VcRR2 regulates chilling-mediated flowering through expression of hormone genes in a transgenic blueberry mutant.

The molecular mechanism underlying dormancy release and the induction of flowering remains poorly understood in woody plants. Mu-legacy is a valuable blueberry mutant, in which a transgene insertion caused increased expression of a RESPONSE REGULATOR 2-like gene (VcRR2). Mu-legacy plants, compared with nontransgenic 'Legacy' plants, show dwarfing, promotion of flower bud formation, and can flower under nonchilling conditions. We conducted transcriptomic comparisons in leaves, chilled and nonchilled flowering buds, and late-pink buds, and analyzed a total of 41 metabolites of six groups of hormones in leaf tissues of both Mu-legacy and 'Legacy' plants. These analyses uncovered that increased VcRR2 expression promotes the expression of a homolog of Arabidopsis thaliana ENT-COPALYL DIPHOSPHATE SYNTHETASE 1 (VcGA1), which induces new homeostasis of hormones, including increased gibberellin 4 (GA4) levels in Mu-legacy leaves. Consequently, increased expression of VcRR2 and VcGA1, which function in cytokinin responses and gibberellin synthesis, respectively, initiated the reduction in plant height and the enhancement of flower bud formation of the Mu-legacy plants through interactions of multiple approaches. In nonchilled flower buds, 29 differentially expressed transcripts of 17 genes of five groups of hormones were identified in transcriptome comparisons between Mu-legacy and 'Legacy' plants, of which 22 were chilling responsive. Thus, these analyses suggest that increased expression of VcRR2 was collectively responsible for promoting flower bud formation in highbush blueberry under nonchilling conditions. We report here for the first time the importance of VcRR2 to induce a suite of downstream hormones that promote flowering in woody plants.

Constitutive Expression of an Apple FLC3-like Gene Promotes Flowering in Transgenic Blueberry under Nonchilling Conditions.

MADS-box transcription factors FLOWERING LOCUS C (FLC) and APETALA1 (AP1)/CAULIFLOWER (CAL) have an opposite effect in vernalization-regulated flowering in Arabidopsis. In woody plants, a functional FLC-like gene has not been verified through reverse genetics. To reveal chilling-regulated flowering mechanisms in woody fruit crops, we conducted phylogenetic analysis of the annotated FLC-like proteins of apple and found that these proteins are grouped more closely to Arabidopsis AP1 than the FLC group. An FLC3-like MADS-box gene from columnar apple trees (Malus domestica) (MdFLC3-like) was cloned for functional analysis through a constitutive transgenic expression. The MdFLC3-like shows 88% identity to pear's FLC-like genes and 82% identity to blueberry's CAL1 gene (VcCAL1). When constitutively expressed in a highbush blueberry (Vaccinium corymbosum L.) cultivar 'Legacy', the MdFLC3-like induced expressions of orthologues of three MADS-box genes, including APETALA1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, and CAL1. As a consequence, in contrast to the anticipated late flowering associated with an overexpressed FLC-like, the MdFLC3-like promoted flowering of transgenic blueberry plants under nonchilling conditions where nontransgenic 'Legacy' plants could not flower. Thus, the constitutively expressed MdFLC3-like in transgenic blueberries functioned likely as a blueberry's VcCAL1. The results are anticipated to facilitate future studies for revealing chilling-mediated flowering mechanisms in woody plants.