Dehydrin variability among rhododendron species: a 25-kDa dehydrin is conserved and associated with cold acclimation across diverse species


Author for correspondence: Rajeev Arora Tel: +1515 2940031 Fax: +1515 2940730 Email:


  • • Here we examine the accumulation pattern of dehydrins in non- vs cold-acclimated leaves of 21 species comprising two divergent groups of Rhododendron, Subgenus Hymenanthes and Subgenus Rhododendron. Individuals from five other Ericaceous genera were also evaluated in the same way. Quantitative comparisons of cold-inducibility of a 25-kDa dehydrin and cold acclimation ability in six Rhododendron species were also performed.
  • • Leaf freezing tolerance assay and dehydrin detection and quantification were performed as previously described.
  • • Eleven dehydrins, ranging from 25- to 73-kDa, were observed among the 21 species, and most were more abundant in winter-collected leaves than in summer-collected leaves. One dehydrin, a 25-kDa protein, was uniquely conserved across most (95%) of the species surveyed, and was absent only in R. brookeanum, a tropical species that may not be capable of cold acclimation. The 25-kDa dehydrin was also identified in Kalmia, a genus closely related to Rhododendron but not in four other less related Ericaceous genera. Comparison of dehydrin profiles in non- and cold-acclimated leaf tissue from six species (three very hardy, and three less hardy, species) indicated a close association (R2 = 0.95) between relative changes in leaf freezing tolerance and 25-kDa dehydrin accumulation.
  • • The taxonomic and physiological comparisons suggest a central, but as yet unknown, function for the 25-kDa dehydrin in protecting rhododendron leaves from freezing injury.


It is well known that the cellular levels of many metabolites increase when plants are exposed to dehydration stresses such as drought, cold, or high salt levels (Levitt, 1980; Ingram & Bartels, 1996). Some of the organic compounds that accumulate in response to these stress factors are proteins called dehydrins (Svensson et al., 2002). Dehydrins have been studied in many plant species, and are characterized by a consensus 15 amino acid sequence known as K-segment, near the carboxy terminus and additional copies upstream of the terminus, in many cases, as a slightly modified 14 amino acid consensus (Close, 1996). Dehydrin proteins and their transcripts have been shown to accumulate during seasonal development of freezing tolerance (cold acclimation) in barks, xylems, buds, shoot apices, and seedlings of a number of woody plant species (Arora & Wisniewski, 1994; Muthalif & Rowland, 1994; Salzman et al., 1996; Cai et al., 1995; Artlip et al., 1997; Welling et al., 1997; Rinne et al., 1998; Levi et al., 1999; Sauter et al., 1999; Kontunen-Soppela et al., 2000; Sarnighausen et al., 2002; among others).

By contrast with herbaceous plant species and model systems such as Arabidopsis, where transformation systems (Artus et al., 1996; Jaglo-Ottosen et al., 1998) or mapping populations (Pan et al., 1994; Choi et al., 1999; Ismail et al., 1999) have been used to establish a more causal relationship between dehydrin gene expression and freezing or chilling tolerance, their presumed function in woody plant winter survival is primarily based on correlative data.

We have been using broadleaf, evergreen members of the genus Rhododendron as a system for studying cold acclimation and freezing tolerance in woody plants (Lim et al., 1998a, 1998b, 1999). These evergreen species and cultivars provide an opportunity whereby cold acclimation physiology could be studied in over-wintering leaf tissues without the interference of endodormancy transitions that occur in other tissues (buds) of deciduous woody perennials (Lang, 1987; Arora et al., 1992). Lim et al. (1999) first studied the role of dehydrins in Rhododendron cold hardiness, using a super-hardy species (R. catawbiense), a less hardy species (R. fortunei) and their F2 progenies. They reported that levels of a 25-kDa dehydrin were closely associated with differences in leaf freezing tolerance among F2 segregants, and suggested that this dehydrin could serve as a genetic marker for cold hardiness in this interspecific population.

