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First published online July 16, 2004; 10.1105/tpc.104.024018 © 2004 American Society of Plant Biologists The SENSITIVE TO FREEZING2 Gene, Required for Freezing Tolerance in Arabidopsis thaliana, Encodes a ß-GlucosidaseSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom 1 To whom correspondence should be addressed. E-mail g.thorlby{at}rhul.ac.uk; fax 01784-470756.
The sensitive to freezing2-1 (sfr2-1) mutation causes freezing sensitivity in Arabidopsis thaliana. By mapping, transgenic complementation, and sequencing, sfr2-1 was revealed to be a mutation in gene At3g06510. A new knockout allele was obtained, and its identical freezing-sensitive phenotype confirmed that the SFR2 gene product is essential for freezing tolerance. Transcription of SFR2 was observed to be constitutive rather than stress inducible and was distributed throughout most aerial tissues. SFR2 encodes a protein homologous to family 1 glycosyl hydrolases (ß-glycosidases), but the predicted AtSFR2 protein is divergent from all other family 1 ß-glycosidases of Arabidopsis, showing closer homology to the sequences of several ß-glycosidases from thermophilic archea and bacteria. After purification from a heterologous expression system, AtSFR2 displayed a specific hydrolytic activity against ß-D-glucosides.
What genes are responsible for the freezing tolerance of cold-acclimated plants? The characterization of genes whose expression is induced during cold acclimation has been a powerful approach to this problem (reviewed in Thomashow, 1999  ). Its success is manifest in the manipulation of freezing and drought tolerance by transgenesis with transcription factors (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Gilmour et al., 2000) and in the molecular genetic description of significant portions of the signaling pathway (Stockinger et al., 2001; Thomashow et al., 2001; Fowler and Thomashow, 2002; Seki et al., 2003). However, the contributions of individual genes to freezing tolerance, except via the control of other genes' expression, have not been readily ascertained by this approach (Xin and Browse, 2000).
A complementary approach has been to discover genes by their effects on phenotype. Natural variation has been exploited in some commercially important species to identify quantitative trait loci affecting freezing tolerance (Teutonico et al., 1995
In one study, mutants were obtained that caused elevated levels of freezing tolerance in the absence of cold acclimation (Xin and Browse, 1998
Mutants deficient in freezing tolerance have also been described. The frs1 mutation was found to be an allele of ABA3 (Llorente et al., 2000
No pleiotropic effects of the sfr2-1 mutation have been observed (McKown et al., 1996
Classical Genetic Mapping of the SFR2 Gene The sfr2-1 mutation was previously mapped to the region between markers GAPC and nga126 on chromosome III (Thorlby et al., 1999 ). From the mapping cross of sfr2-1/sfr2-1 Columbia (Col) x SFR2+/SFR2+ Landsberg erecta, we screened 690 F2 zygotes for crossovers in this region. Among those zygotes showing crossovers, we determined SFR2 genotypes by freeze testing the F3 progeny of each zygote. By screening these lines with publicly available mapping markers, we obtained a position for sfr2-1 between markers mi403 and C6 (Figure 1A).
An assembly of contiguous sequence (contig) was constructed from BAC clones covering this genetic interval. Further mapping markers in this interval were generated as PCR amplicons, using sequence information from members of the contig. (For Arabidopsis, this approach has now been superseded by the creation of an online polymorphism database by Monsanto Company, http://www.arabidopsis.org/Cereon/index.html.) Analysis with the new mapping markers (Figure 1B) allowed us to revise both boundaries of the interval containing sfr2-1. It also showed that the mapping population had been employed to the effective limit of its resolution because markers just inside the new boundaries (by 16 and 17 kb, respectively) had no crossovers with SFR2. Thus, classical genetic mapping placed SFR2 within a 218-kb interval bounded by markers 28L1SP6 and GT10.
Identification of the SFR2 Gene and sfr2-1 Mutation The DNA sequence of this region contained two complete putative genes according to contemporary genome annotations, along with incomplete portions of two other flanking genes (Figure 1D). We sequenced the complete genes in amplicons derived from the sfr2-1 mutant plants and compared them with published genomic data. Gene At3g06510 contained a single base change, a G>A transition consistent with mutagenesis by ethyl methanesulfonate (Figure 1D), whereas gene At3g06520 showed no changes. We noticed that the sequence alteration in gene At3g06510 would create a recognition site for the restriction endonuclease BsrI (ACTGG) and confirmed that this site was present in amplicons obtained from sfr2-1 material and absent in the cognate amplicons from wild-type Arabidopsis. In parallel with the sequence analysis, we subcloned portions of the 11-kb complementing region that contained At3g06510 and At3g06520 individually; these were transformed into the sfr2-1 mutant line. Two independent clones of gene At3g06510 (identified by hatched bars in Figure 1D) restored freezing tolerance to the sfr2-1 mutant, with freezing sensitivity reappearing and segregating as expected in the next generation. A subclone of the other gene, At3g06520, had no effect on the mutant's freezing sensitivity. SFR2 was thus identified with the putative gene At3g06510 both by mutant sequence analysis and by complementation.
