PHYSIOLOGIA PLANTARUM 107: 98–109. 1999 Printed in Ireland —all rights reser6ed
Copyright © Physiologia Plantarum 1999 ISSN 0031-9317
Complementary DNA cloning, sequencing and expression of an unusual dehydrin from blueberry floral buds Amnon Levia, Ganesh R. Pantab,c, Cecile M. Parmentierb, Mubarack M. Muthalifd, Rajeev Arorae, Savita Shankerf and Lisa J. Rowlandb,* a
Vegetable Laboratory, Southern Region, Agricultural Research Ser6ice, Charleston, SC 29414, USA Fruit Laboratory, Belts6ille Agricultural Research Center, Agricultural Research Ser6ice, Belts6ille, MD 20705, USA c Department of Horticulture, Uni6ersity of Georgia, Athens, GA 30602, USA d Department of Pharmacology, College of Medicine, Uni6ersity of Tennessee, Memphis, TN 38163, USA e Di6ision of Plant and Soil Sciences, West Virginia Uni6ersity, Morgantown, WV 26506, USA f DNA Sequencing Core Laboratory, The Biotechnology Program, Uni6ersity of Florida, Gaines6ille, FL 32611, USA *Corresponding author, e-mail:
[email protected] b
Received 19 March 1999; revised 12 May 1999
Levels of three major dehydrins of 65, 60, and 14 kDa have been observed to increase in blueberry (Vaccinium spp.) floral buds during chill unit accumulation and cold acclimation and decrease during deacclimation and resumption of growth. Indeed, levels of the 65-, 60-, and 14-kDa dehydrins increase such that they become the most predominant proteins visible on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The peptide sequence information from the 65- and 60-kDa dehydrins was used to synthesize degenerate DNA primers for amplification of a part of the gene(s) encoding the dehydrins. One pair of primers amplified a 174-bp fragment. The 174-bp fragment was used to screen a cDNA library (prepared from RNA from cold-acclimated blueberry floral buds) and resulted in the isolation of a clone with a 2.0-kb insert. The cDNA was sequenced and found to be a full-length clone encoding a K5-type dehydrin (5 K boxes). Five high-confidence peptide sequences, ranging from 9 to 25 amino acids long, obtained
from the 60-kDa dehydrin exactly matched sequences encoded within the cDNA clone. Furthermore, amino acid composition of the 60-kDa dehydrin agreed well with the expected amino acid composition based on the cDNA sequence. However, the DNA sequence and coupled in vitro transcription/translation reactions of the cDNA clone indicated that it encodes a dehydrin with a native molecular mass of 40 kDa instead of 60 kDa. Experiments to determine if the dehydrins undergo post-translational modifications revealed that the 65- and 60-kDa dehydrins are glycosylated. Thus, our results indicate that the 2.0-kb dehydrin cDNA encodes the native version of the 60-kDa dehydrin. The dehydrin cDNA hybridized on RNA blots to two chilling/cold-responsive messages of 2.0 and 0.5 kb. Both the 2.0- and 0.5-kb messages increased to higher levels more quickly in the cold-hardy cultivar Bluecrop than in the less hardy cultivar Tifblue. In addition, the 0.5-kb message remained at a higher level longer in Bluecrop than in Tifblue.
Introduction Temperate-zone woody perennial plants are exposed to freezing temperatures each winter. They survive these adverse conditions by entering a state of dormancy and developing cold hardiness known as cold acclimation (Powell 1987). Both winter dormancy and cold acclimation are induced by environmental signals, namely, increasingly shorter photoperiods and lower temperatures (Nissila and Fuchigami 1978, Fuchigami and Nee 1987). Exposure to low temperatures, which leads initially to a deeper state of dormancy and to greater cold hardiness than short photope-
riods alone can induce, is required for many species to break dormancy (Kobayashi 1987). This requirement, called the chilling requirement (CR), synchronizes a plant’s growth with exposure to favorable environmental conditions by preventing growth during transitory periods of warm temperatures in a major portion of the winter. The CR is generally defined as the amount of chilling, temperatures in the approximate range of 2 – 9°C, necessary for greater than 50% budbreak upon exposure to temperatures favorable for growth (Richardson et al. 1974). The amount of chilling or
Abbre6iations – CR, chilling requirement; CU, chill units.
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‘‘chill units’’ (CU) a plant is exposed to is calculated slightly differently depending on the model used (Rowland and Arora 1997). In our previous work (Muthalif and Rowland 1994a,b, Arora et al. 1997) and the work reported here, we have used 1 h of exposure to temperatures from 0 to 7°C as equivalent to 1 CU. The CR may range from a negligible amount to as much as 3000 CU depending upon species and genotype of the plant. Despite the fact that seasonal transitions in dormancy and cold hardiness status are integral parts of the life cycle of many woody perennials, fundamental understanding of these two phenological events is limited. This, in part, is due to the development of dormancy coinciding with cold acclimation and the release from dormancy coinciding with deacclimation. Because of the lack of understanding in these areas, we have been using a combination of molecular and genetic approaches to investigate genetic control of dormancy/CR and cold hardiness in a woody perennial, blueberry (Vaccinium, section Cyanococcus). To identify proteins responsive to low temperature exposure or ‘‘chilling’’, previously we examined changes in protein levels associated with CU accumulation in blueberry floral buds (Muthalif and Rowland 1994a,b). From profiles of soluble proteins, the levels of three proteins of 65, 60, and 14 kDa were observed to increase with CU accumulation such that they become the predominant proteins visible on sodium dodecyl sulfate (SDS)-polyacrylamide gels (Muthalif and Rowland 1994a,b). Further characterization of these proteins revealed that they belong to the dehydrin family of proteins (Muthalif and Rowland 1994a,b). Assessment of cold hardiness levels for dormant buds every 300 CU until the resumption of growth revealed that cold hardiness levels are positively correlated with relative levels of these dehydrins (Muthalif and Rowland 1994a). Subsequently, other less-abundant dehydrins have been observed in blueberry floral buds as well, with molecular masses of 40, 22, and 10 kDa. Unlike the former three dehydrins, which have been observed in all blueberry species and cultivars examined to date, the presence and sizes of these additional dehydrins varies, depending on blueberry species and cultivar (Muthalif and Rowland 1994a,b, 1995, Arora