Peng Meng?Xuefeng Bai?Hongdan Li?Xiaodong Song?Xueli Zhang

Cold hardiness estimation of Pinus densiflora var.zhangwuensis based on changes in ionic leakage,chlorophyll fluorescence and other physiological activities under cold stress

Peng Meng1?Xuefeng Bai1?Hongdan Li1?Xiaodong Song1?Xueli Zhang1

Pinus densiflora var.zhangwuensis grows fast, and its droughtand salinity resistance are better than Pinus sylvestris var.mongolica.We compared cold hardiness and mechanisms of cold hardiness between the two species,to provide a theoretical basis for promoting and applying P. densiflora var.zhangwuensis in cold regions.A cold stress experiment was carried out on 3-year-old plantlets of P. densiflora var.zhangwuensis and P.sylvestris var.mongolica after hardening at five temperature regimes,5,-10, -20,-40,and-60°C,respectively.Some indices of needle samples for both species were measured,such as relative conductivity(REL),maximum photochemical efficiency(Fv/Fm),malondialdehyde(MDA),catalase (CAT),proline(Pro),soluble sugar(SS),and stomata density.REL and MDA values of both species after hardening had the same trend of increasing,but the trend was opposite in Fv/Fmvalue with increasing cold stress.Compared with P.sylvestris var.mongolica,the P.densiflora var.zhangwuensis had smaller increases in REL and MDA, and a smaller decline in Fv/Fmduring cold stress.Compared to the control,REL growth of P.densiflora var. zhangwuensis and P.sylvestris var.mongolica at-60°Cwere 0.41 and 0.60,and MDA growth was 29.94 mol g-1FW and 47.80 mol g-1FW,and Fv/Fmdeclines were 0.08 and 0.27.Half-lethal temperatures(LT50)calculated by logistic equation for P.densiflora var.zhangwuensis and P. sylvestris var.mongolica were-58.23 and-50.34°C, respectively.These data suggest that cold resistance of P. densiflora var.zhangwuensis is stronger than that of P. sylvestris var.mongolica.Cold-resistance mechanisms of the two species differed.In response to cold stress,P. sylvestris var.mongolica had strong osmotic adjustment ability because of higher Pro and SS content,while P. densiflora var.zhangwuensis had strong antioxidantability due to stronger CAT activity.Stomata density and diameter of P.densiflora var.zhangwuensis were smaller,as were single leaf area and number of leaves per plant,both characteristics promoting survival in a cold environment. Greater shoot height and total biomass of seedlings of P. densiflora var.zhangwuensis might be another reason for its stronger cold tolerance.

Antioxidantability·Maximum photochemicalefficiency·Osmotic adjustment·Pinus densiflora var.zhangwuensis·P.sylvestris var.mongolica· Relative conductivity

Introduction

Pinus sylvestris var.mongolica,a large tree of the Pinaceae is a geographical variant of Scots pine(Pinus. sylvestris),which grows in Mongolia and Heilongjiang in mainland China where cool summers and cold winters contribute to a low average annual temperature of 0–6°C and extreme minimum temperatures of-40–50°C.In the 1950s,the Liaoning Province Sand Fixation andAfforestation Research Institute(LPSFARI)introduced the species into Horqin sandy land in a successfulreforestation project.Now P.sylvestris var.mongolica has become one of China’s main dune fixation and afforestation conifer species.P.densiflora var.zhangwuensis,another sandy conifer,was also selected by LPSFARI(Zhang etal.1995). Its ratio ofannualheightgrowth to thatof P.sylvestris var. mongolica is 121%in normal years and 130–150%in droughtyears thus ithas potentialfor use in reforestation. Ithas been planted in many regions of China,and a largescale demonstration area has been established in Longjiang County,Heilongjiang province.We hypothesized that P. densiflora var.zhangwuensis could be grown in any areas where P.sylvestris var.mongolica proved successful.To testthis hypothesis,we compared cold hardiness ofthe two species.Drought and salinity resistance of P.densiflora var.zhangwuensis has been reported during recent years (Meng et al.2010,2013),but its cold hardiness has not been studied.

