Figure 1.
Comparison between Vaccinium and Arabidopsis CBF protein sequences.
Protein lineups of V. vitis-idaea from both Northern Sweden and China; V. myrtillus; V. corymbosum; V. uliginosum and the three A. thaliana CBF sequences. The AP2/EREBP and DSAWR regions of the COR gene binding domain identified in Arabidopsis [40] are labelled in green boxes and thick underline along with the recognisable areas of the hydrophobic domains in the COOH region as described in Arabidopsis [42] labelled in a pink box and underline.
Figure 2.
Vaccinium CBF and COR gene expression in transgenic Arabidopsis lines.
Relative transcript abundance was measured in transgenic Arabidopsis lines overexpressing either V. myrtillus, V. uliginosum or V. vitis-idaea CBF (“M”, “U” and “I”, respectively). Four transgenic lines (labelled 1–4) for each of the three constructs were compared to wild type Arabidopsis (WT). Bar charts show expression of Vaccinium CBF (A) and Arabidopsis KIN2 (B), GOLS3 (C) and LTI78 (D). Error bars represent RQMIN and RQMAX and constitute the acceptable error level for a 95% confidence level according to Student’s t-test [31].
Figure 3.
COR414 and COR15A gene expression in transgenic Arabidopsis lines.
Bar charts show expression of the Arabidopsis COR genes COR414 (A) and COR15A (B) in the transgenic and WT Arabidopsis lines. Samples and details as described in legend to Figure 2.
Figure 4.
Effect of Vaccinium CBF overexpression upon freezing tolerance and development in transgenic Arabidopsis.
(A) Photos show transgenic Arabidopsis lines expressing Vaccinium CBF: V. myrtillus (iv,v,ix,x); V. uliginosum (ii,vii); V. vitis-idaea (iii, viii) and wild type (i, vi), before (i-v) and after (vi-x) freezing. (B) Bar chart showing average rosette diameter of wild type (WT) Arabidopsis and lines overexpressing either V. myrtillus, V. uliginosum or V. vitis-idaea CBF (“M”, “U” and “I”, respectively), n = 8, error bars are standard errors of the mean. Transgenic line numbers correspond to those shown in Figures 2 and 3 for ease of comparison.
Figure 5.
Effect of Vaccinium CBF overexpression upon Fv/Fm after freezing.
(A) Representative Fluocam images for each transgenic line measured. Before freezing Fv/Fm measurement images (i-iii) for Arabidopsis lines expressing V. uliginosum (i), V. vitis-idaea (ii) and V. myrtillus (iii) CBFs. After freezing Fv/Fm measurement images (iv-vi) for Arabidopsis lines expressing V. uliginosum (iv), V. vitis-idaea (v) and V. myrtillus (vi) CBFs. (B) Average Fv/Fm values for each transgenic line before (white bars) and after (black bars) freezing. Each line is a transgenic Arabidopsis line expressing CBF from a different Vaccinium species: U = V. uliginosum, V = V. vitis-idaea and M = V. myrtillus construct, WT-wild type.
Figure 6.
Transactivation of CRT::LUC by Vaccinium GFP-CBF fusions.
Bar chart showing relative transient expression levels of CRT::LUC co-expressed with Vaccinium GFP-CBF in Nicotiana benthamiana. “M”, “U” and “I” denote V. myrtillus, V. uliginosum or V. vitis-idaea CBF, respectively. Values are ratios of LUC luminescence normalised for area, and also normalised by dividing by LUC luminescence obtained by co-expressing a free GFP control with CRT::LUC.
Figure 7.
Cellular localisation of Vaccinium GFP-CBF protein fusions.
Confocal microscopy images of Vaccinium GFP-CBF proteins (B–D) compared to a free GFP control (A). GFP fusions to CBFs from V. myrtillus (B), V. uliginosum (C) and V. vitis-idaea (D). White scale bars represent 22 µm.
Figure 8.
Protein expression of Vaccinium GFP-CBF fusions.
Western blot analysis of proteins extracted from Vaccinium GFP-CBF expressed in Nicotiana benthamiana 24, 48 and 72 hours after infiltration. “M”, “U” and “I” refer to V. myrtillus, V. uliginosum or V. vitis-idaea CBF, respectively.