Fig 1.
Key shoot branching regulatory pathways and involvement of the branching genes used in this study of axillary bud outgrowth.
The left panel shows the shoot branching model from what is known in other species, while the right panel shows the branching genes that have been described in chrysanthemum with the genes that were isolated in this study underlined. ① indicates the bud outgrowth regulation that follows the auxin canalisation model where auxin export is required for an axillary bud to grow out. ② indicates the regulation of bud outgrowth according to the second messenger model where auxin indirectly regulates bud outgrowth through strigolactone and cytokinin signalling.
Fig 2.
Branching phenotypes of the cut flower chrysanthemum C17 and C18.
C17 grows vegetatively in week 1 (V1) and starts floral transition in week 2 (T2) after which it shows generative growth and outgrowth of subapical axillary buds (G3-G5). C18 shows vegetative growth throughout week 1–5 (V1 to V5). Sampling zones A, B, and C are indicated in shades of green.
Table 1.
Different zones and the corresponding nodal position from which stem and axillary bud samples were harvested in Chrysanthemum C17 and C18.
For C17 in V1 nodal positions 37 to 31 (Zone A V1) were pooled together to represent inhibited axillary buds under apical dominance. Positions 26 to 16 in week 1 (Zone B V1) contain axillary buds that are further away from the shoot apex and show more outgrowth. Positions 13 to 8 (Zone C V1) represent bottom axillary buds inhibited by correlative inhibition of middle axillary buds. In T2 nodal positions 45 to 42 (Zone A T2) were pooled because the buds in this section showed the strongest outgrowth after T2 and release from apical dominance. Nodal positions 41 to 38 (Zone B T2) were pooled because these buds showed a diminishing outgrowth after week 2 when compared to the nodal positions above them. Nodal positions 37 to 31 (Zone C T2) were pooled to represent buds that show inhibited outgrowth after week 2. For C18 in V1 nodal positions 24–20 represent subapical inhibited buds (Zone A V1), positions 19–14 represent buds that are less inhibited by apical dominance (Zone B V1) and positions 13–8 represent buds furthest away from apical influence. In V2 positions 30–25 represent inhibited subapical buds (Zone A V2), positions 24–20 (Zone B’ V2) and 19–14 (Zone B” V2) represent less inhibited buds with zone B’ and B” the same positions as zone A and B in V1.
Fig 3.
Axillary bud lengths for chrysanthemum C17 in V1, T2 and G3 to G5.
Mean bud/shoot length (±SE; n = 10) is presented on the x-axis for every nodal position presented on the y-axis with 1 being the node closest to the shoot base. The dotted lines mark the nodal positions that were sampled in V1 and T2 according to Table 1.
Fig 4.
Axillary bud lengths for chrysanthemum C18 in V1 to V5.
Mean bud/shoot length (±SE; n = 10) is presented on the x-axis for every nodal position presented on the y-axis with 1 being the node closest to the shoot base. The dotted lines mark the nodal positions that were sampled in V1 and V2 according to Table 1.
Table 2.
Kruskal-Wallis tests comparing the hormone levels between the shoot apex and the different zones of the axillary buds and stems.
Data are fold changes (Zone X/Apex) and the significant difference between means by Kruskal-Wallis test is indicated by * (p-value<0.05).
Fig 5.
Hormone concentrations measured in two Chrysanthemum genotypes C17 and C18 at time point V1 and T2/ V2.
IAA and cytokinin (CK) content of the shoot apex and the axillary buds or stem in different zones are presented. Data are means ± SE (n = 3). * indicates significance at the 0.05 level between V1 and T2/V2.
Fig 6.
CK/IAA ratio for Chrysanthemum genotypes C17 and C18 at two time points V1 and T2/V2.
Total cytokinin/IAA of the shoot apex and the axillary buds or stem in different zones are presented. Data are mean cytokinin levels (n = 3) divided by mean auxin levels (n = 3) + calculated SE.
Table 3.
BlastX search of the isolated cDNA sequences with E-value, % identity and accession numbers.
Fig 7.
RT-qPCR gene expression analysis of bud development genes for Chrysanthemum genotypes C17 and C18 at time points V1 and T2/V2.
CNRQ for bud development related genes CmBRC1, CmDRM1, CmLsL and CmSTM in the shoot apex, axillary buds and stem samples. Data are means ± SE (n = 3) of non log-transformed CNRQ. Fold changes between V1 and T2/V2 are indicated above the grey bars with * indicating significance of the Kruskal Wallis test (p<0.05).
Fig 8.
RT-qPCR gene expression analysis of strigolactone genes for Chrysanthemum genotypes C17 and C18 at time points V1 and T2/V2.
CNRQ for strigolactone genes CmMAX1 and CmMAX2 in the shoot apex, axillary buds and stem samples. Data are means ± SE (n = 3) of non log-transformed CNRQ. Fold changes between V1 and T2/V2 are indicated above the grey bars with * indicating significance of the Kruskal Wallis test (p<0.05).
Fig 9.
RT-qPCR gene expression analysis of cytokinin genes for Chrysanthemum genotypes C17 and C18 at time points V1 and T2/V2.
CNRQ for cytokinin pathway genes CmIPT3, CmRR1, CmHK3a and CmHK3b in the shoot apex, axillary buds and stem samples. Data are means ± SE (n = 3) of non log-transformed CNRQ. Fold changes between V1 and T2/V2 are indicated above the grey bars with * indicating significance of the Kruskal Wallis test (p<0.05).
Fig 10.
RT-qPCR gene expression analysis of auxin transport genes for Chrysanthemum genotypes C17 and C18 at time points V1 and T2/V2.
CNRQ for auxin transport genes CmPIN1 and CmTIR3 in the shoot apex, axillary buds and stem samples. Data are means ± SE (n = 3) of non log-transformed CNRQ. Fold changes between V1 and T2/V2 are indicated above the grey bars with * indicating significance of the Kruskal Wallis test (p<0.05).
Fig 11.
RT-qPCR gene expression analysis of auxin signalling genes for Chrysanthemum genotypes C17 and C18 at time points V1 and T2/V2.
CNRQ for auxin perception genes CmTIR3, AXR1 and AXR6 in the shoot apex, axillary buds and stem samples. Data are means ± SE (n = 3) of non log-transformed CNRQ. Fold changes between V1 and T2/V2 are indicated above the grey bars with * indicating significance of the Kruskal Wallis test (p<0.05).
Fig 12.
RT-qPCR gene expression analysis of auxin response genes for Chrysanthemum genotypes C17 and C18 at time points V1 and T2/V2.
CNRQ for auxin response genes CmAXR2, CmIAA16 and, CmIAA12 in the shoot apex, axillary buds and stem samples. Data are means ± SE (n = 3) of non log-transformed CNRQ. Fold changes between V1 and T2/V2 are indicated above the grey bars with * indicating significance of the Kruskal Wallis test (p<0.05).