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. 2004 Jul;135(3):1654–1665. doi: 10.1104/pp.103.037473

Phylogenetic Analyses in Cornus Substantiate Ancestry of Xylem Supercooling Freezing Behavior and Reveal Lineage of Desiccation Related Proteins

Dale T Karlson 1,1,*, (Jenny) Qiu-Yun Xiang 1, Vicki E Stirm 1, AM Shirazi 1, Edward N Ashworth 1
PMCID: PMC519079  PMID: 15247394

Abstract

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.

Due to their sessile nature, plants have been forced to adapt to the dynamic environmental conditions that surround them. Temperature creates a selective pressure on plants growing in temperate climates and has affected their geographical distribution based upon a capacity to survive seasonal thermal fluctuations (Smithberg and Weiser, 1968; Sakai and Weiser, 1973; George et al., 1974; Becwar et al., 1981; Gusta et al., 1983). In woody plants, two distinct and fundamentally different strategies for the seasonal survival of subzero temperatures have evolved: freeze tolerance (nonsupercooling) and freeze avoidance (supercooling; Burke et al., 1976; George et al., 1982). Freezing behavior strategies employed by a woody plant vary from tissue to tissue and are species specific. For example, cortical tissues are strictly nonsupercooling; however, buds and xylem ray parenchyma may exhibit either strategy. In nonsupercooling tissues, ice formation is initiated within extracellular spaces and generates a dehydrative vapor pressure gradient between extracellular ice and intracellular water. Nonsupercooling cells readily desiccate in response to extracellular ice formation (George et al., 1982; Fujikawa et al., 1999) and are capable of surviving low temperature extremes (Guy et al., 1986) due to an inherent capacity to tolerate desiccation (Ashworth et al., 1993; Fujikawa et al., 1997). In supercooling tissues, ice may also initiate in extracellular spaces; however, cells are thought to resist intracellular desiccation (Burke et al., 1976; George et al., 1982; Wisniewski and Ashworth, 1985; Fujikawa et al., 1994) and maintain intracellular water in a nonequilibrium condition. The supercooling of intracellular water is limited to the approximate point of homogeneous ice nucleation (−40°C; Rasmussen and MacKenzie, 1972). When the capacity for supercooling is exceeded, spontaneous and lethal intracellular ice formation may occur (Ristic and Ashworth, 1993).

Due to the temperature constraints of supercooling, woody plants which exhibit this freezing behavior are generally limited in geographical and altitude distribution to regions warmer than the −40°C isotherm (Smithberg and Weiser, 1968; Sakai, 1970; Sakai and Weiser, 1973; George et al., 1974; Becwar et al., 1981). On the contrary, nonsupercooling species, such as red osier dogwood (Cornus sericea or Cornus stolonifera) are not restricted by low temperature boundaries and may have expansive ranges within the northern hemisphere (George et al., 1974). Due to this disparity, it has been of great interest to decipher the fundamental differences of freezing behavior in woody plants. Surprisingly, no efforts have been devoted to understanding the evolution and mode of inheritance of xylem freezing behavior in woody plants. As a result, the genetic basis for this trait remains poorly understood.

In many plants, dehydrin proteins (group 2 late embryogenesis proteins [LEA D-11 family]) are induced by conditions that affect plant water status such as desiccation, salinity stress, and freezing stress (Close et al., 1993; Close, 1996, 1997; Campbell and Close, 1997). Since freeze tolerance is limited by a cell's capacity to withstand intracellular desiccation, dehydrins have been hypothesized to minimize deleterious desiccative effects associated with extracellular ice formation (Close, 1996; Arora and Wisniewski, 1997; Thomashow, 1999). Genetic studies in Rhododendron revealed that a 25-kD dehydrin may serve as a potential marker for cold hardiness (Lim et al., 1999). Recently, data obtained from wide interspecific sampling within Rhododendron provided clear correlation of the 25 kD to cold acclimation ability (Calin et al., 2004). However, to our knowledge no studies have widely studied their proposed role in relation to extracellular ice formation. In the only investigation that related dehydrins to xylem freezing behavior (Arora and Wisniewski, 1996), nonsupercooling peach cortical tissues were found to accumulate more dehydrin than the supercooling xylem tissue. Due to the likelihood that desiccative effects may differ between woody plant species that exhibit contrasting xylem freezing behavior, it was of interest to classify freezing behavior and compare the accumulation of desiccation related proteins in a representative woody plant genus.