The research presented here was conducted to determine: whether the 25-kDa rhododendron dehydrin (which we propose to term RCA25 for ‘rhododendron cold-acclimation’ protein of 25-kDa) is present in other Rhododendron species or related genera; and whether its accumulation is associated with cold hardiness status across a diverse array of species.

Materials and Methods


Non- and cold-acclimated leaves from a total of 28 taxa comprising 21 Rhododendron species were obtained from The Holden Arboretum's David G. Leach Research Station in Madison, Ohio, USA (Table 1). These species were selected from two Rhododendron subgenera – Subgenus Hymenanthes, the nonscaly leaved or elepidote rhododendrons, and Subgenus Rhododendron, the scaly leaved or lepidote species. For cold hardy species, nonacclimated (NA) leaves were field-collected during summer (July and August) while cold acclimated (CA) leaves were collected from the same individuals during late December and January. For less hardy species unable to tolerate outdoor winter conditions at the site (R. arboreum, R. decorum, and R. brookeanum), plants in containers were held in cold storage (2–4°C) following acclimation outside through late fall. Field and container plants received irrigation and fertilizer as needed throughout the growing season. NA (June) and CA (January) leaves from three evergreen members of Ericaceae (Kalmia latifolia, Leucothoe fontanesiana and Pieris floribunda) were also obtained from the Madison, Ohio, location. Leaf samples from two other genera in the same family (Arctostaphyllos uva-ursi and Vaccinium macrocarpum) were summer and winter collected in the wild, near Morgantown, West Virginia.

Table 1. Rhododendron species used in this study, their corresponding cold-acclimated leaf freezing tolerance (LFT), and various dehydrin proteins that accumulate in acclimated leaves
Rhododendron speciesGenotype used1Leaf freezing tolerance (°C)2Dehydrins (kDa)
  • 1

    Names indicate selected species forms (cultivars). Numbers with letter superscripts indicate accessions from H (The Holden Arboretum collection) or R (originating from Rhododendron Species Botanical Garden with the RSBG accession number).

  • 2

    LFT values in parentheses were estimated in our laboratory as Tmax (the temperature causing maximum rate of injury), whereas those without parentheses are leaf LST (lowest survival temperatures) values from Sakai et al. (1986). ND – no data available.

  • 3

    3 does not increase in cold acclimated leaves compared with the nonacclimated ones (the only case in this study where a dehydrin accumulation was not higher in winter-collection compared with summer-collected leaves).

Subgenus Hymenanthes
R. adenogynum Diels57–1193H(−29)X       X  
R. arboreum Sm.64/118R(−20)X       X  
R. brachycarpumD. Don ex G. Don79/056R−60XX     XXX 
R. brachycarpum(Roslyn form)HNDXX     XXX 
R. brachycarpum ssp. tigerstedtii Nitz.82/109RNDXX     XXX 
R. catawbiense Michaux‘Catalgla’H(−53)X          
R. decorum Franch.75/170RNDXX         
R. dichroanthum ssp. scyphocalyx Cowanno #H(−23)XXXX       
R. oreodoxa var. fargesii (Franch.) DF Chamb.L78-506HNDX       XX 
R. fortunei LindleyL62-560H(−38)XX    XXXX 
R. fortunei64-718-81HNDXX    XXXX 
R. makinoi Tagg79–181HNDX X   X X  
R. maximum L.65–285H(−52)X X     X X
R. maximum84–238HNDX X     X X
R. maximum‘Mt. Mitchell’L51-500HNDX X     X X
R. metternichii[R. degronianum ssp. heptamerum (Carrièrre) Hahn]L82-515H(−48)XX      X  
R. vernicosum Franch.L78-508H(−25)XX   X  X  
R. degronianum ssp. yakushimanum (Nakai) Hara67–76H−40X       X  
R. degronianum ssp. yakushimanum‘Koichiro Wada’HNDX       X  
R. degronianum ssp. yakushimanum‘Mist Maiden’HNDX       X  
Subgenus Rhododendron
R. brookeanum82/210R(−7)      X3    
R. dauricum L.67–143H−50X X     X  
R. hirsutum L.79-259-85HNDX    X     
R. keiskei MiquelL59–63AH−25X    X     
R. minus MichauxL62-570HNDX X     XX 
R. mucronulatum Turcz.L82-551H−50X   X   X  
R. myrtifolium Schott & Kotschy60–336HNDX          
R. russatum Blaf.f & ForrestL78-543H−40X  X   X   

