<i>tcf1-1</i> plants are tolerant to freezing treatment.

<p><b>(A)</b> Schematic presentation of the <i>TCF1</i> gene structure and T-DNA insertions in the <i>TCF1</i> gene (arrowheads). The closed rectangles represent exons and lines between the exons denote introns. <b>(B)</b><i>tcf1-1</i> is a null mutation of <i>TCF1</i>. The levels of <i>TCF1</i> transcripts were determined by RT-PCR using 3-week-old <i>tcf1-1</i> seedlings subjected to low temperature (4°C) for the indicated time periods (h); the <i>ACT2</i> gene was used as loading control. <b>(C)</b> Tolerance of 3-week-old <i>tcf1-1</i> and wild-type (WT) plants at the indicated temperatures below freezing under long-day photoperiod with cold acclimation for 7 days. The pictures were taken 7 days after treatments. <b>(D)</b> Quantification of survival rate of the treated plants in <b>(C)</b>, (*, <i>P</i> < 0.05, <i>t</i>-test). <b>(E)</b> Leakage of electrolytes in <i>tcf1-1</i> and WT plants treated at indicated temperatures below freezing. WT (cold) and <i>tcf1-1</i> (cold): 3-week-old plants were cold-acclimated (4°C for 7 day), WT and <i>tcf1-1</i>: both plants were grown under normal conditions. Error bars are standard deviation (<i>n</i> = 8), (*, <i>P</i> < 0.05, <i>t</i>-test). <b>(F)</b> Tolerance of freezing treatments (-8°C for 2 h) of control and transgenic plants, which were cold-acclimated at 4°C for 7 days before the treatment. The plants included WT, <i>tcf1-1</i>, representative homozygous lines of <i>tcf1-1</i> transformed with an empty vector pEZR(K)LC (<i>tcf1-1Vector</i>-2 (#2–3)) or <i>TCF1</i> gene (<i>tcf1-1TCF1-3</i> (#3–8), <i>tcf1-1TCF1-12</i> (#12–4) and <i>tcf1-1TCF1-13</i> (#13–2)).</p

Gene expression analyses of the CBFs and the CBF regulon in the <i>tcf1-1</i> and wild-type plants.

<p><b>(A)</b> to <b>(C)</b> The levels of <i>CBF1</i>, <i>CBF2</i> and CBF3 transcripts, respectively, in <i>tcf1-1</i> and wild-type plants. The plants were treated with cold (4°C) at the indicated time points. <b>(D)</b> The level of <i>TCF1</i> transcripts in the loss-of-function <i>cbf2</i> mutant plants grown under normal conditions or given a 4°C treatment for 7 day. Transcript levels of the <i>ACT2</i> gene were used as a loading control. <b>(E)</b> to <b>(G)</b> Transcript levels of <i>COR47</i><b>(E)</b>, <i>COR15A</i><b>(F)</b> and <i>RD29A</i><b>(G)</b> in <i>tcf1-1</i> and wild-type plants. The plants were treated with cold (4°C) for the indicated times. <b>(H)</b> Western blot analysis with anti-acetylated H4 antibody (<i>Top</i>) revealed that <i>tcf1-1</i> and WT plants have similar levels of acetylated H4. Histone H4 antibody for core histone H4 was used as the immunoblot control (<i>Bottom</i>).</p

Analysis of <i>TCF1</i> expression and nuclear localization of the TCF1 protein.

<p><b>(A)</b> Three-week-old Col-0 (WT) plants were subjected to low temperature (4°C) and the samples were harvested at the indicated time points for semi-quantitative RT-PCR analysis of <i>TCF1</i> transcripts. <i>ACT2</i> (<i>At3g18780</i>) was used as a loading control. <b>(B)</b> GUS staining of transgenic plants expressing <i>TCF1pro</i>::<i>GUS</i> under normal temperature or treated at 4°C for 7 days. <b>(C)</b> Semi-quantitative RT-PCR for <i>TCF1</i> in different tissues with or without cold treatments for 7 days. R, root; Sh, shoot; L, leaves; F, flowers; Si, siliques. <b>(D)</b> Semi-quantitative RT-PCR analysis for <i>TCF1</i> of three-week-old Col-0 plants treated with 100 μM ABA, 400 mM mannitol, 20% PEG6000, 300 mM NaCl for 3 h and 4°C Cold treatment for 24 h. <b>(E)</b> Localization of fluorescence in <i>tcf1-1</i> plants expressing a <i>TCF1pro</i>::<i>GFP-TCF1</i> fusion at 22°C (<i>Upper</i>) and 4°C for 7 days (<i>Bottom</i>). GFP: GFP-TCF1 fusion protein (<i>tcf1-1TCF1-3</i>), DAPI: DAPI staining, Merge: Merger of GFP and DAPI channels (Scale bars, 20 μm).</p

<i>amiRNA-BCB</i> plants are tolerant to freezing treatment.

