Rapid freezing injury in red spruce: seasonal changes in sensitivity and effects of temperature range

On calm, cold days in winter, sun-exposed needles of red spruce (Picea rubens Sarg.) may warm 10 to 20 degrees C above ambient air temperature, and undergo rapid (>/= 1 degrees C min(-1)) fluctuations in temperature as light breezes or passing clouds alter the energy balance of the foliage. It has been proposed that the resulting rapid freeze-thaw cycles (freezing stress) cause a type of winter injury in montane red spruce that is characterized by necrosis of sun-exposed foliage. In autumn and winter, we monitored rapid freezing stress response of needle sections from 10 montane red spruce trees by subjecting needles to rapid freezing over the temperature span typically recorded in the field. In autumn, experimental rapid freezing stress produced severe injury only at temperatures considerably lower than expected for that time of year. In winter, rapid freezing caused occasional, moderate injury in fully hardened foliage of trees susceptible to both slow and rapid freezing. Seasonal changes in sensitivity to rapid and slow freezing were correlated, suggesting that environmental factors that are known to affect sensitivity to slow freezing may also affect sensitivity to rapid freezing. Experimental manipulation of the start and end temperatures of rapid freezing stress events showed that moderate to severe needle injury can occur in susceptible trees at temperature spans slightly more extreme than those typically recorded in the field. The extent of injury was similar at different starting temperatures if rapid freezing occurred over the same temperature span. Year-old foliage was consistently less sensitive to rapid freezing stress than current-year foliage, but some year-old foliage was damaged when the rapid freezing stress regime caused severe injury in current-year foliage. We conclude that rapid freeze-thaw cycles can explain light to moderate injury of current-year foliage, but they do not explain the more severe and widespread pattern of foliar damage that has occurred intermittently over at least the last 18 years.     

Midwinter needle temperature and winter injury of montane red spruce

To assess the role of solar warming and associated temperature fluctuations in the winter injury of sun-exposed red spruce foliage, we used fine wire thermocouples to monitor midwinter needle temperature in the upper canopy of mature red spruce trees over two winters. In 1989-1990, 15-min mean temperatures were recorded for six needles in a single tree. In 1990-1991, 10-min mean temperatures of six needles in one tree, and 1-min mean temperatures of seven needles in a second tree were recorded during rapid temperature changes. Warming was more frequent and greatest on terminal shoots of branches with a south to southwest aspect. The maximum rise above ambient air temperature exceeded 20 degrees C, and the maximum one minute decrease in temperature was 9 degrees C, with maximum rates of 0.8 and 0.6 degrees C min(-1) sustained over 10- and 15-min intervals, respectively. These data demonstrate that red spruce is subject to rapid temperature fluctuations similar to those known to produce visible injury in American aborvitae, a much hardier species. We concluded that solar warming to temperatures above the freezing point was unlikely to result in dehardening and subsequent freezing injury, because warming was infrequent, of short duration, and did not always raise needle temperature above the freezing point. Parts of branches and some individual shoots were frequently covered by snow or rime that may have prevented injury by reducing the frequency or intensity of needle temperature fluctuations. Radiation load on exposed shoots may have been increased by reflection of short wave radiation from snow and rime deposits on surrounding surfaces, which would exacerbate temperature fluctuations.     

Freezing cycles enhance winter injury in Picea rubens

We examined changes in chlorophyll absorbency in red spruce (Picea rubens Sarg.) foliage in response to simulated freezing cycles. Current-year branch tips were collected from 16 trees on January 8, January 20, February 8 and February 26, 1996. Tissue was subjected to freezing cycle treatments with a minimum of -35 degrees C and a maximum of 3 degrees C for a one-cycle treatment, and -9, -6, -3, 0 or 3 degrees C for four-cycle treatments. Samples were frozen at a rate of 5 degrees C h(-1), and warmed at 12 to 15 degrees C h(-1). Controls were held at -9 degrees C. Temperatures during the three-day periods preceding each sample date averaged -18, 4.7, -9.6 and 3.7 degrees C, respectively. On January 8, treated trees showed no significant (P > 0.1) increase in the breakdown of chlorophyll, as measured by the ratio of chlorophyll a absorbency (435 nm) to phaeophytin a absorbency (415 nm), compared with control branch tips. On later sampling dates, seven trees consistently exhibited needle reddening and nine exhibited few symptoms (< 10% of total needle surface reddened) after four-cycle treatments. On February 26, chlorophyll degradation in trees with needle reddening differed (P < 0.05) from the control by 26, 26, 16, 14 and 15% for the 3, 0, -3, -6 and -9 degrees C maxima, respectively. No detectable chlorophyll degradation occurred after a one-cycle treatment in any trees on any sampling date. Freezing cycles with sub-zero maxima and a -35 degrees C minimum enhanced winter injury in red spruce after a midwinter thaw had rendered the trees susceptible to freezing damage.     

