have been associated with development of freezing tolerance increased during cold acclimation, but there were no statistically differences in carbohydrate concentration between the two ecotypes (Fig. 2). Norway spruce apoplastic extracts examined with antisera raised against winter rye AFPs showed the presence of one 32-kD protein reacting to the GLP antisera, several proteins reacting to the TLP antisera (16–25 kD), while no immuno-interaction was detected with the CLP antisera

Acta Physiol Plant (2015) 37:1717 Fig. 5 Chitinolytic activity in northern (66°N) Norway spruce needles during cold acclimation. Enzyme activity in a apoplastic fluid and b total extracts visualized by PAGE-IEF with a chitin overlay gel. The apoplastic fluid was obtained by vacuum centrifugation of the needles in a 0.1 M sodium citrate buffer at pH 5.0. Equal amount of EWF (5 ll) was applied in each lane. Temperature and photoperiod (PD) during cold acclimation is given in Table 1

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pI

A

B

Apoplastic extraction

Total extraction

pI

9.8 —

—9.8

9.5 —

—9.5

8.5—

—8.5

7.5—

—7.5

6.5—

—6.5

5.5—

—5.5

Day length (h) 24 Day (°C)

20

Night (°C) 20

21 18 20 18

17

16

15

14 13 12

20 20

19

18

19 18 17

18 13

13

12

8

7

7

24

21 18

17

16

15

14 13 12 19 18 17

20

20

20 20

19

18

20

18

18 13

13

12

8

7

7

Fig. 6 Antifreeze activity of the EWF proteins isolated from adult Norway spruce needles. EWF protein solutions were flash frozen and warmed on a freezing stage and monitored by a light microscope. The stage was warmed until crystals were formed, and then cooled to observe changes in the ice morphology. a Protein fractions \10 kDa

did not show antifreeze activity as indicated by the round and flat structure of the ice crystal while protein fractions [10 kDa and total EWF exhibited antifreeze activity as indicated by the formation of different morphologies of ice crystals (b) and (c), respectively. The magnification bars represent *10 lm

(Fig. 4) likely due to the specificity of the antibody, since several chitinolytic isoforms were found to be present both in cold-treated and untreated Norway spruce seedlings (Figs. 4, 5). There were chitinases present in the EWF in Norway spruce needles, and the chitinolytic activity increased during cold acclimation. Several chitinolytic isoforms were found in the extracellular fluid from needles of both cold-treated and untreated Norway spruce seedlings (Fig. 5). There were few differences in chitinolytic activity when plants underwent cold acclimation and the chitinases were mainly extracellular. The chitinases that we found were predominantly basic or acidic; however, there was one acidic band showing up during cold acclimation. In general, there was an increase in chitinolytic activity during cold acclimation. This

notation is consistent with the previously published results obtained in winter rye (Marentes et al. 1993). There was an increase in chitinolytic isoforms with pI closed to neutral values in the total needle extracts (Fig. 5b). The neutral chitinolytic isoforms found in total needle extracts when plants exposed to 20 °C and 24 h day length (Fig. 5b) are probably unrelated to freezing tolerance and could be part of a pathogen defense or a general stress response. Low temperatures have been shown to increase pathogen resistance in barley (Tronsmo et al. 1993; Tronsmo 1984), and in winter, rye b-1,3-glucanase showed both antifreeze and hydrolytic activity at subzero temperatures (Yaish et al. 2006). However, in the case of Norway spruce chitinase, there is no available evidence yet to support a similar situation despite the fact that total

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apoplastic proteins extracted from other mature spruce plants have been found to exhibit antifreeze activity (Jarzabek et al. 2009). Chitinases are found in roots (Sharma 1995; Sharma et al. 1993), buds (Baumann 1996), needles (Ka¨renlampi et al. 1994), bark, and green cones (Lo¨nneborg, unpublished), indicating that they are constitutively and ubiquitously expressed in Norway spruce tissue. The similarity in chitinolytic enzyme patterns between apoplastic and total extracts indicate an extracellular location of most of these proteins—in accordance with the findings of Antikainen et al. (1996), who found winter rye AFPs, including a chitin-like AFP, to be located in the epidermis and in cells surrounding intercellular spaces in coldtreated plants. In winter rye, the concentration of apoplastic proteins increase during cold acclimation (Griffith et al. 1992) and a similar situation was noticed in Norway spruce. The AFPs found in apoplastic extracts of winter rye have been shown to increase the freezing tolerance of the plant but no such relationship was found in Norway spruce seedlings. The lack of this relationship could be due to the relatively high temperatures employed during cold acclimation (Table 1). Development of freezing tolerance in perennial plants such as conifers is mainly induced by photoperiod, and this effect can also be observed in our experiment where the difference in critical photoperiod for growth cessation and bud set between the two latitudinal ecotypes is clearly expressed in their different LT50 values (Fig. 3). These results indicate that the chitinolytic PR proteins we looked at in this study probably were not involved in development of freezing tolerance in the Norway spruce, at least not during the cold acclimation regime we have employed in this study. Many perennial plants such as trees respond to shorter photoperiod which acts together with low temperature to trigger the induction of cold acclimation developmental program. In this study, we have used the inducible photoperiod; however, plants may require a combination of both short day and lower temperature to develop freezing tolerance during a cold acclimation regime. The antifreeze PR proteins might function in freezing tolerance after a low temperature cue, and not be much affected by photoperiod. Low temperatures during cold acclimation are known to increase freezing tolerance in conifers (Glerum 1985), and AFPs could be involved in this process. Although AFPs accumulated in the EWF of cold acclimated winter rye are similar to PR proteins expressed under pathogen infection, pathogen infection under non-acclimating conditions (or in non-acclimated plants) does not induce PR proteins with antifreeze activity (Hiilovaara-Teijo et al. 1999).

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Conclusion Our results suggest that PR proteins similar to AFPs found in certain monocots are found in Norway spruce, and that the activity of PR proteins such as chitinase is induced by low temperature. However, the chitinolytic activity did not explain the great difference in freezing tolerance found between the northern and southern Norway spruce ecotype used in this study. Despite the presence of PR proteins in the EWF of the cold-treated Norway spruce seedlings of the two ecotypes growing under controlled light and temperature conditions, these proteins did not exhibit antifreeze activity. However, the fact that EWF extracted from mature Norway spruce needles harvested during winter showed antifreeze activity suggests that the acquired cold acclimation in Norway spruce is likely due to the accumulation of carbohydrates and/or other unknown proteins through a freezing tolerance mechanism different from the accumulation of PR proteins. These carbohydrates or proteins which may play an antifreeze role at an advanced stage of Norway spruce growth and development are not detectable in the seedlings used in this study. Author contribution Lars Sandved Dalen, Øystein Johnsen, and Anders Lo¨nneborg designed and performed the experiments, analyzed the data, and wrote the paper. Mahmoud W. Yaish wrote the paper. Acknowledgments The skillful help and assistance of technicians Marianne Jensen and Signe Drømtorp is greatly appreciated. Financial support came from the Research Council of Norway and Borregaard AS Research Foundation. This paper is dedicated to the memory of Professor Marilyn Griffith, Department of Biology, University of Waterloo, Canada.

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