The dogwood genus Cornus consists of approximately 55 species that are mostly woody and mainly distributed in northern temperate regions. The freezing behavior in seven Cornus species has been previously determined and found to consist of both nonsupercooling and supercooling species (George et al., 1974; George et al., 1982; Ishikawa and Sakai, 1982; Ristic and Ashworth, 1993). One of the examined nonsupercooling species, C. sericea, is among the most freeze tolerant plants known and is capable of withstanding gradual cooling to the temperature of liquid helium (−269°C; Guy et al., 1986). In addition, our recent study in C. sericea xylem documented the marked winter accumulation (Sarnighausen et al., 2002) of a 24-kD dehydrin-like protein that directly correlates to increased freeze tolerance (Karlson et al., 2003). The Cornus genus provides us with a model system to investigate the evolutionary significance of xylem freezing behavior and the role of dehydrin-like proteins. By utilizing interspecific Cornus hybrids that were previously crossed from non- and supercooling parents, we were able to assess the heritability and, to our knowledge for the first time, suggest the mode of inheritance for xylem freezing behavior. By overlaying differential thermal analysis (DTA) data upon a preestablished Cornus molecular phylogeny (Xiang et al., 1996, 1998; Fan and Xiang, 2001), we performed the first evolutionary analysis, to our knowledge, of freezing behavior in woody plants. Immunoblot analyses with a polyclonal antibody raised against C. sericea's predominant 24-kD dehydrin-like protein allowed us to investigate the relationship of desiccation related proteins to our characterization of xylem freezing behavior among 31 Cornus and 4 outgroup species.

RESULTS

Characterization of Xylem Freezing Behavior and Protein-Blot Analyses

We utilized DTA as a method to distinguish xylem freezing behavior from a wide range of Cornus species and related outgroup species. Twenty-three of the 30 tested Cornus species (excluding the herbaceous Cornus canadensis) exhibited low temperature exotherms (supercooling). The only species found to lack low temperature exotherms were Cornus alba, Cornus amomum, Cornus bretschneideri, Cornus obliqua, Cornus rugosa, and C. sericea. Two interspecific hybrids were also tested, Cornus arnoldiana (C. obliqua × Cornus racemosa) and Cornus horseyi (Cornus macrophylla × C. amomum), and freezing behavior was identical to the maternal parent. C. arnoldiana exhibited the nonsupercooling behavior characteristic of its maternal parent C. obliqua. In contrast, C. horseyi displayed a low temperature exotherm similar to its maternal parent C. macrophylla (Fig. 1).

Figure 1.

Figure 1.

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DTA analysis of hybrid species reveals maternal inheritance of xylem freezing behavior. Freezing profiles obtained from DTA analyses of two interspecific hybrids were plotted to illustrate the maternal influence on xylem freezing behavior. A, Comparison of interspecific hybrid (C. arnoldiana) to maternal and paternal parent species C. obliqua and C. racemosa, respectively. B, Comparison of interspecific hybrid (C. horseyi) to maternal and paternal parent species C. macrophylla and C. amomum, respectively. The occurrence of high temperature exotherms and low temperature exotherms are indicated by HTE and LTE abbreviations, respectively. In respect to the occurrence of LTEs, note the similarity of interspecific hybrid freezing behavior to that of the maternal parent species.

Freezing behavior data were overlaid upon a Cornus molecular phylogeny as a method to enable us to characterize the evolution of the trait. Outgroup genera, the basal lineage of the blue-fruited clade C. oblonga, the alternate-leaved blue-fruited species, and the red-fruited clade all displayed supercooling freezing behavior. Within the later divergent opposite-leaved blue- or white-fruited dogwoods, xylem freezing behavior varied, where five species were found to exhibit nonsupercooling behavior (Fig. 2B). Data obtained from ITS analysis confirmed that the sixth identified nonsupercooling species, C. bretscheideri, also falls within this same category (Q.-Y. Xiang, D. Thomas, T.K. Seo, J.L. Thorne, W. Zhang, S.R. Manchester, C. Fan, and Z. Murrell, unpublished data).

Figure 2.

Figure 2.

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A, Super-tree generated from compilation of molecular phylogenetic trees of Cornus derived from chloroplast DNA restriction site and matK, rbcL, and 26S rDNA sequence data (27–29). Numbers adjacent to nodes (indicated as black circles) are bootstrap support values. Values before the forward slash are from analysis of combined chloroplast and nuclear DNA data (matK, rbcL, 26S rDNA sequences, and cpDNA RS); values after the slash are from analyses of combined chloroplast DNA data (matK-rbcL sequences and cpDNA RS). Circled numbers are bootstrap values for the three subclades within the blue- or white-fruited group identified in the cpDNA RS analysis. This group was sampled for only one to a few species in all gene sequence studies; thus, these nodes were not present in the trees derived from 26SrDNA, matK, and rbcL sequences. B, Trend of evolutionary changes of freezing behavior. Experimentally determined freezing behaviors for Cornus and outgroup species were overlaid to the super-tree, and trends were inferred from Fitch parsimony using MacClade 3.05. Freezing behavior was designated as black or red for supercooling and nonsupercooling, respectively. Green lineages were used to designate species that were not tested for freezing behavior in this study. As designated by a black circle, note the occurrence of nonsupercooling freezing behavior exclusively in the single subclade in opposite-leaved blue- or white-fruited dogwoods. C, Evolutionary trend for the accumulation of proteins similar to C. sericea's predominant 24-kD xylem dehydrin-like protein. Phylogenetic relationships were inferred by using Fitch parsimony on MacClade 3.05. The lineage of species that lack or contain a predominant similar protein was labeled as red or black, respectively. A green lineage was used to identify species that were not tested for the presence of a similar protein in the present study. Note the lack of a similar protein in the Alangium outgroup, the early divergent C. oblonga, and all tested members of the red-fruited dogwood clade. D, Evolutionary changes in size of proteins similar to the 24-kD C. sericea xylem dehydrin-like protein. Protein size characters were specified as ordered and analyzed by using Fitch parsimony on MacCLade 3.05. Protein size lineages were labeled by corresponding color codes, and individual sizes were labeled adjacent to their representative species. Lineages that cannot be determined by this study are designated by green lines. Notice the occurrence of proteins similar to C. sericea's 24-kD protein in the opposite-leaved blue- or white-fruited dogwood clade.