Effects of radiational heating at low air temperature on water balance, cold tolerance, and visible injury of red spruce foliage

Recent studies have shown that winter needle mortality in red spruce (Picea rubens Sarg.) is increased by exposure to direct solar radiation, possibly as a result of photo-oxidative damage, accelerated winter desiccation, or reduced cold tolerance due to heating of sun-exposed needles. In an experiment at controlled subfreezing air temperatures of -10 to -20 degrees C, visible radiation was less effective than infrared radiation in producing needle desiccation and visible injury during freeze-thaw cycles. However, visible radiation produced a red-brown color in injured needles, similar to natural winter injury, whereas injured needles exposed to infrared radiation were yellow and injured needles kept in darkness were dark brown. Thus, visible radiation was necessary to produce the red-brown color of damaged needles, but not the injury itself. Needle desiccation was not strongly correlated with visible injury, but the pattern of variation in visible injury among trees and the positive correlation between electrolyte leakage and visible injury suggested that freezing damage following freeze-thaw cycles might cause the visible injury. This was confirmed by a second experiment that showed loss of cold hardiness in needles thawed by radiational heating for six consecutive days. Even with a constant nighttime temperature of -10 degrees C, six days of radiational heating of needles to above freezing caused a small (2.8 degrees C) mean decrease in needle cold tolerance, as measured by electrolyte leakage. Continuous darkness at -10 degrees C for six days resulted in an estimated 5.6 degrees C mean increase in needle cold tolerance. Freezing injury stimulated desiccation: cooling at 4 degrees C h(-1) to -43 or -48 degrees C increased the dehydration rate of isolated shoots by a factor of two to three during the first day after thawing. Within three days at 15 to 22 degrees C and 50% relative humidity, the mean water content of these shoots fell to 60% or lower, compared to 90% or greater for unfrozen controls or shoots subject to less severe freezing stress. In some but not all severely freeze-stressed shoots, accelerated needle desiccation and abscission were accompanied by a red-brown color typical of red spruce winter needle injury. We conclude that severe winter desiccation in red spruce may often be due to prior freezing injury, increased as a result of exposure to direct solar radiation. Furthermore, freezing injury in red spruce may sometimes cause desiccation and abscission of green needles.     

Cold tolerance and water content of current-year red spruce foliage over two winter seasons

Red spruce (Picea rubens Sarg.) in high elevation forests of northeastern North America suffers from frequent and severe winter injury, leading to apical dieback, decreased growth, and high mortality. To examine the role of winter desiccation and freezing injury in winter damage, weekly assessments of cold tolerance and water content were made on current-year foliage collected from native red spruce trees at a high elevation site over two winter seasons. In both years, foliage maintained high water contents and adequate cold tolerance; nonetheless, slight to moderate injury was observed each year on some trees. Despite brief thaw periods each winter, no mid-winter dehardening sufficient to put foliage at risk of freezing injury was evident. These findings suggest that, at least in some years, winter injury to current-year red spruce foliage is produced by a mechanism other than desiccation or absolute low temperatures.     