Transcription and Splicing of SFR2
To determine the transcriptional initiation site of SFR2, we designed a primer complementary to SFR2 mRNA, 182 nucleotides downstream from the presumed initiation codon. Using a PCR protocol to define the 5' end of the mRNA (Frohman et al., 1988
We compared the abundance of SFR2 transcripts in wild-type plants before and during cold acclimation by RNA gel blotting. RNA samples were extracted from plants and tested in three independently replicated experiments. Blots were probed with a transcript region (the 5' end of the cDNA, amplified by PCR) sufficiently nonhomologous to other Arabidopsis sequences to preclude cross-hybridization. SFR2 transcripts were detected at low levels in all samples: there was no indication of induction by cold, even after 24 h (Figure 2A). As a positive control, the illustrated blot was reprobed with a known cold-inducible gene, KIN1 (Kurkela and Franck, 1990
The expression of SFR2 was characterized further using real-time PCR. Wild-type plants were subjected to various dehydrative stress treatments (dehydration, salt, and abscisic acid application) and the level of SFR2 transcript determined (Figure 3A). None of the treatments had other than a small effect on the level of SFR2 transcript. This suggests that the tested stresses have, at most, a limited effect on the transcriptional regulation of SFR2; this contrasts with many stress-induced genes that show a large amplification. Monitoring of a known stress-induced gene, COR47, demonstrated that the experimental plants were undergoing a normal pattern of gene induction in response to these treatments (data not shown). In the same experiment, it was shown that the level of SFR2 transcript was approximately sixfold lower in the root than in the leaf of wild-type plants. A comparison between wild-type and sfr2-1 plants, both untreated, showed that they had very similar levels of SFR2 transcript.
In a similar set of experiments, real-time PCR was used to determine the levels of transcript in wild-type and sfr2-1 plants over a prolonged period in the cold. Figure 3B extends the results obtained by RNA gel blotting, demonstrating little variation in the level of SFR2 transcript even after 8 d in the cold. Transcript levels in sfr2-1 were similarly stable, suggesting that transcriptional differences between the two are unlikely to explain the mutant phenotype. The localization of SFR2 expression was determined using a translational fusion of a ß-glucuronidase (GUS) reporter to the 31st codon of the SFR2 gene, which was introduced into the wild-type background. GUS activity was observed in 7 out of 11 primary transformants. Activity was seen in most aerial organ types (hypocotyls, cotyledons, stems, leaves, pedicels, sepals, anthers, and pistils) but not in petals or filaments (Figure 4). In support of the real-time PCR experiments, expression in roots was very limited, with only a few plants showing small and weak patches of expression. The distribution of reporter activity appeared heterogeneous in the leaves of older plants (Figures 4C and 4D). Consistently with the result of RNA gel blotting and real-time PCR, we did not observe any differences in localization between untreated and cold-treated plants (Figure 4, comparing A with B, C with D, E with F, and G with H).
Protein Sequence Homologies Public databases were searched for protein sequences homologous with the conceptual translation product of SFR2. The highest similarities that we found were to ß-glycosidase enzymes from thermophilic and halophilic archea and bacteria (Table 1, Figure 5). These enzymes were members of glycosyl hydrolase family 1 (Henrissat, 1991 ), and a characteristic family 1 motif was detected in AtSFR2 protein (residues 264 to 268 and 427 to 431). The various activities reported for the prokaryotic homologs included specific cleavage of ß-D-glucosidic, ß-D-mannosidic, and ß-D-galactosidic bonds and more catholic recognition of multiple types of ß-glycosidic bonds (Table 1). We examined the relationship between all the family 1 ß-glycosidase homologs of Arabidopsis and the two prokaryotic ß-glycosidases most closely homologous to AtSFR2. A multiple protein sequence alignment was calculated and used to derive a cladogram relating the proteins by minimum substitution distances (Figure 6). This confirmed that AtSFR2 and the prokaryotic ß-glycosidases form a distinct clade. Their grouping highlights the unusual extent of AtSFR2's divergence from every other family 1 ß-glycosidase enzyme in Arabidopsis.
