A Role for MORE AXILLARY GROWTH1 (MAX1) in Evolutionary Diversity in Strigolactone Signaling Upstream of MAX2

Strigolactones (SLs) are carotenoid-derived phytohormones with diverse roles. They are secreted from roots as attractants for arbuscular mycorrhizal fungi and have a wide range of endogenous functions, such as regulation of root and shoot system architecture. To date, six genes associated with SL synthesis and signaling have been molecularly identified using the shoot-branching mutants more axillary growth (max) of Arabidopsis (Arabidopsis thaliana) and dwarf (d) of rice (Oryza sativa). Here, we present a phylogenetic analysis of the MAX/D genes to clarify the relationships of each gene with its wider family and to allow the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, there may be a diversity of SL-like compounds, the response to which requires a D14/D14-like protein. This ancestral system could have been refined toward distinct ligand-specific pathways channeled through MAX2, the most downstream known component of SL signaling. MAX2 is tightly conserved among land plants and is more diverged from its nearest sister clade than any other SL-related gene, suggesting a pivotal role in the evolution of SL signaling. By contrast, the evidence suggests much greater flexibility upstream of MAX2. The MAX1 gene is a particularly strong candidate for contributing to diversification of inputs upstream of MAX2. Our functional analysis of the MAX1 family demonstrates the early origin of its catalytic function and both redundancy and functional diversification associated with its duplication in angiosperm lineages.Strigolactones (SLs) are carotenoid-derived terpenoid lactones, which have been identified as signaling molecules in several areas of plant biology. SLs were first identified as germination stimulants for seeds of plants in the genus Striga (Cook et al., 1966). Striga spp. and related Orobanchaceae are parasitic weeds that germinate in response to host plant root exudates and develop haustoria to penetrate the host tissue and draw nutrients. Striga spp. are major agricultural pests across much of tropical and subtropical Asia and are present in two-thirds of arable land in Africa, where they are the greatest biological cause of crop damage (Humphrey and Beale, 2006). The secretion of SLs by roots, despite its exploitation by Striga spp., has been preserved because it also serves to recruit arbuscular mycorrhizal (AM) fungi (Akiyama et al., 2005). AM fungi form symbiotic associations with most land plants, whereby the plant gains access to mineral nutrients, particularly phosphate, absorbed by the fungal hyphae, and in exchange the fungus gains fixed carbon from the plant. In several flowering plant species, SL production is correspondingly increased when phosphate availability is limiting, thereby presumably increasing fungal recruitment (Yoneyama et al., 2007, 2012).AM symbioses can be traced back to the origin of land plants, between 360 to 450 million years ago, and are thought to have facilitated plant colonization of the terrestrial environment (Simon et al., 1993). Although AM symbiosis has been lost from some lineages, such as Brassicaceae, it is still widespread, with 80% of land plants able to form associations with AM fungi (Schüssler et al., 2001). In support of a similarly ancient origin for SL secretion, the liverwort Marchantia polymorpha and the moss Physcomitrella patens, both basal land plant groups, have been shown to produce SLs (Proust et al., 2011; Delaux et al., 2012). Furthermore, the presence of SLs in charophyte algae indicates that SL production may predate the emergence of land plants (Delaux et al., 2012), and Chara corallina responds to SL treatment by producing longer rhizoids (Delaux et al., 2012). In P. patens, SLs appear to act as intercolony coordination signals, regulating colony growth and competition by controlling flexible developmental processes such as protonemal branching (Proust et al., 2011; Delaux et al., 2012). In flowering plants, SLs have also been implicated in development, including several processes regulated in response to phosphate limitation (Kohlen et al., 2011; Ruyter-Spira et al., 2011). In particular, SLs play important roles in the regulation of shoot branching in higher plants (Gomez-Roldan et al., 2008; Umehara et al., 2008). It is through work on their effects on shoot branching that some of the genes in the SL pathway were first identified.Arabidopsis (Arabidopsis thaliana) MORE AXILLARY GROWTH (MAX) mutants show increased branching and reduced stature relative to wild-type plants, and analogous phenotypes have been identified in pea (Pisum sativum; RAMOSUS [RMS]), petunia (Petunia hybrida; DECREASED APICAL DOMINANCE [DAD]), and rice (Oryza sativa; DWARF [D] or HIGH TILLERING DWARF) mutants. So far, six MAX/RMS/DAD/D genes have been identified, with roles in SL biosynthesis or signaling. MAX3/RMS5/HIGH TILLERING DWARF1/D17 (Booker et al., 2004; Johnson et al., 2006; Zou et al., 2006) and MAX4/RMS1/DAD1/D10 (Sorefan et al., 2003; Snowden et al., 2005; Arite et al., 2007) encode carotenoid cleavage dioxygenases (CCD7 and CCD8, respectively). These enzymes are capable of sequentially cleaving the carotenoid 9-cis-β-carotene to produce a novel compound, carlactone, a putative strigolactone intermediate (Alder et al., 2012). Another biosynthetic gene, D27, was originally mutationally defined in rice (Lin et al., 2009), and reverse genetic approaches in Arabidopsis indicate a similar function in this species (Waters et al., 2012a). D27 is an iron-containing protein with isomerase activity that can produce the 9-cis-β-carotene substrate for MAX3 from all-trans-β-carotene (Alder et al., 2012). The fourth gene known to be involved in SL biosynthesis, MAX1, encodes a cytochrome p450 monooxygenase belonging to the CYP711 clan (Booker et al., 2005). Mutant phenotypes associated with this gene have so far only been identified in one species, Arabidopsis, although the gene is present in all tracheophytes (Nelson et al., 2008). The excessive-branching phenotypes associated with mutations in all of these genes can be rescued by exogenous application of SL, while mutants in the two remaining genes in the pathway are SL insensitive. D14 encodes an α/β hydrolase, which is proposed to act in signaling or in the hydrolysis of SLs to an active compound and provides specificity to signaling via MAX2/RMS4/D3, an F-box protein that mediates both SL signaling and signaling of karrikins (Stirnberg et al., 2002, 2007; Ishikawa et al., 2005; Johnson et al., 2006; Arite et al., 2009; Hamiaux et al., 2012; Waters et al., 2012b). Karrikins are compounds structurally related to SLs that are found in smoke and act as germination stimulants for plants that colonize ground cleared by forest fires (Nelson et al., 2010; Waters et al., 2012b).Homology searches described in the original publications for each of the MAX/D genes revealed two general patterns. MAX1, MAX3, and MAX4 are members of widespread gene families and are more closely related to nonplant sequences than to other plant genes (Sorefan et al., 2003; Booker et al., 2005). By contrast, MAX2, D14, and D27 are members of plant-specific gene families (Stirnberg et al., 2002; Arite et al., 2009; Lin et al., 2009). These contrasting patterns of SL pathway gene ancestry and the diverse biological roles of SLs present interesting evolutionary questions. The identification of SLs and SL responses in charophyte algae demonstrate their early evolution, but these species lack many of the genes required for SL synthesis and signaling in angiosperms. In an attempt to trace the evolution of the angiosperm SL pathway, we conducted a phylogenetic analysis of the known SL biosynthesis and signaling genes, allowing the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, the angiosperm pathway seems to have been defined by the rapid evolution of MAX2 in early land plants. Upstream of MAX2, there appears to be much greater flexibility, especially in the requirements for the synthesis of SLs. We present evidence for the contribution of MAX1 to this flexibility. Our functional analysis of MAX1 orthologs from phylogenetically diverse species demonstrates the early origin of its catalytic activity and both redundancy and functional diversification associated with its duplication in angiosperm lineages.