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First published online January 24, 2008; 10.1104/pp.107.108779 Plant Physiology 146:1242-1254 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Comparative Genomic Sequence and Expression Analyses of Medicago truncatula and Alfalfa Subspecies falcata COLD-ACCLIMATION-SPECIFIC Genes1,[W],[OA]Department of Horticulture and Crop Science, The Ohio State University/Ohio Agricultural Research and Development Center, Wooster, Ohio 44691
In Arabidopsis (Arabidopsis thaliana) the low-temperature induction of genes encoding the C-REPEAT BINDING FACTOR (CBF) transcriptional activators is a key step in cold acclimation. CBFs in turn activate a battery of downstream genes known as the CBF regulon, which collectively act to increase tolerance to low temperatures. Fundamental questions are: What determines the size and scope of the CBF regulon, and is this is a major determinant of the low-temperature tolerance capacity of individual plant species? Here we have begun to address these questions through comparative analyses of Medicago truncatula and Medicago sativa subsp. falcata. M. truncatula survived to –4°C but did not cold acclimate, whereas Medicago falcata cold acclimated and survived –14°C. Both species possessed low-temperature-induced CBFs but differed in the expression of the COLD-ACCLIMATION-SPECIFIC (CAS) genes, which are candidate CBF targets. M. falcata CAS30 was robustly cold-responsive whereas the MtCAS31 homolog was not. M. falcata also possessed additional CAS30 homologs in comparison to the single CAS31 gene in M. truncatula. MfCAS30 possessed multiple pairs of closely spaced C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) motifs, the cognate CBF binding site in its upstream region whereas MtCAS31 lacked one CRT/DRE partner of the two proximal partner pairs. CAS genes also shared a promoter structure comprising modules proximal and distal to the coding sequence. CAS15, highly cold-responsive in both species, harbored numerous CRT/DRE motifs, but only in the distal module. However, fusion of the MtCAS15 promoter, including the distal module, to a reporter gene did not result in low-temperature responsiveness in stably transformed Arabidopsis. In contrast, both MtCAS31 and MfCAS30 promoter fusions were low-temperature responsive, although the MfCAS31 fusion was less robust than the MfCAS30 fusion. From these studies we conclude that CAS genes harbor CRT/DRE motifs, their proximity to one another is likely key to regulatory output in Medicago, and they may be located kilobases distal to the transcriptional start site. We hypothesize that these differences in CRT/DRE copy numbers in CAS30/CAS31 upstream regions combined with differences in gene copy numbers may be a factor in determining differences in low-temperature tolerance between M. truncatula and M. falcata.
Many plants increase their capacity to survive freezing temperatures after exposure to a period of low, nonfreezing temperatures, a process known as cold acclimation (Thomashow, 1990  , 1999). In Arabidopsis (Arabidopsis thaliana), a major pathway leading to cold acclimation and freezing tolerance is the C-REPEAT BINDING FACTOR (CBF) response pathway. The CBFs are transcriptional activators that bind to the C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE), which has at its core a 5-bp motif, CCGAC (Stockinger et al., 1997). Initially identified as the DNA regulatory element critical for the low-temperature induction of the COR genes (Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994; Wang et al., 1995; Iwasaki et al., 1997), the CRT/DRE is now known to effect cis-acting regulatory control over a much larger network of regulatory and structural genes referred to as the CBF regulon (Fowler and Thomashow, 2002; Vogel et al., 2005). The CBF regulon in turn functions to impart freezing tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Jaglo et al., 2001). Through mechanisms that have only recently begun to be understood (Chinnusamy et al., 2003; Zarka et al., 2003), the CBFs are themselves induced in response to low temperatures, initiating the regulatory cascade (Gilmour et al., 1998).