Relative cold-hardiness estimations

Leaf freezing tolerance (LFT) was determined according to a leaf disk method previously reported (Lim et al., 1998a,b). For CA samples, the leaf disks were cooled at relatively slow rates following ice nucleation at −1.5°C, and sampled at treatment temperatures ranging from −10°C to −52°C. Cooling rates varied with sample temperatures: −1°C h−1 from −1.5 to −4°C; −2°C h−1 from −4°C to −10°C; and −7°C h−1 thereafter. A similar protocol was used to freeze NA samples, except that they were cooled down to only between −10°C to −12°C. Ion leakage calculations, percentage injury estimations, Gompertz functions fitting, and determination of Tmax (temperature causing maximum rate of injury and defined as leaf freezing tolerance) were performed as described by Lim et al. (1998a).

Tmax was selected (instead of LT50) as the quantitative measure of LFT because we believe that physiologically, Tmax is more descriptive. LT50 is the temperature that causes 50%-injury and is generally considered to be a value that represents the critical temperature (approximates killing temperature) of cold hardiness of the tissue evaluated (Levitt, 1980). It is arguable, however, that LT50 represents a temperature that causes 100% injury to half of the total tissue area or causes all cells to be half-injured. Tmax is the temperature that causes maximum rate of injury where any lowering of temperature beyond Tmax results in diminishing rates (Lim et al., 1998a). Moreover, Lim and coworkers determined that Tmax estimations made by Gompertz function (based on an ion-leakage assay) were highly correlated to the visual LT50 estimates of LFT in Rhododendron. Evidence is accumulating in the literature supporting the use of Tmax as a quantitative cold hardiness index (Lim et al., 1998a and references therein).

Dehydrin detection and quantification

Total protein was extracted from rhododendron leaves, precipitated, washed, and solubilized in SDS-PAGE loading buffer per Lim et al. (1999). The method described by Esen (1978) was used for determining total protein content in the loading buffer samples, using BSA standards. This protein assay (extracts spotted on a filter paper, stained with Coomassie Brilliant Blue R-250, dye-protein complex eluted with SDS, and absorbance measured) is virtually free from interference by common laboratory reagents including SDS-PAGE loading buffer and other nonproteinaceous substances. Separation of proteins by SDS-PAGE electrophoresis and subsequent visualization of Coomassie Brilliant Blue G-250 stained protein bands followed standard protocols (Lim et al., 1999). Proteins were transferred from duplicate gels on to nitrocellulose membranes and immunoblots were obtained using antidehydrin antibody (against the consensus K-segment; Close et al., 1993) provided by Dr Tim Close (UC-Riverside). Immunoblotting proceeded as described (Lim et al., 1999) with the exception that 3% nonfat dry milk in Tris-buffered saline plus Tween 20 was used in place of gelatin as the blocking buffer. Parallel blots were incubated with preimmune serum (provided by Dr Tim Close, UC-Riverside) to verify that the band recognition on antidehydrin immunoblots was not a result of nonspecific binding of secondary antibody.

The immunoblots were recorded using a digital image analysis system (Alpha Innotech Corporation, San Leandro, CA, USA), and integrated optical density (OD) values for the RCA25 band were determined using the system's software. In this procedure, an OD value that quantifies protein band-intensity is an integrated density value (IDV) determined from the number of pixels/band and the sum of intensity of each pixel. In order to establish the relationship between protein abundance (amount loaded) and OD reading, measurements of the RCA25 band intensity were made on a dilution series ranging from 1 µg to 17 µg total protein from CA leaves of R. catawbiense. From these calibration curve data (not shown) it was determined that protein loadings of up to 7 µg had a linear relationship to OD values. 7 µg protein loadings were used for all the immunoblots performed in this study.