Cold hardiness is the ability of a seedling to withstand exposure to freezing conditions.It is an important physiologicalcomponentof seedling quality(Davis etal.2011). The ionic leakage technique is appropriate for studying changes in physical properties of membranes,and it can compare cold hardiness by calculating half-lethal temperatures(LT50)(AslMoshtaghi et al.2009).Since it is improbable that only a single indicator(e.g.relative conductivity,REL)could accurately reflect plant hardiness, use of multiple indices is essential to reveal the complete mechanism of cold hardiness(Koster and Lynch 1992). These indices include maximum photochemical efficiency (Fv/Fm,related to intrinsic capacity for photosynthesis) (Islam etal.2011),malondialdehyde(MDA,related to the extent of lipid peroxidation),proline(Pro)and soluble sugar(SS,related to osmotic regulation capacity),catalase (CAT,related to antioxidantcapacity)(Kuroda etal.1991), and stomata density,which is negatively correlated to overwintering plantsurvivalrate(Cuiand Ma 2007).These indices are closely linked to each other under cold stress. Mochida and Itamura(2009)compared cold tolerance among Japanese persimmon strains and found that one of strains(‘‘Abe’’)had higher REL at low temperatures because SS contentin its stem was significantly lower than in other strains.Wang et al.(2014)compared cold tolerance of six cultivars of Iris germanica and found that REL was negatively correlated with Pro in almost all cultivars with the declining temperature.Yang et al.(2013)found that SS and Pro were significantly positively correlated in walnut shoots under low temperature stress.Liu et al. (2010)studied cold-regulated genes and found that overexpression of a certain gene(AtICE1)could lead to simultaneous accumulation of Pro and SS.

Our study objective was to compare cold hardiness and mechanisms of cold hardiness between P.densiflora var. zhangwuensis and P.sylvestris var.mongolica by measuring the above indices to provide a theoretical basis for incorporating P.densiflora var.zhangwuensis into reforestation programs in cold regions.

Materials and methods

Site description

Ourtestsiteswere located atZhanggutai(42°43′N,122°29′E, at elevation of 226.5 m),Zhangwu County,Liaoning province,China.Mean annual precipitation for this area is 433 mm,mean evaporation is 1570 mm,mean annualtemperature is 6.7°C,frost-free period is 154 days,≥10°C effective accumulated temperature is 2800–3200°C.Soils are sandy and formed from riveralluvium,and the depth of the sand layer is about30 m.

Plant material description

Three-year-old container plantlets of P.densiflora var. zhangwuensis and P.sylvestris var.mongolica were cultured in containers(20 cm diameter upper opening,and 15 cm height)using a prepared growth medium(local sandy soil-peat medium at 5:1 by volume).In April2011, 100 container plantlets per species were placed in the outdoor nursery of LPSFARI and all were exposed to the same environment conditions and the same management regime(irrigation and fertilization).The plantlets were exposed to natural photoperiods during dormancy without cold-proof measures to elicit natural cold resistance responses.

Healthy container plantlets(50 plantlets per species) were chosen and transferred to the laboratory in October 2011(before hardening phase)and March 2012(after hardening phase)for low-temperature experiments.Branches and needles of the plantlets were rinsed with distilled water,then blotted with clean filter paper,and transferred to freezing cabinets and exposed to low-temperatures treatments.Five temperature regimes,5,-10,-20,-40 and-60°C,were set up taking 5°C as the control(CK), according to published literature,which concluded thatthe LT50s of P.sylvestris var.mongolica seedlings before and after hardening were-20.06 and-46.19°C,respectively (Wang et al.2008a,b).We tested five replications per treatment.Every low-temperature treatmentwas conducted for24 h in dark with gradually decreasing temperature ata rate of 5°C h-1followed by a short period of recovery (24 h)at5°C.

Plant growth estimation

Growth indices of the two species after hardening(10 plantlets per species)were estimated before determination of physiologicalindices,including shootheight(SH),total biomass(TB),single leafarea(SLA),and numberofleaves perplant(NLP),in which TB was determined by the ovendrying method and SLA was calculated using Image Analysis System 10.0 software.

Determination of physiological indices of needles

Determination of electrolyte leakage was performed according to the method of Xuan et al.(2009)with some modifications.Ten needles were taken from five plantlets per treatment after thawing,and washed with deionized water and wiped with filter paper.From the centralportion of each needle a 20 mm section was cutinto pieces,which was divided into 3 fresh samples,0.5 g per sample.The samples were placed in a beaker filled with 60 mL deionized waterwhere they soaked for24 h atroom temperature. Initial electrical conductivity(C1)of the solution was measured with a digital conductivity meter(DDS-11C) equipped with platinum black electrodes.To killthe needle tissues,the beakers were placed in a 100°C waterbath and boiled for 20 min,then placed on one shaker,shaking for 2 h.After the solution cooled,final conductance(C2)was measured.For each determination,deionized water was used as the controlto determine the conductance value of a blank(Cblank).Formula(1)was used to calculate REL:

where RELis he relative conductivity,C1isinitialelectrical conductivity,and C2is the final conductance.LT50the lowesttemperature causing 50%damage,was determined by plotting REL(relative conductivity)againsttemperature, using a logistic regression model:

where x represents freezing temperature,and c represents inflection point temperature,which is LT50(°C).Those parameters in Eq.(2)were calculated with SPSS 17.0 software.