Tomato (Solanum lycopersicum), a chilling sensitive plant also harbors CBF genes that respond to low temperatures (Zhang et al., 2004
In alfalfa (Medicago sativa) a freezing-tolerant plant that cold acclimates, transcripts encoding the COLD-ACCLIMATION-SPECIFIC (CAS) proteins are induced and accumulate to very high levels during cold acclimation (Mohapatra et al., 1989
Alfalfa is one of 83 Medicago species (Small and Jomphe, 1989 In this study, we compared M. truncatula and alfalfa subsp. falcata, a diploid subspecies of alfalfa, in their capacity to cold acclimate and in components of the CBF cis-/trans-transcriptional regulatory system. A specific objective was to determine the structure of the Medicago CAS genes and explore whether they might be under a cis- and trans-regulatory control system homologous to that of the Arabidopsis COR genes.
M. truncatula Survives to –4°C But Cold-Acclimating Conditions Do Not Significantly Increase Its Capacity to Survive Freezing To determine whether M. truncatula could cold acclimate and survive freezing, 4-week-old plants of M. truncatula and alfalfa subsp. falcata, hereafter referred to as Medicago falcata, were placed at +2°C for 4 weeks, and then subjected to a series of decreasing temperatures below freezing. M. truncatula survived to about –4°C whether plants were subjected to cold-acclimating conditions prior to freezing or not (Fig. 1B ). In comparison, M. falcata survived to about –6°C without prior exposure to cold; and survived and regrew after exposure to nearly –14°C when cold acclimated (Fig. 1B). When plants were evaluated by electrolyte leakage, M. truncatula had TEL50 values of about –4°C whether plants were subjected to cold-acclimating conditions or not. In comparison, leaves from cold acclimated and nonacclimated M. falcata yielded TEL50 values of –11°C, and –5°C, respectively (Fig. 1C). Cold-treated M. truncatula crown tissues showed a slightly greater TEL50 of –8°C (Fig. 1C). These experiments suggest that there is a marginal depression in the temperature at which electrolyte leakage occurs in M. truncatula in response to a period of low nonfreezing temperatures, however M. truncatula plants are only capable of surviving to –5°C, and cold-acclimating conditions do not impart a significantly greater capacity to survive lower temperatures.
M. truncatula and M. falcata Differ in Their CAS Gene Cold-Responsive Expression
As the CBF transcriptional activators play a central role in the cold-acclimation response of Arabidopsis, we considered that M. truncatula and M. falcata might differ in one or more components of the CBF response pathway. To explore this idea, three Medicago CBF sequences, MtCBF1, MtCBF2, and MtCBF3 (based on the chronological order in which they were identified from database searches) alongside CAS15 and CAS17 (cold acclimation specific; Mohapatra et al., 1989
In both Medicago species, low levels of CAS15 were detected in warm-grown plants (Fig. 2A). Transcript levels increased in abundance 6 h after decreasing the temperature, and continued to accumulate to very high levels 24 h after the temperature decrease (Fig. 2A). After 7 d, CAS15 remained elevated in M. falcata but was diminished in M. truncatula (Fig. 2A). Twenty-four hours after returning plants to 25°C, CAS15 transcripts decreased to levels detected in the warm-grown plants (Fig. 2A). CAS17 showed a similar expression profile as CAS15 in M. falcata but was undetectable in M. truncatula at a lower RNA loading concentration (Fig. 2A); increasing the amount of RNA loaded onto the gel from 5 to 15 µg allowed for its detection (Fig. 2B). This indicated that very low levels of CAS17 were present in M. truncatula at all temperatures, which increased slightly at 12°C and the lower temperatures (Fig. 2B). Faint levels of CAS17 were also detected at all temperatures in M. falcata, and each incremental temperature decrease resulted in CAS17 levels elevated above the previous temperature, which were most pronounced at 8°C and 6°C (Fig. 2B).
