Supplementary Material:
Results and Discussion
Statistical Analysis, p and q values.
The statistical analysis of microarray data poses particular challenges due to the large number of features that are analyzed. The traditional p value thresholds of 0.01 and 0.05 have a potential false positive rates of 1% or 5%. When these numbers are considered in a population of over 5000 features, there could be a substantial number of genes that are declared as significant that actually are not. Higher threshold levels of confidence can be used to reduce the level of false positives. However, in this study and other similar studies, the genes with the highest level of confidence, or lowest p values, tend to be genes that have very high levels of induction or repression of mRNA levels in response to low temperature treatment and also tend to be well-know genes that have previously been reported to be regulated by the low temperature. Candidate genes with likely regulatory and signaling function tend to have moderate levels of induction and repression, lower p values, and higher risk of being false positives.
There is not a universally invoked threshold for p for microarray analysis; rather, p values serve as an estimate of the error involved in the declaration of significance. The false discovery rate, that is the % of features that have been declared to be significant that are likely to be false positives, is a useful gauge of this error. We have used the method of Storey and Tibshirani (2003) to calculate false discovery rate, q, for each p value calculated from ANOVA or Students t test. Each column of p values in Table S2 was used as a population, containing approximately 5600 values, to calculate a corresponding q value, which is the probability that a feature with a p value equal or less than the given value is a false positive. The focus of discussion in this work is features with significant cultivar by treatment interaction effects determined by ANOVA, having a p ≤ .01; such features have a false discovery rate of 3.8%.
Other Cold Acclimation Response Genes
Previously characterized Stress proteins
Among genes significantly regulated by CA, there were 51 probes with sequence similarity to genes annotated as genes responding to stress, many of which are characteristically elicited by exposure to low temperature during CA (Thomashow, 1999). This group is comprised of genes annotated as cold acclimation proteins, cold-responsive proteins, low-temperature induced proteins, ice recrystallisation inhibitor proteins, and dehydrins (Supplementary Table S3). Though there is wide variation in gene sequence among these, they share the feature of being strongly induced by CA. The great majority of these genes were more strongly up-regulated in the winter than in the spring cultivar. There was a subgroup of genes that had substantially lower levels of mRNA in non-acclimated spring wheat plants and higher rates of increase in the spring cultivar, but in spite of the greater rate of increase, they did not achieve steady state levels as high as those of the winter cultivar. The average relative level of mRNA for the whole group of genes previously annotated as cold-regulated genes was about two fold higher in the winter cultivar than the spring cultivar after 1 day of acclimation and increased to 6 fold by day 14 of CA. The differential regulation of stress proteins between the two cultivars highlights the overriding suppression of the cold response in spring wheat that appears to be a general theme in the comparison of gene expression in the two cultivars.
Sugar Metabolism
Accumulation of sugars during CA has been documented for a variety of plant species (Thomashow, 1999; Chen and Murata, 2002). Among enzymes involved in the metabolism of sugars and other carbohydrates, we detected a marked up-regulation of galactinol synthase (Tr003_P22, Tr002_N11), sucrose synthase (Tr008_H11, Tr003_D16, Tr002_B17) and sorbitol dehydrogenase (Tr016_I13). Galactinol synthase, which catalyses the first step in the biosynthesis of raffinose from UDP-galactose, has been documented to increase in response to CA (Fowler and Thomashow, 2002; Taji et at, 2002), and by transgenic technology has been shown to be a key enzyme for the accumulation of raffinose and galactinol under cold stress (Taji et at, 2002). We found differential regulation of galactinol synthase between the two cultivars. Up-regulation of galactinol synthase was detected earlier in the spring than in the winter cultivar, beginning at 6 hr of CA. However, by 24 hours of CA, levels had begun to decrease in the spring wheat and at the same time the winter cultivar showed marked induction that was sustained through CA in the winter cultivar. Sugars and other sugar alcohols are known to act as cryoprotectants in plant cells (Chen and Murata, 2002), but the precise involvement of sucrose synthase remains unclear. Three probes annotated as variants of sucrose synthase genes (Tr003_D16, Tr002_B17, Tr003_D08) were up-regulated in the winter wheat at 1 d CA and remained at elevated levels throughout the time course studied in these experiments, but they were not significantly increased in the spring cultivar. Three other sucrose