Fv/Fm(maximum photochemical efficiency)values for both species before and after hardening was measured using a portable pocket PEA,in which the maximum fluorescence(Fm)was determined in saturation lightintensity of 3500μmol m-2s-1.Needles in the sunny side of both species in the nursery were measured before daybreak on October 2011 and March 2012 using five replications for each species(five plantlets per species).White tags with string were tied to the bottom of needles measured in October 2011 to measure the same needles in March 2012, and avoid variations due to needle arrangement.In addition,needle Fv/Fmvalues were measured for both species after hardening by exposure to different freezing temperatures.Thawed container plantlets were removed from freezing cabinets and their needle samples were dark adapted for30 min,then detection was measured using five replications per species.

MDA(malondialdehyde),Pro(proline),SS(soluble sugar)and CAT(catalase)were determined from needle samples of both species after hardening by exposure to different freezing temperature(5 plantlets per treatment) using the spectrophotometric method,indene three ketone colorimetric method,sulfuric acid anthrone colorimetric method and Na2S2O3titration method(Zhang and Qu 2003).

Stomata density was estimated according to the method of Alvin and Boulter(1974)with some modifications.Five needles per species were taken as specimens from container plantlets of the two species before hardening in the outdoor nursery at 1000 h.These were socked in 5% glutaraldehyde solution(neutralized by 0.025 M Na3PO4buffer)for 3 h,then rinsed and fixed in 1%osmium tetroxide for 5 h.Alcohol series was used for specimen dehydration and Au for coating.Stomata density and diameter of needle specimens were measured using a Leo VP-435 SEM scanning five views on each needle.

Data processing

Alldata were analyzed using SPSS17.0 software,and data between groups were subjected to one-way ANOVA and to Fisher’LSD multiple comparison test(P＜0.05).Figures in this study were drawn with Excel 2003 software.

Results

Growth characters

SH(shoot height)and TB(total biomass)of P.densiflora var.zhangwuensis were 1.79 and 1.49 times greater, respectively,than that of P.sylvestris var.mongolica.P. densiflora var.zhangwuensis had lower SLA and NLP (71.4 and 72.0%of P.sylvestris var.mongolica) (Table 1).Differences in all four parameters were significant.

Relative conductivity and LT50

Logistic regression equations plotting REL against temperature before hardening phase(October)were:

Table 1 Plant growth indices of P.densiflora var. zhangwuensis and P.sylvestris var.mongolica

P.sylvestris var.mongolica:

P. densiflora var. zhangwuensis :

Logistic regression equation plotting REL against temperature after hardening phase(March):

P.sylvestris var.mongolica:

P.densiflora var.zhangwuensis:

The above LT50values showed thatcold resistance of P. densiflora var.zhangwuensis was slightly lower than that of P.sylvestris var.mongolica before hardening with a difference of about 2°C(Fig.1).REL values for P.densiflora var.zhangwuensis were higher,butthe gap between the two species narrowed with decreasing temperature. After hardening,cold resistance of P.densiflora var. zhangwuensis increased and was ultimately significantly higher than that of P.sylvestris var.mongolica with a difference of nearly 8°C.REL values of P.densiflora var. zhangwuensis were lower,and REL value at-60°C was 2.71 times CK,while itreached as high as 3.07 times for P. sylvestris var.mongolica.

Fv/Fm(maximum photochemical efficiency)ratio

Before hardening,Fv/Fmvalues for the two species were similar,0.80 for P.densiflora var.zhangwuensis,and 0.79 for P.sylvestris var.mongolica.After 5 months of hardening,Fv/Fmof both species declined to 92.5%of the prehardening level for P.densiflora var.zhangwuensis and 70.9%for P.sylvestris var.mongolica.The Fv/Fmratio of P.densiflora var.zhangwuensis was 1.32 times that of P. sylvestris var.mongolica(Fig.2).The reduction Fv/Fmvalue after hardening for P.sylvestris var.mongolica (0.18),was greaterthan for P.densiflora var.zhangwuensis (0.06).