To determine whether the Medicago CAS genes harbored CRT/DRE regulatory motifs, and to explore the mechanism behind the difference in CAS17 gene expression we sequenced the M. truncatula and M. falcata CAS15 and CAS17 genomic regions using inserts cloned in bacteriophage
Surprisingly, the M. truncatula and the M. falcata genomic clones isolated with the CAS17 probe were predicted by ab initio gene structure prediction programs FGENESH and GENSCAN to encode polypeptides much larger than 17 kD. The predicted polypeptide encoded by the M. truncatula genomic clone was 31 kD, whereas that of the M. falcata genomic clone was 30 kD (Fig. 3B). Similarity to CAS17 started approximately at amino acid residue 140 of the predicted M. falcata polypeptide (Fig. 3B). Searches of GenBank and the Gene Index Project databases for expressed sequences encompassing the entire length of these ab initio-predicted genes identified an alfalfa cDNA clone, GenBank accession AF411554 (E = 1e–169, score 1,349; Ivashuta et al., 2002 ), and the M. truncatula tentative consensus sequence TC100921 (E = 7.4e–208, score 4,695). No single M. truncatula EST encompassed the entire predicted coding sequence (CDS; Fig. 3C), however the TC100921 sequence assembly comprising multiple ESTs was 100% identical to the ab initio predicted CDS. RNA blot hybridization using only the upstream part of the predicted CDS indicated that a higher Mr species of RNA cross-hybridized with this probe (Fig. 3D). This higher Mr RNA species also cross-hybridized to the CAS17 probe; albeit to a lesser degree (Fig. 3D). As these genes likely encoded polypeptides much larger than 17 kD, we designated them MtCAS31 and MfCAS30. At the polypeptide level, MtCAS31 and MfCAS30 each possessed four of the characteristic dehydrin Lys-rich K-segments and a single Y-segment (Fig. 3B). Over the entire length of the polypeptide MtCAS31 and MfCAS30 were about 55% identical. Most of the dissimilarity occurred in the form of small insertions and deletions in the regions between each of these conserved K- and Y-segments (Fig. 3B).
Alignment of the MtCAS15, MtCAS31, and MfCAS30 genomic sequences to their respective expressed sequences revealed that these genes were interrupted by a single intron (Fig. 4A ; data not shown). In the instance of MtCAS15 the entire protein CDS was contained within Exon 1 (Fig. 4A), whereas MtCAS31 and MfCAS30 were split between Exon 1 and Exon 2 (Fig. 4B). This single intron in MtCAS31 and MfCAS30 occurred at a conserved Gly; residue 89 in MtCAS31, and residue 69 in MfCAS30 (Fig. 3B).
Immediately upstream of the M. falcata and M. truncatula CAS15 CDSs was a shared block of nucleotide identity ending at –270 in MtCAS15 and –325 in MfCAS15 (Fig. 4A; Supplemental Fig. S1). Further upstream, at –1,529 in MtCAS15 and –1,819 in MfCAS15, began a second block of nucleotide conservation (Fig. 4A; Supplemental Fig. S1). This second block continued to –2,586 of MtCAS15 and to –2,807 of MfCAS15 (Fig. 4A; Supplemental Fig. S1). Between the two conserved blocks and beyond the second block, the identity between the two genomic regions was negligible (Fig. 4A). In the distal block, five CRT/DRE and two ABRE motifs were present at conserved positions in the two sequences (Supplemental Fig. S1). However, neither M. falcata nor M. truncatula CAS15 harbored CRT/DRE motifs in the proximal block. Because the distal block was more than 1.5 kb upstream of the predicted Met initiator codon, we conducted 5' RACE experiments to determine if there might be an additional upstream exon, but the data indicated that the transcriptional start site resided 68 bp upstream of the predicted MET initiator codon (Supplemental Fig. S1A). Alignment of the M. falcata CAS30 and M. truncatula CAS31 genomic regions also revealed two blocks of nucleotide identity separated by regions of nonidentity. One block extended to –300 in MtCAS31, and to –313 in MfCAS30 (Fig. 4B; Supplemental Fig. S1B). The distal block began at –947 in MtCAS31 and –1,611 in MfCAS30, and continued almost to the end of our MtCAS31 genomic clone, which corresponded to –1,724 of MtCAS31 and –2,383 of MfCAS30 (Fig. 4B; Supplemental Fig. S1B). In the MfCAS30 proximal conserved block was a pair of CRT/DRE motifs separated by 98 nucleotides and four ABRE motifs (Fig. 5C ). The homologous region of MtCAS31 harbored these same four ABRE motifs and the most proximal CRT/DRE, but three of the five nucleotides in the second CRT/DRE differed from the consensus CCGAC (Fig. S1B; CRT/DRE no. 2). In the MfCAS30 distal conserved block was a second pair of CRT/DRE motifs separated by 40 nucleotides (Supplemental Fig. S1B). The homologous region of MtCAS31 shared one of the CRT/DRE motifs, CRT/DRE 4, but the second motif, CRT/DRE 3, was absent due to altered nucleotide residues in two of the five core CRT/DRE positions (Supplemental Fig. S1B). Otherwise, the flanking region was essentially identical (Supplemental Fig. S1B). Further upstream of the MfCAS30 CDS were five additional CRT/DRE motifs, four of which formed closely spaced pairs (Supplemental Fig. S1B).