In situ experimental freezing produces symptoms of winter injury in red spruce foliage

Two mechanisms have been proposed to explain winter injury to needles of red spruce (Picea rubens Sarg.): (1) desiccation, which is characterized by net loss of foliar water from the needle to the environment, with cell injury resulting from dehydration; and (2) freezing, which is characterized by direct injury to cells resulting from intracellular or extracellular ice formation during exposure to low temperature. To compare the separate and combined effects of freezing and desiccation, branches of a mature red spruce at 1160 m were (a) experimentally frozen in situ to -50 degrees C; (b) cut and tied in their original orientation and allowed to desiccate passively; or (c) both frozen in situ and cut and tied in their original orientation. Needle water content, electrolyte leakage (an index of cell injury), and needle color were monitored for 60 days after treatment. Freezing resulted in immediate increases in electrolyte leakage, rapid water loss, and reddening necrosis of needles similar to that of naturally injured needles. Cutting resulted in more gradual water loss, no significant changes in electrolyte loss until severe desiccation had occurred, and a change in the color of the needles to a dull green. Because freezing produced reddening necrosis, a key symptom of winter injury, whereas desiccation did not, we conclude that freezing is probably the primary cause of winter injury in red spruce, and that desiccation is a secondary effect.     

Calcium addition at the Hubbard Brook Experimental Forest increases sugar storage, antioxidant activity and cold tolerance in native red spruce (Picea rubens)

In fall (November 2005) and winter (February 2006), we collected current-year foliage of native red spruce (Picea rubens Sarg.) growing in a reference watershed and in a watershed treated in 1999 with wollastonite (CaSiO(3), a slow-release calcium source) to simulate preindustrial soil calcium concentrations (Ca-addition watershed) at the Hubbard Brook Experimental Forest (Thornton, NH). We analyzed nutrition, soluble sugar concentrations, ascorbate peroxidase (APX) activity and cold tolerance, to evaluate the basis of recent (2003) differences between watersheds in red spruce foliar winter injury. Foliar Ca and total sugar concentrations were significantly higher in trees in the Ca-addition watershed than in trees in the reference watershed during both fall (P=0.037 and 0.035, respectively) and winter (P=0.055 and 0.036, respectively). The Ca-addition treatment significantly increased foliar fructose and glucose concentrations in November (P=0.013 and 0.007, respectively) and foliar sucrose concentrations in winter (P=0.040). Foliar APX activity was similar in trees in both watersheds during fall (P=0.28), but higher in trees in the Ca-addition watershed during winter (P=0.063). Cold tolerance of foliage was significantly greater in trees in the Ca-addition watershed than in trees in the reference watershed (P<0.001). Our results suggest that low foliar sugar concentrations and APX activity, and reduced cold tolerance in trees in the reference watershed contributed to their high vulnerability to winter injury in 2003. Because the reference watershed reflects forest conditions in the region, the consequences of impaired physiological function caused by soil Ca depletion may have widespread implications for forest health.     

Age-related changes in foliar morphology and physiology in red spruce and their influence on declining photosynthetic rates and productivity with tree age

The contribution of changes in meristem behavior to age-related decline in forest productivity is poorly understood. We studied age-related trends in needle morphology and gas exchange in a population of red spruce (Picea rubens Sarg.) growing in a multi-cohort stand where trees ranged from first-year germinants to trees over 150 years old, as well as in grafted scions from these trees. In the field study, age-related trends in foliar morphology were determined in six cohorts ranging in age from 2 to 120 years, and differences in gas exchange characteristics were compared between 60- and 120-year age classes. In a common-rootstock study, scions from trees representing 20-, 60-, and 120-year cohorts were grafted onto juvenile rootstock and maintained for three growing seasons, after which morphological and physiological foliar attributes were evaluated. The field study revealed significant age-related trends in foliar morphology, including decreasing specific leaf area, and increasing needle width, projected area, and width/length ratio. Similar trends were apparent in foliage from the grafted scions. Both in situ foliage and shoots of grafted scions from the oldest cohort showed significantly lower photosynthetic rates than their counterparts from younger trees; however, differences in stomatal conductance and internal CO(2) concentrations were not significant. These results suggest that: (1) foliage of red spruce exhibits age-related trends in both morphology and physiology; (2) age-related decreases in photosynthetic rates contribute to declining productivity in old red spruce; (3) declines in photosynthetic rates result from nonstomatal limitations; and (4) age-related changes in morphology and physiology are inherent in meristems and persist for at least 3 years in scions grafted to juvenile rootstock.     

