SVP-like MADS Box Genes Control Dormancy and Budbreak in Apple

Abstract

The annual growth cycle of trees is the result of seasonal cues. The onset of winter triggers an endodormant state preventing bud growth and, once a chilling requirement is satisfied, these buds enter an ecodormant state and resume growing. MADS-box genes with similarity to Arabidopsis SHORT VEGETATIVE PHASE (SVP) [the SVP-like and DORMANCY ASSOCIATED MADS-BOX (DAM) genes] have been implicated in regulating flowering and growth-dormancy cycles in perennials. Here, we identified and characterized three DAM-like (MdDAMs) and two SHORT VEGETATIVE PHASE-like (MdSVPs) genes from apple (Malus × domestica 'Royal Gala'). The expression of MdDAMa and MdDAMc indicated they may play a role in triggering autumn growth cessation. In contrast, the expression of MdDAMb, MdSVPa and MdSVPb suggested a role in maintaining bud dormancy. Consistent with this, ectopic expression of MdDAMb and MdSVPa in 'Royal Gala' apple plants resulted in delayed budbreak and architecture change due to constrained lateral shoot outgrowth, but normal flower and fruit development. The association of MdSVPa and MdSVPb expression with floral bud development in the low fruiting 'Off' trees of a biennial bearing cultivar 'Sciros' suggested the SVP genes might also play a role in floral meristem identity.

Keywords: DAM; SVP; apple; biennial bearing; dormancy; ectopic expression.

FIGURE 1

Phylogenetic tree of DAM and SVP predicted proteins. The tree was built from the full-length amino acid sequence alignment using the neighbor-joining method and the bootstrap test (1000 replicates). Bootstrap values above 50% are shown next to the branches. Ac, Actinidia chinensis, At, Arabidopsis thaliana, Ee, Euphorbia esula, Pm, Prunus mume, Ppe, Prunus persica, Ppy, Pyrus pyrifolia, Pt, Poncirus trifoliata, St, Solanum tuberosum, Md, Malus × domestica ‘Royal Gala’ sequences, with indicated accession numbers.

FIGURE 2

Relative expression of MdDAM and MdSVP genes in root, stem, leaf, apical bud, flower, and fruit of apple. The level of expression was normalized to apple reference gene, MdSGL29F. Error bars represent ± SE of three biological replicates.

FIGURE 3

Relative expression of MdDAM and MdSVP genes in apple apical buds during the season. (A) MdDAM and MdSVP genes expression during winter dormancy and active growth in spring. (B) MdDAM and MdSVP genes expression from full bloom to leaf drop. The solid line represents samples collected from the fruit bearing (‘On’) trees. The dotted line represents samples collected from non-fruit bearing (‘Off’) trees. The level of expression was normalized to apple MdSGL29F. Data points represent the mean ± SE of two biological replicates. 50% budbreak is indicated by a vertical line. Stages representing predicted common terminal meristem identity in ‘Off’ trees are presented below. The timing of transitions between meristem identities was adopted from Foster et al. (2003): competent vegetative meristems (VM) transition to inflorescence meristems (IM) around 100 days after full bloom (DAFB) and to terminal floral meristem (FM) identity between 100 and 141 DAFB.

FIGURE 4

Appearance of transgenic MdDAMb and MdSVPa ‘Royal Gala’ lines at the first year spring in the glasshouse. (A) Relative expression of MdDAMb and MdSVPa in leaf tissue of transgenic ‘Royal Gala’ lines. The expression of each gene was normalized against apple MdSGL29F. Error bars represent SE for four technical replicates. (B) Phenotypes of constitutive expression of MdDAMb and MdSVPa in ‘Royal Gala’ compared with controls.

FIGURE 5

Appearance of transgenic MdDAMb and MdSVPa ‘Royal Gala’ lines at the second year spring in the glasshouse. All plants were pruned to induce lateral shoot growth.

FIGURE 6

Delayed budbreak in transgenic MdDAMb and MdSVPa ‘Royal Gala’ lines. Budbreak was recorded as a number of days from 100% leaf drop in late autumn to the first visible leaf in spring and presented as the mean ± SE of individual transgenic lines.

FIGURE 7

Flowers and fruits on 35S:MdSVPa transgenic lines versus controls. (A) The flowers of 35S:MdSVPa transgenic lines harbored on the main stem. (B) Typical position of flowers on control plants. (C,D) Fruit on 35S:MdSVPa transgenic lines and controls.