Fig. 1 The central rosette leaves of Arabidopsis exhibited a greater degree of freezing tolerance than did peripheral rosette leaves. Eighteen-day-old plants were acclimated at 2°C under continuous illumination for 7 d. Top panels show plants before freezing. Non-acclimated (left panel) and cold-acclimated plants (right panel) were then frozen to indicated temperatures, thawed at 4°C in darkness for 1 d (middle plate) and grown further at 23°C for 10 d (bottom plate). From: The Leaf-Order-Dependent Enhancement of Freezing Tolerance in Cold-Acclimated Arabidopsis Rosettes is not Correlated with the Transcript Levels of the Cold-Inducible Transcription Factors of CBF/DREB1 Plant Cell Physiol. 2003;44(9):922-931. doi:10.1093/pcp/pcg117 Plant Cell Physiol |

Fig. 2 Changes of the degree of freezing tolerance, osmotic pressures and sugar and Pro levels in different leaf orders of Arabidopsis rosettes during cold acclimation. The 1st and 2nd (open bars), the 3rd and 4th (gray bars), and leaves younger than the 8th leaf (black bars) were detached from 18-day-old Arabidopsis rosette plants incubated for 0, 1, 3 and 7 d at 2°C. (A) EL₅₀ values. (B) Leaf osmotic pressure measured with a vapor osmometer. (C) Sugar levels quantified by the capillary electrophoresis system. (D) Pro levels quantified by HPLC. Each datum represents an average of three measurements and bars indicate standard deviations. From: The Leaf-Order-Dependent Enhancement of Freezing Tolerance in Cold-Acclimated Arabidopsis Rosettes is not Correlated with the Transcript Levels of the Cold-Inducible Transcription Factors of CBF/DREB1 Plant Cell Physiol. 2003;44(9):922-931. doi:10.1093/pcp/pcg117 Plant Cell Physiol |

Fig. 3 Accumulation of transcripts of stress-inducible genes in different leaf orders of Arabidopsis rosettes during cold acclimation. Total RNA was extracted from the 1st and 2nd leaves (1–2), the 3rd and 4th leaves (3–4), and the leaves younger than the 8th leaf (>8) of Arabidopsis rosette plants incubated for various periods at 2°C, and RNA gel blot analysis was performed using hybridization probes specific to CBF1/DREB1B, CBF2/DREB1C, CBF3/DREB1A, COR78/RD29A and RAB18. (A) Transcript levels within 24 h at 2°C. (B) Transcript levels during 1–7 d at 2°C. Hybridization bands for 25S rRNA were shown as loading controls. From: The Leaf-Order-Dependent Enhancement of Freezing Tolerance in Cold-Acclimated Arabidopsis Rosettes is not Correlated with the Transcript Levels of the Cold-Inducible Transcription Factors of CBF/DREB1 Plant Cell Physiol. 2003;44(9):922-931. doi:10.1093/pcp/pcg117 Plant Cell Physiol |

Fig. 4 Accumulation of COR78/RD29A protein in leaves of Arabidopsis rosettes subjected to 2°C treatment. (A) A typical immunoblot membrane for COR78/RD29A protein. Proteins were extracted from the 1st and 2nd leaves (1–2), the 3rd and 4th leaves (3–4), and from the 8th to 10th leaves (8–10) from non-acclimated and cold-acclimated plants. Each lane contained 100 µg protein. (B) Quantification of the band intensity of COR78/RD29A protein. Quantification of three independent membranes was averaged and the standard deviation is shown by error bars. (C) Total protein content in leaf protein extracts. Open, gray and black bars represent data from the 1st and 2nd leaves, the 3rd and 4th leaves, and from the 8th to 10th leaves, respectively. From: The Leaf-Order-Dependent Enhancement of Freezing Tolerance in Cold-Acclimated Arabidopsis Rosettes is not Correlated with the Transcript Levels of the Cold-Inducible Transcription Factors of CBF/DREB1 Plant Cell Physiol. 2003;44(9):922-931. doi:10.1093/pcp/pcg117 Plant Cell Physiol |

Fig. 5 Correlation between solutes and the degree of freezing tolerance. (A) Average sugar contents (n = 3–5) in samples from non-acclimated (N) and cold-acclimated for 1 d (1), 3 d (3), and 7 d (7) were plotted against EL₅₀ values and bars indicated standard deviations. The solid line represents the linear correlation (y = –9.5263x – 28.377, R² = 0.9863) among a limited number of samples (see text). (B) The sum of average sugar contents (n = 3–5) and average Pro contents (n = 3) in samples from non-acclimated (N) and cold-acclimated for 1 d (1), 3 d (3), and 7 d (7) were plotted against EL₅₀ values and bars indicated the sum of standard deviations for sugar and Pro data. The solid line represents the linear correlation (y = –10.37x – 31.624, R² = 0.977) among a limited number of samples as in (A). Open circles, open squares and closed diamonds represent data from the 1st and 2nd leaves, the 3rd and 4th leaves, and from leaves younger than the 8th leaf, respectively. From: The Leaf-Order-Dependent Enhancement of Freezing Tolerance in Cold-Acclimated Arabidopsis Rosettes is not Correlated with the Transcript Levels of the Cold-Inducible Transcription Factors of CBF/DREB1 Plant Cell Physiol. 2003;44(9):922-931. doi:10.1093/pcp/pcg117 Plant Cell Physiol |