Biochemical and Transcriptional Responses in Cold-Acclimated and Non-Acclimated Contrasting Camelina Biotypes under Freezing Stress

Abstract

Cold-acclimated and non-acclimated contrasting Camelina (Camelina sativa L.) biotypes were investigated for changes in stress-associated biomarkers, including antioxidant enzyme activity, lipid peroxidation, protein, and proline content. In addition, a well-known freezing tolerance pathway participant known as C-repeat/DRE-binding factors (CBFs), an inducer of CBF expression (ICE1), and a cold-regulated (COR6.6) genes of the ICE-CBF-COR pathway were studied at the transcriptional level on the doubled-haploid (DH) lines. Freezing stress had significant effects on all studied parameters. The cold-acclimated DH34 (a freezing-tolerant line) showed an overall better performance under freezing stress than non-acclimated plants. The non-cold-acclimated DH08 (a frost-sensitive line) showed the highest electrolyte leakage after freezing stress. The highest activity of antioxidant enzymes (glutathione peroxidase, superoxide dismutase, and catalase) was also detected in non-acclimated plants, whereas the cold-acclimated plants showed lower enzyme activities upon stress treatment. Cold acclimation had a significantly positive effect on the total protein and proline content of stressed plants. The qRT-PCR analysis revealed significant differences in the expression and cold-inducibility of CsCBF1-3, CsICE1, and CsCOR6.6 genes among the samples of different treatments. The highest expression of all CBF genes was recorded in the non-acclimated frost-tolerant biotype after freezing stress. Interestingly a significantly higher expression of COR6.6 was detected in cold-acclimated samples of both frost-sensitive and -tolerant biotypes after freezing stress. The presented results provide more insights into freezing tolerance mechanisms in the Camelina plant from both a biochemical point of view and the expression of the associated genes.

Keywords: C. sativa; cold acclimation; electrolyte leakage; freezing tolerance; gene expression.

Figure 1

The effect of freezing stress on biochemical properties of two Camelina biotypes (freezing sensitive (FS) and freezing tolerant (FT)) with (AC) and without cold acclimation (NA). EL: electrolyte leakage; CAT: catalase; SOD: superoxide dismutase; GPX: guaiacol peroxidase; MDA: malondialdehyde; GB: glycine betaine. The ns, *, **, and *** show non-significant differences or significant differences at p ≤ 5%, 1%, and 0.1%, respectively.

Figure 1

The effect of freezing stress on biochemical properties of two Camelina biotypes (freezing sensitive (FS) and freezing tolerant (FT)) with (AC) and without cold acclimation (NA). EL: electrolyte leakage; CAT: catalase; SOD: superoxide dismutase; GPX: guaiacol peroxidase; MDA: malondialdehyde; GB: glycine betaine. The ns, *, **, and *** show non-significant differences or significant differences at p ≤ 5%, 1%, and 0.1%, respectively.

Figure 2

The CsICE1, CsCBF1, CsCBF2, CsCBF3, and CsCOR6.6 genes in two Camelina biotypes (freezing sensitive (FS) and freezing tolerant (FT)) with (AC) and without cold acclimation (NA) after freezing stress. The ns, *, **, and *** show non-significant differences or significant differences at p ≤ 5%, 1%, and 0.1%, respectively.

Figure 2

The CsICE1, CsCBF1, CsCBF2, CsCBF3, and CsCOR6.6 genes in two Camelina biotypes (freezing sensitive (FS) and freezing tolerant (FT)) with (AC) and without cold acclimation (NA) after freezing stress. The ns, *, **, and *** show non-significant differences or significant differences at p ≤ 5%, 1%, and 0.1%, respectively.

Figure 3

Synteny analysis of the selected ICE-CBF-COR genes in A. thaliana and C. sativa. The At_chr and Cs_chr show the chromosome number in A. thaliana and C. sativa, respectively. Three sub-genomes of C. sativa represent in different green colors.

Figure 4

The schematic diagram illustrating the experiment and treatment design.