Modeling shoot water contents in high-elevation Picea rubens during winter

During the winter of 1990-1991, a meteorological tower was established at an 880-m elevation site within the spruce-fir zone on Mt. Moosilauke, New Hampshire, USA. Hourly means of air, needle and trunk temperatures, wind velocity, relative humidity and solar radiation were recorded. On a weekly basis, shoots that had elongated during the preceding growing season were collected from four red spruce (Picea rubens Sarg.) trees and their relative water contents (RWC) determined. Cuticular resistances of needles from these shoots were measured four times during the winter.Measured meteorological parameters were used in a previously developed model to simulate changes in red spruce shoot RWC during the winter. The modeled results were compared to measured shoot RWCs. The predictive power of the model was improved when it was modified to include measured values of cuticular resistance and needle and trunk temperatures. The new version of the model accurately predicted RWC from late December 1990 to the beginning of April 1991, after which spring recharge appeared to occur. We conclude that water lost from foliage was easily replaced by stored reserves and that uptake of water by the roots was not required to maintain an adequate foliar water content during the winter.     

Frost hardiness of Picea rubens growing in spruce decline regions of the Appalachians

It has been proposed that pollutants predispose Picea rubens Sarg. growing in the high Appalachians to frost damage. The pattern of autumn hardening of P. rubens growing at Whiteface Mountain, NY, and Newfound Gap, NC, was monitored by detaching shoots at 1-3 weekly intervals, air freighting them to Scotland, and freeze-testing them. The temperatures that produced freezing injury from August 1986 to January 1987 were compared with minimum air temperatures recorded in those months at nearby meteorological stations over 22 previous years. There was only weak evidence that the onset or degree of frost hardening was inadequate to protect the trees from direct freezing injury (as opposed to winter desiccation). Historically, minimum air temperatures occasionally fell below the lethal temperature for a 10% kill (LT(10)), but they rarely fell below the LT(50). The trees hardened rapidly in the autumn (max. 2.2 degrees C day(-1)) to between -30 degrees C and -40 degrees C by January (LT(50)), including trees showing visible decline on Clingman's Dome, TN. Individual trees differed in hardiness by up to 10 degrees C. It is concluded that any pollutant-induced susceptibility to freezing injury is insufficient, on its own, to account for forest decline in the Appalachians.     

Accretion, partitioning and sequestration of calcium and aluminum in red spruce foliage: implications for tree health

Calcium (Ca) is an essential macronutrient in plants and is an important component of many cellular structures and physiological processes as well as overall forest function. Aluminum (Al) in soil solution can inhibit Ca uptake by plants and disrupt many Ca-dependent metabolic and physiological processes of plants. The ratio of Ca to Al in soil solution can be an important indicator of forest health, especially on acid soils. We used sequential chemical extractions (water, acetic acid and hydrochloric acid) to assess the chemical availability of Ca and Al in foliage from mature red spruce (Picea rubens Sarg.) trees growing under ambient environmental conditions. In plants deficient in Ca and with intermediate total foliar Ca concentration ([Ca]), Ca preferentially accrued in labile and physiologically available forms (water- and acetic acid-extractable). In plants with total foliar [Ca] above a "sufficiency" threshold, Ca also accrued in a chemically sequestered form with low solubility (HCl-extractable), suggesting that Ca sequestration is an inducible process in response to excess foliar Ca. Because it has low solubility, it is likely that sequestered Ca is unavailable for Ca-dependent physiological processes. Immobilization of Al in foliage was related to Ca sequestration, suggesting that Ca sequestration may provide a passive mechanism for Al tolerance in the foliage of these trees. Aluminum immobilization was evident based on the ratio of HCl-extractable Al to the more labile (water- and acetic acid-extractable) forms of Al. Sufficient labile Ca combined with Al sequestration was associated with plant health, including enhanced foliar accretion of Mg and Mn, greater tree growth, enhanced foliar cold hardiness and reduced winter injury. These findings demonstrate that not all chemical forms of foliar Ca and Al are of equal physiological significance and underscore the importance of assessing the biologically significant element forms in biogeochemical research.     