Motif Searches Identify the CRT/DRE
As the Medicago CAS genes exhibit similar expression kinetics in response to low temperature as the Arabidopsis COR, barley (Hordeum vulgare) DHN5 (dehydrin) and DHN8, and wheat (Triticum aestivum) WCS120 (wheat cold-specific) genes, we also searched the upstream regions of these genes for common motifs using the motif finding software tool Weeder (Pavesi et al., 2004 Carrying out a similar analysis using only four Medicago CAS upstream sequences and only their distal and proximal conserved regions did not identify the CRT/DRE. The five top-scoring motifs resulting from this search were the sequences flanking the TATA box (motif 1 [M1]), a motif in the distal conserved region (M2), the transcriptional start site (M3), a motif near the 5' end of both proximal conserved regions (M4), and one of the ABRE motifs (M5; Supplemental Fig. S1).
The presence of the clustered CRT/DRE and ABRE motifs in the distal upstream conserved block of M. truncatula and M. falcata CAS15 suggested that this region might function as a regulatory island critical for the low-temperature induction of CAS15. To explore this idea we generated MtCAS15 promoter constructs that were either inclusive of the CRT/DRE and ABRE motifs (pMC7), or exclusive of these motifs (pMC1), fused the promoter regions to the uidA (GUS) reporter gene, and transformed them into Arabidopsis (Fig. 5; Supplemental Fig. S1). Expression analyses indicated that constructs lacking the upstream CRT/DRE and ABRE island were not low-temperature responsive (Fig. 5A). However, constructs harboring the CRT/DRE and ABRE island were also not cold-responsive in either as robust a manner as Arabidopsis COR15 (Fig. 5A), or as Medicago CAS15 was in both Medicago species (Fig. 2). Of the six independent pMC7 lines generated, only pMC7-6 showed low-temperature responsiveness; the other five lines did not (Fig. 5). We also asked whether MtCAS31pro:uidA and MfCAS30pro:uidA fusions would show cold-responsiveness in Arabidopsis. Fusion constructs were generated such that they included two CRT/DRE motifs and all common ABRE motifs immediately proximal to the CDS. The data from these experiments indicated that both the MtCAS31pro:uidA and MfCAS30pro:uidA fusions were cold-responsive but a more robust cold response resulted from a greater proportion of the MfCAS30pro:uidA transgenic lines than from the MtCAS31pro:uidA lines (Fig. 5, B and C). Addition of the MfCAS30 distal segment harboring the additional pairs of closely spaced CRT/DRE motifs (construct pMC2 + 8; Fig. 5B) to the MtCAS31 pMC2 promoter construct resulted in a greater number of lines exhibiting a robust response to low temperatures (Fig. 5B).
