DISCUSSION
In this study, we propose a model in which beneficial rhizobacteriumBacillus spp. released VCs promotes LR branching through modulating oscillation. It increased the frequency of prebranch site formation and further accelerated the emergence of LRP from the PR, resulting in a denser LRP and LR along the PR spatially. This process is dependent on auxin signaling pathway; fully functional machinery of YUCs-mediated auxin biosynthesis and polar auxin transport are also required (Fig. 9).
By detecting the amplitude of the DR5:Luciferase oscillation and counting the numbers of prebranch sites, LRPs and LRs in series of auxin signaling and biosynthesis mutants, W. Xuan et al. (2015) concluded that both aspects of the oscillation, frequency and amplitude, are necessary for LR pre-patterning. In this study, we demonstrated that SQR9 VCs significantly enhanced the signal intensity of the DR5:Luciferaseexpression in the OZ (Fig. 3E) and subsequently formed prebranch sites in shorter period (Fig. 3C). It is suggested that meristem activity can influence the oscillation frequency (Berg & Tusscher, 2018). Likewise, we observed an enhanced expression of CYCB1 in the meristematic zone of SQR9 VCs-treated seedling (Fig. 2D), indicating that SQR9 VCs promote cell division in the root apical meristem. Moreover, the density of the prebranch sites and LR along the primary root was also increased by SQR9 VCs treatment despite the reduced number of prebranch sites with SQR9 VCs treatment for a longer time (Supplemental Fig. S3 B and C); it is conceivable that denser prebranch sites are the result of higher oscillation frequency while the reduced prebranch site number is due to the accelated LRP outgrowth. Together, we proposed that the enhanced LR pre-patterning should be caused by SQR9 VCs-induced stronger intensity and higher frequency of oscillation.
As a result of oscillation, the spatial distribution of LRP is established, followed by LR initiation, outgrowth, and emergence (W. Xuan et al., 2020). Increased early LRP stages (I–II) and decreased late (V–VII) LRP stages suggested SQR9 VCs induced more LR initiation and probably stimulated LRP emergence in advance (Fig. 1i), and that was further confirmed by the reduced number of prebranch sites with SQR9 VCs treatment for a longer time (Supplemental Fig. S3). The precise molecular mechanism by which SQR9 VCs regulate LRP development is still unclear, whereas histochemical staining of pDR5:GUS in LRPs highlighted the role of local auxin accumulation and signal activation in SQR9 VCs-modulated LRP development (Fig. 2A). The SLR/IAA14-ARF7/ARF19 module, which is an auxin signaling component, is vital for LR initiation. LRP and LRs are utterly absent from slr andarf7arf19 mutant roots (Fukaki, Nakao, Okushima, Theologis, & Tasaka, 2005; Fukaki et al., 2002; Y. Okushima et al., 2005; Wilmoth et al., 2005). Our results showed SQR9 VCs treatment could not rescue the lack of LRs in slr-1/iaa14 and arf7arf19 mutants (Fig. 2, B to D), and many Aux/IAAs were up-regulated by SQR9 VCs treatment (Fig. 4A; Supplemental Table S1). These data suggest SQR9 VCs-promoted LR initiation requires integral SLR/IAA14-ARF7/ARF19 module, and other Aux/IAAs may have a potential role in this process. Moreover, an endodermal SHOOT HYPOCOTYL2 (SHY2)/IAA3 auxin signaling module, which mediates the endodermal volume loss or shape change, is reported to be crucial for LR initiation (Vermeer & Geldner, 2015; J. E. Vermeer et al., 2014). Also, a PIN3-dependent hormone reflux pathway is necessary for the progress of LRFCs towards the LR initiation phase, and the pin3 mutation caused a decreased LRP development in stage I–II (Q. Chen et al., 2015). In this study, SQR9 VCs did not affect LR formation in CASP1pro:shy2-2 mutant and had a weaker effect on LR formation in pin3 (Fig. 4, B to D), andPIN3 was up-regulated by SQR9 VCs treatment (Fig. 5, E and G; Supplemental Table S1). It is tempting to speculate that SHY2-mediated endodermis deformation and PIN3-dependent auxin reflux between the endodermis and pericycle might be involved in SQR9 VCs-promoted LR initiation. Tang et al. (2017) reported that the FUSCA3 (FUS3)-LEAFY COTYLEDON2 (LEC2) complex synergistically activates the expression of the auxin biosynthetic gene YUC4 in the pericycle founder cells to function in LR initiation. The GH3.3 , which encodes acyl-acid-amido synthetases to cope with excessive free IAA inArabidopsis (Gutierrez et al., 2012; Staswick et al., 2005), andYUCs were up-regulated by SQR9 VCs treatment (Fig. 6B; Supplemental Table S1). Moreover, the expression site of up-regulatedDR5:GUS induced by SQR9 VCs, protoxylem pole, is consistent with one of the expression sites of GH3.3 (W. Xuan et al., 2015) (Fig. 2C). Considering the attenuated effect of SQR9 VCs on inducing LR formation with yucasin treatment and the enhanced effect of that on IBA-to-IAA conversion deficient mutant ech2ibr1ibr3ibr10 (Fig. 4, C and D; Fig. 6, A and C), these results indicated the potential roles of YUCs-mediated auxin biosynthesis in SQR9 VCs-induced LR initiation.
Altogether, our study provides a novel spatiotemporal regulation model of B. amyloliquefaciens SQR9-produced VCs on the LR formation, which can be instructive to understand how PGPRs or other environment factors reprogramme root architecture. Nevertheless, it remains unclear which chemical in SQR9 VCs play the major role in regulating LR formation. Despite the faint simulative effect of acetoin on LR formation, other substances in SQR9 VCs modulating RSA is worth further exploring.