Nutrient contents and concentrations in relation to growth of Picea abies and Fagus sylvatica along a European transect

Mineral nutrition of Norway spruce (Picea abies (L.) Karst.) and beech (Fagus sylvatica L.) was investigated along a transect extending from northern Sweden to central Italy. Nitrogen (N) concentrations of needles and leaves in stands growing on acid soils did not differ significantly between central Italy and southern Sweden (1.0 +/- 0.1 mmol N g(-1) for needles and 1.9 +/- 0.14 mmol N g(-1) for leaves). In both species, foliar N concentrations were highest in Germany (1.2 mmol N g(-1) for needles and 2.0 mmol N g(-1) for leaves) and decreased by 50% toward northern Sweden (0.5 mmol N g(-1)). Both species showed constant S/N and P/N ratios along the transect. Calcium, K and Mg concentrations generally reflected local soil conditions; however, Mg concentrations reached deficiency values in Germany. Leaf area per unit dry weight varied significantly along the transect with lowest values for Norway spruce recorded in northern Sweden and Italy (3.4 m(2) kg(-1)) and a maximum in central Europe (4.7 m(2) kg(-1)). A similar pattern was observed for beech. Despite the low variation in foliar N concentrations on the large geographic scale, local and regional variations in N concentrations equalled or exceeded the variation along the entire continental transect. Furthermore, nutrient contents (i.e., nutrient concentration x dry weight per needle or leaf) showed a greater variation than nutrient concentrations along the transect. Nitrogen contents of Norway spruce needles reached minimum values in northern Sweden (2.4 micro mol N needle(-1)) and maximum values in Denmark (5.0 micro mol N needle(-1)). The N content of beech leaves was highest in Denmark (242 micro mol N leaf(-1)). At the German site, foliar N content rather than N concentration reflected the seasonal dynamics of foliar growth and N storage of the two species. During foliage expansion, there was an initial rapid increase in N content and a decrease in N concentration. This pattern lasted for about 2 weeks after bud break and was followed by 6 weeks during which dry weight and N content of the foliage increased, resulting in a further decrease in N concentration. During summer, dry weight and N content of mature needles of Norway spruce increased further to reach a maximum in autumn, whereas N concentration remained constant. In spring, reallocation of N from 1- and 2-year-old needles was 1.5 and 1.0 micro mol N needle(-1), respectively. This remobilized N was a major source of N for the development of new needles, which had an N content of 1.5 micro mol N needle(-1) after bud break. The seasonal remobilization of N from old foliage decreased with increasing needle age. Needle N content and dry weight decreased progressively with age (1 micro mol N needle(-1) between age classes 2 and 5), whereas N concentrations remained constant. For Norway spruce, annual stemwood production was correlated with needle N content but not with foliar N concentration or with the total amount of N in the canopy. Interspecific and geographical differences in plant nutrition are discussed on the basis of competitive demands for C and N between growth of foliage and wood.     

Winter desiccation and injury of subalpine red spruce

Montane red spruce (Picea rubens Sarg.) in the northeastern United States has undergone a decline during the past two decades. One symptom associated with the decline syndrome is the episodic browning of first-year foliage in early spring. To examine the potential role of winter desiccation in this browning, the water relations of red spruce foliage in a subalpine forest on Mt. Moosilauke, New Hampshire, USA, were monitored from January to May, 1989. All sampled trees lost water during the winter and the first-year foliage on some trees turned brown in early spring. The relative water content of first-year shoots during the winter was a significant predictor of spring browning; red spruce trees that showed browning had desiccated faster and reached lower relative water contents. Damaged trees also had more closely packed needles and lower cuticular resistances to water loss. The first-year shoots had a significantly lower average relative water content than older shoots before and after browning. Cuticular resistance to water loss decreased with elevation. Sun-exposed shoots lost more water than shaded shoots because of solar heating of needles. Winter desiccation can occur before the decline-related spring browning of red spruce foliage.     

