Abstract

A predominant 24-kD dehydrin-like protein was previously found to fluctuate seasonally within red-osier dogwood (Cornus sericea L.) stems. The current study attempted to determine what environmental cues triggered the accumulation of the 24-kD protein and to assess its potential role in winter survival. Controlled photoperiod and field studies confirmed that photoperiod regulates a reduction of stem water content (SWC), freeze-tolerance enhancement and accumulation of the 24-kD protein. Diverse climatic ecotypes, which are known to respond to different critical photoperiods, displayed differential reduction of SWC and accumulation of the 24-kD protein. A time-course study confirmed that prolonged exposure to short days is essential for SWC reduction, 24-kD protein accumulation, and freeze-tolerance enhancement. Water deficit induced 24-kD protein accumulation and enhanced freeze-tolerance under long-day conditions. In all instances, freeze-tolerance enhancement and 24-kD protein accumulation was preceded by a reduction of SWC. These results are consistent with the hypothesis that C. sericea responds to decreasing photoperiod, which triggers a reduction in SWC. Reduced SWC in turn may trigger the accumulation of the 24-kD protein and a parallel increase in freeze-tolerance.

(Received July 4, 2002; Accepted October 23, 2002)

Introduction

Temperate woody plants have the capacity to survive seasonal temperature fluctuations that are characteristic of their indigenous climate. During late summer and early fall, decreasing day length at warm temperatures induces an initial stage of cold acclimation in woody plants (Hurst et al. 1967, Irving and Lanphear 1967, VanHuystee et al. 1967, Howell and Weiser 1970, Weiser 1970, Fuchigami et al. 1971, Williams et al. 1972, McKenzie et al. 1974a, Chen and Li 1978, among others). In red-osier dogwood (Cornus sericea L.) the first frost rapidly induces a second stage of cold acclimation (VanHuystee et al. 1967, Weiser 1970) and prolonged sub-freezing exposure is hypothesized to initiate a third stage of cold acclimation (Weiser 1970). Once fully acclimated, C. sericea can survive temperatures as low as –269°C (Guy et al. 1986). Warming temperatures during late winter and early spring will induce deacclimation. Following bud burst and the initiation of rapid growth, C. sericea becomes fully deacclimated and is injured at temperatures slightly below zero (°C) (Fuchigami et al. 1982).

C. sericea is a lowland plant, and despite the year round availability of water, it exhibits a reduction of stem water content (SWC) during the fall (McKenzie et al. 1974b). Previous physiological studies have shown that SWC reduction is controlled by short-day (SD) lengths and occurs during the first stage of cold acclimation (McKenzie et al. 1974b, Chen et al. 1975, Parsons 1978, Bray and Parsons 1981). Within this first stage, freeze-tolerance may reach –22°C (McKenzie et al. 1974b). Interestingly, water stress can induce freeze-tolerance in C. sericea grown under inhibitory long-day (LD) conditions (Chen et al. 1975, Chen et al. 1977, Chen and Li 1977). Under these conditions, SWC is directly reduced, irrespective of photoperiod. Since freeze-tolerance can be induced under LD with water stress, it is likely that SWC reduction is an important stimulus for freeze-tolerance induction and is capable of overriding inhibitory LD photoperiod effects.

A common occurrence in both drought and freezing stress is cellular dehydration (Siminovitch and Cloutier 1983). C. sericea, one of the most freeze-tolerant plants known, survives freezing exposure by enduring the dessicative stress that results from extracellular ice formation (Burke et al. 1976). Due to the presence of extracellular ice, a vapor pressure gradient is generated, and intracellular water migrates to the extracellular ice crystals, resulting in intracellular desiccation (Burke et al. 1976, Levitt 1980, Guy 1990, Ristic and Ashworth 1994, among others). Molecular studies have confirmed that both drought and freezing stress induce expression of similar genes and that abscisic acid (ABA) mediates the response in both stresses (Close et al. 1993a, Close et al. 1993b, Campbell and Close 1997, Close 1997). Additionally, exogenous applications of ABA have been shown to enhance freeze-tolerance within previously non-acclimated plants (Chen et al. 1983, Chen and Gusta 1983, Orr et al. 1986, Lang et al. 1989, Lee and Chen 1993, Robertson et al. 1994, Bravo et al. 1998, among others).

Dehydrins (class D-11 of LEA [late embryogenesis abundant] proteins), have been found to accumulate in response to dehydration. These proteins may accumulate during drought, salinization, late stages of seed development, freezing and in response to exogenous ABA (Close et al. 1993a, Close et al. 1993b, Welin et al. 1994, Close 1996, Close 1997, Campbell and Close 1997, among others). Dehydrins are characteristically boiling stable, highly hydrophilic and typically contain at least one lysine-rich consensus sequence similar to EKKGIMDKIKEKLPG. Although dehydrin function remains unknown, the K-consensus sequence may help to stabilize proteins and membranes through interactions of a putative amphipathic α-helix (Close et al. 1993b, Close 1996, Close 1997). Dehydrins may function to limit deleterious effects associated with cellular dehydration (Close 1997).

Woody plants accumulate dehydrin-like proteins during periods of cold acclimation in leaves, buds, and bark (Salzman et al. 1996, Arora et al. 1996, Arora et al. 1997, Wisniewski et al. 1996, Wisniewski et al. 1999, Artlip and Wisniewski 1997, Rinne et al. 1999, among others). The accumulation of dehydrin-like proteins in relation to cold acclimation has been attributed to the occurrence of freeze-induced dehydration (Arora et al. 1997, Close 1997, among others). Seasonal dehydrin fluctuations have been observed in the xylem of multiple Populus species (Sauter et al. 1999), Salixcaprea L. (Sauter et al. 1999) and Prunus persica (Arora et al. 1992, Arora and Wisniewski 1996, Wisniewski et al. 1999).

