BRANCHED1 interacts with FLOWERING LOCUS T to repress the floral transition of the axillary meristems in Arabidopsis

Abstract

Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.

Figure 1.

Protein Interaction between BRC1 and FT. (A) Yeast two-hybrid analysis of protein interaction between BRC1, BRC2, or GRF3 and FT, TSF, or TFL1. Clones containing each combination of bait and prey vectors were grown on nonselective medium (+His) or on selective medium (−His). For the clones containing BRC1, 3-amino-1,2,4-triazole (3-AT) was added to the medium (left two rows). pCUY and pGAD424 empty vectors were used as a negative control (BD [binding domain] and AD [activation domain], respectively). (B) BiFC analysis of the interaction between BRC1 and FT in N. benthamiana leaf epidermal cells. The N-terminal fragment of EYFP was fused to BRC1 (YN-BRC1) and the C-terminal fragment of EYFP to FT (FT-YC). The fusion proteins were transiently expressed under the cauliflower mosaic virus 35S promoter through agroinfiltration. EYFP fluorescence (top panels) and EYFP fluorescence merged with a bright-field (BF) image (bottom panels) are shown. Combinations in which one of the fusion constructs was replaced with a fragment of EYFP (YN or YC) were used as negative controls (central and right columns). Arrowheads indicate complemented EYFP fluorescence in the nucleus. Bar = 200 μm. (C) In vitro pull-down assay demonstrating the interaction between BRC1 and FT. Closed arrowhead, open arrowhead, and arrow indicate T7-FT, GST-BRC1, and GST, respectively. (D) Yeast two-hybrid assay of protein interaction between FT and truncated versions of BRC1. TCP, TCP domain; R, R domain; a.a., amino acids.

Figure 2.

14-3-3 Proteins Do Not Mediate the Interaction between FT and BRC1. (A) Yeast two-hybrid analysis of interaction between BRC1 and mutant FT proteins defective in interaction with 14-3-3 proteins. T66I, P94L, G102D, and P75L are mutant FT proteins, the former three of which do not interact with 14-3-3 proteins. S390A is a mutated BRC1 in which a Ser residue in a putative 14-3-3 recognition site (S390) was substituted by Ala. GRF3 was chosen as a representative 14-3-3 protein. Yeasts were grown on nonselective medium (+His) or on selective medium (−His and −His+3-AT). WT, the wild type. (B) Schematic diagram of BRC1 protein depicting the putative 14-3-3 recognition site. TCP, TCP domain; R, R domain; a.a., amino acids. (C) Yeast two-hybrid assay of interaction between mutant FT proteins and BRC1 or GRF3. Representative results for seven FT mutants defective in interaction with the BRC1 protein and a mutant defective in interaction with GRF3 were shown. These mutants were obtained by screening of Ala-scanned FT mutants (see Supplemental Figure 3 online). Yeasts were grown on nonselective medium (+His) or on selective medium (−His and −His+3-AT). (D) Amino acid residues essential for interaction with BRC1 ([C], indicated by magenta) or 14-3-3 proteins ([A], indicated by yellow; Taoka et al., 2011) on a ribbon model of the FT protein.

Figure 3.

FT Expression Pattern and Movement of FT Protein into Axillary Buds. (A) to (D) FT expression patterns monitored by GUS staining of gFT:GUS plant. Whole-mount preparations of a rosette leaf (A) and the main stem of a plant with the axillary bud subtended by a cauline leaf (B). Resin sections of the axillary buds subtended by a cauline leaf (C) and by a rosette leaf (D). Arrowhead in (B) indicates staining in the peripheral region of the cauline leaf. (E) and (F) Movement of FT-EGFP into an axillary bud from the subtending leaf. EGFP fluorescence merged with a bright-field image of the axillary bud is shown. FT-EGFP expression was induced from the ProHSP18.2:FT-EGFP transgene by heat treatment of the leaf blade of a rosette leaf (see Supplemental Figure 5 online). EGFP fluorescence was observed 24 h after the end of the heat treatment. The treated leaf blade was either left intact (E) or cut off immediately after the heat treatment to prevent FT-EGFP export (F). The cut site is not seen in (F). ap, apical meristem; ax, axillary meristem; cl, cauline leaf; fl, floral bud; pt, petiole of the heat-treated rosette leaf; rl, rosette leaf; st, stem. Bar in (A) = 1 mm and bars in other panels = 100 μm.

Figure 4.