The quantitative association between RCA25 abundance and level of LFT was examined by comparing NA and CA leaf tissue from six Rhododendron species in a replicated experiment. Three very hardy species (R. catawbiense, R. maximum and R. metternichi; with the cold acclimated Tmax of −53°C, −52°C and −48°C, respectively) and three less hardy species (R. arboreum, R. dichroanthum and R. vernicosum with the Tmax of −20°C, −23°C and −25°C, respectively) were selected for comparison. Immunoblots for each species were run in three replications (three separate gels from the same extraction) and the mean optical density values of the RCA25 bands were recorded for non- and cold acclimated leaves.

Results and Discussion

Multiple dehydrins were observed across diverse Rhododendron taxa

A total of 11 dehydrins were observed in the survey of 21 Rhododendron species (Table 1). They ranged in molecular weight from 25-kDa to 73-kDa, and were detected, for the most part, in both non- and cold-acclimated leaf tissue. Species contained from one to as many as six different dehydrins, with a median value of 3.0 per taxon. With the single exception of a 41-kDa dehydrin in R. brookeanum, accumulation of the 11 dehydrins was higher (based on visual assessment) in CA/winter-collected than NA/summer-collected leaves (Table 1).

A few taxonomic similarities and differences were noted based on the dehydrin profiles. The two major groups represented – Subgenus Hymenanthes (13 species) and subgenus Rhododendron (8 species) – shared 7 of the 11 dehydrins (64%). Three proteins were unique to the Hymenanthes group (28-, 46-, and 73-kDa dehydrins), while the 34-kDa form in R. mucronulatum, was unique to the group comprising Subgenus Rhododendron. Dehydrin variability within species was nonexistent in the three instances where multiple accessions were evaluated –R. brachycarpum, R. maximum, and R. yakushimanum (Table 1).

Our data are consistent with previous observations (Close et al., 1993) that dehydrins are typically encoded by a multigene family that can vary among plant taxa. The present finding of interspecific (intrageneric) differences in Rhododendron parallel observations of intergeneric and intraspecific dehydrin variability in other plants (Close et al., 1993; Muthalif & Rowland, 1994; Wisniewski et al., 1996; Sarhan et al., 1997; Nylander et al., 2001; among others).

Among species with the highest level of leaf freezing tolerance, no consistent dehydrin profile was observed (Table 1). In Subgenus Hymenanthes, for example, six dehydrins were distributed among the three hardiest species: one in R. catawbiense (LFT = −52°C), four in R maximum (LFT = −52°C), and five in R. brachycarpum (LFT = −60°C). Less cold hardy species such as R. dichroanthum and R. vernicosum were not characterized by fewer dehydrins than the median value of 3. Cold hardiness therefore appears to be independent of total dehydrin diversity within species, and may be influenced more by the expression of these proteins (abundance) and/or qualitative differences in their function at the cellular level.

A 25 kDa dehydrin is conserved among Rhododendron species with leaf freezing tolerance, and also appears in Kalmia

Only a few dehydrins were detected at high frequency in our survey of species. Most notably, the 25-kDa dehydrin (RCA25) was found in all but one (95%) of the taxa (Table 1) and accumulated at higher levels in CA/winter collected tissues compared to NA/summer-collected ones (Table 1; Figs 1 and 2). A 50-kDa dehydrin was next in frequency, occurring in 13 of 21 (62%) species in both subgenera. The 28-kDa protein was observed in 6 of 21 (28%) of the species, but only in subgenus Hymenanthes.

Figure 1.

(a) Anti-dehydrin immunoblot profile of leaf proteins from six Ericaceae species: (1) R. catawbiense (2) Kalmia latifolia (3) Vaccinium macrocarpon (4) Leucothoe fontanesiana (5) Pieris floribunda (6) Arctostaphyllos uva-ursi. The 25-kDa Rhododendron dehydrin (RCA25) is indicated by an arrow. (b) Cladogram representing phylogenetic relationships within Ericaceae family (adapted from Kron, 1997). All lanes were loaded on an equal protein basis (7 µg/lane). CA, cold acclimated; NA, nonacclimated.