Enzymatic Activity of AtSFR2 Protein The full coding sequence from both SFR2 and sfr2-1 cDNAs were expressed in the yeast Pichia pastoris. In the supernatant of induced cultures, a protein was detected whose migration on SDS-PAGE indicated a size (  70 kD) consistent with AtSFR2 protein. No such protein was detected in control cultures transformed with the empty vector. The secretion of AtSFR2 and AtSFR2.G234D protein into the medium suggests that the N terminus of AtSFR2 functions in yeast as a signal sequence, supporting the prediction of current annotations of At3g06510 by the Munich Information Center for Protein Sequences (München, Germany; http://mips.gsf.de/proj/thal/).
Protein concentrates from culture supernatants were tested for their glycosidic activity against a variety of synthetic glycosides (Table 2). A ß-glucosidase purified from almond emulsin (EC 3.2.1.21; Sigma) was included in the tests as a positive control. Protein concentrated from both the SFR2 and sfr2-1 transformants produced significant hydrolysis of o-nitrophenyl-ß-D-glucoside and of p-nitrophenyl-ß-D-glucoside but not of several other p-nitrophenyl-ß-D-glycosides nor of a p-nitrophenyl-
Mutant Alleles of SFR2 The sfr2-1 mutation affects codon 234 of SFR2, converting a Gly (GGC) into an Asp (GAC) codon; thus, sfr2-1 is a missense mutation (Figure 5). An automatically generated alignment of At3g06510 with its closest homologs was obtained from the Munich Information Center for Protein Sequences (data not shown). This revealed strong conservation of G234 among a wide variety of ß-glycosidases; hence, it is likely that G234 is important for the function of ß-glycosides.
The recessive nature of the sfr2-1 allele led Warren et al. (1996) We used PCR to distinguish mutant homozygotes from heterozygotes. Homozygous sfr2-i2 mutants were selfed, and their progeny were tested for freezing tolerance. All the tested progeny (33/33) showed freezing sensitivity, and the appearance of freezing damage was indistinguishable from that of sfr2-1 plants frozen in parallel (Figure 7). Further progeny were tested under freezing conditions of varying severity, but in no test did we observe any difference between the phenotypes of the sfr2-1 and sfr2-i2 mutants.
Immunoblot Analysis A polyclonal antibody raised against a peptide generated from the C-terminal region of the AtSFR2 protein was used to compare both the abundance of the AtSFR2 protein in different alleles of SFR2 and the effect of cold treatment (Figure 8). The antibody detected a protein with an apparent molecular mass of 65 kD in extracts of wild-type and sfr2-1 plants but not in material isolated from sfr2-i2 plants. This is consistent with the expected early termination of translation in sfr2-i2, predicted from DNA sequence analysis, and suggests that the 65-kD protein detected in wild-type and sfr2-1 plants is AtSFR2. A cross-reacting band (110 kD) present in all plants tested is unlikely to be AtSFR2 because its molecular weight is much higher than predicted for unprocessed AtSFR2 and because of its presence in sfr2-i2 plants.
The abundance of protein detected in plants of sfr2-1 grown at 20°C was slightly reduced compared with that in the wild type, suggesting that the mutation may have some effect on the translation or stability of the protein. After transfer of plants to 4°C for 24 h, there was a reduction in the level of AtSFR2 protein in wild-type plants, but no AtSRF2 protein could be detected in sfr2-1 plants. A time-course experiment (data not shown) showed that AtSFR2 protein was not detected in sfr2-1 plants over a period of at least 5 d after transfer to growth at 4°C. This suggests that the missense mutation in sfr2-1 plants has created a cold-sensitive allele of SFR2. The apparent mobility of the AtSFR2 protein in planta ( 65 kD) was smaller than that produced by expression in P. pastori (70 kD). This is most likely the result of posttranslational modification, possibly involving cleavage of the predicted N-terminal signal sequence.
Our identification of the SFR2 gene was undertaken because SFR2 was expected to encode an essential freeze-protective function in the cold-acclimated plant. This was based on an inference that the sfr2-1 mutant phenotype reflected a loss (rather than a gain or change) of function. However, sfr2-1 was found to be a missense mutation. By isolating a second mutant allele of SFR2, of a type that would be expected a priori to destroy the gene's function absolutely, the rationale for gene identification has been tested. Because the second mutant sfr2 allele had a similarly freezing-sensitive phenotype, we can be confident that SFR2 is indeed essential for freezing tolerance.