Protein-blot analyses of winter xylem protein extracts revealed predominant 24-kD-like proteins in 22 Cornus species and 3 outgroup taxa (Davidia involucrata, Nyssa aquatica, and Nyssa ogeche; Table I). A 22-kD protein was detected in D. involucrata and 36- and 34-kD proteins were found within N. aquatica and N. ogeche, respectively. The alternate-leaved species, Cornus alternifolia and Cornus controversa, accumulated a higher molecular mass protein (32.8 and 31.2 kD, respectively). Cross-reacting proteins of similar size to the C. sericea 24-kD protein were exclusively identified within the opposite-leaved blue- or white-fruited dogwood clade. Species in one well-supported clade (Cornus foemina-C. racemosa-Cornus drummondii; Xiang et al., 1998) accumulated a protein doublet of similar size to the 24-kD C. sericea dehydrin. Comparisons of total xylem protein extracts from nonacclimated and cold acclimated total xylem protein extracts from 24 selected Cornus species confirmed their winter association (Fig. 3). Two species, C. bretschneideri and Cornus poliophylla, were the only species found to contain significant quantities of 24-kD-like proteins under nonacclimated conditions (Fig. 3). In addition to the most closely related outgroup species (Alangium platinifolium), the early divergent blue-fruited C. oblonga, and the species tested species from the red-fruited group (C. angustata, C. capitata, C. canadensis, C. florida, C. kousa, C. mas, C. officinalis, and C. sessilis), no species accumulated a similar 24-kD-like protein. Usage of a polyclonal antibody specific for dehydrin proteins confirmed the lack of 24-kD-like protein accumulation in winter samples for C. oblonga and all species tested in the red-fruited dogwood clade (data not shown).

Table I.

Summarization of xylem freezing behavior and occurrence of 24-kD proteins

Specimen Freezing Behavior Protein (kD)
A. platanifolium S ND
C. alba N 24
C. alternifolia S 32.8
C. amomum N 24.4
C. angustata S ND
C. arnoldiana N 24, 22
C. australis S 24.4
C. bretschneideri N 24.2
C. capitata S ND
C. canadensis Equivocal ND
C. controversa S 31.2
C. coreana S 24.2
C. darvasica S 24.2
C. drummondii S 26, 23.2
C. florida S ND
C. foemina S 24.5, 23.2
C. horseyi S 23.8
C. kousa S ND
C. macrophylla S 26
C. mas S ND
C. obliqua N 25.2
C. oblonga S ND
C. officinalis S ND
C. paucinervis S 25.2
C. poliophylla S 25.2
C. pumila S 24.9
C. racemosa S 24.9, 23.2
C. rugosa N 24
C. sanguinea S 25
C. sericea N 24
C. sessilis S ND
C. walteri S 25.5
D. involucrata S 22
N. aquatica S 36
N. ogeche S 34
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The xylem freezing behavior of 31 Cornus species and out-group genera Alangium, Davidia, and Nyssa was based upon the detection of low temperature exotherms utilizing DTA. Those which displayed low temperature exotherms were categorized as supercooling (S) and those which lacked exotherms were labeled as nonsupercooling (N). Protein-blot analysis with a polyclonal antibody raised against a C. sericea 24-kD dehydrin was used to identify similar proteins throughout the genus and out-group genera. Predominant cross-reacting proteins that accumulated within winter xylem total protein extracts are listed with estimated molecular mass (kD). Species that did not accumulated 24-kD-like proteins were designated as (ND). Alangium, Cornus, Davidia, and Nyssa were abbreviated with A., C., D., and N., respectively. Respective locations and date of harvests are listed (Table II).

Figure 3.

Figure 3.

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Seasonal protein-blot analysis of total xylem protein extracts from 24 Cornus species. Cold acclimated (A) and nonacclimated (B) wood samples were harvested from field conditions on March 3, 1999 and August 5, 1999, respectively. Total protein extracts were separated by SDS-PAGE, electro-transferred to nitrocellulose membranes, and subsequently probed with polyclonal antibodies directed against C. sericea's predominant 24-kD dehydrin-like protein. Comparisons from cold and nonacclimated samples revealed winter-association of immunlogically related proteins in the majority of species tested. C. florida, C. kousa, C. mas, and C. officinalis were the only species found to lack accumulation of related proteins. C. bretschneideri and C. poliophylla were the only species to display significant amounts of related proteins under nonacclimated conditions.