We have identified a 24-kD dehydrin-like protein that is seasonally regulated in C. sericea wood, and its presence directly correlates to cold acclimation (Sarnighausen et al. 2002). The objectives of the present study were: (1) to identify the environmental cue for the seasonal regulation of a 24-kD dehydrin-like protein and (2) to examine its relation to plant water status and freeze-tolerance. Here we present evidence which couples the presence of a 24-kD dehydrin-like protein to reduced stem water content and the photoperiod regulated stage of cold acclimation in C. sericea xylem. The environmental regulation of the 24-kD protein and its implications as an ecological adaptation for the survival of early annual freezing events are discussed.

Results

Response of C. sericea to seasonal changes in photoperiod

Analysis of tissues harvested at hourly incremental changes in day length showed that SWC, freeze-tolerance, and 24-kD protein accumulation changed seasonally (Fig. 1). During the longest day of the year, stem tip SWC was elevated and progressively decreased with shortening day lengths until reaching lowest levels during winter. The following spring, as day length increased from 13 to 14 h, SWC increased (Fig. 1B) coincident with bud burst (not shown). Freeze-tolerance and 24-kD protein abundance were each inversely related to SWC (Fig. 1B). Maximum electrolyte leakage, which corresponds to minimum freeze-tolerance, occurred when day lengths exceeded 13 h. These also corresponded to the time when SWC was elevated and the 24-kD protein was absent (Fig. 1A, B). The accumulation of the 24-kD protein during the transition from 14 to 12 h day length was observed in both plants grown in the field and in the glasshouse where potential effects from low temperature or limited nutrient and water availability were eliminated (Fig. 2).

C. sericea climatic ecotypes vary in their response to photoperiod

Climatic ecotypes growing at a single field site were sampled to determine if the 24-kD protein and SWC were differentially regulated (Fig. 3). During the longest day of the year, northern ecotypes (AK, NW) exhibited reduced SWC and increased 24-kD protein accumulation relative to the more southern ecotypes (UT, MA). Differential regulation was also observed within the hybrids, where 70-2 and 45-12 resembled northern and southern ecotypes respectively. When day length decreased to 14 h, northern ecotypes exhibited conspicuous accumulation of the 24-kD protein and maintained reduced SWC levels. SWC increased for UT and 45-12, but it decreased in the MA ecotype. The 24-kD protein had not accumulated in UT, 45-12 or MA. As day length decreased to 13 h, northern ecotypes continued to accumulate 24-kD protein and maintained reduced SWC. The MA ecotype had reduced SWC and accumulated 24-kD protein. The 45-12 and UT ecotype maintained elevated SWC and slight 24-kD protein accumulation was detected in 45-12. Collectively these data demonstrated that climatic ecotypes responded differentially to photoperiod when grown under uniform field conditions. SWC reduction and 24-kD accumulation were initiated at longer days in northern ecotypes and 24-kD accumulation correlated to reduced SWC levels.

Determination of the critical photoperiod (Massachusetts ecotype)

During the natural transition from 14 to 13 h day length, C. sericea (Massachusetts ecotype) accumulated 24-kD protein, decreased SWC and increased freeze-tolerance (Fig. 1). To further define the critical photoperiod, the effect of prolonged exposure to individual controlled day lengths was evaluated (Fig. 4). Plants grown under 14 and 16 h day lengths continued growing, had elevated SWC, increased freeze susceptibility and lacked 24-kD protein (Fig. 4). Plants grown under photoperiods of 13 h or less stopped shoot elongation, set terminal buds and developed red bark pigmentation (not shown). Substantial 24-kD protein accumulation, SWC reduction, and increased freeze-tolerance were also detected in these treatments. Plants grown under 8-h SD, but exposed to a night interruption treatment had elevated SWC, were susceptible to freezing and lacked 24-kD protein (Fig. 4). Collectively, these data indicate that the critical photoperiod for SWC reduction, freeze-tolerance augmentation, and 24-kD protein induction is less than 14 h for the Massachusetts ecotype. Lastly, the observation that SD plants exposed to NI react as LD plants suggested that these responses are mediated by photoperiod.

Time course of C. sericea response to SD length

To quantify the critical day length for SD responsiveness, SWC, freeze-tolerance and 24-kD protein accumulation were monitored over time (Fig. 5). Average stem tip SWC was greater than 90% during the first 3 weeks of SD exposure but then decreased linearly to a value of 66% by week 8 (Fig. 5). The LT50 estimation of freeze-tolerance decreased to –8.6°C after 5 weeks SD exposure and a slight increase in 24-kD protein was evident (Fig. 5B, C). Freeze-tolerance increased and additional 24-kD protein accumulated after 8 weeks of SD exposure (Fig. 5B, C). Collectively, these data revealed that progressive SWC reduction was initiated by prolonged SD exposure and that SWC reduction preceded both 24-kD protein accumulation and the induction of freeze-tolerance.

Pith senescence correlates to reduced SWC in C. sericea stems

Fluorescein diacetate vital staining was used for assessment of pith cell senescence during the time-course photoperiod study. Fig. 6 shows representative stem tip cross-sections from C. sericea (Massachusetts ecoype) plants treated for 8 weeks with either 8 or 16 h day lengths. Pith cells senesced in uppermost internodes in plants exposed to prolonged 8 h SD, whereas, the pith tissue was alive in uppermost internodes of 16 h LD controls (Fig. 6).

