Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity

Abstract

BackgroundIndole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 μM IBA is more AR-inductive than 10 μM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 μM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 μM) or IAA alone (10 μM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process.ResultsRevealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants.ConclusionsAltogether, results showed that IBA induced AR-formation by conversion into IAA involving NO activity, and by a positive action on IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis. Results are important for applications aimed to overcome rooting recalcitrance in species of economic value, but mainly for helping to understand IBA involvement in the natural process of adventitious rooting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1071-x) contains supplementary material, which is available to authorized users.

EIN2 and COI1 control the antagonism between ethylene and jasmonate in adventitious rooting of Arabidopsis thaliana thin cell layers

“…However, histological analyses in the hypocotyls of dark-grown Arabidopsis seedlings showed that exogenous Kin counteracted auxin action on AR-formation in planta, affecting both auxin transport and auxin maximum definition in the AR-apex (Della Rovere et al 2013). Based on these results, in following experiments IBA was applied alone to intact seedlings, but also to TCLs of Arabidopsis, and AR-formation was successfully obtained in both cases (Veloccia et al 2016;Fattorini et al 2017b). IBA alone favoured AR formation in Arabidopsis TCLs in comparison with IBA + Kin, whereas, in the presence of Kin alone, any morphogenic/organogenic response was obtained, i.e.…”

“…IBA alone favoured AR formation in Arabidopsis TCLs in comparison with IBA + Kin, whereas, in the presence of Kin alone, any morphogenic/organogenic response was obtained, i.e. the explants remained at the initial stage (Fattorini et al 2017b). When added with IBA, Kin favours cell division per se, leading to an increased callus formation and limiting the AR induction due to IBA (Fattorini et al 2017b;Fig.…”

“…the explants remained at the initial stage (Fattorini et al 2017b). When added with IBA, Kin favours cell division per se, leading to an increased callus formation and limiting the AR induction due to IBA (Fattorini et al 2017b;Fig. 2b).…”

“…However, Arabidopsis stem thin cell layers (AtTCLs) form a large amount of ARs when cultured in darkness on MS (Murashige and Skoog 1962) salt medium supplemented with 10 μM IBA and 0.1 μM kinetin (Kin) (Falasca et al 2004). It has been recently shown that in AtTCLs IBA affects AR-formation by its conversion into IAA (Fattorini et al 2017b). However, other hormones may be also important in the control of the AR-response, e.g., jasmonates (Fattorini et al 2018).…”

“…Arabidopsis seedlings exhibit a very limited AR-formation when cultured on MS medium with thiamine and myo-inositol but without hormones (Della Rovere et al 2013), and AtTCLs produce no AR under the same culture condition (Fattorini et al 2017b). However, AR-formation occurs not only with IBA plus Kin, but also with IBA alone both in planta and in TCLs (Veloccia et al 2016;Fattorini et al 2017b), whereas Kin exhibits an inhibitory role when applied alone (Della Rovere et al 2013;Fattorini et al 2017b). Up to now the AR-promoting effect of JA has been demonstrated only in the IBA + Kin condition, not only in Arabidopsis, but also in tobacco TCLs, and always in the presence of thiamine and myo-inositol in the medium (Fattorini et al 2009(Fattorini et al , 2018.…”