Evolution of Xylem Freezing Behavior and Proteins Similar to C. sericea's 24-kD Dehydrin-Like Protein in Cornus

Evolutionary analysis of xylem freezing behavior and protein accumulation from a phylogenetic framework determined that the deep supercooling characteristic is ancestral within Cornus (Fig. 2B). Nonsupercooling evolved in five species of the blue- or white-fruited clade. Given that the relationships within this clade are poorly resolved, it is not clear whether nonsupercooling evolved a single time or had evolved independently in different species. The analysis suggested that nonaccumulation of the 24-kD-like protein represents an ancestral state in Cornus (Fig. 2C). With the exception of the unusual C. oblonga, whose lineage separates from the remainder of the blue- or white-fruited species, the accumulation of proteins similar to the 24-kD C. sericea dehydrin-like protein evolved a single time in the ancestor of the clade consisting of all blue- or white-fruited species (Fig. 2C). Within the same blue- or white-fruited group, an ancestral form of a 24- to 24.9-kD protein was documented and was followed by a trend for an increased size to higher molecular masses (Fig. 2D). At least two independent increases were identified: an increase to 31 to 33 kD in the alternate-leaved dogwoods and an increase to 25 to 27 kD in the opposite-leaved group. It is likely that the later increase occurred several times; however, due to the lack of resolution of relationships and incomplete sampling of species within this group, the number cannot be accurately determined.

Induction of Dehydrin-Like Protein in Species of Contrasting Freezing Behavior

Since the accumulation of 24-kD-like proteins was not detected within any of the tested species from the red-fruited dogwood clade, we attempted to determine if red-fruited species were capable of accumulating similar proteins when placed under conditions (short day length and water deficit) that are known to induce dehydrins in C. sericea (Karlson, 2001) and other woody species (Welling et al., 1997, 2002). Results confirmed that the tested red-fruited species do not accumulate similar dehydrin-like proteins in either condition (Figs. 4 and 5).

Figure 4.

Figure 4.

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The response of four Cornus species to photoperiod in relation to (A) SWC and 24-kD protein accumulation (B). Two supercooling species (C. florida and C. kousa) and two nonsupercooling species (C. amomum and C. sericea) were exposed to either 16-h long days or 8-h short days. In comparison to samples collected at time zero, average SWC from replicates (n = 6) of each species did not dramatically change after prolonged long-day exposure. Following prolonged short-day exposure, however, SWC was substantially reduced in the nonsupercooling species (C. amomum and C. sericea). Error bars indicate ±sd. Note that the 24-kD protein was detected with anti-24-kD protein antibodies only in short day exposed C. amomum and C. sericea.

Figure 5.

Figure 5.

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The response of four Cornus species to water deficit in relation to SWC (A) and 24-kD protein accumulation (B). Replicates (n = 6) of two supercooling species—C. florida and C. kousa—and two nonsupercooling species—C. amomum and C. sericea—were exposed to prolonged water deficit and measured for SWC ±sd. Total proteins were extracted and were used for subsequent protein-blot analysis with anti-24-kD polyclonal antibodies. Note reduction of SWC in all species. Predominant proteins of similar size that cross-reacted with anti-24-kD polyclonal antibodies were only detected in C. amomum and C. sericea after water deficit.

Prior to the initiation of photoperiod treatments, both supercooling and nonsupercooling species tested exhibited high stem relative water content (SWC) and lacked proteins of similar size to the C. sericea 24-kD protein. Subsequent to 8 weeks of short day exposure, the two representative blue- or white-fruited opposite-leaved dogwoods, C. amomum and C. sericea, reduced SWC and accumulated a 24-kD protein. Neither red-fruited dogwoods, C. florida and C. kousa, exhibited a similar SWC reduction, nor accumulation of a similar sized 24-kD protein. Negative control plants that were grown under 16-h-long day did not reduce SWC nor accumulate a similar sized 24-kD protein in any species (Fig. 4). Subsequent to prolonged water deficit, substantial SWC reduction occurred in all species tested. Vital staining with fluorescein diacetate confirmed sublethality of the water deficit treatment (data not shown). Protein-blot analysis revealed that prolonged water deficit stimulated the accumulation of similar proteins only in C. amomum and C. sericea but not in the two red-fruited species analyzed (Fig. 5). Usage of a polyclonal antibody specific for dehydrin proteins provided a secondary means of confirmation for the lack of 24-kD-like proteins in C. florida and C. kousa under the tested conditions (data not shown). These data strengthen the hypothesis that this difference is likely under genetic constraints.

