Strigolactone regulation of shoot branching in chrysanthemum (Dendranthema grandiflorum)

Abstract

Previous studies of highly branched mutants in pea (rms1-rms5), Arabidopsis thaliana (max1-max4), petunia (dad1-dad3), and rice (d3, d10, htd1/d17, d14, d27) identified strigolactones or their derivates (SLs), as shoot branching inhibitors. This recent discovery offers the possibility of using SLs to regulate branching commercially, for example, in chrysanthemum, an important cut flower crop. To investigate this option, SL physiology and molecular biology were studied in chrysanthemum (Dendranthema grandiflorum), focusing on the CCD8/MAX4/DAD1/RMS1/D10 gene. Our results suggest that, as has been proposed for Arabidopsis, the ability of SLs to inhibit bud activity depends on the presence of a competing auxin source. The chrysanthemum SL biosynthesis gene, CCD8 was cloned, and found to be regulated in a similar, but not identical way to known CCD8s. Expression analyses revealed that DgCCD8 is predominantly expressed in roots and stems, and is up-regulated by exogenous auxin. Exogenous SL can down-regulate DgCCD8 expression, but this effect can be overridden by apical auxin application. This study provides evidence that SLs are promising candidates to alter the shoot branching habit of chrysanthemum.

Fig. 1.

Effect of GR24 and NAA on bud outgrowth on single-node isolated stem segments in chrysanthemum. One-node stem segments were excised from chrysanthemum plantlets and inserted between two agar blocks. The apical agar blocks contained either 5 μM NAA or 0 μM NAA (with an equal volume of ethanol as a control). The basal agar blocks contained either 5 μM GR24, or 0 μM GR24 (with an equal volume of acetone as a control). Bud lengths were measured every 24 h and the mean lengths are presented. Error bars represent the standard error of the means, n=19–20. The data presented are typical of three independent experiments.

Fig. 2.

Effect of GR24 and NAA on bud outgrowth on two-node isolated stem segments in chrysanthemum. Two-node stem segments were excised from chrysanthemum plantlets and inserted between two agar blocks. The apical agar blocks contained either 5 μM NAA or 0 μM NAA (with an equal volume of ethanol as a control). The basal agar blocks contained either 5 μM GR24, or 0 μM GR24 (with an equal volume of acetone as a control). The length of the top bud (A) or bottom bud (B) was measured every 24 h and the mean lengths are presented. The arrows indicate the axil for which bud length was measured. Error bars represent the standard error of the mean, n=23–24. The data presented are typical of three independent experiments.

Fig. 3.

Isolation of DgCCD8. (A) Alignment of the predicted amino acid sequences of DgCCD8 compared with Arabidopsis (MAX4), pea (RMS1), petunia (DAD1), and rice (D10). Intron positions corresponding to the genomic DNA sequence are denoted by triangles. (B) Phylogenetic analysis. Maximum likelihood phylogeny of CCD8 orthologues reconstructed using RAxML (Stamatakis, 2006) under the Dayhoff substitution matrix. Node support values were estimated from 100 rapid bootstrap resamplings (Stamatakis et al., 2008). Proteins are labelled with a prefix that represents the species origin of the sequence: Arabidopsis thaliana AtCCD8 (At4g32810.1); Brachypodium distachyon BdCCD8 (Bd2g49670.1); Dendranthema grandiflorum DgCCD8a, DgCCD8b, and DgCCD8c; Medicago truncatula MtCCD8 (CR9563923.4); Oryza sativa OsCCD8a (Os01g38580.1), OsCCD8b (Os01g54270.1); Pisum sativum PsCCD8/RMS1 (AAS66906.1); Petunia hybrida PhCCD8/DAD1 (AAW33596.1); Populus trichocarpa PtCCD8a (eugene 300061708), PtCCD8b (gw1.XV111.1171.1); Physcomitrella patens PpCCD8 (e gw 1.14.69.1); Sorghum bicolor SbCCD8a (Sb05g00950), SbCCD8b (Sb03g034400); Selaginella moellendorffi SmCCD8 (egw1.86.30.1); Vitis vinifera VvCCD8 (GSVIVT0003 2423001); Zea mays ZmCCD8 (Zm2g147254).

Fig. 4.

Complementation of Arabidopsis max4-1 mutant phenotype with DgCCD8. (A) Comparison of phenotypes of wild type, max4-1, and max4-1 transformed with the 35S::DgCCD8a and 35S::DgCCD8b constructs. (B) The number of secondary rosette branches produced by WT, max4-1, and the three most-strongly rescued independent homozygous lines transformed carrying either 35S::DgCCD8a or 35S::DgCCD8b. Branching was assessed using a decapitation assay. The mean number of rosette branches with a length of at least 2 cm 10 d after decapitation is shown (error bars =SEM, n=20). The data presented are typical of two independent experiments. (C) Analysis of DgCCD8 expression for the experiment presented in (B). Transcripts were assayed by reverse transcriptase PCR from total RNA from rosette leaves. One leaf from each of the 20 plants in each sample was collected and pooled after branching had been assessed. Detection of the UBQ transcript was used as a cDNA normalization control. The data presented are typical of two independent experiments. (This figure is available in colour at JXB online.)

Fig. 5.

RT-PCR analysis of total DgCCD8 (DgCCD8t) and DgCCD8b expression in chrysanthemum. Total RNA was extracted from R, root; ST, shoot tips; L, leaf; and S, stem, with tissue samples taken from pools of six in vitro-grown plantlets. Detection of rRNA was used as a normalization control. The data presented are typical of three independent experiments.

Fig. 6.

DgCCD8 transcript response to decapitation, auxin, and GR24. Top panel, DgCCD8 gene expression in the three basal internodes (1I–3I as represented by the lines in the scheme on the right). Pools of six plantlets were sampled either intact (left two lanes) or 6 h after decapitation (right six lanes). Plantlets were treated with 5 μM NAA (NAA) or 0 μM NAA [with an equal volume of ethanol as a control (Eth)] supplied to the decapitated stump, and/or with 5 μM GR24 (GR24) or 0 μM GR24 [with an equal volume of acetone as a control (Ace)] to the basal node. Bottom panel, rRNA gene expression was used as a cDNA normalization control. The data presented are typical of three independent experiments.