Water deficit induces an increase of freeze-tolerance and 24-kD accumulation

A soil water deficit was imposed on LD-grown plants to reduce SWC and to evaluate the subsequent affects on freeze-tolerance and 24-kD protein induction. Stressed plants had significantly lower SWC, increased freeze-tolerance and 24-kD protein accumulation in comparison to well-watered controls (Fig. 7). Vital staining with fluorescein diacetate confirmed that the water stress was sub-lethal to xylem tissues (not shown). These data demonstrated that stress-induced SWC reduction can induce 24-kD protein accumulation and enhance freeze-tolerance under a normally inhibitory LD photoperiod.

The effect of frost exposure upon 24-kD protein accumulation

The effect of freezing exposure on 24-kD protein accumulation was investigated by sampling field-grown plants exposed to a natural frost episode (Fig. 8). Significant quantities of 24-kD protein were present prior to the frost exposure and subsequent levels did not dramatically fluctuate 3 and 11 d following frost exposure.

Photoperiod effects upon Jv (root water flux) and Lp (root hydraulic conductivity)

Replicate plants were exposed to either 8 or 16 h photoperiods for 2 and 4 weeks for quantification of Jvand Lp (Fig. 9). Linear relationships of Jv and hydrostatic pressure were observed within all photoperiod treatments. After 2 weeks, no significant difference in either Jvor Lp was observed. However, roots of plants exposed to 4 weeks SD exhibited a significant reduction of Jvin comparison to LD controls. Linear regression analysis indicated that Lp was not significantly altered in response to SD.

Discussion

Photoperiod is a reliable environmental cue that regulates the first stage of cold acclimation in several woody plant species (Weiser 1970, among others). In C. sericea, the first stage of cold acclimation is photoperiod-mediated (McKenzie et al. 1974a, among others), accompanied by gradual stem dehydration and characterized by moderate levels of freeze-tolerance (–22°C) (McKenzie et al. 1974b). Prolonged SD exposure at warm temperatures (VanHuystee et al. 1967, Smithberg and Weiser 1968, McKenzie et al. 1974b) and development of dormancy (Smithberg and Weiser 1968, McKenzie et al. 1974a) are prerequisites for attaining maximum freeze-tolerance upon subsequent exposure to freezing temperatures. With the present study, we describe the photoperiod-dependent regulation of a 24-kD dehydrin-like protein and its apparent relationship to this photoperiod-mediated cold acclimation stage in C. sericea wood tissue.

In field and controlled studies, the 24-kD protein accumulated as SWC decreased and freeze-tolerance increased. 24-kD protein accumulation and freeze-tolerance was markedly increased in the MA ecotype as the day length decreased from 14 to 13 h (Fig. 1). In C. sericea, temperature, water and nutrient availability affect cold acclimation (Hummel et al. 1982), and thereby complicate interpretations from field data. Moreover, low temperature and water deficit may induce dehydrin-like gene expression (Close 1997, among others). These potential effects were eliminated in a comparative study using glasshouse grown plants (Fig. 2) and by comparing results from controlled photoperiod treatments at warm temperatures (Fig. 4, 5). Similar protein regulation was observed in comparison to field samples (Fig. 2). From September 26, 1999 (12-h day length) through February 23, 2000 (11-h day length), SWC remained relatively unchanged. However, freeze-tolerance progressively increased within this same time period (Fig. 1B). This observed increase in freeze-tolerance is likely to be indicative of the photoperiod independent low-temperature induced third stage of cold acclimation (Weiser 1970). Although additional cold acclimation can be induced by frost episode (Weiser 1970), such conditions did not further induce the accumulation of the 24-kD protein (Fig. 8). During spring, disappearance of the 24-kD protein occurred as day length increased from 13 to 14 h (Fig. 1). Unlike the first stage of cold acclimation and the onset of dormancy, deacclimation (Weiser 1970, among others) and bud-break (Smithberg and Weiser 1968, Fuchigami et al. 1982) are temperature-dependent phenomena within C. sericea stems. Further controlled experiments are necessary to determine if 24-kD protein down-regulation responds to warming temperatures or to increasing day length transitions.

Critical day length is defined as the longest photoperiod that induces growth cessation (Olsen et al. 1997). In our controlled studies, all photoperiod treatments less than 14 h day length induced a cessation of growth in the Massachusetts ecotype, an accumulation of 24-kD protein, a reduction in SWC and increased freeze-tolerance. In addition, analysis of plants that were exposed to 8 h of day light plus a night interruption confirmed photoperiod-mediation for each of these responses (Fig. 4).

Woody plants often evolve unique ecotypes that are adapted to photoperiodic conditions specific to their indigenous environment (Olsen et al. 1997, among others). Diverse C. sericea ecotypes have been studied for assessment of differential cold acclimation behavior and photoperiod responsiveness (Smithberg and Weiser 1968, Bray and Parsons 1981, Hummel et al. 1982). When grown under uniform conditions, northern ecotypes respond to longer days, cease growth (Smithberg and Weiser 1968, Hummel et al. 1982), reduce SWC (McKenzie et al. 1974b, Bray and Parsons 1981) and increase their freeze-tolerance prior to southern ecotypes (Smithberg and Weiser 1968, Hummel et al. 1982). We observed that northern ecotypes accumulated the 24-kD protein prior to southern ecotypes and in all cases, 24-kD protein accumulation was preceded by a reduction in SWC (Fig. 3). Northern ecotypes had longer critical photoperiods for SWC reduction and 24-kD protein accumulation (Fig. 3). Perhaps the differential timing for SWC reduction, and subsequent accumulation of 24-kD protein within C. sericea ecotypes, is related to the observation that northern ecotypes cold acclimate prior to southern ecotypes (Smithberg and Weiser 1968, Hummel et al. 1982) and are less susceptible to early seasonal freeze damage (Smithberg and Weiser 1968, McKenzie et al. 1974a).