Acquired thermotolerance of jack pine, white spruce and black spruce seedlings

The acquired thermotolerance of first-year seedlings of jack pine (Pinus banksiana Lamb.) hardened at 36, 38, 40 or 42 degrees C for 90, 180 or 360 minutes and of black spruce (Picea mariana (Mill.) B.S.P.) hardened at 34, 36, 38 or 40 degrees C for 30, 90, 180 or 360 minutes was determined by comparison of needle damage to that of non-hardened seedlings (25 degrees C) following exposure to temperatures of 49 and 47.5 degrees C, respectively. Compared to seedlings kept at 25 degrees C, heat injury sustained from exposure to high temperatures was markedly reduced following hardening for 180 minutes at 36 and 38 degrees C in jack pine and black spruce, respectively. Increasing the exposure time at 36 degrees C in jack pine, and at 36 to 40 degrees C in black spruce, also reduced needle damage. The duration of increased thermotolerance was investigated in jack pine, black spruce and white spruce (Picea glauca (Moench) Voss) by comparing heat injury from high temperatures in non-hardened seedlings and in seedlings hardened at 38 degrees C for 180 minutes a day for either 1, 3 or 6 days. In all three species, the duration of acquired thermotolerance increased with the number of days of heat hardening. For jack pine and white spruce seedlings hardened at 38 degrees C for 6 days, increased thermotolerance persisted for at least 14 and 10 days, respectively, after the end of the hardening treatment. In contrast, the thermotolerance of black spruce seedlings hardened at 38 degrees C for 6 days remained elevated for only 4 days.     

Freezing tolerance of conifer seeds and germinants

Survival after freezing was measured for seeds and germinants of four seedlots each of interior spruce (Picea glauca x engelmannii complex), lodgepole pine (Pinus contorta Dougl. ex Loud.), Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and western red cedar (Thuja plicata Donn ex D. Donn). Effects of eight seed treatments on post-freezing survival of seeds and germinants were tested: dry, imbibed and stratified seed, and seed placed in a growth chamber for 2, 5, 10, 15, 20 or 30 days in a 16-h photoperiod and a 22/17 degrees C thermoperiod. Survival was related to the water content of seeds and germinants, germination rate and seedlot origin. After freezing for 3 h at -196 degrees C, dry seed of most seedlots of interior spruce, Douglas-fir and western red cedar had 84-96% germination, whereas lodgepole pine seedlots had 53-82% germination. Freezing tolerance declined significantly after imbibition in lodgepole pine, Douglas-fir and interior spruce seed (western red cedar was not tested), and mean LT50 of imbibed seed of these species was -30, -24.5 and -20 degrees C, respectively. Freezing tolerance continued to decline to a minimum LT50 of -4 to -7 degrees C after 10 days in a growth chamber for interior spruce, Douglas-fir and lodgepole pine, or after 15 days for western red cedar. Minimum freezing tolerance was reached at the stage of rapid hypocotyl elongation. In all species, a slight increase in freezing tolerance of germinants was observed once cotyledons emerged from the seed coat. The decrease in freezing tolerance during the transition from dry to germinating seed correlated with increases in seed water content. Changes in freezing tolerance between 10 and 30 days in the growth chamber were not correlated with seedling water content. Within a species, seedlots differed significantly in freezing tolerance after 2 or 5 days in the growth chamber. Because all seedlots of interior spruce and lodgepole pine germinated quickly, there was no correlation between seedlot hardiness and rate of germination. Germination rate and freezing tolerance of Douglas-fir and western red cedar seedlots was negatively correlated. There was a significant correlation between LT50 after 10 days in the growth chamber and minimum spring temperature at the location of seedlot origin for interior spruce and three seedlots of western red cedar, but no relationship was apparent for lodgepole pine and Douglas-fir.     