Fig.1 Change in REL(relative conductivity)of freezing needles in P.densiflora var.zhangwuensis and P.sylvestris var.mongolica seedlings by temperature measured in October(left)and March(right).Mean±SD of five replications(n=5,5 plantlets per treatment)

Fig.2 Comparison of Fv/Fm(maximum photochemical efficiency) by species as measured in October and March(mean±SD,n=5,5 plantlets per species).Different lowercase letters indicate significant difference between the species in the same treatment at 0.05 level. Different uppercase letters indicate significant difference between treatments in the same species at 0.05 level

Fv/Fmdeclines with decreasing temperature were recorded for both species after hardening,and the rate of decline for both species was similar to that of CK at-10, -20 and-40°C.At-60°C Fv/Fmlevels for both species were significantly lower than the level for CK.Fv/Fmat -60°C was 89.2%of the level for CK for P.densiflora var.zhangwuensis and 51.1%for P.sylvestris var.mongolica.Under every treatment,Fv/Fmof P.densiflora var. zhangwuensis was significantly higher than that of P.sylvestris var.mongolica(P＜0.05),by a factor ranging from 1.32 to 2.31 times.With decreasing temperature,gaps between Fv/Fmvalues for the two species increased (Fig.3).

MDA(malondialdehyde)content

Fig.3 Effect of low temperature stress on Fv/Fm(maximum photochemicalefficiency)in needles of two species(mean±SD,n=5,5 plantlets a species).Different lowercase letters indicate significant difference between the species in the same treatment at 0.05 level. Different uppercase letters indicate significant difference

MDA content initially decreased and then increased with declining temperature,which is consistentwith the change in REL(Fig.4).Under temperatures of-40°C or higher, MDAs of both species were similar to thatof CK,showing the extentoflipid peroxidation in theircellmembranes did not rise.MDA content of both species at-60°C was significantly higher than that of CK(P＜0.05).MDA contents of P.densiflora var.zhangwuensis and P.sylvestris var.mongolica at-60°C were 29.94 mol g-1FWand 47.80 molg-1FW higher than CK,respectively.These data suggest that the two species were injured to different extents,while P.densiflora var.zhangwuensis was only slightly injured.

Pro(proline)content

Pro contents of P.sylvestris var.mongolica were higher than those of P.densiflora var.zhangwuensis for all treatments.The difference between them was significantat temperatures in the range of-20–40°C(P＜0.05).With declining temperature,Pro for both species showed a similar trend,increasing to a peak at-40°C (18.98μg g-1FW for P.densiflora var.zhangwuensis, 22.10μg g-1FW for P.sylvestris var.mongolica),which were significantly greater than that of CK(P＜0.05).Pro content for both species at-60°C dropped rapidly to almost the same level as CK(Fig.4).

SS(soluble sugar)content

SS contents of P.sylvestris var.mongolica were significantly higherthan those of P.densiflora var.zhangwuensis for all treatments(P＜0.05).With declining temperature, SS of the two species showed a trend similar to thatof Pro, increasing first then dropping.SS contents reached a peak at-40°C,8.10 mg g-1DW for P.densiflora var. zhangwuensis and 12.24 mg g-1DW for P.sylvestris var. mongolica.Compared to CK,a larger increase was recorded for P.densiflora var.zhangwuensis(6.84 mg g-1DW) and a smaller increase for P.sylvestris var.mongolica (3.42 mg g-1DW),both of which were significantly higher than that of CK(P＜0.05).SS content for both species at-60°C showed decreasing tendency(Fig.4).

CAT(catalase)activity

Mean CAT activity of P.densiflora var.zhangwuensis (3848.59 U g-1FW)was 1.87 times higher than thatof P. sylvestris var.mongolica(2053.41 U g-1FW)in all treatments.In comparison to CK,the CAT amplitudes of P. densiflora var.zhangwuensis were smaller than those of P. sylvestris var.mongolica.CAT decline appeared in both species with decreasing temperature no lower than-20°C. With further decrease of temperature,CAT activity in needle samples of P.densiflora var.zhangwuensis began to rise.Compared to-20°C,its CAT activity increased by 20.0%at-40°C and 25.5%at-60°C,and thedifferences were significant(P＜0.05).CAT activity for P.sylvestris var.mongolica remained constant at temperatures lower than-20°C(Fig.4).

