Circadian and Light Regulated Expression of CBFs and their Upstream Signalling Genes in Barley

Abstract

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light-including its spectra-also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 °C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeumvulgare (Hv) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBFs, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance.

Keywords: HvCBF-phylogenetic subgroups; barley; calcium signalling; circadian rhythm; gene expression; light quality; low red/far-red ratio.

Figure 1

The expression patterns of the HvCCA1 and HvTOC1 (A,B) with white light and low R/FR illumination. In the first two days, the plants were illuminated for 12 h, whereas, in the next two days, they were kept under constant light conditions. Samples were collected every 4 h during four days. Transcript levels were calculated with the ΔC_t method. The values on the X-axes show the time in hours after dawn. The white and black bars symbolise the light and dark periods, while grey bars indicate subjective night. The data and error bars, which represent the standard deviation, originated from three technical replicates.

Figure 2

The expression patterns of HvPITP and HvPI4K genes (A,B) with white light and low R/FR illumination. Conditions are the same as in Figure 1.

Figure 3

The expression patterns of the HvPLD (put.), HvCBL2, HvCaM.2, HvCDPK12 (A–D) with white light and low R/FR illumination. Conditions are the same as in Figure 1.

Figure 4

Gene expression patterns of the HvCBF3 and HvCBF6 (A,B) with white light and low R/FR ratio in the spectra. Conditions are the same as in Figure 1.

Figure 5

Gene expression levels in HvCBF2A, HvCBF4B, HvCBF9 and HvCBF14 (A–D) with white light and low R/FR illumination. Conditions are the same as in Figure 1.

Figure 6

Graphical summary of our results based on the gene expression analyses. The abbreviations (R/FR, CCA1, LHY, PITP, PI4K, PLC, CBL2, CaM.2, CDPK12 and CAMTA) and the references in the brackets are detailed in the text. Lines ending with arrowheads represent induction, while lines with blunt ends mean inhibition.