synthase probes showed significant cultivar x treatment interaction effects, with different patterns of expression; two (Tr008_H11, Tr002_J19) were induced late in winter wheat and were slightly repressed in spring wheat. The third gene (Tr004_G06) showed repression during CA with spring wheat showing somewhat stronger down-regulation. Levels of sucrose synthase have been shown to increase within one hour of wheat seedlings exposure to cold (Calderon et al. 1985). This enzyme is involved in the degradation of sucrose which provides carbon for respiration, synthesis of cell wall polysaccharides, and the synthesis of starch (Amor et al. 1995; Nakai et al. 1999). Since we also observed an increase in transcripts for a starch degrading enzyme, α amylase (Tr016_E18), it is more conceivable that sucrose synthase, in conjunction with α amylase, is involved in the production of substrates that support respiration, which is known to increase during CA (Talts et al., 2004). In addition, products derived from sucrose synthase activity could be supporting cell wall synthesis.
Cell Wall Proteins
As observed for many other classes of genes, there was also differential regulation between the cultivars for 17 probes comprising nine classes of enzymes annotated as cell wall enzymes. Whereas four of the cell wall genes were down-regulated, 13 cell wall genes were up-regulated during CA and had higher transcript levels in the winter than in the spring cultivar. Transcript levels for a member of the glycosyl hydrolase family 1 (Tr002_B03) decreased to lower levels in the winter than in the spring cultivar. Three other members of this gene family (Tr002_J13,Tr002_J17,Tr002_J05) were up-regulated by CA. In general the level of increase was higher in winter wheat than in spring wheat, but each member of the family showed distinct timing of induction. Recently, the sensitive to freezing 2-1 mutation which causes freezing sensitivity in Arabidopsis was found to affect a variant of glycosyl hydrolase (Thorlby et al., 2004). Taken together, these results suggest that there are significant adaptations of the cell wall to low temperature and that the differential regulation of these adaptations contributes to the overall acquired capacity for freezing tolerance. Furthermore, these results complement conclusions derived from morphological comparisons of freezing tolerant and freezing sensitive plants that implicate cell walls in conjunction with the plasma membrane in blocking the propagation of extracellular ice (Yamada et al., 2002).
Actin Cytoskeleton
Dynamic rearrangements of F-actin networks interconnecting endocellular membranes play important roles in signal perception for a variety of plant processes, including mechano- and gravity-sensing, plant host–pathogen interactions, morphogenesis, and wound-healing (Volkmann and Baluska, 1999). These dynamic changes are modulated by actin-binding proteins, such as actin depolymerizing factor (ADF), myosins, profilin, and fimbrin (Volkmann and Baluska, 1999; Chen et al, 2002). The present analysis detected differential regulation for six homologs of ADF, all of which were up-regulated by CA. This up-regulation of ADF was stronger in the first three days of CA and declined later in both cultivars. However, both a higher peak induction as well as a slower subsequent decline were found in the winter relative to the spring cultivar. Similarly, transcript levels for another known actin-binding protein, myosin (Tr016_P03), and those of an actin-related protein (Tr011_A15) were differentially regulated. Myosin up-regulation was detectable early in CA in both cultivars, but later it was sustained through CA in the winter cultivar whereas it declined below the two-fold induction threshold after day 3 of CA in the spring cultivar. Transcript levels of the actin-related protein increased markedly only after day 3 of CA, and its up-regulation was detected in the winter but not in the spring cultivar. There is evidence that the form of growth that distinguishes plant cells from other organisms, cytoplasmic growth leading to cell elongation, is positively correlated with the formation of actin networks (Allwood et al., 2002; Chen et al, 2002). In the best documented case, that of pollen tube elongation, overexpression of ADF results in inhibition of this process (Chen et al, 2002). As a more direct link to our study, cytological and pharmacological evidence have strongly suggested that the dynamic reorganization of the cytoskeleton plays a crucial role in CA and calcium-mediated signal transduction (Orvar et al.,2000). Most likely in connection with this crucial actin network turnover, a CA-induced ADF has been functionally characterized in wheat and its transcripts levels have been shown to increase during CA by northern blots and microarray analysis (Ouellet et al., 2001, Gulick et al, 2005). Taken together, these observations further support an important role of the regulation of microtubule dynamics for low temperature sensing and the development of robust freezing tolerance. Sustained up-regulation of ADF during CA may be related to restricted growth under low temperature stress, which is linked to better freezing tolerance (Thomashow, 1999).
