Cluster roots are specialised lateral roots and therefore, a similar hormonal regulation as in lateral root formation must be expected. Auxin plays a key a role in the regulation of lateral root formation, starting with priming of founder cells in the pericycle, followed by induction, development and outgrowth of lateral root primordia, which involves PIN-mediated transport, gradient formation and signalling of shoot- and root-borne auxin (Chang et al., 2013). Accordingly, RT- qPCR analysis revealed the highest expression of various genes involved in auxin biosynthesis and transport (YUCCA, AUX1, PIN1) in the PE stage, followed by a continuous decline during outgrowth and CR maturation (Fig. 2A). This is in line with previous reports on the expression of auxin-related genes in CRs (Meng et al., 2013). A key role of auxins was also suggested by stimulation of CR formation after external auxin application even in P-sufficient plants (Gilbert et al., 2000; Neumann et al., 2000; Skene & James, 2000; Hocking & Jeffery, 2004), while the application of auxin transport antagonists exerts inhibitory effects (Gilbert et al., 2000).
An interesting new finding was the observation that similar to auxins, the expression of a transcript involved in conversion of the early brassinosteroid (BR) precursor 24- methylenecholesterol to campesterol in the BR biosynthesis pathway was high in PE and JU clusters, but declined dramatically during CR maturation. Lateral root formation can be promoted by BRs via the stimulation of acropetal auxin transport (Fukaki & Tasaka, 2009). Outgrowth of lateral root primordia seems to be particularly dependent on acropetal transport of shoot-borne auxin (Chang et al., 2013) and accordingly, the highest expression of BR-related genes was found in actively-growing JU clusters (Table 1, Fig. 2B).
Low levels of ethylene promote lateral root initiation by stimulation of auxin biosynthesis, while high ethylene concentrations exert inhibitory effects on lateral root initiation (Fukaki & Tasaka, 2009) and also on root elongation (Torrey, 1976). Consequently, the expression of transcripts encoding 1-aminocyclopropane-1- carboxylate (ACC) oxidase, S-adenosylmethionine (SAM) synthase and formamidase, as enzymes mediating ethylene biosynthesis, was low in PE and JU clusters, but a massive up-regulation was indicated by RNA-seq and RT-qPCR data (Table 1, Fig. 2B) in MA clusters, which exhibit no more growth activity. Up- regulation of genes involved in ethylene biosynthesis in CRs of white lupin is highly consistent with the data set of O‟Rourke et al. (2013), although that study did not differentiate between the different developmental stages of CRs. Increased ethylene production seems to be a general characteristic of P-deficient tissues (Lynch & Brown, 1997) and accordingly, Gilbert et al. (2000) reported increased production of ethylene also in root systems with CRs of P-deficient white lupin. This observation is in line with the lower P status of MA root clusters (Neumann et al., 1999; Massonneau et al., 2001) as massive Pi re-translocation occurs from MA to JU and PE stages, which have a higher P requirement. Although Gilbert et al. (2000) did not find effects of ethylene inhibitors on CR formation in Lupinus albus, in other plant species (Casuarina glauca), cluster root development was clearly stimulated by the external application of ethylene precursors (ACC) and inhibited by various ethylene antagonists (Zaid et al., 2003). Moreover, ethylene has also been implicated in the promotion of root hair development (Jung & McCouch, 2013) and accordingly, formation of long and densely-spaced root hairs covering the lateral rootlets is
characteristic for CR maturation and is associated with increased ethylene production (Neumann & Martinoia, 2002).
Cytokinins exert inhibitory effects on lateral root formation by affecting PIN- mediated auxin transport (Fukaki & Tasaka, 2009). In accordance, Neumann et al. (2000) demonstrated the inhibition of CR formation and elongation of lateral rootlets by external application of the synthetic cytokinin kinetin. Moreover, cytokinin-induced inhibition of auxin transport seems to be also involved in the formation of the auxin gradient that is required for the induction of lateral root primordia (Jung & McCouch, 2013). This may explain the preferential expression of cytokinin receptor (CRE) genes particularly in the PE stage of CR development (Table 1, Fig. 2B). Neumann et al. (2000) also reported elevated cytokinin concentrations in the root tissue of P-deficient white lupin, compared with P- sufficient control plants. As cytokinins are preferentially produced in growing root tips (Aloni et al., 2006), it was speculated that auxin-induced formation of JU cluster rootlets in P-deficient plants results in the increased production of cytokinins due to the large number of root tips (Neumann et al., 2000). The enhanced accumulation of cytokinins in the JU clusters may in turn contribute to the inhibition of rootlet elongation during CR maturation. Although inhibition of lateral root formation by high cytokinin levels is mainly discussed in the context of primordia induction (Jung & McCouch, 2013), inhibitory effects on lateral root elongation are also documented, possibly mediated by stimulation of ethylene biosynthesis (Skoog & Miller, 1957; Cary et al., 1995). The increased expression of transcripts encoding cytokinin oxidase (CKX) in MA root clusters (Table 1, Fig. 2B), similarly reported by Uhde- Stone et al. (2003), may indicate that cytokinin degradation occurs as a response to elevated cytokinin production by lateral rootlets in the JU stage. However, interactions of cytokinins and auxins are also required for development and maintenance of meristems (Su et al., 2011). In contrast to the PE stage, high expression of transcripts related to cytokinin oxidase (CKX), mediating cytokinin degradation, and the lack of cytokinin receptor (CRE) transcripts (Fig. 2B) may reflect cytokinin deficiency in the root tips of MA root clusters, finally leading to meristem inactivation, repression of their further elongation and formation of the functional clusters (Watt & Evans, 1999b).
Apart from the hormones mentioned above, the transcriptome studies on CR development also demonstrated distinct changes in transcript levels related to other hormones, such as abscisic and jasmonic acids (up-regulated in MA clusters, Fig. 3N, Appendix Suppl. Fig. S5), suggesting a putative involvement in the regulatory network of CR development, which requires further investigations. The same holds true for nitric oxide (NO) related gene expression and the potential role of NO signalling in CR induction (Wang et al., 2010; Meng et al., 2012).