Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation
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C-Repeat Binding Factors (CBFs) are DNA-binding transcriptional activators of gene pathways imparting freezing tolerance. Poaceae contain three CBF subfamilies, two of which, HvCBF3/CBFIII and HvCBF4/CBFIV, are unique to this taxon. To gain mechanistic insight into HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in spring barley (Hordeum vulgare) cultivar ‘Golden Promise’. The Hv-CBF2A overexpressing lines exhibited stunted growth, poor yield, and greater freezing tolerance compared to non-transformed ‘Golden Promise’. Differences in freezing tolerance were apparent only upon cold acclimation. During cold acclimation freezing tolerance of the Hv-CBF2A overexpressing lines increased more rapidly than that of ‘Golden Promise’ and paralleled the freezing tolerance of the winter hardy barley ‘Dicktoo’. Transcript levels of candidate CBF target genes, COR14B and DHN5 were increased in the overexpressor lines at warm temperatures, and at cold temperatures they accumulated to much higher levels in the Hv-CBF2A overexpressors than in ‘Golden Promise’. Hv-CBF2A overexpression also increased transcript levels of other CBF genes at FROST RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites in their upstream regions, the most notable of which was CBF12. CBF12 transcript levels exhibited a relatively constant incremental increase above levels in ‘Golden Promise’ both at warm and cold. These data indicate that Hv-CBF2A activates target genes at warm temperatures and that transcript accumulation for some of these targets is greatly enhanced by cold temperatures.
KeywordsCold acclimation and freezing tolerance Triticeae cereals Barley CBF transcription factors Gene regulation
This research was supported in part by grants from the NSF Plant Genome Project (DBI 0110124 and DBI 0701709). Katherine Pillman was supported by a fellowship from the Australian Centre for Plant Functional Genomics (Adelaide, Australia). Alfonso Cuesta-Marcos was supported by a postdoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación (MICINN). Salaries and research support in the Stockinger lab provided by state and federal funds appropriated to The Ohio State University, Ohio Agricultural Research and Development Center, the Ohio Plant Biotechnology Consortium, and USDA-CSREES subaward CO396A-F. We thank Dr. Wang Ming-Bo (CSIRO, Australia) for providing pWBVec10a binary vector and Dr. Neil Shirley (The University of Adelaide, Australia) for providing assistance with qRT-PCR. We also thank Dr. Michael F. Thomashow for helpful suggestions and Drs. David Mackey and Esther van der Knaap for critically reviewing the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
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