DISCUSSION

In woody plants, it is well established that two mechanisms have evolved for the survival of annual seasonal temperature fluctuations. Since the extent of supercooling is limited by the homogenous ice nucleation point (−40°C), this typically dictates the lower temperature limit for species that exhibit this characteristic and thus affects the geographical range (Sakai and Weiser, 1973; George et al., 1974) of such species. If the temperature of the homogeneous ice nucleation point is exceeded, the result is presumably lethal intracellular ice formation (Burke et al., 1976; George and Burke, 1977) and subsequent tissue injury (George et al., 1982). Survival of nonsupercooling tissues, however, is dependent upon desiccation tolerance (Ashworth, 1993; Fujikawa et al., 1997), and an absolute physical limitation to low temperature exposure may be nonexistent. As a consequence of dehydration, cellular contents may become highly viscous and nondestructively vitrify. Vitrification may partially explain how C. sericea is capable of surviving the temperature of liquid helium (Guy et al., 1986; Pearce, 2001). From an evolutionary point of view, the acquisition of a nonsupercooling behavior would be advantageous for geographical expansion into harsh low temperature environments. However, little is known about the genetics and the evolution of freezing behaviors in plants. The disparities between supercooling and nonsupercooling freezing behavior creates an interesting scenario to question from both an evolutionary and molecular point of view. By coupling xylem freezing behavior and protein-blot data to a molecular phylogeny, we gained insight into this enigma.

Our analysis provided evidence that freezing behavior is maternally inherited (Fig. 1) and is highly correlated with species phylogeny in Cornus (Fig. 2B). A maternal influence on the inheritance of cold hardiness in various fruit trees (Dorsey and Bushnell, 1925; Harris, 1965; Wilner, 1965; Stushnoff, 1972) has been previously observed; however, there are no previous reports regarding the genetics and evolution of either supercooling or nonsupercooling traits (Ashworth, 1993). Our results suggest that these traits are heritable and may be passed and acquired during woody plant evolution.

In Cornus, the supercooling freezing mechanism was employed by most species and was inherited down from the common ancestor of the genus. On the contrary, the nonsupercooling mechanism evolved in only a few species of a single subclade derived from supercooling ancestor(s). Interestingly, five of the six identified nonsupercooling species (exception C. amomum) inhabit the northernmost ranges of the genus (excluding the three rhizomatous herbaceous species). Phylogenies that are based on ITS and matK sequence data (Q.-Y. Xiang, D. Thomas, T.K. Seo, J.L. Thorne, W. Zhang, S.R. Manchester, C. Fan, and Z. Murrell, unpublished data) from a broad range of species indicate that they are scattered in different subclades within the blue- or white-fruited group. As a result, a shift to nonsupercooling freezing behavior may have evolved repeatedly in independent blue- or white-fruited dogwood lineages and may have played a key role in the adaptive radiation of the largest subgroup of Cornus into higher latitudes.

It has been postulated that dehydrin-like proteins play an important role in providing protection from desiccation resultant from the formation of extracellular ice (Close, 1996; Arora et al., 1997; Thomashow, 1999). Thus, it might be expected that nonsupercooling species would accumulate proteins similar to C. sericea's 24-kD dehydrin during winter conditions. Our data indicated that the accumulation of 24-kD-like proteins was not restricted to nonsupercooling species in Cornus. They were found in the out-group genera, Nyssa and Davidia, and a large blue- or white-fruited clade (excluding C. oblonga) that consist of both supercooling and nonsupercooling species. Our phylogenetic analysis suggested that the trait for the accumulation of similar proteins evolved prior to and independently from nonsupercooling freezing behavior (Fig. 2, B and C).

Our finding that dehydrin-like protein accumulation occurs widely within supercooling species (Table I; Fig. 3) suggests that the relationship of dehydrins to the nonsupercooling mechanism (extracellular ice formation) requires further investigation. Alternatively, these observations cast doubts on the hypothesis that supercooling is strictly a nondesiccative freeze-avoidance strategy (Burke et al., 1976; George et al., 1982; Wisniewski and Ashworth, 1985; Fujikawa et al., 1999). Independent studies in supercooling species have discovered intermediate forms of freezing behavior where supercooling is accompanied by partial intracellular dehydration (Gusta et al., 1983; Wisniewski and Ashworth, 1985; Fujikawa et al., 1997). In an electron microscopic study by Wisniewski and Ashworth (1985), they demonstrated that deeply supercooled Prunus persica xylem cells behave as if they are exposed to a dehydrative stress during freezing. It is important to note that subsequent studies in P. persica have clearly demonstrated the association of dehydrins to periods of cold acclimation (Arora et al., 1992; Artlip and Wisniewski, 1997; Wisniewski et al., 1999). It has been hypothesized that intermediate forms of freezing behavior may have evolved from deep supercooling to extracellular freezing in a continuous process as an adaptation to occupy colder climates (Fujikawa et al., 1997). Recently, Kuroda et al.(2003) reported microscopic evidence to suggest that all boreal woody species undergo deep supercooling in combination with partial desiccation. Collectively, these findings justify the possibility that the supercooling blue- and white-fruited Cornus species experience partial dehydration during winter months and may consequently accumulate dehydrin-like proteins.

