Autumn cold acclimation and freezing tolerance of three oak species in semi-Mediterranean Zagros forests

Introduction

Cold stress is one of the environmental stress that limits the growth and geographical distribution of forest trees across latitudinal and altitudinal gradients (Alberdi & Corcuera, 1991Alberdi M, Corcuera LJ, 1991. Cold acclimation in plants. Phytochemistry30: 3177-3184. 10.1016/0031-9422(91)83172-H). In temperate climates, tree survival frequently depends on withstanding extremely low temperatures both before and after winter hardening (Sakai & Larcher, 2012Sakai A, Larcher W, 2012. Frost survival of plants: responses and adaptation to freezing stress. Ecological Studies, vol 62. Springer Verlag, p 321.). During autumn, plants are exposed to both chilling and freezing stress (Gao et al., 2020Gao CH, Zhao Y, Zhao Y, 2020. Stem water content for crape myrtle in response to drought, cold, and disease stress. J Sens2020: 2893069. 10.1155/2020/2893069). Chilling stress can be defined as occurring above 2°C and is less harmful than freezing stress for plants. Models of climate simulation showed that despite global warming trends, extreme events ‒ such as early and late season frosts ‒ are more frequent (IPCC, 2013IPCC, 2013. Fifth Assessment Report. Climate Change 2013: The Physical Science Basis. Cambridge University Press, UK.) due to increasing autumn and spring air temperatures that can delay dormancy at the end of the growing season or advance bud break at the start (Gu et al., 2008Gu L, Hanson PJ, Mac Post W, Kaiser DP, Yang B, Nemani R, et al.,2008. The 2007 eastern US spring freeze: increased cold damage in a warming world?BioScience58(3): 253-262. 10.1641/B580311). Consequently, forest trees and seedlings may be increasingly exposed to chilling and freezing stress due to rapid temperature shifts (Rixen et al., 2012Rixen C, Dawes MA, Wipf S, Hagedorn F, 2012.Evidence of enhanced freezing damage in treeline plants during six years of CO2 enrichment and soil warming. Oikos121(10): 1532-1543. 10.1111/j.1600-0706.2011.20031.x). Cold hardening is an important mechanism for many tree species to withstand low temperatures and cope with changing environmental conditions (Hamilton et al., 2016Hamilton JA, El Kayal W, Hart AT, Runcie DE, Arango-Velez A, Cooke JE, 2016. The joint influence of photoperiod and temperature during growth cessation and development of dormancy in white spruce (Picea glauca). Tree Physiol36(11): 1432-1448. 10.1093/treephys/tpw061; Zeps et al., 2017Zeps M, Jansons Ā, Matisons R, Stenvall N, Pulkkinen P, 2017. Growth and cold hardening of European aspen seedlings in response to an altered temperature and soil moisture regime. Agric For Meteorol242: 47-54. 10.1016/j.agrformet.2017.04.015), particularly given predicted shifts in species' geographic ranges to higher altitudes and latitudes especially across the Mediterranean areas (Valavi et al., 2019Valavi R, Shafizadeh-Moghadam H, Matkan A, Shakiba A, Mirbagheri B, Kia SH, 2019. Modelling climate change effects on Zagros forests in Iran using individual and ensemble forecasting approaches. Theor Appl Climatol137(1): 1015-1025. 10.1007/s00704-018-2625-z). Moreover, late flushing species like the oaks are less freezing resistant than earlier flushing species in temperate environments (Vitra et al., 2017Vitra A, Lenz A, Vitasse Y, 2017. Frost hardening and dehardening potential in temperate trees from winter to budburst. New Phytol216(1): 113-123. 10.1111/nph.14698), and global warming increases of 1–4 °C are changing the phenology of trees, negatively impacting the development of cold hardiness and autumn freezing resistance (Pagter & Arora, 2013Pagter M, Arora R, 2013. Winter survival and deacclimation of perennials under warming climate: physiological perspectives. Physiol Plant147: 75-87. 10.1111/j.1399-3054.2012.01650.x; Zeps et al., 2017Zeps M, Jansons Ā, Matisons R, Stenvall N, Pulkkinen P, 2017. Growth and cold hardening of European aspen seedlings in response to an altered temperature and soil moisture regime. Agric For Meteorol242: 47-54. 10.1016/j.agrformet.2017.04.015) due to insufficient physiological transitions in the fall (Hamilton et al., 2016Hamilton JA, El Kayal W, Hart AT, Runcie DE, Arango-Velez A, Cooke JE, 2016. The joint influence of photoperiod and temperature during growth cessation and development of dormancy in white spruce (Picea glauca). Tree Physiol36(11): 1432-1448. 10.1093/treephys/tpw061).