As the CAS30 genomic clone encoded a polypeptide substantially larger than that of either the highly identical alfalfa CAS17 and CAS18 cDNAs (Wolfraim and Dhindsa, 1993
CAS30 Exon 2 (the CAS17 homologous region) hybridization resulted in a weak signal in PI502449V at the predicted EcoRI and EcoRV sized fragments, and instead hybridized much more strongly to fragments that were approximately 5 and 6.6 kb (EcoRI) and 23 kb (EcoRV), respectively (Fig. 6, A and B). An XbaI digest produced the predicted fragment plus an additional higher Mr fragment migrating at approximately 15 to 16 kb (Fig. 6A). Hybridization of the MfCAS30 Exon 1 and Exon 2 fragments to PI502449H genomic DNAs resulted in patterns that were for the most part similar, albeit not identical to those exhibited by PI502449V (Fig. 6A). The pattern produced with XbaI digestion and CAS30 Exon 2 probe resulted in one band shared between PI502449V and PI502449H, but there were additional bands present in PI502449H (Fig. 6A). Similarly, the lower 11 kb EcoRI fragment forming the doublet pair in PI502449V with the Exon 1 probe was present in PI502449H, but the higher Mr fragment was not and instead "new" fragments appeared at about 23 and 6.6 kb (Fig. 6A). Hybridization to M. truncatula DNA with the M. truncatula CAS31 proximal-promoter-Exon 1 fragment produced the predicted XbaI fragments of 3,269 and 389 bp, and an additional weaker hybridizing fragment approximately 1.5 kb in size (Fig. 6, A and C). (This latter fragment extended beyond the 5' end of our genomic clone.) The 3,269 bp XbaI fragment was also produced by hybridization with the Exon 2 region. Similarly, a predicted 2,210 bp HindIII fragment of weak signal intensity resulted from hybridization with the Exon 2 region, and an approximately 5-kb fragment, which extended beyond the 5' of our genomic clone and was predicted to be at least 3,424 bp in size (Fig. 6, A and C). Taken together these data indicate that the M. truncatula genome harbors only a single CAS31 gene, whereas the M. falcata genome harbors the CAS30 gene, and at least one additional gene that share the CAS30 Exon 1 and 2 fragments. Adding to the complexity, a single M. falcata accession harbors two allelic forms of these genes.
The 5-bp CRT/DRE motif forms the conserved core of a cis-acting regulatory element to which the CBF transcription factors bind (Baker et al., 1994 ; Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997; Liu et al., 1998). Multiple copies present in the highly induced cold-responsive genes of many dicot and monocot species suggest the CRT/DRE is a plant-universal cis-acting regulatory element. Expression analyses and alignment of the MfCAS30 and MtCAS31 upstream regions indicate that it is not just the presence of the CRT/DRE, however, but of equal importance is the contextual positioning of the CRT/DRE relative to a second motif. A pair of closely spaced CRT/DRE motifs may be more effective at producing a transcriptional output than two distantly spaced motifs. Mechanistically these paired CRT/DRE motifs may function similar to the binding sites in Notch target genes in which "sequence-paired" binding sites act to fine-tune responsiveness to Notch protein levels (Cave et al., 2005; Nam et al., 2007).
M. truncatula and M. falcata also differed in the number of CAS31/CAS30 homologs. M. truncatula harbored a single CAS31 gene whereas the diploid M. falcata harbored at least two homologs. Thus tetraploid alfalfa is likely to harbor a minimum of four CAS30 homologs. The Southern hybridization experiments indicate that there are at least two different genes that possess the MfCAS30 Exon 1 fragment (in addition to each individual possessing two allelic forms of each gene), whereas the northern hybridization experiments suggest two types of genes. One that produces the larger species of transcript recognized by the Exon 1 probe and the second smaller Mr species of transcript recognized by the CAS17/Exon 2 probe. Caution must be exercised in equating the signal intensity with gene copy numbers, however, because the Exon 1 probe is unique whereas the CAS17/Exon 2 region is of low complexity, and consists of multiple partially repetitive units. In essence M. falcata harbors more CAS31/CAS30 homologs, which are more cold-responsive than M. truncatula. The greater low-temperature responsiveness appears to be due to greater numbers of paired CRT/DRE motifs in the M. falcata CAS30 promoter. Both of these scenarios were postulated to account for the differences across alfalfa cultivars in which a positive relationship exists between the expression levels of certain CAS genes detected in an individual cultivar and the low-temperature tolerance limit of that cultivar (Mohapatra et al., 1989 CAS15 was a slightly different story from MfCAS30 and MtCAS31. CAS15 was highly cold-responsive in both M. truncatula and M. falcata. However, introducing the pMC7 MtCAS15PRO:uidA fusions harboring the distal CRT/DRE-ABRE island into the Arabidopsis genome did not result in convincing cold-responsiveness. Nonetheless, the pMC1 constructs lacking the CAS15 CRT/DRE island were completely non-cold-responsive. What these data suggest is that although at the nucleotide level the cis-elements are conserved, the promoter architecture may be interpreted differently in Arabidopsis than in Medicago. Other regulatory elements crucial in facilitating gene expression may also exist in the CAS gene promoters and these may function differently in Medicago than in Arabidopsis. Thus, Arabidopsis may not be the best system for functional analyses of these Medicago promoters despite the relative ease of recovering transgenic Arabidopsis.