Photosynthesis proteins
Transcripts for the majority of the cold regulated genes related to photosynthesis decreased during CA. This general down-regulation of photosynthesis related genes was more markedly detected in the winter than in the spring cultivar. A similar tendency for the decline of transcripts of genes related to photosynthesis during CA has been noted in Arabidopsis (Fowler and Thomashow, 2002) However, our study further points to an apparent inverse correlation between higher capacity to develop freezing tolerance and more pronounced inhibition of genes for photosynthesis. Decreased gene expression for photosynthesis- related genes may reflect a decrease in the light harvesting apparatus which would reduce the imbalance between light harvesting and dark reactions, leading to reduced photoxidative damage and consequently reduced recycling of proteins of the photosynthetic apparatus (Ndong et al., 2001). There were two exceptions to the general down-regulation of components of the photosystems (Tr017_D21, Tr010_N19). The protein encoded by Tr017_D21, a photosystem 1 associated protein, has previously been postulated to increase the efficiency of the photosynthetic apparatus under stress conditions (Rumeau et al., 2005).
Unknown Proteins
Functions for a large proportion of genes have not been determined. In Arabidopsis and rice; these represent approximately one third of the genome (The Arabidopsis Genome Initiative 2000, Goff et. al 2002). Among this group of unclassified EST probes we detected 62 probes with significant cultivar by treatment interaction effects for CA. Marked differences in transcript levels between the cultivars could be seen among the unclassified probes, comprising many genes with levels of induction higher in the winter than in the spring cultivar. Likewise, this group includes genes with a more marked down-regulation in the winter than in the spring cultivar and a small number of genes with more marked regulation in the spring than in the winter cultivar. While the functions of these proteins remain unknown, they constitute a rich pool for the discovery of additional key factors that determine differential development of freezing tolerance among wheat genotypes.
