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Supporting Information
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Materials and methods
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Construction of plant expression vector and plant transformation
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To verify the main responsibility of CBF cold response pathway for the cold-tolerance
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variation among crofton weed populations, the AaCBF1 isolated from HGG was
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transferred to a BSG plant. Both plant expression vector construction and plant
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transformation were performed as described by Xu et al. (2010). AaCBF1 was cloned
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from HGG and BSG. Coding regions of the genes were amplified by PrimeSTAR
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DNA
polymerase
using
the
primer
pairs:
AaCBF1
forward
and
reverse
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5’-CGCGGATCCATGGCTACCTTTATCCAATTC-3’
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5’-CGGGGTACCTTAGAAACTCCATAATGAAGC-3’, and then digested using XbaI.
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The digested PCR products were introduced into the plant expression vector PBI121
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digested with XbaI to produce PBI121-AaCBF1. The final constructs (Fig. S7) were
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introduced into Agrobacterium tumefaciens GV3101 by the freeze-thaw method, and
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subsequently transformed into crofton weed BSG via Agrobacterium-mediated
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transformation.
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Southern-blot analysis of transgenic plants
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Total DNA (10 μg) extracted from plant samples was denatured, electrophoresed in
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1% (w/v) agarose gel, and transferred onto nylon membranes (Hybond-N+) using
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standard blotting techniques described in DIG High Prime DNA Labeling and
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Detection Starter Kit I (Roche). The membranes were pre-hybridized for 30 min at
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60ºC and hybridized with the gene-specific cDNA fragments which were labeled with
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DIG-High Primer for 20h at 60ºC in a rapid hybridization buffer (Roche). Membranes
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were washed twice at room temperature in 2× SSC with 0.1% (w/v) SDS and then
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washed twice in 2× SSC with 0.1% (w/v) SDS for 15 min at 68ºC. The
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immunological identification was recorded according to the operation manual of DIG
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High Prime DNA Labeling and Detection Start Kit I (Roche).
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Determination of content of MDA, soluble sugar, soluble protein, SOD
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and chlorophyll of transgenic plants
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Content of malondialdehyde (MDA) and superoxide Dismutase (SOD) was
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determined using TBA (Heath and Packer 1968) and NBT (Beauchamp and Fridovich
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1971) methods, respectively. Phenol-sulphuric acid method was used to determine the
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content of soluble sugar (Dubois 1956). Coomassie brilliant blue-G250 was used to
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measure the content of soluble protein (Sedmak and Grossberg 1977). After extraction,
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chlorophyll concentration was determined according to Lichtenthale (1987).
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Results
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Comparison of the structure of AaICE1, AaCBFs and AaCORs genes
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To elucidate the factors responsible for the differential expression of AaICE1,
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AaCBFs and AaCORs, their cDNA and DNA sequences from the four populations
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were compared using DNAMAN. AaICE1 contains a typical bHLH structure
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(301-346aa) (Fig. S3A) and AaCOR29 has a dehydration feature structure (172-196aa)
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(Fig. S3B). The 5΄ flank sequences of both AaCBF1 and AaCBF3 genes included a
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TATA box (-99~ -94bp and -69~ -66bp ATG codons upstream, respectively). AaCBF1
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contained a G-box (-116~ -110bp ATG codons upstream), two MYC recognition sites
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(-135~ -129bp and -108~ -103bp ATG codons upstream) and two MYB recognition
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sites (-259~ -255bp and -282~ -287bp ATG codons upstream) (Fig. S5A). AaCBF3
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also had three MYC recognition sites (-271~ -266bp, -217~ -212bp and -171~-166bp
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ATG codons upstream) and one MYB recognition site (-214~ -209bp ATG codons
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upstream) (Fig. S5B). Both AaCBF2 and AaCBF3 had identical cDNA and DNA
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sequences respectively but 13 nucleotides of cDNAs and DNA sequences of AaCBF1
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were different (Table S4). DLY and BSG shared one polymorphic AaCBF1 sequence
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while HGG and JHY shared another that had three base deletions in the 3’ uncoding
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region (Table S4). However, AaCBF1 DNA flank sequences and its coding amino
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acids were 100% identical among accessions from the four populations. As an
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upstream regulator of CBFs, differential AaICE1 expression may contribute to
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AaCBFs expression differences. The 5΄ flank sequence of AaICE1 included a TATA
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box (-108~ -103bp ATG codons upstream) (Fig. S5C), but no AaICE1 gene cDNA
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and DNA sequence differences were found among the compared populations except
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BSG and XCS. Among the accessions detected of BSG and XCS, one accession each
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had two more Histidine at 45th amino acid sites, but there was no difference in
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AaICE1 expression level with other accessions within BSG and XCS, respectively,
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under 4oC treatment (Fig. S6).
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Identification of transgenic plants
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AaCBF1 gene was integrated into 23 crofton weed individuals (Fig. S8A, B) but the
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transgenic plants grew stunted (Fig. S8C).
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AaCBF1 over-expression enhanced freezing-tolerance of crofton weed
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Crofton weed was also transformed with the plasmid via Agrobacterim. Among 23
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transgenic individuals, two were more freezing-tolerant than wild type BSG (TG-1
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and TG-2). Southern-blots showed that both transgenic BSG had two copies of
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AaCBF1 while BSG population had the native one (Fig. S9). The two restriction
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bands of TG-1 and TG-2 digested by EcoRI, BamHI and KpnI were at different
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locations (Fig. S9), indicating that the transgenic AaCBF1 was integrated into
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different positions of the genomic DNA. Under experimental conditions, the freezing
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tolerance of TG-1 and TG-2 increased from 35% in the receipt BSG plant to 83% and
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79% (P<0.01), respectively, but was still 13%-17% lower than that of HGG (P<0.01).