In red-fruited dogwoods, the absence of 24-kD-like proteins and the nonresponsiveness of tested species to normally inductive conditions suggest that a genetically determined basis exists for this trait and is attributed to the evolutionary history of their group. In a previous study, we determined that a photoperiod controlled SWC reduction is highly correlated to freeze tolerance and dehydrin-like protein accumulation in C. sericea and likely serves as a cue for the acquisition of freezing tolerance prior to the onset of winter (Karlson et al., 2003). When two representative red-fruited species were examined under controlled conditions, they failed to respond to photoperiod, and neither reduced stem water content nor accumulated 24-kD-like proteins. In a cold acclimated state, it is tempting to speculate that red-fruited dogwoods resist freeze-induced desiccation and maintain intracellular water in a supercooled state. Due to a lack of desiccation during freezing events, the necessity to program and maintain a winter associated dehydrin-like protein accumulation would be superfluous. Due to a lack of selection pressure to accumulate 24-kD-like proteins, the ancestral absent protein character state (similar to the closely related Alangium outgroup) would remain unchanged in these species during evolution. At this time, we are unable to confirm if the lack of protein accumulation is due to the simple fact that the gene is missing. Alternatively, the red-fruited species may contain a similar gene but have alterations in gene regulation that would render them unresponsive to photoperiodic and/or desiccation related stimuli, thereby preventing the eventual translation of a similar protein product. Future studies are warranted and necessary to clarify these questions.

In conclusion, our study has shed light upon a poorly understood area of plant adaptation to low temperature environments. For the first time, to our knowledge, we have demonstrated that there is an evolutionary separation of freezing behavior in a representative woody plant species and documented a maternal influence for this trait. Interestingly, all out-group species and early divergent dogwood species exhibited supercooling behavior, a freezing behavior that has been documented to impose discrete low temperature limitations to woody plants for over-winter survival. Therefore, a conversion to nonsupercooling freezing behavior, which is highly correlated to dessication tolerance and is not physically limited to low temperature extremes, would be an advantageous acquisition and may have facilitated their expansion into northernmost ranges.

MATERIALS AND METHODS

Plant Material

Current year's growth from stems was randomly harvested from established plantings during winter field conditions for the evaluation of xylem freezing behavior and total protein extractions for 31 Cornus and 4 out-group species. The sources of plant material and sampling dates are respectively listed (Table II). Immediately upon harvest, the cut ends of stem tips were wrapped in parafilm, placed into a plastic bag with a moist paper towel, and packaged on ice. Samples obtained from the Morton Arboretum and the Purdue University campus were immediately brought to the laboratory for freezing behavior determination and total xylem protein extraction as described below. Samples obtained from other sources were similarly packaged and overnight-mailed to Purdue University. Samples were maintained at 4°C in moist paper towels within sealed plastic bag until at least three replicate DTA analyses were completed.

Table II.

Dates and locations of harvests for winter samples

Specimen Source Harvest Accession No. Native Range
A. platanifolium Morris Feb. 13, 2002 91-042-B E. Asia (China, Japan), temperate
C. alba Morton Mar. 3, 1999 773-80 Siberia to Manchuria, N. Korea, cold temperature
C. alternifolia Morton Mar. 3, 1999 231-86 MN to Nova Scotia, south to GA and AL
C. amomum Morton Mar. 3, 1999 279-26 E. and N.C. US and adjacent Canada
C. angustata MHRC Jan. 29, 2002 1997-174 S. and S.W. China, sub-tropical mountains
C. arnoldiana Morton Feb. 9, 2000 372-70 N/A
C. australis Morton Mar. 3, 1999 359-82 Asia Minor around Black and Caspian Seas
C. bretschneideri Morton Mar. 3, 1999 303-90 N. China, colder temperate
C. capitata MHRC Jan. 29, 2002 2002-097 S.W. China mountains, high elevation, India, and Indo-China
C. canadensis Arnold Mar. 21, 2002 843-93 Circumboreal
C. controversa Morton Mar. 3, 1999 640-51 China, Japan, Korea, temperate
C. coreana Morton Mar. 3, 1999 235-90 Korea
C. darvasica Morton Mar. 3, 1999 63-91 Central Asia
C. drummondii Morton Mar. 3, 1999 117-24 S. Ontario to MS, E. TX, and NE
C. florida Purdue Mar. 3, 1999 N/A MA to FL, Mexico, and Ontario, temperate
C. foemina Morton Mar. 3, 1999 279-84 VA to FL, TX, and MO, warm temperate to sub-tropical
C. horseyi Morton Feb. 9, 2000 1026-26 N/A
C. kousa Purdue Mar. 3, 1999 N/A E. China, Japan, and Korea
C. Macrophylla Morton Mar. 3, 1999 681-39 Japan, E., C., S.W. China, warm & sub-tropical mountains
C. mas Purdue Mar. 3, 1999 N/A C. and S. Europe, Asia Minor, Caucasus
C. obliqua Morton Mar. 3, 1999 724-79 Quebec to MN and KS, S. to PA, IL, MO
C. oblonga MHRC Jan. 29, 2002 2002-182 S.W. China to Himalayas, sub-tropical and tropical mountains
C. officinalis Morton Mar. 3, 1999 325-39 E. China, Japan, and Korea, temperate
C. paucinervis Morton Mar. 3, 1999 569-63 S.W. China, sub-tropical mountains
C. poliophylla Morton Mar. 3, 1999 359-72 E., C., S.W. China, warm and sub-tropical mountains
C. pumila Morton Mar. 3, 1999 642-54 Origin unknown
C. racemosa Morton Mar. 3, 1999 1634-25 ME to GA, NE, MN, and Ontario, colder temperate
C. rugosa Morton Mar. 3, 1999 190-83 Nova Scotia to VA, IA, and Manitoba
C. sanguinea Morton Mar. 3, 1999 196-81 Czechoslovakia, Romania
C. sericea Purdue Mar. 3, 1999 N/A Newfoundland to DC, IL, CA, and Yukon, cold temperate
C. sessilis Cornell Jan. 30, 2002 97-162*A N. CA, tropical rain forest, conifer forest, riparian zones
C. walteri Morton Mar. 3, 1999 361-82 EC. S.W. China, warm temperate to sub-tropical mountains
D. involucrata National Feb. 16, 2001 N/A C. and S.W. China, deciduous seasonal mountains
N. aquatica National Feb. 16, 2001 N/A S. IL to Gulf of Mexico, S.E. VA to S. GA, and N.W. FL, swamps
N. ogeche National Feb. 16, 2001 N/A SC to S.E. GA and N. FL, swamps
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Sources were abbreviated as Arnold (The Arnold Arboretum, Boston), Cornell (Cornell Plantations, Ithaca, NY), MHRC (Mountain Horticultural Research Center, Fletcher, NC), Morris (The Morris Arboretum, Philadelphia), and Morton (The Morton Arboretum, Chicago). Specimens that were harvested from general collections on arboretum or campus grounds lacked accession numbers and are indicated by N/A. Range for three species was not documented. The origin for C. pumila is unknown and C. arnoldiana and C. horseyi lack a native range since they are hybrid species.