et al. 1997) and even on the method of protein extraction (Arora et al. 1997). For example, the 10-kDa dehydrin has been observed in cvs Tifblue and Berkeley but not in Bluecrop or Gulfcoast. Because cold acclimation and the development of dormancy, as well as deacclimation and release from dormancy, occur simultaneously, it is impossible from the work described above (Muthalif and Rowland 1994a,b) to conclude unequivocally that the dehydrins are more closely associated with cold acclimation than with development or maintenance of dormancy. More recently, we have carried out a physiological study to better make this distinction (Arora et al. 1997). This study used an approach to trigger deacclimation in dormant plants and, thus, allow the separation of dormancy from cold hardiness. We found that if cold hardy, dormant blueberry plants are given a temperature treatment that results in deacclimation, but not negation, of CU, dehydrin levels decrease to the pre-chilling level. Thus, it Physiol. Plant. 107, 1999
was concluded that dehydrin levels are associated with cold hardiness level rather than with dormancy status per se. Dehydrins are a group of heat-stable, glycine-rich plant proteins that are induced by environmental stimuli that have a dehydration component including drought, low temperature, salinity, and seed maturation (Close et al. 1993, Close 1996). Also indicative of dehydrins is the presence of a highly conserved lysine-rich 15-amino acid sequence (consensus sequence EKKGIMDKIKEKLPG), referred to as the K segment, which is often repeated several times (Close et al. 1993, Close 1996). A number of physiological studies have demonstrated a positive correlation between accumulation of dehydrins and tolerance to environmental stresses with a dehydration component. Indeed, recently, evidence for a causal relationship between dehydrin genes and cold tolerance was reported from work on Arabidopsis (Jaglo-Ottosen et al. 1998). These researchers isolated the regulatory gene CBF1, which is responsible for induction of a family of cold-regulated or COR genes (some of which are dehydrins) in Arabidopsis. CBF1 binds a DNA regulatory element present in the promoter region of many COR genes and stimulates transcription in response to low temperature and water deficit. By making a construct of the CBF1 gene for its constitutive overexpression, researchers were able to develop transgenic plants with elevated COR gene expression in the absence of low temperature or drought stimuli. Transgenic plants that were not cold-acclimated, but were overexpressing CBF1, were shown to be more frost-tolerant than nonacclimated control plants and as tolerant as cold-acclimated plants. Here, we report the isolation of a 2.0-kb full-length cDNA that encodes the 60-kDa dehydrin from blueberry. The clone was sequenced and the sequence compared with that of other dehydrins. Because of a discrepancy between the size of the encoded protein as determined by SDS-polyacrylamide gel electrophoresis (PAGE) and the size as predicted from the cDNA sequence, we also investigated whether the blueberry dehydrins are modified post-translationally by glycosylation. Also, northern hybridizations were performed to examine expression of the dehydrins at the RNA level in two blueberry cultivars with different freezing tolerances, and Southern hybridizations were performed to estimate the number of dehydrin genes in blueberry.
Materials and methods Plant material Young leaf tissue, for extracting DNA, was collected from greenhouse plants of the Vaccinium corymbosum L. cv. Bluecrop and selected plants of our V. darrowi Camp×V. caesariense Mackenzie-derived mapping population, including the original parent plants (V. darrowi clone Fla4B and V. caesariense clone W85-20) and F1s (Fla4B×W85-20-5, -6, and -10). Beginning in September of each year (1993– 1995), floral buds for RNA and protein extractions were collected periodically (approximately every 300 CU) from field plants of the V. corymbosum cv. Bluecrop and the V. 99
ashei cv. Tifblue until the resumption of growth in the spring. CUs, calculated using a biophenometer (Omnidata, Logan, UT, USA) placed in the field, were defined as the number of hours that plants were exposed to temperatures from 0 – 7.2°C (32–45°F). After collection, tissues were frozen in liquid nitrogen and stored at −80°C until analyzed.
Nucleic acid purification Blueberry genomic DNA, for use in polymerase chain reactions (PCRs), was extracted from the V. corymbosum cv. Bluecrop as previously described (Rowland and Levi 1994). Genomic DNA, for use in Southern hybridizations, was extracted from parent plants of the mapping population using the Nucleon Phytopure plant DNA extraction kit from Vector Laboratories, Inc. (Burlingame, CA, USA). Extractions were performed according to the manufacturer’s instructions, with two exceptions. The nonionic detergent Igepal (Sigma Chemical Co., St Louis, MO, USA) was added to reagent 2 at a final concentration of 2% (v/v), and additional chloroform extractions (choroform:isoamyl alcohol, 24:1, v/v) were performed immediately after the one described in the instruction manual until the interphase appeared clear. For construction of the cDNA library, total RNA was extracted from floral buds of field plants of cv. Bluecrop collected on December 20, 1993 (655 CU). Total RNA was extracted by the method of Callahan et al. (1989), with a few modifications. Frozen tissue, rather than lyophilized material, was used; the extraction buffer included 10 mM ascorbic acid in addition to the other components described in Callahan et al. (1989); the extraction buffer was pre-heated to 65°C before it was added to the tissue; and the final potassium acetate/ethanol precipitation was eliminated. The RNA was quantified spectrophotometrically and resuspended at a final concentration of 2 mg ml − 1 in sterile water. The integrity of the RNA was tested visually following electrophoresis through a 1.4% (w/v) agarose gel containing 5 mM methyl mercuric hydroxide (Bailey and Davidson 1975). For northern blots, RNA was isolated from floral buds, collected during the 1995–1996 fall through spring seasons at 0 (9/22/95), 50 (10/18/95), 300 (11/16/95), 600 (12/18/95), 900 (1/29/96), 1200 (3/11/96), and 1500 (4/11/96) CU, from field plants of two blueberry cvs, Bluecrop and Tifblue. Total RNA ( 0.1–0.2 mg RNA g − 1 tissue) was extracted according to the protocol of Levi et al. (1992). The RNA was quantified spectrophotometrically and the quality and quantity of the RNA was confirmed visually by electrophoresis through formaldehyde gels.
