Abstract C-Repeat Binding Factors (CBFs) are DNAbinding
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