In previous studies of C. sericea, multiple SD-dependent alterations of plant water relations were found to precede freeze-tolerance induction. Specifically, increased transpiration rates (Parsons 1978), reduced stomatal resistance, decreased root water flux (McKenzie et al. 1974b, Parsons 1978) and pith cell senescence (McKenzie et al. 1974a) were hypothesized to collectively contribute to controlled stem dehydration. Parsons (1978) suggested that stem dehydration occurs because water loss exceeds water uptake during photoperiod-dependent cold acclimation. Both Parsons (1978) and McKenzie et al. (1974b) reported up to a 3.5-fold reduction of root water flux after prolonged SD exposure. However, their results were not corrected for root surface area and may have also incorporated large osmotic effects due to their experimental approach. Our data, which were corrected for root surface area, also demonstrated a reduction of root hydraulic flux (Jv). In comparison to LD controls, Jv was reduced nearly 50% after 4 weeks SD exposure, however, hydraulic conductivity (Lp) was not significantly reduced. McKenzie et al. (1974b) has correlated suberin deposition to prolonged SD exposure. Therefore our observed reduction of Jv after 4 weeks SD exposure may represent a reduced proportion of conductive root surface area in comparison to LD controls.

Although transpiration was not directly measured in the present study, the observed SWC reductions from the controlled day length experiment (Fig. 4) indirectly support transpirational involvement for photoperiod-dependent SWC reduction. In controlled SD treatments (13, 12, 8 h), SWC reduction was greatest in 13 h replicates. Due to the extended light period, the total available time for transpiration would be greatest within 13 h treatments. Throughout the prolonged study, we speculate that this may have had a cumulative effect, which resulted in the greatest SWC reduction. In addition, photoperiod-dependent cold acclimation is prevented with low temperature exposure (Weiser 1970). In a preliminary study, non-acclimated plants were grown in a growth chamber under 8-h short days at low temperatures (7/4°C day/night). After 6 weeks, SWC was not reduced in comparison to control tissue harvested prior to SD and low temperature treatments (unpublished data). In addition, the 24-kD protein had not accumulated in these plants.

Fluorescein diacetate vital staining confirmed that pith senescence is induced under prolonged SD exposure. Due to the large pith volume within C. sericea stems, it is likely that a significant portion of the observed reduction in SWC (Fig. 1, 3, 4, 5) is related to pith senescence. We suspect that SWC is lowered to a critical level during prolonged exposure to SD at warm temperature, and this critical SWC reduction ultimately triggers 24-kD protein accumulation. A water stress that directly reduced SWC independent of photoperiod, triggered 24-kD protein accumulation and FT enhancement within non-acclimated C. sericea (Fig. 7). This suggests that reduced SWC is a necessary prerequisite for both 24-kD protein accumulation and the first stage of cold acclimation in C. sericea.

Photoperiod has been found to affect woody plant gene expression in numerous independent studies (Rowland and Arora 1997). In poplar, Coleman et al. (1991) observed rapid accumulation of bark storage protein subsequent to SD exposure. The idea that a dehydrin-like protein would accumulate in response to photoperiod seems to be unique at first glance and an example of a unique regulatory pathway for such proteins. However, the current study has correlated the regulation of the 24-kD protein to prerequisites of photoperiod-controlled reduction of SWC and prolonged SD exposure. It is likely therefore, that this photoperiod-induced water deficit is the stimulus for 24-kD protein accumulation. The accumulation of dehydrins in response to water deficit is a pattern similar to that observed in many investigations. Given these results, it is unlikely that C. sericea responds directly to short days and triggers 24-kD protein accumulation. Instead, C. sericea undergoes a series of physiological changes that ultimately results in a SWC reduction, which in turn, may serve a 2-fold function. This controlled reduction of SWC may have a preventative function to minimize injurious mechanical effects resultant from ice formation in the winter season (Burke et al. 1976). Second, it may act as a stimulus for programmed accumulation of desiccation-related proteins prior to the first freezing episodes of the season. The timing of freeze-tolerance acquisition has been stated to be more important for tree survival than its actual lower limit for freeze-tolerance (Rinne et al. 1999). Therefore, this adaptation likely increases the chance for survival of the initial freezing episodes of the winter season.

Materials and Methods

Plant material

An established plantation of Cornus sericea L. (Massachusetts ecotype, 42°N) growing in West Lafayette, IN, U.S.A. was used for most experiments. In addition, diverse C. sericea climatic ecotypes were transplanted to a field plot in West Lafayette, IN, U.S.A. (Spring 1998). Ecotypes originating from Alaska (65°N), Northwest Territories (62°N), Utah (42°N) and two F2 hybrid genotypes (70-2, 45-12) which originated from crossing the Utah and Alaska ecotypes and subsequently making sibling crosses between the F1 hybrids (Hummel et al. 1982) were used for intraspecific ecotype analyses. The 70-2 and 45-12 F2 hybrids used in this study were morphologically similar to the Alaska and Utah parent, respectively. Current year’s growth was sampled for all experiments.