Protein extraction For western blotting and glycoprotein detection, soluble proteins were extracted from floral buds of cv. Tifblue plants that had received 865 CU in a borate buffer (50 mM sodium borate, 50 mM ascorbic acid, 1 mM EDTA, 1% (v/v) b-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, pH 9.0), using a modification of the method described by 100
Arora et al. (1992) and Arora and Wisniewski (1994). Bud samples (200 mg) were ground with a mortar and pestle in liquid nitrogen and 50% (w/w) insoluble polyvinylpolypyrrolidone. Then, 1.5 ml borate buffer was added to the tissue and it was ground further using the mortar and pestle. The extract was centrifuged at 5000 g at room temperature for 15 min. The resultant supernatant was centrifuged at 18000 g at room temperature for 30 min. Proteins present in the supernatant were quantified using a protein assay kit (Bio-Rad, Richmond, CA, USA) based on the method of Bradford (1976). For subsequent protein sequencing and amino acid composition analysis, soluble proteins were extracted from floral buds of cv. Bluecrop plants that had received 600 or 900 CU using the method of Hurkman and Tanaka (1986), with some modifications. One gram of tissue was ground in liquid nitrogen with a mortar and pestle. The ground tissue was mixed with extraction buffer (0.7 M sucrose, 0.5 M Tris, 30 mM HCl, 50 mM EDTA, 0.1 M KCl, 2% [v/v] b-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and the mixture was thoroughly vortexed. Five milliliters of water-saturated phenol, pH 7.0, containing 0.1% (v/v) 8-hydroxyquinoline, was added, and the mixture was shaken vigorously for 10 min. The extract was centrifuged at 10000 g at 4°C for 10 min. The phenol phase (top phase) was collected, mixed with 4 volumes of 0.1 M ammonium acetate in methanol, and incubated overnight at −20°C. Proteins were pelleted by centrifugation at 6000 g at 4°C for 10 min. The pellet was washed twice with 0.1 M ammonium acetate in methanol and once with acetone, dried at room temperature, and resuspended in protein solubilization buffer (1 mM Tris-HCl, pH 8.0, 8 M urea, 8 mM CHAPS, 5% [v/v] glycerol). The protein was then centrifuged at 16000 g for 20 min at room temperature and the supernatant was collected. Proteins were quantified as described above.
PCR amplification, fragment isolation, and cloning Previously, the 65- and 60-kDa dehydrins from blueberry were purified, digested with endoproteinase Lys-C, and selected peptide fragments were sequenced (Muthalif and Rowland 1994a). The amino acid sequences with the least codon degeneracy were chosen for synthesis of degenerate primers (National BioSciences Inc., Plymouth, MN, USA) (Fig. 1). All possible pairs of primers (assuming a 5%–3% and a 3% – 5% orientation) were used in PCRs in an attempt to amplify a region of a dehydrin gene from blueberry. First strand cDNA, synthesized from RNA from cold-hardy floral buds of blueberry, was used initially as a template in PCRs, but without success. Therefore, cv. Bluecrop genomic DNA was used as the template in all subsequent reactions. Amplifications were performed in 50-ml reactions containing 1× PCR buffer (20 mM NaCl, 50 mM Tris-HCl, pH 9.0, 1% [v/v] Triton X-100, 0.1% [w/v] gelatin) (Barry et al. 1991), 1.5 mM MgCl2, 200 mM each of dATP, dCTP, dGTP, and dTTP, 4.0 mM sense and antisense primers, 50 ng genomic DNA, and 4.0 units Taq-DNA-polymerase. Physiol. Plant. 107, 1999
DNA was amplified using a thermal cycler model PTC100 (MJ Research, Watertown, MA, USA) and the following program: an initial denaturation step of 3 min at 94°C, followed by 30 cycles of 1 min at 94°C, 80 s at 59°C, and 2 min at 72°C. Amplification products were separated by electrophoresis through 2.5% NuSieve GTG agarose (FMC Bioproducts, Rockland, ME, USA) gels in 0.5× Tris borate buffer (Sambrook et al. 1989). DNA fragments were visualized under UV light after staining with 0.5 mg ml − 1 ethidium bromide. A 174-bp fragment was amplified from PCR using the primer combination shown in Fig. 1. This fragment was purified from gels using the Prep-A-Gene DNA purification kit from Bio-Rad and cloned into a plasmid vector, using the TA cloning system from Invitrogen (San Diego, CA, USA).
cDNA library construction and screening Total RNA extracted from floral buds of cv. Bluecrop, having accumulated 655 CU, was provided to Stratagene (La Jolla, CA, USA) for purification of poly(A + )-RNA and then construction of a custom cDNA library using the Uni-ZAP™ unidirectional l cloning vector. The estimated titer of the primary unamplified library was 7.2× 105 pfu ml − 1 and the average size of cDNA inserts was \400 bp. The unamplified library was screened using the 174-bp dehydrin clone (produced by PCR and described in the previous section). The clone was radioactively labeled with 32P using the random priming labeling kit, DECA Prime II™, from Ambion, Inc. (Austin, TX, USA). The screening method used was that described in the Uni-ZAP™ custom cDNA library instruction manual (Stratagene) and in Sambrook et al. (1989). In vivo excision of the pBluescript phagemid (containing the cDNA clone) from the Uni-ZAP™ vector was performed using the exassist/solar™ cell system (Stratagene). Phagemid DNA was isolated from bacterial cells and digested with
EcoRI and XhoI restriction enzymes (Promega Corporation, Madison, WI, USA) to release the inserts. DNA sequencing of clones Sequencing of both DNA strands of the 174-bp clone from PCR and the 2.0-kb cDNA clone was carried out at the University of Florida, DNA Sequencing Core Laboratory (Gainesville, FL, USA), using ABI Prism Dye Terminator cycle sequencing protocols (part number 402078), developed by Applied Biosystems (Perkin-Elmer Corporation, Foster City, CA, USA). The fluorescently labeled extension products were analyzed on an Applied Biosystems Model 373 Stretch DNA Sequencer (Perkin-Elmer). For sequencing the 2.0-kb cDNA clone by primer walking, oligo primers were designed using OLIGO 4.0 (National BioSciences Inc.) and synthesized at the DNA Synthesis Core Laboratory (University of Florida). Nucleotide sequences were aligned and assembled using programs in the Sequencher 3.0 software package (Gene Codes Corporation, Ann Arbor, MI, USA). RNA and DNA blotting and hybridizations Total RNA (2 mg lane − 1) was separated by electrophoresis through 1.8% (w/v) agarose, 6% (v/v) formaldehyde gels and transferred onto Zeta-Probe nylon membranes (Bio-Rad). The membranes were washed, air-dried, crosslinked with UV light, and baked for 1 h at 80°C. The 2.0-kb cDNA (dehydrin) insert was gel-purified, 32P-labeled (by random priming), and hybridized to the RNA blots at 68°C. Hybridization and wash conditions were according to Galau et al. (1986). Genomic DNA was digested with restriction enzymes BamHI, EcoRI, EcoRV, HindIII, and KpnI (Promega), and separated through 0.8% agarose gels. DNA was transferred to Zeta-Probe nylon membranes; membranes were baked at 80°C for 1 h and hybridized with 32P-labeled 2.0-kb cDNA insert at 65°C overnight in 0.5 M sodium phosphate buffer, pH 7.2, 7% (w/v) SDS. Blots were washed three times in 0.5 × SSC, 0.5% SDS at 68°C for 30 min (moderate stringency), followed by a wash in 0.1× SSC, 0.1% SDS at 68°C for 30 min (for the high stringency blots only). Densitometry
