Functional and expression analyses of kiwifruit SOC1-like genes suggest that they may not have a role in the transition to flowering but may affect the duration of dormancy

Abstract

The MADS-domain transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) is one of the key integrators of endogenous and environmental signals that promote flowering in the annual species Arabidopsis thaliana. In the deciduous woody perennial vine kiwifruit (Actinidia spp.), environmental signals are integrated to regulate annual cycles of growth and dormancy. Accumulation of chilling during winter is required for dormancy break and flowering in spring. In order to understand the regulation of dormancy and flowering in kiwifruit, nine kiwifruit SOC1-like genes were identified and characterized. All genes affected flowering time of A. thaliana Col-0 and were able to rescue the late flowering phenotype of the soc1-2 mutant when ectopically expressed. A differential capacity for homodimerization was observed, but all proteins were capable of strong interactions with SHORT VEGETATIVE PHASE (SVP) MADS-domain proteins. Largely overlapping spatial domains but distinct expression profiles in buds were identified between the SOC1-like gene family members. Ectopic expression of AcSOC1e, AcSOC1i, and AcSOC1f in Actinidia chinensis had no impact on establishment of winter dormancy and failed to induce precocious flowering, but AcSOC1i reduced the duration of dormancy in the absence of winter chilling. These findings add to our understanding of the SOC1-like gene family and the potential diversification of SOC1 function in woody perennials.

Keywords: Actinidia; budbreak; dormancy; flowering SOC1..

Fig. 1.

Phylogenetic analysis of kiwifruit SOC1-like protein sequences. (A) Alignment of the C-terminal region of SOC1-like amino acid sequences. The amino acid residues in the alignment are shaded according to their similarity scores: white on black, 100% similar; white on grey, 80–100% similar; black on grey, 60–80% similar; grey on white, <60% similar. The conserved SOC1 motif is underlined. The observed deviation of this motif in AcSOC1h appears to be due to a putative 1bp deletion within the motif leading to a frameshift from EVETELFFGLA to EVETELSLALL. (B) Phylogenetic tree based on the amino acid alignment of kiwifruit SOC1-like predicted proteins and SOC1-like proteins from other plant species. The tree was generated using the Geneious Tree Builder plug-in (Geneious 5.5.6) using the Neighbor–Joining method with Arabidopsis AGL6 as an outgroup. Numbers below the branches represent bootstrap support values from 1000 replicates (values ≥50% are indicated). Origins of the genes, with the exception of Arabidopsis, are indicated by a prefix as follows: Ac, Actinidia chinensis; Vv, Vitis vinifera; Pt, Populus tremuloides; Ps, Pisum sativum; Ph, Petunia hybrida; Fv, Fragaria vesca; Pa, Prunus armeniaca; Md, Malus×domestica; their GenBank accession numbers are indicated to the right. Arabidopsis genes SOC1, AGL14, AGL19, AGL42, AGL71, and AGL72 are associated with their unique IDs. Kiwifruit SOC1-like genes in different clades are indicated by arrows. (This figure is available in colour at JXB online.)

Fig. 2.

Constitutive expression of kiwifruit SOC1-like genes affects flowering in Arabidopsis. (A) Flowering time of primary transgenic (T1) Arabidopsis Col-0 plants grown in non-inductive short-day conditions. Flowering time was recorded as the number of rosette leaves when the primary inflorescence stems were 0.5cm long. Each dot represents one line. (B) Flowering time of T1 Arabidopsis soc1-2 plants grown in short-day conditions, recorded and presented as above. (C) Flowering time of hygromycin-resistant progeny (T2) of three independent T1 lines of transgenic Arabidopsis Col-0 plants grown in short-day conditions. (D) Transgene expression in T2 plants. (E) Normal rosette development of wild-type Arabidopsis Col-0. (F, G) Early bolting and small rosette leaves resulting from constitutive expression of AcSOC1 constructs. (H) Small first flower (arrow) in the AcSOC1e early flowering line (I, J) Abnormal flower development in lines expressing AcSOC1 genes. (H) Wild-type Arabidopsis Col-0 flower. Scale bars=1mm.

Fig. 3.

Relative expression of kiwifruit SOC1-like genes in leaf, stem, bud, flower, young fruit, mature fruit, seed, and root, normalized to kiwifruit ACTIN (ACT). Error bars represent standard errors (SE) for three replicates.

Fig. 4.

Relative expression of kiwifruit SOC1-like genes in leaf samples collected at regular intervals over a 24h period in spring (A) and summer (B), normalized to kiwifruit ACTIN (ACT) and expressed as a ratio to the first sample point, which was arbitrarily set to 1. Data points represent the mean ±SE for three replicates. The white and black rectangles indicate the daylight and night-time, respectively.

Fig. 5.

Relative expression of kiwifruit SOC1-like genes in axillary buds during the growth and dormancy cycles, normalized to kiwifruit UBC9 and expressed as a ratio to the sample point with lowest expression, which was arbitrarily set to 1. Data points represent the mean ±SE for two replicates, the solid line represents samples collected from Hamilton, New Zealand, in 1995–1996, and the dotted line represents samples collected from Kerikeri, New Zealand, in 2008–2009. The vertical solid and dashed lines indicate budbreak time in Hamilton and Kerikeri, respectively. Hamilton is the region with colder winters (Supplementary Fig. S2 at JXB online).

Fig. 6.

Summary of kiwifruit SOC1-like protein interactions detected by yeast two-hybrid analysis. (A) Homo- and heterodimerization of kiwifruit SOC1-like and Arabidopsis SOC1 proteins. (B, C) Heterodimerization of kiwifruit SOC1-like, SVP-like, and Arabidopsis SOC1, SVP, and AGL24. Black, very strong interaction; dark grey, strong interaction; light grey, weak interaction; white, no interaction. The summary is based on results presented in Supplementary Fig. S3 at JXB online.

Fig. 7.

Constitutive expression of kiwifruit SOC1-like genes in transgenic kiwifruit Actinidia chinensis. (A) Relative expression of kiwifruit SOC1-like transgenes in A. chinensis transgenic lines. (B) Examples of A. chinensis single-node cuttings at the indicated stages. (C) Percentage of cuttings at the budbreak stage. (D) Average budbreak time of individual transgenic lines. Error bars represent SEs for three cuttings.