Total protein extraction and analysis

Bark and cambium were removed from stems with a scalpel, and remaining wood was cut into small internodal segments and plunged into liquid nitrogen. Samples were lyophilized and subsequently stored at –80°C. Lyophilized tissue was pooled for each treatment and pre-ground in an electric coffee grinder. Samples were subsequently pulverized in a ball mill for 3 min. The powder (100 mg) was transferred to a microcentrifuge tube, mixed with 1 ml of ice cold lauryl sulfate (lithium salt, LiDS) extraction buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w : v) LiDS, 5% (v : v) 2-mercaptoethanol, 2% (v : v) 50 mM PMSF in MeOH), vortexed, and immediately heated for 5 min at 95°C. Samples were centrifuged for 5 min at 14,000 rpm in a microcentrifuge, and 325 µl supernatant was removed and precipitated with 1.625 ml ice cold acetone and stored at –20°C for several h. Additional supernatant was used for protein quantification after treatment with 0.1 M KPO4 buffer for SDS-precipitation. The protein content of KPO4-precipitated extracts were quantified by the Bradford protein assay (Bradford 1976). Acetone-precipitated proteins were briefly centrifuged at 14,000 rpm in a pre-cooled rotor (–20°C) and the supernatant was discarded. After residual acetone evaporated, lauryl sulfate (sodium salt, SDS) buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w : v) SDS, 10% (v : v) glycerol, 5% 2-mercaptoethanol, 0.001% (w : v) bromophenol blue) was added to adjust the final protein concentration to 2 µg µl–1. The pellet was re-suspended and heated for an additional 5 min at 95°C. The supernatant was kept and used for SDS-PAGE electrophoresis. SDS-PAGE and immunoblotting were performed as previously described (Sarnighausen et al. 2002).

Estimation of freeze-tolerance

Freeze-tolerance was estimated using an electrolyte leakage assay. For both field and glasshouse studies, 1 cm sections (n = 10) were cut from the fifth internode from each replicate stem (n = 6), rinsed with nano-pure water, and placed into glass test tubes (2 sections tube–1). Nano-pure water (500 µl) was added to each tube, and samples were maintained at 0°C for 30 min. Ice nucleation was initiated by adding small ice chips to each tube. Samples were subsequently cooled at 2°C h–1 in a NESLAB RTE-120 refrigerated bath and subsets were removed upon reaching –4, –8, –12, and –16°C. Samples were placed into an insulated ice bath to thaw overnight. The following morning, 7 ml nano-pure water was added and specimens were gently agitated at 4°C overnight. Solution conductivity was measured with a YSI Scientific conductance meter. Samples were subsequently boiled for 30 min, allowed to cool to room temperature, and solution conductivity was immediately measured. Percent electrolyte leakage was calculated as (initial/final conductivity) × 100. Average percent electrolyte leakage (n = 6) was plotted for each temperature tested and the LT50 is reported in the results section.

Stem relative water content determination

Stem relative water content (SWC) was measured by sampling internodal cross-sections (1 mm thick) from each internode along the entire shoot axis. Specimen fresh weight (FW) was measured immediately upon harvest, and cross-sections were subsequently immersed in water for at least 8 d at 4°C to allow maximum water uptake. After complete saturation, cross-sections were removed from the tubes, briefly rolled (on edge) across a paper towel to remove excess surface moisture, and immediately weighed to determine saturated sample weight (SW). Sample dry weight (DW) was determined after 4 d of drying at 75°C. SWC was calculated as [(FW–DW)/(SW–DW)] × 100. Average SWC for stem tips (first five internodes) are reported.

Seasonal field photoperiod study

Clonally propagated C. sericea were grown in the field (West Lafayette, IN, U.S.A.) and supplemented with drip irrigation. Current year’s growth was sampled at each hourly change in day length: 15 h – June 23, 1999, 14 h – August 9, 1999, 13 h – September 2, 1999, 12 h – September 26, 1999, 11 h – October 19, 1999, 10 h – November 16, 1999, 9 h 22 min – December 27, 1999, 10 h – January 26, 2000, 11 h – February 23, 2000, 12 h – March 17, 2000, 13 h – April 10, 2000, 14 h – May 9, 2000. Replicate shoots (n = 6) were harvested from the field, wrapped in plastic to minimize desiccation, and brought to the laboratory for immediate analysis.

Field ecotype photoperiod study

Ecotypes originating from Alaska, Northwest Territories, Utah and Massachusetts were used for intraspecific analyses in addition to two F2 hybrids, 70-2 and 45-12. Replicate shoots (n = 4) were harvested and used for subsequent stem tip SWC determination and total protein extractions at each hourly change in day length: 15 h – June 23, 1999, 14 h – August 9, 1999, 13 h – September 2, 1999.

Controlled photoperiod manipulations: determination of the critical photoperiod

C. sericea terminal stem cuttings (approximately 10 cm) were harvested (February 1999) from identical field propagules, dipped in rooting hormone (Hormodin™) and maintained under mist. Rooted cuttings were transplanted into large pots (0.07 m3) containing Scott’s MetroMix 560™ substrate. Six replicate plants were maintained under six different controlled photoperiods: 8, 12, 13, 14, 16 h day length and 8-h day length (+ 1.5 h night interruption) for 8 weeks. All photoperiod treatments consisted of an 8 h exposure to sunlight (black cloths were opened 8 : 00 AM and pulled at 4 : 00 PM daily). The extended day length for the 12, 13, 14, and 16 h treatments was achieved with supplemental incandescent lighting for appropriate time periods. Eight-hour SD plants received no additional incandescent lighting, however, the 8-h SD (+ night interruption) received an additional 1.5 h incandescent lighting during the middle of the dark period (1.5 h was used because shorter interruption periods failed to prevent growth cessation). The extraction and analysis of proteins, freeze-tolerance estimation, and SWC determinations were performed as previously described.