Fig.4 Effect of low temperature stress on some physiological indices.Mean±SD of five replications(n=5,5 plantlets per treatment).Different lowercase letters indicate significant difference between the species in the same treatment at 0.05 level.Different uppercase letters indicate significantdifference between treatments in the same species at 0.05 level

Stomata density

P.densiflora var.zhangwuensis had significantly lower mean stomata density(105.3 stomata mm-2)than P.sylvestris var.mongolica(STOMATA DENSITY)(Fig.5).P. densiflora var.zhangwuensis had significantly smaller mean stomata diameter(18.5μm)than P.sylvestris var. mongolica(MEAN STOMATA DIAMETER).The stomata line of P.sylvestris var.mongolica was dense,often two stomata lines together,and derangement of stomata often formed stomata belts comprised of 2 or 3 stomata lines, which led to greater stomata density.

Discussion

Freezing temperature can result in alterations of cell membranes and the resulting impaired integrity is connected with leakage of electrolytes,which causes REL to increase.Cold stress can often induce oxidative stress (Apostolova et al.2008),which causes the extent of lipid peroxidation to rise and MDA content to increase.Therefore,plants always showed the same tendency in REL and MDA content with increasing cold stress.Cold stress can lead to accumulation ofactivated oxygen species(AOS)in plants(Scebba et al.1998),especially when enduring double stresses of cold and high light intensity during overwintering.This can induce photo inhibition,followed by more AOS production.Photosynthetic structure can also be destroyed resulting in decline of Fv/Fm(O¨quist and Malmberg 1989).This study confirmed the same regularity existed in P.densiflora var.zhangwuensis and P.sylvestris var.mongolica.REL and MDA levels for both species at -60°C began to significantly exceed those of CK.The smaller increase REL and MDA for P.densiflora var. zhangwuensis suggested it suffered from slight injury.Fv/ Fmvalues forthe two species at-60°C were significantly lower than those of CK,and a smaller decrease was recorded for P.densiflora var.zhangwuensis.A smaller decrease in Fv/Fmwas also recorded in this species after hardening as compared with before hardening.Previous research showed that the intensity of freeze damage toconiferous species is related to the rate of reduction of Fv/ Fm,and species with smaller decreases in Fv/Fmoften suffer slight injury under cold stress(Adams and Perkins 1993).Fv/Fmwill decline in conifers during cold acclimation(Bigras and Bertrand 2006)and species maintaining a larger Fv/Fmafter hardening are often plants with strong cold resistance(Woods et al.1991).P.densiflora var. zhangwuensis proved more tolerant of cold stress than P. sylvestris var.mongolica due to its smaller increases in REL and MDA,and smaller decrease in Fv/Fm.LT50of P. densiflora var.zhangwuensis was about 8°C lower than that of P.sylvestris var.mongolica after hardening,confirming that the cold hardiness of P.densiflora var. zhangwuensis was stronger,although P.sylvestris var. mongolica is also a cold-tolerant species because its LT50was less than-50°C.

Fig.5 Stomata size,shape and density of P.densiflora var.zhangwuensis(left)and P.sylvestris var.mongolica(right).Samples were magnified 200 times in the upper panel and 500 times in the lower panel

Cold resistance is considered from the viewpoint of osmotic adjustment and antioxidant ability.Protection of the cell membrane is essential for survival during freezing and it is usually achieved by accumulation of compatible solutes like Pro and SS.SS not only supplies plant energy and nutrients,but also regulates physiological activity of downstream cold-related genes as primary messengers in signal transduction(Ma et al.2009).Therefore,it has been confirmed that levels of the two substances increase in various plants under low-temperature stress(Alberdi et al. 1993;Yano et al.2005).In this study,at temperatures higher than-40°C,Pro and SS content for two species increased with declining temperature,indicating their improved cold-resistance by increasing osmotic adjustment capacity.Higher Pro and SS for P.sylvestris var.mongolica in every treatment suggested that its capacity for osmotic adjustmentto cold stress is stronger than thatof P. densiflora var.zhangwuensis.Pro and SS content of the two species peaked at-40°C,and then decreased, showing that cold stress lower than-40°C is beyond the range ofosmotic adjustment.Pro and SS contentofthe two species peaked before temperatures reached LT50,consistentwith survey results offreezing resistance ofrye(Puma rye)reported by Koster and Lynch(1992).Pro and SS content in both species dropped rapidly at-60°C,especially in P.sylvestris var.mongolica,both indices decreasing to the same level as that of CK.A similar phenomenon has been reported for other plants.Wang etal. (2014)compared cold tolerance within 6 cultivars of Iris germanica and found that with the drop in temperature(from-2 to-23°C),the contentofPro initially increased, then decreased,and finally Pro content at-23°C was not significantly different from that at-2°C.Yang et al. (2013)found that when the stress temperature dropped to -30°C,SS content in walnut was significantly lower than CK level.These results suggest that when the stress temperature exceeds the threshold ofplanttolerance,synthesis of Pro would be blocked due to the loss ofenzyme activity and damage to the antioxidant system,and SS would be consumed excessively as energy.