Figure 2.

Anti-dehydrin immunoblots for the 25-kDa dehydrin (RCA25) (indicated by arrows) of leaf proteins from three super hardy (a) and three less hardy (b) Rhododendron species. All lanes were loaded on an equal protein basis (7 µg/lane). The optical density for the RCA25 band and the Tmax (temperature causing maximum rate of injury, a measure of leaf freezing tolerance) value for non- and cold acclimated leaves of each species are indicated. OD, optical density, a quantitative measure of RCA25 band intensity; NA, nonacclimated; CA, cold acclimated.

The only species that lacked RCA25 was R. brookeanum, a tropical Indonesian plant that appears incapable of cold-acclimation. Leaf-freezing tests indicated that CA R. brookeanum leaves had a Tmax of −7°C (Table 1), which is roughly equivalent to NA freezing tolerances (−3°C to −6°C) determined previously in many rhododendron species, cultivars, and progenies (Holt & Pellet, 1981; Anisko & Lindstrom, 1995; Lim et al., 1998a, 1998b; and also the present study, Fig. 2). Other reports indicate that tropical species belonging to the same taxonomic group, subgenus Rhododendron section Vireya, were injured by slight freezing even after cold hardening at 0–5°C (Sakai et al., 1986). In addition, the single dehydrin observed in R. brookeanum immunoblots, a 41-kDa protein, did not appear to be up-regulated in leaf tissues following fall acclimation and subsequent storage at 2–4°C (Table 1; blots not shown).

A survey of dehydrins from NA and CA leaves of five other ericaceous genera revealed that mountain laurel, Kalmia latifolia, also contained a cold-induced RCA25 (Fig. 1a). The other four genera –Arctostaphyllos, Vaccinium, Leucothoe, and Pieris– lacked the RCA25 but contained other dehydrins that appeared to be more abundant in the CA condition. The band in lane 4/CA (Leucothoe) resolving very close to 25-kDa appears to be due to non-specific immune reaction based on the corresponding immunoblots incubated with preimmune serum (data not shown) and is therefore not RCA25. Of the five genera, Kalmia is the most closely related to Rhododendron (Fig. 1b).

From these taxonomic comparisons, it is possible to postulate a key role for the RCA25 dehydrin in protecting Rhododendron leaves from freezing injury. This protein appears to have been conserved in two subgenera that have probably diverged at least 20 million yr ago (D. Chamberlain, pers. comm.) and have Asian and North American temperate zone distributions, but is absent in a tropical species that appears to be incapable of cold-acclimation. This conserved status is based in our study on the consistent appearance of proteins of equivalent molecular mass, not on any peptide or nucleic acid sequence. In addition, it should be noted that the only super cold-hardy genotype containing a single dehydrin, R. catawbiense‘Catalgla’, has the RCA25. Thus there may have been selection pressure over evolutionary time to maintain this particular dehydrin in Rhododendron. The reasons for its conserved status are unknown, but may involve some unique functional property of the RCA25, either alone or in interactions with other dehydrins, during freeze-induced dehydration stress.

Cold acclimation ability and 25-kDa dehydrin accumulation are closely associated

Anti-dehydrin immunoblots of RCA25 at varying levels of CA and subsequent LFT are shown in Fig. 2. In the comparisons of three very cold hardy species (Fig. 2a) and three less hardy species (Fig. 2b), the RCA25 in each genotype is more abundant in the CA than NA state, based on OD values. To determine whether a general relationship between RCA25 and LFT exists across species, regression of OD values on Tmax was performed, using either absolute values (n = 12), or standardized values given as fold increases in cold-acclimated Tmax and RCA25 O.D. relative to the nonacclimated state (ΔTmax and ΔOD, respectively, n = 5). Only five of the six species could be used for the standardized data set, because R. metternichii had no detectable RCA25 band in nonacclimated leaves at the protein loadings used in these experiments. The regressions using absolute values (OD = 0.14 − 1.2Tmax, R2 = 0.55, df = 11) and standardized data (ΔOD = 0.88 + 0.16ΔTmax, R2 = 0.95, df = 4), were both significant (P < 0.01), although a stronger relationship was obtained by standardizing the cold-acclimated values relative to nonacclimated ones (Fig. 3a,b).