Differential Thermal Analysis, Total Protein Extraction, and Protein-Blot Analyses

Xylem freezing behavior was determined with DTA as previously described (Malone and Ashworth, 1991) and categorized as supercooling or nonsupercooling based upon the presence or absence of low temperature exotherms, respectively. In brief, a scalpel was used to remove bark from current year's growth from stem tips and a 1-cm internode section was attached to the junction of a 40-gauge copper/constantan thermocouple. Debarked stem sections were lowered into a drilled aluminum block that was housed within a −80°C freezer. Block temperature was controlled by resistance heating and monitored with an Omega CN-2010 programmable temperature controller (Stamford, CT). Samples were cooled from 5°C to −60°C at the rate of 20°C/h. Exothermic events were identified by comparing the differential output of thermocouple pairs monitoring individual tissue samples with an oven-dried reference specimen. At least three replicate DTA analyses were conducted on independent stem samples from each species. Cornus canadensis was not tested for xylem freezing behavior because it is an herbaceous species that utilizes an underground rhizome as an overwintering strategy.

Xylem total protein extractions from winter samples were performed as previously described (Sarnighausen et al., 2002). Briefly, bark was removed from current year stems by using a scalpel and xylem tissue was immediately flash frozen in liquid nitrogen. Samples were maintained at −80°C and subsequently lyophilized then pulverized in a ball mill and total proteins were extracted by boiling samples in ice cold LiDS-extraction buffer (62.5 mm Tris-HCl, pH 6.8, 2% [w/v] lauryl sulfate [lithium salt], 5% [v/v] 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride). Total protein extracts were precipitated with acetone (−20°C) and resuspended in SDS sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS [w/v], 10% glycerol [v/v], 5% 2-mercaptoethanol [v/v]). As previously described, equally loaded total protein extracts (15 μg) were separated by SDS-PAGE and subsequently transferred to nitrocellulose membranes for protein-blot analysis with a polyclonal antibody raised against the predominant 24-kD dehydrin-like protein of Cornus sericea xylem (Sarnighausen et al., 2002). The estimated molecular mass of detected proteins was calculated based upon comparison to the migration of protein standards (Bio-Rad, Hercules, CA). A polyclonal antibody that was raised against a peptide which represents a consensus motif specific for dehydrin proteins was used as a secondary method to confirm the lack of 24-kD-like proteins in red-fruited dogwood species. Protein-blot analyses with this antibody was performed as previously described (Sarnighausen et al., 2002).

Total xylem protein extracts were harvested from trees during summer conditions as a negative control to assess the previously observed winter association of dehydrin-like proteins in the majority of sampled species (Table I). Current year's growth from 24 Cornus species was harvested August 5, 1999 from identical specimens and locations that were sampled the previous winter. Protein extraction and blot methods were performed identically as previously described (Sarnighausen et al., 2002).