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The drop percentage of Y(II) and PI(abs) of TG-1 and TG-2 decreased to 66% and
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67%, and 54% and 57% from 90% and 97% of the reciptient BSG plants (P<0.01),
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respectively, which nearly places them into the cold-tolerant group. Trans-HGG
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AaCBF1, AaCOR29, AaCOR15a and AaCOR6.6 of the two transgenic crofton weed
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individual plants expressed at ambient temperature and AaCBF1 always had higher
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expression levels than BSG population (P<0.05) under cold-treatment. The AaCORs
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in transgenic plants had different expression patterns. AaCORs accumulated greatly
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after cold-treatment for 0.5h and reached the peak value at 1h (AaCOR15a and
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AaCOR6.6) or 2h (AaCOR29) cold-treatment, and then the expression level started to
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decline but was still much higher than that in the wild BSG. But after 8h (AaCOR6.6)
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or 12h (AaCOR29 and (AaCOR15a) cold-treatment, HGG had higher expression
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levels than the transgenic plants (Fig. S10).
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Physiological responses of crofton weed over-expressing AaCBF1 under
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low temperature stress
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The enhancement of cold-tolerance of both TG-1 and TG-2 plants is mainly attributed
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to overexpression of trans-HGG-AaCBF1. Hence, only TG-1 was selected for a
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bioassay of freezing-stress physiological parameters. The MDA content of plants
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decreased after exposure to low temperature for one day (BSG and TG-1) or two days
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(HGG) and then increased gradually reaching a maximum four days after treatment.
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But the MDA content of TG-1 was always the lowest while that of BSG was the
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highest (Fig. S11A). At all of the treatment times except at one day, the MDA contents
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differed among the three populations (P<0.05), which implies that transferring
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HGG-AaCBF1 significantly improved the cold-tolerance of transgenic TG-1 plants.
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Soluble sugar concentration of BSG and TG-1 increased after 2d of cold-treatment
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and then decreased, BSG decreasing more than TG-1. After 4 d treatment, the soluble
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sugar content of BSG was only 14% of its peak value while soluble sugar content of
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TG-1 is 49% of its peak (P<0.05). Soluble sugar of HGG population displayed a
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different pattern, increasing gradually with treatment time until experiment
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completion (Fig. S11B). Evidently transgenic TG-1 plants significantly increased the
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soluble sugar content contributing to its freezing-tolerance enhancement compared
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with receipt BSG plants (P<0.05).
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Soluble protein content of crofton weed populations changed differently under
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cold-treatment of -2ºC. After exposure to -2ºC the soluble protein content of BSG and
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TG-1 decreased to a minimum after 3d and then slightly increased with BSG
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accumulating more than TG-1. In contrast, the soluble protein of HGG initially
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increased reaching a maximum at 2d and then decreased until the last evaluation time
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at 4d (Fig. S11C). Thus the soluble protein contents of transgenic TG-1 plants were
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different from those of the receipt BSG plants (P<0.05) and more similar to those of
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HGG plants.
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The SOD content of these three populations all increased after exposure to -2ºC
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with BSG and TG-1 reaching a maximum value at 1d while HGG reaching the
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maximum value at 2d. SOD of HGG maintained at a higher stable value. The content
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of BSG and TG-1 were almost the same 1d before treatment, but the content of TG-1
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stayed significantly higher than BSG since then (P<0.05) (Fig. S11D). This indicates
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that TG-1 enhanced its freezing-tolerance through increase of its antioxidant activities
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and improved ROS scavenging ability.
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Chlorophyll content of all BSG, TG-1 and HGG declined under low temperature
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stress but at different rates. The chlorophyll content of BSG decreased rapidly after 1d
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of treatment and TG-1 decreased significantly after 2d of cold-stress (P<0.05).
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Chlorophyll in the HGG population decreased only slightly after 4d of treatment. The
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chlorophyll of BSG, TG-1 and HGG decreased by 48%, 39% and 24% after exposure
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to -2ºC for 4d (Fig. S11E). Although at a lower level than HGG (P<0.05), TG-1
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maintained higher chlorophyll contents than those of its receipt BSG plant at all
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treatment times (P<0.05), which implies that the transferring of AaCBF1 enhanced its
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tolerance to cold stress.
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References
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134
135
136
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140
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147
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Beauchamp C, Fridovich I (1971) Superoxide dismutase: Improved assays and an
assay applicable to acrylamide gels. Analytical biochemistry, 44, 276-287.
Dubois M, Guilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric
method for determination of sugars and related substances. Analytical chemistry, 28,
350-356.
Heath RL, Packer L (1968) Photoperoxidation in isolated chloro-plasts. I. Kinetics
and stoichiometry of fatty acid peroxidation. Archives of biochemistry and
biophysics, 125, 180-198.
Lichtenthaler HK (1987) Chlorophylls and carotenoids: Pigments of photosynthetic
biomembranes. Methods in enzymology, 148, 350-382.
Sedmak JJ, Grossberg SE (1977) A rapid, sensitive, and versatile assay for protein
using Coomassie brilliant blue G250. Analytical biochemistry, 79(1-2), 544-552.
Xu J, Tian YS, Peng RH et al. (2010) AtCPK6, a functionally redundant and positive
regulator involved in salt/drought stress tolerance in Arabidopsis. Planta, 231(6),
1251-1260.
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