Sequences of novel NAC transcription factors and ice recrystalization inhibition proteins
Tr012_B03: TaNAC020_Group ATAF, NAC transcription factor ATTCCAAGTCTTCCCCCGCGAGGGCGAGCCGCCGCCGCCGCCGATCAGCCGAACCAGCCG
CCGATCCAACCAATCTCCCGAGCGCCCGCCCTCCGAGCTCAAGCCCCGTTCGTGAATGGA
CCACGGCTTCGACGGCGCTCTCCAGCTGCCCCCGGGGTTCAGGTTCCACCCCACGGACGA
GGAGCTGGTGATGTACTACCTTTGCCGCAAGTGCGGCGGCCTGCCCATCGCCGCGCCGGT
GATCGCCGAGGTCGACCTGTACAAGTTCGAGCCGTGGAGGCTGCCGGAGAAGGCGGCGGG
AGGGGGGCCGGACGCCAAGGAGTGGTACTTCTTCTCGCCGCGCGACCGCAAGTACCCCAA
CGGGTCGCGGCCGAACCGCGCCGCCGGGACCGGGTACTGGAAGGCCACCGGCGCCGACAA
GCCCGTGGGGTCGCCCCGCCCCGTGGCCATCAAGAAGGCCCTCGTCTTCTACGCCGGCAA
GCCCCCCAAGGGCGTCAAGACCAACTGGATCATGCACGAGTACCGCCTCGCCGACGTCGA
CCGCTCCGCCGCCGCCCGCAAGAAGTCCAACAACGCGCTCAGGCTGGATGACTGGGTGCT
CTGCCGAATCTACAACAAGAAGGGCGTGATCGAGCGGTACGACACGGCGGACTCCGACGT
GGCCGACGTCAAGCCGGCGCCGGCGCCGGCTGCCAGGAACCCGCGGCCGGGCCAGTACCA
CGCTGCTGGGCCGGCGATGAAGGTCGAGCTGTCCGACTACGGGTTCTACCAGCAGCCGTC
GCCGCCGGCCACGGAGATGCTCTGCTTCGACCGCTCCGGGTCGGCGGACCGGGACTCCAA
CTCGAACCACTCCATGCCGCGCCTGCACACGGACTCCAGCTCCTCGGAGCGCGCGCTGTC
CTCGCCCTCGCCCGACTTCCCGAGCGATATGGACTACGCGGAGAGCCAGCACGCGGCCGG
CCTCGCCGCGGGGTGGCCGGGCGACGACTGGGGCGGCGTCATAGAAGACGACGGGTTCGT
CATCGACGGCTCGCTCATCTTCGACCCGCCGTCGCCGGGCGCCTTCGCCCGCGACGCCGC
CGCGTTCGGGGACATGCTCACGTACCTGCAGAAGCCGTTCTGAATGAACGCGGCATCCGT
CAGACCCCTCCTCCTTAGCAGCCTCCACTAACATGTTCGTCAGGTCTCGTGTAATTCGGT
CTGCAAGCTTCCGAAACCAATGCAGATTAGAGAAAAAAAAAAAAAAAAAAAAAA
Tr012_B03 TaNAC020_Group AtAF, NAC transcription factor
MDHGFDGALQLPPGFRFHPTDEELVMYYLCRKCGGLPIAAPVIAEVDLYKFEPWRLPEKAAGGGPDAKEWYFFSPRDRKYPNGSRPNRAAGTGYWKATGADKPVGSPRPVAIKKALVFYAGKPPKGVKTNWIMHEYRLADVDRSAAARKKSNNALRLDDWVLCRIYNKKGVIERYDTADSDVADVKPAPAPAARNPRPGQYHAAGPAMKVELSDYGFYQQPSPPATEMLCFDRSGSADRDSNSNHSMPRLHTDSSSSERALSSPSPDFPSDMDYAESQHAAGLAAGWPGDDWGGVIEDDGFVIDGSLIFDPPSPGAFARDAAAFGDMLTYLQKPF
Tr003_D11 TaNAC002 Group OsNAC3, NAC transcription factor
CCACGCGTCCGCTCCACACAGCTCGCTCACCACAATCTCAACAGCCATCGGAATCAACAG
CAGCAGCAGCGGAGCGATCTCATCGACCAACTCTCTTCCAAGTTTCGATTCAACGGACAT