Cold hardening is a gradual process that occurs in autumn when tree species are exposed to low temperatures and declining photoperiods (Beck et al., 2004Beck EH, Heim R, Hansen J, 2004. Plant resistance to cold stress: mechanisms and environmental signals triggering frost hardening and dehardening. J Biosci29: 449-459. 10.1007/BF02712118). Cold hardening processes and freezing tolerance are accompanied by a series of biochemical and physiological changes (Alberdi & Corcuera, 1991Alberdi M, Corcuera LJ, 1991. Cold acclimation in plants. Phytochemistry30: 3177-3184. 10.1016/0031-9422(91)83172-H; Rixen et al., 2012Rixen C, Dawes MA, Wipf S, Hagedorn F, 2012.Evidence of enhanced freezing damage in treeline plants during six years of CO2 enrichment and soil warming. Oikos121(10): 1532-1543. 10.1111/j.1600-0706.2011.20031.x) and low photosynthetic productivity that causes growth cessation (Rossi et al., 2008Rossi S, Deslauriers A, Grièar J, Seo JW, Rathgeber CBK, Anfodillo T, et al.,2008. Critical temperatures for xylogenesis in conifers of cold climates. Glob Ecol Biogeogr17: 696-707. 10.1111/j.1466-8238.2008.00417.x; Bauerle et al., 2012Bauerle W, Oren R, Way D, Qian S, Stoy P, Thornton PE, et al.,2012. Photoperiodic regulation of the seasonal pattern of photosynthetic capacity and the implications for carbon cycling. Proc Natl Acad Sci USA109: 8612-8617. 10.1073/pnas.1119131109). For example, osmotic adjustment through the accumulation of total carbohydrates (Morin et al., 2007Morin X, Ameglio T, Ahas R, Kurz-Besson C, Lanta V, Lebourgeois F, et al.,2007. Variation in cold hardiness and carbohydrates concentration from dormancy induction to bud burst among provenances of three European oak species. Tree Physiol27: 817-825. 10.1093/treephys/27.6.817) and proline (Hare & Cress, 1997Hare PD, Cress WA, 1997. Metabolic implications of stress induced proline accumulation in plants. Plant Growth Regul21: 79-102. 10.1023/A:1005703923347) can allow plants to avoid intracellular freezing in cold hardening tissues of forest species trees (Delauney & Verma, 1993Delauney AJ, Verma DPS, 1993. Proline biosynthesis and osmoregulation in plants. Plant J4: 215-223. 10.1046/j.1365-313X.1993.04020215.x). Sodium and potassium can contribute to the regulation of osmotic potential under environmental stress, including chilling and freezing (Bogeat-Triboulot & Lévy, 1998Bogeat-Triboulot MB, Lévy G, 1998. Contribution of different solutes to the cell osmotic pressure in tap and lateral roots of maritime pine seedlings: effects of a potassium deficiency and of an all-macronutrient deficiency. Ann For Sci55(3): 315-327. 10.1051/forest:19980304). Also, chlorophyll fluorescence as a non-invasive tool also is appreciated for the study of tree species hardening such as oak species under low temperatures (Cavender-Bares et al., 2005Cavender-Bares J, Cortes P, Rambal S, Joffre R, Miles B, Rocheteau A, 2005. Summer and winter sensitivity of leaves and xylem to minimum freezing temperatures: a comparison of co-occurring Mediterranean oaks that differ in leaf lifespan. New Phytol168: 597-612. 10.1111/j.1469-8137.2005.01555.x; Corcuera et al., 2005Corcuera L, Morales F, Abadía A, Gil-Pelegrín E, 2005. Seasonal changes in photosynthesis and photoprotection in a Quercus ilexsubsp. Ballota woodland located in its upper altitudinal extreme in the Iberian Peninsula. Tree Physiol25(5): 599-608. 10.1093/treephys/25.5.599; Cavender-Bares, 2007Cavender-Bares J, 2007. Chilling and freezing stress in live oaks (Quercussection Virentes): intra-and inter-specific variation in PSII sensitivity corresponds to latitude of origin. Photosynth Res94(2): 437-453. 10.1007/s11120-007-9215-8). Another reliable physiological indicator for testing the freezing tolerance and cold hardiness of seedlings and trees is electrolyte leakage (EL) (Flint et al., 1967Flint H, Boyce B, Beattie D, 1967. Index of injury - a useful expression of freezing injury to plant tissues as determined by electrolytic method. Can J Plant Sci47: 229-230. 10.4141/cjps67-043; Koehler et al., 2012Koehler K, Center A, Cavender-Bares J, 2012. Evidence for a freezing tolerance - growth rate trade-off in the live oaks (Quercus series Virentes) across the tropical-temperate divide. New Phytol193: 730-744. 10.1111/j.1469-8137.2011.03992.x). This method is used extensively and shows membrane permeability and degradation of membrane integrity by cold stress (Moshtaghi et al., 2009Moshtaghi EA, Shahsavar AR, Taslimpour MR, 2009. Ionic leakage as indicators of cold hardiness in olive (Olea europaeaL.). World Appl Sci J7(10): 1308-1310.).