Early Floral Transition of the Axillary Shoots in brc1 Mutants. (A) Axillary shoot phenotype of the wild type (Col) and brc1-2 mutant. Successive axillary shoots with the subtending leaf of 44-d-old plants are shown. Arrowheads and arrows indicate vegetative nodes and the first flowers in the most apical branches. Plus numbers represent cauline-leaf axils in the acropetal direction, and minus numbers represent rosette-leaf axils in the basipetal direction as shown in the right diagram. In the diagram, open circles, arrows, and bars represent flowers, shoots, and leaves, respectively. (B) Developmental stages of the buds in the axils of cotyledons (c1 and c2) and rosette leaves (L1 to L15) of 10 plants of wild-type Col (left) and brc1-2 (right). Developmental stages were defined according to Aguilar-Martínez et al. (2007). Gray indicates absence of the leaf node such that the position is occupied by a flower. Observations were made on the days when the first floral buds became visible (before bolting), the main shoot became 1-cm long (1 cm bolting), and the main shoot became 5-cm long (5 cm bolting). (C) Number of leaves formed on axillary shoots at the five axil positions of the wild type (Col) and brc1-2 mutant grown in LD. The axil position is defined in (A). Leaves in the basal part of axillary shoots without internodes are classified as axillary rosette leaves (ARL) and those with elongated internodes as cauline leaves (CL). Error bars indicate the sd (n = 9). The differences significant between Col and brc1-2 in two-tailed multiple t test with Bonferroni correction (P < 0.05) are indicated with asterisks. (D) and (E) Expression of AP1 in the axillary buds monitored by ProAP1:GUS. Number of GUS-positive axillary buds in ProAP1:GUS plants in wild-type (Col) and brc1-2 background grown under LD (D). Error bars indicate the sd (n ≥ 21). The differences significant in two-tailed multiple t test with Bonferroni correction (P < 0.05) with respect to the wild type (Col) are indicated with asterisks. Representative 22-d-old ProAP1:GUS plants in wild-type (Col) and brc1-2 backgrounds (E). Arrowheads indicate GUS-positive axillary buds at rosette-leaf axils.

Figure 5.

Delayed Floral Transition of Axillary Shoots in ft and ft tsf Mutants and Genetic Interaction with brc1. (A) Number of leaves formed on the axillary shoots at the five axil positions of the wild type (Col) and brc1-2, ft-2, and brc1-2 ft-2 mutants grown in LD. Error bars indicate the sd (n = 12 for Col and brc1-2; n = 24 for ft-2 and brc1-2 ft-2). The differences significant between ft-2 and brc1-2 ft-2 in two-tailed multiple t test with Bonferroni correction (P < 0.05) are indicated with asterisks. ARL, axillary rosette leaves; CL, cauline leaves. (B) Representative plants of ft-2 and brc1-2 ft-2 mutants grown in LD for 60 d Arrows indicate elongated axillary shoots at rosette axils. (C) Number of leaves formed on the axillary shoots at the three axil positions of the wild type (Col) and brc1-2, ft-2 tsf-1, and brc1-2 ft-2 tsf-1 (triple) mutants grown in LD. Error bars indicate the sd (n = 12). The differences significant between ft-2 tsf-1 and brc1-2 ft-2 tsf-1 in two-tailed multiple t test with Bonferroni correction (P < 0.05) are indicated with asterisks. (D) Axillary shoot phenotype of ft-2 tsf-1 and brc1-2 ft-2 tsf-1 mutants. Top panel shows the rosette part of the plants, and bottom panels show the uppermost rosette leaf with the axillary bud or shoot. Note that most of the primary rosette leaves on the main axis were decaying or senescent. Arrowheads in the bottom panels indicate the senescent rosette leaves. Bars = 5 mm. Designation of the axil positions and classification of leaves in (A) and (C) are as in Figure 4. [See online article for color version of this figure.]

Figure 6.

brc1 and Genes Acting Downstream of Florigen. (A) Expression of FD in the AMs monitored by ProFD:GUS. Longitudinal (left) and transverse (right) sections of ProFD:GUS plants grown for 18 d under LD conditions are shown. Arrowheads indicate AMs at rosette axils. Dotted lines show the outline of the tissues. (B) Number of leaves formed on the axillary shoots at the five axil positions of the wild type (Col) and brc1-2, fd-1, and brc1-2 fd-1 mutants. Error bars indicate the sd (n = 15). The difference significant between fd-1 and brc1-2 fd-1 in two-tailed multiple t test with Bonferroni correction (P < 0.05) is indicated with an asterisk. ARL, axillary rosette leaves; CL, cauline leaves. (C) Relative expression levels of FT-downstream genes AP1, FUL, and SOC1 after the shift from SD to LD. Plants were grown for 24 SDs and transferred to LD. Rosette-axil samples were analyzed (see Methods). Error bars indicate the se for three biological replicates. Differences significant in two-tailed t test (P < 0.05) with respect to the wild type (Col) are indicated with asterisks. (D) Number of leaves formed on axillary shoots at the five axil positions of the wild type (Col) and brc1-2, soc1-2, and brc1-2 soc1-2 mutants. Error bars indicate the sd (n = 9). The differences significant between soc1-2 and brc1-2 soc1-2 in two-tailed multiple t test with Bonferroni correction (P < 0.05) are indicated with asterisks. Designation of the axil positions and classification of leaves in (B) and (D) are as in Figure 4.

Figure 7.

Ectopic Expression of BRC1 Delays Floral Transition in the SAM. (A) Thirty-day-old plants of wild type (Col) and two independent lines of ProFD:BRC1 (#1 and #2) grown in LD. (B) Flowering time measured by the number of rosette leaves (RL) and cauline leaves (CL). Error bars indicate the sd (n = 15). The differences significant in two-tailed Dunnett’s test (P < 0.01) in the total leaf numbers with respect to Col are indicated with asterisks. [See online article for color version of this figure.]