GGGGATGCCGGCGGTGAGGAGGAGGGAGAGGGACGCGGAGGCGGAGCTCAACCTGCCGCC
GGGCTTCCGCTTCCACCCCACCGACGACGAGCTCGTGGAGCACTACCTGTGCCGCAAGGC
GGCCGGCCAGCGCCTGCCCGTGCCCATCATCGCCGAGGTCGACCTCTACCGCTTCGACCC
GTGGGCGCTCCCCGACCGCGCCCTCTTCGGCACCCGCGAGTGGTACTTCTTCACCCCGCG
CGACCGCAAGTACCCCAACGGCTCCCGCCCCAACCGCGCCGCCGGCAACGGATACTGGAA
GGCCACCGGCGCCGACAAGCCCGTCGCGCCCCGCGGTGGGAGGACCATGGGGATCAAGAA
GGCCCTGGTGTTTTACGCCGGCAAGGCGCCCAAGGGGGTTAAGACCGACTGGATCATGCA
TGAGTACCGCCTCGCCGACGCCGGCCGCGCCGCCGCCAGCAAGAAGGGCTCGCTCAGGCT
GGACGACTGGGTGCTCTGCCGGCTCTACAACAAGAAGAACGAGTGGGAGAAGATGCAGCT
CCAGCAGCAGGGGGGAGAGGAGATGATGGTGGAGCCCAAGGAGGAGCACGCCGCGTCGGA
CATGGTGGTCACCTCGCACTCCCACTCGCAGTCCCAGTCGCACTCGCACTCCTGGGGCGA
GGCGCGCACGCCCGAGTCGGAGATCGTCGACAACGACCCGTCGCTGTTCCAGCAGGCGGC
GGCGTTCCAGGCCCAGAGCCCCGCCGCCGCGGCGGCGCACCAGGAGATGATGGCCACGCT
GATGGTGCCCAAGAAGGAGGCGGCGGACGAGGCCGGCAGGAACGACCTGTTCGTGGACCT
CAGCTACGACGACATCCAGAGCATGTACAACGGCCTCGACATGATGCCGCCAGGGGACGA
TCTGCTCTACTCGTCCCTCTTCGCCTCCCCAAGGGTCCGGGGGAGCCAGCCCGGCGCCGG
CGGCATGCCGGCCCCGTTCTAAGCAGAGCCAAGACACAGACCGTCAGATACCGAGATCGA
CAGAAGTGGAACGGATGACTTCTTCGCCGCGAGCGAGCGAGATCGCGGCGCAGTGTAAAT
ACAGCATAGGAAACGGGAGGGAGGTCGCGTCGTCGTGTCGAGAGAGCCGGCGTGGCGCGG
GCGCCGGCGCCGGGGCGTGCCGTTGTACATGGAGAGCCCGGTGCCGGGGCCTGGCCGGCT
GGGTTGATTCTTTTGTTCTTACTACTTTGTACTCTCAATGCGGATGTAGCTCTTTCTTCT
CACTCCTCACTAGTAGAATCGACCGACCAAATACATATGTAGCTCTGTTTCTTCAGTAGA
AATCAACCAAATTTTGCTGTTAAAAAAAAAAAAAAAAAAAAAAAAAA
>Tr012_M24 TaNAC002 Group OsNAC3, NAC transcription factor
MGMPAVRRRERDAEAELNLPPGFRFHPTDDELVEHYLCRKAAGQRLPVPIIAEVDLYRFDPWALPDRALFGTREWYFFTPRDRKYPNGSRPNRAAGNGYWKATGADKPVAPRGGRTMGIKKALVFYAGKAPKGVKTDWIMHEYRLADAGRAAASKKGSLRLDDWVLCRLYNKKNEWEKMQLQQQGGEEMMVEPKEEHAASDMVVTSHSHSQSQSHSHSWGEARTPESEIVDNDPSLFQQAAAFQAQSPAAAAAHQEMMATLMVPKKEAADEAGRNDLFVDLSYDDIQSMYNGLDMMPPGDDLLYSSLFASPRVRGSQPGAG
GMPAPF
>Tr001_B19 ice recrystallization inhibition protein 4 precursor [Triticum aestivum
AGAGAGCACTTGCCAATCACTCACTCGACCTCAATGAATCCCATGGCCAAATGCTGCCTA
CTGCTCCTCCCCATCTTCTTCTTGGCATTTCTCTTGCCCGAGGCGCACGCGACTTCGTGC
CACCCCGACGACCTTCATGCACTGCAGGACTTTGCTGGGAACCTCGGCGGCGGGGGTGTC
CTCCTCCGGGCCACGTGGTTCGCCGCCGCATGCTGCAGCTGGGAAGGTGTGAGCTGTGAT
GCCGCCAGTGGCCGTGTCACGGCACTGCGTCTCCCCAGGCGCGGCCTTGTGGGGCCCATC