The Zagros forests located in the west of Iran cover an area of about 6 million hectares, representing 44% of the country’s forested area (Sagheb-Talebi et al., 2013Sagheb-Talebi KH, Sajedi T, Yazdian F, 2013. Forests of Iran: A treasure from the past, a hope for the future. Research Institute of Forests and Rangelands of Iran Press, p 55. 10.1007/978-94-007-7371-4). Zagros forests are particularly important for water supply, soil conservation, environmental change, and the socio-economical balance of the whole country. Oak species (Quercus brantii Lindl., Quercus infectoria Oliver and Quercus libani Oliver) are dominate in these forests. Also, these species are native to large areas of Mediterranean forests and they are widely distributed in western Asia, namely in Iraq, Syria, Lebanon and Turkey (Mirhashemi et al., 2023Mirhashemi H, Heydari M, Ahmadi K, Karami O, Kavgaci A, Matsui T, et al.,2023. Species distribution models of Brant's oak (Quercus brantiiLindl.): The impact of spatial database on predicting the impacts of climate change. Ecol Eng194: 107038. 10.1016/j.ecoleng.2023.107038).

Seedlings of oak during the first years of their life are very sensitive to environmental stress such as extreme summer drought and low temperatures. Therefore, these unfavorable climatic conditions can limit the distribution of oak species, and also most reforestation projects with these oak species have poor field performance. Some prior research has been conducted on physiological and biochemical responses to low temperatures stress in European or American oak species such as Quercus robur L., Quercus pubescens Willd., Quercus ilex L., Quercus virginiana, Quercus oleoides (Cavender-Bares et al., 2000Cavender-Bares J, Aposto S, Moya I, Briantais JM, Abazzaz F, 2000. Chilling-induced photoinhibition in two oak species: Are evergreen leaves inherently better protected than deciduous leaves?Photosynthetica36(4): 587-596.10.1023/A:1007000406399; 2005; Cavender-Bares, 2007Cavender-Bares J, 2007. Chilling and freezing stress in live oaks (Quercussection Virentes): intra-and inter-specific variation in PSII sensitivity corresponds to latitude of origin. Photosynth Res94(2): 437-453. 10.1007/s11120-007-9215-8; Morin et al., 2007Morin X, Améglio T, Ahas R, Kurz-Besson C, Lanta V, Lebourgeois F, et al.,2007. Variation in cold hardiness and carbohydrate concentration from dormancy induction to bud burst among provenances of three European oak species. Tree Physiol27(6): 817-825. 10.1093/treephys/27.6.817; Andivia, 2012bAndivia E, Márquez-García B, Vázquez-Piqué J, Córdoba F, Fernández M, 2012b. Autumn fertilization with nitrogen improves nutritional status, cold hardiness and the oxidative stress response of Holm oak (Quercus ilex ssp. Ballota [Desf.] Samp) nursery seedlings. Trees26(2): 311-320. 10.1007/s00468-011-0593-3; Koehler et al., 2012Koehler K, Center A, Cavender-Bares J, 2012. Evidence for a freezing tolerance - growth rate trade-off in the live oaks (Quercus series Virentes) across the tropical-temperate divide. New Phytol193: 730-744. 10.1111/j.1469-8137.2011.03992.x), but there is no information for these three oak species (Q. brantii, Q. infectoria and Q. libani) despite the wide distribution of them in Mediterranean forests. On the other hand, understanding the physiological responses to low temperatures and the mechanisms involved in cold acclimation of species originating from altitudinal clines is very limited and it is critical to predicting how species will respond to climate changes (Cheaib et al., 2012Cheaib A, Badeau V, Boe J, Chuine I, Delire C, Dufrene E, et al.,2012. Climate change impacts on tree ranges: model intercomparison facilitates understanding and quantification of uncertainty. Ecol Lett15: 533-544. 10.1111/j.1461-0248.2012.01764.x). This information may also allow the selection of cold-tolerant ecotypes or populations for reforestation.

Therefore, the objectives of our studies were 1) to determine how cold hardening alters physiological responses to low temperature in these three oak species and the traits that contribute to low-temperature resistance after hardening or cold acclimation, and 2) to predict early season freezing resistance in these three species.

Material and methods

Distribution of the oak species studied

 ⌅

Three oak species studies are distributed in the Zagros forests of Iran. The climate of these forests is sub-humid with cold winters according to Emberger's formula (Emberger, 1943Emberger L, 1943. Les limites de l'aire de végétation méditerranéenne en France. Bull Soc Hist Nat Toul78: 158-180.) with a long dry season lasting between 5-7 months. These regions are thus characterized by double stresses of summer drought and winter cold like other Mediterranean environments (Mitrakos, 1982Mitrakos K, 1982. Winter low temperatures in mediterranean-type ecosystems. Ecol Mediterr8(1): 95-102. 10.3406/ecmed.1982.1936). The southern Zagros is both drier and warmer than northern Zagros. These forests are dominated by three oak species, Q. brantii, Q. infectoria, and Q. libani, whose distributions collectively span an area of about 5 million hectares (Fatahi, 1995Fattahi M, 1995. The study of Zagros' Oak forests and the most important factors of its destruction. Research Institute of Forests and Rangelands Press, Tehran, Iran.). Q. brantii has a very wide-ranging distribution and spanning northern and southern Zagros forests, while Q. infectoria and Q. libani are found only in the North of Zagros (Fig. 1). Pure populations of Q. libani are found at higher elevations (from 1500 m asl) than the other two oak species and at north aspects due to humidity conditions. In contrast, Q. brantii may be more adapted to drought and less cold tolerant given its distribution in lower elevation and south aspects. Q. infectoria is located primarily at the middle elevations and north aspects (Sagheb-Talebi et al., 2013Sagheb-Talebi KH, Sajedi T, Yazdian F, 2013. Forests of Iran: A treasure from the past, a hope for the future. Research Institute of Forests and Rangelands of Iran Press, p 55. 10.1007/978-94-007-7371-4).