The predicted AtSFR2 protein is homologous to enzymes with glycosyl hydrolase (glycosidase) activity, in particular the group of ß-glycosidases designated family 1 (Henrissat, 1991
When produced by heterologous expression, AtSFR2 protein displayed hydrolytic activity against two nitrophenyl ß-D-glucosides. Activity was not detected when the glucose moiety of the substrate was replaced by any of several other glycones nor when the ß linkage to glucose was replaced by an The ß-D-glucosidase activity present in the mutated AtSFR2.G234D protein was surprising. It seemed likely that the mutant protein would lack activity because it has the same freezing phenotype as the sfr2-i2 insertional mutant that is expected to be totally deficient in activity. Indeed, it cannot be ruled out that the protein produced in the sfr2-1 mutant, although active against synthetic substrates in vitro, fails to cleave its natural substrate. The results obtained from protein blotting, however, suggest another possible explanation for the freezing sensitivity of the sfr2-1 allele. Given the minor differences in the level of SFR2 RNA transcript between the wild-type and sfr2-1 plants and the conservation of the in vitro ß-D-glucosidase activity of the AtSFR2.G234D mutant protein, the elimination of AtSFR2 protein during the cold acclimation period may explain the freezing sensitivity of the sfr2-1 plants. If sfr2-1 plants do contain biologically active protein before cold acclimation, this is not sufficient to protect the plant from freezing. This suggests a specific requirement for SFR2 during the cold acclimation process rather than the manifestation of a latent lesion that is undetected in the warm. Evidence only exists, however, for ß-glucosidase activity in vitro and not for the appropriate biological activity of the mutant protein in planta.
ß-Glucosidases are ubiquitous. Glucosylation (reversible by the appropriate glucosidase) can affect various characteristics of the glucosylated moiety (the aglycone), including reactivity, solubility, and transport (Li et al., 2001 Both bioinformatic analysis and its secretion from yeast cells indicate that AtSFR2 has a signal peptide at its N terminus. If AtSFR2 is indeed secreted from the protoplast in planta, the range of possible substrates for the AtSFR2 ß-glucosidase would be limited to apoplastic metabolites, membrane components, and structural components of the cell wall.
The constitutive expression of SFR2, both in response to cold and to several dehydrative stresses, indicates that this gene would not have been detected by the approach of characterizing cold-inducible genes. Constitutive expression of the SFR2 gene does not necessarily equate to constitutive activity of AtSFR2 protein; in particular, posttranslational control is conceivable because most plant ß-glucosidases are themselves glycosylated, and glycosylation has been implicated in their stabilization (Cicek and Esen, 1999
Freeze Testing To screen individuals for freezing tolerance, seedlings were grown for 5 weeks at 18 to 20°C with a 9-h photoperiod at 250 µmol m–2 s–1, and then subjected to 11 d of cold acclimation at 4°C, with an 8-h photoperiod at 220 µmol m–2 s–1. They were placed in a freezer with air temperature at a minimum of –6.0°C for 16 h and then returned to their preacclimation growth conditions. Injury was assessed after 5 d.
Generation and Use of Physical Markers in Mapping
DNA samples were isolated from F2 plants (Thorlby et al., 1999 ) and analyzed for cleaved amplified polymorphic sequence marker genotypes (Konieczny and Ausubel, 1993) and simple sequence length polymorphism marker genotypes (Bell and Ecker, 1994) as described. One restriction fragment length polymorphism marker, mi403, was also used. Map positions refer to the May 2001 release by the Nottingham Arabidopsis Stock Centre (Nottingham, UK; http://nasc.nott.ac.uk/) of the recombinant inbred map (Lister and Dean, 1993).
BAC Identification and Alignment
Subcloning for Complementation
Plant Transformation and Selection
Sequencing of SFR2 5' Rapid amplification of cDNA ends was performed using the 5'/3' RACE kit (Roche Molecular Biochemicals, Lewes, UK; http://www.biochem.roche.com), using a gene-specific primer (5'-TGCAGTAGCTACCCAAAGAAG-3') positioned 182 nucleotides downstream of the initiation codon. PCR products were cloned into pGEM-T-Easy. A plasmid clone whose insert matched the size of the PCR amplicon was selected for sequence analysis (accession number AJ491320).