CCAGGAACCTCCCTTGCAGGCCTTGCGCGGCTCGAGGAGCTCGACCTTGGCTCCAACAAC
TTTCAAAACATCTCAGGGGTACTCACCGTGTTGCATGGGTGCCAGAACCTCGCCACGCTG
ATTCTCACCAAGAATTTCGATGGTGAGGAGCTACTAGGTGATGGTATTATTGGCGGGTTC
AAGAGCCTCGAGGTGCTGGCCCTTGGTGATTGTGCTCTCAAGGGCAGGGTTCCGGAATGG
TTGTCTCAATGCAAGAAAATGGAGGTGCTTGATTTGTCCGGCAACCAATTGGTGGGCACC
ATCCCATCGTGGATTGGTGAGCTTCACCACCTTTGCTACTTGGATCTCTCAAACAATTCA
TTGGTTGGCGATGCACCTAAGAGTTTGGCACTGCTAAAGGGGCTCGCCCCTGATGGGCGT
TCACCGGGCATGGTTTTCACTAACATTCCATCGTATGTGAAGCATAACAGAAGCACACTC
GGACGACGACTTAACGACCTCCCAAATGTCATCACAGGGACCAACAACTTTGTAACATCT
GGGAGCAACAATGTTTTATCGGGGAATGACAACACAGTCATATTTGGGGATGAAAACACC
GTATCTGGGAACAACCATGCTATATATGGGGATGGGAACACGGTATCTGGGAACAACCAT
GTCGTATCTGGGAGCAAGCATGTTGTATCCGGGAGTAGGCATGGCGTAACTGGGAGAAGT
AGTGTGGTATCTGGGTTCAACAATGGCGTATCTGGGATCAACCATGTTGTATCGGGGAGT
AACAATGTCGTATCCGGGAGCAACAATGTTGTATCTGGGATGAACCATATTGTGTCTGGA
AACAACAAAGTAATAACATGAGGTTAATGATTTTATAAGAGAGAGGAGGGAGGGGGAGAG
ATTATGCCTAGTATCCGAGGCCGATGAGATGGTTCAATAAATAAGGTACATAAGTTGCCA
CACAAGATGTGCCCTATGCCTCGTATAAGTTGTAGTGAGTATATGTATTGTAATAGTATG
GTTTGGTTTGTATGTTAAGTTTTATTGTGTATCGAGAAGACGCGATCTTGAGTGGTTTGT
TTGTTTTAATTATTTGAGCTCTGATCTCTAATATATTAATTAGATTAGATTAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAA
>Tr001_B19 ice recrystallization inhibition protein 4 precursor [Triticum aestivum]
MNPMAKCCLLLLPIFFLAFLLPEAHATSCHPDDLHALQDFAGNLGGGGV
LLRATWFAAACCSWEGVSCDAASGRVTALRLPRRGLVGPIPGTSLAGLARLEELDLGSNN
FQNISGVLTVLHGCQNLATLILTKNFDGEELLGDGIIGGFKSLEVLALGDCALKGRVPEW
LSQCKKMEVLDLSGNQLVGTIPSWIGELHHLCYLDLSNNSLVGDAPKSLALLKGLAPDGR
SPGMVFTNIPSYVKHNRSTLGRRLNDLPNVITGTNNFVTSGSNNVLSGNDNTVIFGDENT
VSGNNHAIYGDGNTVSGNNHVVSGSKHVVSGSRHGVTGRSSVVSGFNNGVSGINHVVSGS
NNVVSGSNNVVSGMNHIVSGNNKVIT
>Tr001_M19 ice recrystallization inhibition protein 3 precursor [Triticum aestivum]
CCACGCGTCCGCAATTGCACACCAAACAAGGTTTCTTACCGAGAGTTGTTGCGTTTGTGC
ATACATAGCATACTTCAGCTAGATCCATGGCGAAATGCTGGCTGCTGCTCCACTTGTTGG
CGTTTCTCTTGCCAGCGGCGAGCGCTACGTCGTGCCACACCGACGACCTCCACGCGCTGC
AGGGCTTCGCCGGGAACCTCGGTGGTGGTGGCGTGCTCCTACGTGCCGTCTGGTCCGGCG
CCTCGTGCTGCGGCTGGGAAGGTGTGAGCTGCGACGGCACAAGTGGACGCGTCACGGCGC
TTCGGCTCCCCGGGCACGGGCTTGTGGGGCCCATCCCTGGAGCATCCTTGGCGGGCCTCA