  (Alijani et al., 2010).

Figure 1 Distribution of different oak species (Nazemi et al., 2008Nazemi J, Talebi AA, Sadeghi SE, Melika G, Lozan A, 2008. Species richness of oak gall wasps (Hymenoptera: Cynipidae) and identification of associated inquilines and parasitoids on two oak species in western Iran. North-West J Zool4(2): 189-202.) and thermal climatic characteristics in Zagros forests, which are divided into northern and southern sections by the horizontal red line at approximately 33°N (Khalyani & Mayer, 2013Khalyani AH, Mayer AL, 2013. Spatial and temporal deforestation dynamics of Zagros forests (Iran) from 1972 to 2009. Landsc Urban Plan117: 1-12. 10.1016/j.landurbplan.2013.04.014). The two days of persistent frost in October in Iran are depicted by the blue lines (Alijani et al., 2010).

Hardening induction and cold temperature treatments

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The experiment had a completely randomized full factorial design with 15 replicates per each hardening treatment and species. To induce cold hardening, 160 of the 200 seedlings were transferred to the growth chamber in July 2017. Thereafter, 15 seedlings of each of the three oak species were randomly selected and removed at the end of each of the four hardening treatments for chilling and freezing treatments (Fig. 2). The hardening induction conditions were simulated in the growth chamber using shortened photoperiods and decreasing day and night temperatures for two months. The four hardening treatments were designed to match the monthly lowest and highest mean temperature in Marivan, north of Zagros forests, the seed origin, in different months as follows: hardening 1‒ temperatures and photoperiods in September; hardening 2‒ October; hardening 3‒ through November, and hardening 4‒ only decreasing of photoperiod (Fig. 2). The four hardening inductions were reflected in the early autumn conditions before a winter freeze. In the initial phase, seedlings were exposed to a decreasing photoperiod and temperature and then only with a photoperiod below 11 hours.

  

Figure 2 Program of cold hardening and chilling or freezing stress treatments. Chl: chlorophyll content. PSII: chlorophyll fluorescence. EL: electrolyte leakage. RWC: relative water content. Pro: proline content. Glu: glucose content. K: potassium content. The scissors show the seedling harvest time following each hardening stage in order to calculate RWC, EL, Pro, Glu, and K. 

After each hardening induction (Control,hardening 1, hardening 2, hardening 3, hardening 4), 15 seedlings of each of the three species were moved to dark cold storage at a chilling temperature of 4°C for one night. Then, the chlorophyll content and chlorophyll fluorescence of seedlings were measured. After that, seedlings were divided into three groups, and each group was randomly assigned to one of the three low-temperature treatments (control (4°C for 2 hours), -20°C (1 hour), and -20°C (2 hours)) (Fig. 2). The freezing (−20°C) and thawing rate of samples were 2 hours. Samples were wrapped in paper bags and aluminum foil to prevent desiccation during freezing. After low temperature exposure, leaves and stem segments of each seedling were cut and sampled from the refrigerator and freezing chamber for other physiological and element measurements (RWC, EL, Proline, Glucose, and K+) (8 replicates per treatment and species).

Results

All PSII traits were restricted by progressing hardening, but the effect of species was only significant for NPQ (Tables 1and 2). Quercus libani had the largest value of NPQ compared to the other two species (1.22 vs 1.04). We found a significant interaction effect between hardening and species for chlorophyll content (Table 1). Thus that, despite the general decline of chlorophyll content by progressing hardening in the studied oak species, the rate of reduction was different in different species. Q. libani responded to simulated hardening quickly, whereas in Q. brantii and Q. infectoria chlorophyll declined at a slower rate (Fig. 3A).

  

Figure 3 The mean (± SE) of interactive effects of treatments (species, cold temperature, and hardening) for measured traits. Vertical bars are standard errors (SE) and different letters indicate significant differences (p < 0.05) of mean values. EL: electrolyte leakage. RWC: relative water content. 

The results of the three-way ANOVA test of the other physiological traits showed that the main effect of species was a significant factor in explaining variation in all traits except for K contents of the stem (Table 3). Also, RWC, and proline displayed highly significant differences among cold temperature treatments (Table 3). On the other hand, the interaction between species, cold temperature, and hardening for all of them except for leaf K was significant. The significant interaction between species and cold temperatures of leaf EL showed that Q. infectoria had the highest value under the chilling condition (4°C), while leaf EL of the other two oak species showed a larger increase under freezing treatments. The increase of leaf EL under freezing temperature was less pronounced in Q. libani and this species showed the lowest leaf EL value under freezing temperature (Fig. 3B). Also, the interaction of cold temperature and hardening showed that stem EL had a lower value under freezing temperature of hardening 2 compared to hardening 1 (Fig. 3C). There were no significant differences between species for stem EL in the hardening 1 treatment, but Q. libani performed significantly better than Q. infectoria in the hardening 2 treatment due to the higher value of stem EL in Q. infectoria (Fig. 3D). Leaf RWC showed the lowest value in the hardening 1 treatment in Q. brantii and declined with progressive cold hardening in both Q. brantii and Q. libani, while leaf RWC was stable between the hardening 1 and hardening 2 treatments in Q. infectoria (Fig. 3E). Also, stem RWC declined during hardening in Q. infectoria and Q. libani but did not change in Q. brantii. However, the RWC values were lower in Q. libani in both hardening treatments (Fig. 3F). Conversely, biochemical traits like proline and glucose increased during hardening treatments except for glucose, which decreased in Q. brantii. There was no significant change for glucose and proline with hardening treatment in Q. infectoria (Figs. 3G, H).