Fig. 1. Peptide sequences obtained after complete Lys-C digestion of 65- and 60-kDa dehydrins. Asterisks indicate high-confidence primary sequences. Others are lower confidence secondary sequences. Underlined regions are the least degenerate regions selected for synthesis of primers. Because it was not known which peptide sequences were N-terminal and which were C-terminal, e.g., which of the primer sequences were 5% and which were 3%, the combinations A D (where A is assumed to be N-terminal to D), D A, 13, and 31 were all tried in PCRs along with cv. Bluecrop genomic DNA as template. Only the combination A D resulted in amplification of a 174-bp fragment. Physiol. Plant. 107, 1999
After choosing the best autoradiograms from northern hybridizations, RNA band intensities were quantified by densitometry using the Eagle Eye II image analyzer system (Stratagene), equipped with Eagle Sight 3.0 software. To correct for slight differences in the amount of RNA loaded per lane, rRNA bands from the gel photographs were also quantified by densitometry and the results used to adjust the densitometric values from autoradiograms. RNA band intensities were also adjusted relative to the background by subtracting the densitometric value for the background from all other values. Values from triplicate autoradiograms were averaged and standard errors were determined. 101
SDS-PAGE and western immunodetection Equal quantities of protein were precipitated with trichloroacetic acid (TCA) (Bensadoum and Weinstein 1976). Precipitates were collected by centrifugation at 18000 g at room temperature for 10 min. Pellets were rinsed with acetone, air-dried, and solubilized in 30 ml of solubilization buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% [v/v] b-mercaptoethanol, 10% [v/v] glycerol, 0.001% [w/v] bromophenol blue) (Laemmli 1970). Samples were boiled for 5 min and cooled to room temperature. Proteins were separated by discontinuous SDS-PAGE through 12.5% polyacrylamide gels, using the Mini Protean II cell (Bio-Rad). Separated proteins from unstained gels were electroblotted onto 0.45-mm nitrocellulose membranes using the Mini Trans Blot electrophoretic transfer cell (Bio-Rad). Transfer was carried out in Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) (Towbin et al. 1979) at 100 V for 1 h 15 min. Membranes were blocked in 3% (w/v) gelatin in Tris-buffered saline (TBS) buffer (25 mM Tris, 137 mM NaCl, 27 mM KCl, pH 7.4) and probed at 30°C for 2 h with anti-65-kDa antibody (polyclonal antibody from gel-purified 65-kDa blueberry dehydrin, Organon Technika, Durham, NC, USA) at 1:500 dilution in 1% gelatin in TBS buffer. Immunoreactive bands were detected by an alkaline phosphatase assay using the PicoBlue immunoscreening kit from Stratagene.
Amino acid composition analysis and peptide sequencing of the 60-kDa dehydrin Proteins extracted from floral buds of cv. Bluecrop plants (600–900 CU) according to the modified protocol of Hurkman and Tanaka (1986) were diluted with an equal volume of 2× solubilization buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 10% [v/v] b-mercaptoethanol, 20% [v/v] glycerol, 0.002% [w/v] bromophenol blue) (Laemmli 1970). Diluted samples were boiled for 5 min, cooled to room temperature, and subjected to SDS-PAGE, as described in the previous section. Ten micrograms of total protein were loaded in each of 7 lanes of two gels. The gels were run at 200 V for 1 h 15 min for good protein separation. For subsequent amino acid composition analysis, separated proteins from one gel were transferred to a PVDF membrane (0.2 mm) in 10 mM 2-[N-morpholino]ethanesulfonic acid, pH 6.0, 20% (v/v) methanol for 1 h 30 min at 100 V. The membrane was washed three times with purified water, stained 5 min in 0.025% (w/v) Coomassie brilliant blue R-250 in 40% (v/v) methanol, destained by washing three times for 10 min each with 50% (v/v) methanol, and air-dried. The position of the 60-kDa dehydrin was marked with a pencil and the membrane was shipped to the University of Florida, Protein Chemistry and Molecular Biomarkers Facility, where the amino acid composition was determined. For peptide sequencing, the second gel was fixed in 12% (w/v) TCA for 1 h. The gel was stained overnight in Coomassie blue staining solution (0.1% [w/v] Coomassie billiant blue G-250 in 2% [w/v] phosphoric acid, 10% [w/v] ammonium sulfate, 20% [v/v] methanol) and destained in 102
20% (v/v) methanol, according to the method of Neuhoff et al. (1988). The 60-kDa protein band was cut out from the gel and shipped in a tube containing 20% (v/v) methanol to the University of Florida, Protein Chemistry and Molecular Biomarkers Facility. There, the protein was subjected to a partial endoproteinase Lys-C digestion, using one-half the amount of enzyme as that recommended in the protocol of Stone and Williams (1995) for digestion of proteins in gels, and the resulting peptides were separated by reverse phase high-performance liquid chromatography (HPLC). Selected peptides were analyzed by mass spectrophotometry and sequenced in a gas-phase sequenator. Coupled in vitro transcription/translation reactions of dehydrin cDNA clone The protein encoded by the 2.0-kb cDNA was synthesized in an in vitro transcription/translation system. Plasmid DNA was first extracted and purified using the PEG method of Nicoletti and Condorelli (1993) and then digested with EcoRI. The linearized DNA was extracted with an equal volume of phenol:chloroform (1:1, v/v) and ethanol precipitated. The transcript encoded by the cDNA was synthesized using the T3/T7 RNA polymerase system provided as part of the mCAP RNA capping kit from Stratagene. The capped RNA transcript was extracted with an equal volume of acid phenol:chloroform (Ambion) and precipitated with 1/10 volume 3 M Na-acetate and 3 volumes 95% ethanol. The transcript was translated in a rabbit reticulocyte lysate translation system (In vitro Express translation kit, Stratagene) with 35S-methionine (555 MBq ml − 1), according to the manufacturer’s directions. 35S-labeled proteins were separated by SDS-PAGE and transferred from the gel onto nitrocellulose. The membrane was air-dried for 1 h and exposed to medical X-ray film at − 80°C for 5 days. Glycoprotein detection Glycoproteins from floral buds of cv. Tifblue (865 CU) were detected using the Immunoblot Glycoprotein Detection kit from Bio-Rad, according to the manufacturer’s directions, after immobilization of SDS-PAGE-separated proteins onto nitrocellulose membranes.