CGCAGCTGGTGGAGCTCAACCTTGCCAACAACAAACTGATCGGCACCATCCCATCATGGA
TTGGTGAGCTTGATCACCTTTGCTACTTGGATCTCTCCAATAATTCATTGGTTGGCGAGG
TACCGAAGACATTGATACAGCTCAAGGGCCTCGTCTCCACTGGTCGTTCACTGGGTAACC
GAAGAACACTCCAACAACAACAACAACCAAATATCATATCTGGGACAAACAACAAAGTCC
GATCTGGTAGAACCAATGTTGTATCCGGGAACGACAACACTGTCATATCCGGAAACAACA
ACACGGTGGCTGGGAGCAACAATACCATCACAACTGGGAGCGACAATACCGTAACTGGTA
GCAACCATGTCGTATCTGGGAGCAAACATATCGTAACAGACAACAACAATGTTGTTTCCG
GAATTGACAATAATGTATCCGGGAGCTTCCACACCGTATCCGGTAGTCACAATACCGTAT
CCGGGAGCAACAATACCGTATCTGGGAGCAACCATGTCGTGTCTGGGAGCAACAAAGTCG
TGACAGGAGGTTAATAGTTTGTCTGTCGATGTAGCTCACAACCACTTGTTGGGGCGAATC
ATGTTTTGTAACCTCGTGGATGTCCCATCCTTTTTCTACTTTAAATAAAATTTCCAGAAA
TTAAAAAAAAAAAAAAAAAAAAAAA
>Tr001_M19 ice recrystallization inhibition protein 3 precursor [Triticum aestivum]
MAKCWLLLHLLAFLLPAASATSCHTDDLHALQ
GFAGNLGGGGVLLRAVWSGASCCGWEGVSCDGTSGRVTALRLPGHGLVGPIPGASLAGLT
QLVELNLANNKLIGTIPSWIGELDHLCYLDLSNNSLVGEVPKTLIQLKGLVSTGRSLGNR
RTLQQQQQPNIISGTNNKVRSGRTNVVSGNDNTVISGNNNTVAGSNNTITTGSDNTVTGS
NHVVSGSKHIVTDNNNVVSGIDNNVSGSFHTVSGSHNTVSGSNNTVSGSNHVVSGSNKVV
TGG
Experimental Procedures
LT50 determination
The LT50 of the winter wheat Triticum aestivum L. cv CDC Clair and the spring wheat T. aestivum L. cv Quantum was determined by controlled freezing and regrowth tests. Seeds were germinated in 50% coarse vermiculite and 50% black soil mixture at 20°C/ 16°C (day/night) with 16-h photoperiod. White fluorescent and incandescent lighting was combined to provide illumination at 250 µmol m-2s-1. After 8 days, the plants were cold acclimated at 4 °C for 5 weeks. At the end of the acclimation period, plants were transferred to a programmable freezer (CFC free, Sanyo ultra low) and kept at 2 °C for 12 h and the temperature was subsequently decreased at 2°C per hour down to –22°C. Plants were removed at intervals of 2°C, thawed at 4°C overnight and transferred to growth chambers at 20°C, 16 hr day length. Survival of the plants was scored after three weeks and theLT50 was calculated for each cultivar as the temperature required to kill 50% of the plants. The experiment was repeated three times.