Comparison of means between species revealed that the leaf K content of Q. libani was significantly higher than the other two species (Fig. 4A). Leaf proline content did not change significantly from 4°C to -20°C (1 hour) and then increased after two hours of freezing at -20°C (Fig. 4B). Also, the effect of cold hardening on all traits was significant except for leaf EL and glucose (Table 3). The comparison of hardening 1 and 2 treatments showed that leaf and stem K content increased in the hardening 2 treatment (Figs. 4C, D), but the response of species to cold hardening was different for the other physiological and biochemical traits (RWC, EL, glucose, and proline) (Table 3).

  

Figure 4 Mean values of main effects of treatments (species, cold temperature, and hardening treatments) for measured traits. Vertical bars are standard errors (SE) and different letters indicate significant differences (p < 0.05) of mean values. 

Irrespective of the cold hardening and the freezing treatments, significant correlations among all measured traits were analyzed for each species, individually. Most of the traits in Q. libani and Q. brantii were significantly correlated while in Q. infectoria only leaf EL and leaf RWC had a weak direct correlation (r = 0.2).

In Q. libani, leaf EL was positively correlated with both leaf and stem RWC and negatively correlated with F_v/F_m. Also, K content showed a positive correlation with F_v/F_m. Glucose and proline content of Q. libani were highly positively correlated with each other, while there was no significant correlation between glucose and proline in Q. brantii. Stem RWC of Q. libani was also negatively correlated with leaf and stem K content and F_v/F_m, but the correlation between leaf RWC and F_v/F_min Q. brantii was positive (Fig. 5).

  

Figure 5 Pearson correlation between physiological and biochemical variables in Quercus libani and Quercus brantii. Non-significant correlations (p > 0.05) are marked with a cross. Positive and negative correlations are in blue and red, respectively. The intensity of the color and the size of the circles are proportional to the correlation coefficient. EL: electrolyte leakage. fvTOfm: maximum quantum yield of PSII. RWC: relative water content. 

Principal component analysis (PCA) showed that the first two components explained 45.68% of the total variance. An ANOVA and t-test indicated that these two PCs were significant in different treatments. So, there were significant differences among the species and cold stress temperatures by only PC1. While both PC1 and PC2 were significant among hardening 1 and 2 (Table 4). The PC1 was contributed by the positive coefficient of physiological traits like EL and RWC content and a negative coefficient of K content (scores > 0.50). Q. libani seedlings, chilling stress (4°C), and some extent progressive hardening were aligned on the negative position of PC1 in comparison to the other two species (Q. brantii and Q. infectoria), freezing temperature (-20°C) and hardening 1. High osmolyte concentration, such as glucose and proline, was another characteristic that identified the second PC (Table 4and Fig. 6).

  

Figure 6 PCA-Biplot of the seedlings of different oak species treated by hardening 1 and 2, cold temperature. The different species are shown by a circle (Quercus brantii), a triangle (Quercus infectoria) and a square (Quercus libani). The blue, yellow and red symbol indicates the different cold stress (Control: 4°C for 2 hours, mild stress: -20°C (1 hour) and severe stress: -20°C (2 hours), respectively) and empty and filled symbols are related to hardening 1 (H1) and hardening 2 (H2), respectively. The centroid of each treatment (species, cold hardening and cold temperature) is also shown. 