Cold-resistance of plants is correlated with efficiency of their antioxidative systems,since AOS can often be produced during cold stress.CAT is an antioxidative enzyme, and it can scavenge excess AOS produced in the peroxisome,glyoxysome,mitochondria and chlorophyll(Foryer etal.1994;Bowler etal.1992).Some plants improve cold tolerance by inducing expression of CAT1,which encodes CAT(Singh etal.2011).In our study,higher activity and lower amplitude of CAT for P.densiflora var.zhangwuensis was recorded in alltreatments.CAT activity began to rise at temperatures below-20°C,and increased by 25.5%at-60°C over levels at-20°C.This indicates that CAT activity for P.densiflora var.zhangwuensis is strong and steady,and its antioxidant ability is stronger than that of P.sylvestris var.mongolica.

Stomata density is nota factor directly influencing cold hardiness of plants.However,lower stomata density can prevent drying of needles during winter,since trees have great difficulty in absorbing water when the temperature drops below 0°C(Wang et al.2008a,b).In some broadleaved tree studies,stomata density and stomata size indicate cold-resistant genotypes(Roselli et al.1989; Roselliand Venora 1990;Aslamarz and Vahdati2010).We found thatstomata density was lowerand stomata diameter smaller in P.densiflora var.zhangwuensis,SLA and NLP were also smaller,allofwhich are beneficialto survivalin a cold environment.Differences in seedling size can influence subsequentfield performance(South etal.2005). Timmis and Tanaka(1976)found that smaller Douglas-fir (Pseudotsuga menziessii Mirb.)seedlings were less cold hardy than largerones.Davis etal.(2011)also found there was a positive correlation between seedling size of P. palustris and its cold hardiness.In ourstudy,higher SH and TB in seedlings of P.densiflora var.zhangwuensis might be another reason for its stronger cold tolerance.

Cold resistance of P.densiflora var.zhangwuensis before hardening was slightly lower than that of P.sylvestris var.mongolica,because needles of the former still carry on photosynthesis in October while the latter has entered dormancy(Meng et al.2012).After 5 months of hardening,cold hardiness of the two species improved owing to cold acclimation(Thomashow 1999).Some researchers reported thatincrease in sugar contentand new protein synthesis will lead to improvement of cold hardiness(Zhang etal.2005).We found that Pro and SS content for both species increased with decreasing temperature to -40°C,which support the above view.

Conclusion

In this study,post-hardening REL and MDA values ofboth species followed similartrends in variation with decreasing temperature,whereas Fv/Fmfollowed the opposite trend. During cold stress,smaller increase of REL and MDA and smaller decrease of Fv/Fmfor P.densiflora var.zhangwuensis suggested that it is more tolerant to cold than P. sylvestris var.mongolica.LT50for P.densiflora var. zhangwuensis and P.sylvestris var.mongolica were -58.23 and-50.34°C,respectively.Their strategies for adapting to cold stress were slightly different.When encountering cold stress,P.sylvestris var.mongolica had stronger osmotic adjustment capacity,while P.densiflora var.zhangwuensis had stronger antioxidant capacity.Stomata density was lower and stomata diameter was smaller for P.densiflora var.zhangwuensis,while SLA and NLP values were also smaller,all characteristics that benefit survival in a cold environment.Higher SH and TB in seedlings of P.densiflora var.zhangwuensis might be another reason for its stronger cold tolerance.

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11 June 2014/Accepted:20 November 2014/Published online:19 July 2015

?Northeast Forestry University and Springer-Verlag Berlin Heidelberg 2015

Project funding:This work was supported by the National Forestry Public Welfare Industry Research Project(201004023)and Liaoning Agricultural Science and Technology Key Project(2011207002 and 2011207004).

The online version is available at http://www.springerlink.com

Corresponding editor:Zhu Hong

?Peng Meng mengpeng18@163.com

1Liaoning Province Sand-Fixation and Afforestation Research Institute,Fuxin 123000,People’s Republic of China