CBF2A–CBF4B genomic region copy numbers alongside the circadian clock play key regulatory mechanisms driving expression of FR-H2 CBFs

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.

Influence of CNV on transcript levels of HvCBF genes at Fr-H2 locus revealed by resequencing in resistant barley cv. ‘Nure’ and expression analysis

“…In the present study, no evident correlation between CNV and expression level for HvCBF4 and HvCBF2A genes was observed after the cold treatment in dark conditions. Conversely, Dhillon et al [18] reported the association between HvCBF4-HvCBF2A copy numbers and HvCBF2 transcript levels; however, in their experiment plants were subjected to light and cold temperature (6°C) stimuli at the same time. Consequently, this implies that the observed expression was the sum/interaction of both environmental factors, each influencing HvCBF expression.…”

“…The regulation also involves members of the calmodulin-binding transcription activator (CAMTA) family and the activation of phytochromes, which play an important role in light-mediated stimulus. Moreover, under warm temperatures, CBFs expression is regulated by the circadian clock through the action of a central oscillator (namely CCA1 and LHY), and day length (photoperiod) [18,19].…”

“…In the economically most important cereals a total of 20, 13 and 37 elements characterize barley, T. monococcum and bread wheat genomes, respectively [9,21,22]. A differential expansion of the CBF gene cluster together with copy number variation (CNV), instead of mutations within individual gene(s), has been proposed as an explanation for the functional role played by the CBFs at Fr-2 in genotypes showing different levels of frost resistance [18,23]. In particular, Knox et al.…”

Successive evolutionary steps drove Pooideae grasses from tropical to temperate regions

“…However, phylogenetic reconstruction supports the early recruitment of VRN1 in vernalization responsiveness, at least in the ancestor of all Pooideae tribes minus Nardeae and Brachyelytreae, but not before the diversification of Pooideae from Bambusoideae (McKeown et al ., ). An additional possible connection between short‐ and long‐term cold responses could be through changes in circadian clock regulation, which are widely known to have large effects on cold responses and flowering time regulation (Salomé & McClung, ; Yamashino et al ., ; Nakamichi et al ., ; Dong et al ., ; Chow et al ., ; Greenham & McClung, ; Seo & Mas, ; Dhillon et al ., ).…”

“…The genetics of vernalization‐mediated flowering and cold acclimation has been largely elucidated in distantly related model species, each being regulated by a number of genes that form complex, and likely interconnected, networks (Thomashow, ; Yan et al ., , , ; Deng et al ., ; Kippes et al ., ; Zhao et al ., ; Dhillon et al ., ). Specifically, vernalization responsiveness in cereal grasses such as barley and wheat (Pooideae) is controlled by at least three core interacting proteins, VERNALIZATION1 (VRN1), VRN2 and VRN3 (Yan et al ., , , ; Kippes et al ., ).…”