<p><b>(A)</b> Freezing treatment of 3-week-old <i>amiRNA-BCB</i> transgenic plants (#4–9 and #9–2) and wild-type (WT) plants at the indicated temperature below freezing under long-day photoperiod with cold acclimation. The pictures were taken 7 days after treatments. <b>(B)</b> Quantification of survival of the plants in <b>(A)</b>. Error bars are standard deviation (<i>n</i> = 80–100), (*, <i>P</i> < 0.05, <i>t</i>-test). <b>(C)</b> Leakage of electrolytes in <i>amiRNA-BCB</i> transgenic plants and WT plants treated at indicated temperature below freezing. Error bars are standard deviation (<i>n</i> = 8). (*, <i>P</i> < 0.05, <i>t</i>-test). <b>(D)</b> Quantitative determination of lignin content from whole rosettes of three-week-old WT and <i>amiRNA-BCB</i> transgenic plants grown in soil with (<i>Grey</i>) or without (<i>Black</i>) cold treatment (4°C for 7 days). Twelve independent experiments were performed and the data are expressed as mean ± S.E. Means with the same letter are not significantly different at <i>P</i> < 0.05 by One Way ANOVA analysis.</p

A proposed model for TCF1 in the <i>Arabidopsis</i> response to low temperature.

<p>Under normal condition, <i>PAL</i> genes are modulated by the developmental signals to synthesize lignin that favor optimal plant growth and water transport. During cold acclimation, <i>TCF1</i> is rapidly induced to activate <i>BCB</i> transcription and then stimulates expression of <i>PAL1</i>/<i>3/4</i> genes to maintain lignin accumulation of the stressed cells. However, when the plants are exposed to freezing temperature, reduction of lignin deposition within the cell wall of the <i>tcf1-1</i> plants may increase cell wall permeability and protect the cells from freezing damage. Reduction of lignin may also enhance elasticity of the cell wall to increase the capacity to accommodate growth of ice crystals with less damage to both the dehydrated cell and cell wall which is required for plant growth arrest. Lines with arrowheads denote direct regulation, and lines with blunt heads represent indirect regulation.</p

GEF activity assay of TCF1 and analysis of TCF1-histone interactions.

<p><b>(A)</b> Binding of <i>E</i>. <i>coli</i>-expressed GST-TCF1 to a calf thymus histone-agarose column. Western blots with anti-GST antibody showing GST-TCF1 (77 kDa) or control GST (26 kDa). Lane 1, protein applied to the columns; lane 2, unbound material that flowed through; lane 3, protein bound after a 5 min incubation, eluted with 0.3 M NaCl. <b>(B)</b> 0.3 μg of <i>in vitro</i> translated Myc-TCF1 was incubated with 20 μg of the appropriate histone (20-fold excess) for 30 min at room temperature. The samples were then incubated with histone-agarose overnight at 4°C. Myc-TCF1 binding to histone-agarose was analyzed by Western blot. The different histones used as competitors are indicated at the top of each lane. ‘All’ indicates the mixture of histones and ‘None’ indicates the control without competitors. <b>(C)</b> Yeast co-transformant strains carrying both <i>TCF1</i> and vector control (TCF1/BD), <i>histone H3/H4</i> and vector control (HFO2/AD, HFO4/AD and HTR9/AD), <i>TCF1</i> and <i>H3</i>/<i>H4</i> (TCF1/HFO2,TCF1/HFO4 and TCF1/HTR9), negative control (BD/AD) and positive control (BD-53/AD-T) were streaked onto selective media. Activation of the <i>lacZ</i> reporter gene is indicated by the formation of blue or blue-green colonies on plates containing X-Gal (<i>left</i>), the growth state of yeast co-transformant strains in YPD medium is shown on the right (<i>right</i>). <b>(D)</b> Ran-GEF activity of <i>E</i>. <i>coli</i>-expressed GST-RCC1 and GST-TCF1 is shown as the percentage of [³H]GDP remaining at the end of the GEF assay.</p

Reduced lignin content increased freezing tolerance.

<p><b>(A)</b> Quantitative determination of lignin content from whole rosettes of three-week-old WT and <i>tcf1-1</i> plants grown in soil with (<i>Grey</i>) or without (<i>Black</i>) cold treatment (4°C for 7 days). Twelve independent experiments were performed and the data are expressed as mean ±S.E. Means with the same letter are not significantly different at <i>P</i> < 0.05 by One Way ANOVA analysis. <b>(B)</b> Three week-old <i>pal1pal2</i> and wild type (WT) plants with or without a 7-day cold treatment at 4°C were used for freezing treatments at indicated time point. The pictures were taken 7 days after treatments. <b>(C)</b> Quantification of survival of the wild type (<i>Black</i>) and <i>pal1pal2</i> plants (<i>Grey</i>) in <b>(B)</b>; Error bars represent standard deviation (<i>n</i> = 80–100). (*, <i>t</i>-test, <i>P</i> < 0.05). <b>(D)</b> Leakage of electrolytes in <i>pal1pal2</i> and wild type plants treated (see experimental procedures) at indicated time point with and without cold acclimation. The experiments were repeated 3 times. Error bars are standard deviation (<i>n</i> = 6). (*, <i>t</i>-test, <i>P</i> < 0.05).</p