Confirmation of critical photoperiod (field and glasshouse)

Shoots were harvested from replicate field- (n = 6) or glasshouse-grown (n = 3) plants at four times within the natural transition of day length from 14 to 12 h: 14 h – August 9, 1999, 13 h 42 min – August 16, 1999, 13 h 26 min – August 23, 1999, 13 h – September 2, 1999 and 12 h – September 26, 1999. Glasshouse plants were watered daily and fertilized twice weekly with (15 : 5 : 15) fertilizing solution. Day/night temperature was maintained at 24/18°C. Total proteins were extracted and analyzed as previously described.

Prolonged exposure to controlled SD, time-course study

To determine C. sericea’s (Massachusetts ecotype) response rate to short days, rooted cuttings that had previously been maintained under LD conditions were exposed to 8-h day lengths for varying durations. Plants were maintained in a temperature controlled glasshouse (24/18°C day/night), watered daily and supplemented twice weekly with (15 : 5 : 15) fertilizing solution. Replicate samples (n = 6) were harvested and analyzed prior to the initiation of the photoperiod experiment (time zero) and after 1, 2, 3, 4, 5 and 8 weeks of inductive conditions.

Fluorescein diacetate vital staining

Fluorescein diacetate vital staining was used to assess the viability of xylem tissues subsequent to water stress and to monitor pith cell senescence during controlled SD and LD exposure. Fluorescein diacetate was added (0.1 mg ml–1) to semi-thick cross-sections of C. sericea, incubated for 30 min and subsequently viewed using epifluorescent microscopy (Olympus BH-2 light microscope, Melville, NY, U.S.A.). Video images were captured with a Sony video camera (Sony DXC-151A, Tokyo, Japan) and recorded with a Panasonic superVHS video recorder (AG-1970 SVHS, Matsushita Electric Corporation of America, Secaucus, NJ, U.S.A.).

Soil water deficit

Glasshouse grown plants were exposed to a 15 d soil water deficit to determine if a water deficit could induce 24-kD protein accumulation and induction of FT in non-acclimated C. sericea (Massachusetts ecotype) under LD conditions. Replicate propagules from stem tip cuttings were transferred to large pots (0.07 m3), grown to an approximate height of 1.5 m and maintained under 16-h long days. Pots were placed into plastic bags which were wrapped around the base of the stem to limit the evaporative water loss from the soil surface. Control plants were watered regularly (n = 6), while treated plants had water withheld (n = 6) for 15 d. Water was completely withheld until leaves remained wilted for at least two full days. To maintain plants under stressed conditions, small quantities of supplemental water were occasionally added (100 ml). The extent of the drought stress was estimated by measuring SWC as previously described.

Effect of frost exposure

The effect of a natural frost episode on the accumulation of the 24-kD protein was studied. C. sericea plants were sampled from the field prior to the first predicted frost (October 19, 1999), the morning after the frost (–1°C) (October 23, 1999), 3 d post-frost (October 26, 1999) and 11 d post-frost (November 3, 1999). Replicate shoots (n = 6) were collected, pooled and used for analysis.

Estimation of root water flux (Jv) and hydraulic conductivity (Lp)

Replicate plants were propagated from stem tip cuttings as previously described and transferred to (50 : 50) perlite : vermiculite and potted in 15 cm standard pots. Replicate plants (n = 6) were grown under 8 and 16 h controlled day lengths for 2 and 4 weeks in a glasshouse where temperature was maintained 24/18°C day/night. Plants were watered via drip irrigation and were fertilized twice weekly with 15 : 5 : 15 fertilizing solution. A pressure flux approach (Joly 1989) was used to measure the volume flux of water (Jv) through root systems of de-topped plants and to estimate root hydraulic conductivity (Lp). Replicates (n = 6) were simultaneously processed, and all measurements of Jvwere initiated at 7 : 00 AM in order to avoid diurnal effects. After determining Jv, soil media was carefully removed and total root surface area was quantified with a WinRHIZO™ root scanner (Regent Instruments Inc., Quebec, Canada). Lp was evaluated as the slope of the regression of Jv on applied hydrostatic pressure.

Acknowledgments

The authors would like to thank Dr. Rita Hummel, Washington State University, for her generous donation of the C. sericea climatic ecotypes. We thank Rob Eddy and Ed Fischer for their help in controlling the photoperiod and monitoring the plants in the glasshouse, Dan Hahn for his technical assistance in measuring root hydraulic conductivity and Dr. Sylvie Brouder for her assistance and cooperation on root surface area measurements. Publication number 16,618 of the Purdue University Office of Agricultural Research Programs.

1

Present address of Dale T. Karlson is Department of Low Temperature Sciences, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka, Sapporo, 062855 Japan.

2

Present address of Yan Zeng is Veteran’s Administration Medical Center, Indianapolis, IN 46202, U.S.A.

3

Corresponding author: E-mail, ashworth@hort.purdue.edu; Fax, +1-765-494-0391.

Fig. 1 Seasonal regulation of (A) 24-kD protein, (B) stem relative water content (SWC), and freeze-tolerance in field grown C. sericea (Massachusetts ecotype). Current year’s growth was harvested at each incremental hourly change in photoperiod 15 (June 23, 1999), 14 (August 9, 1999), 13 (September 2, 1999), 12 (September 26, 1999), 11 (October 19, 1999), 10 (November 16, 1999), 9 h 22 min (December 27, 1999), 10 (January 26, 2000), 11 (February 23, 2000), 12 (March 17, 2000), 13 (April 10, 2000), 14 (May 9, 2000). Total wood proteins were extracted from pooled samples from current year’s growth, equally loaded (10 µg lane–1) and probed with anti-24-kD polyclonal antibodies. Mean SWC for stem tips (n = 4) and mean electrolyte leakage (EL) from –12°C exposed samples (n = 6) are shown. Error bars indicate SD. Note seasonal regulation of less abundant 13- and 74-kD proteins and constitutive accumulation of a 37-kD protein. Also note occurrence of an 87-kD protein at times of reduced freeze-tolerance and high SWC.