Discussion

In temperate regions, cold hardening processes are critical for resistance to cold and freezing stress in forest trees (Burke et al., 2003Burke MJ, Gusta LV, Quamme HA, Weiser CJ, Li PH, 2003. Freezing and injury in plants. Annu Rev Plant Physiol27(1): 507-528. 10.1146/annurev.pp.27.060176.002451). In this study, the autumn hardening was stimulated by decreasing photoperiods and temperature for oak seedlings during two months as previously described in other studies (Beck et al., 2004Beck EH, Heim R, Hansen J, 2004. Plant resistance to cold stress: mechanisms and environmental signals triggering frost hardening and dehardening. J Biosci29: 449-459. 10.1007/BF02712118; Hamilton et al., 2016Hamilton JA, El Kayal W, Hart AT, Runcie DE, Arango-Velez A, Cooke JE, 2016. The joint influence of photoperiod and temperature during growth cessation and development of dormancy in white spruce (Picea glauca). Tree Physiol36(11): 1432-1448. 10.1093/treephys/tpw061; Homayounfar et al., 2019Homayounfar S, Zolfaghari R, Fayyaz P, 2019. Effect of cold stress on physiological traits of Pistacia atlantica and P. khinjuk during acclimation. Iran J Forest11(2): 207-219.). During progressive hardening, a reduction in chlorophyll content was observed in the three studied oak species. This result is consistent with other studies that found the preliminary chlorophyll degradation is mostly triggered by the shortening of daylight and further reduction occurrs by decreasing temperature (Fréchette et al., 2016Fréchette E, Chang CYY, Ensminger I, 2016. Photoperiod and temperature constraints on the relationship between the photochemical reflectance index and the light use efficiency of photosynthesis in Pinus strobus. Tree Physiol36(3): 311-324. 10.1093/treephys/tpv143). At the beginning of the hardening period, the amount of leaf chlorophyll content in Q . brantii and Q. libani seedlings diminished more in comparison to Q. infectoria which could provide frost tolerance in these two species by preventing photo-degradation (Oberschelp et al., 2020Oberschelp GPJ, Guarnaschelli AB, Teson N, Harrand L, Podestá FE, Margarit E, 2020. Cold acclimation and freezing tolerance in three Eucalyptus species: A metabolomic and proteomic approach. Plant Physiol Biochem154: 316-327. 10.1016/j.plaphy.2020.05.026). All chlorophyll fluorescence traits also progressively declined during hardening, and in contrast, NPQ increased. In many tree species a restriction in photochemical quenching of photosystem II during cold acclimation has been observed (Cavender-Bares et al., 2000Cavender-Bares J, Aposto S, Moya I, Briantais JM, Abazzaz F, 2000. Chilling-induced photoinhibition in two oak species: Are evergreen leaves inherently better protected than deciduous leaves?Photosynthetica36(4): 587-596.10.1023/A:1007000406399; Zhou et al., 2017Zhou X, Chen S, Wu H, Xu H, 2017. Effects of cold stress on the photosynthesis and antioxidant system of Rhododendron chrysanthum Pall. Preprints1-10. 10.20944/preprints201703.0131.v1; Homayounfar et al., 2019Homayounfar S, Zolfaghari R, Fayyaz P, 2019. Effect of cold stress on physiological traits of Pistacia atlantica and P. khinjuk during acclimation. Iran J Forest11(2): 207-219.) due to a decline in metabolic sink capacity mediated by short photoperiod (Hamilton et al., 2016Hamilton JA, El Kayal W, Hart AT, Runcie DE, Arango-Velez A, Cooke JE, 2016. The joint influence of photoperiod and temperature during growth cessation and development of dormancy in white spruce (Picea glauca). Tree Physiol36(11): 1432-1448. 10.1093/treephys/tpw061) or loss of photochemical efficiency of PSII and inhibition of the electron transport as a consequence of low temperature (Cavender-Bares et al., 2005Cavender-Bares J, Cortes P, Rambal S, Joffre R, Miles B, Rocheteau A, 2005. Summer and winter sensitivity of leaves and xylem to minimum freezing temperatures: a comparison of co-occurring Mediterranean oaks that differ in leaf lifespan. New Phytol168: 597-612. 10.1111/j.1469-8137.2005.01555.x). Furthermore, there were no significant variations across oak species in any of the chlorophyll fluorescence traits except of NPQ. The greater NPQ in Q. libani in comparison to the other two species during hardening suggested that Q. libani, which occurs at higher altitudes, has more efficient mechanisms of photo-protective energy dissipation (Murchie & Lawson, 2013Murchie EH, Lawson T, 2013. Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications. J Exp Bot64(13): 3983-3998. 10.1093/jxb/ert208). Consistent with this result, higher NPQ under chilling temperatures was observed in Q. virginiana found in temperate climates compared to Q. oleoides, which occurs in tropical climates that lack a cold winter period (Cavender-Bares, 2007Cavender-Bares J, 2007. Chilling and freezing stress in live oaks (Quercussection Virentes): intra-and inter-specific variation in PSII sensitivity corresponds to latitude of origin. Photosynth Res94(2): 437-453. 10.1007/s11120-007-9215-8).