Figure 3.

Association of freezing tolerance and RCA25 abundance. (a) Regression of 25-kDa OD on Tmax using absolute values from Fig. 2a,b. (b) Regression of OD on Tmax using –fold increase from nonacclimated to cold-acclimated conditions (ΔOD and ΔTmax, respectively) in leaves. OD, optical density, a quantitative measure of RCA25 band intensity; Tmax, temperature causing maximum rate of injury, a measure of leaf freezing tolerance.

Data on the correlation between dehydrin abundance and the level of freezing tolerance in tissues across diverse species are scarce. However, differences in the freezing tolerance among cultivars (for example, Vaccinium sp. and Tritcum aestivum) have been found to be positively correlated with the accumulation levels of specific dehydrins (Arora et al., 1997; Danyluk et al., 1998). This is the first report, to our knowledge, to demonstrate a correlation between the degree of cold acclimation ability (ΔTmax) and the cold-inducibility (ΔOD) of a specific dehydrin.

Lim et al. (1999) suggested that the presence/absence and/or the abundance levels of 25-kDa dehydrin could serve as a biochemical marker to distinguish between super hardy and less hardy Rhododendron genotypes. This work was based on a segregating population from a controlled, interspecific cross, where F2 progeny shared a similar genetic background. The regression data from six diverse Rhododendron species further suggests that accumulation of this particular dehydrin can be used to ‘predict’ the extent of cold acclimation and subsequent winter leaf freezing tolerance. Taken together with the finding that the RCA25 is the only ‘universal’ dehydrin among cold-acclimating rhododendrons in our survey, these data are concordant with our hypothesis that this particular dehydrin plays a central, but unknown, role in rhododendron cold hardiness.

From a physiological perspective, overall plant cold hardiness (or winter survival) is a complex trait that results from the interaction between several biochemical, physiological and morphological factors. Research to date involving transformation strategies has not identified single structural genes that ‘confer’ the cold hardy phenotype (Xin & Browse, 2000 and references therein). Similarly, although constitutive expression of a regulatory gene (CBF) significantly improved cold and drought tolerance at whole plant level (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999), this effect is associated with multiple cellular changes such as increased expression of many cold-regulated genes, including dehydrins, as well as an increase in the cellular pools of proline and carbohydrates (Gilmour et al., 2000).

In vitro studies have shown that many dehydrins (spinach COR85, maize DHN1, wheat WSC120, peach PCA60 and Citrus unshiu CuCor 19) can protect cold-labile LDH enzyme from freeze-thaw deactivation (Kazuoka & Oeda, 1994; Houde et al., 1995; Close, 1996; Wisniewski et al., 1999; Hara et al., 2001) and that a birch dehydrin can preserve α-amylase activity under low water activity (Rinne et al., 1999). Studies of in vitro cryoprotection using purified RCA25, coupled with data on gene sequence, expression profiles, and subcellular localization may provide insights into the functional relevance of this dehydrin in Rhododendron freezing tolerance. In all likelihood, the RCA 25 alone is not sufficient to confer tolerance to freezing stress, but it may be a necessary component of biochemical interactions with other cryoprotectant metabolites or other dehydrins. As noted above, presence and accumulation of greater number of dehydrin proteins in Rhododendron does not necessarily translate into more cold hardy tissue (Table 1). This suggests that different cold-induced rhododendron dehydrins may not contribute equally towards improving cold tolerance – some might be more potent stress-protector than others.


This journal paper of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project no. 3601 was supported by Hatch Act and State of Iowa funds. We thank American Rhododendron Society for its financial assistance to support a portion of this study.