Sequencing the entire bacteriophage
A final important point is that morphological and anatomical differences between M. falcata and M. truncatula are likely to be a contributing factor in the capacity of plants to survive low temperatures. The cold-treated M. truncatula crown tissues showed a slightly greater TEL50 of –8°C in comparison to the –4°C TEL50 of leaves suggesting that the crown has greater capacity for low-temperature tolerance. However, the whole-plant low-temperature tolerance assays were more consistent with the leaf TELs, which may be due to the absence of meristematic cells in the M. truncatula crown region capable of generating new shoots and roots. Whereas M. truncatula has a clearly defined region that separates a primary shoot apex and meristem from the root, M. falcata exhibits a branched structure with multiple stems and a less distinct crown region that consisted of numerous underground stems with lateral buds (Oliver, 1913
Plant Material
Medicago truncatula ‘Jemalong’ and Medicago falcata accessions PI502447 and PI502449 were obtained through the U.S. Department of Agriculture (USDA) GRIN system (http://www.ars-grin.gov/). The USDA Medicago germplasm collection has been rated on a numerical scale of 1 to 10 for numerous traits of agronomic value including frost and winter injury damage. Both accessions PI502447 and PI502449 were ranked as the most resistant to winter injury (USDA rating no. 1) and have been karyotyped as diploid alfalfa (Medicago sativa) subsp. falcata (Brummer et al., 1999
Four-week-old clonally propagated M. falcata plants and 4-week-old M. truncatula seedlings were used to evaluate low-temperature tolerance. Plants were grown in Bacto Promix (Michigan Peat Company) under a 16-h photoperiod at a light intensity of 250 µmol m–2 s–1 photosynthetically active radiation provided by cool-white fluorescent lamps using a 20°C/18°C day/night temperature. Only healthy well-watered plants fertilized once a week with 0.5 g L–1 of an all purpose 20:20:20 plant nutrient solution (Schultz) were used. Plants of each species were divided into two groups. Plants in one group were used to measure low-temperature tolerance without cold-acclimating conditions, and were maintained in an environmentally controlled chamber under the conditions described above. Plants in the second group were transferred to a growth chamber set at constant 2°C with a 12-h photoperiod under 150 µmol m–2 s–1 photosynthetically active radiation for 4 weeks.
Low-temperature tolerance of nonacclimated and acclimated plants of M. falcata and M. truncatula was determined on detached leaf discs and crown segments by electrolyte leakage (Pennycooke et al., 2003 Whole plants were treated and frozen as described above for detached leaves and crowns with the following modifications. Styrofoam chips were added to the pots to simulate snow cover during an artificial freeze (C. Stushnoff, personal communication), plants were misted with tap water at 0°C to initiate extracellular ice nucleation, and then allowed to equilibrate at –3°C for 15 h before further temperature decreases. Whole plants were thawed slowly at 2°C overnight and returned to growth chambers to regrow under the initial establishment conditions described above. Shoots were harvested after a 3-week regrowth period and oven dried at 60°C for dry-weight determination. Regrowth was expressed as a percentage of the nonfrozen control. Three plants per pot and five pots per treatment were used for each test temperature. The temperature (TEL50) that resulted in either 50% ion leakage (calculated using a fitted model plot), or 50% kill as indicated by lack of regrowth was defined as the LT50. These experiments were repeated twice. Statistical analyses were performed with statistical software (SAS).