Also, there was inter-specific variation in physiological responses between studied oak species to freezing stress after hardening. Freezing temperature can impair the integrity of cell membranes, causing an increase in EL (Rihan et al., 2017Rihan HZ, Al-Issawi M, Fuller MP, 2017. Advances in physiological and molecular aspects of plant cold tolerance. J Plant Interact12(1): 143-157. 10.1080/17429145.2017.1308568). We observed increases in leaf and stem EL with more negative freezing temperatures, as has been reported by many studies (Azzarello et al., 2009Azzarello E, Mugnai S, Pandolfi C, Masi E, Marone E, Mancuso S, 2009. Comparing image (fractal analysis) and electrochemical (impedance spectroscopy and electrolyte leakage) techniques for the assessment of the freezing tolerance in olive. Trees23(1): 159-167. 10.1007/s00468-008-0264-1; Homayounfar et al., 2017Homayounfar S, Zolfaghari R, Fayyaz P, 2017. physiological responses to cold stress in different provenances of Pistacia atlanticaseedlings. Zagros For Res3(2): 27-41.). Also, freezing stress duration time had an increasingly negative effect on seedling leaves from one hour to two hours and this result is not surprising since the previous study has shown the leaves are more sensitive tissue to cold stress (Shao et al., 2013Shao YR, Xu JX, Xue L, Zhang R, Wu CQ, Lu GC, 2013. Effects of low temperature stress time on physiological and biochemical and photosynthetic characteristics four plant species. Acta Ecol Sin33(14): 4237-4247. 10.5846/stxb201301150100). Furthermore, the results of ANOVA and PCA demonstrated that different species had different levels of susceptibility to freezing stress. For example, Q. libani seedlings, which are native to colder regions and higher elevations than other species, displayed a lower increase in leaf EL during freezing stress. These results suggest that Q. libani is more freezing tolerant than Q. brantii or Q. infectoria. Also, a smaller increase in stem EL under freezing stress was observed by progressing the hardening period. These results revealed that freezing resistance is enhanced upon hardening, as expected in temperate species (Koehler et al., 2012Koehler K, Center A, Cavender-Bares J, 2012. Evidence for a freezing tolerance - growth rate trade-off in the live oaks (Quercus series Virentes) across the tropical-temperate divide. New Phytol193: 730-744. 10.1111/j.1469-8137.2011.03992.x; Homayounfar et al., 2017Homayounfar S, Zolfaghari R, Fayyaz P, 2017. physiological responses to cold stress in different provenances of Pistacia atlanticaseedlings. Zagros For Res3(2): 27-41.; Oberschelp et al., 2020Oberschelp GPJ, Guarnaschelli AB, Teson N, Harrand L, Podestá FE, Margarit E, 2020. Cold acclimation and freezing tolerance in three Eucalyptus species: A metabolomic and proteomic approach. Plant Physiol Biochem154: 316-327. 10.1016/j.plaphy.2020.05.026). Yet the response of different oak species to hardening treatments was different, and freezing resistance increased earlier in the Q. libani. Prior observations also showed a more positive effect of cold acclimation on freezing responses in tree species from climates where freezing occurs earlier and is more intense (Cavender-Bares, 2007Cavender-Bares J, 2007. Chilling and freezing stress in live oaks (Quercussection Virentes): intra-and inter-specific variation in PSII sensitivity corresponds to latitude of origin. Photosynth Res94(2): 437-453. 10.1007/s11120-007-9215-8; Homayounfar et al., 2017Homayounfar S, Zolfaghari R, Fayyaz P, 2017. physiological responses to cold stress in different provenances of Pistacia atlanticaseedlings. Zagros For Res3(2): 27-41.). There was an inverse relationship between leaf RWC, EL, and Fv/Fm. Water status is known to affect cold hardening and cold resistance (Worland, 1996Worland MR, 1996. The relationship between water content and cold tolerance in the Arctic collembolan Onychiurus arcticus (Collembola: Onychiuridae). Eur J Entomol93: 341-348.). Indeed, the presence of a large amount of RWC under cold stress caused a cellular injury and was accompanied by a reduction of photosynthesis activity (Balamurugan et al., 2018Balamurugan S, Ann JS, Varghese IP, Murugan SB, Harish MC, Kumar SR, et al.,2018. Heterologous expression of Lolium perenne antifreeze protein confers chilling tolerance in tomato. J Integr Agric17(5): 1128-1136. 10.1016/S2095-3119(17)61735-0). We also found that decreasing RWC was accompanied by increasing in K+ content in Q. libani and Q. brantii. Increasing K⁺content, especially in Q. libani, could protect seedlings from low temperatures by osmotic adjustment of leaf cells upon hardening (Hsiao & Laüchli, 1986Hsiao TC, Lauchli A, 1986. Role of potassium in plant-water relations. Adv Plant Nutr2: 281-312.), consistent with the findings of Gleeson et al. (2004Gleeson D, Lelu-Walter MA, Parkinson M, 2004. Influence of exogenous L-proline on embryogenic cultures of larch (Larix leptoeuropaea Dengler), sitka spruce (Picea sitchensis (Bong.) Carr.) and oak (Quercus robur L.) subjected to cold and salt stress. Ann For Sci61(2): 125-128. 10.1051/forest:2004003) and Andivia et al. (2012aAndivia E, Fernández M, Vázquez-Piqué J, Alejano R, 2012a. Two provenances of Quercus ilex ssp. Ballota (Desf) Samp. Nursery seedlings have different response to frost tolerance and autumn fertilization. Eur J For Res131(4): 1091-1101. 10.1007/s10342-011-0578-1). In addition, an increasing K⁺, proline, and glucose content, as osmo-protectants or scavengers, accompanied by progress of cold hardening and freezing, has been found by other researchers (Meng et al., 2015Meng P, Bai X, Li H, Song X, Zhang X, 2015. Cold hardiness estimation of Pinus densifloravar. zhangwuensis based on changes in ionic leakage, chlorophyll fluorescence and other physiological activities under cold stress. J For Res26(3): 641-649. 10.1007/s11676-015-0111-3). Among species, we found highly significant variation in the accumulation of glucose and proline upon hardening. Because of the higher proline and sugar content of Q. libani, the osmotic adjustment ability of this species was stronger than Q. brantii and Q. infectoria, which originated from milder climates at lower altitudes. Similar results have been reported previously in some other Quercus species (Morin et al., 2007Morin X, Améglio T, Ahas R, Kurz-Besson C, Lanta V, Lebourgeois F, et al.,2007. Variation in cold hardiness and carbohydrate concentration from dormancy induction to bud burst among provenances of three European oak species. Tree Physiol27(6): 817-825. 10.1093/treephys/27.6.817; Fernández et al., 2008Fernandez M, Alejano R, Domınguez L, Tapias R, 2008. Temperature controls cold hardening more effectively than photoperiod in four Mediterranean broadleaf evergreen species. Tree For Sci Biotech2: 43-49.; Andivia et al., 2012bAndivia E, Márquez-García B, Vázquez-Piqué J, Córdoba F, Fernández M, 2012b. Autumn fertilization with nitrogen improves nutritional status, cold hardiness and the oxidative stress response of Holm oak (Quercus ilex ssp. Ballota [Desf.] Samp) nursery seedlings. Trees26(2): 311-320. 10.1007/s00468-011-0593-3). A reverse pattern of glucose in Q. brantii during progressive hardening and a lower value of PC2, which is associated with osmolyte content, demonstrated that glucose was excessively consumed as energy under conditions of severe low-temperature stress (Meng et al., 2015Meng P, Bai X, Li H, Song X, Zhang X, 2015. Cold hardiness estimation of Pinus densifloravar. zhangwuensis based on changes in ionic leakage, chlorophyll fluorescence and other physiological activities under cold stress. J For Res26(3): 641-649. 10.1007/s11676-015-0111-3). Q. brantii was still able to increase cell osmotic to some extent concentration by increasing proline content in comparison to Q. infectoria.