Results PCR-generated dehydrin probe One pair of primers derived from peptide sequences from the 65-kDa dehydrin (Fig. 1) resulted in amplification of the genomic DNA template. The amplified 174-bp fragment was cloned and sequenced (Fig. 2). The presence of a K box, indicative of dehydrins (Close 1996), within the sequence confirmed that part of a dehydrin gene had been isolated. In addition, the clone was hybridized to northern blots of RNA extracted from blueberry floral buds that were collected about every 300 CU. The clone hybridized to two chilling-induced messages of 2.0 and 0.5 kb (data not shown). Physiol. Plant. 107, 1999
Fig. 2. The nucleotide and deduced amino acid sequence of the 174-bp PCR product. The presence of a K box within the sequence (underlined) confirmed that a part of a dehydrin gene had been amplified.
Isolation and DNA sequence analysis of a blueberry dehydrin cDNA The 174-bp PCR fragment was used to screen a cDNA library prepared from RNA from dormant, cold-hardy blueberry floral buds. The buds for the cDNA library construction were collected from field plants having acquired 655 CU. Previously, we had shown that dehydrin levels are maximal by 600–900 CU (Muthalif and Rowland 1994a,b). One positively hybridizing plaque was detected out of 4× 104 plaques screened. Purification of the clone, followed by digestion with restriction enzymes to release the insert, revealed that the clone carried a 2.0-kb insert. The complete nucleotide sequence, along with the deduced amino acid sequence, for the dehydrin cDNA is presented in Fig. 3. The cDNA contained a long open reading frame starting at nucleotide 320 and ending at nucleotide 1261 before a TAG stop codon. The cDNA contained 319 bp of a 5%-untranslated sequence and 714 bp of a T-rich 3%-untranslated sequence after the stop codon. It did not include the poly(A + ) tail. The reading frame encoded a polypeptide of 314 amino acid residues with a predicted molecular mass of 34.3 kDa. Like dehydrins (Close 1996), the deduced protein was hydrophilic (based on hydropathy plot results, data not shown), had a preponderance of glycine residues (23.2%), was rich in polar and charged amino acids (glutamine, 16.5%; aspartic acid, 10.5%; lysine, 9.6%; tyrosine, 5.7%; histidine, 5.4%; glutamic acid, 4.5%; and arginine, 4.5%), and contained no phenylalanine or tryptophan (Table 1). Inspection of the cDNA sequence confirmed that it was a member of the dehydrin family of proteins. The deduced protein sequence of the cDNA contained five lysine-rich repeats [K segment consensus sequence EKKGIMDKIKEKLPG, as defined by Close (1996)] typical of dehydrins. These sequences were contained within larger contiguous imperfect repeats composed of 48 – 62 amino acids (consensus sequence for blueberry cDNA QDQQLGGYRQDQRKEGGGLMDKVKDKIHGGGGGSADQHQGGY(K/G)QDQQ(H/L)GGYR). The alignment of these repeats is shown in Fig. 4. The cDNA did not contain a tract of serine residues (S segment) or the consensus amino acid sequence (V/T)DEYGNP (Y segment) present in some dehydrins (Close 1996). Because dehydrin sequences are not colinear, e.g., short consensus sequences are present, but sequences outside the consensus sequences are divergent in terms of length and sequence, reporting the percent homology with other dehydrins is not meaningful. However, a computer search of the GenBank database revealed similarity to other dehydrins, the following five Physiol. Plant. 107, 1999
being the highest scoring matches: alfalfa cold acclimation protein, CAS15 (Monroy et al. 1993); Pistacia inflorescence bud protein, 32 kDa (accession Y07600); spinach cold acclimation protein, CAP85 (Neven et al. 1993); peach dehydrin, PCA60 (Artlip et al. 1997); and citrus cold-stress protein, COR19 (Cai et al. 1995). Interestingly, the first nine amino acids encoded by this cDNA (MAGIMNKIG) are identical to the first nine amino acids of the alfalfa cold acclimation protein, CAS15. A sequence identical to the 174-bp sequence (amplified from degenerate primers derived from peptide sequences from the 65-kDa dehydrin and used as a probe to isolate this cDNA) was not present within the cDNA sequence (Figs. 2 and 3). However, very similar sequences were present as part of the five large imperfect repeats. The two high-confidence primary peptide sequences obtained previously from the 60-kDa dehydrin exactly matched two sequences encoded within the cDNA, whereas peptide sequences unique to the 65-kDa dehydrin were similar to, but did not exactly match, any sequences in the cDNA clone (Figs. 1 and 3). Furthermore, a sequence very similar to a lower-confidence secondary sequence obtained for the 60kDa dehydrin was present within the cDNA (cDNA sequence QHQQQQYNK, extending from residues 28 through 36, as compared to KQQHQHEQQQHNK, obtained from the protein) (Figs. 1 and 3). To further confirm that the cDNA clone encodes the 60-kDa dehydrin, efforts were made to sequence the 60-kDa dehydrin from the N-terminus. However, this failed because the N-terminus was blocked. Therefore, the 60-kDa dehydrin was again digested with endoproteinase Lys-C, this time partially rather than completely, and three additional peptides were sequenced. The three sequences, which ranged in length from 11 to 25 amino acids, exactly matched sequences encoded within the cDNA (Figs. 3 and 5). In addition, the amino acid composition of the 60-kDa dehydrin was determined and found to agree well with the expected amino acid composition based on the cDNA sequence (Table 1).