‘Jemalong’ seed was germinated as described above. One to three seedlings per 4-inch pot were transplanted into high-porosity soilless mix (Bacto). Pots were set inside trays and the trays placed into a BD16 growth chamber (Conviron) under combined metal halide and high-pressure sodium lamps at a light intensity of 500 µmol m–2s–1. Growth conditions were 17 h light, 7 h dark cycle, constant 25°C, and 60% relative humidity for approximately 3 weeks prior to the temperature drop at the indicated times (details in Fig. 2 legend). Vigorous, bushy greenhouse-grown M. falcata plants were cut back to approximately 5 to 8 cm above the soil and placed into the growth chamber concurrently with the transplanted ‘Jemalong’ seedlings. At the time of the temperature drop, the light intensity was simultaneously reduced to approximately 225 µmol m–2s–1. Plants were deacclimated by returning the chamber to 25°C and the original light intensity. T2 seeds of the Medicago CAS promoter-uidA fusion constructs in the Arabidopsis ecotype backgrounds RLD and WS-2 (pMC) were surfaced sterilized in 30% bleach and 0.2% Triton X-100 for approximately 20 min followed by four washes in sterile water. The seeds were stratified at 4°C for 4 d. Seeds were sown on Gamborg's B5 (GibcoBRL Life Technologies) plates containing 0.8% (w/v) phytagar and 2% Suc and incubated at 22°C with a 16-/8-h photoperiod under cool-white fluorescent light at 100 µmol m–2 s–1. Four weeks after germination, seven or eight lines of each construct were transferred to a 4°C cold room or maintained at room temperature for 6 h.
To identify CBF sequence homologs from Medicago we performed a BLAST search of the M. truncatula database (Lamblin et al., 2003
High Mr total plant genomic DNA, isolated as described (Stockinger et al., 1996
Genomic clones cross-hybridizing with MtCAS15 and MfCAS17 PCR-amplified products selected for DNA sequence determination included, MtCAS15:
Both the M. truncatula and the M. falcata CAS15 genomic clones were truncated within the CAS15 coding region;
Total RNA was extracted from Arabidopsis plants using the RNeasy Mini Kit (QIAGEN). Five or 7 µg (as indicated in the figure legends) of total RNA was loaded onto each lane. Fractionation and northern transfers to Hybond-N+ (Amersham Biosciences) were performed as described (Ausubel et al., 1993
Primers were designed to amplify the CAS upstream regions (5' end sites along with the 3' end site of pMC2 + 8 are indicated in Supplemental Fig. S1, A and B). Downstream reverse primers were generated within the CDSs of each CAS gene resulting in a translational fusion between each CAS promoter and uidA. Plasmids pMC1 and pMC7 encode the first 25 amino acid residues of MtCAS15; pMC10 encodes the first 18 amino acid residues of MfCAS30; and pMC2 encodes the first 19 amino acid residues of MtCAS31. Amplified products were then subcloned into either pGEM-T-EASY, pGEM-T (Promega), or pUC119. The resulting cloned PCR products were sequenced and subcloned into the BamHI site of pRD420 (Jefferson et al., 1987
Percent identity plots (PIPs) were generated using the web interface of the PipMaker (Schwartz et al., 2000 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EU139865 to EU139871.
The following materials are available in the online version of this article.
We thank David M. Francis, Esther van der Knaap, and Sophien Kamoun for helpful suggestions during the preparation of the manuscript, Jeff Volenec for insight into alfalfa growth habit, Imed Dami for the use of the Tenney programmable freezer, Raju S. Datla for pRD420, Annie Knox for completing the sequence work, Erik R. Rowley for Arabidopsis transformation, and Kip G. Gardner for technical assistance in RNA blot hybridizations. Received September 8, 2007; accepted January 22, 2008; published January 24, 2008.
1 This work was supported in part by the National Science Foundation plant genome program (DBI 0110124) and the Ohio State University/Ohio Agricultural Research and Development Center seed grant award (OHOA1014). Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. 
2 Present address: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. 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.plantphysiol.org) is: Eric J. Stockinger (stockinger.4{at}osu.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.108779 * Corresponding author; e-mail stockinger.4{at}osu.edu.
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ABSTRACT
RESULTS
. The M. truncatula CAS15 genomic clone encoded a 13-kD polypeptide, as did the best-hit ESTs to alfalfa CAS15, in comparison to the 14.5-kD polypeptide encoded by alfalfa CAS15 (
