Abstract
The C-Repeat Binding Factors (CBFs) are DNA-binding transcriptional activators that were identified using Arabidopsis thaliana. In barley, Hordeum vulgare, a cluster of CBF genes reside at FROST RESISTANCE-H2, one of two loci having major effects on winter-hardiness. FR-H2 was revealed in a population derived from the winter barley ‘Nure’ and the spring barley ‘Trèmois’. ‘Nure’ harbors two to three copies of CBF2A and CBF4B as a consequence of tandem iteration of the genomic region encompassing these genes whereas ‘Trèmois’ harbors single copies, and these copy number differences are associated with their transcript level differences. Here we explore further the relationship between FR-H2 CBF gene copy number and transcript levels using ‘Admire’, a winter barley accumulating FR-H2 CBF gene transcripts to very high levels, and a group of lines related to ‘Admire’ through descent. DNA blot hybridization indicated the CBF2A–CBF4B genomic region is present in 7–8 copies in ‘Admire’ and is highly variable in copy number across the lines related to ‘Admire’. At normal growth temperatures transcript levels of CBF12, CBF14, and CBF16 were higher in lines having greater CBF2A–CBF4B genomic region copy numbers than in lines having fewer copy numbers at peak expression level time points controlled by the circadian clock. Chromatin immunoprecipitation indicated CBF2 was at the CBF12 and CBF16 promoters at normal growth temperatures. These data support a scenario in which CBF2A–CBF4B genomic region copy numbers affect expression of other FR–H2 CBFs through a mechansim in which these other FR-H2 CBFs are activated by those in the copy number variable unit.
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Acknowledgements
We thank Dr. Harold E. Bockelman at the National Plant Germplasm System (NPGS; http://www.ars-grin.gov/npgs/index.html) for providing barley Accessions. We thank Drs. David F. Francis for suggestions in assaying the MO B lines and Erich Grotewold for suggestions in carrying out the ChIP assays.
Funding
This work was supported by grants from the Ohio Plant Biotechnology Consortium (2010-011) and the United States Barley Genome Project (USDA-CSREES subaward CO396A-F), and the OARDC tornado research loss recovery fund (OHOG0490). Salaries and research support in the Stockinger lab were provided by state and federal funds appropriated to The Ohio State University, Ohio Agricultural Research and Development Center.
Author contributions
EJS directed the experiments, TD and EJS designed individual experiments, TD carried out the experiments, KM assisted with ChIP experiments, TD and EJS interpreted the data and wrote the manuscript. TD, KM, and EJS edited the manuscript.
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Dhillon, T., Morohashi, K. & Stockinger, E.J. CBF2A–CBF4B genomic region copy numbers alongside the circadian clock play key regulatory mechanisms driving expression of FR-H2 CBFs. Plant Mol Biol 94, 333–347 (2017). https://doi.org/10.1007/s11103-017-0610-z
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DOI: https://doi.org/10.1007/s11103-017-0610-z