Strigolactones (SLs) are a group of carotenoid-derived phytohormones that were first identified as seed germination stimulants of root parasitic plants, such as Striga and Orobanche (Cook et al. 1966). SLs have also been implicated in other processes, including the stimulation of hyphal branching in arbuscular mycorrhizal fungi (Akiyama and Hayashi 2006), the regulation of leaf morphology (Scaffidi et al. 2013) and root architecture (Koltai 2011), and the inhibition of bud outgrowth to reduce shoot branching (Gomez-Roldan et al. 2008). Studies into the effects of SLs have isolated a series of enhanced shoot branching, and have identified dwarfing mutants including ramosus (rms) of pea, more axillary growth (max) of Arabidopsis, and dwarf (d) mutants of rice (Wang et al. 2018); SL biosynthesis and signal transduction were identified in these mutants. The primary step of SL biosynthesis involves the conversion of all-trans-β-carotene into 9-cis-β-carotene by the isomerase D27 in rice (Alder et al. 2012). Sequential actions of carotinoid cleavage deoxygenase 7 (CCD7) and CCD8, as encoded by DWARF17 (D17) and D10, lead to the formation of carlactone, which is the precursor for SLs (Arite et al. 2007; Umehara et al. 2008). After several further types of catalysis, carlactone is converted to SLs (Al-Babili and Bouwmeester 2015; Zhang et al. 2014). The SL receptor DWARF14 (D14) was initially established from an SL-insensitive rice mutant, d14, which showed dwarf phenotypes and increased shoot branching (Arite et al. 2009). As an α/β-fold hydrolase, D14 is also responsible for both the perception and deactivation of bioactive SLs (Seto et al. 2019). In rice, the complex for the perception of SLs also includes the negative regulators D53 and suppressor of max 2 1-like (SMXL) 6/7/8 in Arabidopsis and the F-box proteins D3 and more axillary growth 2 (MAX2) (Jiang et al. 2013; Mach 2015; Wang et al. 2015; Zhou et al. 2013). SL-responsive genes begin to play their roles after the ubiquitination and subsequent degradation of D53 through the 26s proteasome pathway. In addition to the feedback regulation of D53 and the inhibitor of bud outgrowth BRANCHED1 (BRC1) (Dun et al. 2012), cytokinin oxidase/dehydrogenase 9 (OsCKX9) and the type-A response regulator OsRR5 have been identified as the primary and secondary SL-responsive genes, respectively (Duan et al. 2019).
To date, several artificial SL analogs have been chemically synthesized based on the structures of natural SLs identified from root exudates; rac-GR24 is the most widely used SL analog. SLs have been identified as a member of the terpenoids, which consist of a tricyclic lactone (ABC ring) and a hydroxymethyl butanolide (D-ring) that are connected via an enol ether bond. Studies into SL bioactivity regarding the induction of root parasitic seed germination have revealed that the C-D-ring may be important for biological activity and that modifying the D-ring reduces SL bioactivity (Mangnus et al. 1992; Mangnus and Zwanenburg 1992). Furthermore, 5-(4-chlorophenoxy)-3-methylfuran-2(5H)-one and several of its derivatives have also been found to possess SL-like activity inhibit shoot branching in rice d10-1 mutants (Fukui et al. 2011a). In garden peas, it has been demonstrated that the presence of a Michael acceptor and a methylbutenolide or dimethylbutenolide motif in the same molecule is essential for controlling plant architecture (Boyer et al. 2012). The structure-activity relationships of SLs have been extensively studied regarding inducing the seed germination of root parasitic plants and inhibiting shoot branching (Xie 2016; Zwanenburg et al. 2009). Furthermore, apparent SL biological activity has been greatly influenced by differences in plant species and application methods (Umehara et al. 2015). The stereoisomers of SLs are also critical for hormonal activity (Scaffidi et al. 2014; Umehara et al. 2015).
Recently, SL structural requirements have been further conjectured because new SL signal transductions have been found. Previously, SL has been hydrolyzed into a covalently-linked intermediate molecule (CLIM), which is closely associated with the conformational change of AtD14 (Yao et al. 2016). When the hydrolase receptor D14 receives intact SL molecules, the D14 active signaling state is triggered and SL is hydrolyzed into a CLIM (Seto et al. 2019). Therefore, the intact SL structure, including the ABC ring, is potentially significant for biological activity. Karrikins are also butenolide compounds similar to SLs, and the receptor for karrikins (KARRIKIN INSENSITIVE 2; KAI2) is a homologous protein of D14. Therefore, KAI2-dependent signalling and structure of Karrikins become a hot topic in research. The butenolide methyl group is considered nonessential to KAI2 signalling, and Desmethyl-GR24 is active by KAI2 but not D14 (Yao et al. 2021). However, to date, the necessity of the methyl substituent in the D-ring for bioactivity has not been investigated regarding the inhibition of tillering through SL signalling in rice.
Here, therefore, we synthesized 29 analogs of SL that did not possess methyl groups and evaluated their effects on shoot branching inhibition. To determine whether these compounds acted through the SL pathway, we selected several to treat the SL signaling mutant d14 and investigated whether they could inhibit the extreme tillering of d14. Through these investigations, we aimed to explore the structural requirements of SLs for shoot branching inhibition in rice and provide new insights into SL signaling pathways.