The pea TCP transcription factor PsBRC1 acts downstream of Strigolactones to control shoot branching

Abstract

The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated. The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application. The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes. The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine. The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth. However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth. In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant. Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1. This phenotype indicates a localized regulation of branching at these nodes specific to pea.

Figure 1.

Structure of the PsBRC1 gene and phenotype of the corresponding mutant. A, Gene structure of PsBRC1 and locations of mutations. Bases are numbered from the start codon. The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below. Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence. B, Comparison of a wild-type (WT) Térèse plant (left) with rms4 (middle left), wild-type Caméor (middle right), and Psbrc1^Cam (right). C, Branch length at each node of wild-type Caméor, Psbrc1^Cam, wild-type Térèse, rms1, and Psbrc1^Te. Data are means ± se (n = 12). D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1^Cam × Térèse). n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch. Data are means ± se (n = 12).

Figure 2.

A, Effects of GR24 application on bud growth. Bud length at node 3 of wild-type Térèse (black bars), rms1 (white bars), wild-type Caméor (gray bars), and Psbrc1^Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nm GR24. Data are means ± se (n = 12). B, SL levels in root exudates of wild-type and Psbrc1 plants. Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d₁-fabacyl acetate internal standard. Data are means ± se, based on analyses of two independent pools of 12 plants for each genotype.

Figure 3.

Effects of GR24 on PsBRC1 transcript levels. PsBRC1 transcript levels were determined relative to EF1α in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars). RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR. The data are representative of two to three independent experiments. A, Six and 24 h after GR24 application in wild-type Térèse, rms1, rms2, and rms4 plants. The branching phenotype at node 4 after GR24 application is given below for each genotype. B, Six and 48 h after GR24 application in wild-type Térèse, rms1, rms4, and Psbrc1^Té plants. Data are means ± se (n = 3).

Figure 4.

Effects of BAP on PsBRC1 transcript levels and on bud growth. A, PsBRC1 transcript levels were determined relative to EF1α in axillary buds at node 4 after BAP (50 μm) application in wild-type Caméor, Psbrc1^Cam, wild-type Térèse, rms1, and rms4. RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR. The data are representative of three independent experiments. B, Effects of BAP (50 μm) treatment on bud growth at node 4 in wild-type Térèse, rms4, wild-type Caméor, and Psbrc1^Cam. Measurements were done 5 d after treatment. Data are means ± se (n = 12). C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1^Cam, wild-type Caméor, rms1, and wild-type Térèse were used. Data are means ± se (n = 8).

Figure 5.

The RMS2-dependent feedback signal is not activated in Psbrc1. A, RMS1 transcript levels were determined in epicotyls of rms1, rms2, rms4, and their corresponding wild-type Térèse as well as Psbrc1^Cam and its corresponding wild-type Caméor. RNA was extracted from plants at stage 6. The data are representative of three independent experiments. B and C, CK contents of root xylem sap. tZ, trans-Zeatin; DZ, dihydrozeatin; tZR, trans-zeatin riboside; DZR, dihydrozeatin riboside; cZR, cis-zeatin riboside; IP, isopentenyl adenine; IPR, isopentenyl adenosine. B, Wild-type (WT) Caméor and the Psbrc1^Cam mutant. C, Wild-type Térèse, M3T-884 (rms1), Psbrc1^Té, and F3 plants with wild-type, rms1, Psbrc1, and rms1 Psbrc1 genotypes derived from four F2 (M3T-884 × F2 [Térèse × Psbrc1^Cam]) populations (see “Materials and Methods”). Measurements were made from pools of 3 mL of sap harvested from 20 to 40 plants. Data are means ± se (n = 3).

Figure 6.

Model for the hormonal control of branching in pea integrating the function of PsBRC1 in the axillary bud and the auxin transport canalization-based model (Domagalska and Leyser, 2011). PsBRC1 integrates the SL and CK pathways to control bud outgrowth. CK also increases bud growth via a PsBRC1-independent pathway. Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels. The RMS2-dependent feedback, which up-regulates SL biosynthesis and down-regulates xylem CK, is activated when there is a lack of SL signaling via RMS4 and may be independent of PsBRC1. SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds.