The auxin indole-3-butyric acid controls adventitious rooting in Arabidopsis thaliana thin cell layers by its conversion into indole-3-acetic acid and nitric oxide production

Indole-3-butyric acid (IBA) is the natural precursor of the main plant auxin, indole-3-acetic acid (IAA), and both are able to induce adventitious root (AR) formation in different types of explants, when applied exogenously, alone or combined with other phytohormones. Arabidopsis thaliana thin cell layer (TCL) explant consists of the tissues of the inflorescence stem external to the vascular system, and previous researches have demonstrated that, when cultured in vitro under darkness in the presence of 10 µM IBA and 0.1 µM kinetin (Kin), these explants are able to form ARs and callus (Falasca et al, Plant Cell Rep, 2004). Similar results were also obtained using tobacco TCLs (Fattorini et al, Planta, 2009). It is known that exogenous IBA may induce AR formation better than IAA at the same concentration, e.g. in stem cuttings of A. thaliana including the vascular system, and it has been hypothesized that the promotion of AR formation by exogenous IBA occurs by an interaction with the endogenous IAA content (Ludwig-Müller et al, J Exp Bot, 2005). Although in dark-grown A. thaliana seedlings IBA is able alone to induce AR formation (Veloccia et al, J Exp Bot, 2016), the role of IBA alone in the TCL system has not yet been investigated. Furthermore, it is unknown whether A. thaliana TCLs contain endogenous IBA and/or IAA at culture onset. Results showed undetectable levels of IAA and IBA in TCLs after the excision, and it was an optimal premise to investigate AR formation under the total control of exogenous auxin. Col-0 TCLs were cultured under hormone-free condition, 0.1 µM Kin, 10 µM IBA, 10 µM IAA or 10 µM IBA + 0.1 µM Kin. The IBA alone treatment gave the best results as regards as mean AR production, AR development and limited amount of callus. Besides, the AR response of TCLs from mutant lines impaired in IBA-to-IAA conversion or tryptophan-dependent IAA biosynthesis or IAA polar intercellular transport, and analyses of TCLs from different transgenic GUS lines, allowed us to hypothesize that the peroxisomal conversion of IBA into IAA is the main mechanism by which IBA stimulates the adventitious rooting but not the only one. In fact, nitric oxyde (NO), an early by-product of IBA-to-IAA conversion known to be positively involved in AR formation (Pagnussat et al, Plant Physiol, 2002), might also affect positively IAA biosynthesis and transport to trigger AR induction in the TCL target cells. NO was detected and quantified in Col-0 IBA- and IAA-treated samples during the inductive phase of the rooting process. It was present more widely and at higher levels in IBA-treated TCLs than in IAA-treated ones. Results are important for helping to understand the IBA specific role in adventitious rooting, which is a natural process essential for the survival of numerous plant species but also essential for vegetative propagation via cuttings