Fig. 1 Seasonal regulation of (A) 24-kD protein, (B) stem relative water content (SWC), and freeze-tolerance in field grown C. sericea (Massachusetts ecotype). Current year’s growth was harvested at each incremental hourly change in photoperiod 15 (June 23, 1999), 14 (August 9, 1999), 13 (September 2, 1999), 12 (September 26, 1999), 11 (October 19, 1999), 10 (November 16, 1999), 9 h 22 min (December 27, 1999), 10 (January 26, 2000), 11 (February 23, 2000), 12 (March 17, 2000), 13 (April 10, 2000), 14 (May 9, 2000). Total wood proteins were extracted from pooled samples from current year’s growth, equally loaded (10 µg lane–1) and probed with anti-24-kD polyclonal antibodies. Mean SWC for stem tips (n = 4) and mean electrolyte leakage (EL) from –12°C exposed samples (n = 6) are shown. Error bars indicate SD. Note seasonal regulation of less abundant 13- and 74-kD proteins and constitutive accumulation of a 37-kD protein. Also note occurrence of an 87-kD protein at times of reduced freeze-tolerance and high SWC.

Fig. 2 Coincident accumulation of 24-kD protein in field and glasshouse (GH) grown C. sericea (Massachusetts ecotype). Wood tissue collected from replicate plants at incremental changes of day length from the field (n = 6) or GH (n = 3). Incremental changes of photoperiod and corresponding harvests dates were: 14 (August 9, 1999), 13 h42 min (August 16, 1999), 13 h 26 min (August 23, 1999), 13 (September 2, 1999), and 12 (September 26, 1999). Protein blot analysis of equally loaded total protein extracts (10 µg lane–1) with anti-24-kD polyclonal antibodies revealed a similar progression of 24-kD protein accumulation in field- and GH-grown plants.

Fig. 2 Coincident accumulation of 24-kD protein in field and glasshouse (GH) grown C. sericea (Massachusetts ecotype). Wood tissue collected from replicate plants at incremental changes of day length from the field (n = 6) or GH (n = 3). Incremental changes of photoperiod and corresponding harvests dates were: 14 (August 9, 1999), 13 h42 min (August 16, 1999), 13 h 26 min (August 23, 1999), 13 (September 2, 1999), and 12 (September 26, 1999). Protein blot analysis of equally loaded total protein extracts (10 µg lane–1) with anti-24-kD polyclonal antibodies revealed a similar progression of 24-kD protein accumulation in field- and GH-grown plants.

Fig. 3 Comparison of SWC and 24-kD protein accumulation within climatic C. sericea ecotypes sampled at incremental changes in day length. Alaska (AK), Northwest Territories (NW), 70-2 (hybrid resembling AK), Utah (UT), 45-12 (hybrid resembling UT), and Massachusetts (MA) were harvested from the field at: 15 (June 23, 1999), 14 (August 9, 1999), and 13 (September 2, 1999) h day lengths. Average SWC for stem tips is displayed (n = 4) ± SD. Equally loaded protein extracts (10 µg lane–1) were separated by SDS-PAGE and subsequently analyzed with protein blot analysis utilizing anti-24-kD polyclonal antibodies. Note longer critical day length for Northern ecotypes.

Fig. 3 Comparison of SWC and 24-kD protein accumulation within climatic C. sericea ecotypes sampled at incremental changes in day length. Alaska (AK), Northwest Territories (NW), 70-2 (hybrid resembling AK), Utah (UT), 45-12 (hybrid resembling UT), and Massachusetts (MA) were harvested from the field at: 15 (June 23, 1999), 14 (August 9, 1999), and 13 (September 2, 1999) h day lengths. Average SWC for stem tips is displayed (n = 4) ± SD. Equally loaded protein extracts (10 µg lane–1) were separated by SDS-PAGE and subsequently analyzed with protein blot analysis utilizing anti-24-kD polyclonal antibodies. Note longer critical day length for Northern ecotypes.

Fig. 4 Effect of controlled photoperiod treatments on SWC, freeze-tolerance and 24-kD protein accumulation in C. sericea (Massachusetts ecotype). After 8 weeks exposure to: 8, 12, 13, 14, 16 d or an 8-h day + 1.5 h night interruption (NI), replicate plants (n = 6) were assayed for SWC, freeze-tolerance, and 24-kD protein. (A) Represents average SWC from stem tips ± SD. (B) Equally loaded total proteins extracts from pooled wood tissue were probed with anti-24-kD polyclonal antibodies. (C) LT50 determined from electrolyte leakage assays.

Fig. 4 Effect of controlled photoperiod treatments on SWC, freeze-tolerance and 24-kD protein accumulation in C. sericea (Massachusetts ecotype). After 8 weeks exposure to: 8, 12, 13, 14, 16 d or an 8-h day + 1.5 h night interruption (NI), replicate plants (n = 6) were assayed for SWC, freeze-tolerance, and 24-kD protein. (A) Represents average SWC from stem tips ± SD. (B) Equally loaded total proteins extracts from pooled wood tissue were probed with anti-24-kD polyclonal antibodies. (C) LT50 determined from electrolyte leakage assays.