Characterization of the gene product encoded by dehydrin cDNA To determine the molecular mass of the protein encoded by the dehydrin cDNA from SDS-PAGE, the protein was synthesized in an in vitro transcription/translation system. The protein was radioactively labeled by incorporation of 35 S-methionine in the in vitro translation reaction and fractionated by SDS-PAGE. By comparison to molecular mass 103
standards, the gene product was estimated to have a molecular mass of 40 kDa (Fig. 6).
drins (detected with anti-65-kDa dehydrin antibody), are glycosylated.
Glycosylation of blueberry dehydrins
Induction of dehydrin mRNA with chilling accumulation
Although all five high-confidence peptide sequences from the 60-kDa dehydrin were present within the dehydrin cDNA clone and the amino acid composition agreed well with the expected composition from the cDNA sequence, the clone appeared to encode a protein with a molecular mass of 40 kDa rather than 60 kDa. Therefore, we tested whether the blueberry dehydrins are modified post-translationally by glycosylation. After SDS-PAGE, floral bud proteins were immobilized on nitrocellulose and then treated with periodate to oxidize any hydroxyl groups of carbohydrate moieties to aldehydes. Oxidized sugars were labeled with biotin, and biotin was subsequently detected in a reaction with streptavidin-alkaline phosphatase conjugate. The results, shown in Fig. 7, reveal that proteins of 65 and 60 kDa, which co-migrate with the 65- and 60-kDa dehy-
Previous studies identified three major dehydrins of 65, 60, and 14 kDa, whose levels increase with CU accumulation and cold acclimation in floral buds of the blueberry (Muthalif and Rowland 1994a,b, 1995, Arora et al. 1997). To determine if these changes in protein levels reflect changes at the RNA level, the 2.0-kb dehydrin cDNA was used as a probe to monitor changes in dehydrin mRNAs in field plants of the more cold hardy cv. Bluecrop and the less hardy cv. Tifblue with CU accumulation. Previously, we showed from freeze-thaw tests that floral buds of Bluecrop and Tifblue field plants reach maximum hardiness levels by mid-winter, of about − 29°C for Bluecrop and about − 22°C for Tifblue (Muthalif and Rowland 1994a). Northern blots revealed hybridization of the 2.0-kb probe to two chilling-responsive transcripts of 2.0 and 0.5 kb in
Fig. 3. Nucleotide and deduced amino acid sequence of the 1978-bp dehydrin cDNA. K boxes (bold lettering with underline) and high-confidence peptide sequences from the 60-kDa dehydrin (double underline) are indicated. The stop codon is indicated with an asterisk. In the longest peptide sequence determined from the 60-kDa dehydrin, one amino acid residue (a glycine indicated with a double asterisk) could not be identified with high confidence.
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Table 1. Observed amino acid composition of the 60-kDa dehydrin and expected amino acid composition based on the sequence of the 2.0-kb dehydrin cDNA clone. ND, not determined. Amino acid
% Observed
% Expected
Ala Arg Asx Cys Glx Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
3.3 4.0 13.5 ND 29.2 19.1 6.6 4.1 4.8 5.3 0 0 0 0 0 ND 5.9 4.3
2.2 4.5 11.8 0.3 21.0 23.2 5.4 2.9 3.8 9.6 1.9 0 1.6 1.9 1.0 0 5.7 3.2
both cultivars (Fig. 8), as was seen with the 174-bp PCR product (data not shown). The 2.0- and 0.5-kb messages were detectable at 0 CU; the level of the 0.5-kb message, in particular, was relatively high in Bluecrop at 0 CU. A comparison of their accumulation patterns during fall through spring in the two cultivars revealed that levels of both messages increased by 50 CU and reached maximum levels more quickly in Bluecrop (levels peaked at 300 CU) than in Tifblue (levels peaked at 600 CU). Level of the 2.0-kb message declined significantly during the 300 CU accumulated after reaching the maximum level, i.e., by 600 CU and 900 CU for Bluecrop and Tifblue, respectively. On the other hand, level of the 0.5-kb message remained quite high in Bluecrop until the resumption of growth (by 1500 CU), whereas, in Tifblue, it declined significantly by 900 CU, like the 2.0-kb message. Densitometric results (Fig. 8) indicated that maximum level of the 2.0-kb message was about the same in Bluecrop and Tifblue, about 4 – 5-fold higher than the 0 CU level. On the contrary, maximum level of the 0.5-kb message was about 1.6 times higher in Bluecrop than in Tifblue. Estimation of the number of dehydrin genes in blueberry The number of dehydrin genes in blueberry was estimated by hybridization of the 2.0-kb cDNA to gel blots of genomic DNA from the original parent plants and the F1s of our diploid V. darrowi×V. caesariense-derived mapping population. Genomic DNAs were digested with several dif ferent restriction enzymes, the sites of which were not present in the cDNA, and washes were carried out under
Fig. 5. Three additional high-confidence primary peptide sequences obtained after partial Lys-C digestion of 60-kDa dehydrin.
highly stringent (4°C below Tm) and moderately stringent conditions (15°C below Tm). Results from digestions with EcoRI, EcoRV, and HindIII and washes under highly stringent conditions are shown in Fig. 9. Each of these digestions resulted in two or three strongly hybridizing fragments (not including allelic fragments, when possible to determine) and two to five weaker hybridizing fragments. Of the two largest strongly hybridizing fragments resulting from digestion of the parent DNAs with HindIII, only one was present in two of the three F1s examined (F1 c 6 shown in Fig. 9), suggesting that these two fragments are alleles of each other and not separate genes. These F1s apparently inherited the larger allele from each of the parents. The third F1 carried both of the alleles, like the parents (data not shown), suggesting that it inherited the larger allele from one parent and the smaller allele from the other parent. Digestion with EcoRI resulted in strongly hybridizing polymorphic fragments in the parents – a 5.8-kb fragment in the V. darrowi parent Fla4B and a 5.4-kb fragment in the V. caesariense parent W85-20. Both fragments were present in the F1s, as expected. Less stringent washes resulted in no significant difference in the number of fragments hybridizing, but some of the weakly hybridizing fragments (from stringent conditions) hybridized more strongly under these conditions (data not shown). These results suggest that blueberry dehydrins are encoded by a multigene family, about two to three genes with good homology to the 2.0-kb cDNA and a few other less related genes.