Seasonal differences in freezing stress resistance of needles of Pinus nigra and Pinus resinosa: evaluation of the electrolyte leakage method

Seasonal changes in freezing stress resistance of needles of red pine (Pinus resinosa Ait.) and Austrian pine (Pinus nigra Arnold) trees were measured by an electrolyte leakage method and by visual observation. During most of the year, freezing stress resistance determined by the two methods gave similar results. The electrolyte leakage method provided a good estimate of seasonal changes in freezing stress resistance except for red pine needles in their most winter-hardy state. To obtain a reliable estimate of freezing stress resistance in winter-hardy red pine needles it was necessary to combine the electrolyte leakage method with visual observations. When red pine needles survived exposure to -80 degrees C or lower, electrolyte leakage was never more than 30% even when the needles were exposed to a slow freeze-thaw stress of -196 degrees C. However, rapid freezing of red pine needles to -196 degrees C resulted in electrolyte leakage of over 80%. Red pine needles attained a much higher freezing stress resistance during the winter than Austrian pine. Red pine needles also acclimated and deacclimated faster than Austrian pine needles. An index of injury was developed based on the electrolyte leakage method ((R(2) + R(1))/2, where R(1) is the minimum % electrolyte leakage from noninjured tissue and R(2) is the maximum % electrolyte leakage at the highest injury) that reliably predicted freezing stress resistance of pine needles for most of the year. Important aspects for developing a successful index of injury for pine needles are: use of cut needles, vacuum infiltration and shaking during incubation in water.We conclude that: (1) during cold acclimation the cell wall properties of the pine needles changed and these changes, which appeared to differ in the two species, might explain the very low leakage of electrolytes from winter-hardy needles of red pine; (2) pine needles survive winter by developing the ability to tolerate extracellular ice formation, because after rapid freezing the needles were severely injured; and (3) red pine is adapted to a shorter growing season and colder winters than Austrian pine.     

Effects of simulated thaw on xylem cavitation,residual embolism,spring dieback and shoot growth in yellow birch

Yellow birch seedlings (Betula alleghaniensis Britt.) that had lost more than 90% of their stem hydraulic conductivity during ambient winter temperatures were exposed to 0 and 20 days of a simulated winter thaw followed by a 48-h freezing treatment at 0, -5, -10, -20 and -30 degrees C. After measuring freezing injury to shoots and roots, the seedlings were placed in a greenhouse where recovery of xylem conductivity and new growth were measured. Shoot xylem cavitation was measured as percent loss of hydraulic conductivity. Shoot freezing injury was assessed by electrolyte leakage (EL) and root freezing injury was assessed by EL and triphenyl tetrazolium chloride reduction. Seedlings pretreated with thaw had higher stem water contents and suffered more freezing damage to roots and shoots (at -20 and -30 degrees C, respectively) than unthawed seedlings. After 3 weeks in a greenhouse, seedlings from the 0, -5 and -10 degrees C freezing treatments showed complete recovery of xylem conductivity, with substantially increased stem water contents. Poor recovery of hydraulic conductivity was observed only in seedlings that were subjected to freezing treatments at -20 and -30 degrees C, regardless of thaw treatment. Of these embolized seedlings, however, only those not previously thawed showed recovery of hydraulic conductivity or regained stem water content after 9 weeks in the greenhouse. Shoot dieback, bud burst and length of new shoots were significantly related to the extent of stem xylem cavitation and freezing injury. We conclude that (1) the simulated winter thaw predisposed yellow birch seedlings to freezing damage in shoots and roots by dehardening tissues and increasing their water content; (2) root freezing damage in turn affected the seedlings' ability to refill embolized stem xylem, resulting in considerable residual xylem embolism after spring refilling; (3) further recovery of stem xylem conductivity was attributable to growth of new vessels; (4) and the permanent residual embolism, together with root and shoot freezing injury, caused increased dieback, bud mortality and reduced growth of new shoots.     

The effects of Arceuthobium sichuanense infection on needles and current‐year shoots of mature and young Qinghai spruce (Picea crassifolia) trees

