Strigolactones stimulate internode elongation independently of gibberellins

Abstract

Strigolactone (SL) mutants in diverse species show reduced stature in addition to their extensive branching. Here, we show that this dwarfism in pea (Pisum sativum) is not attributable to the strong branching of the mutants. The continuous supply of the synthetic SL GR24 via the root system using hydroponics can restore internode length of the SL-deficient rms1 mutant but not of the SL-response rms4 mutant, indicating that SLs stimulate internode elongation via RMS4. Cytological analysis of internode epidermal cells indicates that SLs control cell number but not cell length, suggesting that SL may affect stem elongation by stimulating cell division. Consequently, SLs can repress (in axillary buds) or promote (in the stem) cell division in a tissue-dependent manner. Because gibberellins (GAs) increase internode length by affecting both cell division and cell length, we tested if SLs stimulate internode elongation by affecting GA metabolism or signaling. Genetic analyses using SL-deficient and GA-deficient or DELLA-deficient double mutants, together with molecular and physiological approaches, suggest that SLs act independently from GAs to stimulate internode elongation.

Figure 1.

Reduced height of the rms SL mutants is not caused by their strong shoot branching. A, Phenotypes of wild-type (WT; cv Térèse) and rms1-10 plants, intact or with axillary buds removed. B, Internode lengths between node 3 and node 11 were measured when plants were 30 d old. Axillary buds were manually removed every 2 or 3 d. Data are means ± se (n = 7–8). Asterisks denote significant differences from the wild type (***P < 0.001, Student’s t test). [See online article for color version of this figure.]

Figure 2.

Inhibiting bud growth by directly treating buds with GR24 does not restore reduced rms1 plant height. Axillary buds of wild-type (WT) or rms1-1 plants (cv Parvus) were either left untreated (nontreated) or treated with 0 or 1 μm GR24 when the leaf was first open and retreated the following day. Bud lengths (Dun et al., 2013) and plant height were measured when the plants were 35 d old. Data are means ± se (n = 14–16). The asterisk denotes a significant difference from the rms1 0 μm GR24-treated plant (*P < 0.05, Student’s t test).

Figure 3.

SL supplied via hydroponics increases the internode length of rms1 SL-deficient mutant plants but not of rms4 SL-response mutant plants. Six-day-old wild-type (WT), rms1-10, and rms4-3 plants (cv Térèse) were supplied with 0 or 3 μm GR24 via hydroponics for 16 d, after which the sum of all lateral buds and branches at nodes 1 to 5 (A) and the internode length between nodes 1 and 7 (B) were measured. Data are means ± se (n = 7–8). Asterisks denote significant effects of GR24-treated versus control-treated plants (*P < 0.05, Student’s t test).

Figure 4.

SL treatment to the shoot tip does not affect the growth of the main stem. The main shoot apex of 7-d-old rms1-11 plants (cv Térèse) with 2.5 expanded leaves was treated on two consecutive days with 0 or 10 μm GR24 or 1.4 mm GA₃. Internode lengths were measured 23 d after treatment. Data are means ± se (n = 7–10). Asterisks denote significant differences from the control (**P < 0.01, ***P < 0.001, Student’s t test).

Figure 5.

The effect of the rms1-10 and le mutations on both internode length and branching is additive. A, Fifteen-day-old wild-type (WT), rms1, le, and le rms1 plants. White arrows indicate the basal branching. B, Internode length between nodes 1 and 9. Data are means ± se (n = 18). Values with different lowercase letters are significantly different from one another (ANOVA, P < 0.01). C, Ratio of total branch length to total stem length (n = 18). Values with different lowercase letters are significantly different from one another (ANOVA, P < 0.001). D, Number of buds or branches from nodes 1 to 5 with a length greater than 1.5 mm (n = 18). Values with different lowercase letters are significantly different from one another (ANOVA, P < 0.001). [See online article for color version of this figure.]

Figure 6.

SLs regulate internode elongation by affecting cell number. A, Epidermal cells of internode 4 of the wild type (WT; top left), rms1 (top right), le (bottom left), and le rms1 (bottom right) in scanning electronic microscopy. B, Epidermal cell length (µm) in internode 4 of the wild type, rms1, le, and le rms1. Data are means ± se (n = 7–8 plants with about 100 cells per plant). Values with different lowercase letters are significantly different from one another (ANOVA, P < 0.05). C, Internode 4 length (mm) of the wild type, rms1, le, and le rms1. Values with different lowercase letters are significantly different from one another (ANOVA, P < 0.01). D, Estimation of cell number in internode 4 of the wild type, rms1, le, and le rms. Values with different lowercase letters are significantly different from one another (ANOVA, P < 0.001).

Figure 7.

GA₁ levels in expanding stem tissue of wild-type (WT; Torsdag), rms1-2, and rms4-1 15-d-old plants. Data are means ± se (n = 3). The asterisk denotes a significant difference from wild-type plants (*P < 0.05, Student’s t test).

Figure 8.

SLs control internode elongation in a della background. Internode length between nodes 1 and 8 were measured in the della line HL178 (la cry-s) and in F3 della families fixed for la cry-s and fixed for RMS1 (1–5) or rms1 (9 and 10) or segregating for RMS1/rms1 (6–8). For families 6 to 8, the gray bars correspond to a mixture of RMS1/RMS1 and RMS1/rms1 plants (n = 11–28 except for rms1 F3 6–8, for which n = 6–8). Asterisks denote significant differences from corresponding rms1 plants (**P < 0.01, ***P < 0.001, Student’s t test).

Figure 9.

GA₃ but not GR24 treatment affects the expression of the GFP-RGA protein in root apex of Arabidopsis. Roots of transgenic plants (rga/ga1-3 background) expressing the pRGA::GFP-RGA fusion were observed using confocal laser microscopy. Shown are three-dimensional projections of the fluorescence images of root tips treated with control solution (left column), 100 µm GA₃ (middle column), or 10 µm GR24 (right column) at different times as indicated.

Figure 10.

SL-related mutants respond to GA₃. Length of internode 4 of wild-type (diamonds), rms1 (squares), and rms4 (triangles) plants after treatment with various quantities of GA₃ (µg plant⁻¹). GA₃ was applied on stipules at node 3 when the plants were 8 d old. Measurements were made 8 d after treatment. Data are means ± se (n = 8–12). Values with different lowercase letters are significantly different from one another (ANOVA, P < 0.05).

Figure 11.

Model integrating the role of auxin (IAA), BR, GA, and SL on internode elongation in pea. Arrows indicate activation, and bars indicate repression. IAA and BR promote internode elongation by activating cell elongation; GA promotes internode elongation by activating both cell elongation and cell division; SL promotes internode elongation by cell division only independently from GA, via RMS4 and a transcription factor that, very likely, is not PsBRC1. In axillary buds, SL inhibits cell division via RMS4 and PsBRC1.