Discussion To date, we have isolated one 2.0-kb dehydrin clone from our blueberry cDNA library (prepared from RNA from cold-acclimated floral buds) using the 174-bp PCR fragment as a probe, which was amplified from degenerate primers derived from peptide sequences from the 65-kDa dehydrin. The nucleotide sequence of the dehydrin clone is unusual in several ways. First, the cDNA contains rather long 5%- and 3%-untranslated sequences of 319 and 714 bp (not including the stop codon), respectively (Fig. 3). In contrast, most dehydrin cDNAs contain less than 100 bp 5% to the ATG start codon and T-rich 3% noncoding regions of 200–300 bp. Also, the repetitive structure of the blueberry dehydrin is remarkable, even for dehydrins. Others have reported the K segment of certain dehydrins to be contained within a larger
Fig. 4. Alignment of the five large 48–62 amino acid-long contiguous repeats found encoded within the dehydrin cDNA. Sequences are aligned to give the least number of mismatches. K boxes found within the repeats are underlined. Physiol. Plant. 107, 1999
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Fig. 6. Gene products from the coupled in vitro transcription/ translation reactions separated by SDS-PAGE. In lane 1 is the negative control (no RNA was included in the in vitro translation reaction), and in lane 2 is the 40-kDa product from the dehydrin cDNA clone. Sizes of molecular mass markers, which were included on the protein gel, are indicated to the left of the autoradiogram.
repeat. For example, the K segment of CAP85 is contained within a larger 22-amino acid repeat (Neven et al. 1993). However, in this blueberry dehydrin, the five 48- to 62amino acid long contiguous repeats (Fig. 4) encompass the entire protein, with the exception of 44 amino acids at the amino terminus and 2 amino acids at the carboxy terminus. The presence of 5 K segments, but no Y or S segments,
Fig. 8. Northern blot of total RNA extracted from blueberry cvs Tifblue and Bluecrop and hybridized with the 2.0-kb dehydrin cDNA probe. (A) RNA for northern was extracted from floral buds collected from field plants after different lengths of chilling (from 0 to 1500 CU). Chill units are given above each lane. The probe hybridized to two messages of 2.0 and 0.5 kb. (B,C) Comparison of changes in levels of the 2.0- (B) and 0.5-kb (C) dehydrin messages in floral buds of Tifblue and Bluecrop with CU accumulation. Data were derived by averaging densitometric results from the northern presented in (A) and two other northerns. Standard errors were smaller than the symbols used in the figures and, therefore, are not shown.
Fig. 7. Results from western blot using anti-65-kDa antibody (A) and from glycoprotein detection (B) experiments. Proteins were extracted from duplicate floral bud samples of blueberry cv. Tifblue, having received 865 CU (lanes 1 and 2), and separated by SDSPAGE. Proteins were then transferred to nitrocellulose membranes for (A) detection of the 60- and 65-kDa dehydrins using anti-65kDa antibody (30 mg protein lane − 1) and (B) for detection of glycoproteins (20 mg protein lane − 1). In the lane marked M are pre-stained molecular mass protein standards (their sizes, in kDa, are shown to the left).
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indicates that this blueberry dehydrin should be classified as a K5-type of dehydrin, according to the YSK classification scheme of Close (1996). We propose that the gene represented by this clone be named bbdhn1 for blueberry dehydrin 1. From DNA sequence information, the cDNA encodes a protein with a molecular mass of 34.3 kDa, provided there are no post-translational modifications. From coupled in vitro transcription/translation reactions followed by SDSPAGE, the cDNA appears to encode a protein with a molecular mass of 40 kDa. In the case of dehydrins, discrepencies between the sizes of the proteins, as predicted from the nucleotide sequences, and the sizes as determined Physiol. Plant. 107, 1999
from SDS-PAGE are often found. Some examples include CAP85 from spinach (85 kDa from SDS-PAGE, 61.5 kDa from DNA sequence) (Neven et al. 1993), 50-kDa coldshock protein from wheat (50 kDa from SDS-PAGE, 39 kDa from DNA sequence) (Houde et al. 1992), and PCA60 from peach (60 kDa from SDS-PAGE, 50 kDa from DNA sequence) (Artlip et al. 1997). In the case of the wheat dehydrin, expression of the cDNA in E. coli results in a protein that co-migrates on SDS gels with the wheat 50-kDa protein (Houde et al. 1992). This suggests that the discrepancy in size is not due to post-translational modifications in the plant, because this cannot occur in E. coli, but may be due to altered mobility through SDS gels because of the shape or some other property of the protein. In support of this argument, maize G50 dehydrin has a molecular mass of 42 kDa from gel filtration chromatography (Ceccardi et al. 1994), which is affected by size and shape of a protein, but a molecular mass of only 17.8 kDa from sedimentation velocity centrifugation, which is influenced to a lesser extent by shape (Close 1996). Thus, it is not surprising that we observe a small discrepancy between the size of this blueberry dehydrin predicted from the DNA sequence (34.3 kDa) and the size as determined from SDS-PAGE (40 kDa). Of the three major blueberry dehydrins of 14, 60, and 65 kDa, we can rule out the possibilities that the 2.0-kb dehydrin cDNA encodes either the 14-kDa or 65-kDa dehydrin. The dehydrin cDNA is too large to encode the 14-kDa dehydrin, and the absence of peptide sequences unique to the 65-kDa dehydrin within the cDNA rules out the possibility that the cDNA encodes the 65-kDa dehydrin. Furthermore, the amino acid composition of the 60-kDa dehydrin
Fig. 9. Southern blots of blueberry genomic DNA hybridized with the 2.0-kb dehydrin cDNA. DNA was extracted from the original V. darrowi parent Fla4B (1), the original V. caesariense parent W85-20 (2), and the F1 c 6 (3) of our diploid blueberry mapping population and digested with restriction enzymes EcoRI, EcoRV, and HindIII. In lane M are the molecular mass standards with the molecular masses in kb shown to the left. The Southerns shown are those from highly stringent washes. Physiol. Plant. 107, 1999
