How does indole-3-butyric acid induce adventitious root formation in Arabidopsis thaliana thin cell layers?

Adventitious roots (ARs) are post-embryonic roots essential for plant survival and vegetative propagation via cuttings. In different types of explants, the auxin indole-3-acetic acid (IAA), and its natural precursor indole-3-butyric acid (IBA), when applied exogenously, are the main inducers of AR formation. In many cases, exogenous IBA is more effective in inducing ARs than exogenous IAA, but the reason needs investigation. Arabidopsis thaliana thin cell layers (TCLs) consist of stem inflorescence tissues external to the vascular system. Previous researches demonstrated that in TCLs excised from A. thaliana and tobacco exogenous IBA induces ARs when combined with kinetin (Falasca et al., Plant Cell Rep, 2004; Fattorini et al., Planta, 2009). However, in dark-grown A. thaliana seedlings IBA is able alone to induce AR formation (Veloccia et al., J Exp Bot, 2016). In cuttings as in planta, the endogenous IAA/IBA contents are determinant for the AR process, and differences in response to exogenous IAA/IBA may depend on differences in endogenous contents. It is unknown whether A. thaliana TCLs contain endogenous IAA/IBA at culture onset. Results showed that IAA and IBA were at undetectable levels at culture onset, and this was an optimal premise to investigate AR formation under the total control of exogenous auxin, revealing possible differences between IAA and IBA. The AR response of TCLs from various ecotypes, transgenic lines and knockout mutants showed that IBA was an AR inducer better than IAA. IBA positively affected IAA cellular influx, mediated by AUXIN RESISTANT1 (AUX1) and LIKE AUXIN RESISTANT3 (LAX3), and efflux, due to PIN-FORMED1 (PIN1), and expression of ANTHRANILATE SYNTHASE-alpha1 (ASA1), a gene of the tryptophan-dependent IAA biosynthesis. Results support that ASA1 and ANTHRANILATE SYNTHASE-beta1 (ASB1), the other subunit of the enzyme, are required for AR formation in the presence of exogenous IBA. The AR response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA conversion, was strongly reduced, showing that IBA action is indirect, i.e. IBA acts mainly by conversion into IAA. Nitric oxide (NO), a downstream signal of IAA, but also a by-product of the conversion process, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter ones. Altogether results showed that exogenous IBA induced AR formation in TCLs by conversion into IAA involving NO, IAA transport, and ASA1/ASB1-mediated IAA biosynthesis.