Conclusions

Variation in cold tolerance in three studied oak species corresponds well with the habitat of their origin and provides evidence in support of adaptive plasticity in cold and freezing tolerance (Cavender-Bares, 2007Cavender-Bares J, 2007. Chilling and freezing stress in live oaks (Quercussection Virentes): intra-and inter-specific variation in PSII sensitivity corresponds to latitude of origin. Photosynth Res94(2): 437-453. 10.1007/s11120-007-9215-8; Koehler et al., 2012Koehler K, Center A, Cavender-Bares J, 2012. Evidence for a freezing tolerance - growth rate trade-off in the live oaks (Quercus series Virentes) across the tropical-temperate divide. New Phytol193: 730-744. 10.1111/j.1469-8137.2011.03992.x). Q. libani, which originates from colder climates and higher altitudes has evolved a survival strategy of higher tolerance to freezing stress through earlier and more effective cold acclimation and hardening than other species. The accumulation of osmotic metabolites such as glucose, proline, and K⁺appears to play an important role in seasonal increases in freezing tolerance in Q. libani seedlings. Also, NPQ measurement due to vast variation between species under cold stress is in agreement with the results Hussain et al. (2022Hussain MA, Li S, Gao H, Feng C, Sun P, Sui X, et al., 2022. Comparative analysis of physiological variations and genetic architecture for cold stress response in soybean germplasm. Front Plant Sci13. 10.3389/fpls.2022.1095335) and can be usefully applied as a non-destructive tool to foresters for assessing the development of dormancy status and predicting freezing tolerance in oak species and selecting the genotypes or mother trees of oak species most likely to persist in a changing environment. Moreover, based on PC1 results, measuring EL and RWC could be the next choice for future evaluation, as already reported by other researchers as important indicators of cold tolerance (Wu et al., 2012Wu L, Zhou M, Shen C, Liang J, Lin J, 2012. Transgenic tobacco plants over expressing cold regulated protein CbCOR15b from Capsella bursa-pastoris exhibit enhanced cold tolerance. J Plant Physiol169: 1408-1416. 10.1016/j.jplph.2012.05.016; Balamurugan et al., 2018Balamurugan S, Ann JS, Varghese IP, Murugan SB, Harish MC, Kumar SR, et al.,2018. Heterologous expression of Lolium perenne antifreeze protein confers chilling tolerance in tomato. J Integr Agric17(5): 1128-1136. 10.1016/S2095-3119(17)61735-0).

It appears that Q. infectoriaand Q. brantii are susceptible to early frost based on physiological data. In reality, though, Q. brantii is expected to die at a higher rate than Q. infectoria because early frost danger is higher in lower altitude species with wider geographic ranges. Fortunately, the PCA bi-plot analysis and other studies (Alikhani et al., 2014Alikhani L, Rahmani MS, Shabanian N, Badakhshan H, Khadivi-Khub A, 2014. Genetic variability and structure of Quercus brantii assessed by ISSR, IRAP, and ScoT markers. Gene552(1): 176-183. 10.1016/j.gene.2014.09.034) suggested a high intraspecific variation within Q. brantii. Therefore, artificial screening for frost tolerance based on physiological responses to cold and freezing stress in Q. brantiifamilies and ecotypes is important for the reforestation of Zagros forests.