agrees very well with the predicted composition from the cDNA sequence. In addition, five high-confidence peptide sequences available to us for the 60-kDa dehydrin, ranging in length from 9 to 25 amino acids, exactly match sequences found encoded within the cDNA, indicating that the cDNA clone does, in fact, encode the 60-kDa dehydrin. The size of the gene product encoded by the dehydrin cDNA, as judged from in vitro transcription/translation reactions followed by SDS-PAGE, is only 40 kDa, however, rather than 60 kDa. This discrepency can be explained if this dehydrin undergoes extensive post-translational modification such as heavy glycosylation. Using a commercially available glycoprotein detection system, it appears that the 65- and 60-kDa dehydrins are glycosylated. This is quite interesting, as it has not been previously reported that other plant dehydrins are glycosylated. Whether other researchers have explored this possibility is unknown. The 2.0-kb dehydrin cDNA does not encode any of the Asn-XXX-Ser potential sites for N-linked glycosylation, but does encode six serine and three threonine residues, which could serve as potential sites of O-linked glycosylation. Efforts to deglycosylate floral bud proteins and determine the sizes of the native forms of the 65- and 60-kDa dehydrins, using our anti-65-kDa antibody (which reacts to both the 60- and 65-kDa dehydrins), have so far proven unsuccessful. We have observed a less abundant 40-kDa dehydrin in floral buds of the blueberry cv. Bluecrop, in addition to the other three abundant dehydrins, but only once during a series of experiments using a sodium borate-based protein extraction procedure (Arora et al. 1997). This protein has not been observed in subsequent extractions using this procedure or when using our customary phenol-based extraction procedure (Muthalif and Rowland 1994a). Therefore, we believe the 40-kDa protein seen in these experiments was most likely a breakdown product of the 65- or 60-kDa dehydrin, or possibly the deglycosylated version of the 60-kDa dehydrin, which, for some reason, has only been observed with one set of tissue samples using one extraction procedure. Hybridization of RNA blots with the 2.0-kb cDNA revealed homology to two chilling-responsive messages of 2.0 and 0.5 kb. The 2.0-kb message is the same size as the cDNA itself and, thus, we assume it is the message encoding the 60-kDa dehydrin. The 174-bp PCR fragment that was amplified from degenerate primers based on peptide sequences from the 65-kDa dehydrin, thus, presumably is derived from the gene encoding the 65-kDa dehydrin, also hybridized on RNA blots to two chilling-responsive messages of 2.0 and 0.5 kb (data not shown). Consequently, it seems reasonable to conclude that the 2.0-kb size class of messages, which hybridizes to these two probes, actually represents two messages, one encoding the 65-kDa dehydrin and one encoding the 60-kDa dehydrin. The 0.5-kb message, which hybridizes to both the cDNA and PCR probe also, is of an appropriate size to encode the abundant 14-kDa dehydrin, but whether or not this is the case remains to be determined. Results from densitometric scans of RNA blots hybridized with the dehydrin cDNA revealed that levels of both the 2.0- and 0.5-kb transcripts were noticeably higher as early as 50 CU, as compared with the 0 CU levels, in the two cultivars examined. In addition, the messages reached 107
maximum levels more quickly in the hardier cv. Bluecrop (by 300 CU) than in Tifblue (by 600 CU). The level of the 0.5-kb message also remained higher for longer in Bluecrop than in Tifblue, not declining dramatically until resumption of growth in the spring. The overall maximum level of the 2.0-kb message was about the same in both cultivars, whereas maximum level of the 0.5-kb message was higher in Bluecrop than in Tifblue. In comparison, from previous protein work (Muthalif and Rowland 1994a), the 65-, 60-, and 14-kDa dehydrins accumulate to higher levels quickly and remain at higher levels longer in Bluecrop than in Tifblue (maximum levels reached by about 900 CU in Bluecrop and by about 600 CU in Tifblue). The maximum level of all three proteins is higher in Bluecrop than in Tifblue, although the major difference seen is in the level of the 14-kDa dehydrin. The lag in reaching maximum protein levels (900 CU) in Bluecrop as compared to RNA levels (300 CU) suggests that the dehydrin proteins are quite stable. This lag is not seen in Tifblue, where protein and RNA levels both peak at about 600 CU. Thus, the dehydrins of Bluecrop may be more stable than those of Tifblue. Our studies have also demonstrated that levels of the dehydrins are closely associated with cold hardiness levels in three different cultivars examined: Bluecrop, Tifblue, and Gulfcoast (Arora et al. 1997). If dehydrins do play a causal role in determination of cold hardiness in blueberry, then the difference in hardiness between Bluecrop and Tifblue could probably be explained by a combination of earlier expression, overall higher expression (especially for the 14kDa dehydrin), and greater stability of the dehydrins in Bluecrop than in Tifblue. The number of dehydrin genes in blueberry was estimated from genomic blots using the 2.0-kb cDNA as a probe. The results suggest that blueberry dehydrins are encoded by a multigene family, consistent with the presence of approximately four to five dehydrins (Muthalif and Rowland 1994a,b, 1995, Arora et al. 1997). The identification of a polymorphic restriction fragment length polymorphism (RFLP) marker (EcoRI fragment) homologous to the dehydrin cDNA will enable us to map the dehydrin gene in our V. darrowi ×V. caesariense-derived mapping population. In conclusion, we have isolated and sequenced a somewhat unusual cDNA that encodes the 60-kDa dehydrin from blueberry. The demonstration that the dehydrin is glycosylated explains the discrepancy in size between the protein from floral buds (60 kDa) and the size of the cDNA gene product, as predicted from either DNA sequence (34.3 kDa) or in vitro transcription/translation followed by SDSPAGE (40 kDa). Our future plans include cloning and sequencing the cDNAs encoding the remaining dehydrins and determining if there is a regulatory gene that controls expression of the entire gene family. We would also like to characterize the nature of the carbohydrate moieties attached to the 65- and 60-kDa dehydrins and determine their function. Acknowledgements – We would like to thank Drs A. Callahan and J. Hancock for their critical reviews of the manuscript. We would also like to acknowledge E. Ogden for her assistance in collecting flower buds for these experiments. This work was supported by the US Department of Agriculture, Agricultural Research Service, and
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by a grant from the National Research Initiative Competitive Grants Program, US Department of Agriculture.
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