Vernalization regulatory network identifies potential novel functions for genes in the HvVRN2 locus

Flowering is controlled by environmental and genetic factors. Vernalization requirement in barley (Hordeum vulgare L.) is determined by the allele in HvVRN1 and the presence of locus HvVRN2, which consists of two zinc-finger and CONSTANS-like domains (ZCCT) genes: HvVRN2a and HvVRN2b. While the effect of HvVRN2 presence is well known, its outcome on the transcriptome and other genes outside the flowering/vernalization pathway remains unknown. Near-isogenic lines, with presence/absence of HvVRN2, were subjected to vernalization treatments of different lengths, enabling the assessment of HvVRN2 expression dynamics as well as broader transcriptional responses regulated by VRN2. Unknown differences in expression levels of the two HvVRN2 genes were identified, as well as an effect on tillering. A regulatory network analysis pointed at new candidate genes for flowering regulation related to the vernalization pathway and suggested additional biological roles for HvVRN2b. Finally, protein modelling resulted in higher VRN2-Nuclear Factor-Y (NF-Y) stability than other CCT-NF-Y complexes. This study advances in the understanding of HvVRN2 in barley adaptation. The ZCCT genes within the HvVRN2 locus should be regarded as distinct genes and may possess broader adaptive functions than previously recognized. Flowering repression by HvVRN2 may take place at the protein level, potentially inhibiting formation of other CCT/NF-YB/NF-YC complexes that activate HvFT1 transcription.