Antagonistic action of strigolactone and cytokinin in bud outgrowth control

Abstract

Cytokinin (CK) has long been implicated as a promoter of bud outgrowth in plants, but exactly how this is achieved in coordination with other plant hormones is unclear. The recent discovery of strigolactones (SLs) as the long-sought branch-inhibiting hormone allowed us to test how CK and SL coordinately regulate bud outgrowth in pea (Pisum sativum). We found that SL-deficient plants are more sensitive to stimulation of bud growth by low concentrations of locally applied CK than wild-type plants. Furthermore, in contrast with SL mutant plants, buds of wild-type plants are almost completely resistant to stimulation by CK supplied to the vasculature. Regardless of whether the exogenous hormones were supplied locally or to the xylem stream, SL and CK acted antagonistically on bud outgrowth. These data suggest that SLs do not affect the delivery of CK to axillary buds and vice versa. Rather, these data combined with dose-response experiments suggest that SLs and CK can act directly in buds to control their outgrowth. These hormones may converge at a common point in the bud outgrowth regulatory pathway. The expression of pea BRANCHED1, a TCP transcription factor expressed strongly in buds and thought to act downstream of SLs in shoot branching, is regulated by CK and SL without a requirement for protein synthesis and in a manner that correlates with observed bud growth responses.

Figure 1.

SL-deficient plants are more responsive to CK than wild-type (WT) plants. The synthetic CK, BA, was supplied to the stem vascular stream below node 3 (A) or applied directly to the bud at node 3 (B) of 9-d-old wild-type and rms1-2 (Torsdag) pea plants. Bud length at node 3 was measured 7 d after treatment. Data are means ± se (n = 7–12).

Figure 2.

SL reduces the stimulatory effect of BA on bud outgrowth in an RMS4-dependent manner. The synthetic SL, GR24 (1 μm), and/or BA (50 μm) were supplied to the vasculature below node 3 (A) or applied directly to the bud at node 3 (B) of 10-d-old wild-type (WT), rms1-2, or rms4-1 (Torsdag) pea plants. Bud growth at node 3 was measured 7 d after treatment. Data are means ± se (n = 7–12).

Figure 3.

SL reduces the stimulatory effect of CK on bud outgrowth in SL-deficient plants in a dose-dependent manner. GR24 and/or BA were supplied to the vasculature below node 3 of 9-d-old (A) and 10-d-old (B) rms1-2 (Torsdag) pea plants. Bud length at node 3 was measured 7 d after treatment. Data are means ± se (n = 14 [A] and 9–12 [B]).

Figure 4.

SL reduces CK-induced outgrowth whether supplied locally or to the vasculature of SL-deficient plants. A, Zero or 1 μm GR24 was supplied to the vasculature below node 3, while 0 or 10 μm BA was supplied directly to the bud at node 3, of 10-d-old rms1-2 (Torsdag) pea plants. B, Zero or 1 μm GR24 was supplied to the bud at node 3, while 0 or 10 μm BA was supplied to the vasculature below node 3, of 10-d-old rms1-2 (Torsdag) pea plants. Bud length at node 3 was measured 7 d after treatment. Data are means ± se (n = 14).

Figure 5.

SL and CK act antagonistically on the same target gene, PsBRC1. The bud at node 3 of 8-d-old wild-type (WT), rms1-2, or rms4-1 (Torsdag) pea plants was treated for 6 h with or without GR24 (1 μm) and/or BA (50 μm). Expression of PsBRC1 in the bud at node 3 is represented relative to the wild-type control; EF1α was used as the internal reference gene. Data are means ± se (n = 3 pools of 30 plants).

Figure 6.

SL and CK regulate PsBRC1 without the need for de novo protein synthesis. The bud at node 3 of 8-d-old wild-type (WT; A) and rms1-2 (Torsdag; B) pea plants was treated for 6 h with or without GR24 (1 μm), BA (50 μm), and/or CHX (10 μm). Expression of PsBRC1 in the bud at node 3 is represented relative to controls; EF1α was used as the internal reference gene. Data are means ± se (n = 3 pools of 30 plants) and are from the same experiment as in Figure 5.

Figure 7.

CK supplied locally or from a distance decreases the expression of PsBRC1 and bud dormancy markers 24 h after treatment. Zero or 50 μm BA was supplied to the vasculature below node 3 or applied directly to the bud at node 3 of 9-d-old wild-type Torsdag pea plants. Expression of PsBRC1, PsDRM1, PsDRM2, and PsAD1 in the bud at node 3 24 h after treatment is represented relative to the vascular supply control treatment; EF1α was used as the internal reference gene. Data are means ± se (n = 3 pools of 28–30 plants).

Figure 8.

PsIPT1 but not PsIPT2 expression is increased in SL mutant stem tissue. Internode 3 and node 3 (including bud) were harvested from 12-d-old (four to five leaves expanded) wild-type (WT), rms4-1, and rms5-3 (Torsdag) pea plants. Expression is represented relative to the wild type; 18S was used as the internal reference gene. Data are means ± se (n = 7–8).

Figure 9.

GR24 does not affect PsIPT1 or PsIPT2 expression in isolated wild-type stem tissue segments within 4 h. Twelve-millimeter segments from internode 4 of 12-d-old wild-type Torsdag pea plants were incubated for 4 h with 0 or 1 μm GR24 or with 0 or 10 μm IAA; equivalent internode tissue was also harvested from intact plants as another control (Intact). Expression is represented relative to the incubated segment control; 18S was used as the internal reference gene. Data are means ± se (n = 3 pools of seven plants).