Characterization of BdCBF genes and genome-wide transcriptome profiling of BdCBF3-dependent and -independent cold stress responses in Brachypodium distachyon

- Cold acclimation is critical in acquiring freezing tolerance and we demonstrated Brachypodium can successfully cold acclimate.
- We generated and characterized RNAi mutants for BdCBF1 and 3, two key transcription factors regulating CBF regulon and freezing tolerance.
- The BdCBF1 and 3 RNAi mutants resulted in drastically reduced freezing tolerance.
- We conducted comparative RNA-seq analysis of BdCBF3 and wild type under both cold and non-cold conditions.
- We identified differentially expressed genes in the BdCBF3-dependent, -independent or -compensatory categories.

Freezing stress substantially reduces crop yields and limits plant distribution. The identification of genes critical for cold acclimation is thus of great importance. C-repeat binding factors (CBFs) are transcription factors that play key regulatory roles in the cold acclimation process, which dramatically increases freezing tolerance in plants. We report here that B. distachyon can successfully cold acclimate and we identified a CBF gene family consisting of eight genes in a tandem array and are designated as BdCBF1-8. Expression analysis indicated that all the eight BdCBF genes are induced by cold. Freezing tolerance experiments showed that the knockdown of BdCBF3 gene in RNAi cbf3 mutant plants results in a significant reduction in survival after an exposure to freezing temperatures. RNA-seq transcriptomic analysis was conducted using the wild type and cbf3 mutant plants under both normal and cold conditions. We identified 460, 3213, 2839 and 1871 differentially expressed genes exhibiting different expression levels by pairwise comparisons of cbf3 (23 °C) vs. WT (23 °C), WT (23 °C) vs. WT (4 °C), cbf3 (23 °C) vs. cbf3 (4 °C), and cbf3 (4 °C) vs. WT (4 °C), respectively. These differentially expressed genes were enriched in several biological pathways. Combined analyses of differentially expressed genes in some of the enriched pathways provide insights into mechanisms of plant response to cold in the BdCBF3-dependent, -independent or -compensatory categories.