Arceuthobium sichuanense is a hemiparasitic angiosperm that infects Qinghai spruce (Picea crassifolia Kom.) in Qinghai province, China, and causes severe damage to spruce forests in Qinghai‐Tibet Plateau. In this study, the impact of A. sichuanense infection on mature and young trees of Qinghai spruce was evaluated by examining needle and current‐year shoot morphology, needle water and nitrogen‐use efficiency (NUE) and needle nitrogen concentration. The most apparent effect of A. sichuanense infection was a significant reduction in both needle size distal to infection and current‐year shoot length in the infected branches (p < 0.001). Per cent reductions in needle and current‐year shoot length were similar between mature and young trees (58.9 vs. 56.3%; 59.7 vs. 62.9%). There was a high degree of correlation in foliar δ¹⁵N values between the dwarf mistletoe and its host trees (R² = 0.9017, p < 0.001), while the foliar δ¹³C values of A. sichuanense were similar to those of infected mature and young spruce trees. The dwarf mistletoe infection also resulted in a significant decrease in host needle N concentration and δ¹³C values (p < 0.001). The per cent reduction in needle N concentration in young trees was nearly twice as much as that in mature trees (20.49 vs. 11.54%), while the per cent reduction in needle δ¹³C values was similar between young and mature trees (?0.98 vs.?1.1‰). The NUE in mature trees was not affected by A. sichuanense infection, but the NUE in young trees was increased by the infection.     

Relationship between freezing tolerance and shoot water relations of western red cedar

Freezing tolerance and shoot water relations parameters of western red cedar (Thuja plicata Donn) seedlings were measured every 2 weeks from October 1989 to April 1990. Freezing tolerance, measured by freeze-induced electrolyte leakage, showed seasonal shifts in the temperature causing 50% foliage electrolyte leakage (LT(50)). The LT(50) value was -4 degrees C in October, it decreased to -20 degrees C in February and then increased to -6 degrees C in April. The foliage index of injury at -10 degrees C (II(-10)) also showed seasonal shifts from a high of 98% in October to a low of 18% in February followed by an increase to 82% in April. Osmotic potentials at saturation (Psi(s(sat))) and turgor loss point (Psi(s(tlp))) were, respectively, -1.07 and -1.26 MPa in October, -1.57 and -2.43 MPa in January, and -1.04 and -1.86 MPa in April. Dry weight fraction (DWF) increased and symplastic volume at full turgor (V(o)) decreased during the fall-winter acclimation phase, whereas DWF decreased and V(o) increased during the late winter-spring deacclimation phase. Relationships between seasonal patterns of freezing tolerance and shoot water relations parameters showed that LT(50) and II(-10) decreased linearly as Psi(s(tlp)) and V(o) decreased and DWF increased. There was no discernible difference in the relationship during fall acclimation or spring deacclimation. The freezing dehydration index at -10 degrees C (FDI(-10)) declined from 0.69 in November to 0.41 in February and increased to 0.56 in April. The value of II(-10) decreased linearly as FDI(-10) decreased, although a measurement made on actively growing spring foliage did not fit this relationship. The results indicate that seasonal changes in freezing tolerance of western red cedar are partially due to changes in tissue water content, symplastic volume, passive osmotic adjustment and FDI(-10).     

Frost resistance and susceptibility to ice formation during natural hardening in relation to leaf anatomy in three evergreen tree species from New Zealand

Foliar frost resistance of three endemic New Zealand land trees, Nothofagus menziesii (Hook. f.) Oerst. (Fagaceae), Pittosporum eugenioides A. Cunn. (Pittosporaceae) and Griselinia littoralis Forst. f. (Cornaceae), was examined as the trees hardened from late summer to midwinter in a lowland forest site. The lowest temperatures causing 50% damage (LT(50)) occurred in late winter and were similar to those recorded for other forest trees native to New Zealand (-11.7 degrees C in N. menziesii, -10.7 degrees C in P. eugenioides, and -10.6 degrees C in G. littoralis). All three species hardened by 4-7 degrees C, with G. littoralis showing the least frost resistance in summer and hence the greatest degree of hardening. Thermal analysis during freezing indicated that all three species became more tolerant of extracellular ice formation in winter. Measurements of chlorophyll a fluorescence correlated well with visible injury. The differing patterns of frost damage development in the three species were related to leaf anatomy: visible injury was localized within the small compartments formed by the highly septate leaves of the most resistant species, N. menziesii, and was somewhat localized in the partially septate leaves of P. eugenioides, whereas damage could be initiated anywhere in the aseptate leaves of G. littoralis,which was the least frost resistant species, particularly in summer.