RNA Gel Blotting
Real-Time RT-PCR Primers were designed such that one of the pair spanned the position of an exon/intron border (At1g67090 and At3g06510) and did not amplify genomic DNA, or the amplicon contained an intron and amplified a different sized genomic DNA and cDNA fragments (At1g49240). Fragments were amplified from the 3' region of the gene transcript and were between 100 and 150 bp in length. The following primers were used for real-time PCR experiments: SFR2/At3g06510 (5'-GCAATGCTAAAGGGTGTTCC-3' and 5'-AAGATCATGGGATCGGTCAA-3'), ribulose bisphosphate/At1g67090 (5'-TTCCTGACCTTACCGATTCC-3' and 5'-ACAAATCCGTGCTCCAACTC-3'), and actin2/At1g49240 (5'-CTTCCCTCAGCACATTCCAG-3' and 5'-CCCAGCTTTTTAAGCCTTTG-3'). The Quantitect SYBR Green PCR system (Qiagen) was used according to the manufacturer's recommendations. Diluted cDNA (2 µL) was added to a reaction mix containing primers (0.3 µM) and 1x master mix in a total volume of 25 µL. The following Smartcycler program was used in all experiments. An initial 15 min at 95°C for Taq activation followed by 45 cycles consisting of 15 s at 94°C, 30 s at 56°C, and 30 s at 72°C. At the end of the PCR, a melt curve analysis (60 to 95°C) was performed to verify the fidelity of the amplification. For each test condition, cDNA derived from three independent experiments was used, and each reaction was run in duplicate.
For relative quantification the method of Pfaffl (2001)
Reporter Gene Analysis
Heterologous Expression and Enzymatic Assay of AtSFR2 Pichia transformants were cultured for 48 h in 200 mL of buffered methanol complex medium at 28°C. Culture supernatants were collected after 10 min centrifugation at 3000g. Protein was precipitated with 60% saturated ammonium sulfate, redissolved in 20 mL of 20 mM sodium phosphate, pH 7.0, 1 M ammonium sulfate, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, and loaded onto an octylsepharose hydrophobic interaction column. Protein was eluted by a three-step gradient from 0.75 to 0.0 M ammonium sulfate in the same buffer. Eluted fractions were analyzed by SDS-PAGE, pooled, concentrated by size exclusion chromatography (Vivaspin column, 50,000 kD mass cutoff; Vivascience, Hannover, Germany) and diluted in 100 mM citrate/50 mM phosphate, pH 6.0. Enzymatic assays were performed in triplicate. Twenty-five microliters of purified protein was mixed with 25 µL of the appropriate chromogenic substrate in 100 mM citrate/50 mM phosphate, pH 6.0, and 250 µL of 100 mM citrate/50 mM phosphate buffer, pH 7.0. Reactions were stopped by addition of 700 µL of 0.4 M sodium carbonate after incubation at 37°C for 0 or 15 min. Release of p-nitrophenol or o-nitrophenol was measured by optical absorbance at 410 nm, assuming extinction coefficients of 18,400 M–1 cm–1 and 3500 M–1 cm–1, respectively.
Isolation of the sfr2-i2 Allele
Protein Immunoblot Analysis For the production of polyclonal antibody against AtSFR2, a cDNA fragment, encoding 109 amino acids at the C terminus of AtSFR2 protein, was cloned by PCR into the expression vector pET28a (Merck Biosciences, Nottingham, UK) in frame with a 6x His tag. The protein was expressed in Escherichia coli strain BL21 and purified using a nickel affinity column (BD Talon; Clontech, BD Biosciences, Cowley, UK). Polyclonal antibodies, raised in rabbit, against the recombinant peptide were used at a dilution of 1:200. Secondary goat anti-rabbit horseradish peroxidase–conjugated antibody (Promega) was used at a 1:5000 dilution and detection performed using 3,3'-diaminobenzidine peroxidase substrate (Sigma-Aldrich, Dorset, UK).
Bioinformatics Sequence data from his article have been deposited with the EMBL/GenBank data libraries under accession numbers AJ491320, AJ491321, and AJ491322.
This research was supported by the Biotechnology and Biological Science Research Council (UK) under Grants A05555 and P10187. Emma Veale made technical contributions. David Bouchez provided physical map information before publication.
2 Gareth Warren died during the final preparation of this manuscript.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Glenn Thorlby (g.thorlby{at}rhul.ac.uk). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.024018. Received May 6, 2004; accepted May 11, 2004.
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ABSTRACT
INTRODUCTION
-D-glucoside. No activity against any of these substrates was detected in the preparations from the empty-vector control. This seems to indicate that AtSFR2 protein is an enzymatically active ß-D-glucosidase with strong glycone selectivity. The presence of comparable ß-D-glucosidase activity in the protein produced from the mutated sfr2-1 transcript suggests that enzymatic activity is maintained, at least in vitro, when the protein is tested against synthetic glycosides. 