Fig. 5 Prolonged exposure to 8-h short day (SD) reduces SWC, increases freeze-tolerance and induces 24-kD protein accumulation in C. sericea (Massachusetts ecotype). Replicates (n = 6) GH grown plants were maintained under 8-h days for 0, 1, 2, 3, 4, 5, and 8 weeks. (A) Average SWC from stem tips ± SD. (B) Protein blot analysis of equally loaded (10 µg lane–1) total wood protein extracts probed with anti-24-kD polyclonal antibodies. (C) LT50 from electrolyte leakage assays. Note greatest SWC reduction, enhancement of freeze-tolerance and 24-kD protein accumulation occurs after 8 weeks SD exposure.

Fig. 5 Prolonged exposure to 8-h short day (SD) reduces SWC, increases freeze-tolerance and induces 24-kD protein accumulation in C. sericea (Massachusetts ecotype). Replicates (n = 6) GH grown plants were maintained under 8-h days for 0, 1, 2, 3, 4, 5, and 8 weeks. (A) Average SWC from stem tips ± SD. (B) Protein blot analysis of equally loaded (10 µg lane–1) total wood protein extracts probed with anti-24-kD polyclonal antibodies. (C) LT50 from electrolyte leakage assays. Note greatest SWC reduction, enhancement of freeze-tolerance and 24-kD protein accumulation occurs after 8 weeks SD exposure.

Fig. 6 Vital staining of C. sericea (Massachusetts ecotype) stem cross-sections with fluorescein diacetate. GH grown plants were exposed to either 8 weeks of (A) 16-h long days or (B) 8-h short days. Cross-sections were cut from the first internode, stained with fluorescein diacetate and viewed with epifluorescent light microscopy. Note substantial fluorescence within the pith of LD stems and the corresponding absence of staining in pith of SD plants.

Fig. 6 Vital staining of C. sericea (Massachusetts ecotype) stem cross-sections with fluorescein diacetate. GH grown plants were exposed to either 8 weeks of (A) 16-h long days or (B) 8-h short days. Cross-sections were cut from the first internode, stained with fluorescein diacetate and viewed with epifluorescent light microscopy. Note substantial fluorescence within the pith of LD stems and the corresponding absence of staining in pith of SD plants.

Fig. 7 Artificial drought stress reduces SWC, increases freeze-tolerance, and induces 24-kD protein accumulation. C. sericea (Massachusetts ecotype) plants were grown in a GH under LD conditions. Water was withheld from six replicate plants for 15 d. Six control plants were watered during the same testing period for comparison. At the end of this period, SWC and freeze-tolerance of stem tips were determined. Mean % SWC (± SD) and LT50 are reported (n = 6). Total proteins were extracted from control (c) and drought stressed (s) stem tissues for analysis. Xylem protein extracts were equally loaded (10 µg lane–1) and probed with anti-24-kD polyclonal antibodies.

Fig. 7 Artificial drought stress reduces SWC, increases freeze-tolerance, and induces 24-kD protein accumulation. C. sericea (Massachusetts ecotype) plants were grown in a GH under LD conditions. Water was withheld from six replicate plants for 15 d. Six control plants were watered during the same testing period for comparison. At the end of this period, SWC and freeze-tolerance of stem tips were determined. Mean % SWC (± SD) and LT50 are reported (n = 6). Total proteins were extracted from control (c) and drought stressed (s) stem tissues for analysis. Xylem protein extracts were equally loaded (10 µg lane–1) and probed with anti-24-kD polyclonal antibodies.

Fig. 8 Effect of frost exposure on 24-kD protein accumulation in C. sericea (Massachusetts ecotype). Replicate stems (n = 6) were harvested from field-grown plants just prior to the frost, the morning after the frost, plus 3 and 11 d after a natural frost episode. Wood tissue was pooled and total proteins were equally loaded (10 µg lane–1) and probed with anti-24-kD polyclonal antibodies. Note substantial accumulation of 24-kD protein prior to the first frost and lack of additional 24-kD protein accumulation.

Fig. 8 Effect of frost exposure on 24-kD protein accumulation in C. sericea (Massachusetts ecotype). Replicate stems (n = 6) were harvested from field-grown plants just prior to the frost, the morning after the frost, plus 3 and 11 d after a natural frost episode. Wood tissue was pooled and total proteins were equally loaded (10 µg lane–1) and probed with anti-24-kD polyclonal antibodies. Note substantial accumulation of 24-kD protein prior to the first frost and lack of additional 24-kD protein accumulation.

Fig. 9 Relationship of Jv (root water flux) and applied hydrostatic pressure for roots grown under 16 h (LD) or 8 h (SD) controlled photoperiods for (A) 2 and (B) 4 weeks duration. Roots were harvested from glasshouse-grown C. sericea (Massachusetts ecotype). Means (n = 6 ± SD) were plotted at each pressure increment and analyzed with linear regression. Note reduction of Jv in the 4-week SD treatment and slight reduction of Lp (hydraulic conductivity) (slope of line).

Fig. 9 Relationship of Jv (root water flux) and applied hydrostatic pressure for roots grown under 16 h (LD) or 8 h (SD) controlled photoperiods for (A) 2 and (B) 4 weeks duration. Roots were harvested from glasshouse-grown C. sericea (Massachusetts ecotype). Means (n = 6 ± SD) were plotted at each pressure increment and analyzed with linear regression. Note reduction of Jv in the 4-week SD treatment and slight reduction of Lp (hydraulic conductivity) (slope of line).

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