Recent studies using highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones, a group of terpenoid lactones, act as a new hormone class, or its biosynthetic precursors, in inhibiting shoot branching. Here, we provide evidence that DWARF14 (D14) inhibits rice tillering and may act as a new compo-nent of the strigolactone-dependent branching inhibition pathway. The d14 mutant exhibits increased shoot branch-ing with reduced plant height like the previously characterized strigolactone-deficient and -insensitive mutants d10 and d3, respectively. The d10-1 d14-1 double mutant is phenotypically indistinguishable from the d10-1 and d14-1 single mutants, consistent with the idea that D10 and D14 function in the same pathway. However, unlike with d10, the d14 branching phenotype could not be rescued by exogenous strigolactones. In addition, the d14 mutant contained a higher level of 2′-epi-5-deoxystrigol than the wild type. Positional cloning revealed that D14 encodes a protein of the α/β-fold hydrolase superfamily, some members of which play a role in metabolism or signaling of plant hormones. We propose that D14 functions downstream of strigolactone synthesis, as a component of hormone signaling or as an enzyme that participates in the conversion of strigolactones to the bioactive form.

Significance: Strigolactones (SLs) were initially characterized as root-derived signals for parasitic and symbiotic interactions with root parasitic plants and arbuscular mycorrhizal fungi, respectively. SLs were later shown to act as endogenous hormones that regulate shoot branching. Carlactone (CL) was identified as a product of three SL biosynthetic enzymes in vitro, and therefore a putative biosynthetic precursor for SLs. However, it was neither detected from plant tissues, nor was the conversion of CL to SL demonstrated in vivo. In this paper, we show that¹³C-labeled CL is converted to SLs in vivo, and that endogenous CL is successfully identified from rice andArabidopsis. These results demonstrate that CL is a true biosynthetic precursor for SLs.

Strigolactones (SLs) or SL-derived metabolite(s) have recently been shown to act as endogenous inhibitors of axillary bud outgrowth. SLs released from roots induce hyphal branching of arbuscular mycorrhizal (AM) fungi that facilitate the uptake of inorganic nutrients, such as phosphate (Pi) and nitrate, by the host plants. Previous studies have shown that SL levels in root exudates are highly elevated by Pi starvation, which might contribute to successful symbiosis with AM fungi in the rhizosphere. However, how endogenous SL levels elevated by Pi starvation contribute to its hormonal action has been unknown. Here, we show that tiller bud outgrowth in wild-type rice seedlings is inhibited, while root 2′-epi-5-deoxystrigol (epi-5DS) levels are elevated, in response to decreasing Pi concentrations in the media. However, the suppression of tiller bud outgrowth under Pi deficiency does not occur in the SL-deficient and -insensitive mutants. We also show that the responsiveness to exogenous SL is slightly increased by Pi deficiency. When Pi-starved seedlings are transferred to Pi-sufficient media, tiller bud outgrowth is induced following a decrease in root epi-5DS levels. Taken together, these results suggest that elevated SL levels by Pi starvation contribute to the inhibition of tiller bud outgrowth in rice seedlings. We speculate that SL plays a dual role in the adaptation to Pi deficiency; one as a rhizosphere signal to maximize AM fungi symbiosis for improved Pi acquisition and the other as an endogenous hormone or its biosynthetic precursor to optimize shoot branching for efficient Pi utilization.

Recent studies of highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones (SLs) act as hormones that inhibit shoot branching. The identification of genes that work downstream of SLs is required for a better understanding of how SLs control the growth of axillary buds. We found that the increased tillering phenotype of fine culm1 (fc1) mutants of rice is not rescued by the application of 1 μM GR24, a synthetic SL analog. Treatment with a high concentration of GR24 (10 μM) causes suppression of tiller growth in wild-type plants, but is not effective on fc1 mutants, implying that proper FC1 functioning is required for SLs to inhibit bud growth. Overexpression of FC1 partially rescued d3-2 defects in the tiller growth and plant height. An in situ hybridization analysis showed that FC1 mRNA accumulates in axillary buds, the shoot apical meristem, young leaves, vascular tissues and the tips of crown roots. FC1 mRNA expression was not significantly affected by GR24, suggesting that transcriptional induction may not be the mechanism by which SLs affect FC1 functioning. On the other hand, the expression level of FC1 is negatively regulated by cytokinin treatment. We propose that FC1 acts as an integrator of multiple signaling pathways and is essential to the fine-tuning of shoot branching in rice.

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/β-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14^D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.