Phylogenetic Analyses of Freezing Behavior and Dehydrin-Like Proteins

Phylogenetic analyses of Cornus using different molecular data (chloroplastic DNA restriction sites and sequences of matK, rbcL, ITS, and 26S rDNA) have been conducted previously by Xiang and colleagues (Xiang et al., 1996, 1998; Fan and Xiang, 2001). Although major lineages were included in each of these studies, species and number of taxa sampled varied among the three independent studies. However, the revealed lineages and phylogenetic relationships were remarkably consistent between all studies. To elucidate the evolution of freezing behavior and proteins similar to the winter associated 24-kD dehydrin from C. sericea, we constructed a supertree which combined all species from the previous studies (Fig. 2A). The supertree represents the consensus of all phylogenetic trees derived from previous independent molecular phylogenetic analyses and illustrates well-supported major lineages and their relationships. The supertree is also congruent with the unpublished ITS and matK trees that include sampling from nearly all Cornus species (Q.-Y. Xiang, D. Thomas, T.K. Seo, J.L. Thorne, W. Zhang, S.R. Manchester, C. Fan, and Z. Murrell, unpublished data), suggesting that it is a robust estimation for the phylogenetic relationships of species analyzed in the present study. This tree served as the basis for character optimization using the maximum parsimony method implemented within MacClade 3.05 software (Maddison and Maddison, 1992). Transformations between supercooling and nonsupercooling, and between the presence and absence of proteins similar to the C. sericea 24-kD dehydrin-like protein were coded as unordered in the analysis. As variable sizes of immunologically related proteins were detected in different species, an ordered character analysis of protein size was performed to gain insights into the evolutionary trend of protein size within the genus (see Fig. 2D legend).

Test for Induction of Dehydrin-Like Proteins under Controlled Conditions

Previous studies in C. sericea confirmed that 24-kD protein accumulation was highly correlated to water deficit and short day length. We were interested to determine if representative red-fruited dogwood species have the capacity to accumulate similar 24-kD-like proteins under known inductive conditions. Therefore, we reproduced similar controlled experiments as previously described (Karlson et al., 2003). Two supercooling species (Cornus florida and Cornus kousa) from the red-fruited group that did not accumulate 24-kD-like proteins in winter conditions were exposed to prolonged controlled short day and long day photoperiod treatments as previously described (Karlson, 2001). Two nonsupercooling species (Cornus amomum and C. sericea) that are known to accumulate the protein in winter conditions were exposed to identical conditions and used as positive controls.

Small 1-year-old seedlings of C. amomum, C. florida, and C. kousa were used as plant material for the study. Replicates of C. sericea were propagated as stem cuttings as previously described (Karlson et al., 2003). All plants were transferred into large pots (0.07 m3) containing Scott's MetroMix 560 substrate (Marysville, OH) and grown in a computer controlled glass house where temperature was maintained at 24°C/18°C day/night during the summer of 2000. Six replicate plants for each species were exposed to 8 weeks of artificial 8-h-short day and 16-h-long day conditions. Short day conditions were created by pulling black cloths daily at 4 pm and reopening the following morning at 8 am. Long day conditions were produced by extending the natural photoperiod with incandescent lighting (photosynthetic photon flux = 2.2 μmol m−2 s−1 at canopy height). All replicates were watered daily and supplemented with 20-20-20 NPK fertilizer regimes. Six replicates of each species were harvested prior to initiation of controlled photoperiod conditions for a time zero determination of SWC (as described by Karlson et al., 2003) and total protein extraction and blotting analyses as previously described (Sarnighausen et al., 2002). Six replicates from each photoperiod treatment were sampled after 8 weeks exposure and processed for SWC determination and total protein extraction.

Response to water deficit was also tested for these species using replicate seedlings of C. amomum, C. florida, C. kousa, and propagated C. sericea stems. Plants were grown in the temperature controlled glass house (24°C/18°C day/night) and maintained under a controlled long day (16-h) photoperiod regime. Pots were wrapped in plastic bags to minimize evaporative water loss from the soil. Control replicates (n = 6) of each species were watered daily, whereas additional replicates of each species (n = 6) were exposed to a water deficit by withholding water until leaf wilting was observed. After 2 to 3 d of persistent wilting, plants were harvested for SWC determination and subsequent xylem protein extraction. Watered control plants were harvested at the same time for comparative analysis of SWC and total xylem proteins. Total xylem protein extraction and subsequent blotting analyses were performed as previously described (Sarnighausen et al., 2002).

Acknowledgments

The authors thank the following individuals and their corresponding arboretum for the kind donation of material used within the study: Tony Aiello (Morris Arboretum), Mary Hirshfield (Cornell Plantations Arboretum), Tom Ranney (Mountain Horticultural Research Center), Kevin Tunison (National Arboretum), and Tom Ward (Arnold Arboretum). We also thank the Morton Arboretum for allowing us to harvest multiple species from their vast Cornus collection. For our controlled environment studies, we thank Heritage Seedlings for their kind donation of C. kousa seedlings. Likewise, we thank the Indiana Division of Natural Resources for their generous donation of C. amomum and C. florida seedlings. Lastly, we thank Dr. Timothy Close (University of California, Riverside) for graciously donating the antidehydrin specific polyclonal antibody for protein-blot analyses.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037473.

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